[a Fuchs] Inulin and Inulin-containing Crops(BookZZ.org)

Inutin and Inulin-containing Crops

Basedon the proceedings of the International Congress on Food and Non-Food Applications oflnulin and Inulin-containing Crops held in Wageningen, The Netherlands, 17-21 February 1991

Studies in Plant Science, 3

InuUn and Inuttn-containing Crops

Edited by

A. Fuchs Netherlands Organisation for Applied Scientific Research TNO Postal address: Department of Phytopathology Agricultural University Wageningen P. 0. Box 8025, 6700 EE Wageningen, The Netherlands

ELSEVIER

Amsterdam — London - New York — Tokyo 1993

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PREFACE

This book is mainly based on papers and posters presented at the International Congress on Food and Non-Food Applications of Inulin and Inulin-containing Crops, held in Wageningen, The Netherlands, 17-21 February 1991. However, in addition, it contains a number of other contributions, for the greater part by authors who had planned to attend the Congress, but for various reasons had to cancel. Though emphasis was put on inulin and inulin-containing crops, in fact the Congress' scope was wider, encompassing other fructans and fructan-containing plants as well. Even microorganisms producing and/or degrading fructans were dealt with, because their study very essentially contributes to our insight and understanding of fructan metabolism in general. The book clearly reflects this broad interest; beside many contributions on inulin- and inulin-containing crops there is quite a substantial number of papers on occurrence and metabolism of fructans in general.

In recent years, fructans, and in particular the inulins among them, are drawing attention not only for merely scientific reasons, especially within the EC they also enjoy an increasing interest for their potential to be utilized in food and non-food applications. Like the carbohydrates in general, fructans nowadays are considered a new challenge in the endeavour to develop a carbohydrate-based chemistry. In Belgium and The Netherlands this has already led to a new industrial activity embodied in several industries which produce inulin from chicory, with a total capacity which will soon be 100,000 tons per year. At present, inulin and its hydrolysis products (fructo-oligosaccharides and fructose) are mainly applied by the food industry, but forthcoming applications of inulin and inulin derivatives are also to be found in the non-food sector, for instance as dicarboxy-inulin, to be used as a biodegradable co-builder in detergents, and as a raw material for the production of hydroxymethylfurfural, a key-compound in furan chemistry.

These achievements have certainly been made possible by the concerted action of fundamental and applied research. In fact, our knowledge on the agronomy, breeding, biochemistry and molecular biology of fructan-containing plants, as well as on their processing, and on the chemistry and microbial conversion of fructan is rapidly expanding. The results of current research efforts, together with the progress being made in the area of industrial application are, indeed, distinct proof for the importance of this, until recently, neglected group of polysaccharides.

The Editor

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vii

ORGANIZING COMMITTEE

A. Fuchs Netherlands Organisation for Applied Scientific Research (chairman) TNO

(Postal address: Department of Phytopathology, Agricultural University, Wageningen)

J . C M . Smeekens Department of Molecular Cell Biology, (secretary) University of Utrecht, Utrecht

A J . Balfoort Dutch Sugar Foundation, Amsterdam

H. van Bekkum Department of Organic Chemistry, Delft Technological University, Delft

A.P.G. Kieboom Royal Gist-Brocades N.V., Delft

A.P.M. van der Veek Netherlands Organisation for Applied Scientific Research TNO, Delft

A.G.J. Voragen Department of Food Science, Section of Food Chemistry and Microbiology, Agricultural University, Wageningen

ADVISORY COMMITTEE

J. Barloy G. Bärwald N.J. Chatterton J. Fockedey A.D. French P. Galzy G. Gosse H. Hidaka H. Klaushofer N. Kosaric A. Margaritis M. Mesken E. Nitsch C.J. Pollock H. Schiweck H. Schnyder R.J. Simpson T. Uchiyama the late K. Vukov

Rennes, France Berlin, Germany Logan, UT, U.S.A. Pecq-Warcoing, Belgium New Orleans, LA, U.S.A. Montpellier, France Thiverval-Grignon, France Kawasaki, Japan Wien, Austria London, Ontario, Canada London, Ontario, Canada Dronten, The Netherlands Linz, Austria Aberystwyth, Wales, U.K. Grünstadt, Germany Bonn, Germany Melbourne, Victoria, Australia Osaka, Japan Budapest, Hungary

IX

ACKNOWLEDGEMENTS

The International Congress on Food and Non-Food Applications of

Inulin and Inulin-containing Crops

has been organized under the auspices of the Netherlands Organisation for Applied Scientific Research TNO, the Royal Dutch Chemical Society, and the Programme Committee on Carbohydrates of the Ministries of Agriculture, Nature Conservation and Fisheries, and of Economic Affairs, The Netherlands

The Organizing Committee gratefully acknowledges the financial support of the following organizations and industries:

- Netherlands Organisation for Applied Scientific Research TNO, The Netherlands

- Programme Committee on Carbohydrates of the Ministries of Agriculture, Nature Conservation and Fisheries, and of Economic Affairs, The Netherlands

- National Council for Agricultural Research, The Netherlands

- Cebeco Handelsraad, The Netherlands

- DSM, The Netherlands

- Generale Sucriere, France

- Royal Gist-Brocades N.V., The Netherlands

- Laevosan GmbH, Austria

- Meiji Seika Kaisha, Ltd., Japan

- Royal Dutch Chemical Society, The Netherlands

- Südzucker AG, Germany

- Suiker Unie, The Netherlands

- Tiense Suikerraffinaderij, Belgium

- Unilever, The Netherlands

X

Without naming them all, the Editor acknowledges the scientific help of many referees in the Netherlands and abroad. He also likes to thank Sjef Smeekens, Dick Smit, Liesbeth Hotke, Jetty Kentie and Piet van Egmond for their organizational and technical support before, during and after the Congress. In addition, he wants to express his gratitude towards the staff of TNO-Zeist for extending hospitality for many months, and last but not least towards Dorien Mertens for retyping most of the manuscripts submitted.

XI

CONTENTS

PREFACE V

ORGANIZING COMMITTEE VII

ADVISORY COMMITTEE VIII

ACKNOWLEDGEMENTS IX

AGRONOMY AND PROCESSING

Variability in agronomic and compositional characteristics of Jerusalem artichoke 1 F.A. Kiehn and B.B. Chubey

Characteristics of growth and development of different Jerusalem artichoke 11 cultivars J. Zubr and H. S. Pedersen

Multiannual, multilocational trials of Jerusalem artichoke in the south of Ireland: 21 soil, pH and potassium A. C. Cassells and M. Deadman

Crop characteristics and inulin production of Jerusalem artichoke and chicory 29 W.J.M. Meijer, E.W.J.M. Mathijssen and G.E.L. Borm

Leaf nitrogen, photosynthesis and crop productivity in Jerusalem artichoke 39 (Helianthus tuberosus L.) G. Soja, T. Samm and W. Praznik

Jerusalem artichoke productivity modelling 45 P. Denoroy

Experiences in cultivation, processing and application of Jerusalem artichoke 51 (Helianthus tuberosus L.) in Yugoslavia D. Pejin, J. Jakovljevic, R. Razmovski and J. Berenji

Xll

The effect of long-term storage on the fructo-oligosaccharide profile of Jerusalem 57 artichoke tubers and some observations on processing H.W. Modler, J.D. Jones and G Mazza

A process for the production of inulin and its hydrolysis products from plant 65 material M. Vogel

Pilot-scale production of inulin from chicory roots and its use in foodstuffs 77 E. Berghofer, A. Cramer, U. Schmidt and M. Veigl

Jerusalem artichoke costs of production in Canada: implications 85 for the production of fuel ethanol L. Baker, J. C Henning and P.J. Thomas sin

ANALYSIS, CHEMISTRY AND NON-FOOD APPLICATIONS

Separation and quantification of fructan (inulin) oligomers by anion exchange 93 chromatography N.J. Chatterton, P.A. Harrison, W.R. Thornley and J.H. Bennett

The applicability of enzymatic methods for the quantitative analysis 101 of fructan-containing plant extracts E. Stefanovits, W. Praznik, G. Soja, J. Kosäry, S. Kammerer, E. Cseke, J. Bocsi and L. Boross

Extraction and purification of preparative amounts of 1-kestose, 6-kestose, 107 neokestose, nystose and inulin-pentasaccharide H. Smouter and R.J. Simpson

Some colligative properties of fructans: implications for ryegrass 115 (Lolium perenne L.) grown under cool conditions S.C. Ilgoutz, G.D. Bonnett and R.J. Simpson

Recent advances in the structural chemistry of inulin 121 A.D. French

X l l l

Structure analysis of small inulin oligosaccharides by ID- and 2D-NMR 129 J.W. Timmermans, B.R. Leeflang and H. Tournois

Chemical modification of chicory root inulin 135 E. Berghofer, A. Cramer and E. Schiesser

Cycloinulo-oligosaccharides; structure and enzymatic synthesis 143 T. Uchiyama

Hydroxymethylfurfural, a possible basic chemical for industrial intermediates 149 M. Kunz

Enzymatic synthesis of hydroxymethylfurfural esters 161 A.C. Besenter, J.P. van der Lugt and H.J. Doddema

BIOCHEMISTRY, MICROBIOLOGY AND MOLECULAR BIOLOGY

Unresolved problems in the enzymology of fructan metabolism 167 C.J. Pollock, A.M. Winters and A.J. Cairns

Purification and properties of sucrose:sucrose fructosyltransferases 173 from barley leaves and onion seeds G.C. Angenent, M.J.M. Ebskamp, P.J. Weisbeek and J.C.M. Smeekens

Fructan accumulation in cell suspension cultures of Phleum pratense L. 185 M. Frehner, M. Lascher and J. Nosberger

Fructan polymerization and depolymerization during the growth of chicory 191 {Cichorium intybus L.) plants A. Limami and V. Fiala

Fructan exohydrolase from Lolium rigidum Gaud. 199 G.D. Bonnett and R.J. Simpson

Regulation of activity and properties of inulinases from roots of Cichorium 205 intybus L. A.K. Gupta, H. Jain, N. Kaur and R. Singh

XIV

Thermostable inulinases from Aspergillus ficuum: biochemical characterization 211 and biotechnological applications J. Baratti and M. Ettalibi

Comparative studies of soluble and immobilized inulin-hydrolysing enzymes 217 L. Boross, W. Praznik, J. Kosary and E. Stefanovits

Immobilization of inulinase in activated carbon for the production 223 of very-high-fructose syrups from Jerusalem artichoke tuber extracts J.M. Magro, M.M.R. da Fonseca and JM. Novais

Preparation and characterization of aqueous inulin/exo-inulinase systems 231 A. Huber, W. Praznik, T. Spies, S. Kammerer and C. Knabl

Production and localization of inulinase in Kluyveromyces yeast 241 M.C.M. Hensing, R.J. Rouwenhorst, W.A. Scheffers andJ.P. van Dijken

Production of high-fructose-containing syrups from Jerusalem artichoke extracts 251 with fructose enrichment through fermentation A. Fontana, B. Hermann and J.P. Guiraud

Modification of the transfructosylation activity of Bacillus subtilis levansucrase 259 by solvent effect and site-directed mutagenesis R. Chambert and M.-F. Petit-Glatron

A biotechnological and ecophysiological study of thermophilic inulin-degrading 267 clostridia WJ. Drent, G.J. Both and J. C. Gottschal

Inulin degradation by Pediococcus pentosaceus 273 W.J. Middelhoven, P.F.L.A. van Adrichem, M.W. Reij and M. Koorevaar

In vitro synthesis of inulin by the inulosucrase from Streptococcus mutans 281 A.S. Ponstein and M.B. van Leeuwen

XV

The cloned Bacillus subtilis levanase gene as a potent system for the exploitation 289 of inulin in biotechnological processes H. Schwab and E. Wanker

Modelling inulin metabolism 297 M. W. Shaw, K. Lodge and P. John

FOOD AND MEDICAL APPLICATIONS

The occurrence of fructan in food plants 309 L.D. lncoll and G.D. Bonnett

Jerusalem artichoke as a multipurpose raw material for food products 323 of high fructose or inulin content J. Barta

Preparation of pure inulin and various inulin-containing products 341 from Jerusalem artichoke for human consumption and for diagnostic use K. Vukov, M. Erdelyi and E. Pichler-Magyar

Production and characteristics of fructo-oligosaccharides 347 M. Hirayama, K. Nishizawa and H. Hidaka

Production of fructo-oligosaccharide-rich fructose syrup 355 H. Yamazaki and K. Matsumoto

Potential medicinal and nutritional uses of chicory roots and inulin 359 Λ.Κ. Gupta, N. Kaur, M. Kaur and R. Singh

Valorization of an inulin-rich by-product of chicory 367 E. Leclercq and G. Hageman

Activity of inulinase of some strains of Bifidobacterium and their effects 373 on the consumption of foods containing inulin or other fructans Pudjono, G. Bärwald and S. Amanu

XVI

Inulin fermentation in germ-free rats associated with a human intestinal flora 381 from methane or non-methane producers C. Andrieux, S. Lory, C. Dufour-Lescoat, R. de Baynast and O. Szylit

The use of Jerusalem artichoke flour in pig and chicken diets 385 E.R. Farnworth, J.D. Jones, H.W. Modler and N. Cave

The use of inulin for the determination of renal function: applicability 391 and problems N. Gretz, M. Kirschfink and M. Strauch

MISCELLANEOUS

Some aspects of research on inulin and inulin-containing crops in the Ukraine 397 G. Lezenko, L. Bobrovnik, I. Grinenko, R. Grushetsky, I. Guly, J. Tsokur and O. Vdovenko

INDEX 401

AGRONOMY AND PROCESSING


A. Fuchs (Ed.) Inulin and Inulin-containing Crops 1993 Elsevier Science Publishers B.V. 1

VARIABILITY IN AGRONOMIC AND COMPOSITIONAL CHARACTERISTICS OF JERUSALEM ARTICHOKE

F.A. KIEHN and B.B. CHUBEY Agriculture Canada Research Station, P.O. Box 3001, Morden, Manitoba, Canada ROG 1 JO

ABSTRACT

Thirty selected accessions of Jerusalem artichoke {Helianthus tuberosus L.) from North America and Europe were investigated under southern Manitoba conditions to determine the existing variability of several agronomic characteristics. Early, mid-season and late maturing lines were grown to determine if differences in the characteristics were evident between groupings.

Fresh tuber yields ranged from 4.4 to 76.5 t ha"1, while dry matter yields ranged from 2 to 13 t ha"1. The early maturing group produced the lowest dry matter yield with a trend to increased yields through mid-season to late maturity. No real differences in reducing sugar content were found between the groups. Plant height varied from 115 to 275 cm, with the early maturity group being shorter than either the mid-season or late group.

A large degree of variability was found in all groups in maturity and plant height as well as in tuber, dry matter, and reducing sugar yield.

1 INTRODUCTION Jerusalem artichoke {Helianthus tuberosus L.), a close relative of the common

sunflower, is native to a large area of North America including the prairie region. It produces dense top growth with underground tubers on the ends of stolons. Two cultivars have been released in Canada, Columbia for high tuber yield and Challenger, for both tuber and top growth yield. High tuber yields have been produced under southern Manitoba conditions, ranging from 46 to 60 t ha"1 for Columbia (Chubey and Dorrell, 1982) and 31.5 to 51 t ha"1 for Challenger (Kiehn and Chubey, 1985).

Jerusalem artichoke has been under investigation as a potential crop for over a period of fifty years in this century, primarily on a trial or small-scale basis and at intermittent intervals. Each time this crop has been considered, the lack of agronomic and processing technology, lack of markets or generally low, variable yields from existing cultivars (Boswell et aL, 1936; Chubey and Dorrell, 1974) have placed the crop at a competitive disadvantage when compared with alternative crops.

2

In recent years with the availability of higher yielding cultivars, this crop has been shown to have good potential for the production of fructose sugar (Chubey and Dorrell, 1974) and protein and flour (Mazza, 1985). It also has been shown to have potential as a feedstock for the production of ethanol (Margaritis and Bajpai, 1982). The top growth is an excellent animal feed and a good source of protein (Rawate and Hill, 1985). The tops are a good source of fiber and can also be utilized as a feedstock for the production of ethanol.

The presently available cultivars Columbia and Challenger, although having high tuber yields, could be improved in tuber shape and composition, disease resistance and storage quality. The improvement of these characteristics requires knowledge of germplasm variability that exists. Apart from some Dutch reports (Mesken and Spitters, 1988; Van Soest and De Jong, 1988; Mesken, 1989), there presently is very little information, in scientific literature, on the agronomic and biological variability available within the present world gene pool.

A study was undertaken to investigate the existing variability of agronomic characteristics of several Jerusalem artichoke accessions grown under southern Manitoba conditions. The Jerusalem artichoke accessions used in this study were selected from a collection obtained over the past fifteen years, from world-wide sources. They were grown on sandy loam soil at the Agriculture Canada Research Station, Morden, Manitoba in 1981, 1982 and 1983. They were grown in single-row plots 3 m long, at a plant density of three plants per meter, with 1 m between rows and replicated three times. The trials were planted on April 28 in 1981 and on May 9 in 1982 and 1983. The plot area was fertilized with 200 kg ha"1 23-23-0 applied broadcast and incorporated into the soil prior to planting. In early August of 1981 and 1982, irrigation at 45 mm and 50 mm, respectively, was applied to the plots after the plants had displayed severe water stress symptoms. No irrigation was applied in 1983.

Thirty Jerusalem artichoke accessions were divided into three maturity groups based on the number of days to full flower or absence of flowering within the growing season. These maturity groups were: a) early - those that reached full flower within 100 days, b) medium - those that reached full flower in 100 to 135 days, or c) late - those that required over 135 days to reach full flower or did not flower within the growing season.

Tubers form each plot were harvested in the second or third week of October in each year and yields were recorded. The tubers were then placed in polyethylene bags and stored at 2 ± 1 °C for 30 days at which time total reducing sugars and dry matter content were determined.

3

Table la. Influence of early, medium and late maturity on dry matter and reducing sugar yields and percent, and plant height, of Jerusalem artichoke in three years (1981-1983).

Maturity

Early

Medium

Late

Year

1981 1982 1983 Mean

1981 1982 1983 Mean

1981 1982 1983 Mean

Dry matter yield (t ha'1)

5.96 4.39 4.23 4.86 a

6.10 6.44 3.01 5.18 a

8.54 7.64 3.69 6.62 a

Dry matter (%)

22.1 24.4 24.7 23.7 a

23.2 26.6 24.1 24.6 a

18.0 21.1 18.6 19.2 b

Reducing sugar yield (t ha"1)

4.11 2.74 2.14 3.00 a

3.98 4.06 1.76 3.27 a

5.99 5.12 2.22 4.44 a

Reducing sugar % of dry matter

67.4 61.9 51.7 60.3 a

63.9 61.5 56.9 60.8 a

69.1 67.6 58.3 65.0 a

Plant height (cm)

156 156 173 162 b

186 215 219 207 a

198 238 208 215 a

Table lb. Influence of three years 1981-1983 on dry matter and reducing sugar yields and percent, and plant height, of all three maturity ranges of Jerusalem artichoke.

Year

1981

1982

1983

Maturity

Early Medium Late Mean



Dry matter yield (t ha"1)

5.96 6.10 8.54 6.87 a

4.39 6.44 7.64 6.16 ab

4.23 3.01 3.69 3.64 b

Dry matter (%)

22.1 23.2 18.0 21.1 b

24.4 26.6 21.1 24.0 a

24.7 24.1 18.6 22.5 ab

Reducing sugar yield (t ha"1)

4.11 3.98 5.99 4.69 a

2.74 4.06 5.12 3.97 a

2.14 1.76 2.22 2.04 b

Reducing sugar % of dry matter

67.4 63.9 69.1 66.8 a

61.9 61.5 67.6 63.7 a

51.7 56.9 58.3 55.6 b

Plant height (cm)

156 186 198 180 a

156 215 238 203 a

173 219 208 200 a

a-b The same letters are not significantly different at P < 0.05 according to Duncan's Multiple Range Test.

2 RESULTS AND DISCUSSION In 1981 and 1982 overall tuber yields increased from early through medium to late

maturing groups. Relatively low moisture levels at the beginning of the 1983 season

4

Table 2. Tuber yield, dry matter and sugar content of 30 Jerusalem artichoke cultivars.

Entry

Early

NC10-3 NC10-11 NC10-12 NC10-23 NC10-31 NC 10-35 NC 10-38 NCI 0-52 NC 10-55 NC 10-59

7305 7513 HMHYA HM-12 DHM-14 DHM-21 W-106 DHM-143 7513A W-1061

Country of origin

Canada Canada Canada Canada Canada Canada Canada Canada Canada Canada

Days to full flower

90 98 98 90 91 89 90 85 93 88

Tuber skin colour

white white white white white white white white white white

Tuber

1981

38.34 28.44 31.50 26.63 23.51 26.92 17.24 11.60 29.35 31.42

yield (t ha"

1982

19.61 16.49 17.86 22.26 15.57 19.32 13.87 17.31 20.50 17.10

l)

1983

7.00 15.03 21.86

5.73 9.41

10.21 24.11 22.38 21.45 32.89

Medium

NC10-2 NC10-8 NC10-15 NC 10-24 NC 10-37 NC 10-40 NC 10-48 NC 10-58 NCI 0-62 NC10-69

A-A 7310 HM-2 HM-13 W-97 Comber A-36 W-97-K1A 266 Columbia

Canada Canada Canada Canada Canada U.S.A. Canada Canada France Canada

131 135 132 126 120 134 121 109 123 105

white white white white white red white white red white

23.91 11.46 30.19 20.44 20.33 39.39 16.33 29.35 13.64 76.52

20.67 23.53 28.19 27.03 21.29 21.12 18.17 21.17 17.99 46.25

16.59 10.74 8.83 6.56 4.40

13.53 7.84 8.23

12.78 45.08

Late

NC10-4 NC 10-20 NC 10-39 NC 10-42 NC 10-43 NC 10-67 NC 10-71 NC 10-72 NC 10-73 NCI 0-75

7306 HM-9 Challenger Red I.B.C. PI-4 Yankton-1 Rizskij Interess Volzskij-2 Leningradskij

Canada Canada Canada Canada U.S.A. U.S.A. U.S.S.R U.S.S.R U.S.S.R U.S.S.R

white white white red white white white white white white

44.05 38.32 67.04 54.30 39.48 46.47 47.00 52.73 45.09 41.77

40.15 35.12 49.23 28.91 36.90 29.58 30.58 38.98 37.91 32.41

9.30 24.34 36.93 6.14

13.07 15.34 23.46 36.62 16.82 15.36

* Over 135 days, or no flowering to end of season.

combined with continued low rainfall and very high temperatures produced conditions that severely reduced tuber dry matter (DM) yields. The early lines had the highest DM yields and were the least affected by the stress conditions that became more severe as the season

Table 2. (continued)

5

Entry

Early

NCI 0-3 NC10-11 NC10-12 NC10-23 NCI 0-31 NC 10-35 NC 10-38 NCI 0-52 NCI 0-55 NCI 0-59


Dry i

1981

22 24 22 23 27 20 20 23 19 21

natter content (%)

1982

27 23 23 25 26 22 23 26 24 25

1983

29 22 22 24 26 21 26 28 22 27

Sugar

1981

19 17 18 13 18 13 9

17 12 15

content

1982

16 11 15 17 17 15 13 12 15 18

(%)

1983

13 9

10 15 16 12 13 12 12 14

Medium

NCI 0-2 NCI 0-8 NC10-15 NCI 0-24 NCI 0-37 NCI 0-40 NC 10-48 NC 10-58 NCI 0-62 NCI 0-69


25 24 22 24 28 20 26 24 22 17

27 26 23 27 27 26 30 27 30 23

24 30 20 22 24 22 24 28 23 19

16 12 14 14 18 13 18 14 15 12

13 16 16 21 18 14 19 15 15 16

12 16 14 13 13 12 12 14 14 14

Late

NCI 0-4 NCI 0-20 NC 10-39 NC 10-42 NC 10-43 NCI 0-67 NC 10-71 NCI 0-72 NC 10-73 NCI 0-75


21 23 18 19 17 17 18 16 17 16

21 23 22 20 22 22 23 20 21 17

16 22 18 19 17 22 19 18 17 18

15 15 15 12 10 11 13 10 11 10

13 13 15 14 13 18 15 15 14 12

9 14 13 11 10 11 10 10 10 10

progressed. No irrigation was applied and many of the medium maturity lines dried down prematurely resulting in tuber DM yields being only half those of previous years. The later maturing lines had improved environmental conditions at the time of tuber sizing, due to

6

Table 3. Dry matter, tuber yield, sugar content and plant height of 30 Jerusalem artichoke cultivars.

Entry Dry matter tuber Dry matter content Reducing sugar yields (t ha"1) (%) (t ha'1)

1981 1982 1983 1981 1982 1983 1981 1982 1983

Early


8.28 6.45 6.93 6.02 6.35 5.38 3.52 2.70 7.67 6.57

5.29 3.79 4.11 5.56 4.05 4.25 3.19 4.50 4.92 4.27

2.03 3.31 4.81 1.37 2.47 2.14 6.27 6.31 4.72 8.88

22 24 22 23 27 20 20 23 19 21

27 23 23 25 26 22 23 26 24 25

29 22 22 24 26 21 26 28 22 27

7.21 4.97 5.67 3.46 4.33 3.63 1.59 2.02 4.92 4.62

3.23 1.90 2.73 3.87 2.68 2.92 1.75 2.11 3.09 3.11

0.90 1.41 2.18 0.85 1.47 1.28 3.23 2.68 2.62 4.77

Medium


5.90 2.76 6.24 4.91 5.71 8.07 4.31 7.16 2.97

12.93

5.58 6.12 6.94 7.30 5.57 5.49 5.45 5.72 5.40

10.64

3.98 3.22 1.76 1.44 1.06 2.97 1.88 2.30 2.94 8.56

25 24 22 24 28 20 26 24 22 17

27 26 23 27 27 26 30 27 30 23

24 30 20 22 24 22 24 28 23 19

3.94 1.39 4.19 2.93 3.72 4.97 2.92 4.22 2.09 9.41

2.77 3.76 4.69 5.70 3.85 2.96 3.38 3.29 2.72 7.48

1.99 1.72 1.20 0.85 0.58 1.60 0.98 1.20 1.75 5.76

Late


9.32 8.77

12.14 10.15 6.67 7.80 8.32 8.33 7.44 6.47

8.43 8.43

10.84 5.78 8.12 6.51 7.04 7.79 7.96 5.51

1.48 5.35 6.65 1.17 2.22 3.37 4.46 6.59 2.86 2.77

21 23 18 19 17 17 18 16 17 16

21 24 22 20 22 22 23 20 21 17

16 22 18 19 17 22 19 18 17 18

6.86 5.90

10.09 6.67 4.15 5.30 6.25 5.59 4.87 4.25

5.24 4.74 7.34 4.00 4.76 5.24 4.74 5.92 5.34 3.92

0.80 3.36 4.84 0.63 1.31 1.77 2.46 3.84 1.70 1.47

lower temperatures and increased moisture which may have resulted in slightly higher yields, although they also only attained approximately one-half the yield obtained in the two previous years.


7

Entry Sugar content Plant height (% of DM) (cm)

1981 1982 1983 1981 1982 1983

Early

7305 7513 HMHYA HM-12 DHM-14 DHM-21 W-106 DHM-143 7513 A W-1061

87 73 82 57 68 67 45 75 64 70

61 50 66 70 66 69 55 47 63 73

44 43 45 62 60 59 52 42 55 54

144 140 136 149 132 116 211 133 202 201

153 143 145 148 122 135 227 126 153 205

175 163 163 173 162 133 200 162 170 232

Medium


67 50 67 60 65 61 68 59 70 73

50 61 68 78 67 54 62 57 50 70

50 53 68 59 55 54 52 52 60 67

154 194 140 185 206 251 169 188 196 180

203 227 195 200 223 273 202 195 232 200

202 218 190 235 212 267 211 190 270 193

Late


73 67 81 66 62 68 75 67 65 66

62 56 68 68 58 80 67 76 67 71

54 63 73 56 59 52 55 58 59 53

201 223 211 200 190 178 197 202 197 183

280 257 247 222 193 240 250 248 223 218

220 227 225 197 203 207 213 203 212 178

The early maturing accessions produced the lowest DM tuber yields with a trend to increased yields from medium to late maturing accessions over the three-year period. There was, however, no significant difference in DM yield between the maturity groups (Table

8

la). There were significant differences in % of DM, between years and between maturity groups. Percent DM in 1982 was significantly higher than in 1981 while neither was significantly different from 1983. The late maturing group was significantly lower in % of DM than either the early or medium maturing group.

Reducing sugar yield and reducing sugar % of DM were significantly lower in 1983 than in either of the two previous years (Table lb). There were, however, no significant differences between the maturity groups. This could possibly be explained by the unfavourable environmental conditions in 1983 which adversely affected the medium and late maturing lines reversing the trend of the two previous years. Plant heights were similar over all years with the early accessions being significantly shorter than the two later groups.

Tuber yields of the individual cultivars varied between years with a few exceptions. Columbia, a medium maturity and Challenger, a late maturity cultivar, fluctuated much less than many other cultivars and were consistently the two highest yielding lines for both dry matter and reducing sugars. However, they were among the lowest for dry matter content (Table 2). Average dry matter yields of Columbia tubers ranged from 12.93 t ha"1 in 1981, to 10.64 t ha"1 in 1982, and 8.56 t ha"1 in 1983; while for Challenger the dry matter yields were 12.14 t ha"1, 10.84 t ha"1 and 6.65 t ha"1 for the three years, respectively (Table 3). This represented a decrease of 45 % for Challenger and only 34 % for Columbia. Breeding programs which would concentrate on increasing the dry matter percentage in high yielding cultivars, such as Challenger or Columbia, should therefore be expected to produce lines with even higher tuber yields.

From this limited study we were able to demonstrate a fairly wide variation in the observed characteristics within and between maturity groups. The knowledge of available germplasm and the range of variability which exists at present in the world gene pool for selected agronomic characteristics in Jerusalem artichoke is essential in any successful breeding program. Such information is required for the selection of parents or genetic material that could be utilized in the improvement of several traits of present cultivars. Knowledge of the range of germplasm variability is also important in the development of new cultivars for a specific end use that require the transfer and incorporation of high yield and improved quality factors or disease resistance.

3 REFERENCES

Boswell, V.R., Steinbauer, C.E., Babb, M.F., Burlison, W.L., Alderman, W.H. and Schoth, H.A., 1936. Studies of the culture and certain varieties of the Jerusalem artichoke. U.S., Dep. Agric, Tech. Bull., 514: 1-69.

Chubey, B.B. and Dorrell, D.G., 1974. Jerusalem artichoke, a potential fructose crop for the prairies. Can. Inst. Food Sei. Technol. J., 7: 98-100.

Chubey, B.B. and Dorrell, D.G., 1982. Columbia Jerusalem artichoke. Can. J. Plant Sei., 62: 537-539.

9

Kiehn, F.A. and Chubey, B.B., 1985. Challenger Jerusalem artichoke. Can. J. Plant Sei., 65: 803-805. Margaritis, A. and Bajpai, P., 1982. Ethanol production from Jerusalem artichoke tubers (Helianthus

tuberosus) using Kluyveromyces marxianus and Saccharomyces rosei. Biotechnol. Bioeng., 24: 941-953.

Mazza, G., 1985. Distribution of sugars, dry matter and protein in Jerusalem artichoke tubers. Can. Inst. Food Technol. J., 18: 263-265.

Mesken, M., 1989. Induction of flowering, seed production, and evaluation of seedlings and clones of Jerusalem artichoke (Helianthus tuberosus L.). In: G. Grassi and G. Gosse (Eds.), Topinambour (Jerusalem artichoke), Proc. EC Workshop, 30 Sep. - 1 Oct. 1987, Madrid, Report EUR 11855. CEC, Luxembourg, pp. 137-143.

Mesken, M. and Spitters, C.J.T., 1988. De veredeling van de aardpeer. In: A. Fuchs (Ed.), Versl. Tweede Themadag Inuline, 30 Oct. 1987, Wageningen. NRLO-rapport nr. 88/5, The Hague, pp. 18-32.

Rawate, P.D. and Hill, R.M., 1985. Extraction of a high-protein isolate from Jerusalem artichoke (Helianthus tuberosus) tops and evaluation of its nutrition potential. J. Agric. Food Chem., 33: 29-31.

Van Soest, L.J.M. and De Jong, W., 1988. Genetische variatie in Helianthus tuberosus L. In: A. Fuchs (Ed.), Versl. Tweede Themadag Inuline, 30 Oct. 1987, Wageningen. NRLO-rapport nr. 88/5, The Hague, pp. 86-92.


A. Fuchs (Ed.) Inulin and Inulin-containing Crops © 1993 Elsevier Science Publishers B.V. All rights reserved. 11

CHARACTERISTICS OF GROWTH AND DEVELOPMENT OF DIFFERENT JERUSALEM ARTICHOKE CULTIVARS

Josef ZUBR* and Henning Schjoler PEDERSEN** Department of Agricultural Sciences, Royal Veterinary and Agricultural University, Rolighedsvej 23, 1958 Frederiksberg, Denmark Botanical Laboratory, Copenhagen University, Gothersgade 140, 1123 Copenhagen, Denmark

ABSTRACT

A field trial with eleven cultivars of Jerusalem artichoke (Helianthus tuberosus L.) was carried out in 1990. During the season, quantitative and qualitative parameters of growth and development were recorded at intervals of two weeks. Large differences were noticed in distribution of photosynthetic products between underground and aerial plant parts of early and late cultivars. Tuber yields amounted to about 12 t dry matter (DM) ha"1 for early cultivars, and about 7 t DM ha"1 for late cultivars. Tuber characteristics were evaluated qualitatively. At harvest in autumn, the largest amounts of inulin with a degree of polymerization (DP) > 4 were found in late cultivars with red tubers, and in tubers of the dwarf cultivar.

1 INTRODUCTION Jerusalem artichoke is known as a crop with a high production potential (Stauffer et

al., 1981; Kosaric et al., 1984). Under the climatic conditions of Northern Europe, the potential yield of tubers from early cultivars was assumed to be about 10 t DM ha"1 (Zubr, 1990). Local field experiments showed that the yield of tubers was determined by inherent properties of the cultivars, and by various exogenous factors, such as nutrition, plant density, water supply, evapotranspiration, etc. Early cultivars usually produced higher and less variable yields of tubers than late cultivars. Long experience with cultivation of Jerusalem artichoke under the local climatic conditions has shown that in late cultivars the translocation of photosynthetic products from aerial plant parts into tubers is not complete before the end of the vegetation period (Zubr, 1989).

The present investigation was designed to evaluate the characteristics of growth and development of different Jerusalem artichoke cultivars, particularly with regard to tuber yield.

12

2 EXPERIMENTAL CONDITIONS AND METHODS The investigation was carried out at the Experimental Station of the Royal Veterinary

and Agricultural University, Tästrup, 55°40' N, 12° 18' E, close to Copenhagen. The experimental field was a clay soil; it was partly protected against the prevailing (westerly) wind. The 1990 season was characterized by a low photosynthetically active radiation (PAR) in June, and a period of drought during the early summer (Table 1).

The plant bed was prepared by rototilling. Calibrated seed tubers were planted by hand on March 24, in rows 60 cm apart. Spacing between plants was about 35 cm in early cultivars (Urodny, Mari, Nora, Columbia, clone 2071-63) and in the dwarf cultivar, and about 53 cm in the late cultivars (K 24, Karina, Refla and Reka). Macronutrients (50 kg P and 300 kg K ha"1) were supplied in the form of fertilizers. Nitrogen was added in the form of urea (46% N), sprinkled on the soil surface before the first interrow cultivation in the beginning of May. In the beginning of June, the cultivation treatment was completed by earthing.

Representative samples of plants were taken for analysis from June 25 to October 20 at two-week intervals. Sampling in October was restricted to late cultivars and the dwarf cultivar. Height of plants, number of stalks, branches, leaves, buds and flowers and tubers, as well as fwt of all plant parts separately, were recorded.

DM was determined using standard methods (after drying at 105 °C, for 24 h). Leaf

Table 1. Local weather conditions (Hansen, 1981; Jensen, 1990).

Parameter

PARb

Normal E m"2

1990 E m"2

Temperature (air Normal °C 1990 °C

Rainfall0

Normal mm 1990 mm

temperature

Evapotranspirationc

Normal mm 1990 mm

Aa

787 788

M

1139 1178

at a height of 2 5.7 7.8

36 47

59 66

11.2 12.0

44 21

94 87

J

1126 980

m)c

14.2 14.9

66 48

95 94

J

1189 1146

16.1 15.8

64 37

108 116

A

948 940

15.9 16.8

64 50

86 96

S

588 518

12.6 11.5

61 112

49 47

O

308 285

9.3 9.5

62 72

25 20

Total

6086 5835

12.1d

12.6d

398 387

514 525

a A - O, April - October (the growing season in Denmark). b Normal PAR (= photosynthetically active radiation, 400-700 nm), for the period 1975-1990. c Normal, for the period 1955-1990. d Mean.

13

area was measured using a Li-Cor M 3100 area meter. Tuber slices were investigated microscopically using a Wild-5 stereo microscope. Growth of cortex, radial parenchyma, xylem rays, and pith were measured at two-week intervals.

The sugar content of tubers was determined using a Carl Zeiss refractometer. Samples of tuber juice for carbohydrate analysis were obtained from sliced tubers, boiled at 1.5 bar pressure for 60 min. The juice was cleaned using an ion exchanger to remove substances which could interfere with the analysis. Composition of carbohydrates was determined using HPLC, equipped with Barien autosampler, Aminex HPX-87C column, Altex RI detector, and Hewlett Packard integrator. The analyses were carried out at a constant temperature of 85 °C and a flow of 0.7 ml min"1, in 5 μΐ-samples.

3 RESULTS AND DISCUSSION The main experimental results are presented in histograms to visualize the differences

between cultivars. The results were evaluated according to typical parameters of growth and development.

3.1 Growth of plants In the 1990 season, shooting started at the end of April. Rapid growth of plants was

observed in the beginning of June. At maximal growth, 18-22 branches were found on the stalks of early cultivars, except for the clone 2071-63, which had 27 branches per plant. Late cultivars with white tubers had 30-33 branches, whereas those with red tubers (Refla and Reka) had 22 and 40 branches, respectively. In dwarf cultivars, only few branches developed from the base of the stalks. Small branches (< 3 cm) appeared during late summer.

The early cultivars, except for the clone 2071-63, attained their final height of about 140 cm at the end of July. In late cultivars, longitudinal growth was continuous, and ceased only in the middle of October when the plants were about 280 cm high. In the dwarf cultivar, the growth of stalks continued up to the beginning of September. The tops were then only about 60 cm high.

In early cultivars the amount of DM in stalks increased up to the end of July, and in late cultivars up to the end of September (Fig. 1). During summer, in late cultivars the photosynthates were mainly stored in the aerial plant parts. At the end of the growth period, the amount of DM in the stalks of late cultivars was more than twice as large as in early cultivars (Fig. 1).

3.2 Growth of leaves Leaves of plants are generally considered to be the major site of photosynthesis.

14

Fig. 1. Growth of stalks in different cuitivars. The dates of sampling (indicated by different ways of shading) were June 25, July 12, July 30, August 13, August 27, September 10, September 24, October 15, and October 29, 1990. Abbreviations used: UR, Urodny; MA, Mari; NO, Nora; CO, Columbia; CL, clone 2071-63; DW, Dwarf; KA, Karina; RF, Refla; RK, Reka. TS (= DM), total solids.

In early cuitivars, growth of leaves was almost completed in the middle of July, while in late cuitivars growth continued up to the end of September. The total amount of green leaves, however, decreased during late summer (Fig. 2). New leaves, on the tips of stalks and branches, partly replaced the foliage lost in the lower part of the canopy. Except for the dwarf cultivar, the photosynthetically active leaf area was limited by competition for light among plants within the canopy. The effects of light competition were especially pronounced in late cuitivars with robust tops.

Leaf area index (LAI) reflects the photosynthetically active area of leaves (Fig. 2). Up to the end of May, LAI in all cuitivars was less than 1. This indicates a proportionally

U R MA N O D I G CO CL· DW K 2 4 KA R F RK CULTIVARS

Fig. 2. Leaf area index in different cuitivars. Dates of sampling and abbreviations used as in Fig. 1.

15

low exploitation of PAR during spring. Rapid growth of leaves started in the beginning of June. In the middle of June, LAI in early and late cultivars, except for the dwarf cultivar, was about 1.5, and thereafter rapidly increased.

The seasonal maximum in LAI, of about 4.2 in early cultivars and 6.2 in late cultivars, was attained in the middle of August. During the subsequent period of growth, the area of photosynthetically active leaves decreased (Fig. 2). In the dwarf cultivar, LAI achieved its maximum of about 2.5 in the beginning of September (Fig. 2). During early autumn there were only small losses of the lower leaves in the dwarf cultivar.

3.3 Flowering of plants Flowering in Jerusalem artichoke marks the culmination of vegetative growth. This

was clearly demonstrated in early cultivars, where growth of tops ceased at the end of July, with the beginning of flowering. Between 9 and 12 flowers developed on each plant. The period of flowering was about two weeks. In clone 2071-63 the period of flowering was about three weeks, from the middle of August to the beginning of September. On an average, each plant developed 18 flowers.

Among the late cultivars, cv. Reka - with red tubers - usually started flowering in the middle of October. In the 1990 season, however, only flower buds developed as in the other late cultivars. Since the time of flowering in late cultivars seems to be determined by the sum of solar hours during early summer, this observation suggests that the low solar radiation during June 1990 obviously had an irreversibly inhibitory effect on the development of plants. In the dwarf cultivar, flower buds did not appear at all.

3.4 Growth of tubers Tuber yield is the most important measure of performance in Jerusalem artichoke.

The yield depends on several factors, such as PAR, evapotranspiration, nutrition, competition of plants for light within the canopy, etc. In the 1990 season, the effect of weather conditions on tuber yield was significantly reduced by the location of the experimental field, and by irrigation of the crop during early summer. The major growth-determining factors were close to the optimum. No specific effects of weather conditions on growth and development of tubers were observed.

During the growth period, histogenetic studies were carried out on tubers. The growth of cortex, pith, radial parenchyma and xylem was recorded periodically. During tuber formation, a strong expansion of xylem elements took place. Actual growth was confined to the parenchyma. Unusual secretory cavities were found in tubers of the dwarf cultivar.

In the 1990 season, tuber initiation in all cultivars was markedly enhanced. In early

16

UR MA NO Die CO CL DW K24 KA RF RK C U L T I V A R S

Fig. 3. Growth of tubers in different cultivars. Dates of sampling and abbreviations used as in Fig. 1.

cultivars, a large number of stolons and the first tubers (diam. > 1 cm) appeared already before the end of June (Fig. 3). Formation of tubers proceeded rapidly, and growth was completed in the beginning of October. Tuber yields varied between 11.38 t DM ha"1 for cv. Columbia and 12.54 t DM ha"1 for cv. Mari.

In late cultivars, the first tubers (diam. > 1 cm) were recorded in the middle of July (Fig. 3). The subsequent growth of tubers was slow. Translocation of assimilates from the tops into the tubers was apparently discontinued in the middle of November. Tuber yields varied between 6.27 t DM ha"1 for cv. Reka - with red tubers - and 8.45 t DM ha"1 for cv. Karina - with white tubers. The dwarf cultivar yielded 6.36 t DM ha"1 (Fig. 3).

3.5 Proportion of DM in tops and tubers As usually, the proportion of DM in tops and tubers was different in early and late

cultivars. At the end of the growth period, in early cultivars DM accumulation in tubers amounted to 78.37% of total DM in tubers and tops. In late cultivars with white tubers the amount of DM in tubers was 54.14% and in late cultivars with red tubers it was 51.77%. In the dwarf cultivar the proportion of DM in the tubers was 61.69% of total DM yield.

The distribution of DM between tubers and tops was most favourable in early cultivars. With a proportionally small amount of DM in aerial parts, tuber yields were almost twice as high as those of late cultivars with the robust tops. In late cultivars, about 50% of the photosynthetic products accumulated in the tops. The tops of Jerusalem artichoke can be valorized as soil conditioner and manure. However, if the amounts of nutrients and water supplied, and the proportional expenses for cultivation and harvesting are considered, early cultivars have the advantage of saving resources.

17

Table 2. Composition of carbohydrates (in %) in Jerusalem artichoke tubers.

Cultivar

Urodny Mari Nora D19 Columbia 2071-63 Dwarf K24 Karina Refla Reka

DMa

19.46 19.52 20.04 19.54 21.26 20.04 24.84 20.38 20.02 20.16 20.01

RS

18.41 19.16 19.33 18.64 20.42 19.15 22.32 19.72 18.66 19.15 19.10

DP > 4

57.92 64.99 64.77 55.78 63.76 63.72 70.21 65.12 66.24 72.89 77.93

DP 3

15.07 12.23 12.95 16.54 12.85 13.91 10.77 14.84 14.47 12.32 9.69

DP 2

17.94 15.35 15.12 18.34 15.37 14.06 10.89 13.88 12.31 10.19 8.18

Glu

2.15 1.91 1.74 2.30 1.88 1.92 2.09 1.66 1.63 1.31 1.28

Gal

1.24 1.17 1.05 1.29 1.12 1.14 1.27 0.95 0.90 0.79 0.67

Fru

5.68 4.35 4.37 5.75 5.02 5.25 4.77 3.55 4.45 2.50 2.25

a Abbreviations used: DM, dry matter in fresh tubers; RS, "refractometrically measured" solids in extracted juice; DP > 4, inulin with a degree of polymerization > 4; DP 3, trisaccharides; DP 2, disaccharides, Glu, glucose; Gal, galactose; Fru, fructose (all sugars given as a percentage of total carbohydrates).

3.6 Quality of tubers Tuber shape is one of the important characteristics of Jerusalem artichoke cultivars.

Besides the genetic background, many different factors, such as soil quality, nutrient levels, water availability, plant density, cultivation methods, etc., affect the shape of tubers. Nutrient and water supply in the 1990 season apparently had no specific effects on tuber shape.

In the early cvs. Urodny, Mari, Nora, D19 and Columbia the tubers were round and very different in size. The largest tubers formed intricate clusters, which made them difficult to harvest and process. The tubers of clone 2071-63 had a characteristically protracted shape with a smooth surface. Apparently due to the clay soil and the nearly optimal water supply, in the 1990 season, the stolons of cv. 2071-63 were exceptionally short.

Late cvs. K24 and Karina - with white tubers - had characteristically pear-shaped tubers with deformations mainly in the form of expanded buds. In late cultivars with red tubers the shape of the tubers was semiprotracted to round. In cv. Refla, enlarged buds were present on the surface of the tubers. A preferable shape with a relatively smooth surface of the tubers was found in cv. Reka - with red tubers. Tubers from the dwarf cultivar were oblong, but very different in size. The majority of tubers grew from the old seed tuber, without stolons being formed.

At harvest in autumn, the highest DM content of tubers was found in the dwarf cultivar (24.84%). Among early cultivars, a maximal content of 21.26% DM was found in tubers from cv. Columbia. The average DM content of tubers from late cultivars was

18

20.14% (Table 2). The amounts of sugars in the tubers were proportional to DM. In tubers from early cultivars the sugar content was on an average 19.19%, and in late cultivars it was 19.16%. Within the trial, the largest sugar content (22.32%) was found in tubers of the dwarf cultivar.

The composition of carbohydrates in the tubers was significantly different in early and late cultivars. The average inulin content (DP >4) in tubers of early cultivars was 61.82%. In late cultivars with white tubers the inulin content amounted to 65.68%, and in late cultivars with red tubers to 75.41%, whereas in the dwarf cultivar it was 70.21% (Table 2). The monosaccharide content in tubers of early cultivars was, on an average, 8.22%, in late cultivars 5.49%, whereas in the dwarf cultivar it was 8.13% (Table 2).

4 CONCLUSIONS Comparison of different Jerusalem artichoke cultivars shows that under the climatic

conditions of Northern Europe, cultivars with a short growth period, and with a small proportion of DM in tops, are preferable to late cultivars with robust tops. In late cultivars, the translocation of photosynthates from tops into tubers was slow. Consequently, tuber yields were significantly lower than those of early cultivars.

In all cultivars, a small leaf area index during spring indicated a low exploitation of the photosynthetically active solar radiation. Intensive vegetative growth of plants started in the beginning of June. In all cultivars, maximal LAI was attained in the middle of August. Flowering of plants occurred only in early cultivars. Late cultivars, except for the dwarf cultivar, developed flower buds.

The largest tuber yields were obtained from early cultivars. The most favourable shape of tubers was found in the early cv. 2071-63, and in the late cv. Reka, with red tubers. The tubers of the dwarf cultivar had the highest content of refractometrically measured sugars. The largest amounts of inulin with DP > 4 were found in late cultivars with red tubers, and in the dwarf cultivar.

5 REFERENCES

Hansen, S., Jensen, S.E. and Aslyng, H.C., 1981. Jordbrugsmeteorologiskeobservationer Statistik analyse og vurdering 1955-1979, Hydroteknisk Laboratorium, Den Kongelige Veterinaer og Landbohojskole, Kobenhavn, 414 pp.

Jensen, S.E., 1990. Klima og vandbalance Hojbakkegärd. Institut for Jordbrugsvidenskab, Den Kongelige Veterinaer og Landbohojskole, K0benhavn (internal communication).

Kosaric, N., Cosentino, G.P., Wieczorek, A. and Duvnjak, Z., 1984. The Jerusalem artichoke as an agricultural crop. Biomass, 5: 1-36.

Stauffer, M.D., Chubey, B.B. and Dorreil, D.G., 1981. Growth, yield and compositional characteristics of Jerusalem artichoke as they relate to biomass production. In: D.L. Klass and G.H. Emert (Eds.), Fuels from Biomass and Wastes. Ann. Arbor Sei. Publ., Inc., Ann Arbor, pp. 79-97.

19

Zubr, J., 1989. Jerusalem artichoke as a field crop in northern Europe. In: G. Grassi and G. Gosse (Eds.), Topinambour (Jerusalem artichoke), Proc. EC Workshop, 30 Sep. - 1 Oct. 1987, Madrid, Report EUR 11855. CEC, Luxembourg, pp. 105-117.

Zubr, J., 1990. Energy from Field Crops, Institute of Agricultural Engineering, The Royal Veterinary and Agricultural University, Copenhagen, 68 pp.



MULTIANNUAL, MULTILOCATIONAL TRIALS OF JERUSALEM ARTICHOKE IN THE SOUTH OF IRELAND: SOIL, pH AND POTASSIUM

A.C. CASSELLS and M. DEADMAN Plant Biotechnology (UCC) Ltd., University College, Cork, Ireland

ABSTRACT

Jerusalem artichoke {Helianthus tuberosus L.) has been evaluated as a European biomass crop from Denmark to Greece and east to Yugoslavia but it has not been studied in detail in the cool wet maritime regions of the Community. In this project the potential of Jerusalem artichoke as a local biomass crop for use in Ireland as a fermentation substrate, pig feed and cattle silage was evaluated.

The results for three years of trials will be reported with reference to the determinants of yield and the incidence of disease in the field. A summary of the biotechnological methods used to produce and clone disease-free stock is presented.

1 INTRODUCTION The European Communities' initiatives in the area of biomass crops have resulted

in a transnational network of multilocational, multiannual trials of Helianthus tuberosus L. (Jerusalem artichoke, syn. topinambour) (Grassi and Gosse, 1989). Yield data has been reported form Spain and Italy (c. 40 °N) to Denmark (55 °N) and from France (Rennes) to Romania with highest yields reported for irrigated crops at southerly latitudes (Barloy and Fernandez, 1988). The suitability of this crop for the cool wet maritime regions of Europe was not investigated.

Biotechnology has been successfully applied to potato (Bajaj, 1987) which, like Jerusalem artichoke, is a tuber-propagated crop. In potato, meristem culture has been used to produce disease-free stocks. These have been multiplied rapidly in vitro. The planting of microplants, microtubers and minitubers derived from axenic stocks, can bypass many years of slow field multiplication of new varieties^. Indeed, automation of the production stages may result in the establishment of ware crops from micropropagated propagules at an economic price in the future.

# The loose term "variety" is used throughout to cover cultivars and lines.

22

Here, we report on a continuing programme started in 1987 to evaluate the potential of Jerusalem artichoke in the Atlantic maritime regions of Europe. The model conformed to the parameters of an EC demonstration project in that agricultural, economic and environmental aspects were covered. Further, biotechnological procedures as outlined above for potato were incorporated in the study.

2 MATERIALS AND METHODS 2.1 Varieties

The varieties trialled and their characteristics are given in Table 1.

2.2 Tissue culture The media and procedures used were based on Cassells (1987) and Cassells et al.

(1987).

2.3 Trials material Small quantities of tubers were obtained from INRA (Rennes), FAL (Braunschweig)

and Agriculture Canada (Morden). Meristem cultures were set up and the cultures screened for cultivable contaminants (Cassells, 1991). Axenic cultures were micropropagated via nodal culture and microplants established in the glasshouse. Established progeny plants were either potted up to 21-cm pots and grown outdoors or planted in the field. Progeny tubers were subsequently used in trials.

2.4 Equipment Potato equipment was used to plant and harvest conventional tuber crops. Microplants

were established in paper chain pots and transplanted as brassicas.

Table 1. Jerusalem artichoke varieties used in field trials.

Name

K8 Violet de Rennes Kharkowskii Fuseau 60 D19-63-122 Nahodka D19 Columbia

Origin

German French Russian French French Russian French Canadian

Habit

tall tall tall tall tall tall bushy bushy

Maturity

medium late very late early late medium early medium late early early

Line number

1 2 3 4 5 6 7 8

23

2.5 Pathology Methods were as described previously (Cassells et al., 1988).

3 RESULTS AND DISCUSSION 3.1 Varietal trials

The first season's planting date was as for potato locally. Yield data for varieties 1 to 7 (see Table 1) are given in Fig. 1. Columbia was received too late for inclusion. The initial trends showed that early maturing varieties give highest yields.

3.2 Early spring planting The reported frost tolerance and low temperature threshold for growth of Jerusalem

artichoke was the basis for an early planting trial. The data (Fig. 2) for Nahodka (medium late maturing) and Kharkowskii (early) show a strong positive correlation between planting date and yield potential.

Fig. 1. Mean tuber yield per plant for the 1987 Jerusalem artichoke trial (figures on the bars represent equivalent yields in t ha"1).

y (Kharkowskii) = 84.253 - 0.46982x P < 0.05

y (Nahodka) = 107.71 - 0.67891 x P < 0.05

D Nahodka ■ Kharkowskii

60 70 80 90 100 110 120 130 Planting date

Fig. 2. The effects of planting date on yield for tuber-derived planting material of the Jerusalem artichoke varieties Kharkowskii (early) and Nahodka (medium late) grown during the 1987 and 1988 field trials.

24

3.3 Autumn planting The data presented above (3.2) suggested the feasibility of an autumn planting under

local conditions with a mean winter temperature of + 6 °C. The data are presented in Fig. 3 from which it can be seen that yields of the order of 100 t ha"1 are possible in overwintered crops.

3.4 Field performance of microplants and small tubers Potato microplants and microtubers can be difficult to establish in cool wet conditions

and arguably minitubers less than c. 10 g also fail to give acceptable yields. Here, in line with the objectives stated in the Introduction (1) microplants and minitubers are evaluated.

The results (Fig. 4) show that while microplants give high yields in overwintering trials they perform less well in spring plantings than conventional tubers (c. 50 g) except

100

80

60

40

20

0

y (spring planting) = 84.803 - 0.40508X P < 0.05

y (all dates) = 83.574 - 0.39562x P < 0.05

I I I I I I I I I -50 -25 0 25 50 75 100 125 150 175

Planting day

Fig. 3. Relationship between planting date and tuber yield for the Jerusalem artichoke variety Nahodka grown in field trials between 1987 and 1990. Open symbols represent yields taken from spring-sown crops, the closed symbol represents the yield from an overwintered trial plot.

y (microplants) = 83.788 - 0.38799x P < 0.05

y (tubers) = 107.71 - 0.67891 x P < 0.05

D Tubers ■ Microplants

-50 -25 25 50 75 100 125 150 175 Planting day

Fig. 4. Relationship between planting date and yield for the Jerusalem artichoke variety Nahodka grown during field trials between 1987 and 1990. Open symbols represent tuber-derived planting material, closed symbols represent tissue culture-derived planting material.

25

Fig. 5. The effect of seed tuber size on the yield of varieties of Jerusalem artichoke used in the 1988 field trial. Figures on the bars represent equivalent yields in t ha"1.

100 150 Potassium

200

Fig. 6. Relationship between soil potassium (in mg per 100 g dwt of soil) and yield for the variety Nahodka in field trials from 1987 to 1990.

at late planting dates. With the exception of varieties 3 and 7, minitubers (c. 10 g) yield as conventional

tubers (c. 50 g) (Fig. 5).

3.5 Soil status and yield No relationship was found between soil pH and growth in the pH range 4.9 to 7.1.

However, a relationship was confirmed between growth and soil potassium (Fig. 6) as reported by Mimiola (1988). Soil potassium at 183 mg per 100 g dwt of soil gave a yield of 120 t ha"1 in a spring-planted crop.

4 PATHOLOGY Diseases affecting Jerusalem artichoke observed in these trials are given in Table 2.

Tuber rots and seed tuber diseases have been discussed previously (Cassells et al., 1988). Here, we report the incidence of powdery mildew which affected the 1990 trial. The

26

Table 2. Putative pathogens limiting production of Jerusalem artichoke in the south of Ireland.

Frequency (%)

Stored and seed tubers; microbes isolated from diseased tubers Fusarium avenaceum 35 F. sporotrichoides 35 F. culmorum 22 Sclerotinia sclerotiorum 4 Trichoderma sp. 2 Rhizopus sp. 2 Pseudomonas syringae pv. tagetis 100a

Stem and foliar diseases Erysiphe cichoracearum 100b

S. sclerotiorum 1

a Only recorded in one batch of imported seed. b 1990 trial.

outbreak was first recorded in September and 100% cover was reached by October. No significant deviation from the predicted yield was recorded and the disease may develop too late to pose a threat to the crop. The incidence of Sclerotinia sclerotiorum, however, while low did result in significant yield reduction of c. 50% in affected plants. Cultural control, including the use of disease-free seed is indicated.

5 DISCUSSION Jerusalem artichoke is able to exploit the long growing season in Ireland and yields

of up to 120 t ha"1 are achievable in overwintered crops. These yields are comparable to the best reported from southern Europe where the crop has to bear the cost of irrigation.

A serious problem in this climate is seed tuber losses due to fungal rots. The strategy of above-ground or in-soil storage may not be feasible here. For the former, high ambient winter temperatures may facilitate rots whereas workability of the soil in spring will not allow early planting with consequent reduction in yield. Autumn lifting and replanting may be a local option. Alternatively, micropropagation, in addition to facilitating clean stock maintenance and rapid introduction of new varieties, may also be used to provide planting material for direct establishment of the crop if the propagules can be produced at an economic price.

Pig feed trials (Zoccarato et al.9 1989) and cattle silage (Moule et al., 1967) have been previously reported and have been evaluated here (to be published). The preponderance of mixed farming in the region may facilitate a phased increase in crop acreage to the levels required for fermentation substrate extraction.

27

6 REFERENCES

Bajaj, Y.P.S. (Ed.), 1987. Biotechnology in Agriculture and Forestry, Vol. 3, Potato. Springer-Verlag, Berlin, 000 pp.

Barloy, J. and Fernandez, J., 1988. Synthesis on Jerusalem artichoke projects. Synthesis on genetic improvement, ecophysiology and crop agronomy. EEC - DG XII - 2nd Workshop on Jerusalem Artichoke, 6-8 Dec. 1988, Rennes (in press).

Cassells, A.C., 1987. In vitro induction of virus-free potatoes by chemotherapy. In: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry, Vol. 3, Potato. Springer-Verlag, Berlin, pp. 40-50.

Cassells, A.C., 1991. Problems in tissue culture: culture contamination. In: R.H. Zimmerman and P.C. Debergh (Eds.), Micropropagation of Horticultural Crops. Kluwer, Dordrecht, pp. 31-44.

Cassells, A.C., Austin, S. and Goetz, E.M., 1987. Variation in tubers in single cell-derived clones of potato in Ireland. In: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry, Vol. 3, Potato. Springer-Verlag, Berlin, pp. 375-391.

Cassells, A.C., Deadman, M.L. and Kearney, N.M., 1988. Tuber diseases of Jerusalem artichoke (Helianthus tuberosus L.): production of bacterial-free material via meristem culture. EEC -DG XII - 2nd Workshop on Jerusalem Artichoke, 6-8 Dec. 1988, Rennes (in press).

Grassi, G. and Gosse, G. (Eds.), 1989. Topinambour (Jerusalem Artichoke). Proc. EC Workshop, 30 Sep. -1 Oct. 1987, Madrid, Report EUR 11855. CEC, Luxembourg, 213 pp.

Mimiola, G., 1988. Test of topinambour cultivation in southern Italy. EEC - DG XII - 2nd Workshop on Jerusalem Artichoke, 6-8 Dec. 1988, Rennes (in press).

Moule, C , Tsvetoukhine, V., Dupuis, G. and Renault, M., 1967. Contribution a l'^tude du topinambour -ensilage. INRA, Rennes, 83 pp.

Zoccarato, I., Tartari, E., Bosticco, A., Benatti, G. and Destefanis, G., 1989. L'impiego dei tuberi di topinambour {Helianthus tuberosus) in razioni per suini all'ingrasso. In: Production Porcine en Europe Mediterraneenne. Quelles Strategies pour le Pore Mediterranean? R&umes des Communications, Coll. 14-16 Nov. 1989, Ajaccio. INRA, pp. 00-00.



CROP CHARACTERISTICS AND INULIN PRODUCTION OF JERUSALEM ARTICHOKE AND CHICORY

W J . M . MEIJER*, E.W.J.M. MATHIJSSEN* and G.E.L. BORM** * Centre for Agrobiological Research (CABO-DLO), P.O. Box 14, 6700 AA

Wageningen, The Netherlands Research Station for Arable Farming and Field Production of Vegetables (PAGV), P.O. Box 430, 8200 AK Lelystad, The Netherlands

ABSTRACT

Crop growth and development, and yield formation of Jerusalem artichoke and chicory were studied in field trials harvested periodically. The annual course of light interception, the total dry matter production and, consequently, the light use efficiencies of the two species were similar. Chicory plants remain vegetative in the first year and the pattern of crop growth and inulin storage is simple and efficient. Jerusalem artichoke is quite different: inherent to the plant type and developmental pattern a relatively large fraction of the total production is diverted to structural stem dry matter and a smaller part to inulin storage.

Using a crop growth model, the potential of these species was calculated for the climatic conditions of each year from 1954 to 1990. Assuming an optimal supply of water and nutrients and no losses at harvest, inulin yields of Jerusalem artichoke varied from 4.5 to 8.3 t ha"1 with an average of 6.5 t ha"1. The potential yields of chicory ranged from 9.8 to 16.11 ha"1 and averaged 12.5 t ha"1. The factors that influence yield levels and stability are briefly discussed.

1 INTRODUCTION

In temperate regions three plant species can be grown for the production of inulin, viz. chicory, dahlia and Jerusalem artichoke. These crops accumulate inulin in their storage organs to levels attractive for processing. For many years, Jerusalem artichoke has drawn most attention not only for its potential as a crop, but also because it is particularly appropriate to study processes like flower induction, tuber dormancy and tissue differentiation. In spite of the continuous attention for this species, breeding work has been done only occasionally and most cultivars are not well developed.

Chicory has a long history as a vegetable crop for the production of chicons, the leafy heads forced in the dark from the tap-roots. In some countries the roots are processed and used as a coffee substitute or additive. The limited but steady breeding for the "coffee-

30

type" chicory has led to cultivars appropriate for inulin production, because the aims are similar: large root yields with a high dry matter content.

Many dahlia cultivars are available, which all have been selected for their flowers and not for inulin production. The tuberous roots have no buds and can be propagated only if attached to a piece of stem tissue. This hampers crop establishment from tubers. If propagated from seed, sowing has to be delayed until late in spring because dahlia is very sensitive to frost. Dahlia, therefore, can not be considered as a promising inulin crop.

Haber et al. (1941) have compared the suitability of the three species for fructose production. They estimated chicory to be the easiest and cheapest producer. In a comparative evaluation of potential energy root crops, O'Hair (1982) grew Jerusalem artichoke and chicory under a range of conditions. Though average yields of both species were similar they varied greatly in Jerusalem artichoke. In Germany, Thome and Kühbauch (1987) tested the yield abilities of two cultivars of both species and concluded that chicory, and to a lesser extent Jerusalem artichoke, equalled or even surpassed the productivity of sugar beet.

This paper reports on field trials with chicory and Jerusalem artichoke carried out at two locations in the Netherlands (Wageningen and Lelystad), to study crop development and yield formation. The objective did not include a direct comparison of the potential of these species, because proper evaluation of such a comparison is often cumbersome due to the different agronomic methods having to be used for the two species. Thus, the results always have to be related to the specific experimental conditions. Our aim was to collect basic data on plant and crop growth and development, to determine the relations between growth processes and to quantify the influences of environmental conditions. In the case of Jerusalem artichoke we derived much of this information from recent work done by Spitters (1989). A crop growth model (Spitters, 1990a, b) was applied to extrapolate the experimental results on the effects of temperature and radiation to the conditions prevailing in the years 1954 to 1990. This produced estimates of the potential yields of the species under climatic conditions in the Netherlands.

2 EXPERIMENTAL In 1987, a growth analysis was done on both species. In the case of Jerusalem

artichoke an early (Columbia) and a late cultivar (Violet de Rennes) were studied. In the case of chicory the effects of sowing date and row width were investigated, using the cv. Pevele. Green leaf area, weight of green and dead leaves, and weight and inulin content of stems, tubers and roots were determined, at harvests every 2 to 3 weeks from emergence onwards.

In the 1988 growth analysis special attention was paid to some processes expected

31

to be constraints to productivity or that needed to be elucidated for modelling. In Jerusalem artichoke, the relation between flower initiation and the onset of rapid tuber filling was determined (Meijer and Mathijssen, 1991). In chicory, on the other hand, the inter-cultivar variation in leaf expansion and dry matter distribution was examined. Moreover, at Lelystad the effects on yield of means to stimulate early crop closure, viz. by forcing plants or by covering the crop with perforated plastic foil, were studied. All crops were well supplied with minerals and received supplementary irrigation during dry periods.

3 RESULTS AND DISCUSSION 3.1 Crop characteristics 3.1.1 Leaf area duration and light interception

Under good growing conditions the inulin production of chicory and Jerusalem artichoke crops depends on three variables: a) the amount of light intercepted; b) the efficiency of the conversion of light energy to dry matter; and c) the distribution of dry matter over the plant organs and their constituents.

The amount of light intercepted by a crop largely depends on the leaf area duration (the period that a green and assimilating foliage is maintained). In most years, chicory and Jerusalem artichoke can be sown or planted in the Netherlands from the beginning of April. Because the tubers of Jerusalem artichoke contain more energy than chicory seed, leaf area expansion was expected to be correspondingly faster. Yet, the experiments in 1987 and 1988 suggest that in both species temperature has a dominant effect - which, moreover, is similar for both species -, because the variation in leaf expansion between years was more pronounced than between the species (Fig. 1). If chicory and Jerusalem artichoke emerge at the same time, therefore, the light interception during the early stages of crop growth will be similar (Table 1). Chicory and late Jerusalem artichoke cultivars maintain their active foliage until late in autumn. The foliage of early Jerusalem artichoke cultivars can deteriorate too early, especially in warm years, and then a substantial part of the solar radiation is wasted.

3.1.2 Utilization of intercepted light In many crops the biomass produced is linearly related to the amount of light

intercepted (Monteith, 1977; Gosse et al., 1986; Kiniry et al. 1989). Light use efficiency is defined as the average value over the growth cycle of dry matter production per unit of intercepted, photosynumerically active light. The light use efficiency depends on the composition of the dry matter produced from the primary assimilates. A greater content of fat or protein decreases the light use efficiency because of the respiration energy needed, and the weight losses that occur when carbohydrates are converted to these constituents.

32

leaf area index 8

4 +

2 +

/ / D > / / / /

U*rf D

l-K%

100 150 200 250 300 350 daynumber

Δ

A

O

♦

D

■

Columbia

Columbia

'87

'88*

Violet de Ft.'87

Violet de R.'88*

Pevele

Orchies

'87

'88

*data C.Spitters

Fig. 1. Leaf area index as a function of time in chicory (cvs. Pevele and Orchies) and Jerusalem artichoke (cvs. Columbia and Violet de Rennes) in 1987 and 1988.

Table 1. Cumulative light interception (expressed as MJ m 2 photosynthetically active radiation) of chicory (cv. Pevele) and Jerusalem artichoke (cvs. Columbia and Violet de Rennes) crops in 1987 and 1988.

1987 1988

Chicory

Pevele

701 779

Jerusalem artichoke

Columbia

649 712

Violet de Rennes

745 821

Lower efficiencies sometimes have trivial explanations. Normally, the fibrous roots are not included in the total biomass. These roots are mainly formed during the early stages of plant life. If that part of the biomass is ignored, the efficiency is slightly reduced. Towards the end of the growth period the efficiency is often underestimated because the dead and deteriorating leaves - as part of the total biomass - are difficult to collect and thus can not be readily estimated. Spitters (1990a) discussed the effects of crop photosynthesis on the apparent constancy of the light use efficiency and concluded that the application of this parameter in production estimates is fairly satisfactory.

33

In our experiments with various cultivars, dates of sowing and sowing densities of chicory, we found a linear relation between biomass production and intercepted light (Fig. 2a). The light use efficiency of Jerusalem artichoke (Fig. 2b) is derived from the 1987 experiment with an early and a late cultivar and from unpublished 1988 data from C.J.T. Spitters. As with chicory, these data suggest a constant light use efficiency over the growing season. Barloy and Poulain (1988) found a comparable relation for the main growth period of the shoot, but an increased efficiency during rapid tuber growth. These authors suggest that the higher sink activity during the latter phase stimulates photosynthesis and increases light use efficiency.

The average light use efficiency of the species considered here was calculated from the regression lines in Fig. 2. The values of 2.66 and 2.53 g dry matter MJ'1 intercepted light for chicory and Jerusalem artichoke, respectively, are well within the range found in other carbohydrate-storing C-3 plants (Monteith, 1977; Gosse et al., 1986; Kiniry et al., 1989).

3.1.3 Distribution of dry matter The seed-to-seed life cycle of chicory is biennial. During the first season chicory

plants remain in the vegetative phase and make only leaves, tap-roots and fibrous roots. In our trials, during the first 10 weeks after emergence the leaves and storage roots grew at similar rates. Thereafter, most assimilates were allocated to the tap-roots (Fig. 3a). Though the data suggest the presence of a constant mass of leaves during the later growth stages,

total DM t/ha (a) Chicory 25

20

15 f

10

5 + -pr

Y = 0.0266X - 0.244 corr. = 0.99

0 X< 0 200 400 600 800

total DM t/ha (b) J.artichoke 25

0 200 400 600 800 cumulative light interception MJ/m2

Fig. 2. The relationship between total dry matter production and cumulative light interception of chicory (a) and Jerusalem artichoke (b) crops in 1987 and 1988. The various symbols refer to different cultivars, dates of sowing or sowing densities.

34

DM t/ha 20

(a) Chicory DM t/ha (b) J.artichoke 20 T

15 +

10 j

H 1 H- u 13 Til V ^ » — ^ — 1 H 120 160 200 240 280 320 120 160 200 240 280 320

daynumber D total DM roots/tubers "O- stems "Δ- leaves

Fig. 3. The seasonal pattern of dry matter production of crops of chicory (cv. Pevele) and Jerusalem artichoke (cvs. Violet de Rennes and Columbia) and its distribution over the plant organs.

in fact new leaves continuously emerged and older leaves deteriorated. The developmental cycle of Jerusalem artichoke is very different. After emergence, a vegetative stem, leaves and often some side-branches are formed. Early cultivars seem insensitive to day length and initiate flowers after a certain temperature sum has been attained. The apexes of late cultivars become reproductive after the length of the dark period has reached a certain threshold value, between mid-August and mid-September. About 20 primordia and leaves in various stages of development are then found on the apex. The lower temperatures in autumn prevent the flower buds of these late cultivars from emerging (Meijer and Mathijssen, 1991). From the onset of flower initiation the number of developing leaves is fixed and the lifespan of the foliage is limited. In all cultivars, tuber initiation starts about 6 weeks (or about 500 degree-days in thermal time) after emergence, probably after the juvenile period has ended. The number of tubers per plant increase until flowering, or in late cultivars until the end of the growing season.

Until the reproductive phase Jerusalem artichoke allocates most dry matter to the stem (Fig. 3b). Much ofthat is structural stem material, the remainder being temporarily stored carbohydrates. After flower initiation, the stems lose their sink activity and the stem inulin is relocated to the tubers. Stem and leaf growth continues until some time after flower initiation and, therefore, late cultivars produce more stems and leaves than early cultivars. In late cultivars, the duration of green leaf area is longer and total production greater, but tuber filling depends nevertheless most on the relocation of stem reserves.

35

3.2 Actual yields In the preceding sections it is concluded that leaf area development in chicory and

Jerusalem artichoke is similar. Because light interception is a function of leaf area, and since emergence of both species is assumed to take place simultaneously, the cumulative light interception of the species over each growing season will also be very similar. From Fig. 2a and b almost equal light use efficiencies have been derived for both species and, therefore, similar levels of total dry matter production are to be expected. The crucial difference between the species is to be found in the plant type and in the distribution of the biomass over the plant organs. In the first year of its phenological cycle, chicory is a very efficient plant. Above ground only leaves are formed, not stems, and their production is stored in the tap-roots. Jerusalem artichoke, on the other hand, invests 4 to 9 t ha"1 in structural stem dry matter. At tuber harvest, these stems contain cellulose, hemicellulose and lignin almost exclusively. Their feed and fibre quality is poor and, unless a better use is found, they will serve to maintain the organic matter content of the soil.

In the experiments with chicory and Jerusalem artichoke the total dry matter yields varied from 15 to 20 t ha"1 (Table 2). With chicory, the lower figures were obtained from late sowings or low plant densities. With Jerusalem artichoke, lower total yields were achieved in the early cv. Columbia. In chicory, inulin yields varied from 8.0 to 12.2 t ha"1, comprising 57% of total dry matter and 74% of root weight on average. The inulin yields

Table 2. Yields of total biomass, stems, tubers or roots, and inulin from various cultivars of chicory and Jerusalem artichoke (t ha"1), as obtained in field trials in 1987 and 1988.

Cultivar Year Total Stems Roots/ Inulin % Inulin of total biomass biomass tubers

Chicory: Pevele Fredonia Pevele Dageraad Orchies Tilda Orchies

Jerusalem artichoke: Columbia Violet de Rennes Columbia Violet de Rennes Violet de Rennes

1987 1987 1988 1988 1988 1988 1988

1987 1987 1988a

1988a

1988

16.6 15.3 18.9 17.6 16.6 16.8 19.9

16.4 19.3 16.8 18.8 15.7

-------

5.3 6.3 5.6 9.7 8.9

12.6 10.6 15.5 13.7 12.9 12.6 16.5

8.5 8.3 8.3 4.6 4.1

9.5 8.0

12.2 10.1 9.3 8.9

12.0

6.4 6.0 6.0 3.0 2.7

57 52 65 57 56 53 60

39 31 36 16 17

a Data C.J.T. Spitters.

36

of Jerusalem artichoke were much lower (4.0 to 6.71 ha"1), thus, on an average, comprising 28% of the total dry biomass and 71 % of the tubers.

3.3 Simulated yields Spitters (1990a, b) has described a relatively simple crop growth model that can be

applied to analyse experimental results or to extrapolate such results to different environmental conditions. The model is based on the strict proportionality between light interception and growth rate of crops under favourable conditions. The average light use efficiency was derived from the experiments with periodic harvests. The fraction of light intercepted by the foliage was calculated from the green leaf area. The leaf area development is mainly affected by temperature and can be described as a function of the cumulative daily average temperatures. Data on daily incoming solar radiation and temperature were obtained from a nearby weather station. Total dry matter growth was calculated from the intercepted light and from the average light use efficiency.

In the model, the dry matter is partitioned to the plant organs according to a phenological pattern and sink-source relations. In Jerusalem artichoke, tuber growth proceeds in two distinct phases: the initiation phase with slow tuber growth and a second rapid growth phase during which the stem reserves are relocated. Tuber growth is assumed to be sink-limited and for both stages maximal tuber growth rates per unit area were determined (Spitters, 1990b). In our experiment with cv. Violet de Rennes, tuber growth per unit area appeared to be related to tuber number, and the number of tubers per unit area appeared to be a linear function of the temperature sum. These relations were integrated in the model. In the model for the early, day length-neutral cultivars an effect of senescence on the foliage, as induced by high temperatures, was introduced.

In chicory, the calculation of dry matter distribution could be simplified because during most of the growth period a constant part of the dry matter is allocated to the roots and to the leaves. Two phases are distinguished: a phase of establishment during which 52% of total dry matter is partitioned to the roots, and a second phase which starts after a certain crop mass has been attained, and in which 74% of the dry matter is distributed to the roots. Finally, the inulin yields of both species were calculated from root or tuber yields, applying constant factors for the inulin content.

These growth models were used to calculate the yields that could have been attained in the Netherlands for both species under the climatic conditions of the years 1954-1990. In the models the crops are assumed to be well supplied with water and nutrients and not hampered by pests or diseases. The year-to-year variations are caused by temperature and radiation only. According to these calculations, the greatest total dry matter production is attained with chicory (Table 3), mainly because, on average, this crop maintains its green

37

Table 3. Simulated yields (in t ha"1) of chicory (cv. Pevele) and Jerusalem artichoke (cvs. Columbia and Violet de Rennes) under the climatic conditions in the Netherlands. Means of the years 1954-1990.

Chicory Jerusalem artichoke

Pevele Columbia Violet de Rennes

Total biomass 23.7 16.3 20.6 Dry roots/tubers 16.6 8.8 8.5 Inulin yield

mean 12.5 6.5 6.4 highest 16.1 8.3 8.3 lowest 9.8 4.5 5.3 variation % 11.0 12.6 11.5

leaf area until late in autumn. The foliage of Jerusalem artichoke, on the other hand, deteriorates gradually, starting some time after the transition to the reproductive state. Because chicory allocates a larger part of the total production to the storage organs, its calculated average inulin yield is almost twice that of Jerusalem artichoke.

3.4 Agronomic potential The experimental results show that the calculated yields are not always attained in

practice. It will be impossible to protect the crops from all stresses. Small tubers, small roots and root-tips will be lost during machine harvesting. Under adverse weather or soil conditions, insufficient plant emergence will be a major problem in chicory. As happened in the 1987 experiment, plant densities can be too low. Late emergence of the re-seeding then reduces yields (Table 2). Because Jerusalem artichoke crops are established from tubers, it is easier to attain good emergence and the intended plant density. Above that, tubers of Jerusalem artichoke can be planted before winter; this saves storage costs, advances emergence and probably increases yield (Morrenhof and Bus, 1990).

With both species, breeding should be aimed at obtaining genotypes with faster leaf growth at the low temperatures in early spring. Breeding of Jerusalem artichoke certainly has to be aimed at reducing the part of biomass used for structural stem growth. It would be even better to find genotypes that store most or even all of the inulin in the tubers from tuber initiation onwards. The temporary storage in the stem is unfavourable to inulin production because of the metabolic costs of relocation and of the formation and maintenance of the extra storage tissue needed.

In the experiments with Jerusalem artichoke the ratio between structural stem matter and temporarily stored inulin appeared remarkably variable. It is not yet clear which factors govern this important ratio. The inulin content of the stems increases until the onset of the

38

reproductive phase; only then, relocation begins. These reserves make up a substantial part

of the ultimate tuber and inulin yield, especially in late cultivars.

Barloy (1989) has shown that high rates of nitrogen fertilization reduce tuber yields

considerably. Spitters (1990b) suggested that mild moisture and nitrogen stress will reduce

structural growth more than the accumulation of reserves and thus will benefit inulin yields.

If such effects could be translated into applicable and reliable agronomic methods, the

potential yield of Jerusalem artichoke might be enhanced. Even then, it will be difficult to

bridge the gap with chicory because the plant type of Jerusalem artichoke demands a

considerable part of the assimilates to be invested in structural stem matter.

4 REFERENCES

Barloy, J., 1989. Techniques of cultivation and production of the Jerusalem artichoke. In: G. Grassi and G. Gosse (Eds.), Topinambour (Jerusalem artichoke), Proc. EC Workshop, 30 Sep. - 1 Oct. 1987, Madrid, Report EUR 11855. CEC, Luxembourg, pp. 45-57.

Barloy, J. and Poulain, D., 1988. Ecophysiological studies on Jerusalem artichoke and agricultural implications. In: J. Kisgeci and U. Wuensche (Eds.), Producing Agricultural Biomass for Energy. Proc. Workshop, Uppsala, Sweden, 21-23 Sep. 1987. FAO-CNRE Bull., 17: 47-58.

Gosse, G., Varlet-Grancher, C , Bonhomme, R., Chartier, M., Allirand, J.-M. and Lemaire, G., 1986. Production maximale de matiere seche et rayonnement solaire intercepts par un couvert v£g£tal. Agronomie, 6: 47-56.

Haber, E.S., Gaessler, W.G. and Hixon, R.M., 1941. Levulosefrom chicory, dahlias and artichokes. Iowa State Coll. J. Sei., 16:291-297.

Kiniry, J.R., Jones, CA. , O'Toole, J.C., Blanchet, R., Cabelguenne, M. and Spanel, D.A., 1989. Radiation-use efficiency in biomass accumulation prior to grain filling for five grain-crop species. Field Crops Res., 20: 51-64.

Meijer, W.J.M. and Mathijssen, E.W.J.M., 1991. The relation between flower initiation and sink strength of stems and tubers of Jerusalem artichoke. Neth. J. Agric. Sei., 39: 123-135.

Monteith, J.L., 1977. Climate and the efficiency of crop production in Britain. Phil. Trans. R. Soc. London, 281:277-294.

Morrenhof, H. and Bus, C.B., 1990. Aardpeer, een potentieel nieuw gewas - teeltonderzoek 1986-1989. Verslag nr. 99, Research Station for Arable Farming and Field Production of Vegetables (PAGV), Lelystad, 66 pp.

O'Hair, S.K., 1982. Root crop evaluation, selection and improvement in Florida for energy applications. In: Energy from Biomass and Wastes VI. Symp. Institute of Gas Technology, Florida, pp. 135-165.

Spitters, C.J.T., 1990a. Crop growth models: their usefulness and limitations. Acta Hortic, 267: 349-368. Spitters, C.J.T., 1990b. Modelling the seasonal dynamics of shoot and tuber growth of Helianthus tuberosus

L. In: A. Fuchs (Ed.), Proc. Third Seminar on Inulin, 1 March 1989, Wageningen, NRLO report 90/28, The Hague, pp. 1-8.

Spitters, C.J.T., Lootsma, M. and Van de Waart, M., 1989. The contrasting growth pattern of early and late varieties in Helianthus tuberosus. In: G. Grassi and G. Gosse (Eds.), Topinambour (Jerusalem artichoke), Proc. EC Workshop, 30 Sep. - 1 Oct. 1987, Madrid, Report EUR 11855. CEC, Luxembourg, pp. 37-43.

Thome, U. and Kühbauch, W., 1987. Alternativen für die Fruchtfolge? Topinambur und Wurzelzichorie -zwei Fruchtarten für die Zuckergewinnung. D.L.G. Mitt., 102: 978-981.


LEAF NITROGEN, PHOTOSYNTHESIS AND CROP PRODUCTIVITY IN JERUSALEM ARTICHOKE (HELIANTHUS TUBEROSUS L.)

G. SOJA*, T. SAMM* and W. PRAZNIK** Department of Agriculture and Biotechnology, Research Centre Seibersdorf, 2444 Seibersdorf, Austria Department of Chemistry, Agricultural University Vienna, Gregor-Mendel-Str. 33, 1180 Wien, Austria

ABSTRACT

The role of photosynthetic performance in the productivity of Jerusalem artichoke was studied with respect to the influence of nitrogen nutrition and partitioning. Despite of significant cultivar differences in both photosynthetic capacity and tuber yield, no positive correlation was found between leaf photosynthetic rate and tuber yield. In view of these findings it is argued that in breeding programs, rather than using photosynthetic parameters for screening, primarily assimilate translocation should be improved to increase the tuber yield of new varieties. Seasonal variations in photosynthetic capacity could be ascribed to varying nitrogen levels in the leaves, resulting from a decrease in sink strength of the leaves for nitrogen allocation during plant development. Chlorophyll concentrations in the leaves were found to be more closely correlated to photosynthetic rates than concentrations of soluble protein in the leaves.

1 INTRODUCTION Jerusalem artichoke (Helianthus tuberosus L.) has been classified photosynthetically

as a C3-plant (Ergashev, 1976). Detailed knowledge about the photosynthetic performance of Jerusalem artichoke is largely lacking. In sunflower (Helianthus annum L.) Blanchet et al. (1986, 1987) observed a distinct dependence of carbon assimilation and productivity on nitrogen nutrition. In Jerusalem artichoke moderate to large productivity improvements due to a higher nitrogen supply have been noticed (Dorrell and Chubey, 1977; Zvara and Herger, 1983; Soja et al., 1990), but changes in photosynthetic behaviour have not yet been studied.

A relationship between photosynthetic capacity and leaf nitrogen levels has been recognized for a wide range of crop plants (Evans, 1989). In view of the significance of nitrogen for photosynthesis and, consequently, for productivity, we considered it worthwhile to investigate their mutual dependency in Jerusalem artichoke. This paper presents

40

some experimental results on the effects of nitrogen nutrition and nitrogen partitioning on photosynthesis and yield in Jerusalem artichoke.

2 MATERIAL AND METHODS Relations between photosynthesis and varietal productivity were investigated in field

experiments. Whole tubers of all varieties tested were planted at 0.63 X 0.40 m spacing in plots of 8 m2 with four replicates. The plots received 84 kg N ha"1, 14 kg P ha"1 and 52 kgKha"1.

Leaf gas exchange was estimated with a LI-COR 6000 Portable Photosynthesis System (LI-COR Inc., Lincoln, Nebraska, U.S.A.). Measurements were taken at full sunlight between 10.00-14.00 h on the uppermost fully expanded leaf.

Chlorophyll concentrations were determined spectrophotometrically in a 80% acetone solution using the equations given by Arnon (1949). Soluble protein was analysed in extracts of fresh leaf material which was homogenized in a cooled buffer (4 °C) containing 25 mM HEPES, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 1 mM Na-EDTA and 2% PVP. Protein was quantitated according to the method of Bradford (1976) with reagents from the Bio-Rad Protein Assay (Bio-Rad Laboratories GmbH, München, Germany) and with bovine serum albumin as protein standard. Total nitrogen was estimated with the Kjeldahl method, after digestion with H2S04.

3 RESULTS A comparison of cultivar-specific tuber yields and average C02-fixation rates under

field conditions showed no relationship between photosynthesis and tuber productivity (Table 1). Between the cultivars tested there was a remarkable variability in leaf photosynthetic rates, the highest mean value being 46% larger than the lowest. The differences in leaf area were considerably smaller (< 18%) and in the first four months of development no significant differences between the cultivars could be detected. Thus, the photosynthetic carbon gain capacity of the cultivars tested could hardly be responsible for the high differences in tuber yield (3.8-10.8 t DM ha"1).

The seasonal course of carbon assimilation in Jerusalem artichoke is characterized by an increasing decline of photosynthetic rates after midsummer; only then, the period of the most intensive assimilate translocation to the tubers commences. During plant development leaf photosynthetic rates and leaf nitrogen concentrations decrease similarly (Fig. la). In fact, there was a close correlation (r = 0.87) between photosynthetic rates and leaf nitrogen levels in differentially nitrogen-fed leaves (Fig. lb).

Despite this close correlation, the C02-fixation rate was not positively related to the concentration of soluble protein (Fig. 2a). However, chlorophyll concentrations of the

41

Table 1. Tuber yields, photosynthetic rates and leaf areas in ten cultivars of Jerusalem artichoke. Tuber yield was analysed 190 days after emergence (d.a.e.), leaf photosynthetic rates are mean values of measurements at 28, 56 and 98 d.a.e., and leaf area was determined 127 d.a.e. Values in columns followed by different letters are different at P = 0.05 (Duncan's multiple range-test).

Cultivar

Bianca Rote Zonenkugel Waldspindel Topianka Kärntner Landsorte Niederösterr. Landsorte Medius Fuseau K-8 Violet de Rennes

Yield (t DM ha"1)

5.5 e 8.6 bc 3.8 f 6.6 de 7.1 cde

10.8 a 8.6 bc 6.8 de 9.6 ab 8.1 bed

Leaf photosynthesis (μΜ m"2 s"1)

35 b 33 bc 41 a 32 c 33 bc 34 bc 33 c 31 c 31 c 28 d

Leaf area (m2 per plant)

1.76 a 1.96 a 1.74 a 1.91 a 1.73 a 1.91 a 1.95 a 2.04 a 1.91 a 1.94 a

^ ^ = 0 .87 o'8

60 90 120 150 days a f t e r emergence

_L 10 20 30

-2 - 1 pmol C0p m s 40

Fig. 1. Leaf photosynthetic rate and total leaf nitrogen in Jerusalem artichoke, cv. Topianka. (a) Changes during plant development, (b) Correlation in differentially nitrogen-fed plants.

leaves were closely correlated to photosynthetic rates as is evident from a comparison of

different cultivars as well as of plants of the same cultivar but grown at different nitrogen

levels (Fig. 2b).

4 DISCUSSION

The combination of a long growth period, a large leaf area and a remarkably high

photosynthetic rate explains the high total dry matter yields of Jerusalem artichoke cultivars.

Tuber yield differences between the cultivars tested can not be attributed to differing

42

ε 2.0

•Η 1 . 5 ω ■Ρ ο u

1.0

D 0.5

Γ = - 0 . 6 7

ΟΟ Ο Ο \

10 20 30 40 - 2 - 1 μπιοΐ CO« m s

~ 0.6

■Q 0 . 4

a 0 .2 o (-. o

O /

r = 0 .91 / '<b 0 .79

10 20 30 2 - 1 μπιοΐ CO« m s

40

Fig. 2. (a) Leaf photosynthetic rate and leaf soluble protein in differentially nitrogen-fed plants of Jerusalem artichoke, cv. Topianka. (b) Leaf photosynthetic rate and leaf chlorophyll a + b in different cultivars of Jerusalem artichoke (see Table 1, O—O) and in differentially nitrogen-fed plants of cv. Topianka ( · — · ) .

photosynthetic capacity. Thus, other factors such as carbon gain during the whole growth period and assimilate translocation to the tubers seem to be limiting to tuber yield. Not only leaf photosynthetic rates but also tuber-forming ability could be useful in achieving progress in breeding as both physiological processes show a wide intraspecific variability. Without an increase in tuber sink capacity, improvements in photosynthetic features will hardly be able to influence harvestable tuber yield. For similar reasons, potato breeding programs probably did not take advantage of C 0 2 uptake characteristics as selection parameters (Dwelle et al., 1981) although to some extent positive correlations to tuber yield could be found (Moll and Henniger, 1978).

A promising improvement in photosynthetic carbon gain of Jerusalem artichoke could result from a delay in the autumnal decrease in photosynthetic intensity. This decline of C 0 2

uptake takes place during the period of the fastest tuber growth (Fig. la). Therefore, during the most important months for tuber filling, September and October, the plants have to rely increasingly on temporarily stored assimilate reserves from the stem (Soja et al., 1989) whereas new photoassimilates become scarce. A possible explanation for this behaviour can be seen in the parallel decrease of leaf nitrogen concentrations which indicates a net export of nitrogen that is supposedly transported to the tubers. So, even under sufficient supply, nitrogen in the leaves becomes limiting for carbon gain as other plant parts exert a strong sink activity. The dependence of the photosynthetic rate on leaf nitrogen is not only evident during plant development but can also be seen in leaves of similar age grown at different nitrogen levels (Fig. lb). The results show that the nearly linear relationship between C 0 2

43

assimilation and total leaf nitrogen for a given species (Evans, 1989) is also found in Jerusalem artichoke. On the basis of this relationship, it should be possible to predict the actual photosynthetic rate in dependence on leaf nitrogen level and plant age. A fine-tuning of this relationship according to the cultivar-specific differences in photosynthesis might be desirable.

The concentrations of soluble protein and of chlorophyll in the leaf may function as estimates for the proportions of soluble and thylakoid-bound nitrogen. Both fractions constitute less than the total leaf nitrogen, the remainder being attributed to nucleic acids (Evans, 1989). Concentrations of soluble protein are generally thought to be closely correlated to the concentrations of ribulose bisphosphate carboxylase, but in Jerusalem artichoke no positive correlation could be found with photosynthetic rate (Fig. 2a). Plants apparently partition a relatively constant amount of nitrogen to ribulose bisphosphate carboxylase, even under moderate nitrogen stress. However, partitioning of nitrogen to the thylakoids is reduced at low nitrogen levels. The decline of the photosynthetic rate in plants under nitrogen stress is at least partly caused by the shortage of chlorophyll and, thus, of components of the electron transport chain. The importance of thylakoid nitrogen for photosynthesis in Jerusalem artichoke can be deduced from the significant correlation between C02 uptake and chlorophyll concentrations (Fig. 2b). Ageing leaves lose chlorophyll only slowly whereas the concentrations of soluble protein remain constant or even increase (data not shown). The increment of soluble protein during plant development could be a side-effect of the intensified nitrogen export from the leaves and probably does not reflect the amount of ribulose bisphosphate carboxylase in older leaves. Additionally, a lower specific activity of the enzyme in these leaves could be assumed.

The results show that differences in the photosynthetic capacity of Jerusalem artichoke can be traced back to differences in nitrogen concentrations in the leaves, resulting from nitrogen allocation at the plant level, and to the amount of thylakoid nitrogen, resulting from nitrogen partitioning at the cellular level. But even if agronomical practice and breeding progress will be able to improve photosynthetic performance of this crop, substantial increases in tuber yield will depend on changes in the assimilate translocation pattern.

5 ACKNOWLEDGEMENTS The authors thankfully acknowledge the financial support of the Fonds zur Förderung

der wissenschaftlichen Forschung (Projekt Nr. P7104-C) for part of this work.

44

6 REFERENCES

Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24: 1-15.

Blanchet, R., Gelfi, N., Piquemal, M. and Amiel, C , 1986. Influence de Γ alimentation azotoe sur revolution de l'assimilation nette au cours du cycle de deVeloppement du tournesol (Helianthus annuus L.). C. R. Acad. Sei., Paris, Ser. Ill, 302: 171-176.

Blanchet, R., Gelfi, N. and Puech, J., 1987. Alimentation azotoe, surface foliaire et formation du rendement du tournesol (Helianthus annuus L.). Agrochimica, 31: 233-244.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.

Dorrell, D.G. and Chubey, B.B., 1977. Irrigation, fertilizer, harvest dates and storage effects on the reducing sugar and fructose concentrations of Jerusalem artichoke tubers. Can. J. Plant Sei., 57: 591-596.

Dwelle, R.B., Kleinkopf, G.E., Steinhorst, R.K., Pavek, J.J. and Hurley, P.J., 1981. The influence of physiological processes on tuber yield of potato clones (Solanum tuberosum L.): stomatal diffusive resistance, stomatal conductance, gross photosynthetic rate, leaf canopy, tissue nutrient levels, and tuber enzyme activities. Potato Res., 24: 33-47.

Ergashev, A., 1976. Intensity and dynamics of photosynthesis products formation in Jerusalem artichoke Fiziol. Biokhim. Kul't. Rast., 8: 299-303. (in Russian)

Evans, J.R., 1989. Photosynthesis and nitrogen relationships in leaves of Cj plants. Oecologia, 78: 9-19. Moll, A. and Henniger, W., 1978. Genotypische Photosyntheserate von Kartoffeln und ihre mögliche Rolle

für die Ertragsbildung. Photosynthetica, 12: 51-61. Soja, G., Dersch, G. and Praznik, W., 1990. Harvest dates, fertilizer and varietal effects on yield,

concentration and molecular distribution of fructan in Jerusalem artichoke (Helianthus tuberosus L.). J. Agron. Crop Sei., 165: 181-189.

Soja, G., Haunold, E. and Praznik, W., 1989. Translocation of 14C-assimilates in Jerusalem artichoke (Helianthus tuberosus L.). J. Plant Physiol., 134: 218-223.

Zvara, J. and Herger, P., 1983. Einfluß von Klärschlamm und NPK auf Ertrag und Zuckergehalt bei Topinambur (Helianthus tuberosus L.). Bodenkultur, 34: 13-21.


JERUSALEM ARTICHOKE PRODUCTIVITY MODELLING

P. DENOROY INRA, Station de Bioclimatologie, 78850 Thiverval-Grignon, France

ABSTRACT

A model describing the growth of Jerusalem artichoke and parametrized for Dutch conditions has been tested using data from 38 trials with Jerusalem artichoke in the Netherlands, France, Spain and Italy. Discrepancies between experimental data and simulation outcomes are probably due to poor simulation of the following processes: leaf area extension, ontogenetic development, and distribution of assimilates. Propositions are made for improvement of the modelling of Jerusalem artichoke productivity.

1 INTRODUCTION Jerusalem artichoke {Helianthus tuberosus L.) is known to show a large interannual

and geographical variation in productivity, which to a large extent depends on weather conditions (Barloy et al., 1990). The existence of a scientific network did allow the collection of experimental data which were obtained using a common growth analysis method (Barloy, pers. comm.). This set of data was used to test an already existing model (Spitters, 1989) and could be used to develop new ones.

2 MATERIAL AND METHODS 2.1 The experimental data

The late cv. Violet de Rennes and the early cv. D19 (Blanc Precoce) were chosen as reference cultivars. Each cultivar was grown for one to several years as follows: four crops were grown in Wageningen (51.6 °N, 5.4 Έ ) , four in Rennes (48.6 °N, 1.5 °W), six in La Miniere-Grignon (48.5 °N, 1.8 °E), one in San Esteban (41.3 °N, 3.1 °W), two in Madrid (40.3 °N, 3.4 °W), one in Polignano (41.0 °N, 17.3 °E), and one in Policoro (40.1 °N, 16.4 Έ ) . In Wageningen, cv. D19 was replaced by cv. Columbia which has a similar growth pattern. The crops were irrigated and fertilized so as to avoid related stresses. Dry matter of the various plant parts (at least shoots and tubers) as well as leaf area index (LAI) were measured at various days during the growing season. Daily weather

46

data were recorded in a meteorological station nearby (generally a few kilometers from the experimental location).

2.2 The LINTUL "sink-limited" model This dynamic model (Spitters, 1989; unpublished) is similar to the one described by

Spitters (1989), with only one modification, viz. for sink-driven assimilate distribution. It was chosen for the following reasons: - it is more complete than the models of Becquer (1985) or Allirand et al. (1989), - it is consistent with the physiological hypothesis of growth pattern determinism (Denoroy etal., 1990), - there is good agreement between the experimental and simulated results obtained at Wageningen (Spitters, 1989).

Its main features are: - the time axis is the temperature sum (that is the sum of daily average temperatures above 0 °C), since the day of emergence, - light interception is defined as the minimum of a logistic equation (increasing LAI phase) and a linear equation (decreasing LAI phase), both being dependent on the temperature sum, - light efficiency is defined as the product of two processes: assimilate production (coefficient: 3.1 g CH20/intercepted MJ PAR [= photosynthetically active radiation]), and conversion to dry matter (coefficients: 0.7 for structural DM, 0.8 for DM of reserves), - first priority is given to aerial structural ("shoot") growth; the potential shoot growth varies as a logistic function of the temperature sum, - second priority is given to tuber growth; the potential growth of this part is defined by two growth phases each of them being described as a linear function of the temperature sum, - if there are assimilates in excess, this surplus constitutes a reserve pool that will be depleted at the end of the growing season, when assimilate supply is lower than potential growth of shoots and tubers, - the model was calibrated with Dutch experimental results; there are two sets of parameters: one for the early cultivar and the other for the late one.

3 RESULTS 3.1 Prediction of total dry matter and of dry matter of tubers

The discrepancy between experiment and simulation is often large (Table 1) and varies in direction. Hence, an attempt was made to discover the weak parts of the model.

47

Table 1. Characteristics of the ratio "simulated yield/experimental yield" (S/E).

Early cv. Late cv.

Total dry matter

Mean (S/E)

1.089 0.976

SDa (S/E)

0.284 0.220

Dry matter of tubers

Mean (S/E)

1.117 0.867

SD (S/E)

0.374 0.373

a SD = standard deviation.

3.2 LAI (or light interception) The LAI was calculated from simulated light interception with the equation given by

Spitters et al. (1989). The average climate at Wageningen being considered as cool, the simulation results differ from the experimental data as follows: - in case of a moderately warm climate (France or Wageningen, 1988): underestimation of early growth (during 1 to 2 months), - in case of a warm climate (Spain or Italy): overestimation of early growth, underestimation of lifetime of foliage (1 to 2 months), - in case of an arid climate, even with irrigation (Madrid): overestimation of maximal LAI.

3.3 Developmental stages Characteristic developmental stages considered were: tuber appearance, moment of

shift in tubers' growth rate, and end of shoot growth. For early as well as for late cultivars, simulations fit well with the data of the trial in the Netherlands and of trials with an early planting date in France. For normal or late planted crops in France, the actual developmental stages appear earlier than as assumed in the simulation. In Spain or Italy the trend is opposite to that found in France.

3.4 Assimilate distribution The over- or underestimation in total dry matter and of LAI are in general positively

correlated. Assimilate distribution between shoots and tubers show a large variability during plant growth. There is no constant trend as for the discrepancy between simulated and experimental shoot/tuber ratios. As simulated growth patterns depend on the stage of development, this discrepancy is partly due to inaccuracies in the simulation of plant development.

4 DISCUSSION 4.1 The light use efficiency

In spite of a large scattering in the data, total dry matter production is closely

48

correlated with the sum of intercepted radiation (calculated with actual LAI). The light use efficiency coefficients of the model appear to be consistent with most of the experimental results. Nevertheless, there is a trend toward a lower efficiency under arid climate conditions. This is probably due to a high respiration rate and to near light saturation of leaves which together yield a lower ratio of carbon assimilation to intercepted radiation than in the other locations. A more detailed evaluation of light interception, respiration and biochemical conversions could help to better investigate the origin of variations in light use efficiency.

4.2 Some ways to improve Jerusalem artichoke productivity modelling 4.2.1 The role of photoperiodism

In the LINTUL model, the occurrence of the developmental stages as defined in section 3.3 is in fact dependent on the cumulated daily average temperatures. However, photoperiodism is known to have a strong effect on the development of late cultivars (Meijer and Mathijssen, 1991). For early cultivars, the temperature regime, particularly extreme temperatures, play an important role (Courduroux, 1966), although photoperiodism has also some effect (Nitsch, 1965). Hence, daylength should be taken into account in the model, at least for late cultivars.

4.2.2 The definition of efficient temperature The existence of a minimal and a maximal efficient temperature is very likely to

explain the discrepancies between the model outputs and the experimental data for LAI dynamics. As the rate of many biological processes depends on temperature, a dynamic model aimed to be applied under very different climatic conditions must, as a rule, carefully take the efficiency of temperature into account.

4.2.3 Assimilate balance as a determinant of developmental stages The developmental stages considered in section 3.3 are in fact the expression of shifts

in assimilate (or reserve sugar) distribution. Appearance of stolons and above all a change in tubers' growth rate could result from excessive assimilate availability (Meijer and Mathijssen, 1991). The possible role of the stem as an active sink competing with tubers for sugar storage, instead of a passive overflow storage organ, should be investigated. As an alternative hypothesis, the relation between flower induction and tuberization, involving growth regulators, could also explain the temporary inability of stolons to grow as tubers.

4.2.4 The determinism of aerial structural growth The priority of assimilates for the aerial part of the plant is likely, but the extent of

49

the related demand is still unclear and probably not simply correlated with the temperature sum. A strong aerial growth results in a relatively low reserve content and as a consequence in a low tuber yield. Plant density induces more or less branching and can have some effect on structural sinks.

4.2.5 The determinism of relative sink effects According to the preceding hypothesis, sink effects of the various plant organs can

not only be defined by the temperature sum reached at a given moment but could also depend on the size, metabolic activity and relative position of the sinks. Assimilate and reserve (re-)distribution could depend on the relative sink strength of the various active organs and could be simulated like the distribution of electrical charges.

5 CONCLUSION A model like LINTUL proves to be efficient as far as forcing functions vary only to

a limited extent. To be relevant under various environmental conditions a dynamic model should rely more on detailed knowledge of the biological processes. The hypothesis discussed is presently tested in a joint research project involving INRA (Thiverval-Grignon, France), the Department of Theoretical Production Ecology of the Agricultural University and the CABO (both in Wageningen, The Netherlands).

6 ACKNOWLEDGEMENTS Experimental data were kindly provided by J.-M. Allirand and M. Chartier (INRA,

Grignon, France), J. Barloy (ENSA, Rennes, France), G. De Mastro (Universita degli Studi, Bari, Italy), J. Fernandez (ETSIA, Madrid, Spain), the late C.J.T. Spitters and W.J.M. Meijer (CABO, Wageningen, The Netherlands).

7 REFERENCES

Allirand, J.-M., Chartier, M., Gosse, G., Lauransot, M. and Bonchretien, P., 1989. Jerusalem artichoke productivity modelling. In: G. Grassi and G. Gosse (Eds.), Topinambour (Jerusalem artichoke), Proc. EC Workshop, 30 Sep. - 1 Oct. 1987, Madrid, Report EUR 11855. CEC, Luxembourg, pp. 17-27.

Barloy, J., Curt, M.D., Martinez, M. and Mimiola, J.C., 1990. Productivite du topinambour dans TEurope communautaire. In: G. Grassi, G. Gosse and G. Dos Santos (Eds.), Biomass for Energy and Industry, Vol. 1, Proc. 5th Conf., Lisbon. Elsevier Applied Science, London, pp. 1.223-1.227.

Becquer, T., 1985. Contribution a une modelisation de Γ elaboration de la biomasse chez le topinambour. Memoire D.A.A, ENSA, Rennes, 43 pp.

Courduroux, J.C., 1966. Etude du mecanisme physiologique de la tuberisation chez le topinambour. These doctorat es-sciences, Univ. Clermont-Ferrand, Masson, Paris, 355 pp.

Denoroy, P., Allirand, J.M., Chartier, M. and Gosse, G., 1990. Modelisation de la production de tiges et de tubercules chez le topinambour. In: G. Grassi, G. Gosse and G. Dos Santos (Eds.), Biomass for

50

Energy and Industry, Vol. 1, Proc. 5th Conf., Lisbon. Elsevier Applied Science, London, pp. 1.489-1.493.

Meijer, W.J.M. and Mathijssen, E.W.J.M., 1991. The relation between flower initiation and sink strength of stem and tubers of Jerusalem artichoke. Neth. J. Agric. Sei., 39: 123-135.

Nitsch, J.P., 1965. Existence d'un stimulus photoporiodique non spocifique capable de provoquer la tuberisation chez Helianthus tuberosus L. Bull. Soc. Bot. Fr., 112, 333-340.

Spitters, C.J.T., 1989. Modelling crop growth and tuber yield in Helianthus tuberosus. In: G. Grassi and G. Gosse (Eds.), Topinambour (Jerusalem artichoke), Proc. EC Workshop, 30 Sep. - 1 Oct. 1987, Madrid, Report EUR 11855. CEC, Luxembourg, pp. 29-35.

Spitters, C.J.T., Lootsma, M. and Van de Waart, M., 1989. The contrasting growth pattern of early and late varieties in Helianthus tuberosus. In: G. Grassi and G. Gosse (Eds.), Topinambour (Jerusalem artichoke), Proc. EC Workshop, 30 Sep. - 1 Oct. 1987, Madrid, Report EUR 11855. CEC, Luxembourg, pp. 37-43.


EXPERIENCES IN CULTIVATION, PROCESSING AND APPLICATION OF JERUSALEM ARTICHOKE (HELIANTHUS TUBEROSUS L.) IN YUGOSLAVIA

D. PEJIN*, J. JAKOVLJEVIC*, R. RAZMOVSKI* and J. BERENJI** Faculty of Technology, University of Novi Sad, Novi Sad, Yugoslavia Institute of Field and Vegetable Crops, Faculty of Agriculture, Novi Sad, Yugoslavia

ABSTRACT

Investigations were carried out on three topics: (i) cultivation of Jerusalem artichoke, (ii) optimization of analytical procedures, and (iii) optimization of ethanol production.

Agronomic investigations on the cultivation of Jerusalem artichoke has shown this crop to have a significant advantage over other ones with respect to biomass yield not only in fertile areas, but especially on marginal, i.e. sandy soils. In a number of experimental trials, an average fresh tuber yield of 40-80 t ha"1 together with an above-ground dry matter yield of 4 to 24 t ha"1 was recorded. Cultivar differences, a.o. regarding tuber yield, indicate possibilities of further improvement by plant breeding. Furthermore, yields could be increased by more adequate growing practices.

Advanced analytical procedures, such as HPLC, were used to analyse the synthesis of inulin and its hydrolytic breakdown during the vegetation period and the post-harvest storage of Jerusalem artichoke tubers. Extraction of the tubers and purification of the raw juice resulted in a juice which, apart from high-polymer inulin, contained significant amounts of fructo-oligosaccharides as well as free glucose and fructose. Glucose usually appeared during inulin synthesis, whereas free fructose was found when degradation of polymers took place during tuber storage.

Optimization of the process of ethanol production was focused on procedures with minimized energy consumption as well as on sources of enzymes needed. The highest yields of ethanol were obtained with Kluyveromyces marxianus. Using dense inocula an average ethanol yield of more than 90% of the theoretical could be attained. GC analysis showed larger inocula to lead to an increased content of acetaldehyde, but a decreased amount of ethylacetate. Concentrations of w-propanol, wo-butanol and wo-amylalcohol were largely independent of inoculum size. The composition of the stillages obtained after fermentation by K. marxianus and by Saccharomyces cerevisiae proved to be similar.

1 INTRODUCTION

Jerusalem artichoke can be found on small plots throughout the whole country but it was not until its recent rediscovery as a distinct food, feed, and raw material for industry and as an "energy crop", that its large-scale production was recommended. Today, Jerusalem artichoke is even being considered as the pre-eminent raw material of the future. Therefore, our studies included a comparison of Jerusalem artichoke with well-known crops

52

from an agronomic point of view. Investigations were also directed to an improvement of analytical procedures, especially with respect to tuber analysis. Potential industrial application of Jerusalem artichoke, for instance as to ethanol production, was also part of our research.

2 MATERIALS AND METHODS Jerusalem artichoke was compared with grain sorghum, sugar beet, corn and potato.

The variety performance trial included six cultivars of Jerusalem artichoke of different origin. A density experiment was performed with cv. Violet Commune.

Field trials were carried out on different soil types (chernozem, black sand and brown sand), in replicated blocks. The polar coordinate grid design la of Neider (1962; see also Bleasdale, 1967) was used to study the effect of plant density.

Extraction of fructan from sliced roots was carried out according to the method described by Jakovljevic et al. (1986). The methods used in the Chromatographie separation of saccharides have been reported earlier (Jakovljevic et al., 1981).

The process conditions used in the fermentation of Jerusalem artichoke raw juice by Saccharomyces cerevisiae and Kluyveromyces marxianus, have been described by Pejin et al. (1985).

Gas-chromatographic analyses were carried out according to the method of Adam and Postal (1987).

ANOVA and regression analysis were used to interpret the experimental data.

3 RESULTS Jerusalem artichoke cultivated on high-quality soils as well as under less favourable

conditions significantly outyielded all the other crops examined (with one exception only, viz. sugar beet on black sand), with respect to biomass and carbohydrate yield (Fig. 1). Its advantage is especially worth attention on marginal, i.e. sandy soils unsuitable for growing conventional food and feed crops (Berenji and Kovac, 1983; Pekic et al., 1983). The cultivar differences with respect to yield of tubers and above-ground plant parts (Table 1) indicate possibilities of further improvement by using the most well-adapted cultivar and/or by plant breeding.

The results of the plant density experiment showed, that the total fresh tuber yield per m2 was lowest (2 kg m"2) at the lowest plant density and was largest (3.2 kg m"2) at a density of 6-7 plants m"2, which could therefore be considered optimal for this trial (Fig. 2). The optimal plant density corresponds to a plant distance, within and between rows, of 35-40 cm. A further increase in plant density leads to yield reduction (Berenji and Kisgeci, 1988).

53

en

n Ί

Ή fl-J ?J 6 1 5-J H 3H

chernozem

Π H H H M H M H

|—|

H H H H M JLd

n M M M M 1Λ

ΡΊ Mm MM MM Ld i z U

JA So Sb Cr Po

black sand

M

ΗΗΗΜΜ

PI

ι JA So Sb Cr Po c r o p s

brown s a n d

Ζ\ΓΊΓΆ 0 Π JA So Sb Cr Po

Fig. 1. Carbohydrate yield of Jerusalem artichoke (JA) as compared with grain sorghum (So), sugar beet (Sb), corn (Cr) and potato (Po) grown on different soil types including highly fertile chernozem, less fertile black soil and very poor brown soil.

Table 1. Agronomic performance of different Jerusalem artichoke cultivars at Podravska Slatina, 1988.

Cultivar

Violet de Rennes Bela Bianca BT-3 BT-4 Waldspindel

Tubers

Fresh yield (t ha"1)

70.10 53.14 52.18 56.69 62.52 46.77

DM content (%)

17.10 20.80 21.27 19.00 18.37 25.17

DM yield (tha1)

11.99 11.05 11.10 10.77 11.48 11.77

Stems and leaves

Fresh yield (t ha1)

60.34 6.72 6.78

49.17 61.72 39.06

DM content (%)

35.53 60.31 57.84 36.62 35.79 38.72

DM yield (tha'1)

21.44 4.05 3.92

18.01 22.09 15.12

Total DM yield (tha1)

33.43 15.10 15.02 28.78 33.57 26.90

Mean LSD1%

LSD5%

56.90 26.38 18.56

20.29 11.36 5.13 3.61

37.30 25.18 17.72

44.14 14.11 8.73 6.14

25.47 13.45 9.46

The raw juice of Jerusalem artichoke, apart from inulin, contained a significant amount of different fructo-oligosaccharides, the elution order of which from Chromatographie columns corresponds to their degree of polymerization (DP) (Fig. 3). Free monosaccharides, viz. D-glucose and D-fructose, also proved to be present in the raw juice of Jerusalem artichoke. Glucose usually appears during fructan synthesis when the tubers are growing, whereas free fructose is formed during enzymatic degradation of high-polymer inulin, during tuber storage (Edelman and Jefford, 1968).

HPLC-chromatographic analysis suggested that hydrolysis of raw juice by Novo

54

3,5

_ 3,0

T3 <U

2,5

2,0 oo

Y=2006x26?x-18x2

0T= 0,6704

LSD 5% - ' V n 1 1 1 1 1 1 i 1 1—

1 2 3 if 5 6 7 8 9 10 plant populationsiplants in m )

Fig. 2. Tuber yield of Jerusalem artichoke as affected by plant density at Baöki Petrovac, 1985.

Timelmin]

Fig. 3. HPLC chromatograms of Jerusalem artichoke raw juice. Stationary phase: ZORBAX-NH2, Du Pont (4.6 mm [i.d.] x 25 cm); mobile phase: acetonitrile/water (72:28, v/v), flow rate: 1 ml min"1; column temperature: 22 °C; peaks: 1, fructose; 2, glucose; 3, sucrose; 4, DP 3; 5, DP 4; 6, DP 5; 7, DP 6; 8, DP 7; 9, DP 8.

55

Table 2. The effect of inoculum size on fermentation parameters of Jerusalem artichoke juice.

Yeast species

S. cerevisiae*

K. marxianus

Inoculum (g dry matter/ 100 g of!

0.250 0.500 1.000

0.250 0.500 1.000

sample)

Maximal concentration of ethanol (%,

8.44 9.38

10.10

7.73 8.62 9.64

w/w)

Total productivity (g g1 h"1)

0.34 0.45 0.46

0.31 0.44 0.47

Yield (% of theoretical)

66.7 88.2 90.2

60.8 86.3 92.2

a In the case of S. cerevisiae inulin was pre-hydrolysed; to this end, Novo-inulinase was added (2 units g"1

inulin).

Table 3. By-products formed during the production of raw ethanol from Jerusalem artichoke juice upon fermentation by different yeasts.

Yeast Inoculum Acet- Ethyl-species (g DM per aldehyde acetate

100 g of (mg Γ1) (mg Γ1) sample)

wo-Butanol (mg Γ1)

wo-Amy 1- /i-Propanol Methanol alcohol (mg Γ1) (mg Γ1) (mg Γ1)

S. cerevisiae

K. marxianus

0.250 0.500 1.000

0.250 0.500 1.000

311.5 375.8 381.7

192.6 256.8 299.5

141.4 176.3 182.5

traces 98.5

151.6

18.5 20.2 25.6

_ 7.2

17.8

115.4 141.5 250.1

-157.3 189.5

---

. 51.2 98.3

-traces traces

traces 2.33 3.50

inulinase first led to degradation of fructan polymers with DP > 10, with the formation of D-fructose and fructo-oligosaccharides.

Table 2 shows the results on fermentation of Jerusalem artichoke juice. These data clearly indicate that for ethanol production K. marxianus is preferable, as the addition of inulinase is not required. It is also important to point out that it gives higher yields than S. cerevisiae.

Table 3 summarizes the data of gas-chromatographic analysis of unpurified ethanol, as produced in fermentations by S. cerevisiae and K. marxianus. Depending on the yeast employed different amounts of various by-products were formed.

CONCLUSIONS

Our agronomic investigations showed cultivation of Jerusalem artichoke on various

56

soils to result in higher yields than standard crops. HPLC was very successfully applied in the determination of the carbohydrate content of Jerusalem artichoke, during ripening, storage and processing. Novo inulinase seemed to hydrolyse inulin with higher DP ( > 10) first; only mono- and disaccharides were used as fermentation substrates. The inulinase-producing yeast species K. marxianus proved to be more convenient than S. cerevisiae, which does not produce inulinase, to ferment Jerusalem artichoke juice to ethanol. The composition of the fermentation liquid appeared to depend strongly on the inoculum size and on the yeast species used.

5 REFERENCES

Adam, L. and Postal, W., 1987. Gaschromatografische Bestimmung von Ethylcarbamat (Urethan) in Spirituosen. Branntweinwirtschaft, 1987 (5): 66-68.

Bleasdale, J.A., 1967. Systematic designs for spacing experiments. Expl. Agric, 3: 73-85. Berenji, J. and Kisgeci, J., 1988. Plant density experiment with Jerusalem artichoke. EEC - DG XII - 2nd

Workshop on Jerusalem Artichoke, 6-8 Dec. 1988, Rennes (in press). Berenji, J. and Kovafc, V., 1983. Jerusalem artichoke as a potential energy crop. CNRE (FAO) Bull., 2:

41-44. Edelman, J. and Jefford, T.G., 1968. The mechanism of fructosan metabolism in higher plants as

exemplified in Helianthus tuberosus. New Phytol., 67: 517-531. Jakovljevio, J., BoSkov, Z. and Nikolov, Z., 1981. Mogu<5nost primene visoko pritisneteöne hromatografije

u analizi skrobnih hidrolizata. Hem. Ind., Supl. Ind. Sedera, 35: 45-48. Jakovljevio, J.B., Nikolov, Z.L. and BoSkov, Z.M., 1986. Some analytical aspects of enzyme degradation

of starch and inulin into malto- and fructooligosaccharides. Nahrung, 30: 171-176. Neider, J.A., 1962. New kinds of systematic designs for spacing experiments. Biometrics, 18: 283-307. Pejin, D., Ga<5eSa, S. and Razmovski, R., 1985. Proizvodnja etanola iz topinambura (Jerusalem artichoke).

Prehrambeno-Tehnol. Rev., 23: 11-18. Peki<5, B., Kova£, V. and Berenji, J., 1983. Mogu<5nosti gajenja öiöoke u Vojvodini kao potencijalne

sirovine za proizvodnju alkohola, Savremena Poljopr. Teh., 9: 83-86.


THE EFFECT OF LONG-TERM STORAGE ON THE FRUCTO-OLIGOSACCHARIDE PROFILE OF JERUSALEM ARTICHOKE TUBERS AND SOME OBSERVATIONS ON PROCESSING

H.W. MODLER*, J.D. JONES* and G. MAZZA** Agriculture Canada, Food Research Centre, Ottawa, Ontario, Canada K1A 0C6 Agriculture Canada, Research Station, Morden, Manitoba, Canada ROG 1J0

ABSTRACT

Jerusalem artichoke tubers, stored at various temperatures (5 °C, 2 °C, -10 °C, program-cooled to -10 °C and ambient), were analysed for relative amounts of fructo-oligosaccharide (FOS) components, using HPLC, to monitor shifts in the sugar profile. Results showed that trimmed and washed Jerusalem artichoke tubers can be stored in 6-mil polyethylene bags for one year at 2 °C, with virtually no microbial or fungal spoilage. Small buds and rootlets developed on the tubers stored at 5 °C for four months, however, this was not evident in tubers stored at 2 °C. The tendency was for inulin to shift to the shorter-chain FOS (degree of polymerization [DP] 2-6) during storage, even in the products stored at -10 °C. Tubers stored for 16 months had virtually no FOS with DP beyond 10, but had accumulated a substantial amount of DP 1-4. Difficulties in spray-drying of tubers (sticking) do not appear to be related to the content of short-chain FOS, but are dependent on hydration of the insoluble fiber component.

1 INTRODUCTION Considerable interest has developed concerning the production, processing and

utilization of Jerusalem artichoke tubers and tuber fractions in both food and feed products. This interest is focused on the importance of both the soluble and insoluble fiber in the human diet and of long-chain fructo-oligosaccharides (FOS) as bifidogenic factors. The short-chain polymers, with a degree of polymerization (DP) of 2-5, are commonly referred to as neosugars (Modler et al., 1990), while the longer-chain polymers (DP > 30) are referred to as inulin.

Food and feed ingredient manufacturers who process Jerusalem artichoke tubers are faced with two basic problems: (a) The harvest time is limited in Canada to 2 to 3 weeks in the autumn and 1 to 2 weeks

in the spring. Early harvest in the autumn increases the chances of spoilage and reduces the amount of inulin in the tubers while sprouting can be encountered in late-spring

58

harvesting, (b) Capital costs for processing equipment are high when the window for harvesting and

processing is narrow. Being able to store tubers for extended periods and processing over a 12-month period is much more cost-effective.

The objectives of this research were: (a) To determine the conditions for long-term storage of Jerusalem artichoke tubers, (b) To determine if the short-chain FOS were responsible for a difficulty encountered in

spray-drying, i.e. sticking of tuber powder, to the dryer walls.

2 METHODOLOGY 2.1 Planting and storage

Most of the experiments were based on the use of Columbia and Challenger cultivars; however, a limited quantity of Fusil and Sunroot was also grown. All varieties were planted as early as possible in the spring and in sandy soil, where possible. Treatments receiving maleic hydrazide were sprayed at a rate of 5.65 kg acre"1. The chemical was coded MH60 and was supplied by Uniroyal, Calgary, Canada. Maleic hydrazide was mixed at a rate of 5.65 kg per 300 litre of water, prior to application.

Tubers were harvested by first cutting the tops with a forage harvester and then digging the crop with a potato harvester. The crop was then transported to cold storage (5 °C) and held at this temperature prior to the trimming and washing operation. The tubers were trimmed and washed in 25-kg lots and stored in 6-mil polyethylene bags in one of five temperature treatments. Three of the treatments consisted of storage at 5 °C, 2 °C and -10 °C, while in the fourth treatment the product was cooled at 1 °C per week (starting at 4 °C) until the temperature reached -10 °C. The final treatment involved placing the tubers in a burlap bag and storing at ambient temperature in an unheated building exposed to winter frost. These storage trials lasted for a period of up to 16 months.

2.2 Processing The methodology for the processing of the tubers, into flour, has been described in

detail by Yamazaki et at. (1989). Basically, the process consists of dicing (1-cm cubes), macerating, heating to inactivate the polyphenol oxidase enzymes and then spray-drying the macerate using an atomizing nozzle.

3 RESULTS AND DISCUSSION 3.1 Tuber preparation and storage

Jerusalem artichoke tubers of cv. Columbia proved to be the highest yielding (Chubey and Dorrell, 1982; Kiehn and Chubey, 1985), but these were difficult to clean;

59

Table 1. Condition of Jerusalem artichoke tubers after various periods of storage.

Treatment

5 °Ca

2 °Ca

-10 °C(P)a'b

-10 °Ca

Ambient0

Time (months)

4

no change

no change --soft, partially spoiled

12

slight sprouting with occasional spoilage no change soft on thawing soft on thawing

16

not recommended

no change --

a Stored in 6-mil polyethylene bags. b Program-cooled. c Stored in burlap bags.

cvs. Challenger, Sunroot and Fusil were easier to wash and trim; however, the yield was substantially less than that of Columbia.

All tubers, regardless of cultivar, kept well for six months at 5 °C with no spoilage or microbial growth (Table 1). Some sprouting and spoilage did occur after 12 months storing at 5 °C. Storing the tubers at 2 °C was a very effective method for long-term storage. Virtually no spoilage or microbial growth was evident after 12 months storage. The tubers, regardless of cultivar, kept well under these conditions. The tubers maintained their firmness and deterioration due to microbial action was not a problem except in tubers which were frost-damaged prior to storage. Presumably, the tubers kept well due to accumulation of C02 in the polyethylene bags, however, this aspect is still under investigation at Kentville, Nova Scotia. It should also be pointed out that the tubers were free from Sclerotinia when placed in storage and this undoubtedly prolonged storage life (Huang and Stauffer, 1979).

3.2 FOS analysis Results of analysis of tubers subjected to various storage treatments, for FOS content,

appear in Table 2. The main difference between the initial analysis and the treatments is in the amount of long-chain FOS (DP > 10). The long-chain FOS tend to decrease in storage while the amount of short-chain FOS varied with treatment: DP 2-6 increased at 5 °C; DP 1 increased from 1.0 to 11.0% in the 2 °C treatment; DP 2-5 increased substantially in the -10 °C program-cooling of tubers, while the -10 °C and the ambient temperature treatment were similar to control. This is not surprising considering that the mean ambient winter (December to March) temperature is not substantially different from that (-10 °C) in Ottawa (soil temperature is claimed not to go below -1 °C in Ottawa).

These results indicate that at 5 °C there is sufficient metabolic activity to utilize the

60

Table 2. Relative percentage of soluble fructo-oligosaccharide components in Jerusalem artichoke tubers (cv. Columbia) stored under various conditions for a period of 18 weeks8.

DPb

1 2 3 4 5 6 7 8 9

10 > 10

Storage treatment (°C)

5

1.0 16.4 14.0 13.0 12.0 10.5 8.0 6.0 4.7 4.0

10.3

2

11.0 11.0 7.0 6.0 7.0 8.0 8.0 7.3 7.0 7.0

21.4

-10PC

5.0 19.0 15.0 11.4 10.0 8.5 6.7 5.0 4.0 3.5

11.2

-10

1.3 11.5 10.3 10.0 10.6 10.3 9.3 8.0 7.0 6.4

15.2

Ambientd

3.6 11.3 11.0 8.7 9.7 9.2 7.9 6.6 5.8 5.4

19.8

Initial

< 1 to 1.0 10.0-12.0 7.5- 9.0 6.5- 8.5 7.0- 9.0 8.0- 9.0 7.5- 8.0 6.5- 7.0 6.0 5.0- 6.0

25.0-33.0

a Trimmed, washed and stored in 6-mil polyethylene bags. Harvested November 15/89 and analysed April 3/90. b Degree of polymerization. c Program-cooled 1 °C per week, starting at 5 °C. d Kept in an unheated storage building in a burlap bag.

Table 3. Relative percentage of soluble fructo-oligosaccharide components in Jerusalem artichoke tubers (cv. Columbia) stored at 5 °C for up to 16 months8.

DPb

1 2 3 4 5 6 7 8 9

> 9

Duration of storage (5

2 wks

< 1 to 1.0 10.0-12.0 7.5- 9.0 6.5- 8.5 7.0- 9.0 8.0- 9.0 7.5- 8.0 6.5- 7.0 6.0

30.0-39.0

°C)

12 months

6.0 32.7 16.1 12.4 9.4 7.2 5.1 3.6 2.4 5.2

16 months

12.3 40.9 17.6 10.9 6.8 4.7 2.9 1.6 1.1 1.3

Standard deviation0

1.08 1.08 1.11 0.44 0.26 0.19 0.20 0.22 0.26 2.07

8 Trimmed, washed and stored in 6-mil polyethylene bags. Planted in April/87 and harvested in Nov/87. b Degree of polymerization. c Standard deviation based on 15 samples.

D P 1 formed from the breakdown of long-chain FOS. As the temperature drops to 2 °C ,

the rate of respiration slows which results in accumulation of DP 1 (11%) and only a slight

61

Table 4. Comparison of the relative amounts (in %) of soluble fructo-oligosaccharides in four varieties of Jerusalem artichoke tubers, following spring harvest.

DPa Variety

Fusilb Columbia0 Sunroot0 Challenged

1 2 3 4 5 6 7 8 9

10 > 10

3.4 8.9 8.1 7.4 7.6 8.4 8.2 7.2 6.5 5.9

28.3

2.3 25.0 15.0 13.2 11.0 8.8 6.6 5.2 3.8 3.0 6.2

2.5 11.2 7.8 7.7 7.9 8.2 7.8 7.3 6.4 5.9

27.4

4.8 12.4 9.0 8.0 7.8 7.7 7.2 6.1 5.7 5.1

25.1

a Degree of polymerization. b Harvested May 12/89. c Harvested April 24/89. d Harvested May 1/89.

drop in DP > 10, i.e. from the initial range of 25-33 to 21.4%. Holding tubers at 5 °C in long-term storage results in a significant decrease in the

amount of long-chain FOS (DP > 9). Initially, the Columbia tubers contained 30-39% of DP > 9 and this dropped to approximately 1.3% at the end of 16 months storage. As expected there was a large increase in the amount of FOS in the DP 1-4 range (Table 3).

Table 4 shows the results of a comparison of FOS profiles of four varieties of Jerusalem artichoke. Although Columbia was harvested the earliest, it still had the lowest content of the long-chained FOS and the greatest accumulation of DP 2-5.

3.3 Observations on cultivar selection Columbia is the highest-yielding cultivar; however, considerations with respect to

cultivar selection should go beyond yield since Columbia: (a) Is difficult to clean, (b) Requires more trimming than other varieties, (c) Is not readily adaptable to continuous mechanical washing.

Challenger is much easier to clean than Columbia; however, the yield is considerably less. Both Fusil and Sunroot winter well, but the yield of these tubers is substantially lower than that of Columbia. Fusil is the cultivar of choice for the authors because of its ease in washing and trimming, wintering ability and relatively high content of DP > 9 at spring

62

harvesting. There is some indication that both Columbia and Challenger are more susceptible to rotting at the stem-end during overwintering in the soil; however, these instances were isolated.

3.4 Processing conditions Macerate of Jerusalem artichoke tubers is a difficult product to spray-dry. There are

two main reasons for this: (a) The fibrous material is very abrasive and is extremely erosive to even tungsten carbide

nozzles. (b) The spray-dried product tends to stick to the walls of the dryer chamber during

processing. Initially, it was thought that the sticking problem was related to the content of short-

chain FOS in the macerate. This aspect was investigated by substantially hydrolysing the FOS with an inulinase preparation which reduced all the long-chained FOS to DP 5 or less. This treatment did not increase the difficulty in drying Jerusalem artichoke tuber macerates. After further investigation it was observed that hydration of the insoluble fiber component is the main factor which contributes to difficulty in drying, i.e. powder sticking to the dryer walls. This can be circumvented by reducing the time the macerate is held in between heat-treatment and spray-drying. Cooling the macerate prior to drying appears to have little influence on slowing hydration of the insoluble fiber.

During the spray-drying of Jerusalem artichoke tuber macerate it is helpful to operate the equipment at outlet air temperatures of 95 to 102 °C. This tends to reduce sticking, but also means that measures must be taken to remove the flour from the box as it is produced, in order to reduce browning. The authors have also observed that pressure atomization is the method of choice rather than attempting to use rotary disc atomization.

Substantial improvements can be made to both the colour and flavour of the products prepared from Jerusalem artichoke tuber flour, if the tubers are blanched prior to macerating. This was achieved by one of two methods in the laboratory: (a) Autoclaving at 10 psi for 20 min (2 x 25-kg lots per autoclaving). Sunroot was easier

to blanch than Columbia and only required 20 min at 10 psi steam pressure. (b) Use of the continuous ABCO blancher (Timbers et al., 1984; Cumming et al., 1984).

The ABCO blancher proved to be excellent equipment for continuous blanching of Jerusalem artichoke tubers. This blancher is designed for regeneration of heat and can process 8 kg of product kg"1 of steam which is substantially better than hydrostatic blanchers at 3:1 or hot-water blanchers at 2:1. Autoclaving is a satisfactory technique for small-scale production of Jerusalem artichoke flour product; however, it does yield a darker product due to caramelization. Drain liquor from the ABCO blancher was of excellent quality in

63

terms of clarity and colour. Upon cooling, inulin rapidly precipitated from this fraction.

3.5 Potential uses of Jerusalem artichoke flour and fractions Jerusalem artichoke tubers are an excellent source of both soluble and insoluble

fibers. The insoluble fiber component, with proper bleaching and reduction in off-flavour, has potential for application in white-bread.

Numerous other uses of FOS exist in both the food and pharmaceutical industry and these have been documented by Kim (1988) and Modler et al. (1990). The major impediment of utilizing Jerusalem artichoke tuber flour in food products is the undesirable flavour. Constituents of this flavour have been described by Chan (1983).

3.6 Feed application Jerusalem artichoke tubers can be used in animal feed as flour, dried cubes or

macerated and co-extruded in pelleted rations as a source of bifidogenic factors. Incorporating Jerusalem artichoke tuber fructo-oligosaccharides, probiotic bacteria (lactobacilli, bifidobacteria) and gamma globulin in milk replacers for neonatal pigs, appears to have some potential in reducing the cost of rearing specific-pathogen-free (SPF) pigs.

Recent work by Farnworth et al. (1993) has shown some potential for the application of Jerusalem artichoke flour to the rations of swine and poultry for the purpose of improving feed efficiency, reducing diarrhoea (swine) and manure odour.

4 ACKNOWLEDGEMENTS The authors of this paper are deeply indebted to Dr. F.A. Kiehn and Dr. B.B.

Chubey (Agriculture Canada, Morden, Manitoba) for their invaluable advice and cooperation during the course of this project. The authors also acknowledge the valuable technical assistance of Alan Payne, Ralph Cooligan and Duncan Stewart.

H.W.M. and G.M. respectfully dedicate this paper to the late Dr. J.D. Jones who died September 10, 1991.

5 REFERENCES

Chan, CM., 1983. Flavor components of Jerusalem artichoke tubers. M.Sc. Thesis, University of Manitoba, Winnipeg.

Chubey, B.B. and Dorreil, D.G., 1982. Columbia Jerusalem artichoke. Can. J. Plant Sei., 62: 537-539. Cumming, D.B., Stark, R., Timbers, G.E. and Cowmeadow, R., 1984. A new blanching system for the

food industry. II. Commercial design and testing. J. Food Process. Preservation, 8: 137-150. Farnworth, E.R., Jones, J.D., Modler, H.W. and Cave, N., 1993. The use of Jerusalem artichoke flour

in pig and chicken diets. In: A. Fuchs (Ed.), Inulin and Inulin-containing Crops, Studies in Plant Science, Vol. 3. Elsevier, Amsterdam, pp. 385-389.

Huang, H.C. and Stauffer, M.D., 1979. Sclerotinia wilt on Jerusalem artichoke. Canadex 164.630.

64

Kiehn, F.A. and Chubey, B.B., 1985. Challenger Jerusalem artichoke. Can. J. Plant Sei., 65: 803-805. Kim, H.S., 1988. Characterization of lactobacilli and bifidobacteria as applied to dietary adjuncts. Cult.

Dairy Prod. J., 23 (3): 6-9. Modler, H.W., McKellar, R.C. and Yaguchi, M., 1990. Bifidobacteria and bifidogenic factors. Can. Inst.

Food Sei. Technol. J., 23: 29-41. Timbers, G.E., Stark, R. and Cumming, D.B., 1984. A new blanching system for the food industry I:

Design, construction and testing of a pilot plant prototype. J. Food Process. Preservation, 8: 115-133.

Yamazaki, H., Modler, H.W., Jones, J.D. and Elliot, J.I., 1989. Process for preparing flour from Jerusalem artichoke tubers. U.S. Patent 4.871.574.


A PROCESS FOR THE PRODUCTION OF INULIN AND ITS HYDROLYSIS PRODUCTS FROM PLANT MATERIAL

Manfred VOGEL Südzucker AG Mannheim/Ochsenfurt, Zentrallabor, 6718 Grünstadt, Germany

ABSTRACT

The main objective of the work reported here was the production of fructo-oligosaccharides or rather inulo-oligosaccharides from inulin-containing plant material, especially from chicory roots. Later, also the extraction of inulin as such was looked into as well as the production of fructose from it, using the same process with minor modifications.

1 PRODUCTION OF INULO-OLIGOSACCHARIDES Two processes for the large-scale production of mixtures of fructo-oligosaccharides

are known. In the first process fructo-oligosaccharides are synthesized by the enzymatic action of a fructosyltransferase on sucrose. The product of this process, developed originally by the Japanese company Meiji Seika Kaisha, Ltd., has been given the trade name "Neosugar" (Hidaka, 1982). It is a mixture of glucose, sucrose and 0-2,1-linked fructo-oligosaccharides with a terminal glucose unit. In this case, the enzyme transfers the fructosyl part of a donor sucrose molecule onto an acceptor sucrose molecule. Since the resulting 1-kestose, and its fructosylation product nystose in its turn, act as acceptor molecules the final product contains 1-kestose, nystose and fructosylnystose. Oligosaccharides with longer chains than DP 5 are produced in minor amounts only.

The other process for the production of fructo-oligosaccharides, which will be described here in more detail, starts with the polysaccharide inulin. In this case, the linear ß-2,1 -linked fructose polymers built on a sucrose residue are partially hydrolysed by the enzyme endo-inulinase. As shown in Fig. 1, the enzymatic inulin hydrolysis yields two types of fructo-oligosaccharides; hetero-oligomers identical with those mentioned above for Neosugar, and homo-oligomers built up of 0-2,1 -linked fructose molecules but lacking the terminal glucose moiety. As evident from Chromatographie separation (Fig. 2), this product contains more fructo-oligosaccharides with longer chains (up to DP 7) than Neosugar; the

66

HOH2C

HO HOH:C ό η~30-35

HOH.C

Endo-lnulinase

+

HO 0 Jn=1-6 I H

GF-saccharide

HOH2C 9H

CH2 HO

HOH2C O

CH:

L· HO o Jn=1-6 I H

FF-saccharide

Fig. 1. Enzymatic preparation of GF- and FF-inulo-oligosaccharides by the hydrolytic cleavage of inulin with endo-inulinase.

amount of mono- and disaccharides, on the other hand, is lower, whereas inulobiose, a component of special interest, is included besides sucrose. In addition to endo-inulinases, also exo-inulinases are known; they hydrolyse inulin by sequential removal of the terminal fructose residue.

Both products, Neosugar and inulo-oligosaccharides, have the same characteristics. This is shown in Table 1. Apart from the fact that they are naturally occurring and that inulin is consumed to about 6 g daily in southern European countries, these oligosaccharides possess a sweetness about 0.4 to 0.6 times that of sugar. Furthermore, they are scarcely digestible in the alimentary tract (Ziesenitz and Siebert, 1987; Nilsson et aL, 1988; F. Heinz, unpubl. results). Therefore, fructo-oligosaccharides can be regarded as a kind of soluble "dietary fibre" with a reduced caloric value.

In the large intestine fructo-oligosaccharides are fermented, preferentially by bifidobacteria, to carbon dioxide, methane and volatile fatty acids. These fructo-

67

GF GF3GF4

%DS DP 1 DP 2 DP 3 DP 4 DP 5 DP 6 DP 7 DP > 7 Inulo-oligosaccharides 8.3 12.7 17.7 17.9 15.0 10.1 8.9 9.4

Fig. 2. The composition of an unrefined inulo-oligosaccharide mixture as shown by gel filtration.

Table 1. Important characteristics of fructo-oligosaccharides.

Fructo-oligosaccharides are: - naturally occurring - sweet - non-digestible in stomach and small gut - soluble dietary fibre - fermented by intestinal microorganisms - selectively utilized by bifidibacteria - suppressing the production of intestinal putrefactive substances

oligosaccharides are believed to exert a beneficial health effect due to the colonization of the large intestine with bifidobacteria thereby eliminating other, noxious bacteria like Clostridium and Staphylococcus spp. which are responsible for the production of putrefactive substances (Hidaka et al., 1986). Based on these properties the fructo-oligomers can be applied as a healthy, low-caloric ingredient in food. Additional positive effects of these oligosaccharides as an ingredient in feed are known (EP-O, 1988).

The first step of the process developed by Südzucker (patent pending) consists of a

68

separation of the exo-type inulinase from the endo-inulinase. The starting enzyme mixture inulinase was obtained from the Danish Novo company, where it is produced by fermentation by Aspergillus ficuum (Zittan, 1981).

The purified endo-inulinase was shown to have also a good stability with respect to incubation conditions in the presence of macerated chicory roots; the temperature optimum for endo-inulinase activity was near 60 °C. The effect of the pH on the composition of the inulo-oligosaccharides is shown in Fig. 3. As can be seen the smallest amount of mono- and disacchandes was obtained at pH 5.4. At this pH, the amount of DP 3 and DP 4 oligomers was maximal. A shift to lower pH-values gave larger amounts of short-chain components. At pH-values > 5.4 the activity decreased. Thus, the optimal pH was between 5.3 and 5.4, and therefore this pH was chosen in all further experiments. The incubation temperature and time in these experiments was 55-58 °C and 16 h, respectively, whereas the enzyme dosage was 2 units of endo-inulinase per g of inulin.

The process for the production of inulo-oligosaccharides as shown in Fig. 4, consists of a combination of enzymatic treatment with maceration and pasteurization, followed by some purification steps. Cleaned and washed plant material like chicory roots or Jerusalem artichoke tubers is chopped, macerated and quickly heated to 93-95 °C and kept at this temperature for about 15 min. The resulting pulp is pasteurized to inactivate the enzymes. Then, it is cooled down to 56-58 °C, the pH is adjusted to 5.3-5.4 and the amount of endo-inulinase required is added. During incubation, the pH is continuously monitored and

Fig. 3. Degradation of inulin with endo-inulinase: the effect of pH on the composition of inulo-oligosaccharides.

chicory roots I

washing

69

chromatography

(Alternative II)

chopping

I maceration/pasteurization

X cooling

X pH- adjustment

X enzymatic treatment

pH-adjustment

X filtration

neutralization

concentration

filtration

_|_ demineralization/.

decolorization I

microfiltration

concentration

drying

oC-Glucosidase plus chromatography (Alternative III)

Fig. 4. Process flow-sheet for the production of inulo-oligosaccharides.

controlled by adding lime. The reaction is stopped after 16 h by increasing the pH to 10.5-11.0, the residue is filtered off and the inulo-oligosaccharide-containing filtrate is neutralized, concentrated up to 35 to 45% dry substance content and filtered again.

At this point, there are three alternatives for the further processing of the solution, the choice depending upon the final purity of the product required, whereby the relative amounts of glucose, fructose and sucrose remaining in the final product are taken as a measure of purity. These alternatives are: a) Demineralization and decolorization of the above-mentioned solution, giving a final product with about 15% glucose, fructose and sucrose.

70

Fig. 5. Separation of inulo-oligosaccharides on a cation exchanger in the Ca-form.

b) A Chromatographie step on a cation exchanger in the Ca-form, leading to a reduction of glucose and fructose content. Fig. 5 shows the result of such an Chromatographie separation of inulo-oligosaccharides. As is evident, the main part of the oligosaccharides are eluted first followed by sucrose, glucose and fructose in that order. The different fractions of this Chromatographie separation were analysed by thin-layer chromatography (Fig. 6). As can

Fig. 6. A thin-layer chromatogram of fractions obtained by separation on a cation exchanger in the Ca-form.

71

be seen, some components of special interest, especially those with a shorter chain length, are eluted relatively late. The latter oligosaccharides seem to be those built up of fructose molecules only. The difference in Chromatographie behaviour between glucose-containing inulo-oligosaccharides and those consisting of fructose only especially applies to the DP 2 components sucrose and inulobiose. The sucrose is eluted very much earlier than the inulobiose. This seems to be also true for the analytical column (Fig. 7). c) The Chromatographie step described under b) yields inulo-oligosaccharides of higher purity but causes also a decrease in yield. Therefore, the possibility to eliminate sucrose from the solution prior to the Chromatographie step was investigated. This objective was achieved using an enzyme known as α-glucosidase which can be removed by the following Chromatographie step so that the product obtained is free from sucrose, glucose and fructose. Therefore, the addition of the latter enzyme can be considered as a third alternative for further processing. Fig. 8 shows two gel chromatograms, with and without treatment with α-glucosidase. As can be seen the DP 1 peak increased and the DP 2 peak decreased after incubation with this enzyme. The residual DP 2 peak is that of inulobiose.

The process for the production of inulin is very similar to that described above for the preparation of inulo-oligosaccharides; however, the enzyme treatment and the Chromatographie separation are omitted.

The process flow-sheet is shown in Fig. 9. The product composition obtained can easily be analysed by high-performance anion exchange chromatography using a Na-acetate

i r ■ I -i i- I i 1 ■ ·» ■ ■ 1 i i I I I I i i i I ■ i i l . I .1 . I .1 .1. 1 .1. I - I I 1 I i i ■ I I I

Fig. 7. HPAE chromatogram of inulo-oligosaccharide mixture; pulsed amperometric detector with gold electrode; elution conditions: 0.36 M NaOH and 0-0.2 M Na-acetate gradient in 45 min.

72

«- & -Glucosidase

-4-oC-Glucosidase

Fig. 8. Effect of treatment of a mixture of inulo-oligosaccharides with α-glucosidase as shown by gel filtration.

gradient in NaOH-solution as shown in Fig. 10. Oligosacchandes with chain lengths up to DP 40 can be determined. The high amounts of mono-, di- and oligosacchandes present can be readily eliminated by a single precipitation in aqueous solutions of ethanol. The precipitate obtained contains only components with DP > 10 (as shown in Fig. 11).

Summarizing, a process for large-scale production of inulin, inulo-oligosaccharides and fructose/glucose syrup from renewable plant material like chicory roots or Jerusalem artichoke tubers has been described (Fig. 12). For the production of fructose/glucose syrup a mixture should be used of exo- and endo-inulinase instead of solely endo-inulinase.

For the preparation of inulo-oligosaccharides three alternatives are mentioned, giving final products with different amounts of glucose, fructose and sucrose.

73

chicory roots

washing

_E chopping

T maceration/pasteurization

coaling

pH- adjustment

filtration

neutralization I ~

concentration I

filtration

demineralization/ decolorization

concentration

drying

Fig. 9. Process flow-sheet for the production of inulin.

ΐ ΐ

llJLJJ -A JL ILJk/LllJlj.lj I . . . . I . . . . I . . . . I . . . . I . . . . I . . . . I

Fig. 10. HPAE chromatogram of inulin extracted from chicory roots; elution conditions: 0.1 M NaOH and 0-0.45 M Na-acetate gradient in 45 min.

74

, v — A . . V V ^ ~ - - A Ü L . \j L W Hfc/V-·-,

I I I I . I I . . I I I I ■ I 1 , 1 , 1 I I I 1 T s

Fig. 11. HPAE chromatogram of precipitated inulin; elution conditions as indicated in Fig. 10.

enzymatic treatment (Inulinase)

Fructose

plant material

washing

chopping

maceration/ pasteurization

cooling

enzymatic treatment

(Endo-lnulinase)

I0S

pressing

I I t

Inulin

Fig. 12. Overall process flow-sheet for the production of fructose, inulo-oligosaccharides and inulin.

75

2 REFERENCES

EP-O, 1988. European Patent application No. 0293935 A2 (1988) (Mitsui Toatsu Chemicals Incorporated). Hidaka, H., 1982. Neosugar - manufacturing and properties. Proc. 1. Neosugar Res. Conf., Tokyo. Meiji

Seika Kaisha, Ltd., Tokyo, pp. 3-13. Hidaka, H., Eida, T., Takizawa, T., Tokunaga, T. and Tashiro, Y., 1986. Effects of fructooligosaccharides

on intestinal flora and human health. Bifidobact. Microflora, 5: 37-50. Nilsson, U., Öste, R., Jägerstad, M. and Birkhed, D., 1988. Cereal fructans: in vitro and in vivo studies

on availability in rats and humans. J. Nutr., 118: 1325-1330. Ziesenitz, S.C. and Siebert, G., 1987. In vitro assessment of nystose as a sugar substitute. J. Nutr., 117:

846-851. Zittan, L., 1981. Enzymatic hydrolysis of inulin - an alternative way to fructose production. Starch/Stärke,

33: 373-377.



PILOT-SCALE PRODUCTION OF INULIN FROM CHICORY ROOTS AND ITS USE IN FOODSTUFFS

E. BERGHOFER, A. CRAMER, U. SCHMIDT and M. VEIGL Institute of Food Technology, University of Agriculture, Peter-Jordanstraße 82, 1190 Vienna, Austria

ABSTRACT

For the isolation of inulin from chicory roots first a raw juice was produced, on the one hand, by aqueous extraction of sliced roots and, on the other, by grinding and wet milling of whole roots. After filtration, the juices obtained were evaporated under vacuum to about 40% (w/w). After being subjected to slow cooling inulin precipitated in a crystalline form and was separated and dried. Though isolation of inulin in this way appeared to be possible, purification in its crystalline form proved to be difficult. For that reason yield obtained as well as purity achieved were not satisfactory. The residual syrup was found to be rich in low-molecular carbohydrates which had escaped crystallization.

As another process variation the raw juice that had been purified by normal filtration was processed further by ultrafiltration. In this way, it was possible to obtain a retentate which consisted of nearly pure inulin (degree of purity 98.6%). This retentate proved to be immediately ready for spray-drying.

The samples of inulin isolated were examined for their chemical, physical and functional properties. With this inulin it was possible, for example, to produce a low-fat chocolate cream, that was sensorily comparable with one commercial product and with some products obtained by adding maltodextrins to the cream.

1 INTRODUCTION

Only few, mostly older publications describe technical procedures for isolation of inulin in a native form (Daniel, 1916; Hoche, 1926; Anonymous, 1947). However, also the inulin isolated according to these procedures was not used as such but hydrolysed in a further step. This may be the reason why hardly anything is known about the chemical, physical and functional properties of inulin in its native form. Only recently, the potential use of inulin in foodstuffs in its native, non-hydrolysed form has been fully recognized (Anonymous, 1989). For that purpose, it is first of all necessary to closely investigate the functional properties of inulin in an isolated form. As a consequence, the author's own research work was aimed at extensively testing several variants of the technical extraction of inulin from chicory roots in order to investigate the properties of the inulins isolated.

78

2 MATERIALS AND METHODS Chicory roots: The Sugar Research Institute Fuchsenbigl, Austria kindly provided

us with chicory roots. Extraction: Extraction of inulin from the roots was carried out using a pilot

countercurrent screw-conveyor slope extractor (De Danske Sugarfabrikker, Denmark; extraction temperature 75-80 °C; time 54 min; rate of root slicing 75 kg h"1; raw juice draw off 117.3%.

Ultrafiltration: Ultrafiltration pilot equipment, UFS1 (Alfa-Laval, Austria), was employed, using hollow-fibre membrane cartridges (Romicon, U.S.A.), PM-2 and PM-5.

Spray-drying: For spray-drying, a laboratory spray-drier with nozzle atomizer (Anhydro, Denmark) was utilized.

Dry matter analysis: Dry matter of solid and liquid samples was determined gravimetrically at a temperature of 105 °C.

Total sugar analysis: Total sugar is defined here as the sum of glucose and fructose resulting from enzymatic hydrolysis of inulin with inulinase (Novozym 230).

Ash analysis: Ash which remained after organic substances were burnt at a temperature of 550 °C in a muffle furnace, was determined gravimetrically.

Raw protein analysis: Nitrogen was determined by the Kjeldahl method. Size exclusion chromatography: For size exclusion chromatography, PW 5000 and

PW 4000 columns (Toso Haas) were used, with 0.05 M NaCl as the solvent system; pullulan served for calibration.

Viscosity measurements: For all rheological measurements a viscosimeter, Viscolab LC 10 (Physica Meßtechnik GmbH, Stuttgart, B.R.D.; measuring system MS-22 DIN) was used.

3 RESULTS AND DISCUSSION 3.1 Aqueous extraction of sliced roots and crystallization

The first method for the technical isolation of inulin tested was more or less similar to the classical way of extraction of sucrose from sugar beets. According to this method, the roots were cleaned, then sliced in a beet slicer and finally extracted with hot water. In order to separate solid particles and colouring matter active carbon and siliceous earth were added to the raw juice, which was then filtered and evaporated under vacuum to about 40% (w/w). The resulting thick juice was heated up to 95 °C and then, over a period of 30 h, cooled down to 4 °C without stirring. This caused the inulin to precipitate or crystallize as a pasty substance. In order to prevent intensive shear forces from destroying the crystal mash, pumping or stirring were avoided as much as possible; only then, filtration on a rotary filter was possible. However, when the crystal mash prior to filtration was stirred

79

Table 1. Composition of the various "fractions" obtained in isolating inulin from chicory roots as dependent upon the process employed.

Dry matter (%)

Total sugar (% d.m.)

Ash (% d.m.)

a) By aqueous extraction and crystallization Cossettes Raw juice Purified thick juice Crystallized inulin Mother syrup

Raw protein (% d.m.)

22.7 14.3 44.6 92.4 9.8

84.2 92.4

96.7 30.3

b) By grinding and wet milling, solid separation and crystallization Pulp 22.9 84.1 Raw juice 7.0 92.4 Purified thick juice 29.6 Crystallized inulin 92.1

c) By aqueous extraction, ultrafiltration and spray-drying Purified raw juice 10.2 Permeate 7.7 Retentate 7.2 Spray-dried inulin 96.6

5.4 5.3 1.6 7.3

4.7

4.3 4.1 0.5

37.9

4.5

92.4 87.4 98.6 98.6

4.8 6.5 0.7 0.7

3.7 4.9 0.6 0.6

more strongly or pumped around for longer periods, satisfactory filtration could no more be achieved, nor could sedimentation by means of a decanter effectively separate off the inulin. The composition of the various "fractions" obtained in isolating inulin from chicory roots as dependent upon the process employed is given in Table 1. The inulin isolated by a one-stage crystallization showed a total sugar content of 96.7%. As compared with sucrose or starch the inulin isolated still contained a considerable amount of impurities.

3.2 Grinding and wet milling of whole roots, solid separation and crystallization As an alternative to extraction a procedure was used in which the roots instead of

being sliced were ground, as it was done with sugar beets in the early days of sugar technology and is still done until now in the isolation of potato starch. The chicory roots were ground in two successive steps, using a rasp. The pulp was immediately suspended in water of 90 °C to destroy the inulinase present. Subsequently, the suspension was subjected to wet fine milling in a colloid mill and the solid particles were separated on a rotary filter. At this point, any cooling of the raw juice has to be avoided since the inulin that would then precipitate would instantly clog the filter pores. After the juice had been subjected to carbon refining to eliminate colouring matter, it was evaporated and crystallized as in the previous procedure. In this case, the crystallized inulin was separated by using a

80

disk separator instead of a filter. As shown in Table 1, the inulin obtained by this procedure was not as pure as that obtained by the previous one. Furthermore, this procedure required two successive separation steps that were very difficult to carry out.

3.3 Aqueous extraction of sliced roots, ultrafiltration and spray-drying In the third process variant it was tried to isolate inulin from the raw juice obtained

after extraction of sliced roots not by letting it crystallize but rather by ultrafiltration and subsequent spray-drying of the retentate. Using an adequate membrane it proved to be possible to retain the high-molecular inulin particles in the retentate while at the same time the greater part of the ash and the nitrogenous substances passed into the permeate. Spray-drying of the retentate was then possible without any problem. As shown in Table 1, according to this method it was possible to isolate inulin with a degree of purity of 98.6%, this being the highest value obtained.

3.4 Properties of the isolated inulin samples As a result of the pilot-scale tests sufficient inulin was available to examine its

properties with regard to its potential food use more closely. (i) Appearance. All inulin samples obtained looked like a powder that depending on purity was white or light grey. The powders were odourless and mostly tasteless; however, samples containing major impurities had a slightly bitter taste. Under the light microscope spheroidal particles could be observed. Under a scanning electron microscope, however, these particles showed a disk-like shape, with a diameter of about 1-5 /xm. (ii) Composition. Strictly speaking the microscopic images did not show the material to be crystalline. X-ray diffraction diagrams, on the other hand, clearly indicated the crystalline nature of the material (Fig. 1).

As to the degree of polymerization (DP) crystalline inulin samples obtained as described above (see 3.1 and 3.2) did not differ from those isolated on a laboratory scale (Beck and Praznik, 1986). In both instances the DP was about 20 to 25. By contrast, upon ultrafiltration higher-molecular-weight inulins were enriched. The average DP in these samples was found to be as high as 40-45. Using a smaller pore-sized membrane for ultrafiltration the average DP even increased further to 66, however, at the cost of the yield obtained, (iii) Functional properties. (a) Solubility: No doubt, the solubility of inulin is one of the most important properties if use in foodstuffs is concerned. The solubility curve as shown in Fig. 2 illustrates that only between 40 and 60 °C considerable amounts of inulin can be dissolved in water. (b) Viscosity: If suspended in cold water inulin precipitates relatively slowly if at all.

2ΘΠ

Fig. 1. X-ray diffraction diagram of inulin from chicory roots.

20 40 60

temperature (°C)

Fig. 2. Solubility of inulin, from chicory roots, in water.

82

Though inulin is insoluble in cold water it swells in it. Therefore, viscosity measurements with aqueous suspensions of inulin produce interesting results. At high temperatures (e.g. at 80 °C) when almost all inulin is dissolved the solutions behave like Newtonian liquids. By contrast, with suspensions with a larger percentage of undissolved inulin characteristic hysteresis curves become apparent the shapes of which are very similar to the rheograms of rheopex-plastic substances. These hysteresis effects become more clear the higher the concentration of inulin (Fig. 3). In comparison, suspensions of corn starch at the same concentration show a much lower viscosity and are characterized by the absence of hysteresis, (c) Adsorption: Fig. 4 shows the adsorption isotherm of spray-dried inulin.

3.5 Tests for a technical application of inulin Although inulin as such is hardly soluble in cold water, as mentioned above, aqueous

suspensions at higher concentrations of inulin are of a gel-like nature. Therefore, it was tempting to investigate whether this property could be used in the field of foodstuffs. To start with, inulin was added to various low-calorie sandwich spreads, types of mayonnaise and chocolate creams which were then subjected to sensory evaluation. For comparison, special kinds of maltodextrins that form thermoreversible gels were added to the same products. Only the results obtained with the chocolate cream samples will be dealt with here. The inulin-containing sample was produced by adding a 30% inulin suspension instead of the usual maltodextrin gel. One sample was a commercially available chocolate cream. The sensory evaluation was based on a sensory test that used a graphical linear scale (13 persons, two repetitions). With regard to taste only one maltodextrin sample was given a better rating than the inulin sample. As to mouth-feel the inulin sample was rated as being an average of the samples tested. In general, the rating of the inulin-containing sample was satisfactory.

4 CONCLUSIONS Three process variants were tested on their suitability for pilot-scale isolation of

inulin from chicory roots. The purest inulin preparation was produced by subjecting a raw juice, obtained by aqueous extraction, to ultrafiltration.

The functional properties of the inulin isolated principally differed from those of other polysaccharides. This is true in particular for the solubility and viscosity characteristics.

Tests concerning potential technical applications permit the conclusion that inulin indeed has interesting possibilities for use in foodstuffs.

83

20,0 % starch

9,9 % inulin

15,9 % Inulin

20,0 % inulin

200 400 600 800

shear rate (1/sec) 1000

Fig. 3. Flow curves of native inulin and corn starch (25 °C).

.4 .6

water activity

Fig. 4. Adsorption isotherm of inulin.

84

5 ACKNOWLEDGEMENT We want to thank the Sugar Research Institute at Fuchsenbigl, Austria, for providing

us with chicory roots and for allowing us to conduct the experiments on inulin extraction there.

6 REFERENCES

Anonymous, 1947. Verfahren zur Darstellung von nicht hauptsächlich zur Ernährung bestimmter Lävulose aus inulinhaltigen Pflanzen. Schweizer Patent 271.102.

Anonymous, 1989. Zuckerfabrik Warcoing entwickelt neue Inulin-Produkte aus Zichorien. Zuckerindustrie, 114: 849.

Beck, R.H.F. and Praznik, W., 1986. Inulinhaltige Pflanzen als Rohstoffquelle. Biochemische und pflanzenphysiologische Aspekte. Starch/Stärke, 38: 391-394.

Daniel, A., 1916. Verfahren zur Gewinnung von Inulin und Lävulose aus Pflanzen. Deutsches Reichspatent 313.986.

Hoche, B., 1926. Fabriksmäßige Gewinnung von Lävulose. Z. Ver. Dtsch. Zucker-Ind., 76: 821-833.


JERUSALEM ARTICHOKE COSTS OF PRODUCTION IN CANADA: IMPLICATIONS FOR THE PRODUCTION OF FUEL ETHANOL

Laurie BAKER, John C. HENNING and Paul J. THOMASSIN Department of Agricultural Economics, McGill University, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 1C0

ABSTRACT

The environmental implications of using transportation fuels have recently been receiving much attention following the World Commission on Environment and Development's report Our Common Futun (1987) and in Canada with the Energy, Mines and Resources' report Energy and Canadians into the 21s

Century (1988). The result in Canada is that the addition of tetra-ethyl lead to gasoline as an octant enhancer was phased out in December, 1990. This opens up opportunities for more environmentally friendlj additives for inclusion in transportation fuels.

The additives which could be used include: isopropyl alcohol, isobutyl alcohol, tertiary buty alcohol, methyl tertiary butyl ether, ethyl tertiary butyl ether, methanol or ethanol. The last two have received special attention in Canada; methanol because it is producible from natural gas based in Alberta, and ethanol as it can be produced from grain and other agricultural feedstocks. Most of the ethanol work completed to date in North America has concentrated on ethanol produced from surplus grain. Although there is adequate excess grain in Western Canada for this purpose, there is none in the Eastern provinces.

This paper presents results of an investigation into the possibilities of producing Jerusalem artichoke as an agricultural feedstock for fuel ethanol production. This crop has certain agronomic properties that make it potentially attractive for the alleviation of soil degradation problems that exist in Quebec. The results provided in this paper are presented in two areas: the costs of production of Jerusalem artichoke at the farm level, and the costs of processing the ethanol from the agricultural feedstock. The per litre cost of producing ethanol is estimated using various Jerusalem artichoke feedstock yields and conversion factors. The results indicate that ethanol can be produced at a competitive price to be used as an octane enhancer in gasoline for transportation fuel. Cost implications of various processing plant sizes are also presented.

1 INTRODUCTION

The use of tetra-ethyl lead as an octane enhancer was phased out in Canada in

December, 1990 for environmental reasons. This has opened the door for other more

environmentally friendly additives to be produced and marketed for transportation fuels.

Many of these have been suggested for this purpose, such as: isopropyl alcohol, isobutyl

alcohol, tertiary butyl alcohol, methyl tertiary butyl ether, ethyl tertiary butyl ether,

methanol or ethanol.

86

Methanol has received attention as it is producible from natural gas based in Alberta. On the other hand, ethanol is of interest because it is producible from agricultural feedstocks, such as grains and inulin-containing crops. The crop which has been investigated for this purpose in this research is Jerusalem artichoke (Helianthus tuberosus L.).

2 FEDERAL AND PROVINCIAL GOVERNMENT INTEREST IN ETHANOL 2.1 Federal government

The present minister of energy has been quoted as saying that the government is "supporting research that would lead to lower alternative fuel and vehicle costs" (Epp, 1990a). Ethanol is not receiving much attention at the federal level, however, as it is seen as a medium-term solution to the energy and environmental problems associated with transportation fuels. On the other hand, methanol is receiving some attention due to the profile of the natural gas industry and its potential for immediate economic pay-off.

The federal government has spent approximately $140 million per year supporting nuclear energy, particularly for the development of deep rock disposal of nuclear wastes (Pollution Probe, 1990). Also, there has been a commitment of $15,000 million over ten years for oil programs (Foody, 1989). Compared to these sums, the federal government has only allocated about 2% of all energy R&D expenditures on all bioenergy, of which ethanol receives a small percentage. The annual expenditure on all bioenergy R&D was under $4 million in 1979, and increased to $22 million by 1983. This figure was reduced to approximately $8 million per year over the next three years, and is approximately $5 million in 1991.

The future level of support for ethanol by the federal government is not clear. According to the minister of energy: "The pursuit of alternatives to gasoline is one of the Canadian government's many efforts to implement a strategy of sustainable development ... we are looking at a number of alternative transportation fuels and we are sponsoring several demonstration projects" (Epp, 1990b). Extensive demonstration projects, including fuel efficiency competitions, have been carried out for methanol. There have been some small fleet trials for ethanol, but not on the same scale as for methanol. If methanol becomes the chosen fuel, there could be limited benefits to agriculture from biomass to methane to methanol. On the other hand, if ethanol receives more attention Canadian farmers would be major beneficiaries.

2.2 Provincial governments The interest in ethanol has been greater at the provincial government level than at

the federal level as they have encouraged its development and sale through tax relief on ethanol sold for transportation fuel. As these subsidy supports only apply to ethanol

87

produced within each province, the difference in level of support between provinces has led to processing plant locations designed to take advantage of the subsidies. One firm producing ethanol based on an agricultural feedstock in Canada is Mohawk Oil Co., based in Burnaby, British Columbia, which produces grain-based ethanol in a plant at Minnedosa, Manitoba, with the aid of government subsidies1.

Mohawk Oil Co. made a $10 million commitment in 1989 to expand its processing facility in Manitoba. It could have increased the processing capacity even more to take advantage of an expansion into the Saskatchewan market. Due to a higher tax exemption on the ethanol portion of transportation fuels in Saskatchewan, $0.40 per litre in Saskatchewan versus $0.35 per litre in Manitoba (Miller, 1987; Government of Alberta, 1988), the company chose to enter into a joint venture with another company to construct a 10-M1 processing facility in Lanigan, Saskatchewan (New Fuels Report, 1990).

3 ETHANOL PRODUCTION IN CANADA Currently, the agricultural feedstock of choice in North America for ethanol

production is grain. Adequate grain supplies are available at this time in Western Canada due primarily to the depressed world grain market. There is thus a potential problem of basing a fuel industry on a feedstock that is bound in price to a market as volatile as that for world grain. Studies have shown that even at these record low prices, grain is not an economically viable feedstock for ethanol production without government subsidies (Gavett et al., 1986; Ahmed and Rask, 1987, 1988; LeBlanc, 1988). When the world grain price rises again, the situation will be even worse. Therefore, there is doubt that such an ethanol industry can be effectively set up in Canada. Inulin-containing crops have been suggested as an alternative feedstock source that would provide for income stabilization in agriculture as well as for ethanol production, while at the same time being sheltered from world markets.

4 ETHANOL PRODUCTION FROM JERUSALEM ARTICHOKE This crop has certain agronomic characteristics that make it attractive as an ethanol

feedstock, particularly in those areas of Canada that do not have surplus grain, such as Quebec. It is a crop that is suitable for marginal land so that there is reason to suggest that it could improve farmers' income potential in those regions. It could be used as a break-crop, if cut as hay rather than if the tubers were lifted each year, to offset soil degradation problems being encountered in regions of the country that produce grain using monoculture methods.

Although this crop is produced commercially in Europe and the U.S.A., other than in specific locations it is not a commercial-scale crop in Canada. In deriving costs of

production and returns for this crop it was necessary to derive an acceptable "supply price" for the crop since a reliable market price for the crop is not available. This supply price includes a reasonable return to the farmer, and can therefore be looked upon as the purchase price of the crop by the processing company at the plant gate.

5 RESULTS 5.1 Field costs of production

Annual costs of production for this crop were derived for Quebec and Western Canada for both tubers and for tops, with costs generated for tops gathered as small and large bales (Frappier et al.9 1990). For the crop to be harvested as tubers, it is assumed that the crop is both planted and harvested each year. As this would require the soil to be disturbed each year, there are assumed to be no beneficial effects for soil problems from this cultural practice. If the crop is to be harvested as hay, it would be cut each year for five years before being eradicated. Thus, the soil would be relatively undisturbed for these years. The analysis indicates that tops could be produced more economically than the tubers which is an important result for this crop if it is to be considered as a break-crop solution for soil degradation problems. Sample results are presented in Table 1 for tops in large bales, as these could be produced more cheaply than small bales.

These results indicate that Western Canada has a cost advantage when compared to Quebec, with the Western Canadian average supply price being $15.30 per ton for tops on a fresh matter basis. It should be noted that the average yields assumed for this study differ markedly by region; 41 t ha"1 in Quebec, and 100 t ha'1 in Western Canada. In arriving at these yield estimates, yield data from many sources were considered and found to be

Table 1. Annual cost of production of Jerusalem artichoke tops in large bales ($CDN).

Land cost per ha (Quebec)

Total variable cost per ha Total fixed cost per ha Storage and transportation per ha

Total costs per ha Cost per ton (fresh matter) t ha"1 (fresh matter)

Land cost per ha (Western Canada)

$2500

503 792 198

1493 36.41 41

$1750

503 669 198

1370 33.41 41

$1100

503 563 198

1264 30.83 41

$1100

$ 675

503 494 198

1195 29.15 41

$ 500

Cost per ton (fresh matter) t ha"1 (fresh matter)

15.78 14.82 100 100

89

Table 2. Ethanol feedstock cost of production using Jerusalem artichoke tops ($CDN).

Land cost per ha (Quebec) $2500 Yield (t ha1) 30 41 55 80 100

Conversion factor (If1) ($ per litre) 90 0.55 0.40 0.30 0.21 0.17

100 0.50 0.36 0.27 0.19 0.15 110 0.45 0.33 0.25 0.17 0.14

Land cost per ha (Western Canada) $1100 Yield (t ha1) 60 80 100 120 130

Conversion factor (11"1) ($ per litre) 90 0.29 0.22 0.18 0.15 0.13

100 0.26 0.20 0.16 0.13 0.12 110 0.24 0.18 0.14 0.12 0.11

highly variable. This leads to some uncertainty with respect to ultimate costs of production. Assuming various conversion factors of feedstock to ethanol, these field costs (per

t and per ha) can be expressed as feedstock costs for ethanol (per 1) produced from tops, as presented in Table 2.

The results indicate that, for the most expensive land type in each region, the feedstock cost could be as low as $0.11 per litre in Western Canada. This would only be reduced by $0.01 per litre if the feedstock was to be produced on land of lower quality in Western Canada. If "average" yields (411 ha'1 in Quebec and 1001 ha'1 in Western Canada) and conversion factors (100 1 t'1) are chosen, the costs are $0.36 per litre and $0.16 per litre in Quebec and Western Canada, respectively. These costs only represent delivered feedstock costs and do not include conversion costs, therefore it is clear that the final cost of ethanol produced from this feedstock will be higher.

5.2 Processing costs of production The costs of processing the feedstock into ethanol were estimated for a plant

producing 100 Ml of ethanol per year. These costs were adjusted for by-product credits which are: animal feed (residual feedstock valued at an equivalent soy-meal price of $240 per ton), and C 0 2 (valued at $150 per ton). The resulting net ethanol cost was calculated to be $0,203 per litre if produced in Western Canada. Assuming the rack price for regular unleaded gasoline to be $0,221 per litre and following an approach reported by Heath (1989), an analysis was carried out to determine the pricing of ethanol blends such as M5E32, gasohol3, and premium plus gasoline4.

This analysis is based on the rack price of gasoline and is set up to determine the cost

90

of the ethanol portion of blended fuels if ethanol is used in the blend as (a) a gasoline extender, or (b) an octane enhancer5. The results of this analysis indicate that for ethanol to be competitive as a gasoline extender it must have a per litre price no greater than: -$0.0433 for M5E3, $0,121 for gasohol, and $0.03 for premium plus gasoline. If the ethanol is to be used as an octane enhancer, the per litre ethanol price must be no greater than: $0.37 for M5E3, $0,252 for gasohol, and $0,262 for premium plus gasoline.

Given the per litre cost of ethanol produced from this agricultural feedstock of $0.36 in Quebec and $0.16 in Western Canada, it appears that: (a) in Quebec it could be produced competitively only for a M5E3 blend, and only as an octane enhancer; (b) in Western Canada it could be produced competitively for each blend, but again only as an octane enhancer.

An investigation of the effects of plant size was carried out using data from LeBlanc and Prato (1983) and, compared to the 100-M1 plant situated in Western Canada, a plant with an annual production of 227 Ml could produce ethanol for $0,162 per litre, and if the plant had an annual capacity of 454 Ml the cost of ethanol could be lowered to $0,139 per litre. These cost reductions would not be enough to make this feedstock competitive for ethanol production as a gasoline extender, but would provide a cost "cushion" for ethanol as an octane enhancer under conditions of falling gasoline prices.

6 CONCLUSIONS The results of this research indicate that Jerusalem artichoke could be produced

economically as the feedstock for ethanol to be produced as an octane enhancer for transportation fuels, certainly in Western Canada. Whether this will in fact happen will depend to a great extent on governmental regulations. Even though lead has been phased out as a fuel additive, the petrochemical industry has the technological capacity and knowledge to replace it without having to use an additive such as ethanol.

Two of these other additives which have been used in Canada are MTBE and MMT6. Refiners have also improved the processing of gasoline to increase the octane rating, with an increase in aromatic content. One of these, benzene, is of concern as it is a known health hazard but is not directly regulated. Thus, consumer awareness must be focused on the fuel question to aid the introduction of compounds such as ethanol.

7 NOTES

1 Provincial government tax exemptions for ethanol range from $0,083 per litre in Ontario to $0.40 per litre in Saskatchewan for those provinces to the west of Quebec. There are no exemptions available in Quebec and in the Eastern provinces. There are no federal tax exemptions for ethanol in Canada (Miller, 1987; Government of Alberta, 1988).

2 This is a blend of 5% methanol and 3% ethanol.

91

3 Gasohol is a 10% ethanol and 90% gasoline blend. 4 This is gasoline containing 5% ethanol. 5 Example for gasohol: Rack price of regular unleaded gasoline = $0.2210 per litre. Gasoline portion =

90% = $0.1989 per litre. Ethanol portion = 10% = $0.0221 per litre of gasohol. A $0.01 per litre market incentive is subtracted for the ethanol portion. The adjusted ethanol portion is $0.0121 per litre. Therefore the maximum price for ethanol to be competitive to the rack price of gasoline is $0.1210 per litre.

6 MTBE - methyl tertiary butyl ether. MMT - methylcyclopentadienyl manganese tricarbonyl. This additive has been banned in the U.S.A. but is still permitted in Canada.

8 REFERENCES (* not explicitly cited in the text)

Ahmed, H. and Rask, N., 1987. Ethanol fuel as an octane enhancer in the US fuel market: potential demand and policy considerations. Paper prepared for the Annual Meeting of the American Agricultural Economics Association, August.

Ahmed, H. and Rask, N., 1988. Ethanol's future: burning bright as an octane enhancer, Ohio's Challenge, Winter.

*Baker, L., Thomassin, P.J. and Henning, J.C., 1990. The economic competitiveness of Jerusalem artichoke (Helianthus tuberosus) as an agricultural feedstock for ethanol production for transportation fuels, Can. J. Agric. Econ., 38: 981-990.

*Energy, Mines and Resources, 1986. Discussion paper on oxygenated gasolines. Communications Branch, Ottawa: Supply and Services Canada.

Energy, Mines and Resources, 1988. Energy and Canadians into the 21st Century: a report on the energy options process. Communications Branch, Ottawa: Supply and Services Canada.

*Energy, Mines and Resources, 1990. Rack prices for four centres - Montreal, St. John, Winnipeg, and Toronto, February. Personal communication.

Epp, J., 1990a. Personal Communication, Office of the Minister of Energy, Mines and Resources, Ottawa, October.

Epp, J., 1990b. Press Release, Sarnia, Ontario, April 6. Foody, P., 1989. Transportation Energy in 2010 AD: A Technology Perspective, Proceedings of the

Seventh Canadian Bioenergy R&D Seminar, Energy, Mines and Resources Canada, Ottawa. Frappier, Y., Baker, L., Thomassin, P.J. and Henning, J.C., 1990. Farm level costs of production for

Jerusalem artichoke: tubers and tops (Quebec, Western and Eastern Canada). Working Paper 90-2, Department of Agricultural Economics, Macdonald College of McGill University, Ste. Anne de Bellevue, Quebec, November.

Gavett, E.E., Grinnell, G.E. and Smith, N.L., 1986. Fuel ethanol and agriculture: an economic assessment. Office of Energy, USDA, Agricultural Economics Report 562, August.

Government of Alberta, 1988. Ethanol fuels for Alberta: a discussion paper. Ethanol Fuels Committee, Edmonton, January.

Heath, M., 1989. Towards a Commercial Future: Ethanol and Methanol as Alternative Transportation Fuels. Canadian Energy Research Institute, The University of Calgary Press, Calgary, January.

'Henning, J.C., Baker, L.B.B., Thomassin, P.J. and Fischer, L.A., 1988. Fuel ethanol policy and developments in Quebec. Proceedings of the 1988 CANPAC Conference, Canadian Biomass Research Institute, Winnipeg, October.

*Henning, J.C., Thomassin, P.J. and Baker, L., 1990. Processing costs for a fuel ethanol industry based on an agricultural feedstock. Working Paper 90-3, Department of Agricultural Economics, Macdonald College of McGill University, Ste. Anne de Bellevue, Quebec, November.

LeBlanc, M., 1988. Ethanol: economic and policy trade-offs, USDA, January. LeBlanc, M. and Prato, A., 1983. Ethanol production from grain in the United States: agricultural impacts

92

and economic feasibility. Can. J. Agric. Econ., 31: 223-232. Miller, P., 1987. Regional feed grain price impacts with increased ethanol production. Agriculture Canada,

Ottawa, November. New Fuels Report, 1990. Mohawk Oil to open 10-million litre/year ethanol plant in Saskatchewan, Canada,

April 16. Pollution Probe, 1990. A ten point plan for government action on global warming: a message to Canada's

Energy Ministers, Toronto, March 19. World Commission on Environment and Development, 1987. Our Common Future. Oxford University

Press, Oxford.

ANALYSIS, CHEMISTRY AND NON-FOOD APPLICATIONS



SEPARATION AND QUANTIFICATION OF FRUCTAN (INULIN) OLIGOMERS BY ANION EXCHANGE CHROMATOGRAPHY*

N.J. CHATTERTON, P.A. HARRISON, W.R. THORNLEY and J.H. BENNETT USD A-Agricultural Research Service, Forage and Range Research, Utah State University, Logan, Utah 84322-6300, U.S.A.

ABSTRACT

Accurately quantifying inulins according to their degree of polymerization (DP) has long challenged fructan researchers. This paper describes recent advances in anion exchange Chromatographie separation and improved quantification of inulin oligomers up to DP 8. Pure inulin standards were prepared by fractionating water-soluble carbohydrate extracts of Jerusalem artichoke (Helianthus tuberosus L.) tubers using gel permeation chromatography. Samples of each pure inulin oligomer were freeze-dried and used to prepare aqueous standards of known concentrations. Elution times in a Dionex anion exchange (HPAE) system run isocratically increased non-linearly with increasing DP. Separation times for polysaccharides up to DP 40 were reduced by the use of a sodium acetate gradient. Responses by the pulsed amperometric detector (PAD) per /*g of inulin decreased in a regular manner as DP increased. Inulin polysaccharides become increasingly difficult to quantify as DP increases. In spite of this limitation, high-performance anion exchange chromatography with pulsed amperometric detection provides a convenient and rapid method to separate and quantify inulins of low to moderate DP.

1 INTRODUCTION Research on plant fructan has lagged behind that of starch because rapid and

definitive methods of analysis have not been available (Pollock and Chatterton, 1988). Separation and quantification of oligosaccharides has traditionally been incomplete and time-consuming (Praznik et al., 1984; Hardy, 1989). The ratio of fructose to glucose following hydrolysis of water-soluble carbohydrates has been used to quantify total fructan (Pollock, 1984; Housley and Daughtry, 1987). Individual fructans have been separated by paper or thin-layer chromatography (Bacon, 1959; Collins and Chandorkar, 1971; Schäffler and Morel du Boil, 1972; Hansen, 1975; Shiomi et al., 1976; Spollen and Nelson, 1988).

* Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and Utah State University, and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

94

Others have extracted individual sugars following Chromatographie separation in attempts to quantify each oligomer (Shiomi et al., 1976; Wagner et al., 1983). Permethylation techniques (Hakomori, 1964) have been used effectively with gas chromatography and high-performance liquid chromatography to separate and identify oligosaccharides (Shiomi et al., 1976; Pollock et al, 1979; Selosse and Reilly, 1985; Ivin and Clarke, 1987).

Individual oligosaccharides have been resolved in high-performance liquid chromatography (HPLC) systems using amino-bonded silica- and calcium-based ion exchange resin columns and also in reverse-phase systems (Pollock, 1982; Praznik et al., 1984; Ivin and Clarke, 1987; Bancal and Gaudillere, 1989). However, even the most effective HPLC methods fail to separate some linkage isomers (Honda, 1984).

High-performance anion exchange (HPAE) chromatography combined with pulsed amperometric detection (PAD) has recently been used to separate and detect monosaccharides (Hardy and Townsend, 1988), trisaccharides (Chatterton et al., 1989), and selected oligosaccharides (Hardy and Townsend, 1988). Subsequent papers demonstrated the utility of HPAE-PAD to separate other oligo- and polysaccharides (Hardy, 1989; Townsend etal., 1989; Chatterton etal., 1990a, b). This paper describes the use of HPAE-PAD to separate and quantify inulins. Additionally, it provides data on the response characteristics of the HPAE-PAD system to changes in oligosaccharide DP and sample concentration, and effects of eluant gradients. Quantification (DP 3-8) and separation of inulins up to DP 40 are demonstrated.

2 MATERIALS AND METHODS Pure inulin standards were prepared by fractionating water-soluble carbohydrate

extracts of Jerusalem artichoke (Helianthus tuberosus L.) tubers using gel permeation chromatography. The column (5 x 120 cm) was packed with Fractogel (Supelco TSK Toyopearl HW-40 F, 30-60 micron) and eluted with degassed, deionized water (Chatterton et al., 1989). The separations were monitored using an in-line refractometer (Waters Model R403). Collected fractions were concentrated in vacuo and repeatedly chromatographed until each fraction contained a single oligomer as determined by HPAE-PAD chromatography (Figs 1, 2). Samples of each purified inulin DP 5-8 (50 to 100 mg) were freeze-dried and used to prepare aqueous standards of known concentrations. Neokestose and 6-kestose were prepared from the action of yeast invertase on sucrose (Chatterton et al., 1989). 1-Kestose and nystose were purified from Neosugar (Meiji Seika Kaisha, Ltd., Japan). Neokestose-based oligosaccharides (DP 4-6), fructosylraffinose and fructosylstachyose were obtained from N. Shiomi (Hokkaido University, Sapporo, Japan). Planteose and loliose were purified from tomato (Lycopersicon esculentum Mill) and ryegrass (Lolium perenne L.) seeds, respectively (Chatterton et al., 1990a). All other carbohydrate standards were purchased

95

commercially. High-performance anion exchange (HPAE) chromatography was performed using a

Dionex Series 4000 ion Chromatograph with a pulsed amperometric detector (Chatterton et al., 1989). Flow rate was 1 ml min"1 at ca. 1400 psi operating pressure. Carbohydrate elution was carried out under alkaline conditions (150 mM NaOH). The high pH (13-14) of the eluant converts hydroxyl groups on the oligosaccharides into oxyanions. The degree of oxyanion interaction with the anion exchange column resin determines carbohydrate retention times. Adding a competing ion such as acetate (15-500 mM NaOAc) to the eluant reduces retention times. All eluants were pre-filtered through 0.45-μηι membranes. Helium was applied to a Dionex Eluant Degas Module to sparge the system of C0 2 and maintain positive pressure on the eluants.

The PAD system oxidizes and detects separated carbohydrates as they pass through the detector. Applied potentials were held constant at 0.04 V (El), 0.60 V (E2), and -0.80 V (E3) for 300, 120 and 300 ms, respectively. The output range was 3mA. An AI-450 Dionex Automated System Controller was used to access, integrate and record the data. Figures were generated using SigmaPlot 4.0 software and printed on a Hewlett Packard Laser-Jet Series II printer.

3 RESULTS Anion exchange chromatography effectively separates monosaccharides,

oligosaccharides, and inulin oligomers through DP 8 extracted from Jerusalem artichoke

RETENTION TIME (min) RETENTION TIME (min)

Fig. 1. (left) HPAE-PAD separation of standards on a 15-300-mM acetate gradient. Concentrations (jxg ml"1) were: G, glucose, 40; F, fructose, 60; S, sucrose, 140; 1 K, 1-kestose, 240; Ny, nystose, 160; DP 5, 180; DP 6, 220; DP 7, 220; DP 8, 240.

Fig. 2. (right) Water-soluble carbohydrates from Jerusalem artichoke tubers eluted by a non-linear NaOAc gradient (100-500 mM). Sample (2.1% solutes) contained the equivalent of 50 mg of tissue extracted in 1 ml of water. Ny, nystose.

96

T I I I I Γ

" S H g G L U

" ( y 0 0 ö 0 FRU

J i 1 i I i I i I i I J 0 50 100 150 200 250

CONCENTRATION (>Jg/ml)

Fig. 3. (left) Effects of carbohydrate concentration on PAD peak area. Separation time: 15 min; eluant: 15-300 mM NaOAc gradient. G, glucose; F, fructose; S, sucrose; 1-K, 1-kestose; NY, nystose; DP 5-8.

Fig. 4. (right) Carbohydrate concentration vs. PAD response per g sugar for DP 1-8 carbohydrates eluted by a 15-300-mM NaOAc gradient. Curves below 1-K, 1-kestose, are DP 4-8 in increasing order. GLU, glucose; FRU, fructose; SUC, sucrose.

tubers (Fig. 1). Inulins up to at least DP 40 can also be separated from a water-soluble extract (Fig. 2), but quantification is limited by the availability of appropriate standards and reduced sensitivity of PAD to high-DP polymers. The detector response (peak area) increased linearly as the concentrations of each individual sugar or oligosaccharide increased (Fig. 3). However, each had a unique slope and required different calibration curves. Detector sensitivity decreased as DP increased (Fig. 3). In Fig. 4 the peak area per μg carbohydrate is plotted for a range of concentrations. The ratio remained constant for DP 2-8, but the detector response per μg carbohydrate decreased with increasing DP.

PAD output per μg sugar decreased as DP increased within a series (Fig. 5). The malto-oligosaccharide series, neokestose series, and inulin series have unique peak area/DP relationships. If one were to assume that the peak area response for sucrose is derived equally from glucose and fructose, then the response for 1-kestose (DP 3) should be about 11/2 times the sucrose response (projected, Fig. 6). This was not the case. The observed response fell short of the "projected" response for all degrees of polymerization larger than 2. The discrepancy increased along with DP. Each additional fructose subunit in successively larger oligomers contributed a smaller net increase in the detector signal. Going from DP 7 to DP 8, the addition of a fructose subunit appeared to have very little, if any, effect on the detector response. Inasmuch as the detector measures electrons released to the gold electrode during oxidation of the carbohydrates, it may be inferred from the increasing discrepancy between projected and observed values with increasing DP, that as carbohydrates become larger, proportionally fewer electrons are released per fructosyl unit in the detector reaction.

< LJ a: < <

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97

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SIZE (DP)

Fig. 5. (left) PAD response per μg sugar. Includes fructans of the inulin series, fructans built on neokestose, and glucans built on maltose. Eluted by 15-300-mM NaOAc gradient. G, glucose; F, fructose; S, sucrose; Ma, maltose; Mz, melezitose; P, planteose; 6 K, 6-kestose; L, loliose; R, raffinose; FR, fructosylraffinose; FS, fructosylstachyose; St, stachyose.

Fig. 6. (right) PAD response per nmole carbohydrate (DP 1-8). Eluant: 15-300-mM NaOAc gradient. G, glucose; F, fructose; S, sucrose; 1-K, 1-kestose; NY, nystose.

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Fig. 7. (left) Effects of DP on retention time at seven isocratic NaOAc concentrations.

Fig. 8. (right) Effects of four NaOAc eluant concentrations on PAD response by five carbohydrates eluted isocratically.

Retention times for large inulins are reduced by adding acetate to the eluant (Fig. 7). The reduction is a function of DP. Because the retention times of saccharides larger than DP 3 are long, the addition of acetate to the eluant is recommended for most analyses. Presence of low concentrations of acetate in the eluant during the initial minutes of elution permits the separation of small sugars. Increasing acetate in a concentration gradient progressively elutes larger DP molecules. No significant loss in PAD sensitivity was observed due to increased acetate concentration in the eluants (Fig. 8).

98

4 CONCLUSIONS HPAE-PAD shows great promise for the rapid separation and quantification of water-

soluble carbohydrates. Although not demonstrated in this study, the PAD can measure pico-mole quantities of sugar without derivatization and requires only small (10-μ1) samples for injection. The relative sensitivity of the PAD per μg carbohydrate is: glucose > fructose > sucrose > 1-kestose, and continues to decline with increasing DP. PAD response per nmole of carbohydrate is: glucose > fructose = sucrose < 1-kestose < nystose and continues to increase per ^mole carbohydrate with increasing DP. However, large polysaccharides are less responsive and more difficult to quantify. Appropriate standards are required for each carbohydrate. However, their individual responses are linear for a relatively wide range of concentrations.

Neutral oligosaccharides are separated as their oxyanions at alkaline pH. Differences in the accessibility of ionizable sites influence the effective negative charge causing molecules to differ in their retention times within the anion exchange column. Retention time does not necessarily increase in proportion to an increase in molecular weight. However, in the case of a given series such as inulin, retention times are positively correlated with molecular size. Sodium acetate in the eluant displaces large carbohydrate molecules that are strongly retained by the anion exchange resin column.

The HPAE-PAD system effectively separates and quantifies inulins through DP 8. Larger inulins can also be quantified if appropriate standards are available. Inulins to DP 40 were resolved and detected by HPAE-PAD procedures. The system can be completely automated, is economical to operate, and is versatile. When used with acetate gradients, the method can be adapted to meet a variety of analytical needs. Retention times and response characteristics of PAD may vary over time. Both potential errors are minimized by the frequent use of calibration standards.

5 REFERENCES

Bacon, J.S.D., 1959. The trisaccharide fraction of some monocotyledons. Biochem. J., 73: 507-514. Bancal, P. and Gaudillere, J.-P., 1989. Oligofructan separation and quantification by high performance

liquid chromatography. Application to Asparagus officinalis and Triticum aestivum. Plant Physiol. Biochem., 27: 745-750.

Chatterton, N.J., Harrison, P.A., Thornley, W.R. and Bennett, J.H., 1989. Purification and quantification of kestoses (fructosylsucroses) by gel permeation and anion exchange chromatography. Plant Physiol. Biochem., 27: 289-295.

Chatterton, N.J., Harrison, P.A., Thornley, W.R. and Bennett, J.H., 1990a. Sucrosyloligosaccharides and cool temperature growth in 14 forb species. Plant Physiol. Biochem., 28: 167-172.

Chatterton, N.J., Harrison, P.A., Thornley, W.R. and Draper, E.A., 1990b. Oligosaccharides in foliage of Agropyron, Bromus, Dactylis, Festuca, Lolium and Phlewn. New Phytol., 114: 167-171.

Collins, F.W. and Chandorkar, K.R., 1971. Thin-layer chromatography of fructo-oligosaccharides. J. Chromatogr., 56: 163-167.

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Hakomori, S., 1964. A rapid permethylation of glycolipid, and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. J. Biochem. (Tokyo), 55: 205-208.

Hansen, S.A., 1975. Thin-layer Chromatographie method for the identification of mono-, di- and trisaccharides. J. Chromatogr., 107: 224-226.

Hardy, M.R., 1989. Liquid Chromatographie analysis of the carbohydrates of glycoproteins. Liquid Chromatography-Gas Chromatography (Bio Separations), 7: 242-246.

Hardy, M.R. and Townsend, R.R., 1988. Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection. Proc. Natl Acad. Sei., 85: 3289-3293.

Honda, S., 1984. High-performance liquid chromatography of mono- and oligosaccharides. Anal. Biochem., 140: 1-47.

Housley, T.L. and Daughtry, C.S.T., 1987. Fructan content and fructosyltransferase activity during wheat seed growth. Plant Physiol., 83: 4-7.

Ivin, P.C. and Clarke, M.L., 1987. Isolation of kestoses and nystose from enzyme digests by high-performance liquid chromatography. J. Chromatogr., 408: 393-398.

Pollock, C.J., 1982. Oligosaccharide intermediates of fructan synthesis in Lolium temulentum. Phytochemistry, 21: 2461-2465.

Pollock, C.J., 1984. Sucrose accumulation and the initiation of fructan biosynthesis in Lolium temulentum L. New Phytol., 96: 527-534.

Pollock, C.J. and Chatterton, N.J., 1988. Fructans. In: J. Preiss (Ed.), The Biochemistry of Plants, A Comprehensive Treatise, Vol. 14, Carbohydrates. Academic Press, Inc., San Diego, pp. 109-140.

Pollock, C.J., Hall, M.A. and Roberts, D.P., 1979. Structural analysis of fructose polymers by gas-liquid chromatography and gel filtration. J. Chromatogr., 171: 411-415.

Praznik, W., Beck, R.H.F. and Nitsch, E., 1984. Determination of fructan oligomers of degree of polymerization 2-30 by high-performance liquid chromatography. J. Chromatogr., 303: 417-421.

Schäffler, K.J. and Morel du Boil, P.G., 1972. Thin-layer Chromatographie separation of oligosaccharides isolated from sucrose-enzyme mixtures. J. Chromatogr., 72: 212-216.

Selosse, E.J.-M. and Reilly, P.J., 1985. Capillary column gas chromatography of trifluoroacetyl trisaccharides. J. Chromatogr., 328: 253-258.

Shiomi, N., Yamada, J. and Izawa, M., 1976. Isolation and identification of fructo-oligosaccharides in roots of asparagus (Asparagus offlcinalisL.). Agric. Biol. Chem., 40: 567-575.

Spollen, W.G. and Nelson, C.J., 1988. Characterization of fructan from mature leaf blades and elongation zones of developing leaf blades of wheat, tall fescue, and timothy. Plant Physiol., 88: 1349-1353.

Townsend, R.R., Hardy, M.R., Cumming, D.A., Carver, J.P. and Bendiak, B., 1989. Separation of branched sialylated oligosaccharides using high-pH anion-exchange chromatography with pulsed amperometric detection. Anal. Biochem., 182: 1-8.

Wagner, W., Keller, F. and Wiemken, A., 1983. Fructan metabolism in cereals: induction in leaves and compartmentation in protoplasts and vacuoles. Z. Pflanzenphysiol., 112: 359-372.



THE APPLICABILITY OF ENZYMATIC METHODS FOR THE QUANTITATIVE ANALYSIS OF FRUCTAN-CONTAINING PLANT EXTRACTS

E. STEFANOVITS*, W. PRAZNIK**, G. SOJA***, J. KOSÄRY*, S. KAMMERER**, E. CSEKE*, J. BOCSI* and L. BOROSS*

Department of Chemistry and Biochemistry, University of Horticulture and Food Industry, P.O. Box 53, 1502 Budapest, Hungary Department of Chemistry, Agricultural University Vienna, Gregor-Mendel-Str. 33, 1180 Wien, Austria Department of Agriculture and Biotechnology, Research Centre Seibersdorf, 2444 Seibersdorf, Austria

ABSTRACT

In the framework of our investigations on the carbohydrate metabolism of plants we studied the glucose, fructose, sucrose and inulin content of Helianthus tuberosus treated with titanium ascorbate. The enzymatic Boehringer UV tests used (hexokinase and glucose-6-phosphate dehydrogenase for glucose and hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase for fructose) proved to be excellent for the determination of monosaccharides. However, estimation of sucrose and inulin using another enzymatic Boehringer test (with invertase) and Novozym 230 inulinase, respectively, was problematic because the invertase preparation exhibited some inulin-hydrolysing activity as well, whereas Novozym 230 proved to contain both exo- and endo-inulinase, as well as invertase.

A simple approach was used to estimate the ratio of inulin to sucrose. This ratio could be characterized by the quotient of the concentrations of bound fructose and bound glucose in the extracts.

The kinetic characteristics of the Novozym 230 inulinase preparation were determined for both sucrose and inulin. Simultaneous estimation of sucrose and inulin with glucose dehydrogenase proved to be impossible because the relatively high Km of this enzyme for glucose resulted in a too low reaction rate.

The molecular distribution of the inulins present in various plant extracts - as measured by HPLC -did not differ significantly.

1 INTRODUCTION Enzymes have been used in chemical analysis for a long time, because of their high

substrate and reaction specificity. In particular, dehydrogenases are being used, alone or together with other enzymes. For instance, simple enzymatic tests are now available for glucose, fructose and sucrose (cf. Methods of Enzymatic Food Analysis; Boehringer Mannheim GmbH). Fully automated flow-injection analytical systems using immobilized

102

enzymes have been developed (Johansson et ah, 1983; Simon et al.9 1988). The effects of titanium on different plants have been known for many years (cf. Pais,

1983). In the course of our investigations on the effects of various metal ions on carbohydrate metabolism we therefore treated Jerusalem artichoke plants with titanium ascorbate, and compared the carbohydrate contents of treated and untreated plants. In this paper, we report some preliminary results of our experiments with Jerusalem artichoke and discuss some problems which emerged during our investigations regarding the enzymatic analysis of the extracts.

2 MATERIALS AND METHODS A Novozym 230 inulinase preparation was a gift of Novo Industri A/S (Copenhagen,

Denmark). Microbial glucose dehydrogenase was a product of Sigma Chemical Company (U.S.A.). Dahlia inulin was a gift of Reanal Fine Chemical Co. (Budapest, Hungary). Glucose, fructose and sucrose were also Reanal products.

Boehringer enzymatic UV tests (Boehringer Mannheim GmbH, Germany) were used for the determination of glucose (hexokinase 4- glucose-6-phosphate dehydrogenase), fructose (hexokinase + phosphoglucose isomerase + glucose-6-phosphate dehydrogenase) and sucrose (invertase).

Jerusalem artichoke tubers were planted at the beginning of June. The plants were sprayed with titanium ascorbate solutions containing 0.5 or 5.0 mg Γ1 (pH 5.2) 7 and 48 days after planting, respectively. After being harvested at the beginning of September, the tubers were homogenized and extracted with cold or hot water. The extracts were frozen and stored at -18 °C until analysis. Then, the thawed suspensions were centrifuged at 10,000 g at 25 °C using a Sorvall RC 28S centrifuge, and the supernatants analysed for their carbohydrate content.

The glucose and fructose contents of the samples were measured as described in the Boehringer UV test, using a Varian DMS 100 spectrophotometer. The bound glucose and fructose contents (from sucrose and inulin) of the samples were determined in the same way after hydrolysis with either Novozym 230 inulinase in 0.2 M sodium acetate buffer (pH 4.2) or a Boehringer invertase preparation in 0.2 M citrate buffer (pH 4.6) at room temperature. Bound glucose and fructose contents were defined as the difference between total and free glucose and fructose content, respectively. Total reducing sugar in the samples was determined using 3,5-dinitrosalicylate (Smith and Cheng, 1978).

In kinetic experiments, reaction mixtures containing inulinase were incubated with the substrate (dahlia inulin or sucrose) and aliquots were taken for determinations of glucose, fructose or total reducing sugar content at various time intervals.

Exo- and endo-inulinase-containing fractions of Novozym 230 were obtained by

103

Chromatographie separation according to the - slightly modified - ion exchange method of Azhari et al. (1989) (cf Boross et al., 1993).

3 RESULTS AND DISCUSSION 3.1 Analysis of various mixtures of glucose, fructose, sucrose and inulin

To start with, we investigated the possibility of applying enzymatic methods for the quantitative estimation of glucose, fructose, sucrose and inulins (both oligo- and polymeric inulins) in various mixtures. For the determination of glucose and fructose content of mixtures the Boehringer UV test proved to be very useful. However, the invertase-containing Boehringer UV test system proved to exhibit some inulin-hydrolysing activity as well so that not only sucrose but also part of the oligo-inulin content was measured. The Novozym 230 preparation, on the other hand, hydrolysed both sucrose and inulins to such an extent that with this enzyme total glucose and fructose of the mixtures could be determined.

Since the pH range for maximal hydrolysis of both sucrose and inulin (pH 4.6 and 4.2, respectively) was lower than the optimal pH for the Boehringer enzymatic test for glucose and fructose (pH 7.6) the hydrolysis and quantitative determination of sucrose and inulin could not be carried out in a "one pot" system.

The ratio of inulin to sucrose in the mixtures could be readily established without further analysis by calculating the quotient of the bound fructose and glucose (F/G) contents. This quotient was 1 for sucrose and about 30 for dahlia inulin. However, this approach did not give any information about the molecular weight distribution of the inulins present.

Glucose dehydrogenase proved to show a relatively low affinity for its substrate (Km

1.4 mM) and therefore complete dehydrogenation of glucose in dilute solutions required a long reaction time. However, in this case a linear relationship between the initial reaction rate (V0) and the glucose concentration was established (Fig. 1).

In a series of experiments the apparent Km values of the Novozym 230 inulinase and the separate exo- and endo-inulinase fractions of this enzyme preparation were determined for both sucrose and dahlia inulin. The specific activities of these enzyme preparations for both substrates were also measured. Data on the separate exo- and endo-inulinase fractions of Novozym 230 were determined by measuring the total reducing sugar contents.

The results are summarized in Table 1. The separate exo- and endo-inulinase fractions of Novozym 230 behaved kinetically quite different from the mixture.

The pH optimum of exo- as well as endo-inulinase proved to be in the acidic range (no data given), therefore the continuous registration of NAD product formation as a result of the three-step reaction (hydrolysis, phosphorylation and dehydrogenation) was not

104

Vo

12

9

6

3

0.1 θ'.2 θ'.3 mM Glucose

Fig. 1. The relationship between the initial rate of the glucose dehydrogenase reaction and the glucose concentration. The reaction mixture contained 0.05 U enzyme and 1 ^mole NAD+ in 1 ml 0.1 M phosphate buffer (pH 7.6). The initial rate of the reaction is expressed in nmoles of NADH formed during the first minute of the reaction.

possible. After the hydrolysis in acidic solution, it therefore was necessary to change the

pH of the reaction mixture by dilution with an appropriate buffer (0 .33 M triethanolamine,

pH 7.6) and to add hexokinase and glucose-6-phosphate dehydrogenase to continue and

terminate the enzymatic transformation process.

As an alternative to hexokinase + glucose-6-phosphate dehydrogenase for the

quantitative determination of glucose we also employed glucose dehydrogenase for the

(spectrophotometric) measurement of glucose. However, the latter enzyme proved to have

Table 1. Kinetic characteristics of Novozym 230 inulinase and the separate exo- and endo-inulinases in it.

Enzyme

Inulinase Inulinase Inulinase Exo-inulinase

fraction Exo-inulinase

fraction Endo-inulinase

fraction Endo-inulinase

fraction

Substrate

inulin inulin sucrose

inulin

sucrose

inulin

sucrose

Buffer (0.1 M)

sodium acetate sodium acetate sodium acetate

sodium acetate

sodium acetate

sodium acetate

sodium acetate

pH

4.2 5.3 4.2

4.2

4.2

4.2

4.2

Km (mM)

6.7 2.0 4.0

8.3

8.0

833a

10.0

Specific activity (U mg"1)

580.7 347.4 377.5

33.3

308.3

91.0

21.1

a Calculated on the basis of "total fructose content", i.e. the total number of fructosyl bonds in inulin molecules.

105

a relatively low affinity for its substrate (Km 1.4 mM), so that the full dehydrogenation of glucose in dilute solutions required a long reaction time. However, this enzymatic test enabled the quantitative determination of glucose by measuring the initial reaction rate. Under well-defined reaction conditions there was a linear relationship between the V0 values and the substrate concentrations (Fig. 1).

3.2 The effect of titanium treatment on the carbohydrate content of Jerusalem artichoke tubers The data on the carbohydrate content of the extracts of titanium-treated and untreated

(control) Jerusalem artichoke tubers, as determined enzymatically, are shown in Table 2. All the analyses were done in triplicate, that means using the separate extracts of three plants. Each entry represents the averaged value of the three determinations concerned. The differences between the various plant groups proved not to be significant.

Table 2. Glucose and fructose contents and fructose/glucose ratio before and after hydrolysis with invertase or inulinase of extracts of Jerusalem artichoke tubers (/ moles g"1 fresh tubers).

Sample8

l.A.I. l.A.II. l.B.I. l.B.II. 2.A.I. 2.A.II. 2.B.I. 2.B.II. 3.A.I. 3.A.II. 3.B.I. 3.B.II.

Before hydrolysis of

Glu

4.38 0 3.53 1.06 5.74 0 3.35 1.68 6.22 0 3.68 0.76

Fru

5.72 0 4.23 1.36 5.65 0 3.13 2.07 7.63 0.95 3.86 0.84

After sucrose and inul

by invertase

30d

Glu

3.70 1.37 2.22 0.33 4.70 1.40 2.54 1.19 6.27 1.35 4.06 0.30

Fru

18.86 9.61

10.67 2.24

23.33 9.52

13.10 8.15

31.32 10.80 19.91 3.12

in

360

Glu

. 1.89 -1.22 -1.66 -1.63 -1.84 -1.07

Fru

_ 18.92 -11.71 -19.10 -21.88 -25.76 -13.98

by inul

30

Glu

17.65 2.08

11.24 4.41

15.85 4.82

10.63 8.10

18.67 4.85

11.13 4.03

inase

Fru

135.95 36.31 85.42 90.85

134.76 86.83 90.05

168.51 204.27 93.60

118.59 113.16

Fructose/glucoseb

Invertase0

30

5.1 7.0 4.8 6.8 5.0 6.8 5.2 6.8 5.0 8.0 4.9

10.4

360

_ 10.0 -9.6

-11.5 -13.4 -14.0 -13.1

Inulinasec

30

7.7 17.5 7.6

20.6 8.5

18.0 8.5

20.8 10.9 19.3 10.7 28.1

a Treatments of plants were 1. control, 2. one treatment, 3. two treatments with 5 mg Γ1 titanium ascorbate solution. Plants were first extracted with cold water (A), and then with hot water (B). Before analysis all samples were centrifuged, the supernatants (I) and the hot water extracts (II) of the pellets were assayed. b Fructose/glucose: the ratio of total fructose to total glucose in sucrose + inul in present in the extracts as measured after hydrolysis with c invertase or inulinase, respectively. d Time in min.

106

On the basis of F/G values the inulin content of hot water extracts proved to be

higher than that of cold water extracts.

The molecular weight distribution of the inulin in the various plant extracts was

studied by HPLC analysis. There proved to be no significant difference between the

distribution of titanium-treated and control plants.

4 ACKNOWLEDGEMENTS

The authors are grateful to Novo Company for Novozym 230 and to Reanal Fine

Chemicals for dahlia inulin. The authors want to express their thanks to the Hungarian

Academy of Sciences for financial support of part of the above experiments.

5 REFERENCES

Azhari, R., Szlak, A.M., Ilan, E., Sideman, S. and Lotan, N., 1989. Purification and characterization of endo- and exo-inulinase. Biotechnol. Appl. Biochem., 11: 105-117.

Boross, L., Praznik, W., Kosäry, J. and Stefanovits, E., 1993. Comparative studies of soluble and immobilized inulin-hydrolysing enzymes. In: A. Fuchs (Ed.), Inulin and Inulin-containing Crops, Studies in Plant Science, Vol. 3. Elsevier, Amsterdam, pp. 217-221.

Johansson, G., Ögren, L. and Olsson, B., 1983. Enzyme reactors in unsegmented flow injection analysis. Anal. Chim. Acta, 145: 71-85.

Pais, I., 1983. The biological importance of titanium. J. Plant Nutr., 6: 3-131. Simon, L.M., Kotormän, M., Boross, L. and Szjajäni, B., 1988. Determination of glycolytic intermediates

in a flow injection system using immobilized enzymes. Acta Biochim. Biophys. Hung., 23: 247-254. Smith, W.T. and Cheng, C , 1978. Use of 3,5-dinitrosalycilate reagent for glucose determination in mixed

solvents. Anal. Lett., B l l : 191-194.


EXTRACTION AND PURIFICATION OF PREPARATIVE AMOUNTS OF 1-KESTOSE, 6-KESTOSE, NEOKESTOSE, NYSTOSE AND INULIN-PENTASACCHARIDE

H. SMOUTER* and R J . SIMPSON School of Agriculture and Forestry, The University of Melbourne, Parkville, Victoria 3052, Australia * Present address: Netherlands Institute of Ecology, Centre for Terrestrial Ecology, P.O. Box 40, 6666 ZG Heteren, The Netherlands

ABSTRACT

Studies of the enzymology of fructan synthesis and hydrolysis are restricted by the lack of commercially available fructans for use as substrates. This has necessitated the development of relatively simple and cheap procedures for the purification of fructan oligosaccharides. Extraction and purification of 1-kestose, 6-kestose and neokestose from a commercial fructan oligosaccharide preparation (Neosugar P), from a digest obtained by enzymatic conversion of sucrose and from bulbs of Alliwn cepa L., respectively, is described. The efficiency and success of these procedures was dependent on (i) the source of the fructan oligosaccharides, (ii) an enrichment step prior to reversed-phase high-performance liquid chromatography (RP-HPLC) and (iii) the good separation of isomers of the fructan trisaccharides that could be achieved by elution from a preparative RP-HPLC column with water.

1 INTRODUCTION The synthesis of fructans in higher plants is thought to proceed by the concerted

action of the enzymes sucrose:sucrose fructosyltransferase (SST, EC 2.4.1.99), which converts sucrose into trisaccharide and fructan:fructan fructosyltransferase (FFT, EC 2.4.1.100), which utilizes trisaccharide as the principal substrate for the synthesis of higher-molecular-weight fructans (Pollock, 1986). In vitro assay of the activities of enzymes involved in fructan metabolism is complicated because many of the substrates are not available commercially. Only SST activity can be assayed readily and studies of FFT are documented for only a few liliaceous species (Allium cepa L., Henry and Darbyshire, 1980; Asparagus officinalis L., Shiomi, 1989). For the assays of FFT, 1-kestose was extracted from tubers of Helianthus tuberosus L. and purified using laborious techniques (carbon-celite chromatography). However, in studying FFT it is also likely that 6-kestose and

108

neokestose will be required as substrates because both 6-kestose and neokestose are potentially the basic units of different series of fructans in grasses (Pollock, 1986). Smouter and Simpson (1989) in a survey of grasses from a temperate region of Australia, found that 1-kestose was present in most of the grasses that accumulated fructans and in some cases together with 6-kestose (e.g. Triticum aestivwn L., Hordeum spp.) or with neokestose (e.g. Agrostis avenacea Gmel., Lolium rigidum Gaud.) (see also Sims et al., 1991).

Hydrolysis of high-molecular-weight fructans is reported to be catalysed by fructan hydrolase(s) (FH, EC 3.2.1.80), but the characteristics of this enzyme are also not well established, due to the unavailability of a standard substrate. Following the First International Symposium on Fructan (Bonn, 1988) and during the International Congress on Inulin and Inulin-containing Crops (Wageningen, 1991) there has been a call for the use of a fructo-oligosaccharide, such as a fructo-pentasaccharide with few branches, as a standard substrate for assaying fructan hydrolase (C.J. Pollock, pers. comm.). However, no such substrate is commercially available.

Improved Chromatographie methods, as described in a previous paper (Sims et al., 1991), offer procedures for the isolation of substrates for fructan enzymes. This paper reports modification of these techniques for preparation of gram quantities of 1-kestose, 6-kestose, neokestose, nystose and inulin-pentasaccharide for use as enzyme substrates.

2 MATERIALS AND METHODS 2.1 Sources of fructans

Stems of Hordeum vulgäre L. near anthesis and bulbs of A. cepa were suitable sources of 6-kestose and neokestose, respectively, because these compounds were present in relatively high concentrations in these plant tissues (Sims et al., 1991). Fructo-oligosaccharides were extracted from bulbs and stems by mechanical homogenization in boiling 80% ethanol and dried under vacuum at 37 °C. The crude carbohydrate extract was dissolved in water and proteins and ions were removed by ion exchange (Serdolit CS-2 (H+) and Serdolit AS-6 (HCOO), Serva, Germany). Lipids and hydrophobic material were removed on Bond Elut C18 columns (Analytichem International, U.S.A.). The neutral carbohydrates were dried under vacuum at 37 °C and dissolved in water to about 10% (w/v).

Alternatively, incubation of 1 M sucrose with invertase from Saccharomyces cerevisiae (Sigma, U.S.A.) in 10 mM sodium acetate buffer (pH 4.8) for 45 min at 24 °C as reported by Straathof et al. (1986) was used to synthesize 6-kestose. Invertase activity was stopped by boiling for 2 min and the proteins and ions in the reaction mixture were removed as above. This procedure for preparing 6-kestose was more rapid than extraction from stems of H. vulgäre and was used in preference subsequently.

109

A commercial preparation of fructan oligosaccharides containing a high proportion of 1-kestose, nystose and inulin-pentasaccharide (Neosugar P, Meiji Seika Kaisha, Ltd., Japan) proved to be the most convenient source of these compounds. The fructo-oligosaccharide mixture was dissolved in water and used at a concentration of approximately 10% (w/v).

2.2 Gel filtration Carbohydrates extracted from A. cepa at an approximate concentration of 10% (w/v)

in water were crudely separated by loading 40 ml on a 60 x 10 cm (diam.) column containing Sephadex G-15 (Pharmacia, Sweden, flow rate 10 ml min'1) to obtain an enriched trisaccharide fraction prior to reversed-phase high-performance liquid chromatography (RP-HPLC). The trisaccharide fraction was identified by elution volume.

Trisaccharides in enzyme digests were separated from sucrose by gel filtration on the large Sephadex G-15 column followed by gel filtration on a 240 x 2.2 cm (diam.) glass column containing Fractogel TSK HW-40 (Superfine; Merck, Germany) prior to RP-HPLC. Aliquots (0.5 ml) of the enriched trisaccharide fraction eluting from the Sephadex G-15 column were loaded onto the Fractogel column at intervals of 3 h and chromatographed at a flow rate of 1.5 ml min"1. Trisaccharide peaks eluting from the Fractogel column were identified using a refractive index detector (model 401; Millipore Waters, U.S.A.).

2.3 Reversed-phase high-performance liquid chromatography All carbohydrate preparations were separated in the final stage of the purification

procedure by preparative RP-HPLC. The HPLC system consisted of an automatic sample injector (WISP model 710B, Millipore Waters), a solvent delivery system (model SP 8700; Spectra Physics, U.S.A.) and a differential refractometer (model R410, Millipore Waters). Samples of 1 ml, containing 100 mg total carbohydrate were repeatedly injected onto a preparative reversed-phase cartridge (Dextro-Pak C18, 100 x 25 mm (diam.), Millipore Waters), pressurized in a radial compression module (RCM 100 x 25, Millipore Waters) and eluted with water at a flow rate of 5 ml min"1. The separated oligosaccharides were collected with a fraction collector (Gilson, France) using the peak detection mode. Fractions were routinely analysed by thin-layer chromatography (TLC) to confirm the identity of the trisaccharides using silicagel plates (Silicagel 60 F254, layer thickness 0.2 mm, Merck) with one development in a mobile phase of propan-1-ol/ethyl acetate/water (5:3:3, v/v/v) (Sims et aL, 1991). The purified fructans were frozen and lyophilized and stored at -20 °C under desiccant. Analytical separations of fructan extracts were made using the same HPLC system fitted with a reversed-phase cartridge (Dextro-Pak C18, 100 x 10 mm (diam.), Millipore Waters), pressurized in a radial compression module (RCM 110 X 10, Millipore

110

Waters), and eluted with water at a flow rate of 0.5 ml min'1. Amounts of carbohydrates were quantified by anthrone (Yemm and Willis, 1954).

3 RESULTS AND DISCUSSION The most useful sources of fructans suitable for preparation of substantial quantities

of 1-kestose, 6-kestose and neokestose proved to be Neosugar P, a digest obtained by enzymatic conversion of sucrose by yeast invertase, and bulbs of A. cepa, respectively. 6-Kestose could also be extracted from stems of H. vulgäre. However, compared with the

LU C/)

CL

UJ

cr DC

B

JJ 5

15

TIME

30

(min)

45

Fig. 1. Analytical RP-HPLC separation of fructans up to DP 5: (A) extracted from Allium cepa, (B) from Neosugar P, and (C) a preparative RP-HPLC separation of fructans up to DP 5 from Neosugar P. Monosaccharides (1), sucrose (2), 1-kestose (3a), 6-kestose (3b), neokestose (3c), some tetrasaccharides (4), nystose (4a), and pentasaccharides (5).

I l l

yield of 6-kestose from an enzymatic digest of sucrose, the yield from stems of H. vulgäre was relatively low (data not shown) and extraction of plant material was slow and expensive.

RP-HPLC provided sufficiently good separations of 1-kestose, neokestose, nystose and inulin-pentasaccharide and their purification from the above-mentioned fructan sources using a preparative column was easily achieved (Fig. 1). Two difficulties were encountered. Firstly, fructans of degree of polymerization (DP) > 5 were strongly retained on the reversed-phase columns and would have required removal by back-flushing with a weak organic solvent {e.g. 10% acetonitrile) to prevent interference with subsequent separations. This was only a problem in extracts from plant sources because products of the incubation of invertase with sucrose, or Neosugar P did not contain significant quantities of fructans with DP > 5. Secondly, sucrose and 6-kestose were not sufficiently separated to allow purification of 6-kestose from enzymatic digests (which contained 1 M sucrose) without prior removal of the sucrose. These problems were overcome by preparing an enriched trisaccharide fraction from extracts of A. cepa or from enzymatic reaction mixtures by gel chromatography on Sephadex G-15 (Fig. 2). The elution position of a suitable enriched fraction was determined by prior HPLC analysis of the composition of the elution profile. For purification of 6-kestose, it was necessary to further remove sucrose from the enzyme reaction mixtures by gel filtration on Fractogel (see also Sims et al., 1991).

Extracts of carbohydrates from A. cepa and reaction mixtures containing 6-kestose and sucrose, contained about 1-5% trisaccharide. Sucrose was the major contaminating

20 30 40 50 60 70 FRACTION NUMBER

Fig. 2. Separation of fructans extracted from Helianthus tuberosus by preparative gel filtration on Sephadex G-15. Detection by refractive index after separation of fraction components by HPLC. A typical enriched fraction of trisaccharides would include fractions 40 to 55. Sucrose (■), DP 3(D), DP 4 ( · ) , DP 5 (O) and DP 6 (A).

112

component in both preparations (50 and 95% of the total sugars, respectively). Enrichment of the trisaccharide fraction from A. cepa by gel filtration increased the trisaccharide content of the fructan preparation to 30% or more of the total carbohydrates. Successful removal of all sucrose from enzymatic digests yielded a carbohydrate sample containing usually 95% 6-kestose, contaminated only with 1-kestose and neokestose. When separated by RP-HPLC the trisaccharide fraction of A. cepa contained approximately equal proportions of 1-kestose and neokestose. By using an automatic sample injector, about 300 mg neokestose of near-100% purity could be collected over 24 h. Other oligosaccharides (1-kestose, nystose and other DP 4 isomers) were also separated. Purification of 6-kestose from enzymatic digests also resulted in a substrate of near-100% purity.

Separation of Neosugar P on a preparative Dextro-Pak column resulted in a similar separation to that on an analytical Dextro-Pak column (Fig. 1). Optimal column loadings were approximately 100 mg total carbohydrate. With these loadings isomeric fructans of DP 3 (neokestose) and DP 4, present in Neosugar P, were also completely separated. Repeated automated injection of samples (20 runs of 100 mg carbohydrate in 24 h) resulted in the isolation of about 600 mg 1-kestose of near-100% purity. In addition, approximately 800 mg nystose and 200 mg pentasaccharide suitable as substrates for FFT and FH assays were typically collected.

4 CONCLUSIONS Preparative RP-HPLC has provided a rapid way to separate gram quantities of

fructan oligosaccharides up to DP 5. Oligosaccharides of the inulin series (1-kestose, nystose and inulin-pentasaccharide) were rapidly and efficiently separated from the commercially available Neosugar P. However, for other oligosaccharides it was necessary to use less convenient sources of fructans. Of these, the most time-consuming and expensive was extraction and purification of neokestose from bulbs of A. cepa.

The success and efficiency of the substrate preparation procedures firstly relied on selection of an appropriate fructan source. To illustrate this point, had stems of H. vulgäre been the only source of 6-kestose it would have been necessary to expend considerable time in isolating a pure trisaccharide fraction by gel filtration on Fractogel before the trisaccharide isomers could be separated. This is because tetra- and pentasaccharides of this species co-elute with trisaccharides on RP-HPLC (Sims et al., 1991). Secondly, it relied on the enrichment of the trisaccharide fraction from A. cepa and enzymatic digests of sucrose, which was essential for the efficient operation of the preparative RP-HPLC column. Finally, it relied on the good separation of the trisaccharide isomers after elution from RP-HPLC columns with water, allowing a rapid and simple procedure for collection of substrates of high purity.

113

5 REFERENCES

Henry, R.J. and Darbyshire, B., 1980. Sucroseisucrose fructosyltransferase and fructanifructan fructosyltransferase from Alliwn cepa. Phytochemistry, 19: 1017-1020.

Pollock, C.J., 1986. Fructans and the metabolism of sucrose in vascular plants. New Phytol., 104: 1-24. Shiomi, N., 1989. Properties of fructosyltransferases involved in the synthesis of fructan in liliaceous plants.

J. Plant Physiol., 134: 151-155. Sims, I.M., Smouter, H., Pollock, C.J. and Simpson, R.J., 1991. The separation of complex mixtures of

fructo-oligosaccharides from plants. Plant Physiol. Biochem., 29: 257-267. Smouter, H. and Simpson, R.J., 1989. Occurrence of fructans in the Gramineae (Poaceae). New Phytol.,

I l l : 359-368. Straathof, A.J.J., Kieboom, A.P.G. and Van Bekkum, H., 1986. Invertase-catalysed fructosyl transfer in

concentrated solutions of sucrose. Carbohydr. Res., 146: 154-159. Yemm, E.W. and Willis, A.J., 1954. The estimation of carbohydrates in plant extracts by anthrone.

Biochem. J., 57: 508-514.



SOME COLLIGATIVE PROPERTIES OF FRUCTANS: IMPLICATIONS FOR RYEGRASS (LOLIUM PERENNE L.) GROWN UNDER COOL CONDITIONS

S.C. ILGOUTZ, G.D. BONNETT and R.J. SIMPSON School of Agriculture and Forestry, The University of Melbourne, Parkville, Victoria 3052, Australia

ABSTRACT

Fructan oligosaccharides were extracted from tubers of Helianthus tuberosus L. and subsequently fractionated by gel permeation chromatography. The freezing point depression, osmotic potential and refractive index of fructan solutions were determined. At concentrations considered to be physiologically relevant (< 0.1 molal), the colligative properties of fructan solutions were similar to solutions showing ideal behaviour. Fructan accumulation was measured in perennial ryegrass {Lolium perenne L.) growing under cool conditions (6 °C). The concentrations of fructans accumulated in these plants were calculated to be insufficient to confer significant cryoprotection to the leaves.

1 INTRODUCTION Fructan accumulation in plants is reportedly confined to temperate species (Hendry,

1987; Smouter and Simpson, 1989). These observations have led to suggestions that fructan accumulation may enhance the resistance of plant tissue to cold stress (Labhart et al., 1983). Few studies of the colligative properties of fructans have been performed.

In order to explore the role of fructans in plant metabolism, some physical properties of fructan oligosaccharides in aqueous solution were determined. Also, accumulation of fructans was measured in plants grown under cool conditions.

2 MATERIALS AND METHODS 2.1 Plant materials

Tubers of Helianthus tuberosus L. were obtained from local markets. Lolium perenne L. (cv. Aurora) was grown at 23 °C day/16 °C night to the 5-leaf stage with a photon flux density of 250 ^moles m"2 s"1 (400-700 nm) and a 14-h photoperiod. Some plants were then transferred to cool conditions (6 °C day and night) for three weeks and returned to warm conditions (23 °C day/16 °C night), thereafter. Control plants were maintained under the

116

original growth conditions.

2.2 Extraction and purification offructans Finely ground tubers of H. tuberosus were extracted twice with boiling 80% (v/v)

ethanol and twice with water (60 °C). High-molecular-mass fructans (DP > 10) were separated from oligosaccharides by gel permeation chromatography (P-4, Bio-Rad) and discarded. The remaining oligomers were deionized using cation and anion exchange resins (Serdolit CS-2, AS-6, Serva) and passed through a C18 modified silica cartridge (Sep-PAK Vac, Millipore Waters) to remove hydrophobic material.

Individual oligosaccharides were separated by gel permeation chromatography (Fractogel TSK HW-40 (S), Merck) and carbohydrates were detected with a differential refractometer (R401, Millipore Waters). Fractions eluting from the gel permeation column were separated by thin-layer chromatography (TLC) and fractions enriched with oligosaccharides of interest were selected. Oligosaccharides of DP 3 to DP 5 were passed through the column twice whereas oligosaccharides of DP 6 to DP 10 were passed through the column three times before oligosaccharides were of sufficient purity. Soluble carbohydrate was extracted from L. perenne and measured with anthrone (Yemm and Willis, 1954).

2.3 Colligative properties and refractive index The freezing-point depression (FPD) caused by oligosaccharides in solution was

measured using an automatic micro-osmometer (Roebling) which estimates the osmotic potential (OP) of a solution from its freezing point according to: OP = FPD/KC, where Kc

= 1.858 for aqueous solutions (Wolf et al., 1988). The osmotic potential of each solution at 21 °C was measured directly using a dew point psychrometer (HR 33T, Wescor) and sample chamber (C-51, Wescor). Refractive index (RI) of each solution at 20 °C was measured using a refractometer (Abbe model G, Carl Zeiss).

2.4 Purity offructan oligosaccharides Thin-layer chromatograms of the purified fructan oligosaccharides were developed

three times in propan-1-ol/ethyl acetate/water (5:3:2, v/v/v) and visualized after spraying with urea-phosphoric acid (Wise et al., 1955). Fructans from tubers of H. tuberosus were used as a reference. TLC was used to determine the purity of each oligosaccharide. Oligosaccharides were eluted in water and quantified by anthrone (Yemm and Willis, 1954). Recovery (all fructan recovered from TLC as a proportion of the amount spotted) and purity (amount of particular oligosaccharide as a proportion of the amount of fructan recovered) of each oligosaccharide was calculated.

117

2.5 Determination of zone of leaf elongation The zone of elongation of expanding leaves was determined using the technique of

Schnyder et al. (1987), whereby the displacement over time of fine holes pierced into leaf bases was measured.

3 RESULTS AND DISCUSSION 3.1 Fructan oligosacchandes

The purities of the oligosacchandes (Table 1) ranged between 74 and 95% and due to the limitations of the separation technique, generally decreased with increase in DP. TLC of fructans of DP 3 to DP 10 indicated that most oligosacchandes were contaminated with approximately equal amounts of fructans of + 1 DP. Fructans of DP 3 and DP 4 were only contaminated with DP 4 and DP 5, respectively.

3.2 Refractive index and colligative properties of fructans The refractive index of solutions of oligosacchandes of equal concentration increased

with DP (Table 1). The refractive index of solutions containing equivalent masses of fructan was calculated to decline with increasing degree of polymerization.

The freezing point of DP 10 solutions was consistently lower than that of DP 5 solutions over the range of concentrations tested (Fig. 1(a)). The FPD caused by oligosacchandes departed from linearity with increasing concentration. This disagreed with the equation describing ideal behaviour of molecules (such as sucrose) which states: FPD = Kc X molality, where Kc = 1.858 for aqueous solutions. At physiologically relevant concentrations (< 0.1 molal), all FPD curves converged and approximated ideal behaviour. At the relatively high concentration of 0.6 molal, the divergence of fructans from ideal behaviour was positively correlated with degree of polymerization (Table 1).

Table 1. Recoveries, purities and some colligative properties of 0.6 molal fructan solutions.

DP

2 3 4 5 6 7 8 9

10

Recovery (%)

„

96 95 85 99 96 97 92 93

Purity (%)

— 94 93 88 95 86 79 74 77

RI - RIH2o

0.026 0.035 0.044 0.049 0.061 0.065 0.074 0.079 0.086

R i - R ^ o i 1

of carbohydrate

0.128 0.117 0.110 0.099 0.102 0.095 0.094 0.090 0.088

FPD (°Q

1.25 1.13 1.32 2.30 2.97 2.61 2.89 3.32 3.85

OP (MPa)

-1.59 -1.76 -2.01 -2.53 -2.67 -2.82 -2.97 -3.25 -4.34

118

(b) 01

'ca Q_

δ 2 15 1 Φ

fc-4 .a i-6 (0 O

" ^ ^ 1 ^c^^r^^^ ^ O S v T ^ ^ ^ ^Γ^Γ^*-^ ν ^ ^

\ ^ ^ η \ ^ k X \ χ .

Ν. ^ .

i 1 1 1 1 0.2 0.4 0.6 0.8

Concentration (mol kg-1 H2O)

Fig. 1. Relation between (a) FPD or (b) OP and concentration of sucrose (Δ), fructan DP 5 (O) and DP 1 0 ( · ) .

The OP of fructan oligomer solutions also deviated from those expected of solutions showing ideal behaviour (Fig. 1(b)). This indicates that the FPD measurements were not merely an artefact of the way in which the solutions were frozen in the automatic micro-osmometer. Within the range of concentrations judged to be of physiological relevance (< 0.1 mol kg"1 H20), the OP of fructan solutions were similar to those of sucrose solutions of the same concentration.

3.3 Soluble carbohydrates in L. perenne grown in cool conditions Plants subjected to cool conditions (6 °C) accumulated biomass more slowly than

plants growing at room temperature (23 °C) (Fig. 2). Soluble carbohydrate per plant, however, accumulated at similar rates irrespective of treatment. This indicates that high soluble carbohydrate concentrations observed in plants in cool conditions resulted from slow plant growth rather than enhanced rates of fructan synthesis.

The elongation zone in leaf blades of L. perenne extended to 20 mm from the leaf base which is similar to Festuca arundinacea Schreb. (Schnyder et al., 1987). Soluble carbohydrates were present in higher concentrations in the expanding zone of leaves than in the expanded zone of both control and treated plants (Table 2). The bulk of the soluble carbohydrates were fructans as confirmed by TLC.

Vacuolar concentrations and FPD of oligosaccharides in leaf tissue of plants grown at 6 °C were calculated by assuming a vacuolar cell volume of 85% (of total cell volume) and a difference between fresh and dry material approximating the mass of solvent (Cairns et al, 1989) (Table 3). For example, an FPD of 1.22 °C would be conferred to the expanding zone of cooled leaves of L. perenne if all of the soluble carbohydrate in the vacuole was in the form of fructose. If the average DP of soluble carbohydrate was assumed

119

10 20 Time (d)

Fig. 2. Total plant dry mass (solid line) and soluble carbohydrate content (broken line) in L. perenne when grown at 6 °C (O) and 23 °C ( · ) . Arrows indicate the time at which cooled plants were returned to 23 °C. Bars represent 2 x SE (n = 4).

Table 2. Soluble carbohydrate concentrations (mg g"1 dry mass) of expanding and expanded zones of leaf tissue in control plants and plants cooled for three weeks. All concentrations were significantly different (P < 0.05).

Treatment Expanding zone (0-20 mm from leaf base)

Expanded zone (20 mm to leaf tip)

Control (23 °C/16 °C) Cooled (6 °C)

283.59 341.02

144.18 242.34

Table 3. Estimated FPD conferred by the soluble carbohydrate content of leaf tissue of L. perenne grown at low temperatures (6 °C).

Expanding zone (0-20 mm from leaf base)

Expanded zone (20 mm to leaf tip)

Soluble carbohydrate concentration (mg g"1 fresh mass)

65.51 37.69

Equivalent fructose concentration (mol Γ1)

0.53 0.29

FDP conferred for fructose (°C) 1.22

Equivalent fructan DP 10 concentration 0.06 (mol Γ1)

0.67

0.03

FDP conferred for DP 10 (°C) 0.19 0.11

120

to be 10, then the freezing point conferred by the soluble carbohydrates would be only 0.19

°C.

4 CONCLUSIONS

Within the range of concentrations tested, the colligative properties of fructan oligomers

were not those expected of solutions showing ideal behaviour. Nonetheless, at concentrations

considered to be physiologically relevant ( < 0.1 molal), the colligative properties of fructan

solutions did not depart significantly from ideal behaviour.

Fructans did not accumulate in sufficient concentrations in cell sap to account for more than

a small change in the freezing point. Our results support the hypothesis that the accumulation of

fructans alone does not play a major role in plant cryoprotection (Hendry, 1987; Pollock et al.,

1988).

5 ACKNOWLEDGEMENTS

L. perenne cv. Aurora was supplied by M.O. Humphries of the Welsh Plant Breeding

Station, Aberystwyth, Wales, U.K. We thank Dr. A.A. Humffray (Department of Chemistry) for

discussion concerning the colligative properties of sugar solutions.

6 REFERENCES

Cairns, A.J., Winters, A. and Pollock, C.J., 1989. Fructan biosynthesis in excised leaves of Lolium temulentum L. III. A comparison of the in vitro properties of fructosyl transferase activities with the characteristics of in vivo fructan accumulation. New Phytol., 112: 343-352.

Hendry, G., 1987. The ecological significance of fructan in a contemporary flora. New Phytol., 106 (Suppl.): 201-216.

Labhart, C , Nösberger, J. and Nelson, C.J., 1983. Photosynthesis and degree of polymerization of fructan during reproductive growth of meadow fescue at two temperatures and two photon flux densities. J. Exp. Bot., 34: 1037-1046.

Pollock, C.J., Eagles, C.F. and Sims, I.M., 1988. Effect of photoperiod and irradiance changes upon development of freezing tolerance and accumulation of soluble carbohydrate in seedlings of Lolium perenne grown at 2 °C. Ann. Bot., 62: 95-100.

Schnyder, H., Nelson, C.J. and Coutts, J.H., 1987. Assessment of spatial distribution of growth in the elongation zone of grass leaf blades. Plant Physiol., 85: 290-293.

Smouter, H. and Simpson, R.J., 1989. Occurrence of fructans in the Gramineae (Poaceae). New Phytol., I l l : 359-368.

Wise, C.S., Dimler, R.J., Davis, H.A. and Rist, C.E., 1955. Determination of easily hydrolyzable fructose units in dextran preparations. Anal. Chem., 27: 33-36.

Wolf, A.V., Brown, M.G. and Prentiss, P.G., 1988. Concentrative properties of aqueous solutions: conversion tables. In: R.C. Weast (Ed.), Handbook of Chemistry and Physics. CRC Press, Boca Raton, Florida, p. D219.

Yemm, E.W. and Willis, A.J., 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem. J., 57: 508-514.


RECENT ADVANCES IN THE STRUCTURAL CHEMISTRY OF INULIN

Alfred D. FRENCH Southern Regional Research Center, U.S. Department of Agriculture, P.O. Box 19687, New Orleans, Louisiana 70179, U.S.A.

ABSTRACT

Computer models and crystal structures that contain fructofuranose residues and that are related to inulin are reviewed. Then, models of inulin were built, based on the dominant values for the interresidue linkage conformations and the geometry of the furanose rings found in those studies.

1 INTRODUCTION Since the physics and chemistry of fructans were reviewed about three years ago

(French, 1989), much has been learned about the nature of the inulin molecule. The application of ion chromatography gives a nearly complete description of molecular size, at least for dahlia and chicory inulin (Dionex Corporation, 1989). Perhaps the most important achievements were the discovery of the cyclic oligomers of inulin (Kawamura et al., 1989) and the determination of a crystal structure of cycloinulohexaose (Sawada et al., 1990). Also, the crystal structure of nystose has been solved using low-temperature X-ray diffraction. By examining these and other crystal studies, and by reviewing the modelling and NMR work on fructofuranose (FF) and inulin oligomers (Fig. 1), likely conformations of inulin can be predicted.

2 GENERAL CHEMISTRY Inulin undergoes many of the reactions that are performed on other polysaccharides.

These reactions, however, are often carried out to alter the physical properties of the molecule, such as to make it into an easily soluble product. It is not clear whether such treatment of inulin is likely to be useful, however, because most inulin is low in molecular weight. A possible exception might be for making biodegradable materials, because inulin is easily broken apart in mild acid. This well-known trait seems to conflict with more recent information that inulin is not digested in the human stomach. This prompted an informal

122

experiment by Earl Roberts of Sugar Processing Research, Inc. Inulin was added to solutions of 1 N and 0.1 N HC1 at 37 °C. After 1 h, all the inulin in the 1 N solution had been hydrolysed to FF but more than 3 h were required to hydrolyse inulin in 0.1 N HC1. Stomach acid is about 0.05 N, so much of the inulin would have travelled past the stomach into the intestines before being hydrolysed.

3 PROPERTIES OF THE FRUCTOFURANOSE RING The combination of crystallography and modelling gives a nice picture of the FF

ring. First, the nomenclature must be described. FF rings can be described in terms often different envelope (E) forms, wherein four of the five ring atoms are in a plane, and the remaining atom is either above or below it. The ten symmetrical twist (T) forms have three atoms in a plane and the other two adjacent atoms displaced in opposite directions above or below the plane. There are also intermediates to these 20 characteristic conformations. Conversion among the various forms occurs through "pseudorotation", wherein the ring remains puckered in low-energy forms. Imagine, for example, that C3 is below the plane of the other four atoms (an E3 form). When pushing C3 toward the plane, C4, previously in the plane, would rise, making a 4T3 form. Further pushing on C3 would bring it to the plane, leaving C4 at the highest point above the plane, a 4E form. Progressing from there, all conformations can be visited. A circular list of the 20 conformations is called a conformational wheel (Fig. 2).

Fig. 1. (left) Nystose fragment of inulin, showing sub-fragments corresponding to sucrose, inulobiose, and 1-kestose.

Fig. 2. (right) Conformational energy map for 0-D-FF. The energy was calculated at 720 points with ΜΜ3 and a dielectric constant of 4. Contours are at 0.5 kcal mol"1. (*)'s show shapes found in crystals.

123

Ring puckering is more precisely described by its amplitude, q, and phase angle, φ. These parameters form a polar coordinate system, with q as the radius and φ the position on the conformational wheel. Thus, the centre of the conformational wheel represents a structure with all ring atoms co-planar. To predict the likely shapes of the furanose ring, the energies of different model rings were calculated. The nine combinations of staggered primary alcohol positions were studied with the MM3 molecular mechanics program (Allinger et aL, 1990) at 720 φ-q combinations within a radius of q = 1.0 A. Then, contours of energy were drawn. The energy map (Fig. 2) has two main minima. The elongated upper, or "northern" minimum near the ^Ύ^ shape predicts that a range of different E and T shapes will be found. The "southern" minimum, near E4, is 1.5 kcal mol" 1 higher. The barrier to facile pseudorotation between the two minima is only about 2.5 kcal mol"1. This barrier would be crossed often by a molecule in solution at room temperature. While the map is similar to results obtained with MMP2(85) (French and Tran, 1990), the energy of the planar (q = 0 A) structure is higher with MM3. Therefore, the conversion would occur through pseudorotation and not through a planar ring. Also, a slightly lower relative energy is found with MM3 for models with 4T5 forms.

Also shown in Fig. 2 are the ring shapes found in crystals of molecules such as sucrose that contain FF rings. The finding of all but two points within 1.6 kcal mol'1

suggests that the energy calculations are approximately correct. The two points that are at highest energy are both from the same crystal structure studied at low temperature. This structure of stachyose (Jeffrey and Huang, 1991) has disorder of C3 (and 03), giving it puckering amplitudes of 0.26 A and 0.60 A. In the crystal structure of anhydrous lactulose at -150 °C (Jeffrey et aL, 1983), the reducing FF residue takes three forms: the ß-pyranose, and the a- and ß-FF. While this conflgurational disorder is for a reducing residue, and would not happen in inulin, it dramatically shows the interconvertibility of fructose rings. For lactulose trihydrate at room temperature (Jeffrey et aL, 1991), only the ß-FF form is found, showing that 0-FF can be stabilized in the solid state.

The above shows that the most likely furanose ring form is 4T3, but that a continuum of ring shapes occurs, requiring little energy for conversion. Therefore, modelling software should allow adjustment of all atomic coordinates so FF residues can take the optimal shape for each model of inulin, and not produce artificially high energies.

4 CONFORMATIONS OF THE LINKAGES BETWEEN FRUCTOSE RINGS Three bonds separate adjacent FF rings of inulin. This spaces the rings far apart from

each other and allows them to rotate with little atomic collision. To predict the torsion angles about these bonds for inulin, models of related oligosaccharides were studied and compared with the crystal structures of three available compounds. In all these studies, the

124

presence of three bonds made it impractical to test all combinations of all variables.

4.1 Inulobiose The model (Calub et al., 1990b) was composed of two FF rings. Even though the

reducing residue of this molecule probably exists mostly in the pyranose form, our goal was to learn the structure of the linkage in inulin. We found that the central bond (described in Fig. 1 by the torsion angle C2"-01'-C1'-C2') always preferred a conformation with ψ = about 180°. Therefore, the conformational analysis was carried out based on φ and ω (Cl"-C2"-01'-C1' and 01"-C1"-C2"-01', respectively). A simple analogy explains the preference for ψ = 180°. Inulobiose can be thought of as a derivative of /z-butane, with bulky substituents that take a trans orientation. The preferred conformations from the study with MMP2(85) are listed in Table 1.

4.2 1-Kestose Two main issues were considered in this study (Waterhouse et al., 1991). The first

was whether the addition of glucose to the inulobiose model would alter the conformation of the linkage in inulobiose. The second was whether the unusual southern conformation found in the crystal (Jeffrey and Park, 1972) for the intermediate FF ring would be found in the model and in solution. Several hundred trisaccharide models were tested with MM2(87), including optimized versions of the crystal structure.

This work points out a weakness of modelling studies. Isolated models often have lowest energy when intramolecular hydrogen bonds are formed, even though intermolecular bonds are more likely in the crystal. In 1-kestose, there are 18 hydrogen bonds per trisaccharide in the crystal, all intermolecular. In the isolated model there are four major intramolecular hydrogen bonds. Despite this problem, the values of φ, ψ and ω in both the crystal and the model of 1-kestose were roughly similar. These ω values differed from ω in inulobiose (values listed in Table 1). The intermediate rings in the best models have northern shapes, suggesting that the southern form in the crystal arises because of the intermolecular hydrogen bonds in the crystal.

To determine the solution conformation of the FF rings in 1-kestose, the complete proton NMR spectra were assigned (Calub et al., 1990a). Through coupling constant and NOE data, it was confirmed that the FF rings both had northern conformations (Calub et al., 1990b).

4.3 Nystose Models of nystose, a tetrasaccharide containing two inulin linkages, were constructed

by adding a third FF residue to the 1-kestose model. This model, optimized with MM3,

125

achieves even more intramolecular hydrogen bonds than 1-kestose by curling up into a ball. The crystal structure is extended, so it can form a maximum number of intermolecular hydrogen bonds. The FF ring shapes are all northern, confirming that the southern shape in crystalline 1-kestose was not an intrinsic feature of its position between a glucose residue and a FF residue. Because of the extended molecular shape, the values of ω differ from those of the models; both are given in Table 1.

4.4 Cycloinulins For many years, cycloamyloses have served as model compounds, giving information

on amylose and its interactions with other molecules. Now, there is a similar source of information on inulin. Before seeing the crystal structures (Sawada et al., 1990, 1991), I tried to build computer models of the molecules. However, I assumed that all residues and their linkages in inulohexaose would be identical, a common assumption when modelling polymers. Those models had unsuitably high energy. When the crystal structure was solved, the error of the assumption became clear. The ω torsion angles alternate between 60° and 180°, giving a structure with three-fold symmetry instead of the assumed six-fold symmetry. Once the alternate ω values were set to 180°, the energy of the model dropped to a value comparable to the non-cyclic inulin chains. Further, the puckerings of two individual furanose rings adjusted slightly from the ideal shape, matching almost perfectly the puckerings found in the crystal.

The models of cycloinuloheptaose and octaose are not as symmetric as the hexaose, perhaps because of my limited imagination. However, the energy values are suitable, even though those rings appear slightly distorted. The cavity in the hexamer is not large enough to entertain a guest, but the octamer may be able to form a complex.

5 THEORETICAL ADVANCES FROM THE STUDY OF COMPOUNDS CONTAINING FRUCTOFURANOSE FF has especially strong anomeric effects. These effects include distortion of the

bond lengths around the anomeric carbon (C2), and orientational preferences for substituents attached to 02 (the exocyclic oxygen attached to C2). DuBois and co-workers (Cosso-Barbi et al., 1989) have proposed that the strengthening of these effects arises because the hybridization of electrons on the ring oxygen (05) is sp2, not sp3 as more commonly thought. The formulae for anomeric effects in modelling software would then be insufficient. At any rate, the errors in bond lengths in the models, compared to crystal structure values, are larger for these bonds than for others in the model. In particular, the energies calculated for some observed conformations of the sucrose linkage may be too high because the bonds are too short.

126

Table 1. Torsion angles of inulin oligomers.

Species

Inulobiose model Kestose model Kestose crystal Nystose model Nystose crystal Cycloinulin model Cycloinulin crystal Preferred value

*

52 52 89 55,64 171, 72 54,48 57,48 60

*

164 177 -162 174, 180 -133,-165 170,-178 178, 178 180

ω

-58 52 43 55,77 176,66 165, 59 163, 52 60, 180

Fig. 3. Inulin models optimized with MM3 and a dielectric constant of 4. Starting models had preferred values of φ and ψ (Table 1) and (from left to right) ω of 60, -60 and 18(F. From left to right, the numbers of residues per turn and rise per residue values are about -4.5, 1.3 A; 2, 2.75 A and 4, 2.75 A. The minus sign indicates a left-handed helix. Hydroxyl groups and primary alcohol groups are not shown, for increased clarity.

6 MODELS OF INULIN Based on the preferred values of φ and ψ (Table 1), and all three staggered values

of ω, models of inulin can be constructed (Fig. 3). Given the lack of consideration of intermolecular factors, and the possible errors associated with anomeric centres, these models are still just models. The energies of the models are all quite similar; none has intramolecular hydrogen bonding.

An attempt to build a regular helix (one with identical torsion angles at every other linkage) from the torsion angles in cycloinulohexaose was not successful.

127

7 ACKNOWLEDGEMENTS

This short review is based on the work of many colleagues. Purified nystose was

supplied by Masao Hirayama of BioScience Labs, and a crystal was grown by Serge Perez

and his colleagues. Professor George Jeffrey and De-bin Huang solved the crystal structure

of nystose and Jeffrey also forwarded unpublished studies of stachyose and lactulose.

Professor Andrew Waterhouse and his student modeled inulobiose and 1-kestose and did the

NMR work, and Earl Roberts did the hydrolysis rate studies. Professor Takao Uchiyama

kindly sent his papers before publication.

8 REFERENCES

Allinger, N.L., Rahman, M. and Lii, J.-H., 1990. A molecular mechanics force field (MM3) for alcohols and ethers. J. Am. Chem. Soc , 112: 8293-8307.

Calub, T.M., Waterhouse, A.L. and Chatterton, N.J., 1990a. Proton and carbon chemical-shift assignments for 1-kestose, from two-dimensional n.m.r.-spectral measurements. Carbohydr. Res., 199: 11-17.

Calub, T.M., Waterhouse, A.L. and French, A.D., 1990b. Conformational analysis of inulobiose by molecular mechanics. Carbohydr. Res., 207: 221-235.

Coss^-Barbi, A., Watson, D.G. and Dubois, J.E., 1989. Anomeric effects in carbohydrates: non equivalence of endocyclic oxygen lone pairs. Tetrahedron Lett., 30: 163-166.

Dionex Corporation, 1989. Technical Note 20, page 17. Dionex Corporation, P.O. Box 3603, Sunnyvale, California 94088-3603.

French, A.D., 1989. Chemical and physical properties of fructans. J. Plant Physiol., 134: 125-136. French, A.D. and Tran, V., 1990. Analysis of fructofuranose conformations by molecular mechanics.

Biopolymers, 29: 1599-1611. Jeffrey, G.A. and Huang, D.-b., 1991. Hydrogen bonding in the crystal structure of the tetrasaccharide

stachyose hydrate: a 1:1 complex of two conformers. Submitted to Carbohydr. Res. Jeffrey, G.A., Huang, D.-b. and Pfeffer, P.E., 1991. Crystal structure and solid-state NMR analysis of

lactulose trihydrate. Manuscript in preparation. Jeffrey, G.A. and Park, Y.J., 1972. The crystal and molecular structure of 1-kestose. Acta Crystallogr.,

B28: 257-267. Jeffrey, G.A., Wood, R.A., Pfeffer, P.E. and Hicks, K.B., 1983. Crystal structure and solid-state NMR

analysis of lactulose. J. Am. Chem. Soc , 105: 2128-2133. Kawamura, M., Uchiyama, T., Kuramoto, T., Tamura, Y. and Mizutani, K., 1989. Formation of a

cycloinulo-oligosaccharide from inulin by an extracellular enzyme of Bacillus circulans OKUMZ 31B. Carbohydr. Res., 192: 83-90.

Sawada, M., Tanaka, T., Takai, Y., Hanafusa, T., Hirotsu, K., Higuchi, T., Kawamura, M. and Uchiyama, T., 1990. Crystal structure of cycloinulohexaose. Chem. Lett., 2011-2014.

Sawada, M., Tanaka, T., Takai, Y., Hanafusa, T., Taniguchi, T., Kawamura, M. and Uchiyama, T., 1991. The crystal structure of cycloinulohexaose produced from inulin by cycloinulo-oligosaccharide fructanotransferase. Carbohydr. Res., 217: 7-17.

Waterhouse, A.L., Calub, T.M. and French, A.D., 1991. Conformational analysis of 1-kestose by molecular mechanics and n.m.r. spectroscopy. Carbohydr. Res., 217: 29-42.



STRUCTURE ANALYSIS OF SMALL INULIN OLIGOSACCHARIDES BY 1D-AND2D-NMR

J.W. TIMMERMANS*, B.R. LEEFLANG** and H. TOURNOIS* ATO Agrotechnological Research Institute, P.O. Box 17, 6700 AA Wageningen, The Netherlands

** RUU Department of Chemistry, State University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

ABSTRACT

The *H chemical shifts of sucrose, 1-kestose and nystose and the 13C signals of nystose have been completely assigned by using ID- and 2D-NMR techniques. Accurate values of 3JH H coupling constants were obtained for these molecules.

1 INTRODUCTION Many interesting possible applications for inulin have been investigated during the

last decade. However, degradation of the polymer is involved in most of them. Development of inulin as a biopolymer requires information about its molecular structure in relation to its physicochemical properties as well as a number of (bio)chemical methods of modification.

We are presently working on the structural analysis of sucrose, 1-kestose, nystose, GF4 and of longer inulin homologues by means of advanced ID- and 2D-NMR techniques.

2 MATERIALS AND METHODS 1-Kestose and nystose were enzymatically synthesized as described by Hidaka et al.

(1988) and purified in large (gram) amounts according to Ivin and Clarke (1987) on a semi-preparative RP 18 HPLC column (22 x 250 mm) and quantitated by refractive index. GF4

was purified by RP 18 HPLC from a fraction obtained from inulin (Jerusalem artichoke) after removal of the larger inulins by ethanol precipitation (85%, v/v).

NMR experiments were performed with Bruker AMX 400, AM 500 and AM 600 MHz NMR spectrometers.

130

3 RESULTS Fig. 1 depicts the molecular structures of 1-kestose and nystose. The anomeric proton

H-1 of the glucose ring and the anomeric carbon atoms of the glucose and fructose rings can be assigned unambiguously because their chemical shifts can be clearly distinguished from all other signals due to the vicinity of two oxygen atoms. From 2D HOHAHA and DQF-COSY experiments all lH signals (H-2, H-3, H-4, H-5, H-6 and H-6') of the glucosyl ring of sucrose, 1-kestose and nystose have been assigned by taking the G H-1 signal as a starting point. This assignment is straightforward because all protons of the glucosyl unit form a single spin system and only one glucosyl unit is present. In each fructosyl unit, protons form two separate spin systems, viz. the H-1 system formed by H-1 and Η-Γ, and the H-3/H-6 system comprised of H-3, H-4, H-5, H-6 and H-6'. The easily identifiable doublet of the H-3 proton of the fructosyl moieties can in turn be used as a starting point for the assignment of all protons of the H-3/H-6 spin system. This enables complete assignment of sucrose.

Data on 1-kestose (Calub et ah, 1990) served to discriminate between the signals of the individual fructose rings. For nystose, additional HMBC experiments have been

CH20H CH20H

H0CH2^0·

HO H

H0CH2/°v ?

HO H CH20H

HO H

HOCH2 / ° v ?

HO H

H0CH2^°\ ?

HO H CH2OH

Fig. 1. 1-Kestose (left) and nystose (right).

131

performed (Fig. 2). HMBC is a powerful technique for detecting long-range (two- and three-bond) l3C,l¥i connectivities and can be applied to relate individual spin systems by detecting such connectivities in the backbone of the inulin oligosaccharides. In the HMBC spectrum one of the fructosyl C-2 signals shows cross signals with GH-1 and one of the fructosyl Η-1/Η-Γ spin systems. This enables identification of Fl C-2, Fl H-l and Fl H-1'. One of the C-2 signals of the remaining fructosyl units is evidently correlated with both remaining Η-1/Η-Γ spin systems of fructosyl units 2 and 3. Obviously, this is the F3 C-2 atom. Because only one of these Η-1/Η-Γ spin systems shows a correlation with the remaining C-2, the F2 C-2 and the F2 H-l and F2 Η-Γ signals can be assigned.

The remaining Η-1/Η-Γ signals have to originate from F3 H-l and F3 Η-Γ. This assignment is further corroborated by the fact that they are correlated with only one C-2 signal as expected. Because correlation between C-2 and H-3 signals can only be due to the presence of atoms within the same fructose ring, based on the assignment of the C-2 all protons of each individual H-3/H-6 spin system can be assigned. This completes the

Fig. 2. 400 MHz HMBC spectrum of nystose.

132

assignment of all proton frequencies of sucrose, 1-kestose and nystose. In order to correlate proton signals with 13C signals, additional heterocorrelation

experiments (HETCOR and HMQC) with 13C and *H detection were performed (data not shown). All remaining 13C frequencies of nystose have been assigned. This assignment shows several discrepancies with previously reported data (Jarrell et al., 1979; Heyraud et al., 1984).

By comparing the 2D-NMR spectra of sucrose, 1-kestose and nystose with 600 MHz ID JH spectra (Fig. 3), accurate values for frequencies could be determined. These values have been used in computer simulations of the experimental spectra. Using this simulation reliable 3J proton-proton coupling constants were determined (Table 1). Chemical shifts and coupling constants are given in Table 1.

NOE's have been measured by use of ROESY experiments (data not shown). These can be used as distance constraints in molecular dynamics calculations to obtain three-

1-KEST0SE 600 MHz

Fig. 3. 600 MHz NMR spectrum of 1-kestose.

133

Table 1. Chemical shifts (in ppm) and coupling constants (in Hz) of sucrose, 1-kestose and nystose.

ppm HI HI' H2 H3 H4 H5 H6 H6'

Hz Hl-Hl' H1-H2 H2-H3 H3-H4 H4-H5 H5-H6 H5-H6'

Sucrose

Glu

5.408 -3.553 3.755 3.465 3.840 3.809 3.809

-3.9 9.8

10.4 10.0 3.3 3.3

Fru

3.672 3.672 -4.209 4.046 3.884 3.819 3.807

---8.7 8.6 2.9 7.4

1-Kestose

Glu

5.429 -3.539 3.749 3.467 3.840 3.807 3.807

-3.9

10.0 9.5 9.8 3.3 3.3

Fru 1

3.823 3.714 -4.273 4.041 3.868 3.813 3.785

-10.5 --

8.7 8.3 3.4 7.2

Fru 2

3.734 3.679 -4.186 4.079 3.863 3.830 3.770

-12.3 --

8.5 8.1 3.0 7.1

Nystose

Glu

5.431 -3.537 3.75 3.468 3.83 3.81 3.81

-3.9

10.3 9.0

10.0 3 3

Fru 1

3.840 3.741 -4.268 4.044 3.87 3.81 3.79

-10.8 --

8.7 8.6 3 7

Fru 2

3.859 3.722 -4.222 4.074 3.86 3.82 3.75

-10.6 --

8.4 8.1 3 7

Fru 3

3.752 3.685 -4.182 4.104 3.86 3.84 3.75

-12.2 --

8.6 7.9 3 7

dimensional structures. From coupling constants, dihedral angles have been determined (data not shown) from which glucosyl and fructosyl ring structures will be calculated.

4 CONCLUSIONS Using ID and 2D techniques, lU signals of sucrose, 1-kestose and nystose and 13C

signals of nystose have been assigned. Accurate values of 3JH ,H coupling constants were obtained for sucrose, 1-kestose and nystose. Coupling constants and NOE's will be used for calculation of the structure of the molecules in solution.

5 ACKNOWLEDGEMENTS This work was supported by the Netherlands Foundation for Chemical Research

(SON) with financial aid of the Netherlands Organization for Scientific Research (NWO). The authors would like to thank Dr. A.S. Ponstein (ATO-DLO) for the preparation of 1-kestose and nystose, and Dr. P. de Waard (ATO-DLO) for assistance with the NMR experiments.

134

6 REFERENCES

Calub, T.M., Waterhouse, A.L. and Chatterton, N.J., 1990. Proton and carbon chemical-shift assignments for 1-kestose, from two-dimensional n.m.r.-spectral measurements. Carbohydr. Res., 199: 11-17.

Heyraud, A., Rinaudo, M. and Taravel, F.R., 1984. Isolation and characterization of oligosaccharides containing D-fructose from juices of the Jerusalem artichoke. Kinetic constants for acid hydrolysis. Carbohydr. Res., 128: 311-320.

Hidaka, H., Hirayama, M. and Sumi, N., 1988. A fructooligosaccharide-producing enzyme from Aspergillus niger ATCC 20611. Agric. Biol. Chem., 52: 1181-1187.


Jarrell, H.C., Conway, T.F., Moyna, P. and Smith, I.C.P., 1979. Manifestation of anomeric form, ring structure, and linkage in the 13C-n.m.r. spectra of oligomers and polymers containing D-fructose: maltulose, isomaltulose, sucrose, leucrose, 1-kestose, nystose, inulin, and grass levan. Carbohydr. Res., 76: 45-57.


CHEMICAL MODIFICATION OF CHICORY ROOT INULIN

E. BERGHOFER, A. CRAMER and E. SCHIESSER Institute of Food Technology, University of Agriculture, Peter-Jordanstraße 82, 1190 Vienna, Austria

ABSTRACT

Some methods of derivatization used for chemically modifying starch were applied to inulin in order to improve its functional properties. Attempts were made to ester ify inulin with sodium trimetaphosphate and with a mixed acetic/adipic acid anhydride. Using these bifunctional reagents esterification of inulin proved to be possible. In a series of tests the characteristic properties of the inulin esters were determined and compared with those of native inulin. The viscosity properties of the inulin derivatives were significantly improved and their solubility reduced.

1 INTRODUCTION Recently, it has been shown that inulin can be technically isolated in native form

from chicory roots (Berghofer et al., 1993). Like other polysaccharides, inulin could be used in foodstuffs as a thickener or filling agent. In both cases, the particular advantage of inulin would be that it is not digestible. Until recently, the average degree of polymerization (DP) of inulin in chicory roots has been assumed to be only 23 (Beck and Praznik, 1986). However, with the inulin preparations we obtained in pilot-scale tests we found an average DP of up to 66. Still, this DP is far below that found in some other polysaccharides now used as additives in consumer goods. Therefore, the functional properties of inulin will not be sufficient for a great deal of technical applications.

In the case of starch it has become common practice for quite a number of years to eliminate a shortage in functional properties by having recourse to chemical modification. In a similar way, some methods of derivatization used to chemically modify starch were therefore applied to inulin.

2 MATERIALS AND METHODS Inulin was obtained from chicory roots by means of extraction and crystallization on

a pilot scale (Berghofer et al., 1993).

136

2.1 Derivatization of inulin with adipic acid anhydride Derivatization of inulin with a mixed acetic/adipic acid anhydride was carried out on

a laboratory scale as described by Tessler and Rutenberg (1972) for starch. According to this method inulin was suspended in cold water, the pH adjusted to 7.5 and the inulin suspension then heated to the temperature required (20 or 60 °C). After the reactant was added the temperature and pH were both maintained at the initial level over the whole reaction period. After completion of the reaction, the mixture was cooled down to 4 °C, and the precipitated inulin derivative separated by centrifugation and subsequently dried in a drying chamber. Table 1 shows the test conditions in detail.

The inulin adipic acid ester was quantitated spectrophotometrically according to the method of McComb and McCready (1957). As a reference dimethyl adipate was used. The degree of substitution was determined according to the following formula:

DS_ 162.14x^/146.14) 100-(145.14/146.14χΛ)

A = % (w/w) adipic acid / dry substance 146.14 = mol wt of adipic acid 145.14 = mol wt of adipic acid minus 1 H+

162.14 = mol wt of one anhydro-fructose (glucose)-unit

Rheological measurements were carried out using a rotational viscosimeter Viscolab LC 10 (Physica Meßtechnik GmbH, Stuttgart, B.R.D.). To measure gelation and gel firmness the inulin samples were weighed into 250-ml glass beakers and then dissolved in

Table 1. Test conditions for the derivatization of inulin with mixed acetic/adipic acid anhydride (reaction time 12 h).

Sample code

Variable A: Adipic acid concentration (%)a

Variable B: Inulin concentration (%, w/w)

Variable C: Temperature (°C)

A-l

0.2 (-)

20 (-)

20 ( - )

A-2

2.0 (+)

20 (-)

20 (-)

A-3

0.2 ( - )

25 (+)

20 ( - )

A-4

2.0 (+)

25 (+)

20 ( - )

A-5

0.2 ( - )

20 ( - )

60 (+)

A-6

2.0 (+)

20 ( - )

60 (+)

A-7

0.2 ( - )

25 (+)

60 (+)

A-8

2.0 (+)

25 (+)

60 (+)

a Expressed on an inulin dry weight basis.

137

boiling water. To allow gel formation to take place the solutions were stored overnight at 4 °C. A subsequent classification of the substance whether it be liquid, pasty or solid was based on visual observation. After keeping the gels for 2 h at 20 °C an Instron universal testing instrument (load transducer 500 N) was used to measure the maximum force needed for a cylindrical plunger (diam. 35 mm, penetration distance 15 mm) to sink into the gel.

2.2 Derivatization of inulin with sodium trimetaphosphate Esterification of inulin with sodium trimetaphosphate was carried out as described

by Kerr and Cleveland (1962) for the derivatization of starch. First, the sodium trimetaphosphate was dissolved at 60 °C; the solution was then

cooled to 4 °C, the inulin added and the pH adjusted to 7.5. After 1 h, the inulin was separated off by centrifugation. A small part of the phosphate reactant remained in the aqueous phase of the inulin precipitate. After drying the precipitate at 60 °C to a residual water content of 8-10% the mixture of inulin and sodium trimetaphosphate was heated to 120 °C under vacuum to enable esterification to take place. Table 2 shows the test conditions in detail. The derivative obtained was then separated from the inactive reactant by three times repeated suspension and centrifugation in distilled water.

To determine the phosphate content the spectrophotometric method according to Bergthaller (1971) was used. In the case of esterification with sodium trimetaphosphate both mono- and diesters may be formed; this has to be kept in mind when determining the degree of esterification. However, when determining the total phosphate content it will not be possible to distinguish between these two forms. Since there was almost no difference between the results obtained with the formulas for monoester or diester binding the degree of substitution was calculated using only the formula for the diester binding.

DS 162.14xP * 100x30.97-83.9xP

DS = degree of substitution P = % (w/w) phosphate / dry substance 162.14 = mol wt of one anhydro-fructose (glucose)-unit 30.97 = atomic wt of phosphorus

All further tests were carried out as described for the adipic acid derivatives.

3 RESULTS AND DISCUSSION 3.1 Derivatization of inulin with adipic acid anhydride

As can be seen from the results listed in Table 3 in all cases an esterification with

138

Table 2. Test conditions for the derivatization of inulin with sodium trimetaphosphate (reaction temperature 120 °C).

Sample code

Variable A: Sodium trimetaphosphate concentration (%)a

Variable B: Reaction time (h)

MP-1

15

1

MP-2

45

3

MP-3

15

1

MP-4

45

3

a Expressed on an inulin dry weight basis.

adipic acid was possible. The degrees of substitution varied between 0.0014 and 0.014 and thus approximately equalled those of corresponding starch derivatives. From Table 3 it is evident that derivatization equally affected the viscosity properties. As can be seen, at both 25 and 50 °C the derivatives had a higher viscosity than that of the initial sample. Fig. 1 shows the viscosity/temperature curves for native inulin and an inulin adipate derivative. In both cases, 10% (w/v) suspensions were heated from 25 to 80 °C and subsequently cooled down again to 25 °C. Clearly, the initial viscosity of the derivative was remarkably higher than that of the native inulin. Furthermore, upon raising the temperature the viscosity of the derivative did not drop as much as that of the native inulin, indicating a decrease of solubility in the former case. Upon cooling the solutions, in general the initial viscosities were not reached again. This was due to the fact that cooling took place very fast so that complete recrystallization of the inulin could not take place. In both cases the initial viscosity could be reached again only after several hours had elapsed.

Table 3. Degrees of substitution and viscosity characteristics of inulin samples esterified with mixed acetic/adipic acid anhydride.

Sample code Adipic acid content Degree of Viscosity (mPa sec"1)8

(% dry matter, w/w) substitution

Native inulin A-l A-2 A-3 A-4 A-5 A-6 A-7 A-8

-0.25 1.24 0.27 0.93 0.08 0.13 0.16 0.28

0 0.0028 0.0140 0.0030 0.0104 0.0009 0.0014 0.0018 0.0031

25 °C

13.3 19.9 15.3 13.4 19.0 13.4 18.4 14.5 16.8

50 °C

9.7 20.8 16.0 10.7 18.3 16.9 12.5 18.1 20.7

a Concentration of inulin adipate derivative 10% (w/w).

139

20 30 τ 40 50 60

temperature (°C)

Fig. 1. Viscosity/temperature curves for native inulin (a) and for the inulin adipate derivative A-1 (b) (10%, w/w).

140

3.2 Derivatization of inulin with sodium trimetaphosphate As can be seen in Table 4, the degrees of substitution of the inulin trimetaphosphate

derivative as determined from the phosphate content varied between 0.016 and 0.039. As with the inulin adipate derivatives, the phosphate derivatives showed an increased viscosity as compared with native inulin. In comparison with the adipate derivatives the initial viscosity was much higher. As distinct from the inulin adipate derivatives, the phosphate derivatives did not show a steady decrease in viscosity upon increasing the temperature, but the viscosity - apart from a small initial decrease - remained relatively constant over quite a large temperature range (Fig. 2). Only at temperatures beyond 60 °C the phosphate derivatives started to dissolve quite fast as well.

Not only did phosphate derivatives show changes in their viscosity and solubility characteristics, but - much more pronounced than native inulin - they were also able to form "thermoreversible gels". When inulin suspensions were stored for some time at a temperature of 4 °C, depending on the concentration gels were formed the consistency of which varied from pasty to rigid. These gels remained stable at room temperature and melted again when the temperature was increased beyond 60 °C. Phosphate derivatives, on the other hand, at similar concentrations formed gels that were considerably firmer than those of native inulin and inulin adipate derivatives (Table 5). These gels showed a firmness that resembled that of gels made from so-called "gel-forming starch hydrolysis products".

Table 4. Degrees of substitution and viscosity characteristics of inulin samples esterified with sodium trimetaphosphate.

Sample code

Native inulin MP-1 MP-2 MP-3 MP-4

Phosphi (% dry

n.n. 0.20 0.50 0.22 0.52

ite content matter, w/w)

Degree of substitution

0 0.011 0.027 0.012 0.028

Viscosity (mPa

25 °C

13.3 19.2 20.3 20.8 23.8

. sec"1)8

50 °C

9.7 20.0 23.3 19.0 21.9

60 °C

7.2 18.0 17.6 14.4 10.1

a Concentration of inulin trimetaphosphate derivative 10% (w/w).

Table 5. Gel strength of inulin gels.

Sample code Maximum force (Newton) Gel property

Inulin (prepared by ultrafiltration) 5.5 pasty Inulin (prepared by crystallization) 6.0 pasty Inulin adipate derivative 6.0 pasty Inulin trimetaphosphate derivative 15.0 rigid

141

τ 50 40 50 60

temperature (°C)

- r 70 80

Fig. 2. Viscosity/temperature curves for the inulin trimetaphosphate derivatives M-1 (a) and M-4 (b) (10%, w/w).

142

4 CONCLUSIONS

Esterification of inulin has been shown to be possible in principle when using mixed

acetic/adipic acid anhydride and sodium trimetaphosphate. In a series of tests the

characteristic properties of the derivatives were determined and compared with those of

native inulin. In part, it was possible to significantly improve the viscosity properties of the

inulin derivatives and to reduce their solubility. The low stability of the inulin molecules

was found to be disadvantageous. From these observations it can be deduced that only very

gentle methods should be used in preparing inulin derivatives.

5 REFERENCES

Beck, R.H.F. and Praznik, W., 1986. Inulinhaltige Pflanzen als Rohstoffquelle. Biochemische und pflanzenphysiologische Aspekte. Starch/Stärke, 38: 391-394.

Berghofer, E., Cramer, A., Schmidt, U. and Veigl, M., 1993. Pilot-scale production of inulin from chicory roots and its use in foodstuffs. In: A. Fuchs (Ed.), Inulin and Inulin-containing Crops, Studies in Plant Science, Vol. 3. Elsevier, Amsterdam, pp. 77-84.

Bergthaller, W., 1971. Untersuchungen über die Herstellung von Phosphorsäureestern der Stärke. 1. Mitteilung. Einfluß der Phosphatzugabe und der Hitzebehandlungszeit auf den Phosphatgehalt und das Theologische Verhalten. Stärke/Starch, 23: 73-79.

Kerr, R.W. and Cleveland, F.C., 1962. U.S. Patent No. 3.021.222. McComb, E.A. and McCready, R.M., 1957. Determination of acetyl in pectin and in acetylated

carbohydrate polymers. Hydroxamic acid reaction. Anal. Chem., 29: 819-821. Tessler, M.M. and Rutenberg, M.W., 1972. U.S. Patent No. 3.699.095.

A. Fuchs (Ed.) Inulin and Inulin-containing Crops © 1993 Else vier Science Publishers B.V. All rights reserved. 143

CYCLOINULO-OLIGOSACCHARIDES; STRUCTURE AND ENZYMATIC SYNTHESIS

T. UCHIYAMA Department of Biology, Osaka Kyoiku University, Tennoji-ku, Osaka 543, Japan

ABSTRACT

Cycloinulo-oligosaccharides, viz. 0-2,1-linked cyclofructohexaose, -heptaoseand -octaose, have been produced from inulin by the extracellular cycloinulo-oligosaccharidefructanotransferase of Bacillus circulans OKUMZ 3IB. The chemical structure of cycloinulohexaose has been determined by enzymatic, spectroscopic and Chromatographie analysis. The crystal structure of cycloinulohexaose and the conformation of the 18-crown-6 moiety were determined. The cycloinulo-oligosaccharidefructanotransferase was purified and its properties were investigated. The enzyme catalyses four reactions: cyclization, coupling, disproportionation and hydrolysis.

1 INTRODUCTION In 1970, we have started research work on the enzymatic degradation of fructans.

In the course of our studies, we found a novel enzyme, inulin fructotransferase (depolymerizing) (EC 2.4.1.93) (Uchiyama et al., 1973), which produces di-D-fructofuranose dianhydride III from inulin. Ever since, our efforts aimed at finding related enzymes and at elucidating the interesting molecular structure of the cyclic di-D-fructose. Since then, we have found three enzymes which either hydrolyse or produce difructose anhydrides, viz. a difructose anhydride III hydrolysing enzyme (Tanaka et al., 1975), a difructose anhydride I producing enzyme (Matsuyama et al., 1982) and a difructose anhydride IV producing enzyme (Tanaka et al., 1981). We have also elucidated, by single crystal X-ray diffraction, the molecular structures of difructose anhydride III (Taniguchi et al., 1982) and difructose anhydride II (Taniguchi et al., 1988).

Our studies led us to presume that another type of fructotransferase which would catalyse the formation of a cycloinulo-oligosaccharide from inulin might exist. In 1987, we isolated a bacterial strain which produced a new type of fructo-oligosaccharide from inulin. The bacterium has been identified as a strain of Bacillus circulans. The oligosaccharide was isolated from a culture broth by active charcoal column chromatography followed by Bio-gel

144

P4 gel filtration. The sugar was non-reducing and consisted solely of D-fructose. The molecular size suggested it to be a hexasaccharide. Whereas open-chain fructo-oligosaccharides in this molecular size range are readily attacked by the common ß-fructofuranosidase, the novel oligosaccharide was stable to exo-type enzymatic breakdown. However, using an endo-type inulinase of Aspergillus niger the oligosaccharide proved to be hydrolysed to inulohexaose, inulotetraose and inulobiose. From these results it became clear that the oligosaccharide had a cyclic structure.

2 CHEMICAL STRUCTURE OF CYCLIC FRUCTO-OLIGOSACCHARIDE After the necessary preparative methods had been improved the oligosaccharide was

obtained in a sufficiently large amount to determine its chemical structure. The oligosaccharide was non-reducing and consisted solely of D-fructose. FD-MS analysis showed it to have a molecular weight of 972 m/z; 995 (M + Na)+, in agreement with the molecular weight of D-fructohexaose anhydride. Elemental analysis yielded C, 42.04; H, 6.42%, which values agreed well with theoretical values for D-fructohexaose anhydride C36H60O30.3H20. To characterize the fructofuranosyl linkages in the molecule, methylation analysis was carried out. After acid hydrolysis, the partially methylated alditol acetate was subjected to GLC-MS. It showed peaks at m/z 234, 205, 190 and 161. These data indicated that methylation analysis of the oligosaccharide led to acetylated 2-deuterio-3,4,6-tri-0-

13

methylglucitol, which is consistent with a 2,1-linked cyclofructo-oligosaccharide. The C-NMR spectrum of the oligosaccharide showed six signals. The signal for the anomeric atom at 103.7 ppm is indicative of polymeric β-Ό- fructofuranosyl residues. The other five signals were assigned as follows: 82.3, 79.0 and 75.2 ppm are C-5, C-3 and C-4. The resonances at 63.3 and 61.4 ppm were assigned to C-6 and C-l, respectively. These data indicated that the extracellular enzyme produced cycloinulohexaose from inulin. The enzyme was arbitrarily designated as cycloinulo-oligosaccharide fructanotransferase (Kawamura et al., 1989).

3 CRYSTAL STRUCTURE OF CYCLOINULOHEXAOSE The crystal data for cycloinulohexaose are as follows: empirical formula, C36H60O30.

3H20; mol wt, 1026.9; trigonal space group R3 with hexagonal axes a = b = 34.688(17) Ä, c = 6.477(3) Ä, a = ß = 90.0°, y = 120.0°, Vol. = 3419(4) Ä3, Z = 3 molecules/ cell, Dc = 1.50 g cm3. A Rigaku AFC-5FOS four-circle diffractometer recorded 4871 reflections with 20 < 55° using graphite-monochromated Mo-Κα radiation. Of the 4871 recorded reflections, 1698 had F0> 3σ (F) and were used to solve the structure. One third of the molecule (an inulobiosyl unit with empirical formula C12H20O10. H20) was revealed by MULT AN 78. Block-diagonal least squares refinement with anisotropic temperature

145

Fig. 1. ORTEP drawing of cycloinulohexaose. (a) top view (H atoms solved are included); (b) side view (H atoms are excluded).

factors for non-hydrogen atoms converged successfully to R = 0.059. Eighteen of 22 hydrogen atoms were located through difference synthesis. The Fl and F2 fructofuranosyl moieties in the inulobiosyl unit (l-0-j3-D-fructofuranosyl jS-D-fructofuranose) have almost identical *% conformations. Fig. 1 is an ORTEP drawing of cycloinulohexaose. The torsion angle 01-Cl-C2-Or in the 18-crown-6 skeleton in Fl has a value of +52.3°, and the ΟΓ-Cl'-C2'-01 torsion angle in F2 is +163.4°. Therefore, the cyclohexaose molecule has an alternating, gauche-trans-gauche-trans-gauche-trans conformational arrangement for the six sequential -0-C-CH2-0- units. In this structure, the three fructofuranosyl residues F2, F4 and F6 are located on the upper side, and the other three fructofuranosyl residues Fl , F3 and F5 on the lower side of the 18-crown ring. Whereas the a-cyclodextrin (cyclomaltohexaose) has a somewhat cone-shaped structure, the cycloinulohexaose has an 18-crown-6 skeleton with a bowl-shaped structure. The upper side of the bowl is capped with the three 0-3' hydroxyl groups, and the bottom side of the molecule is lipophilic because the methylene groups of the gauche -0-C-CH2-0- units project downward (Sawada etaL, 1990).

4 CYCLOINULO-OLIGOSACCHARIDE FRUCTANOTRANSFERASE Cycloinulo-oligosaccharide fructanotransferase (CFT-ase) produces cycloinulohexaose

(CF6) as the main product from inulin. Simultaneously with CF6 a small amount of cycloinuloheptaose (CF7) and a trace of cycloinulo-octaose (CF8) are formed. The yields of CF6 and CF7 are 60 and 20%, respectively, based on inulin.

146

Fig. 2. Polyacrylamide disc gel electrophoresis of CFT-ase from B. circulans OKUMZ 3IB.

For enzyme preparation, the cultured medium of Bacillus circulans (Kawamura et al., 1989) was centrifuged and the supernatant solution was purified by column Chromatographie separation on DEAE-Toyopearl, Toyopearl HW65 and Sephacryl S-200. The enzyme was purified about 216-fold. The purified enzyme preparation showed a single protein band upon polyacrylamide gel electrophoresis as shown in Fig. 2.

The molecular weight of CFT-ase is 132 kDa, the pH optimum is at 7.0 to 7.5, the enzyme is stable in the pH range 5.5 to 8.0, and below 40 °C.

Fig. 3 shows the time course of product formation from inulin by CFT-ase, as revealed by thin-layer chromatography. First, CF6 and CF7 are formed, followed by various fructo-oligosaccharides. The data suggest that the CFT-ase, like the cyclodextrin glucanotransferase of Bacillus macerans, in addition to the cyclizing reaction catalyses many

Fig. 3. Pattern of inulin degradation by CFT-ase; IH, partial acid hydrolysate of inulin.

147

Fig. 4. Action of CFT-ase on some oligosaccharides.

side-reactions. Fig. 4 shows that beside inulin also fructo-oligosaccharides of the 1-kestose series such as nystose (GF3) and the corresponding pentaose (GF4) were attacked by CFT-ase. From the pentaose CF6 and CF7 were formed, while sucrose (GF) and 1-kestose (GF2) remained in the reaction mixture. When CF6 was incubated with the enzyme inulotetraose (F4), inulotriose (F3) and inulobiose (F2) were produced. These results indicate that the CFT-ase catalyses disproportionation and hydrolysis reactions. To make sure that the CFT-ase catalyses intermolecular transfructosylation reactions (coupling reactions) as well, GF, GF2, GF3 and GF4 were incubated together with CF6. The results indicated that CF6 was decyclized and the fhictohexaose residues formed were transferred to GF, GF2, GF3 and GF4, thus giving rise to GF7, GF8, GF9 and GF10, respectively. These results clearly show that CFT-ase also catalyses a coupling reaction.

5 CONCLUDING REMARKS Although the synthesis and structures of the cycloinulo-oligosaccharides have now

been elucidated, as have some properties of the responsible enzyme CFT-ase, very little is known about the potential usefulness of these compounds and the enzyme. Once the types of complexes and other intermolecular interactions are known, then a variety of uses is likely.

So far, we have found that CF6 forms a very specific complex with barium ions (Ba2+), whereas other divalent cations with almost the same ionic radius do not form complexes in aqueous solution. However, in aqueous methanol (MeOH/H20 1:1, v/v), some cations (K+, Rb+, Cs+, Ag+ and Pb2+) may show some interaction with cyclic fructans (Uchiyama et aL, 1992). The mechanism of complexation could be indicated by X-ray crystal structure analysis or NMR spectroscopy.

148

6 REFERENCES

Kawamura, M., Uchiyama, T., Kuramoto, T., Tamura, Y. and Mizutani, K., 1989. Formation of a cycloinulo-oligosaccharide from inulin by an extracellular enzyme of Bacillus circulans OKUMZ 31B. Carbohydr. Res., 192: 83-90.

Matsuyama, T., Tanaka, K., Mashiko, M. and Kanamoto, M., 1982. Enzymic formation of di-D-fructose 1,2'; 2,Γ dianhydride from inulobioseby Aspergillus fumigatus. J. Biochem. (Tokyo), 92: 1325-1328.

Sawada, M., Tanaka, T., Takai, Y., Hanafusa, T., Hirotsu, K., Higuchi, T., Kawamura, M. and Uchiyama, T., 1990. Crystal structure of cycloinulohexaose. Chem. Lett., 2011-2014.

Tanaka, K., Kawaguchi, H., Ohno, K. and Shoji, K., 1981. Enzymic formation of difructose anhydride IV from bacterial levan. J. Biochem. (Tokyo), 90: 1545-1548.

Tanaka, T., Uchiyama, T., Kobori, H. and Tanaka, K., 1975. Enzymic hydrolysis of di-D-fructoruranose 1,2';2,3' dianhydride with Arthrobacter ureafaciens. J. Biochem. (Tokyo), 78: 1201-1206.

Taniguchi, T., Sawada, M., Tanaka, T. and Uchiyama, T., 1988. Crystal structure of di-/S-D-fructofuranose 2\l:2,3,-dianhydride. Carbohydr. Res., 177: 13-20.

Taniguchi, T. and Uchiyama, T., 1982. The crystal structure of di-D-fructose anhydride III, produced by inulin D-fructotransferase. Carbohydr. Res., 107: 255-262.

Uchiyama, T., Kawamura, M., Uragami, T. and Okuno, H., 1992. Complexing of cycloinulo-oligosaccharides with metal ions. Carbohydr. Res. (in press).

Uchiyama, T., Niwa, S. and Tanaka, K., 1973. Purification and properties of Arthrobacter ureafaciens inulasell. Biochim. Biophys. Acta, 315: 412-420.


HYDROXYMETHYLFURFURAL, A POSSIBLE BASIC CHEMICAL FOR INDUSTRIAL INTERMEDIATES

Markwart KUNZ* Institut fur Landwirtschaftliche Technologie und Zuckerindustrie, Technische Universität Braunschweig, Langer Kamp 5, 3300 Braunschweig, Germany * Present address: Südzucker AG Mannheim/Ochsenfurt, Zentrallabor, 6718 Grünstadt, Germany

ABSTRACT

Fructose ex inulin can be readily converted to the basic chemical hydroxymethylfurfural (HMF). Due to its various functionalities HMF, in its turn, could be utilized to produce a wide range of chemical intermediates or end-products. Among the reactions possible, some are discussed to illustrate the potential to open up important fields of industrial application of these HMF-derived chemicals, for instance as polymers, surfactants, solvents, pharmaceuticals and plant protection agents.

In particular polymers seem to constitute a very interesting area of potential applications. Among these polymers, polyesters and polyamides, the latter being comparable with the terephthalic acid- and isophthalic acid-based polyamides Kevlar and Nomex, are worth mentioning. In addition, conducting polyene-like furan polymers seem to be promising, especially for their potential application in batteries, sensors and switches.

However, prerequisite for a substantial future role of HMF as a basic chemical is a low price. It means that, if fructose ex inulin should be used for HMF production, the price level for inulin should be roughly DM 1000 per ton.

1 INTRODUCTION The main raw materials for the organic chemical industry are by-products available

from refining crude oil, coal or gas for the energy market. Therefore, the raw material supply to the chemical industry is very sensitive to price fluctuations and availability on the energy market. To escape from this dependence, numerous investigations have been made to open up alternative sources of raw materials for the chemical industry, looking mainly for the possibilities of biomass from naturally occurring crops.

The main end-products of organic chemical industry are polymers, surfactants, plasticizers, dyes and pharmaceuticals. These end-products are normally produced in a stepwise reaction sequence, as follows:

150

raw materials, like naphta from oil refining, are converted to the basic chemicals, e.g. ethylene, these are then converted to so-called intermediates, e.g. ethyleneoxide, acetaldehyde or acetic acid, based on the intermediates the end-products, e.g. surfactants are made.

The same holds for the production of polymers like polyesters or polyamides.

Raw material

Basic chemical

Intermediate

Product

Petroleum (Naphta)

Ethylene

Ethylene oxide

Surfactants

Petroleum

Xylene

Terephthalic acid

Polymers

Biomass (Inulin)

HMF

Furan-dicarboxylic acid

Polymers

Fig. 1.

Applying similar reaction sequences (Fig. 1) to biomass, for instance inulin-containing chicory roots, as the raw material, one has to consider: a) Biomass is not available the year round and worldwide at favourable prices. Therefore, at first a developed industry, e.g. food industry, is required to convert, on a large scale, biomass to a cheap, storable and well-transportable product, for instance in the example chosen into inulin. b) Inulin could be converted by a lot of different known processes (Fig. 2) to the basic chemical hydroxymethylfurfural (HMF).

Fig. 2. Inulin

H7H20

151

The main aim of this paper is to show the potential of this possible basic chemical to be converted into chemical intermediates or end-products.

The HMF molecule has various functionalities (Fig. 3): a) the hydroxyl and the aldehyde groups at the exocyclic carbon atoms 1 and 6, b) the furan ring which makes HMF a heterocyclic compound with an aromatic character and a diene-like structure.

Fig. 3.

Instead of discussing all the different possibilities, only an overview will be given.

2 REACTIONS OF THE ALCOHOL FUNCTION (Fig. 4)

Fig. 4.

1. Esterification

R = -CH3, -CH2CH3, -CH2Ph

2. Dehydration - condensation of two HMF units

A hydroxyl group offers a lot of different reaction possibilities of which, in the case of HMF, for example the esterification with different types of organic acids could be important. Such products could be used as selective solvents in the chemical industry and moreover as antibacterial compounds in pharmaceuticals and foods.

Intermolecular dehydration of two HMF molecules leads to an "HMF dimer". Such a compound reacts as a "dialdehyde" and is a potential intermediate for surfactants, polymers and solvents.

152

3 HALOGENATION

Fig. 5. ΘΟ^. X = Cl, Br

Halogenated HMF derivatives are very important compounds for further organic reaction, e.g.:

1. Etherification

2. Amination

Η2Ν^ρ<ζ^ ^ ^ ^ Η

3. Reduction

HjC

4. Friedel-Crafts-reaction

5. Dimerization

\

Exchange of the hydroxyl group with halogens (Fig. 5) opens up important reaction sequences, for example to produce ethers or amines. Reduction of halogenated HMF yields methylfurfural. Friedel-Craft-reactions enable the synthesis of products containing aryl and heterocyclic aromatic ring systems. Important intermediates for pharmaceuticals or plant protection agents do contain such structural features.

153

4 REACTIONS OF THE ALDEHYDE FUNCTION

Fig. 6. HO

1. Reduction

HO- Λ OH

2. Reductive amination

>-~<>Q) Besides oxidation - to be discussed hereafter - reduction is possible (Fig. 6). The

product is e.g. 2,5-bis-(hydroxymethyl)furan. Important fields of application for such products could be once again in polymer chemistry or in the synthesis of surfactants or solvents.

Reductive amination is also possible. The resulting amines could be used as alkaline hydrophilic compounds for anionic surfactants, in shampoos or in corrosion inhibitors.

5 OXIDATION OF HMF

Fig. 7.

2,5-Furandicarboxylic acid 2,5-Furandicarboxaldehyde

Two oxidation products may especially be of future interest, viz. 2,5-furandicarboxylic acid and 2,5-furandicarboxaldehyde (Fig. 7). Furandicarboxylic acid in particular may be used for the synthesis of polymers. Examples will be given below.

154

DECARBONYLATION

Fig. 8. / — x

H O ^ - 4 : J ^C^H J V H -οκ^) ^ 0 '

HMF Furfural

- C0 2 \ / ♦ H2

Decarbonylation \ / Reduction

As the last reaction dealing with the exocyclic carbon atoms the decarbonylation of HMF and its conversion product, furfurylalcohol, should be mentioned (Fig. 8). However, it should be realized that this product can be even more easily made from furfural which is produced on the basis of very cheap agricultural by-products, e.g. of corn cobs. As a basic chemical, furfural has a big market of 100,000 tons per year. It is used as a solvent, and in the manufacture of resins, polymers, and pharmaceuticals. At the moment, the price of furfural is below DM 3000 per ton. HMF will not be cheap enough to compete with furfural in such applications.

7 HMF AS A HETEROCYCLIC COMPOUND WITH AROMATIC CHARACTER

Fig. 9. ^ ^ ^

The reactions discussed above dealt with the exocyclic carbon atoms. However, HMF has also a diene-like structure and an aromatic character (Fig. 9). A number of very important reaction sequences could make use of the reactivity of the quasi-aromatic ring system. The most important types of the reactions involving the ring structure are substitutions, especially halogenation, nitration and sulphonation, and Friedel-Crafts alkylation or acylation. Comparable reactions are being industrially applied at a large scale for other aromatic or heterocyclic compounds today. The diene-like structure enables HMF to react with dienophiles by Diels-Alder cycloaddition reactions which opens up another

155

wide field of synthesis possibilities. Halogenated aromatic compounds are very important industrial intermediates too.

Pharmaceuticals and plant protection agents very often contain heterocyclic structures but not always furan rings.

From HMF, a lot of different other types of cyclic or heterocyclic compounds could be prepared, for example, the carbo- and heterocyclic structures shown in Fig. 10.

Fig. 10.

XT Pyridine derivatives Pyrrole derivatives

HO-~^"3^R

Cyclopentenone derivatives Thiophene derivatives

8 HMF IN POLYMER CHEMISTRY Other possible roles of HMF are to be found in polymer chemistry. For instance,

HMF could be used in the production of resins, poly condensates and polyaddition products or in the synthesis of special polymers.

8.1 Resins The production of resins, for example based on formaldehyde and phenol together

with HMF results in resins of special types (Fig. 11).

Fig. 11. HMF

+ phenol + HCHO

or sulfonamide, ketones

phenolic resins

As compared to conventional resins such products are characterized by a) lower energy costs and a more convenient manufacturing process, and b) will result in a lower amount of residual monomers and reduces the phenol and formaldehyde content in the end-product.

156

As to potential application, the HMF-based resins are comparable to current commercial ones and could be used to make foundry products and laminate resins, or as adhesives, coating materials and binders.

8.2 Polycondensation products based on HMF

Fig. 12. / ^

Polyamides

o o - 0(CH2)2-0-^M^nJVÔ--(CH2)20

Polyesters

Theoretically, both reduction and oxidation products of HMF are possible intermediates for polyester formation. However, as far as known, the reduction products do not form polymers stable enough for commercial usage. Polymers based on furandicarboxylic acid, on the other hand, could be of commercial interest. Besides the polyesters, poly amides (Fig. 12) with aromatic amines received most interest in research. With respect to important application parameters like decomposition and glass temperature these products are comparable with the well-known terephthalic acid- and isophthalic acid-

Φ Φ based poly amides Kevlar and Nomex (Fig. 13).

Fig. 13.

=N K > ^ „ 4 decomp.

385-480

1 glass

325

Furan aramide

^>— HN-CO — ^ ^>—CO-NH-j- 5 5 0

Kevlar

340-360

- L ^ ^ ^ N H - CO ^ ^ . C O - N H « \xy xy 370 280-290

Nomex

157

The problem for industrial application of such a product is mainly the price level of furandicarboxylic acid as compared with that of terephthalic acid and isophthalic acid, which now cost less than DM 3000 per ton.

8.3 Special polymers In addition to those already mentioned, numerous other special polymers, e.g.

urethanes or Diels-Alder products, are known. A very new but rapidly expanding area is the field of conducting polymers.

Fig. 14. o 0, S, NH

One important condition for polymers to be conducting is a polyene-like structure. For such products, HMF could be the basic raw material. Application possibilities for conducting polymers are in batteries, sensors and switches.

Although only a few examples of the possible usage of HMF in polymer chemistry have been given, one may assume that in the future HMF may play an important role in polymer chemistry. One of the main problems up till now is to find unique application niches for HMF-based polymers with acceptable prices. Another problem is the instability of HMF. On one hand, the high reactivity is a disadvantage, on the other hand an advantage. In fact, the chemical instability is the real basis for the reaction possibilities discussed. However, depending on the nature of substitution, in some applications HMF-based derivatives are not stable enough to become commercial products. For example, the furan ring structure is to some extent sensitive to oxidation, especially to peroxide formation. On the other hand, this phenomenon may offer a chance for the production of better degradable products, especially polymers.

9 PHARMACEUTICALS Another important field of industrial organic chemistry is the production of

pharmaceuticals. One of today's most important industrially made pharmaceuticals is the

158

product named Zantac from Glaxo. This is an anti-ulcer drug and was the first prescription drug to exceed US $ 109 annual sales. It contains the structural feature of HMF, but is produced at present from furfural.

Fig. 15. Possible Way Usual Way

0

NH I CH3

The traditional synthetic pathway starts with reduction of furfural to furfurylalcohol (Fig. 15) followed by a Mannich-type addition reaction. The same intermediate is available using the reductive amination of HMF in a one-step reaction. This reaction would be cheaper in energy demand, more economic and may be higher in yield. However, the price difference between HMF and furfural still limits the usage of HMF in this big pharmaceutical production.

10 THE ECONOMICS OF HMF For their potential as an industrial raw material or basic chemical, respectively, the

prices of inulin and HMF, in comparison with those of competitive raw materials and basic chemicals are of paramount importance.

The prices for naphta and ethylene have been fluctuating in the past at levels between DM 300 and DM 800. To build up an industry on the basis of biomass as raw material which is competitive with such low-priced raw materials may be difficult.

Because inulin is claimed to be a raw material for fructose production it must be cheaper than fructose. At the moment, the price of fructose is below DM 2000 per ton. Therefore, if inulin is to be used for fructose production and is to be a large-scale product, the price level must be roughly DM 1000 per ton. Though, with this price level for products based on inulin like HMF, a big cost problem is the relatively low yield of about half a kilo HMF per kg inulin. That means, the raw material costs for HMF based on inulin are in the range of DM 2000 per ton. At such a low yield and without a possible utilization

H O — O

HO ^ y N^ C " 3

CH,

-s O — H<™3

^ Ο ^ Θ CH3

159

of the by-products (such as humic acids), the waste disposal will cause additional costs. That means the marketing price for HMF will have to be at least DM 5000 or - in the beginning of a marketing phase - more realistically DM 10,000 per ton. That is very expensive for a large-scale industrial product. Moreover, the price level will depend very much on the purity of HMF required for further chemical processing steps. Because HMF is sensitive to oxidation, polymerization or self-condensation as mentioned above, it is only storable for longer periods as pure crystalline compound at or below ambient temperatures. Therefore, a better and cheaper possibility for the production of end-products based on HMF very often will be to produce stable intermediates like the ones discussed above without the recovery of HMF as such.

11 CONCLUSIONS HMF has unique chemical properties. Depending on the kind of substitution HMF-

derived products are more or less stable. HMF-based products could be used for the production of a. polymers, b. surfactants, c. solvents, and d. pharmaceuticals.

To enhance the chances for HMF as an industrial basic chemical there is a need for more research and developmental work to find special applications for HMF-derived products.

However, prerequisite for a substantial future role of HMF as a basic chemical is a low price, for

at a low price - HMF will have a big market, at a high price - HMF will have a small market.

12 REFERENCES FOR FURTHER INFORMATION ON HMF

Elming, N. and Clauson-Kaas, N., 1956. Transformation of 2-(hydroxymethyl)-5-(aminomethyl)-fliran into 6-methyl-3-pyridinol. Acta Chem. Scand., 10: 1603-1605.

Faury, A., Gaset, A. and Gorrichon, J.-P., 1981. Reactivite et valorisation chimique de l'hydroxymethyl-5-furannecarboxaldehyde-2. Inf. Chim., 214: 203-209.

Gandini, A., 1977. The behaviour of furan derivatives in polymerization reactions. Adv. Polym. Sei., 25: 47-96.

Gandini, A., 1986. Furan polymers. Encycl. Polym. Sei. Eng., 2nd ed., Vol. 7. Interscience Publishers, New York, pp. 454-473.

Kharchenko, V.G., Gubina, T.I., Voronin, S.P. and Markushina, I.A., 1983. About the reaction of the conversion of furans into thiophenes and selenophenes. In: I. Kovac (Ed.), Topics Furan Chem., Proc. 4th Symp. Furan Chem., Slovak Tech. Univ. Bratislava, pp. 142-145.

Koch, H., Krause, F., Steffan, R. and Woelk, H.U., 1983. Herstellung von Phenolharzen unter Verwendung von Stärkeprodukten. Starch/Stärke, 35: 304-313.

Mitiakoudis, A., Gandini, A. and H. Cheradame, 1985. Polyamides containing furanic moieties. Polymer

160

Comm., 26: 246-249. Moore, J.A. and Kelly, J.E., 1978. Polyesters derived from furan and tetrahydrofuran nuclei.

Macromolecules, 11: 568-573. Moore, J.A. and Kelly, J.E., 1979. Polymerization of furandicarbonyl chloride with bisphenol A poly(2,5-

furandiylcarbonyloxy-1,4-phenylenedimethylmethylene-l ,4-phenyleneoxycarbonyl). Polymer, 20: 627-628.

Piancatelli, G., Scettri, A., David, G. and D'Auria, M., 1978. A new synthesis of 3-oxocyclopentenes. Tetrahedron, 34: 2775-2778.

Snyder, F.H., 1957. U.S. 2.804.445, Dendrol Inc. Snyder, F.H., 1959. U.S. 2.875.180, Dendrol Inc. Stone, M., 1987. Keeping pace with surging market. Performance Chem. 1987 (Aug.): 50, 52. Zech, J.D., 1986. U.S. Patent 3,392,148.


ENZYMATIC SYNTHESIS OF HYDROXYMETHYLFURFURAL ESTERS

A.C. BESEMER, J.P. van der LUGT and H.J. DODDEMA TNO, Netherlands Organization for Applied Scientific Research, Nutrition and Food Research, Department of Biotechnology, P.O. Box 360, 3700 AJ Zeist, The Netherlands

ABSTRACT

In the framework of our investigations concerning the preparation of detergents from renewable resources we have considered the possibility of using hydroxymethylfurfural (HMF) as a substrate. This compound can be esterified, e.g. with stearic acid, resulting in a new compound with a hydrophilic and a hydrophobic part. Since HMF is susceptible to degradation during chemical transformation, enzymatic conversion is desirable. Starting with vinyl esters the HMF-esters can be readily synthesized by transesterification in tetrahydrofuran using a lipase. In view of the easy synthetic procedure, large-scale preparation is likely to be feasible.

1 INTRODUCTION There is an increasing demand for non-ionic detergents, which preferentially should

be prepared from renewable resources. A possible pathway is the derivatization of carbohydrates (hydrophylic part) with a long alkyl chain (hydrophobic part). Several compounds have been developed such as alkyl glucosides and sucrose palmitate and stearate. A new and cheap carbohydrate is inulin. Because it is expected to be available in bulk quantities in the near future, also derivatives of inulin might be of interest as starting material. A suitable derivative in this respect is hydroxymethylfurfuraldehyde (HMF, see Fig. 1). This compound has a hydrophylic character and may be easily converted e.g. by esterification with a long-chain fatty acid. If desired, further transformation is possible by

.o

\ XT + H XC=c/H jj > \ Γ 1 + H3C—/

o II

Fig. 1. The transesterification reaction of HMF with vinyl acetate (R' = R - C -).

162

reaction of the aldehyde group. Since the molecule is susceptible to degradation during chemical transformation, we have studied the possibility of an enzymatic synthesis of short-and long-chain alkyl-HMF-esters. Because of the solubility of HMF in organic solvents, lipase is used for the transesterification reaction.

2 EXPERIMENTAL 2.1 Materials

HMF has been prepared in our laboratory. The melting point of this red-brown substance is 30-31 °C (lit. 31.5 °C). The structure has been confirmed by NMR spectroscopy. As acyl donors butyl acetate (Merck) and vinyl acetate (Fluka) have been used. The latter compound has the advantage that the primary product, vinyl alcohol, rearranges to acetaldehyde. Consequently, the reverse reaction will not take place. Three lipases have been investigated: pig pancreas lipase, type II (Sigma), Pseudomonas fluorescens lipase (Hoechst), and subtilisin from Bacillus subtilis (Boehringer). Vinyl stearate was a gift from Hoechst AG. The solvents used were tetrahydrofuran (THF) and pyridine (Merck).

2.2 Methods The reaction was followed by means of TLC on DC-Alufolien Kieselgel 60 F2$4

(Merck) with diethylether as eluant. Spots were detected by UV-light (254 nm) and I2-vapour. The structure of the products obtained were confirmed by NMR spectroscopy (100 MHz, C-13 and 400 MHz, H-l) on a Varian VXR-400.

The reactions were carried out in a closed vessel, kept at a constant temperature of 40 °C and stirred magnetically.

3 RESULTS 3.1 Small-scale experiments

In a number of experiments 100 mg lipase, 100 mg HMF, 0.45 ml solvent and 0.45 ml butyl acetate or vinyl acetate were mixed and incubated during 17 h. The reaction was followed using thin-layer chromatography. In Fig. 2 the chromatograms of the reaction mixtures are shown (THF and pyridine as solvent, butyl acetate and vinyl acetate as acyl donor and several types of enzymes, respectively). Prolonged incubation of the reaction mixture did not change the pattern of the chromatograms.

It is obvious that the best results were obtained with lipase from P. fluorescens (see spots A2-S, A2-H and A2-E). Subtilisin gave only poor results. As expected, vinyl acetate proved to be a suitable acyl donor in both solvents (compare e.g. Al-S with A2-S, Al-H with A2-H, or Bl-H with B2-H). Both solvents considered can be used (see A2-H and B2-

163

H, in which the conversion is complete) but in connection with the bad smell of pyridine and the volatility of THF the latter solvent is preferred.

3.2 Preparation of HMF-acetate In 25 ml THF and 25 ml vinyl acetate, 5.11 g HMF was dissolved; the mixture was

incubated with 1 g lipase {P. fluorescens) during 16 h at 40 °C. Thin-layer Chromatographie analysis showed that the conversion was complete and that indeed HMF-acetate was formed. The lipase was removed by centrifugation (9000 g). After removal of the solvent, about 5 g (80%) of a yellow compound was obtained. Its structure was confirmed by NMR spectroscopy. The melting point was 54-55 °C (lit. 54.5 °C).

σ O o O O Q 0 o o o 0

Al-S A2-S Bl-S B2-S Al-H A2-H

o o ° o o o o o o o o

Bl-H B2-H Al-E A2-E Bl-E B2-E blank 1 blank 2

Fig. 2. Thin-layer chromatograms of reaction mixtures (see text). Variables were: solvents used: A = THF, B = pyridine; acyl donors used: 1. butyl acetate, 2. vinyl acetate; and sources of lipase used: E = subtilisin, H = Pseudomonas fluorescens lipase, and S = porcine pancreas lipase. The controls ("blanks") used were 1. HMF in THF, and 2. HMF in pyridine.

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3.3 Preparation of HMF-stearate In 3 ml THF 266 mg HMF (2.12 mmoles) and 658 mg vinyl stearate (2.12 mmoles)

were dissolved. After addition of 100 mg P. fluorescens lipase the mixture was incubated at 40 °C. It appeared from TLC that after 24 h reaction time HMF was converted completely. To avoid crystallization upon cooling of the HMF-stearate 8 ml THF was added. Then, the mixture was centrifuged and the supernatant solution concentrated. The precipitated ester was collected and recrystallized from THF and dried. The yield was 150 mg; the melting point of this faint yellow substance was 74.5-75 °C. The structure was confirmed by NMR spectroscopy (see Fig. 3). From the mother liquor an additional fraction of about 300 mg product was isolated.

4 CONCLUSION It is possible to prepare HMF-esters in an organic medium with commercially

available Upases by a transesterification process. The most suitable acyl donors are vinyl esters; the reverse reaction does not occur because the primary product (vinyl alcohol) rearranges resulting in the formation of acetaldehyde. The preparation proceeds in a simple way and the reaction mixture consists of the desired ester only. Therefore and because of the simplicity of enzyme removal scaling-up seems feasible. Further research is needed for the characterization of the compounds as detergents. Derivatization is possible by chemical reactions using the aldehyde group of the various compounds.

BIOCHEMISTRY, MICROBIOLOGY AND MOLECULAR BIOLOGY



UNRESOLVED PROBLEMS IN THE ENZYMOLOGY OF FRUCTAN METABOLISM

CJ . POLLOCK, A.M. WINTERS and A.J. CAIRNS AFRC-IGER, Welsh Plant Breeding Station, Aberystwyth, Dyfed SY23 3EB, Wales, U.K.

ABSTRACT

The evidence for the participation of sucrose as the initial fructosyl donor for fructan synthesis in higher plants is reviewed. The current theories of fructan enzymology suggest that fructosyl transfer from sucrose to fructans and their hydrolysis to yield free fructose are both mediated by specific enzymes each catalysing a single reaction. The possibility is discussed that enzymes may catalyse multiple reactions, and that the balance between these reactions may be altered by pH, temperature, substrate concentrations and the chemical activity of water. Evidence is presented that such effects can be observed in studies on the purified 0-fructofuranosidase from yeast, which does not accumulate fructan in vivo. The importance of compartmentation in regulating the environment around such enzymes is also considered.

1 INTRODUCTION Fructans are the most widespread members of the classes of oligo- and

polysaccharides apparently derived from sucrose without the direct intervention of sugar nucleotides or phosphorylated intermediates. They are thought to occur in about 10% of the world flora (Hendry, 1987). There is direct evidence for the central role of sucrose in fructan synthesis in Gramineae (Pollock, 1979) and in Asterales (Dickerson and Edelman, 1966; Pontis, 1970). Strong circumstantial evidence exists for other accumulators (Pontis and Del Campillo, 1985; Pollock and Chatterton, 1988). Although all fructans may be synthesized from a common precursor, the final products differ markedly in both size and structure (Pollock and Chatterton, 1988). In this review we consider the enzymology of fructan synthesis and the extent to which current models can account for synthesis of the structures observed in vivo.

2 THE ENZYMES OF FRUCTAN METABOLISM 2.1 Studies on the tubers ofHelianthus tuberosus L.

The fructans of H. tuberosus consist essentially of a homologous series of linear

168

oligosaccharides containing fructose residues bonded in the 0-2,1 -position. They terminate in a parent sucrosyl moiety. A model for the synthesis and breakdown of fructan in H. tuberosus was proposed by Edelman and Jefford (1968). They suggested that synthesis proceeded via the concerted action of two fructosyltransferases, with sucrose as the primary fructosyl donor, sucrose:sucrose fructosyltransferase (SST, EC 2.4.1.99) (1) G-F + G-F > G-F-F + G fructan:fructan fructosyltransferase (FFT, EC 2.4.1.100) (2) G-F-(F)n + G-F-(F)m τ± G-F-(F)n+1 + G-F-OF)^

The donor and acceptor specificities of FFT were considered to be such that continuous production of the trisaccharide 1-kestose by SST generated fructosyl donors for FFT and thus permitted the progressive elongation of acceptor fructan chains. Fructan breakdown was thought to be catalysed by a specific fructan exohydrolase (FEH, EC 3.2.1.80). All of these activities can be measured in vitro and attempts at purification were made (Edelman and Jefford, 1964; Edelman and Dickerson, 1966; Scott, 1968). The purification entailed differential salt precipitation and ion exchange chromatography. It seems unlikely that the preparations were homogeneous and the properties should, therefore, be viewed with caution (Pollock and Cairns, 1991). The salient properties of the SST as proposed by Edelman and Jefford (1968) were that it was irreversible (i.e. it would not catalyse the cleavage of sucrose), that it produced only a single trisaccharide under the assay conditions employed and that it had an apparent Km for sucrose of 57 mM (Scott et al., 1966). By its nature, FFT catalysed both chain elongation and chain shortening, although sucrose could only function as an acceptor. Thus, this enzyme also would not apparently catalyse sucrose hydrolysis. The exohydrolase from H. tuberosus was also inactive against sucrose (Edelman and Jefford, 1968). If these properties, measured in vitro in partially purified extracts, reflect the true properties of the enzymes in vivo, then all three enzymes appear to catalyse distinct reactions in which sucrose can only function either as a fructosyl donor (SST) or as an acceptor (SST and FFT) but where there is no competing hydrolytic activity against sucrose.

2.2 Fructan-metabolizing enzymes in Asparagus officinalis L. In roots of A. officinalis, three synthetic activities have been purified, each of which

yields a single band on native PAGE following staining with amido black (Shiomi, 1989 and references therein). The SST activity would not hydrolyse sucrose under the conditions employed in vitro and catalysed trisaccharide synthesis with an apparent Km for sucrose of 110 mM. In addition, this preparation catalysed fructosyl transfer from sucrose to higher oligosaccharides of the neokestose series. An FFT activity (designated 1-F FT) catalysed

169

fructosyl transfer to the C1 position of a fructose moiety in a manner analogous to the FFT described from H. tuberosus. A second FFT activity (6-G FT) catalysed reversible fructosyl transfer to the C6 position of a glucose moiety. The concerted action of these three enzymes led to the synthesis of a range of low-molecular-weight isomeric oligosaccharides similar to those which occur in vivo (Shiomi, 1989). Natural fructans from A. officinalis do, however, include high-molecular-weight forms (Shelton and Lacy, 1980; Cairns, 1992). As in the studies on H. tuberosus (Edelman and Jefford, 1968), it was proposed that these contrasting activities resided on different separable proteins, that none of these enzymes were capable of hydrolysing sucrose and that all three could be purified free from invertase activity. At the time of writing, these studies on H. tuberosus and A. officinalis represent the only attempts to purify and fully characterize all the enzymes responsible for fructan synthesis in a particular species. In neither case have modern criteria for protein purity been fully met (Pollock and Cairns, 1991).

2.3 Fructosyltransferase activities in temperate Gramineae Based on measurements of the in vitro activities isolated from leaves of temperate

grasses, we conclude that it is difficult to assign unambiguously a specific individual enzyme activity to crude or partially purified protein preparations. We suggest that this is because the reactions of fructan metabolism and of sucrose hydrolysis are similar (Table 1). Under the conditions we use, in vitro trisaccharide synthesis from sucrose alone only occurs at non-physiological sucrose concentrations and does not produce the range of oligosaccharides observed in vivo (Cairns et al., 1989; Pollock and Cairns, 1991). In the presence of acceptor fructans, however, transfer of radioactivity from [U-14C]-sucrose occurs at low sucrose concentration and results in the labelling of the entire range of structures observed in vivo. It is not yet known whether or not this is accompanied by net synthesis of high-molecular-weight fructan (Cairns, 1989; Pollock and Cairns, 1991).

When net fructose transfer is measured using crude leaf extracts of Lolium temulentum L., we observe that the ratio of sucrose hydrolysis to fructosyl transfer, together with the identity of the products of such transfer, is sensitive to pH, temperature and sucrose concentration (Cairns et al., 1989; Pollock et al., 1989). Such variability has been used as evidence for the presence of distinct invertase and SST activities in leaves of barley (Wagner and Wiemken, 1987). We believe that the experiments describing separable monofunctional activities have not been performed under a wide enough range of assay conditions to demonstrate this property unequivocally. It should be noted that the enzymes involved in bacterial fructan synthesis exhibit multiple activities and may be capable, dependent upon the assay conditions, of catalysing all the reactions shown in Table 1 (Dedonder, 1972).

170

Table 1. The reactions of fructan metabolism in plants.

Type reaction RÔ-R2 + R3-0-R4 ±? RÔ-R4 + R3-0-R2

Fructan hydrolysis F(n)"0"H + H-O-H *± F(n.1}-0-H + F-O-H Sucrose hydrolysis G-O-F + H-O-H ^ G-O-H + F-O-H Trisaccharide synthesis GF-O-H + G-O-F ** GF-O-F + G-O-H Fructosyl transfer Fn-0-F + Fm-0-F <* F(n+1)-0-F + F(m_irO-F

2.4 Studies on purified yeast ß-fructofuranosidase (invertase; EC 3.2.1.26) It is known that a single purified enzyme from baker's yeast (an organism which does

not accumulate fructan in vivo) is capable of catalysing all the reactions shown in Table 1 (Pollock and Cairns, 1991 and references therein). This indicates that at least some eukaryotic proteins do have innate multifiinctionality. The factors which determine the ratio of transferase to hydrolase activity have been described (Cairns and Ashton, 1991). The transferase reaction showed a Km of 224-308 mM, which is within the range reported for higher plant SST (Pollock, 1986). This contrasted with the hydrolytic activity which had a Km of 11-34 mM. Hydrolase and transferase activities could be distinguished on the basis of response to temperature and pH. The differences observed were similar to those reported for comparable activities extracted from grass leaves. These were assigned to different proteins (Wagner et al., 1983; Chatterton et al, 1988) (Table 2). Our data suggests that differences in pH and temperature optima are not necessarily associated with the presence of distinct and separable proteins, each catalysing only one activity. Other studies on yeast invertase (Selisko et al., 1990) and on bacterial levansucrase (Chambert and Petit-Glatron, 1989) have indicated the potential role of the chemical activity of water in regulating fructosyl transfer in multifunctional systems. As yet, no consideration has been made of the importance of this effect in vivo, although it has been argued that fructan synthesis in H.

Table 2. Contrasting kinetic parameters of hydrolase and transferase activities from different sources.

Source

Purified yeast invertase*

Barley leaf extract0

Agropyron leaf extract0

Activity

hydrolase transferase



pH optimum

4.8 5.2

5.7 5.2

5.0-5.5 4.6

Temperature optimum (°C)

59 50

28 45

30 50

(Sue; mM)

11- 34 224-308

not reported

100 15

a Data from Cairns and Ashton (1991). b Data from Wagner et al. (1983). c Data from Chatterton et cd. (1988).

171

tuberosus occurs in microvesicles (Kaeser, 1983). Here, the chemical activity of both sucrose and water could be radically different from that pertaining in the central vacuole and consequently might facilitate the transferase activities of multifunctional enzymes which would assay as hydrolases in vitro under more dilute conditions.

3 CONCLUDING REMARKS There is compelling, if not entirely conclusive, evidence that fructan synthesis in

higher plants occurs by successive fructosyl transfer from sucrose without the direct mediation of enzymes using phosphorylated or nucleotide-linked sugar derivatives.

Although there are two detailed studies, among others, which suggest that a number of separable fructosyltransferase/hydrolase enzymes are involved in fructan metabolism, we suggest that none of these enzymes has been purified to homogeneity and shown unequivocally to be monofunctional under a wide range of assay conditions.

There is good evidence that multifunctional enzymes do exist which can catalyse the reactions of fructan metabolism. Their importance may be considerable, particularly in grasses and cereals, but has yet to be firmly established.

Compartmentation of fructan metabolism in the vacuole of higher plants is generally accepted (Matile, 1987). The effects of smaller-scale inhomogeneities in concentration of water, sucrose and enzyme proteins need to be considered more carefully.

4 ACKNOWLEDGEMENTS We gratefully acknowledge support from NATO (CRG 0706/87) and AFRC (PG

11/52).

5 REFERENCES

Cairns, A.J., 1989. Fructan biosynthesis in excised leaves of Loliwn temulentwn L. IV. Cell-free 14C labelling of specific oligofructans at low sucrose concentration. New Phytol., 112: 465-473.

Cairns, A.J., 1992. A reconsideration of fructan biosynthesis in storage roots of Asparagus offlcinalis L. New Phytol. 120: 463-473.

Cairns, A.J. and Ashton, J.E., 1991. The interpretation of in vitro measurements of fructosyl transferase activity: an analysis of patterns of fructosyl transfer by fungal invertase. New Phytol., 118: 23-34.

Cairns, A.J., Winters, A. and Pollock, C.J., 1989. Fructan biosynthesis in excised leaves of Loliwn temulentwn L. III. A comparison of the in vitro properties of fructosyl transferase activities with the characteristics of in vivo fructan accumulation. New Phytol., 112: 343-352.

Chambert, R. and Petit-Glatron, M.-F., 1989. Study of the effect of organic solvents on the synthesis of levan and the hydrolysis of sucrose by Bacillus subtilis levansucrase. Carbohydr. Res., 191: 117-123.

Chatterton, N.J., Harrison, P.A., Thornley, W.R. and Bennett, J.H., 1988. Characterization of sucrose: sucrose fructosyltransferase from crested wheatgrass. New Phytol., 109: 29-33.

Dedonder, R., 1972. Role and mechanisms of transglycosylation reactions. In: R. Piras and H.G. Pontis (Eds.), Biochemistry of the Glycosidic Linkage. Academic Press, New York, pp. 21-78.

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Dickerson, A.G. and Edelman, J., 1966. The metabolism of fructose polymers in plants VI. Transfructosylation in living tissue of Helianthus tuberosus L. J. Exp. Bot., 17: 612-619.

Edelman, J. and Dickerson, A.G., 1966. The metabolism of fructose polymers in plants. Transfructosylation in tubers of Helianthus tuberosus L. Biochem. J., 98: 787-794.

Edelman, J. and Jefford, T.G., 1964. The metabolism of fructose polymers in plants 4. ß-Fructofuranosidases of tubers of Helianthus tuberosus L. Biochem. J., 93: 148-161.

Edelman, J. and Jefford, T.G., 1968. The mechanism of fructosan metabolism in higher plants as exemplified in Helianthus tuberosus. New Phytol., 67: 517-531.


Kaeser, W., 1983. Ultrastructure of storage cells in Jerusalem artichoke tubers (Helianthus tuberosus L.). Vesicle formation during inulin synthesis. Z. Pflanzenphysiol., I l l : 253-260.

Matile, P., 1987. The sap of plant cells. New Phytol., 105: 1-26. Pollock, C.J., 1979. Pathway of fructosan synthesis in leaf bases of Dactylis glomerata. Phytochemistry,

18: 777-779. Pollock, C.J., 1986. Fructans and the metabolism of sucrose in vascular plants. New Phytol., 104: 1-24. Pollock, C.J. and Cairns, A.J., 1991. Fructan metabolism in grasses and cereals. Annu. Rev. Plant Physiol.

Plant Mol. Biol., 42: 77-101. Pollock, C.J., Cairns, A.J., Collis, B.E. and Walker, R.P., 1989. Direct effects of low temperature upon

components of fructan metabolism in leaves of Lolium temulentum L. J. Plant Physiol., 134: 203-208.

Pollock, C.J. and Chatterton, N.J., 1988. Fructans. In: J. Preiss (Ed.), The Biochemistry of Plants, A Comprehensive Treatise, Vol.14, Carbohydrates. Academic Press, Inc., San Diego, pp. 109-140.

Pontis, H.G., 1970. The role of sucrose and fructosylsucrose in fructosan metabolism. Physiol. Plant., 23: 1089-1100.

Pontis, H.G. and Del Campillo, E., 1985. Fructans. In: P.M. Dey and R.A. Dixon (Eds.), Biochemistry of Storage Carbohydrates in Green Plants. Academic Press, London, pp. 205-227.

Scott, R.W., 1968. Transfructosylation in higher plants containing fructose polymers. Ph.D. Thesis, University of London.

Scott, R.W., Jefford, T.G. and Edelman, J., 1966. Sucrose fructosyltransferase from higher plant tissues. Biochem. J., 100: 23P-24P.

Selisko, B., Ulbrich, R., Schellenberger, A. and Müller, U., 1990. Invertase-catalyzed reactions in alcoholic solutions. Biotechnol. Bioeng., 35: 1006-1010.

Shelton, D.R. and Lacy, M.L., 1980. Effect of harvest duration on yield and on depletion of storage carbohydrates in asparagus roots. J. Am. Soc. Hortic. Sei., 105: 332-335.

Shiomi, N., 1989. Properties of fructosyltransferases involved in the synthesis of fructan in liliaceous plants. J. Plant Physiol., 134: 151-155.


Wagner, W. and Wiemken, A., 1987. Enzymology of fructan synthesis in grasses. Properties of sucrose-sucrose-fructosyltransferase in barley leaves (Hordeum vulgäre L. cv Gerbel). Plant Physiol., 85: 706-710.

A. Fuchs (Ed.) Inulin and Inulin-coniaining Crops 1993 Elsevier Science Publishers B.V. 173

PURIFICATION AND PROPERTIES OF SUCROSE:SUCROSE FRUCTOSYLTRANSFERASES FROM BARLEY LEAVES AND ONION SEEDS

G.C. ANGENENT, M.J.M. EBSKAMP, PJ . WEISBEEK and J.C.M. SMEEKENS Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3583 CH Utrecht, The Netherlands

ABSTRACT

Fructan biosynthesis in primary leaves of barley {Hordeum vulgäre L. cv. Gerbel) was induced by continuous illumination of excised leaves. No fructans were synthesized during the first 8 h of induction, although enzymes were present which were able to synthesize 1-kestose from sucrose. In this first period no fructan:fructan fructosyltransferase (FFT) activity was detectable in an in vitro assay. Fructan accumulation occurred simultaneously with the appearance of FFT activity. Using FPLC ion exchange chromatography we were not able to separate a sucrose:sucrose fructosyltransferase (SST) activity from the hydrolytic activity caused by acid invertase. Km's of 10 mM and 300 mM were found for the hydrolytic and fructosyltransferase activity, respectively. Both activities were inhibited to the same extent by addition of pyridoxal or pyridoxin. These results provide additional evidence that both activities in barley could be the result of only one enzyme. A distinct enzyme with only SST activity was isolated from onion seeds. The enzyme had a molecular weight of 68,000, a pH optimum of 5.5 and a Km of 250 mM.

1 INTRODUCTION Various species of Gramineae, but also members of the Asterales and Liliales,

accumulate large amounts of fructan (polyfructosylsucrose), serving as an important reserve carbohydrate in these species (for reviews see Nelson and Smith, 1986; Pollock and Chatterton, 1988). Fructans can be stored in sink as well as in source tissue, depending on environmental conditions and developmental stage. In sink tissue like bulbs and roots of the Liliales (Shiomi et al., 1979; Shiomi, 1989) or tubers of Helianthus tuberosus L. (Edelman and Jefford, 1968) fructans serve as a long-term storage carbohydrate, while in developing seeds fructans are more temporary intermediates between sucrose and starch (Housley and Daughtry, 1987). Accumulation of assimilates in photosynthetically active tissue results in the synthesis of fructans. In the primary leaves of barley, fructans may account for up to 70% of the dry weight (Wagner et al., 1983). Fructan biosynthesis in barley can be induced by illumination of excised leaves or by feeding detached leaves with several sugars (Wagner

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et al, 1983; Wagner and Wiemken, 1986). These results demonstrate that photosynthates can act both as substrate (sucrose) and as inducer for fructan biosynthesis in leaves. Consequently, fructan biosynthesis is closely related to the synthesis and partitioning of photosynthetic products.

Fructan biosynthesis in plants is the result of the sequential action of at least two enzymes. Sucrose:sucrose fructosyltransferase (SST, EC 2.4.1.99) catalyses the synthesis of a trisaccharide and glucose from two molecules of sucrose (Scott, 1968). Although several types of trisaccharides are found in nature, 1-kestose is thought to be the main product of SST. Several groups have reported the partial or complete purification of SST from a number of sources (for reviews see Pollock, 1986; Praznik et al., 1990). Studies on the properties of SST have mainly been done on partially purified SST. SST has a molecular weight of 65-70 kDa and a pH optimum between 5.0 and 5.7, which is consistent with a vacuolar localization of the SST activity (Wagner et al., 1983). Comparison of the Km

values for sucrose shows a large variation, probably due to contamination of the various SST preparations with invertase (EC 3.2.1.26).

The second enzyme involved in fructan biosynthesis in plants is fructan:fructan fructosyltransferase (FFT, EC 2.4.1.100). This enzyme catalyses the transfer of fructosyl residues from one fructan molecule to another. In addition, it can utilize sucrose as a fructosyl acceptor but not as a fructosyl donor (Edelman and Dickerson, 1966). FFT is not only involved in chain elongation, but is probably the key enzyme responsible for the structural diversity of fructans occurring in nature (Shiomi, 1989). 6-Kestose and neokestose, which accumulate in barley (Wagner and Wiemken, 1987) and onion bulbs (Shiomi, 1989), respectively, can be synthesized by the action of FFT using 1-kestose as a donor and sucrose as an acceptor.

Recent studies with Lolium temulentum L. support the notion that the mechanism for fructan biosynthesis in plants might be more complicated than the SST/FFT model described above (Cairns et al., 1989). In addition to their hydrolytic activity, invertases are able to catalyse fructosyl transfer yielding all three isomers of monofructosylsucrose (Straathof et al., 1986; Ivin and Clarke, 1987; Pollock et al, 1989). Although this bifunctional activity is known for some time, the role of invertase in fructan biosynthesis is not clear and might be underestimated.

To study the possible role of invertase in fructan biosynthesis, we analysed the kinetics of a partially purified invertase from induced primary leaves of barley. Here, we report new evidence supporting the view that acid invertase may account for sucrose:sucrose fructosyl transfer in barley. In addition, we isolated SST from onion seeds which was free from detectable hydrolytic activity. A new purification protocol was developed which was less time-consuming than a purification protocol previously used by Shiomi et al. (1985).

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Features of this enzyme preparation were in agreement with earlier results obtained by Shiomi et al (1985).

2 MATERIALS AND METHODS 2.1 Induction offructan biosynthesis in barley leaves

Barley seedlings (Hordeum vulgäre L. cv. Gerbel) were grown in a growth cabinet with a 14h/10h day/night cycle at 22 °C. Fructan synthesis was induced by excision of 10-day-old primary leaves at the beginning of the photoperiod. Standing in water, the excised leaves were continuously illuminated for 24 h.

2.2 Extraction and analysis of soluble carbohydrates Leaf blades of barley, bulbs of onion or tubers of Helianthus tuberosus (1 g fwt)

were homogenized in 2 ml of 25% ethanol. The homogenates were briefly centrifuged (1 min, 12,000 g) and the volume of the supernatants was reduced to one third by vacuum evaporation. Extracts were qualitatively analysed by thin-layer chromatography on silica gel TLC foils (Schleicher and Schuell) as described by Wagner and Wiemken (1987). Chromatograms were stained with the fructose-specific urea-phosphoric acid spray according to Wise et al. (1955).

2.3 Protein extraction and separation by FPLC chromatography Primary leaves of barley seedlings and onion seeds (Allium cepa L. cv. Jumbo,

provided by Zaadunie, The Netherlands) were used as sources for enzyme isolation. The primary leaves were excised 24 h prior to extraction and continuously illuminated. The onion seeds were germinated for 4 days in order to improve protein extraction. Seeds and leaves were homogenized in a mortar with 1 ml 50 mM citrate-phosphate buffer (pH 5.7) per g material. The homogenate was centrifuged at 10,000 g, the supernatant saturated to 80% with ammonium sulphate and incubated for 2 h at 4 °C. Precipitated proteins were collected by centrifugation at 10,000 g for 15 min and dissolved in 20 mM sodium acetate buffer (pH 4.6). For small-scale protein extractions, the ammonium sulphate precipitation was omitted. The protein solution was dialysed against 20 mM sodium acetate buffer (pH 4.6) for 20 h at 4 °C. The precipitate was removed by centrifugation (5 min, 12,000 g) and the supernatant was passed through a 0.22-μπι membrane filter before being subjected to FPLC chromatography. The first two steps were carried out by ion exchange chromatography using prepacked Mono-S and Mono-Q columns (Pharmacia) equilibrated with 20 mM sodium acetate buffer (pH 4.6) and 20 mM sodium phosphate buffer (pH 7.0), respectively. Proteins were eluted from the Mono-S column (HR 5/5) with a linear gradient of 0 to 0.75 M NaCl and 50-μ1 aliquots of the fractions (1 ml) were assayed for SST and

176

invertase activity. Fractions containing enzyme activity were pooled and dialysed overnight against 20 mM sodium phosphate buffer (pH 7.0). The dialysed solution was loaded onto a Mono-Q column (HR 5/5) and the proteins were eluted from the column with a linear gradient of 0 to 0.5 M NaCl. Subsequently, the fractions containing SST activity were dialysed overnight against 20 mM phosphate buffer (pH 5.7) containing 1 M (NH4)2S04 and subjected to hydrophobic interaction chromatography using a phenyl-Superose column (Pharmacia). Proteins were eluted from the column with a linear gradient of 1 to 0 M (NH4)2S04. SST was eluted from the column at the end of the gradient and was subsequently loaded onto an affinity chromatography column. For affinity chromatography a raffinose-coupled epoxy-Sepharose 6B column was prepared according to the instructions supplied by Pharmacia. Proteins were eluted from the column with a linear gradient of 0 to 0.2 M NaCl in 10 mM phosphate buffer (pH 5.7). SST-containing fractions were dialysed overnight against 5 mM sodium phosphate buffer (pH 6.5) and subsequently freeze-dried. The lyophilized material was dissolved in 100 μΐ water and subjected to gel filtration chromatography using a prepacked Superose-6B column (Pharmacia) equilibrated with 50 mM phosphate buffer (pH 6.5) containing 1 % Triton X-100. The active fractions (3 X 0.5 ml) were concentrated to 50 μΐ and the proteins were analysed by SDS-PAGE (Laemmli, 1970). Protein bands were stained by either 0.2% Coomassie Brilliant Blue or Ag.

2.4 Enzyme assays Enzyme preparations were incubated with an equal volume of 50 mM phosphate

buffer (pH 5.7) containing 0.3 M sucrose as substrate. Reaction mixtures were incubated for 1-4 h at 30 °C and the SST and invertase activities were separately measured by the colorimetric microtitre plate method of Cairns (1987). One μιτιοΐ of glucose or fructose produced per h at 30 °C was referred to as 1 unit of SST or invertase, respectively. Products from the in vitro enzyme assays were analysed by thin-layer chromatography.

3 RESULTS 3.1 Induction offructan synthesis in barley leaves

Excised leaves of barley seedlings were illuminated over a 48-h period. Cell-free extracts of the induced leaves were incubated with sucrose and the carbohydrates produced were analysed on TLC plates (Fig. 1, panel A). Fig. 1, panel B, shows the in vivo accumulation of fructans in the excised leaf blades. Excision of the leaves prevented export of photosynthates resulting in a rapid increase in sucrose concentration (Fig. 1, panel B, lanes 1-8). Trisaccharides and higher polymers of fructose appeared 12 h after excision and continued to accumulate during the incubation period. The barley leaves exclusively produced the trisaccharide 6-kestose, whereas 1-kestose was the major trisaccharide

177

produced in vitro. In contrast, 1-kestose was predominantly found in barley leaves by Wagner and Wiemken (1987). Fig. 1, panel A, shows no significant change in hydrolytic activity and only a slight increase in fructosyltransferase activity during the 48 h after excision. Enzymes catalysing the synthesis of 6-kestose and higher polymers of fructose appeared after a lag of about 12 h and their activities increased simultaneously with the accumulation of fructans in vivo.

To examine the effect of protein synthesis on changes in enzyme activities and carbohydrate accumulation, the leaf blades were excised and incubated for 24 h with different concentrations of cycloheximide (CHI). Fig. 2 shows that the in vivo synthesis of fructans was completely inhibited by the addition of 5 μΜ CHI. Also the accumulation of sucrose and fructose was dramatically reduced. It is not clear whether the absence of fructan accumulation was due to the inhibition of enzyme synthesis or to a lack of substrate (sucrose) for the initial step in fructan biosynthesis. Undoubtedly, CHI blocks fructan synthesis at concentrations reported to inhibit protein synthesis in barley leaves (Stewart et al., 1986). On the other hand, the in vitro synthesis of 1-kestose was less affected by the treatment with CHI (Fig. 2, panel A). Extracts of leaves treated with 5 μΜ CHI were still able to produce small amounts of 6-kestose and higher oligofructans, although treatment with higher concentrations of CHI abolished the synthesis of these carbohydrates completely.

The results obtained with the time-course and inhibition experiments led us to suggest that fructan biosynthesis in barley is induced at the level of 6-kestose production and

Fig. 1. TLC analysis of fructan accumulation in excised barley leaves after various times of illumination. Proteins extracted from the leaves were incubated in vitro with sucrose (panel A). Panel B shows the accumulation of soluble carbohydrates extracted from the excised leaves. Extracts of bulbs of Allium cepa and tubers of Helianthus tuberosus are used as standards. Markers F, S, N, 1-K, 6-K, DP4 and DP5 represent mobilities of fructose, sucrose, neokestose, 1-kestose, 6-kestose, tetrasaccharide and pentasaccharide, respectively.

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Fig. 2. Effect of different concentrations of cycloheximide (CHI) on the induction of fructan accumulation in excised barley leaves. Extracts of the leaves were incubated with sucrose and the products were analysed by TLC (panel A). Panel B presents the in vivo accumulation of carbohydrates in the excised leaves. Extracts of tubers of Helianthus tuberosus, bulbs of Alliwn cepa and leaves of Hordewn vulgäre were used as standards. For markers, see Fig. 1.

synthesis of longer fructose polymers rather than at the level of 1-kestose synthesis.

3.2 Purification and properties of acid invertase from barley leaves In Fig. 1 it is shown that cell-free extracts of barley leaves contain both invertase and

SST activity. Attempts to separate both activities by subsequent cation and anion ion exchange chromatography were not successful. Table 1 summarizes the steps used for the partial purification of a barley invertase containing an additional 'SST' activity in an in vitro assay system. The overall purification of the isolated invertase is approximately 50-fold. The invertase was released from the Mono-S column by elution with 0.4 M NaCl (pH 4.6) and with 0.45 M NaCl (pH 7.0) from the Mono-Q column.

The final invertase preparation and commercially available yeast invertase (Boehringer) were incubated with 0.15 M sucrose. Both invertases produced the trisaccharide 1-kestose exclusively (results not shown). In contrast, in vitro synthesis of substantial proportions of neokestose and 6-kestose by fungal and plant invertases have been reported (Wagner and Wiemken, 1987; Pollock et at., 1989).

Both activities of acid invertase showed different kinetics with respect to pH optimum and substrate concentration. Fig. 3 shows the pH response curves for SST and invertase activities of the isolated barley invertase preparation. SST activity was measured as glucose release over the fructose production. The pH optima for in vitro SST and invertase activity were 5.5 and 5.0, respectively. These values are similar to the pH optima reported for

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crested wheatgrass (Chatterton et al., 1988) and very near the optima measured for barley (Wagner and Wiemken, 1986). The acidic pH optima for both activities are consistent with the conclusion of Wagner et al. (1983) that these activities are located in the vacuole, although acid invertases are also found in the cytosol (Karuppiah et al., 1989). Under the conditions used in these in vitro experiments, the invertase activity was approximately four times as high as the fructosyltransferase activity.

The substrate concentration dependence of invertase and SST activity was determined in the range of 5-500 mM sucrose. The Km value for the invertase activity was 10 mM, while the SST activity exhibited a much higher Km, near 300 mM sucrose (results not shown). Consequently, the additional fructosyltransferase activity from the partially purified acid invertase is only significant at relatively high sucrose concentrations.

Pyridoxal and pyridoxin are compounds known to react with invertases and to inhibit their hydrolytic activity (Krishnan et al., 1985). The effect of pyridoxal and pyridoxin on invertase and SST activity was analysed in an in vitro assay (Table 2). Both compounds were added during the incubation of the barley acid invertase preparation with sucrose (0.15

Table 1. Steps in the purification of acid invertase from barley leaves.

Treatment

(NH4)2S04

Acid precipitation Mono-S Mono-Q

Total

210 165 70 51

activity (U) Total protein (mg)

125 41

1.6 0.64

Purification (fold)

1 2.4

26 48

a c t i v i t y CU/mg}

70 |

60 h

50 h

40 l·

30 r

20 l·

10 p

o ' 0 2 4 6 Θ 10

PH

Fig. 3. pH response curves for SST and invertase activities of the partially purified invertase preparation from barley leaves.

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Table 2. Effect of different concentrations of pyridoxin and pyridoxal on the in vitro SST and invertase activity of the partially purified barley invertase.

Treatment

Pyridoxal 10 mM 50 mM

Pyridoxin 10 mM 50 mM

Inhibition relative to control (%)

SST activity

57 86 43 82

Table 3. Steps in the purification of SST from

Treatment

(NH4)2S04 80% Acid precipitation Mono-S Mono-Q Phenyl-Superose Epoxy-Sepharose Superose-6B

Total activity (U)

508 440 236

97 36 27 18

Invertase activity

54 76 51 74

onion seeds.

Total protein (mg)

525 220

19 1.9 0.230 0.11 0.015

Purification (fold)

1 2.1

12.8 53

163 312

1250

M). Invertase as well as fructosyltransferase activity were inhibited in a similar way by pyridoxal and pyridoxin. These results are in disagreement with data reported for leaf extracts of L. temulentum, where a complete inhibition of invertase and a stimulation of SST activity was observed using the same concentrations of pyridoxal and pyridoxin (Cairns, 1989).

3.3 Purification of SST from onion seeds SST was completely purified from mature seeds of A. cepa using five successive

FPLC chromatography columns. Table 3 summarizes the steps used to purify SST. The overall purification was approximately 1250-fold and the final recovery was 3.2%.

Attempts to bind SST to sucrose-coupled epoxy-Sepharose were not successful, while almost all SST activity was bound to the raffinose-coupled epoxy-Sepharose. The final step in the purification of SST was gel filtration chromatography on a Superose-6B column, which was equilibrated with phosphate buffer (pH 6.5) containing 1% Triton X-100 (step G). Omission of Triton resulted in an elution of SST in three protein peaks, while all SST activity was found in a single peak when 1% Triton was added. Three SST-containing fractions (0.5 ml each) were collected and analysed by SDS-PAGE. Silver staining of the proteins revealed a single protein band with a molecular weight of approximately 68 kDa

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(data not shown).

3.4 Properties ofSST The final SST preparation was incubated with sucrose for 4 h and the in vitro

products were analysed by TLC (results not shown). 1-Kestose was the only detectable trisaccharide product and no hydrolysis of sucrose (release of fructose) was measured by the enzyme-linked colorimetric assay (Cairns, 1987). No change in carbohydrate production was observed when the enzyme was incubated with sucrose and inulin (1%) used as fructosyl donor, suggesting that the FFT activity of this onion SST preparation was negligible. Next, the specificity of SST was tested using maltose, raffinose and stachyose as substrates. None of the substrates were affected by the enzyme as was shown by TLC analysis and enzymatic measurement of glucose and fructose release. This demonstrates that no hydrolytic or transfer activity was observed with these saccharides as substrate.

The optimal pH for in vitro SST activity was 5.5 and resembled well the pH optimum found by Shiomi et al. (1985). Furthermore, measurement of the influence of sucrose concentration on the SST activity revealed a Km value of approximately 250 mM. In contrast with this Km value, Shiomi et al. (1985) have calculated a three times lower Km

for SST using the same source for enzyme purification.

4 DISCUSSION This paper reports the purification of sucrose: sucrose fructosyltransferase (SST) from

onion seeds using five successive Chromatographie steps. The purity of the preparation was assessed by means of SDS-PAGE followed by high-sensitive silver staining. A single band of a protein with an apparent molecular weight of 68 kDa was observed. The only in vitro products of SST were 1-kestose and glucose, thus showing that SST was free from hydrolytic or FFT activity. However, onion seed extracts did contain FFT activity, producing neokestose and higher fructose polymers in an in vitro assay. This activity could be separated from SST by FPLC Mono-S chromatography (Angenent, unpublished data). The occurrence of two distinct enzymes, one catalysing the transfer of a fructose unit to sucrose and the other catalysing the subsequent elongation of the fructose chain led us to suggest that the general SST/FFT model for fructan biosynthesis holds for onion. However, our results obtained with barley demonstrate that the mechanism might be more complex in grasses.

We were not able to separate invertase and SST activity from extracts of excised primary barley leaves using high-resolution FPLC columns. Similar results were obtained in studies using enzymes extracted from detached illuminated leaves of L. temulentum (Cairns et al., 1989). Our results are in contrast with those of Wagner and Wiemken (1987)

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who reported the separation of SST and invertase activity from barley protoplast extracts. Attempts to isolate a plant SST free from invertase activity using protoplast extracts of induced barley leaves were unsuccessful in our hands. In addition to activities of plant invertases, a substantial fructosyltransferase activity was observed which originated from the pectolyase preparation used for protoplast isolation.

Surprisingly, our results show that in excised leaves no synthesis of 1-kestose was induced, but that accumulation of 6-kestose and higher fructan polymers started 12 h after excision of the leaves. Furthermore, only 1-kestose but no detectable amount of fructans accumulated when cytoplasmatic protein synthesis was inhibited by cycloheximide. For barley, it was suggested by Wagner and Wiemken (1986) that fructan biosynthesis is induced at the level of SST. However, our results indicate that fructan synthesis might be regulated at the level of FFT activity. This conclusion is in agreement with results recently reported by Cairns et al. (1989) showing that fructan accumulation could be induced in the absence of any detectable rise of SST activity using the L-isomer of 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamide (L-MDMP). In addition to induction of FFT activity a surplus of photosynthates (sucrose) is necessary to allow accumulation of fructan in leaves. Under non-induced conditions the endogenous sucrose concentration in barley ranges between 10 and 20 mM, while excision and illumination of the leaves induced the accumulation of free sucrose to at least 10-fold higher levels (Wagner and Wiemken, 1989). The SST activity, which was accompanied by invertase activity in a partially purified preparation from barley leaves, had an apparent Km value of 300 mM sucrose. Consequently, the discrepancy between the absence of 1-kestose in vivo, on one hand, and its accumulation in an in vitro assay, on the other, during the first 8 h after excision could be explained by the difference in sucrose concentration. Fructosyl transfer to sucrose was only detectable at relatively high sucrose concentrations. The sucrose concentration used in the in vitro assays (150 mM) resembles the physiological sucrose concentration present in excised illuminated leaves suggesting that the same activity might be present in vivo.

The nature of the protein catalysing the initial in vivo fructosyl transfer in barley to produce the trisaccharide is, at present, unclear. Our results indicate that an acid invertase is responsible for this activity. Several other reports have demonstrated that invertase itself can catalyse fructosyl transfer at high sucrose concentrations (Straathof et al., 1986; Ivin and Clarke, 1987; Pollock et al., 1989). Similar bifunctional enzymes are also found in prokaryotic microorganisms possessing distinct sites for hydrolase and transferase activities (Yamashita et al., 1989). Differences in pH optima and Km values could be assigned to the different active sites of such a protein.

Our results suggest that fructan biosynthesis in barley is different from the mechanism found in onion. Probably, a constitutively expressed acid invertase is involved

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in the initial step in fructan synthesis, however further investigations are necessary to proof this hypothesis. It is clear that fructan metabolism in vivo not only depends on the presence or absence of enzymes but is also strongly regulated by the relative rates of the different enzyme activities (hydrolysis vs. transfer) which can be directed by changes in substrate concentration.

5 ACKNOWLEDGEMENTS These investigations were supported in part by the Netherlands Foundation for

Chemical Research (SON) with financial aid from the Netherlands Technology Foundation.

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fructosyltransferase from tubers of Helianthus tuberosus L. Agric. Biol. Chem., 54: 2429-2431. Scott, R.W., 1968. Transfructosylation in higher plants containing fructose polymers. Ph.D. Thesis,

University of London. Shiomi, N., 1989. Properties of fructosyltransferases involved in the synthesis of fructan in liliaceous plants.

J. Plant Physiol., 134: 151-155. Shiomi, N., Kido, H. and Kiriyama, S., 1985. Purification and properties of sucrose:sucrose lF-/3-D-

fructosyltransferase in onion seeds. Phytochemistry, 24: 695-698. Shiomi, N., Yamada, J. and Izawa, M., 1979. Synthesis of several fructo-oligosaccharides by asparagus

fructosyltransferases. Agric. Biol. Chem., 43: 2233-2244. Stewart, C.R., Voetberg, G. and Rayapati, P.J., 1986. The effects of benzyladenine, cycloheximide, and

cordycepin on wilting-induced abscisic acid and proline accumulations and abscisic acid- and salt-induced proline accumulation in barley leaves. Plant Physiol., 82: 703-707.

Straathof, A.J.J., Kieboom, A.P.G. and Van Bekkum, H., 1986. Invertase-catalysed fructosyl transfer in concentrated solutions of sucrose. Carbohydr. Res., 146: 154-159.


Wagner, W. and Wiemken, A., 1986. Properties and subcellular localization of fructan hydrolase in the leaves of barley {Hordeum vulgäre L. cv Gerbel). J. Plant Physiol., 123: 429-439.

Wagner, W. and Wiemken, A., 1987. Enzymology of fructan synthesis in grasses. Properties of sucrose-sucrose-fructosyltransferase in barley leaves {Hordeum vulgäre L. cv Gerbel). Plant Physiol., 85: 706-710.

Wagner, W. and Wiemken, A., 1989. Fructan metabolism in expanded leaves of barley {Hordeum vulgäre L. cv. Gerbel): change upon ageing and spatial organization along the leaf blade. J. Plant Physiol., 134: 237-242.

Wise, C.S., Dimler, R.J., Davis, H.A. and Rist, C.E., 1955. Determination of easily hydrolyzable fructose units in dextran preparations. Anal. Chem., 27: 33-36.

Yamashita, Y., Hanada, N., Itoh-Andoh, M. and Takehara, T., 1989. Evidence for the presence of two distinct sites of sucrose hydrolysis and glucosyl transfer activities on 1,3-a-D-glucan synthase of Streptococcus mutans. FEBS Lett., 243: 343-346.


FRUCTAN ACCUMULATION IN CELL SUSPENSION CULTURES OF PHLEUM PRATENSE L.#

Marco FREHNER, Marcel LÜSCHER and Josef NÖSBERGER Swiss Federal Institute of Technology, Institute for Plant Sciences, ETH-Zentrum, CH-8092 Zürich, Switzerland

ABSTRACT

We present a model on fructan accumulation using cell suspension cultures of Phlewn pratense L. These well-established cultures (> 24 months) produce fructan as a stable characteristic. Cultures subjected to cold induction at 4 °C showed enhanced fructan production in comparison to control cultures at 22 °C. No fructan was released into the culture medium.

1 INTRODUCTION Photosynthate is stored as transient starch in leaves and may be diverted into

fructans. In some of the temperate forage grasses and C-3 cereals, fructans functionally complement or even replace starch. In a few cases, the biochemistry and subcellular organization of fructan metabolism are known (Edelman and Jefford, 1968; Frehner et al., 1984; Wagner and Wiemken, 1986; Pollock and Chatterton, 1988; Shiomi, 1989). But only little is known of fructan metabolism in the economically important Gramineae (Wagner et al., 1983; Schnyder and Nelson, 1987; Pollock et al, 1989).

To study the biochemistry of fructan metabolism in grasses and its regulation, we prepared cell suspension cultures of several temperate grass species. A culture of Phlewn pratense L. was shown to produce fructans. A similar culture has been described previously (Hale et al., 1987). Additionally, fructan accumulation of our culture is inducible and fructans are completely contained within the cells.

# Abbreviations used: 2,4-D, 2,4-dichlorophenoxyaceticacid; DP, degree of polymerization; frc eq, fructose equivalent.

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2 METHODS 2.1 Liquid cell suspension culture

The culture was obtained from chopped Phleum pratense embryos as described earlier (Dalton, 1988) using P-medium (Potrykus etal., 1979) containing 2% (w/v) sucrose and 2 mg ml"1 2,4-D (hereafter simply referred to as culture medium). The cultures were maintained by transferring one volume of stationary phase culture into ten volumes of fresh sterile culture medium at three-week intervals. Cells were collected on a nylon filter, the filtrate was kept for analysis (see below). The cells were washed with fresh medium (with sucrose replaced by equimolar mjo-inositol), frozen and dried by lyophilization.

2.2 Carbohydrate extraction and analysis Carbohydrates were extracted by boiling a weighed portion of dry powdered cells in

80% (v/v) ethanol and re-extracting the residue with water by sonication in a water bath. The combined extracts were desalted (Dowex 50, H+ ; Dowex 1, acetate), dried under reduced pressure, dissolved in water and used for analysis by HPLC. Culture filtrate samples were desalted similarly before being analysed by HPLC.

To estimate the total fructan content of a sample, the HPLC carbohydrate fraction with DP > 3 was collected and hydrolysed in 1 M HC1 at 80 °C for 1 h, neutralized with 1 M NaOH, desalted and analysed as above.

HPLC separation of sugar samples was carried out using a Benson BC 100/Ca column at 80 °C with water at 0.6 ml min"1 as the eluant. An electrochemical detector

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Fig. 1. Growth of the Phleum suspension culture. The cell dry weight (rectangles) and the conductivity of the culture medium (triangles) are inversely related in both the control experiment at 22 °C (solid symbols) and the cold-induced experiment (open symbols).

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(model 400, EG&G Princeton Applied Research) in the pulsed mode (E{ = 0 mV, E2 = 600 mV, E3 = -800 mV, t{ = 300 ms, t2 = 200 ms, t3 = 200 ms) and post-elution addition of 1 M NaOH at 0.1 ml min"1 were used to detect and quantify carbohydrates.

3 RESULTS We tried to obtain fructan-producing cell suspension cultures from several species

of the Gramineae. About 6 months after their initiation from embryos, a Phleum pratense culture and zDactylis glomerata L. culture contained detectable fructan levels. The Dactylis culture contained fructosylsucrose. In addition to that, the Phleum culture contained higher DP fructans. It was competent in fructan metabolism and fructan production was a stable characteristic of this cell line. Two-year-old cultures were used here.

3.1 Growth of the Phleum cultures Growth of the cells was monitored, and conductivity and carbohydrate content of the

medium and the cells was measured in both control and cold-induced cells. A typical growth curve of the cells and the conductivity profile of the medium are shown in Fig. 1. When cultures were grown at 4 °C the growth rate was reduced (Fig. 1). The carbon source sucrose was split into glucose and fructose almost immediately after subculture (Fig. 2) and these subsequently used for growth, similar to the observation of Hale et al. (1987). Cleavage is probably due to an extracellular invertase. The glucose in the medium was utilized more efficiently than fructose (Fig. 2). Glucose and fructose were consumed more

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Fig. 2. Mono- and disaccharides in the medium of Phleum suspension cultures. Sucrose (diamonds) was converted rapidly to glucose (rectangles) and fructose (triangles). The carbon sources were consumed more rapidly in the control experiment (solid symbols) than in the cold-induced experiment (open symbols).

188

slowly in the cultures grown at 4 °C, reflecting their lower growth rate (Fig. 1). Some polysaccharides were released by the cells as reported by Hale et al. (1987) but no fructan was detected in the culture medium.

3.2 Intracellular carbohydrates The carbohydrate contents of washed Phleum cells were analysed. Glucose

concentration did not vary significantly during the culture period (Fig. 3). Fructose, the predominant sugar, accumulated during the first two weeks of culturing and its concentration declined thereafter (Fig. 3). Sucrose was barely detectable except in the last third of the culture period, then its concentration increased as the fructose concentration decreased (Fig. 3). Changes in the concentrations of glucose, fructose and sucrose followed a similar pattern in cultures grown at 4 °C, except that the changes in concentrations of fructose and sucrose were slower than in the control experiment (Fig. 3). In both experiments the intracellular fructose appeared to be converted to intracellular sucrose towards the end of the culture period.

There was a low level of fructans (DP > 3) and trisaccharide present in the Phleum cells at all times. During the first 14 days the fructan concentration was around 15 jumoles frc eq g"1 dwt and increased thereafter (Fig. 4). The trisaccharide concentration showed a similar pattern as the fructans but during the first two weeks it was < 5 jLtmoles g'1 dwt (Fig. 4). The cold-induced cultures accumulated fructans and trisaccharide steadily during the third week of growth and did not cease to do so before the end of the experimental period (Fig. 4). The increase of fructan and trisaccharide concentration in both the induced

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and the control cultures paralleled the increase of sucrose concentration (Figs. 3 and 4). Although fructan accumulated at the end of the growth period and the fructose level decreased, the most prominent neutral, soluble carbohydrate in the cells was always fructose. In the control, at the very end of the three-week culture period, the intracellular concentrations of all the carbohydrates analysed were decreasing. This was probably due to carbon source depletion in the medium (Fig. 2) forcing the cells to use intracellular carbon sources to survive.

4 DISCUSSION AND CONCLUSION We obtained a fructan-producing cell culture from Phleum embryos which always

contained a minimal amount of fructans (about 15 /xmoles frc eq g"1 dwt; Figs. 1 and 4). This suggests that fructan synthesis kept pace with cellular growth; before the cells entered the stationary phase, the rate of fructan synthesis surpassed the declining growth rate. Hence, the cells increased in fructan content (Fig. 4). Cold-induced cells slowed their growth rate down before the external carbon source was depleted (Figs. 1 and 2) and were therefore able to sustain a continued high rate of net fructan accumulation (Fig. 4). Cells entering the stationary phase in presence of external substrate were diverting carbon into fructan reserves.

This cell suspension system will be useful to investigate the regulation of fructan synthesis and breakdown as their rates obviously change during a culture cycle (Fig. 4).

190

5 REFERENCES

Dalton, S.J., 1988. Plant regeneration from cell suspension protoplasts of Festuca arundinacea Schreb., Loliwn perenne L. and L. multiflorum Lam. Plant Cell Tissue Organ Culture, 12: 137-140.


Frehner, M., Keller, F. and Wiemken, A., 1984. Localization of fructan metabolm in the vacuoles isolated from protoplasts of Jerusalem artichoke tubers {Helianthus tuberosus L.). J. Plant Physiol., 116: 197-208.

Hale, A.D., Pollock, C.J. and Dalton, S.J., 1987. Polysaccharide production in liquid cell suspension cultures of Phleumpratense L. Plant Cell Rep., 6: 435-438.

Pollock, C.J., Cairns, A.J., Collis, B.E. and Walker, R.P., 1989. Direct effects of low temperature upon components of fructan metabolism in leaves of Lolium temulentum L. J. Plant Physiol., 134: 203-208.


Potrykus, I., Harms, C.T. and Lorz, H., 1979. Callus formation from cell culture protoplasts of corn (Zea mays L.). Theor. Appl. Genet., 54: 209-214.

Schnyder, H. and Nelson, C.J., 1987. Growth rates and carbohydrate fluxes within the elongation zone of tall fescue leaf blades. Plant Physiol., 85: 548-553.



Wagner, W. and Wiemken, A., 1986. Regulation of fructan metabolism in barley. Experientia, 42: 655.


FRUCTAN POLYMERIZATION AND DEPOLYMERIZATION DURING THE GROWTH OF CHICORY (CICHORIUMINTYBUS L.) PLANTS

A. LIMAMI and V. FIALA FNPE and INRA, Laboratoire du Metabolisme et de la Nutrition des Plantes, Route de St Cyr, 78026 Versailles cedex, France

ABSTRACT

Inulin, ß-(2,l)-linked fructan, is a major reserve carbohydrate in Cichorium intybus L. roots. Biosynthesis of fructan in the tap-root is assumed to be the result of the concerted action of two enzymes, sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyltransferase (FFT). Extracts of growing roots contain fructans and free glucose (but only little fructose) whereas extracts of mature roots contain only small quantities of glucose. The decrease of free glucose, a product of SST action, could indicate a decline of fructan synthesis. The amount of free fructose increases in the mature root probably due to inulinase activity in the root.

SST activity was assessed, at physiological sucrose concentrations, by supplementing endogenous sucrose with [U-14C]-sucrose at various times during the growing period. Variations of the amounts of the two hexoses and the relation between SST activity and root growth and fructan accumulation are discussed with regard to the transition of the tap-root from sink to source.

1 INTRODUCTION

During its growth in the field, from spring to late summer, Witloof chicory {Cichorium intybus L.), a biennial composite, produces a tap-root. This storage organ operates mainly as a sink for photosynthates which are converted into and stored as fructan (Fiala and Jolivet, 1980, 1984) in the vacuole (Kaeser, 1983). The roots are harvested and forced in a hydroponic system (Lesaint and Co'ic, 1983) in the dark, at 16-18 °C, to produce an etiolated bud, the chicon. The growth of the chicon, in the absence of photosynthesis, occurs at the expense of remobilized carbohydrates from the roots. Thus, during forcing the latter operates as a source organ. The transition of the root from sink to source, during root maturation, is induced at the end of the growing period. It is accompanied by biochemical changes such as hydrolysis of fructan with a high degree of polymerization (DP) (Fiala and Jolivet, 1980) and probably a decrease in fructan synthesis (data not published). In Helianthus tuberosus L. tubers, which also accumulate fructan,

192

Edelman and Jefford (1968) observed that sucrose:sucrose fructosyltransferase (SST) which catalyses the first step of fructan synthesis, disappears from the tissue when the tubers stop growing. These authors suggested that SST may be the controlling factor for the rate of growth. Our aim has been to characterize the transition of the chicory root from sink to source by biochemical markers and to study the role of SST in the control ofthat transition.

2 MATERIALS AND METHODS 2.1 Plant material

Witloof chicory (Cichorium intybus L. cv. Flash) seeds were sown in a field in the north of France at the end of April. Plants were harvested at various times after planting. The aerial parts were excised and weighed. The storage roots were weighed, cut into small pieces which were immediately frozen in liquid nitrogen and then freeze-dried, ground, homogenized and stored at -80 °C for subsequent analysis.

2.2 Extraction and analysis of carbohydrates by HPLC and TLC Carbohydrates were extracted from an aliquot of the freeze-dried tissue with ethanol

(80%) following the method described by Fiala and Jolivet (1984). The tissue aliquot was taken from a sample of ten plants and experiments were duplicated. The ethanol was evaporated off under vacuum. The carbohydrates were resuspended in demineralized water and quantified by isocratic high-performance liquid chromatography (HPLC) on a 300 X 7.8 mm Aminex HPX 42 C column (Bio-Rad) at 75 °C, with water as the eluant at a flow rate of 0.4 ml min"1. Eluated carbohydrates were detected by differential refractometry.

Ascending thin-layer chromatography (TLC) was performed on pre-coated 20 x 20 silica gel plates (F 1500, Schleicher and Schuell, FRG). Chromatograms were developed twice with butan-1-ol, propan-2-ol, water (3:12:4, v/v/v) as solvent (Kanaya et al., 1978). Carbohydrates were visualized by spraying with ketose-specific urea-phosphoric acid (Wise et al., 1955). A mixture of fructose, sucrose, 1-kestose, nystose and an inulin pentasaccharide (DP 5) served as a standard (Mejoligo, Bio Sciences Laboratories, Japan).

2.3 Preparation of cleared homogenate of root tissue Hundred mg of freeze-dried tissue was mixed for 30 min at 4 °C in 50 ml

homogenization medium, containing 50 mM sodium acetate buffer (pH 5.5) with 1 mg ml"1

bovine serum albumin (Cairns, 1989). Mercaptoethanol was added to prevent oxidation. The homogenate was centrifuged at 10,000 g for 15 min and used immediately in labelling experiments.

193

2.4 Assay offructosyl transfer by l4C-labelling and autoradiographic detection [U-14C]-sucrose (20 GBq mmole1, CEA, France) was dried under vacuum to remove

ethanol and resuspended in demineralized water at a final radioactive concentration of 3.33 MBq ml"1. The labelling reaction was started by adding [U-14C] to 1 ml of freshly prepared cleared homogenate (0.2 MBq ml"1) and carried out at 25 or 30 °C. The reaction was stopped by heating at 70 °C. After centrifugation an aliquot of the homogenate was fireeze-dried and resuspended in demineralized water. Labelled carbohydrates were separated on silica gel TLC plates and detected by autoradiography.

3 RESULTS Fig. 1 shows that the growth of the chicory plant in the field ceased in late summer-

early autumn. At that time, the root fresh weight reached a plateau while the fresh weight of the aerial part of the plant began to decrease. Free fructose in the root tissue was initially very low but subsequently increased to approximately 1 mg g"1 dwt. Free glucose, on the other hand, was more abundant than free fructose in the beginning, but later decreased and then remained almost constant until harvest (Fig. 2a). The root sucrose content only slightly increased, in the early autumn, whereas the DP > 2 fructan content did not vary significantly during the whole growth period. However, total fructan quantity increased due to the increase in root mass (Fig. 2b and c).

Silica gel TLC of chicory root fructan allowed separation of oligosaccharides (Fig. 3). The extracts contained hexose, sucrose, a single trisaccharide species, which co-chromatographed with 1-kestose, a tetrasaccharide, a pentasaccharide which co-

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Fig. 1. Changes in Witloof chicory biomass during growth in the field. Biomass is expressed as g fvvt (root: open symbols; aerial part of the plant: closed symbols). Results are the mean of four replicates often plants each. The confidence limits given are for P = 0.05.

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Fig. 2. Changes in carbohydrates in the roots of Witloof chicory during the growing period. Amounts of a: glucose (closed symbols) and fructose (open symbols); b: sucrose; and c: DP > 2 fructans, are expressed as mg g"1 dwt. Results are the mean of two replicates.

Fig. 3. Time-dependent incorporation of 14C into oligofructan by a cleared homogenate of Witloof chicory root, supplemented with [U-14C]-sucrose, at 25 °C (time is given in min). Ten μΐ, equivalent to 40 μg dry matter, were loaded on each lane. The X-ray plate was exposed for 3 days. For comparison, urea-phosphoric acid-stained plates are also depicted.

chromatographed with an inulin tetrasaccharide (nystose) and an inulin pentasaccharide.

195

Fig. 4. Time-dependent incorporation of 14C into oligofructan by a cleared homogenate of Witloof chicory root, supplemented with [U-14C]-sucrose, at 30 °C (time is given in min). Ten μΐ equivalent to 40 μg dry matter were loaded on each lane. The X-ray plate was exposed for 3 days.

High DP fructans extracted from the root tissue were not well separated. At two different times during plant growth, fructosyltransferase activity (SST) was

assessed, in a cleared homogenate of the root tissue (Cairns, 1989) by 14C-labelling and silica gel TLC coupled with autoradiographic detection. A time-dependent incorporation of 14C into 1-kestose at 25 °C (Fig. 3) and in fructan of higher DP at 30 °C (Fig. 4) was found. The Chromatographie distribution of the labelled fructans did not differ from that of fructans normally present in the root.

4 DISCUSSION The first step of fructan biosynthesis is assumed to be carried out by the enzyme

sucrose:sucrose fructosyltransferase (Edelman and Jefford, 1968; Cairns and Pollock, 1988a). The activity of the latter results in synthesis of a trisaccharide and release of glucose. In line with this hypothesis, in our experiments levels of free glucose in chicory root extracts can be expected to reflect the rate of fructan synthesis. A decrease in free glucose in the root in early autumn could thus suggest a decline in fructan synthesis. As reported by Edelman and Jefford (1968) free glucose decreased to reach a very low level in mature Jerusalem artichoke tubers. According to the same authors, the slight increase in

196

free fructose could indicate an increased activity of inulinase, the enzyme which is also responsible for fructan hydrolysis in the chicory root (Ciaessens et al., 1989). In our experiments, fructose increased but remained low, probably due to its conversion to sucrose. Thus, amounts of free fructose in the tissue did not reflect fructan hydrolysis.

Upon termination of root growth the decline in free glucose, assumed to be due to a decrease in fructan synthesis, could characterize the transition of the root from sink to source. Indeed, in roots with a low level of free glucose fructan hydrolysis allows the mobilization of carbohydrates for chicon growth, in the dark, during root forcing.

In order to assess the relation between SST and root growth, fructan biosynthesis and accumulation, the activity of the enzyme was assayed in vitro in a cleared homogenate of root tissue at two times: in the summer when the plant was growing and accumulating fructans, and in the early autumn when the root was mature and growth had ceased. Cleared homogenates prepared at both times were capable of catalysing fructosyl transfer since label was transferred from [U-14C]-sucrose to 1-kestose and oligofructans (Figs. 3 and 4). The Chromatographie pattern of the labelled fructans, identical to those which accumulate in the root, indicated that fructan synthesis in vitro at physiological substrate concentrations, as shown by Cairns (1989), is not an artifact. Unlike Gramineae where three trisaccharides, 1-kestose, 6-kestose and neokestose, are synthesized (Cairns and Pollock, 1988a, b; Bancal and Gaudillere, 1989), chicory root appears to produce 1-kestose as the single intermediate between sucrose and fructan. This trisaccharide might be synthesized by fructosyl transfer from donor to acceptor sucrose, by the action of SST and serves, in turn, as fructosyl donor for the polymerization of fructan chains by the action of fructan:fructan fructosyltransferase (FFT) according to the model described by Edelman and Jefford (1968) for Jerusalem artichoke. The role of SST, however, appears to be different in both types of plants. The latter authors have claimed that both tuber growth and fructan synthesis in Jerusalem artichoke are controlled by SST and found that the latter disappeared rapidly from the tissue when tubers stopped growing. In chicory, at any time during growth and also when the amount of free glucose declined, SST activity was detectable in the root tissue. Thus, SST may not be a controlling factor for the rate of growth and similarly the transition of the root from sink to source may not be dependent upon the activity of the latter enzyme.

5 REFERENCES

Bancal, P. and Gaudillere, J.P., 1989. Rate of accumulation of fructan oligomers in wheat seedlings (Triticum aestivwn L.) during the early stages of chilling treatment. New Phytol., 112: 459-463.

Cairns, A.J., 1989. Fructan biosynthesis in excised leaves of Loliwn temulentwn L. IV. Cell-free 14C labelling of specific oligofructans at low sucrose concentration. New Phytol., 112: 465-473.

Cairns, A.J. and Pollock, C.J., 1988a. Fructan biosynthesis in excised leaves of Loliwn temulentwn L. I. Chromatographie characterization of oligofructans and their labelling patterns following 14C02

197

feeding. New Phytol., 109: 399-405. Cairns, A.J. and Pollock, C.J., 1988b. Fructan biosynthesis in excised leaves of Lolium temulentum L. II.

Changes in fructosyl transferase activity following excision and application of inhibitors of gene expression. New Phytol., 109: 407-413.

Claessens, G., Van Laere, A. and De Profit, M., 1990. Purification and properties of an inulinase from chicory roots (Cichorium intybus L.). J. Plant Physiol., 136: 35-39.


Fiala, V. and Jolivet, E., 1980. The aptitude of roots of Witloof chicory for chicon production studied by their carbohydrate composition. Sei. Hortic, 13: 125-134.

Fiala, V. and Jolivet, E., 1984. Mise en evidence d'une nouvelle fraction glucidique dans la racine de chicoroe et son Evolution au cours de la formation des reserves. Physiol. Veg., 22: 315-321.

Kaeser, W., 1983. Ultrastructure of storage cells in Jerusalem artichoke tubers {Helianthus tuberosus L.). Vesicle formation during inulin synthesis. Z. Pflanzenphysiol., I l l : 253-260.

Kanaya, K., Chiba, S. and Shimomura, T., 1978. Thin-layer chromatography of linear oligosaccharides. Agric. Biol. Chem., 42: 1947-1948.

Lesaint, C. and Coic, Y., 1983. Cultures Hydroponiques. La Maison Rustique, Flammarion, Paris, 215 pp. Wise, C.S., Dimler, R.J., Davis, H.A. and Rist, C.E., 1955. Determination of easily hydrolyzable fructose

units in dextran preparations. Anal. Chem., 27: 33-36.



FRUCTAN EXOHYDROLASE FROM LOLIUM RIGIDUM GAUD.

G.D. BONNETT and RJ. SIMPSON School of Agriculture and Forestry, The University of Melbourne, Parkville, Victoria 3052, Australia

ABSTRACT

Fructan exohydrolase (FEH) was extracted from mature leaves and stems of Loliwn rigidwn Gaud, and partially purified by salt precipitation, affinity chromatography and anion exchange chromatography. The partially purified FEH was incubated with a range of substrates and found to be most active against fructan extracted from L. rigidwn. When FEH was incubated with individual /5-2,1-1 inked fructans, the Km of the exohydrolase activity increased with the increase in degree of polymerization of the substrate.

1 INTRODUCTION

In pastures of annual ryegrass (Lolium rigidum Gaud.) growing in southern Australia the amount of soluble carbohydrate present in the sward decreases after anthesis and this decrease results in loss of digestibility and reduced animal production during summer (Ballard et al., 1990). The major component of the soluble carbohydrate in L. rigidum is fructan (Smouter and Simpson, 1989).

In plants, fructan has only been shown to be hydrolysed by removal of the terminal fructose residues {i.e. exohydrolase activity) (Rutherford and Deacon, 1972; Smith, 1976; Henson, 1989). Little is known, however, about the activity of fructan exohydrolase (FEH) against substrates of different molecular mass. In this study FEH from L. rigidum was partially purified and activity against different substrates was characterized.

2 MATERIALS AND METHODS 2.1 Plant material

Plants of L. rigidum were grown in a potting mixture of soil, sand and peat (3:2:1, v/v/v), in a glasshouse at 21 °C day/13 °C night. Shoots were harvested after they had flowered by cutting at ground level. Inflorescences were removed and discarded.

200

2.2 Extraction and purification offructan exohydrolase Harvested plant material (1.5 kg) was homogenized in 50 mM phosphate-citrate

buffer (pH 5.5, at 5 °C) at a ratio of 1:2 (w/v), with polyvinylpolypyrrolidone (0.05 g g'1) and acid-washed sand. The homogenate was clarified by centrifugation at 22,000 g for 10 min and the supernatant was retained. Initial enzyme activities were measured in subsamples that were desalted (Biogel P-30, Bio-Rad).

From the crude extract, proteins precipitating between 30% and 80% of saturation with ammonium sulphate were applied to an affinity column, 220 mm by 22 mm (Concanavalin A, Pharmacia) in 50 mM phosphate-citrate buffer (pH 5.5) with 1 mM CaCl2

and 1 mM MnCl2 at a rate of 1.5 ml min"1. Proteins bound to the column were eluted in two steps, with 40 mM and 250 mM methyl mannopyranoside (MMP) (Sigma). Proteins eluting with the higher sugar concentration were concentrated by filtration (Centriprep 30, Amicon), desalted into 20 mM Tris-HCl with 1 mM EDTA disodium salt and applied to an anion exchange column (Mono-Q HR 5/5, Pharmacia) in the same buffer at 1 ml min"1. Proteins bound to the column were eluted in a gradient 0-500 mM NaCl over 60 min and collected in 1-ml fractions.

Protein was measured by the procedure of Bradford (1976).

2.3 Enzyme assays As invertase extracted from fungal sources can hydrolyse plant fructans (Nilsson et

al., 1987; Gonzalez et al., 1989) FEH and invertase activities were measured at all stages of the purification procedure.

FEH activity was measured as the release of fructose at 25 °C for up to 4 h from a 5% (w/v) solution of inulin extracted from Cichorium intybus L. (Sigma) in 50 mM phosphate-citrate buffer (pH 5.5) with 1 mg ml"1 bovine serum albumin added.

Invertase activity was measured as the release of fructose at 25 °C for up to 2 h from a 100-mM sucrose solution in 50 mM phosphate-citrate buffer (pH 4.8) with 1 mg ml"1

bovine serum albumin added. Fructose was measured by coupling enzymes to the reduction of NADP as described

by Jones et al. (1977).

2.4 Preparation of a series offructan oligosaccharides Fructan oligosaccharides were prepared from tubers of Helianthus tuberosus L. by

gel filtration. Both their preparation and determination of purity are described by Ilgoutz et al (1993).

201

3 RESULTS 3.1 Purification offructan exohydrolase

The purification of FEH is summarized in Table 1. Partial separation of invertase and FEH was obtained with both affinity chromatography and anion exchange chromatography. Both activities bound to the affinity column indicating that they were glycoproteins. FEH required a higher concentration of MMP for its release than most of the invertase (Table 1). From anion exchange chromatography FEH eluted just ahead of invertase. The peaks, however, overlapped (185 mM and 220 mM NaCl, respectively). The first few fractions containing FEH activity had relatively low invertase activity and were combined (viz. 185 mM NaCl, Table 1). The remaining fractions with FEH activity were also combined (viz. 220 mM NaCl, Table 1). The fraction eluted with 185 mM NaCl was used for determination of the Km of FEH activity with various substrates.

The specific activity of FEH in the partially purified enzyme preparation was 27 nkat mg"1 protein. The ratio of fructan exohydrolase activity to invertase activity was 1:1.5 (Table 1). This ratio was 1:0.375 when FEH activity was measured against a 5% (w/v) solution of fructan extracted from leaves of L. rigidum.

Table 1. Purification offructan exohydrolase (FEH) from combined stems and leaves (1.5 kg) of L. rigidum.

Procedure

Crudec

Precipitation

Con A 40 mM MMPd

250 mM MMP

Concentration

Mono-Q 185 mM NaCl 220 mM NaCl

FEH specific activity8

(nkat mg"1)

0.19

0.30

2.91 7.98

9.3

27 35

Invertase specific activity0

(nkat mg"1)

5.0

83 44

105

40 429

Ratio of FEH to invertase

1:17

1:29 1: 6

1:11

1: 1.5 1:12

Purity of FEH (fold)

1

1.6

41

48

140

Recovery of FEH (%)

100

44 26

11

5 1.5

Total protein (mg)

421

19 4.1

1.5

0.25 0.06

a Measured against a 5% solution of chicory inulin. b Measured against a 100-mM solution of sucrose. c Activity in the crude extract was not measured in this case. However, salt precipitation usually gives a 3-fold purification with 70% recovery. The specific activity of FEH shown is an average value from similar extractions. d Methyl mannopyranoside.

202

3.2 Fructan exohydrolase activity against different substrates FEH activity was 3- to 4-fold larger against fructan extracted from L. rigidum than

commercially available inulin from H. tuberosus or C. intybus (Table 2). High FEH activity was also measured against fructan extracted from Urginea maritima Baker. The partially purified FEH, however, was not active against a 1 % solution of fructan extracted from either Aerobacter levanicum or Streptococcus salivarius. Activity measured against sucrose and raffmose was probably mostly due to the contaminating invertase activity (Table 2).

Each of the fructan substrates from different species (Table 2) contained no fructans of low DP as determined by thin-layer chromatography (silica gel plates developed twice in propan-1-ol/ethyl ethanoate/water (5:3:2, v/v/v)). No fructan was detected above the origin. The fructan substrates did, however, contain a range of fructans with different degrees of polymerization. Some of the fructans have branched structures and contain both 0-2,1- and 0-2,6-linkages (e.g. from U. maritima, Nitsch et al., 1979; A. levanicum, Feingold and Gehatia, 1957; and S. salivarius, Simms et al., 1990). Thus, although it is clear that some substrates are more useful for assay purposes, the relative FEH activities were difficult to interpret because the concentration of terminal fructose residues in a 1 % solution of each substrate was unknown.

In order to overcome these problems FEH activity was measured against a series of linear 0-2,1-linked fructan oligosaccharides prepared from H. tuberosus. Because the partially purified protein preparation had both FEH and invertase activity (Table 1), the substrates were also incubated with a similar activity of invertase from the invertase peak eluting from the anion exchange column. This was free from FEH activity when measured against 5% (w/v) fructan from C. intybus. The Km of FEH activities shown in Table 3 was corrected to account for the hydrolysis of fructan due to invertase (this was usually less than

Table 2. Activity of a partially purified FEH from L. rigidum against fructans extracted from various species at a concentration of 1% (w/v), unless specified, in 50 mM phosphate-citrate buffer.

Sources of fructan substrate Activity as a percentage of that using fructan extracted from L. rigidum

Lolium rigidum 100.0 Cichorium intybus 32.0 Helianthus tuberosus 26.0 Urginea maritima 85.6 Aerobacter levanicum 3.0 Streptococcus salivarius 4.0 Sucrose (20 mM)a 121.6 Raffmose (20 mM) 28.0 a The ratio of FEH to invertase activities is 1:1.2 in this table and not 1:0.375 as reported in the text. This is because FEH and invertase activity reported here were measured at 1 % and 20 mM, respectively, as opposed to 5% and 100 mM; see text.

203

Table 3. Km of a partially purified FEH with different fructans as substrate.

DP

3 4 5a

6 7

30b

*m (mM)

0.44 1.91 5.51 9.61

16.67 18.54

a DP 5 was purified from Neosugar P (Meiji Seika Kaisha, Ltd.). All other oligosaccharides were extracted and purified from tubers of H. tuberosm. b Km calculated assuming that the average DP of inulin from C. intybus was 30.

15% of the total fructose released in the assay). Sufficient quantities of fructans of DP 3, 4, 6 and 7, had been prepared to test the activity of FEH against a range of substrate concentrations. FEH activity exhibited Michaelis-Menten-like kinetics. FEH activity was saturated at substrate concentrations above 10-25 mM. The Km values estimated were lowest for the trisaccharide and increased as the DP was increased to 7 (Table 3). When FEH was incubated with inulin extracted from C. intybus the activity was not saturated for concentrations up to 10% (w/v). If the average DP of this substrate is assumed to be 30 the apparent Km was calculated to be 18.54 mM (Table 3).

4 DISCUSSION There may be differences between plant species in the relative ease with which FEH

and invertase are displaced form concanavalin A. In L. rigidum invertase was displaced at a lower MMP concentration than that required to elute FEH. This contrasts with FEH and invertase from barley where the reverse was found (Henson, 1989).

The partially purified FEH exhibited higher affinity for fructans of lower degrees of polymerization. At saturating concentrations of substrate, however, the velocity of fructose release was faster for fructans of increasing DP (data not shown).

The Km of 18.54 mM calculated from incubations with fructan extracted from C. intybus was similar to that calculated for FEH extracted from stems of barley (15.2 mM; Henson, 1989). By contrast, Yamamoto and Mino (1985) calculated an apparent Km of 91 mM for FEH from stem bases of DactyUs glomerata L. They also reported that the velocity of FEH activity against partially fractionated mixtures of fructans with mean degrees of polymerization ranging from 34 to 314 was influenced only by concentration, and not by DP.

The Km for ß-2,1 -linked fructan oligosaccharides reported here are the lowest for FEH extracted from a plant. These results show that the Km of FEH is likely to approach the concentrations of oligosaccharides in plant cells and highlight the need to use well-

204

defined substrates for the study of FEH activity.

5 ACKNOWLEDGEMENTS Steven Ilgoutz is thanked for the preparation of fructan oligosaccharides. Levan from

Streptococcus salivarius was a gift from Dr. N. Jacques and sinistrin extracted from Urginea maritima was a gift from Dr. E. Nitsch of Laevosan-Gesellschaft, Linz, Austria.

6 REFERENCES

Ballard, R.A., Simpson, R.J. and Pearce, G.R., 1990. Losses of the digestible components of annual ryegrass (Loliwn rigidum Gaudin) during senescence. Aust. J. Agric. Res., 41: 719-731.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.

Feingold, D.S. and Gehatia, M., 1957. The structure and properties of levan, a polymer of D-fructose produced by cultures and cell-free extracts of Aerobacter levanicum. J. Polym. Sei., 23: 783-790.

Gonzalez, B., Boucaud, J. and Langlois, J., 1989. Comparative estimation of non-structural carbohydrate contents in perennial ryegrass by enzymatic and high performance liquid chromatography. J. Plant Physiol., 134: 251-253.

Henson, CA. , 1989. Purification and properties of barley stem fructan exohydrolase. J. Plant Physiol. 134: 186-191.

Ilgoutz, S.C., Bonnett, G.D. and Simpson, R.J., 1993. Some colligative properties of fructans: implications for ryegrass (Lolium perenne L.) grown under cool conditions. In: A. Fuchs (Ed.), Inulin and Inulin-containing Crops, Studies in Plant Science, Vol. 3. Elsevier, Amsterdam, pp. 115-120.

Jones, M.G.K., Outlaw, W.H. and Lowry, O.H., 1977. Enzymic assay of 10"7 to 10"14 moles of sucrose in plant tissues. Plant Physiol., 60: 379-383.

Nilsson, U., Öste, R. and Jägerstad, M., 1987. Cereal fructans: hydrolysis by yeast invertase, in vitro and during fermentation. J. Cereal Sei., 6: 53-60.

Nitsch, E., Iwanov, W. and Lederer, K., 1979. Molecular characterization of sinistrin. Carbohydr. Res., 72: 1-12.

Rutherford, P.P. and Deacon, A.C., 1972. The mode of action of dandelion root /3-fructofuranosidases on inulin. Biochem. J., 129: 511-512.

Simms, P.J., Boyko, W.J. and Edwards, J.R., 1990. The structural analysis of a levan produced by Streptococcus salivarius SS2. Carbohydr. Res., 208: 193-198.

Smith, A.E., 1976. /3-Fructofuranosidase and invertase activity in tall fescue culm bases. J. Agric. Food Chem., 24: 476-478.

Smouter, H. and Simpson, R.J., 1989. Occurrence of fructans in the Gramineae (Poaceae). New Phytol., I l l : 359-368.

Yamamoto, S. and Mino, Y., 1985. Partial purification and properties of phleinase induced in stem base of orchardgrass after defoliation. Plant Physiol., 78: 591-595.


REGULATION OF ACTIVITY AND PROPERTIES OF INULINASES FROM ROOTS OF CICHORIUMINTYBUS L /

Anil K. GUPTA, Harsh JAIN, Narinder KAUR and Rangil SINGH Department of Biochemistry, Punjab Agricultural University, Ludhiana-141004, India

ABSTRACT

Inulin is a reserve carbohydrate in the roots of Cichorium intybus L. It is hydrolysed by inulinase during flowering and seed formation. Seventy to eighty five percent of inulinase was found to be firmly bound to the cell wall. The quantity of cell wall-bound inulinase proved to be too low to be used for commercial purposes. The level of soluble inulinase in the vascular bundles was generally higher than that in the root cortex. The activity of bound inulinases of vascular bundles and cortex was characterized by a sigmoidal velocity curve with increasing inulin concentration. Sucrose was a non-competitive inhibitor of bound inulinase from vascular bundles. It may play an important role in regulating the activity of this enzyme under in vivo conditions. Metabolites like phosphoenol pyruvate, 2-phosphoglycerate, 6-phosphogluconate, and nucleotides (ADP, ATP, UDP, UTP and UDP-glucose) changed the sigmoidal pattern of inulinase activity of vascular bundles to a near-hyperbolic pattern, thus suggesting enhancement of inulinase activity at low and inhibition at high inulin concentrations. Hg2+ was a very strong inhibitor of inulinases. The temperature optimum of bound inulinases from vascular bundles (45 °C) was higher than that of other inulinases (37 °C).

1 INTRODUCTION Sucrose has been commonly used as a sweetener in the food industry for a very long

time. However, in the last decade, D-fructose has gained popularity as a sweetener probably because of its nutritional properties. Inulin, a polymer of ß-2,1 -linked fructose residues, is the principal reserve carbohydrate of chicory (Cichorium intybus L.) roots. This polymer on complete hydrolysis by inulinase yields 95% fructose. Inulinase has been purified from a number of microbial sources (Vandamme and Derycke, 1983; Gupta et al., 1988) especially in attempts to get a potent inulinase for preparing fructose syrups from inulin. However, relatively few reports on this enzyme from plant sources are available.

# This work was supported by a grant from the Indian Council of Agricultural Research, New Delhi.

206

2 MATERIALS AND METHODS Chicory cv. Kalpa No. 1 was sown in the first week of November 1988. In the next

year's months of May-June, an erect shoot emerged from the reduced stem followed by flowering. After the shoot had reached a length of 15-20 cm, the plants were tagged and this stage was indicated as 0 day after shooting (DAS).

2.1 Enzyme preparation The roots of chicory, when collected after the emergence of the erect stem, have two

distinct regions: an inner fibrous part constituted primarily by vascular bundles and an outer non-fibrous one constituted mainly by the cortex. These regions were separated using a sharp razor. Five-g aliquots of each of these tissues were crushed in a chilled pestle and mortar and inulinase was extracted with 10 ml of 0.02 M phosphate buffer (pH 7.2) containing 0.1 M cysteine and 0.05 M sodium diethyldithiocarbamate. After the pH had been readjusted to 7.2 the extract was passed through two layers of muslin cloth. This extraction procedure was repeated three times with the residue. All filtrates were centrifuged separately at 5000 g for 10 min and assayed for inulinase activity. The ionically bound inulinase left after the extraction of soluble inulinase was solubilized using extraction buffer containing 1 M NaCl. This process was repeated two times. Attempts to solubilize cell wall-bound inulinase from the remaining residue with extraction buffer (pH 7.2) containing either 0.1 % Triton X-100 or 10 mM mercaptoethanol or 10 mM mercaptoethanol + 1 M NaCl or with 0.2 M sodium acetate buffer (pH 5.4) 4- 1 M NaCl were unsuccessful. Therefore, the residue (cell wall) was repeatedly washed with an excess of 0.2 M acetate buffer + 1 M NaCl (pH 5.4), then dried with filter paper, and subsequently used for measuring the cell wall-bound inulinase activity.

2.2 Estimation of cell wall-bound inulinase Reaction mixtures consisting of 100 mg of cell wall material of appropriate tissue,

10 ^moles of inulin (with an estimated MW of 5000) and 200 jumoles of acetate buffer (pH 5.4) in a total volume of 2 ml were incubated either at 45 or 37 °C (depending upon the temperature optimum of inulinases) for 30 min. The fructose liberated was estimated according to the method of Nelson (1944).

2.3 Estimation of soluble and ionically bound inulinases Soluble and ionically bound inulinase preparations were passed through a Sephadex

G-25 column by using 0.1 M acetate buffer (pH 5.4) to remove low-MW sugars. Assay mixtures consisting of 10 μπ ιο^ of inulin, 50 ^moles of acetate buffer (pH 5.4) and 300 μΐ of enzyme in a total volume of 1 ml were incubated at 37 °C for 30 min and fructose

207

so liberated was measured (Nelson, 1944).

3 RESULTS AND DISCUSSION The major part of the inulinase activity (70-85%) proved to be firmly bound to the

cell wall (Table 1) and was not extractable with extraction buffer (pH 7.2) containing 0.1 % Triton X-100 or 10 mM mercaptoethanol, or 10 mM mercaptoethanol + I M NaCl or with 0.2 M acetate buffer (pH 5.4) + 1 M NaCl. Though soluble inulinase constituted only 10-30% of the total inulinase activity, its activity was relatively high in vascular bundles as compared with that in the cortex (Table 1). Flood et al. (1973) had also observed that approximately 60% fructan hydrolase in the roots of chicory was present in a bound form. A related enzyme, phleinase or rather levanase, from timothy haplocorm was also found to be localized in the cell wall fraction (Mino and Maeda, 1974). On the contrary, in the tubers of Helianthus tuberosus L. and in the leaves of Hordeum vulgäre L., fructan exohydrolase was exclusively present in the vacuoles (Frehner et al., 1984; Wagner and Wiemken, 1986) which are also the storage sites for fructans in these plants (Edelman and Jefford, 1968; Wagner et al., 1983). The question arises which function the cell wall-bound inulinase should have in the roots when its substrate is not present in its immediate environment. Though only soluble inulinase appears to be physiologically important as it, along with inulin, is localized in the vacuoles, however, bound inulinases, depending upon the developmental stage of the plant, may determine the level of vacuolar inulinase. Since the vacuoles do not have any machinery to synthesize enzymes, it may be that cell wall inulinase under some unknown controlled mechanism is liberated from the cell wall and passes into the vacuole through the cytoplasm.

Cell wall material from 1 g of vascular bundles was found to be capable of forming

Table 1. Distribution of inulinase activity (nmoles of fructose liberated min"1 perlOO g tissue) in roots of C. intybus at different days after shoot emergence (DAS).

Tissue Inulinase Inulinase activity at DASa

0 20 40 60

soluble covalently bound ionically bound

soluble covalently bound ionically bound

433 3896

157

746 3970

203

195 696

0

743 1786

139

235 2620

0

597 1994

0

199 1278

0

201 2036

0

a Data represent mean of values obtained from four plants.

Cortex

Vascular bundles

208

around 20 nmoles of fructose min"1 (Table 1). Cell wall material obtained from 50 g of tissue will therefore be able to form only approximately 250-270 mg fructose in 24 h which appears to be too low for commercial exploitation. In C. intybus the content of free fructose in the roots increases during flowering and seed formation (Gupta et al., 1985) and can rise to as high as 18% of dwt (Gupta et al., 1989). Efforts need to be directed to separate this fructose from the rest of the root material.

The activity of cell wall-bound inulinases was characterized by a sigmoidal velocity curve with an increase in inulin concentration; and from a Hill plot the number of sub-units have been calculated to be 2 (data not shown). The present data are insufficient to rule out an alternative possibility implicating that calculated numbers of subunits and allosteric behaviour of cell wall-bound inulinase may be an artifact arising from covalent linkage of this enzyme to the cell wall components especially because of the monomeric nature of soluble inulinase which has been shown to obey Michaelis-Menten kinetics (Ciaessens et al., 1990).

Effects of various metabolites and metal ions on the activity of this enzyme were studied. Phosphoenol pyruvate, 2-phosphoglycerate, 6-phosphogluconate and nucleotides like ADP, UDP, UTP and UDP-glucose changed the sigmoidal pattern of inulinase activity

Fig. 1. Effect of different metabolites (1 mM) on the activity of bound inulinase from vascular bundles, ■, control; · , phosphoenol pyruvate; D, UTP; O, 6-phosphogluconate; A, 1,3-diphosphoglycerate; Δ, 3-phosphoglycerate and x, pyruvate. Effect of ADP, UDP-glucose and 2-phosphoglycerate was similar to that of phosphoenol pyruvate. Effect of ATP was similar to that of UTP.

209

of vascular bundles to a near-hyperbolic pattern, thus suggesting enhancement of inulinase activity at low and inhibition at high inulin concentrations (Fig. 1). Pyruvate was a potent inhibitor of inulinase (Fig. 1). Sucrose was found to be a non-competitive inhibitor of cell wall-bound inulinase of vascular bundles (Fig. 2). Edelman and Jefford (1964) have also reported a non-competitive inhibition of soluble fructan exohydrolase from H. tuberosus by sucrose whereas Rutherford and Deacon (1972) reported that none of two fructan exohydrolases separated by DEAE-cellulose chromatography from Taraxacum qfficinale Weber was inhibited by sucrose. Experiments with 14C incorporation from 1 4 C0 2 supplied to leaves have shown that during flowering and seed formation, the supply of sucrose from

Fig 2. Lineweaver-Burk plot for bound inulinase from vascular bundles in the presence of 25 mM sucrose. O, control and · , in presence of sucrose. Velocity is calculated as /*moles of fructose released.

Table 2. Effect of various metal ions on activity of inulinases.

Ion (1 mM) % Inulinase activity

Bound inulinase

Cortex Vascular bundles

Soluble inulinase8

Nil Zn2+

Mn2+

Cu2+

Hg2+

100 100 61 48 0

100 100 60 57 0

100 32 0

12 0

a This was a partially purified fraction obtained after ammonium sulphate precipitation and Sephadex G-150 column chromatography.

210

leaves to roots is significantly restricted (Gupta et al., 1991). This might be an important factor in the activation of inulinase during late stages of plant development. Soluble inulinase of vascular bundles was more sensitive to inhibition by metal ions than bound inulinases of vascular bundles and cortex (Table 2). EDTA did not inhibit inulinases. Bound inulinase of vascular bundles had a temperature optimum at 45 °C whereas that of bound inulinase of cortex and of soluble inulinase of vascular bundles was found at 37 °C.

4 REFERENCES

Claessens, G., Van Laere, A. and De Proft, M., 1990. Purification and properties of an inulinase from chicory roots (Cichorium intybus L.). J. Plant Physiol., 136: 35-39.

Edelman, J. and Jefford, T.G., 1964. The metabolism of fructose polymers in plants 4. ß-Fructofuranosidases of tubers of Helianthus tuberosus L. Biochem. J., 93: 148-161.


Flood, A.E., Price, R. and Rowe, H.F., 1973. Effect of 2,4-dichlorophenoxyacetic acid treatment on the soluble and cell wall bound protein in Cichorium intybus root tissue. Phytochemistry, 12: 1005-1008.

Frehner, M., Keller, F. and Wiemken, A., 1984. Localization of fructan metabolism in the vacuoles isolated from protoplasts of Jerusalem artichoke tubers {Helianthus tuberosus L.). J. Plant Physiol., 116: 197-208.

Gupta, A.K., Kaur, N. and Singh, R., 1989. Fructose and inulinase production from waste Cichorium intybus roots. Biol. Wastes, 29: 73-77.

Gupta, A.K., Mamta and Bhatia, I.S., 1985. Glucofructosan metabolism in Cichorium intybus roots. Phytochemistry, 24: 1423-1427.

Gupta, A.K., Mann, P., Kaur, N. and Singh, R., 1991. Profiles of enzymes of sucrose metabolism in the leaves of chicory {Cichorium intybus) during development. Plant Sei., 77: 191-196.

Gupta, A.K., Nagpal, B., Kaur, N. and Singh, R., 1988. Mycelial and extracellular inulinases from Fusarium oxysporum grown on aqueous extract of Cichorium intybus roots. J. Chem. Technol. Biotechnol., 42: 69-76.

Mino, Y. and Maeda, K., 1974. Metabolism of sucrose and phlein in the haplocorm of timothy. J. Jpn. Grassl. Sei., 20: 6-10.

Nelson, N., 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153: 375-380.

Rutherford, P.P. and Deacon, A.C., 1972. /5-Fructofuranosidases from roots of dandelion {Taraxacum offlcinale Weber). Biochem. J., 126: 569-573.

Vandamme, E.J. and Derycke, D.G., 1983. Microbial inulinases: fermentation process, properties, and applications. Adv. Appl. Microbiol., 29: 139-176.


Wagner, W. and Wiemken, A., 1986. Properties and subcellular localization of fructan hydrolase in the leaves of barley {Hordeum vulgäre L. cv Gerbel). J. Plant Physiol., 123: 429-439.


THERMOSTABLE INULINASES FROM ASPERGILLUS FICUUM: BIOCHEMICAL CHARACTERIZATION AND BIOTECHNOLOGICAL APPLICATIONS

J. BARATTI and M. ETTALIBI* Centre National de la Recherche Scientifique, Laboratoire de Chimie Bacterienne, BP 71, 13277 Marseille Cedex 9, France * Present address: Institut Agronomique et Veterinaire Hassan II, Rabat-Instituts, Morocco

ABSTRACT

Nine /5-fructosidases were purified from a commercial inulinase preparation of the thermotolerant fungus Aspergillusflcuum. All enzymes were active towards inulin and sucrose, with an I/S ratio lower than 1 (except two enzymes). They were all glycoproteins with a high sugar content. According to their mode of action towards inulin (exo or endo attack on fructosyl bonds) and to their a value (catalytic power on inulin divided by catalytic power on sucrose) they were classified as one invertase, five exo-inulinases and three endo-inulinases. The a value is proposed as a major criterion for inulinase definition. The crude mixture was immobilized by covalent linkage on porous glass beads. The resulting immobilized preparation showed interesting properties for inulin hydrolysis in batch and continuous reactors.

1 INTRODUCTION Enzymes degrading inulin (inulinases) are classified as 2,1-ß-D-fructan

fructanohydrolases (EC 3.2.1.7). These enzymes may be utilized for the preparation of high-fructose syrups (Fleming and GrootWassink, 1979) used as sweetener in the food industry or as fermentation medium for biofuel production (ethanol or acetone-butanol). The first enzymes isolated were of plant origin (Jerusalem artichoke, dahlia or dandelion), they were called inulinases since they hydrolysed inulin at a high rate and had very low activity towards sucrose. Later on, microbial inulinases have been isolated and studied (Vandamme and Derycke, 1983). In contrast with the plant enzymes they displayed high sucrose-hydrolysing activity in addition to inulinase activity and the difference with invertase (EC 3.2.1.26) was only based on the I/S ratio (activity towards inulin divided by activity towards sucrose). However, this classification is rather difficult to handle since most of the known inulinases have I/S ratios lower than 1 and, in addition, this ratio is dependent upon the assay conditions (Allais et ai, 1987).

212

In this work, the thermostable inulinases of the fungus Aspergillus ficuwn were purified to homogeneity and their molecular and kinetic properties determined. Immobilization by covalent binding on porous glass beads was also investigated to define conditions of inulin hydrolysis in bioreactors.

2 RESULTS 2.1 Enzyme purification

The commercial inulinase preparation from Novo Industries (Novozym 230) was subjected to ammonium sulphate precipitation, DEAE-Sephacel and DEAE-Trisacryl chromatography and filtration on Ultrogel ACA34. This resulted in the separation of nine proteins with inulinase activity. These 0-fructosidases were characterized (see later) as one invertase (Inv), five exo-inulinases (Exo I to Exo V) and three endo-inulinases (Endo I to Endo III). All proteins showed one peak upon analytical chromatography on a Mono-Q column (FPLC), one band on SDS-PAGE and one precipitation line with an antiserum raised against the crude preparation.

2.2 Molecular properties The molecular properties of the nine enzymes are shown in Table 1. All enzymes

(except invertase) had a high specific activity towards inulin (20-328 IU mg"1); they were purified 3.9 to 64.5-fold. They were all also active towards sucrose with I/S ratios ranging from 0.01 to 2.92. Only two enzymes (Endo I and III) were more active towards inulin than towards sucrose. Their molecular mass was determined by SDS-PAGE and gel filtration. It was higher for invertase (84 kDa) than for exo-inulinases (around 75 kDa) or endo-inulinases (64 kDa) (Table 1). Staining of the gels with the periodic acid-Schiff reagent

Table 1. Properties of the nine inulinases isolated from Aspergillus ficuwn.

Enzyme

Invertase Exo-inulinase I Exo-inulinase II Exo-inulinase III Exo-inulinase IV Exo-inulinase V Endo-inulinase I Endo-inulinase II Endo-inulinase III

Total protein (mg)

12.0 16.0 7.5 6.6 4.5 7.0

12.0 2.1 2.9

Specific (IU mg

Inulin

1.6 78.2

155.0 123.0 20.0 40.0 61.6

328.0 50.3

activity ')

Sucrose

139.0 499.0 427.0 650.0

66.0 127.0 27.8

381.0 17.2

I/S ratio

0.01 0.16 0.36 0.19 0.30 0.32 2.22 0.86 2.92

Molecular mass (kDa)

84 75 76 74 74 74 64 64 64

Sugar content (%, w/w)

31 41 39 24 23 24 25 22 33

213

revealed a positive band for all proteins. Therefore, the sugar content was estimated using the purified fractions. The inulinases were all glycoproteins with a high sugar content, ranging from 22 to 41% (Table 1).

The mechanism of inulin hydrolysis was investigated by incubation of the purified enzymes with inulin and analysis of the reaction products by thin-layer chromatography. Two characteristic patterns (not shown) were obtained. Inulinases Exo I-V liberated only fructose while inulinases Endo I-III liberated first oligofructosides and later on fructose. The invertase showed an exo-inulinase pattern for inulin hydrolysis.

The amino acid composition of inulinases Exo I, Exo II and Endo I was determined (results not shown). They were different but all showed a high content of Ser+Thr (21-22%), Asx+Glx (7-18%) and Gly (16%) and a low content of Arg+Lys+His (5%) and Met+Cys (1%). The TV-terminal sequence of inulinase Exo I was Phe-Asn-Tyr-Asp-Gln-Pro-Tyr-Arg.

Sedimentation coefficients were determined by using high-speed ultracentrifugation. Values of 5.3, 5.1 and 4.1 were found for inulinases Exo I, Exo II and Endo I, respectively.

2.3 Kinetic properties Measurement of the effect of temperature on activity towards inulin and sucrose

showed enzyme activity to be maximal at 60 °C for the three enzymes tested, with activation energies in the range of 22.1 to 33.9 kJ mole"1. That means that the A. ficuum inulinases were thermostable, which is of interest in relation to their biotechnological applications. The three enzymes were active from pH 3 to 8 with a maximum at pH 4.7. It should be noted that almost 40% of the maximal activity was retained at pH 8, which makes the use of these enzymes, under neutral or alkaline conditions, very attractive for either inulin or sucrose hydrolysis.

Since both endo- and exo-inulinases were present in the culture medium of A. ficuum a possible synergistic action between the two kinds of inulinases was investigated. For this purpose, inulin (20 g Γ1) was hydrolysed with Exo I (activity 32.7 IU ml"1) or Endo I (19 IU ml"1) or with a mixture of the two enzymes (544 IU ml"1). A strong synergism was observed (more than 10-fold) which may explain the high inulinolytic activity of the fungus.

Activity of the nine inulinases was investigated at different concentrations of inulin or sucrose. In all cases, Michaelis-Menten kinetics were obtained and the catalytic constant (&cat) and Michaelis constant were determined (Table 2), using an alleged molecular mass of 5000 Da for inulin. The highest Km value was observed for invertase and the lowest for endo-inulinase. The catalytic constant (kcat) was estimated using the molecular mass determined previously; it was maximal for exo-inulinase V and endo-inulinase III and

214

Table 2. Kinetic parameters for inulin and sucrose hydrolysis.

Enzyme

Invertase Exo-inulinase I Exo-inulinase II Exo-inulinase III Exo-inulinase IV Exo-inulinase V Endo-inulinase I Endo-inulinase II Endo-inulinase III

Inulin hydrolysis

^cat (sec-1)

0.050 0.078 0.195 0.127 0.290 0.458 0.060 0.162 0.350

Km (M)

0.047 0.015 0.011 0.010 0.012 0.007 0.001 0.010 0.005

UKm (M'1 sec"1)

1.0 5.2

17.7 12.7 24.9 65.5 60.0 16.2 27.0

Sucrose hydrolysis

^cat (sec"1)

0.223 0.113 0.207 0.175 0.098 0.345 0.038 0.053 0.035

*m (M)

0.037 0.032 0.034 0.045 0.027 0.045 0.085 0.048 0.050

KJKm (M"1 sec"1)

6.0 3.5 6.1 3.9 3.6 7.7 0.5 1.1 0.7

a*

0.18 1.5 2.9 3.3 6.8 8.5

133.3 14.4 37.7

■ a = {[kcJKJ^JKJs).

minimal for invertase. Very different results were obtained for sucrose hydrolysis. Michaelis constants were similar for all enzymes but the catalytic constant was highest for invertase, and exo-inulinases II and V. For all enzymes (except invertase) the catalytic power (ratio of &cat to Km) was higher for inulin than for sucrose. Therefore, we propose to use the ratio of catalytic powers for inulin and sucrose hydrolysis, which we named the a value, to determine whether an enzyme is an inulinase or an invertase. According to this definition, inulinases will be enzymes with an a value higher than 1, whereas invertases will be enzymes with an a value lower than 1. This method was used to classify the nine inulinases purified from A. ficuum. Another interesting feature of the a value is that it enables to distinguish between exo- and endo-inulinases. The former showed lower a values than the latter.

Compounds affecting enzyme activity were also tested. A strong inhibition was observed in the presence of 0.02 mM HgCl2, 0.3 mM AgN0 3 and 0.2 mM MnCl2.

Table 3. Immobilization of inulinases from Aspergillusficuum on porous glass beads of different porosities.

Pore Activity Activity in Immobilization Immobilized activity Retention diameter added washings yield (IU g"1) of activity (A) (iu) (iu) (%) (%)

Theoretical Experimental

120 763 260 66 503 300 60 375 763 260 66 503 265 53 875 763 400 48 363 148 41 1170 763 660 14 103 37 36

215

Activities towards inulin and sucrose were affected similarly. A slight activation was noticed in the presence of calcium ions.

2.4 Immobilization The crude inulinase preparation was directly used for covalent immobilization onto

porous aminated glass beads using glutaraldehyde. Supports with average pore diameters of 120, 375, 875 and 1170 A were used and the results are shown in Table 3.

The support with the lowest pore diameter showed the highest immobilization yield, highest activity and highest retention of activity. Thus, it was selected for all further experiments. Its activity per g of support is one of the highest reported in the literature on immobilized inulinases.

2.5 Properties of immobilized inulinases The pH-activity profiles were very similar for free and immobilized enzymes. In

contrast, the temperature activity profiles were different. The immobilized preparation showed maximal activity at 70 °C as compared with 60 °C for the free enzyme. Thus, some stabilization occurred during immobilization. Activation energies were similar for free and immobilized enzymes, suggesting the absence of diffusional limitations for inulin inside the beads of support.

Michaelis-Menten kinetics were observed for both free and immobilized enzymes with inulin as well as sucrose as substrates. The ^-values were similar for inulin (9.6 and 6.1 itiM for free and immobilized inulinases) and for sucrose (28.6 and 17.9 mM). This result further confirms the lack of diffusional limitation inside the beads.

2.6 Enzymatic bioreactors Solutions containing 5, 10 and 15% (w/v) inulin were hydrolysed using the

immobilized inulinase preparations at temperatures of 40, 50 and 60 °C. For an inulin

Table 4. Inulin hydrolysis in batch reactors with immobilized inulinases as affected by temperature.

Inulin concentration (%, w/v)

5 10 15

40 °C

Via

13.3 19.0 24.2

tb

4.1 5.5 6.5

Pc

13.5 20.2 25.6

50 °C

V|

36.9 44.4 49.3

t

34 37 41

P

1.6 3.0 4.1

60 °C

v i

18.7 24.7 31.5

t

<45 <50 >68

P

<1.2 <2.2 <5.3

a Vj, initial reaction rate in g of reducing sugar liberated per litre and per hour (g Γ1 h'1). b t, time (h) for complete hydrolysis. c P, overall productivity in g of reducing sugar liberated per litre and per hour (g Γ1 h"1).

216

Table 5. Comparison of performances of continuous reactors for inulin hydrolysis.

Inulin concentration (%)

7 0.5 6.5a

10a

5 5 5 5 5

Space velocity Or1)

0.26 2.25 1.24 0.22 0.11 0.21 0.30 0.34 0.42

Inulin conversion (%)

90 100 100 90

100 100 100 100 99

Volumetric productivity (g r1 h-1)

18.0 1.2

88.5 2.4 6.1

11.7 16.7 18.9 23.1

Specific productivity (g g"1 h"1)

0.12 0.005 1.35 0.018 0.55 1.05 1.49 1.69 2.07

Half life (days)

13.9 >21 >10 >14 17.9 ----

Reference

Kim et al, 1982 Guiraudeftf/., 1981 B. and M.b, 1985 Kim and Rhee, 1989 this paper this paper this paper this paper this paper

a Inulin from Jerusalem artichoke. b B. and M., Bajpai and Margaritis.

concentration of 5% (w/v) 100% hydrolysis was observed at 40 and 50 °C, but not at 60 °C (Table 4), most probably because of enzyme inactivation at the latter temperature. The temperature of 40 °C was selected for continuous reactor operation in a fixed-bed reactor (total volume 21.2 ml, H/D ratio = 8, filled with 11.2 g of immobilized inulinase preparation). The effect of different flow rates (expressed as space velocity) is shown in Table 5. For space velocities lower than 0.42 h"1 99% hydrolysis was obtained, resulting in a volumetric productivity of 23.1 g sugar Γ1 h"1 {i.e. 2 g sugar g"1 support h"1). These values compared favourably with data in the literature. The operational stability of the continuous bioreactor was 17.9 days.

3 REFERENCES

Allais, J.-J., Hoyos-Lopez, G., Kammoun, S. and Baratti, J., 1987. Isolation and characterization of thermophilic bacterial strains with inulinase activity. Appl. Environ. Microbiol. 53: 942-945.

Bajpai, P. and Margaritis, A., 1985. Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 2. Application for high fructose syrup production. Enzyme Microb. Technol., 7: 459-461.

Fleming, S. E. and GrootWassink, J.W.D., 1979. Preparation of high-fructose syrup from the tubers of the Jerusalem artichoke (Helianthus tuberosus L.). CRC Crit. Rev. Food Sei. Nutr., 12: 1-28.

Guiraud, J.P., Demeulle, S. and Galzy, P., 1981. Inulin hydrolysis by the Debaryomycesphaffii inulinase immobilized on DEAE cellulose. Biotechnol. Lett., 3: 683-688.

Kim, C.H. and Rhee, S.K., 1989. Fructose production from Jerusalem artichoke by inulinase immobilized on chitin. Biotechnol. Lett., 11: 201-206.

Kim, W.Y., Byun, S.M. and Uhm, T.B., 1982. Hydrolysis of inulin from Jerusalem artichoke by inulinase immobilized on aminoethylcellulose. Enzyme Microb. Technol., 4: 239-244.



COMPARATIVE STUDIES OF SOLUBLE AND IMMOBILIZED INULIN-HYDROLYSING ENZYMES

L. BOROSS*, W. PRAZNIK**, J. KOSÄRY* and E. STEFANOVITS* Department of Chemistry and Biochemistry, University of Horticulture and Food Industry, P.O. Box 53, 1502 Budapest, Hungary Institute of Chemistry, Agricultural University, Gregor Mendel Straße 33,1180 Vienna, Austria

ABSTRACT

The possible use of inulin-hydrolysing enzymes in the qualitative and quantitative analysis and the preparative hydrolysis of fructans were studied. Whereas the cleavage of the terminal 0-2,1-fructosyl-fructose bond could be easily achieved by different enzymes such as inulinase, invertase, and glucoamylase preparations, the special enzymic removal of the terminal glucose moiety was not accomplished.

The inulin-hydrolysing enzymes were immobilized by covalent binding onto various supports and the kinetic properties of the preparations were characterized. The exo- and endo-inulinase components of the commercial Novo inulinase preparation were partially purified by ion exchange fractionation, using a modified procedure of the method described by Azhari et al. (1989). The formation of intermediate and end-products of the hydrolysis of inulin by the separated enzymes was analysed by spectrophotometric and thin-layer Chromatographie methods. Beside mono- and oligosaccharides, two by-products of more apolar character were found in the reaction mixtures of both enzymes.

Some of the immobilized inulinase preparations could be used effectively in bioreactors for the hydrolytic degradation of inulin.

1 INTRODUCTION Hydrolysis of inulin to fructose can be easily accomplished with inulinase. The

enzyme (EC 3.2.1.7, 2,1-0-D-fructan fructanohydrolase), is of either microbial or plant origin (Adams et al., 1943; Snyder and Phaff, 1962; Edelman and Jefford, 1964; Avigad and Bauer, 1966; Flood et al., 1967; Zittan, 1981; Vandamme and Derycke, 1983). Since the industrial potential of fructose, as the sweetest naturally occurring nutritive sweetener, is being more and more emphasized, fructans and fructan-hydrolysing enzymes seem to become increasingly important with regard to the large-scale industrial production of fructose.

Fructan-hydrolysing enzymes seem to be also of importance in the analysis of various

218

fructans, in particular because enzymatic hydrolysis can be carried out under very mild conditions and formation of by-products is negligible. Within the framework of our investigations concerning the application of enzymes in the analysis of plant sugars, we performed comparative studies on some soluble and immobilized inulin-hydrolysing enzymes.

2 MATERIALS AND METHODS Dahlia inulin was a gift of Reanal Fine Chemical Co., Budapest. Inulinase (Novozym

230, Novo Industri A/S, Copenhagen), α-glucosidase (Sigma Chemical Co., U.S.A.), amyloglucosidase (Sigma Chemical Co., U.S.A. and Serva Feinbiochemica GmbH Co., Heidelberg), invertase (from baker's yeast, Sigma Chemical Co., U.S.A.), N-hydroxysuccinimide-agarose (Sigma Chemical Co., U.S.A.), Acrylex P-100 (Reanal Fine Chemical Co., Budapest), and CM-cellulose (Serva Feinbiochemica GmbH Co.) were commercial preparations. All other chemicals were commercial products of analytical grade.

For TLC analysis of sugars DC-Alufolien Kieselgel 60 F254 (E. Merck, Darmstadt) plates were used, with butanol/propanol/ethanol/water (20:30:30:20, v/v/v/v) as the solvent system. In the quantitative analysis of sugars the Boehringer (Mannheim) UV test was used.

Immobilization of enzymes on Acrylex P-100 gel was performed as described by Kaiman et al. (1983). For covalent immobilization on A/-hydroxysuccinimide-agarose the suspension was filtered on a glass filter by suction, and washed twice with cold distilled water; the enzyme solution was added to the gel. After mixing the suspension was transferred into a test tube and incubated in the cold (5 °C) during one night. Thereafter, it was filtered and washed twice with 0.1 M phosphate buffer (pH 7.0), and then once with an equal volume of 1.0 M ethanolamine solution (pH 7.0). Finally, the gel was washed, in five equal portions, with a double volume of the appropriate buffer solution.

3 RESULTS AND DISCUSSION Novozym 230 is a complex enzyme preparation. It contains both exo- and endo-

inulinases. For the separation of the two different enzymes we used the ion exchange Chromatographie method described by Azhari et al. (1989), with some minor alterations. The enzyme solutions, gel-filtered on a Sephadex G-25 column or dialysed against 0.01 M sodium acetate buffer (pH 4.0) were applied to a CM-cellulose column, previously equilibrated with the same buffer. Protein concentrations in the effluent fractions were determined by the biuret method. After all of the non-bound protein was washed out from the column, the bound protein was eluted with 0.1 M acetate buffer (pH 5.0).

We have found, in good agreement with the results of Azhari et al. (1989), that the non-bound protein fraction {i.e. the first peak) contained the endo-inulinase and the eluted

219

peak the exo-inulinase enzymes (Fig. 1). To be sure that the fractionation obtained was not an apparent one, we repeated the fractionation in an experiment with two 5-ml CMC columns connected one after the other: the effluent from the first column flowed to the top of the second column. After the non-bound protein was washed out, the two columns were disconnected and eluted separately. The eluant of the first column contained practically all of the bound enzyme (exo-inulinase, fraction B in Fig. 1), but the solution eluted from the second column (fraction C) contained very little protein and a very low inulinase activity. Also in this experiment, the non-bound protein fraction (A in Fig. 1) contained the endo-inulinase-type enzyme. When this enzyme was added to a 5% inulin solution, the reducing sugar content increased faster than the fructose content, whereas with the bound and eluted enzyme in a comparable experiment fructose and reducing sugar concentrations were similar all the time. This indicates that the latter enzyme is a real exo-inulinase, and the non-bound enzyme an endo-inulinase. However, the rate of formation of reducing sugars was only about twice as high as that of fructose. This suggests that the endo-inulinase has also relatively strong exo-inulinase-type activity (i.e. it has a relatively high affinity to the terminal fructoside bond of the inulin chain), or this fraction contains, in addition to the endo-inulinase activity, an exo-inulinase type impurity, which shows ion exchange characteristics different from those of the exo-inulinase of peak B.

Effluent volume

Fig. 1. Ion exchange Chromatographie separation of the endo- and exo-inulinase components of Novozym 230 preparation. Solid line: protein concentration, dotted line: inulinase activity. Peak A (non-bound fraction) corresponds to the endo-inulinase component and peak B and C to the exo-inulinase component(s). The twin 5-ml CMC columns were loaded with 7 ml Novozym solution dialysed against 0.01 M acetate buffer (pH 4.0).

220

Analysis of the kinetics of the separated exo- and endo-inulinase using sucrose as a substrate showed that both enzymes had a similar affinity towards sucrose (Km = 8.3 and 8.0 mM for exo-inulinase and endo-inulinase, respectively). A similar Km value was obtained for inulin as the substrate when the exo-inulinase enzyme reactions were analysed (Km = 8.3 mM). These results indicate that this enzyme exhibits practically the same affinity for the fructoside bond of sucrose and that between the terminal fructose units in the inulin chain. All of our calculated Km values are lower than those published by Azhari et at. (1989). Our experiments showed that the ion exchange separation of the exo- and endo-inulinase components of Novozym 230 preparations could be performed as well in a batch process using the same buffers as above.

In another experiment we tried to specifically remove the glucose moiety from the other end of the inulin chain. Neither α-glucosidase (maltase) nor amyloglucosidase gave positive results. However, the latter enzyme exhibited some inulinase-like activity. Presumably, the commercial preparation contained some inulinase impurity. However, the Sigma and the Serva preparations showed similar inulinase activities.

The properties of various immobilized forms of the enzymes were also investigated. The continuous hydrolytic activities of some column microreactors, filled with different immobilized inulinases, are shown in Fig. 2. From these results the conclusion can be drawn that these preparations could be used effectively in bioreactors for the production of fructose from inulin. The catalytic properties of our previously immobilized ISB and ASB

7 r— 1 1 S Ί0 ^5 2.0 _v

Fig. 2. The efficiency of the hydrolysis with different immobilized inulinase-containing microreactors as a function of relative flow rate. Substrate: 5% dahlia inulin in distilled water. Fillings: ISB- (·), agarose-bound (O), ASB- (Δ) and Acrylex-bound inulinase (x).

221

inulinase preparations will be described in detail in a separate paper. Our results, obtained with 5-ml microreactors, match those described by Kim et al. (1982), Guiraud et al. (1983), and Parekh and Margaritis (1986), for inulin hydrolysis.

TLC analysis of the products formed in the reaction mixtures containing inulin and inulinases showed two relatively weak spots at higher Rf values (0.77 and 0.74) than fructose or glucose. Investigations on the chemical nature of these by-products are in progress.

4 REFERENCES

Adams, M., Richtmyer, N.K. and Hudson, C.S., 1943. Some enzymes present in highly purified invertase preparations; a contribution to the study of fructofuranosidases, galactosidases, glucosidases and mannosidases. J. Am. Chem. Soc, 65: 1369-1380.

Avigad, G. and Bauer, S., 1966. Fructan hydrolases. Methods Enzymol., 8: 621-628. Azhari, R., Szlak, A.M., Ilan, E., Sideman, S. and Lotan, N., 1989. Purification and characterization of

endo- and exo-inulinase. Biotechnol. Appl. Biochem., 11: 105-117. Edelman, J. and Jefford, T.G., 1964. The metabolism of fructose polymers in plants 4. ß-

Fructofuranosidases of tubers of Helianthus tuberosus L. Biochem. J., 93: 148-161. Flood, A.E., Rutherford, P.P. and Weston, E.W., 1967. Effects of 2 : 4-dichlorophenoxyacetic acid on

enzyme systems in Jerusalem artichoke tubers and chicory roots. Nature (London), 214: 1049-1050. Guiraud, J.P., Bajon, A.M., Chautard, P. and Galzy, P., 1983. Inulin hydrolysis by an immobilized yeast-

cell reactor. Enzyme Microb. Technol., 5: 185-190. Kälmän, M., Szajäni, B. and Boross, L., 1983. A novel polyacrylamide-type support prepared by p-

benzoquinone activation. Appl. Biochem. Biotechnol., 8: 515-522. Kim, W.Y., Byun, S.M. and Uhm, T.B., 1982. Hydrolysis of inulin from Jerusalem artichoke by inulinase

immobilized on aminoethylcellulose. Enzyme Microb. Technol., 4: 239-244. Parekh, S.R. and Margaritis A., 1986. Continuous hydrolysis of fructans in Jerusalem artichoke extracts

using immobilized nonviable cells of Kluyveromyces marxianus. J. Food Sei., 51: 854-855. Snyder, H.E. and Phaff, H.J., 1962. The pattern of action of inulinase from Saccharomyces fragilis on

inulin. J. Biol. Chem., 237: 2438-2441. Vandamme, E.J. and Derycke, D.G., 1983. Microbial inulinases: fermentation process, properties, and

applications. Adv. Appl. Microbiol., 29: 139-176. Zittan, L., 1981. Enzymatic hydrolysis of inulin - an alternative way to fructose production. Starch/Stärke,

33: 373-377.



IMMOBILIZATION OF INULINASE IN ACTIVATED CARBON FOR THE PRODUCTION OF VERY -HIGH-FRUCTOSE SYRUPS FROM JERUSALEM ARTICHOKE TUBER EXTRACTS

J.M. MAGRO*, M.M.R. da FONSECA and J.M. NOVAIS Laboratorio de Engenharia Bioquimica, Instituto Superior T^cnico - 1096 Lisboa Codex, Portugal * Permanent address: Faculdade de Farmäcia de Lisboa, Av. das Forgas Armadas, 1699 Lisboa Codex, Portugal

ABSTRACT

Activated carbon and tannic acid are cheap materials with a relatively low toxicity and therefore with potential applications in immobilization techniques used in large-scale food processes. An immobilization method developed for a fungal inulinase which was based on adsorption/cross-linkage on activated carbon with tannic acid as a cross-linking agent performed well, both in terms of activity of the immobilized preparation and of operational stability in continuous packed-bed reactors. The method may be of interest in the enzymatic hydrolysis of Jerusalem artichoke tuber extracts for the production of very-high-fructose syrups.

ABBREVIATIONS

C: concentration in g Γ1; INU: unit of inulinase activity; NRS: non-reducing sugars; RS: reducing sugars; TS: total sugars. Subscripts: i: inlet stream to the reactor, o: outlet stream from the reactor.

DEFINITIONS

Degree of conversion: X = (CRS -CRS.)/CNRS.. Instability factor: ratio of the RS produced during the first 20 min after removal of the immobilized preparation from the reaction medium and the RS initially present. Retention of activity: ratio of the specific activities of immobilized and soluble inulinase. Unit of inulinase activity: amount of enzyme which produces 1 micromole of RS per min at 50 °C, pH 4.7.

1 INTRODUCTION Fructose is the sweetest natural sugar and is reported to have 1.5 to 2.0 times the

sweetness of sucrose. In addition, fructose has a higher solubility in water, and is less viscous and less canogenic than sucrose. Low levels of fructose can be metabolized without

224

a need for insulin, thus enabling its use by diabetics. The fructose polymer inulin replaces starch as the reserve carbohydrate in some

Compositae, where it accumulates either in the tubers, as e.g. in Jerusalem artichoke, or in the roots, like in chicory. In Jerusalem artichoke and chicory these inulin analogues are linear polymers of 0-2,1 -linked fructose units and a terminal glucose unit. Very-high-fructose syrup (75-95%) can be prepared by direct - either acid or enzymatic - hydrolysis of Jerusalem artichoke tuber and chicory root extracts. Enzymatic hydrolysis is to be preferred since, due to the mild reaction conditions, unfavourable side-reactions and the formation of an undesirable taste and odour are avoided.

Activated carbon displays many attractive properties for use as support for immobilized enzymes: (a) it is reasonably priced, (b) it can be obtained with high surface areas (600-1500 m2 g"1), (c) 10-30% of its pore volume is in the 300-1000 Ä range and is therefore suitable for

enzyme immobilization, and (d) it has a high mechanical strength which is useful for operation in a packed-bed mode.

Therefore, attempts were made to use activated carbon as an immobilization matrix for inulinase, with the aim of achieving hydrolysis of Jerusalem artichoke tuber extracts. Since adsorption is a rather weak immobilization method for continuous operation, adsorption/cross-linkage and covalent linkage were investigated. A certain emphasis was given to the use of tannic acid as cross-linking agent due to its ample availability, low price and reduced toxicity. The different immobilization methods were compared in terms of activity of the immobilized preparation and operational stability.

2 MATERIALS AND METHODS 2.1 Materials

a. Enzyme: Inulinase (2,1-ß-D-fructan fructanohydrolase, EC 3.2.1.7) from Aspergillus in the form of a crude extract ("Novozym 230") was kindly made available by Novo Nordisk, Denmark.

b. Substrates: Inulin (analytical grade) was purchased from Merck, Germany; inulin-containing extracts were prepared from the tubers of Jerusalem artichoke (Helianthus tuberosus L.) clone Braganga (which had been kept frozen since the time of harvest).

c. Immobilization matrix and reagents: Activated granulated carbon (20-40 mesh) was from Aldrich Chemical Co. Inc., U.S.A.; gallotannic acid was from J.T. Baker Chemical Co., U.S.A.; carbodiimide was from Sigma Chemical Co., U.S.A. and glutaraldehyde was from Merck, Germany.

225

2.2 Extraction After thawing, the tubers were washed, diced and extracted in a juice extractor. The

turbid extract obtained was filtered through cheese-cloth and Whatman no. 2 filter paper on a Büchner funnel. A typical extract contained 300 g TS Γ1.

2.3 Immobilization ofinulinase (a) For batch operation, immobilization mediated by carbodiimide was carried out

using the method of Cho and Bailey (1978) with modifications. With the glutaraldehyde method, 0.5 g of carbon was added to 0.5 ml ofinulinase stock solution in phosphate buffer (pH 7) and this agitated in a shaking bath (100 rpm) at room temperature for 1.5 h. The preparation was then rinsed with 3 x 30 ml phosphate buffer. The wet cake was mixed with 10 ml of 2% glutaraldehyde solution in phosphate buffer and agitated for 1 h at 100 rpm. The immobilized preparation was finally rinsed with 3 X 30 ml phosphate buffer and stored at + 5 °C. The tannic acid method was carried out using the same conditions described for the glutaraldehyde method, except that borate buffer and a 5% tannic acid solution were used.

(b) For continuous reactor operation, the immobilization was carried out by the reactor loading process (in situ) with recirculation of cross-linking agent and inulinase solutions through the packed-bed reactor which contained granulated activated carbon; the flow direction was changed periodically.

2.4 Analytical methods Total and reducing sugars were assayed by the method of Somogyi-Nelson (Somogyi,

1952), with and without acid hydrolysis, respectively (pH 2, 100 °C, 30 min); protein was assayed by the method of Lowry et al. (1951).

2.5 Activity of immobilized inulinase The initial activity was assayed by adding 2.5 ml of substrate solution (2.5% NRS)

to 10 mg of the immobilized preparation in 2-cm diam. test tubes. The tubes were placed in a shaker (100 rpm) at 50 °C. After 10 min the reaction was stopped by boiling the liquid for 10 min in a water bath. After cooling, the samples were assayed for TS and RS. Blanks were prepared using carrier without enzyme. The results were expressed in INU g"1 dried immobilized preparation.

2.6 Operational stability In situ immobilization of inulinase was carried out by three different methods on 1

g of granulated carbon in identical columns which were immersed in a water bath. These

226

small reactors were operated at 50 °C for 20 days; the feed was tuber extract (80 g NRS l"1) at a flow rate of 1.2 ml h1.

2.7 Reactor operational studies Jacketed glass columns (18 cm high, 1 cm inner diam.) fitted with plates of sintered

glass at the bottom, were packed with granulated carbon to attain a bed height of 15 cm approximately. Constant temperature operation was achieved through recirculation of water of constant temperature (50 °C) through the jacket. The pressure drop introduced by the catalytic bed was measured by means of a simple manometer which was connected to the ends of the column. The inlet stream (a tuber extract containing 252 g NRS Γ1), was pumped downflow at a rate of 4 ml h"1. The feed reservoir was kept at 4- 5 °C. Size separation of the carbon particles was carried out with a set of ASTM sieves.

3 RESULTS 3.1 The choice of the immobilization method

Simple adsorption of inulinase onto activated carbon was examined first. The activity of the immobilized preparation was compared with that obtained by covalent binding mediated by a carbodiimide reagent and by adsorption followed by cross-linkage with glutaraldehyde and tannic acid.

The preparation obtained by the diimide method gave the lowest activity and the highest instability factor for both substrates. A much better performance was obtained with the two methods involving a cross-linking agent (Table 1). The activity of the immobilized preparation was always higher towards the Jerusalem artichoke tuber extract than towards pure inulin. These two substrates have average molecular weights of 1000 and 5000, respectively. The lower activity with inulin might be due to impediment of substrate transfer through the pores of the immobilization matrix, as compared to much smaller internal mass transfer limitations occurring in the case of the tuber extract. The difference in activity of the immobilized enzyme prepared with tannic acid against the two substrates was, however, much smaller. This might be explained in one or both of the following ways: a) Due to the very high activity of the tannic acid preparations, the reactions catalysed by them could have been substrate-limited, since the reactions of all preparations were performed under standard conditions (cf. 2.3). b) The high activity of the tannic acid preparations is probably due to the fact that the network formed by enzyme and cross-linking agent is more open and mainly located on the support surface, rather than in the pores, as compared to that obtained with glutaraldehyde. Thus, internal diffusion problems would have been less crucial for both the pure inulin and the tuber extract.

227

Table 1. Performance of the immobilized preparations with respect to hydrolysis of pure inulin (I) and Jerusalem artichoke tuber extract (E).

Immobilization method

Simple adsorption Diimide

Glutaraldehyde

Tannic acid

Substrate

E

I E I E I E

Activity (INU g"1 of immobilized preparation)

733.0

97.6 172.8 281.6 819.6 901.4 976.3

Immobilized protein (mg g"1 of immobilized preparation)

-

21.9 21.2

131.4 133.0 63.2 62.7

Specific activity (INU mg"1

protein)8

-

4.4 8.2 2.1 6.2

14.3 15.6

Retention of activity

l o f (%)

-

7.9 14.6 3.8

11.1 25.5 27.9

Instability factor (%)

20.2 18.4 5.2 4.8 2.0 2.0

8 Specific activity of soluble inulinase: 56 INU mg"1 of protein.

Table 2. Optimization of coupling conditions for the adsorption of inulinase onto activated carbon with cross-linkage.

Glutaraldehyde Tannic acid

Concentration of cross-linking agent solution (%, w/v) 2 pH of cross-linking agent solution 78

pH of enzyme solution 3C

Temperature during contact between support 40 and cross-linking agent (°C) Temperature during contact between support/cross-linking 10 agent complex and inulinase (°C)

5 5b

7d

0

10

a,b,c,d Buffers used were, respectively, (a) 0.2 M phosphate buffer, (b) 0.02 M acetate buffer, (c) 0.2 M phthalate buffer, and (d) 0.2 M borate buffer.

This preliminary screening led to the selection of the two cross-linkage methods for subsequent studies.

3.2 Optimization of immobilization conditions The influence of the order of addition of cross-linking agent and enzyme to the

carrier was investigated next (data not shown). For both cross-linking agents the highest activity and retention of activity were obtained by contacting the cross-linking agent with the carbon before the addition of the enzyme solution.

Immobilization tests under the above conditions were carried out in order to optimize the immobilization method (Table 2). The optimal contact time between the cross-linking

228

agent and the carrier and between the resulting complex and the inulinase solution was found to be 40 min, in both cases.

3.3 Operational stability The operational stability of the immobilized preparations obtained with the use of

cross-linking agents under optimized conditions was compared with that of inulinase immobilized by simple adsorption. The degrees of conversion observed during 20 days of continuous operation were expressed in terms of relative activity, i.e. as a percentage of the initial packed-bed activity. The use of a cross-linking agent is clearly advantageous (Fig. 1).

3.4 Reactor operational studies The influence of particle size on the degree of conversion and the pressure drop was

studied for enzyme reactors where the immobilization had been carried out by the optimized tannic acid method. When the particle size increased from 180-250 μνα to 750-1000 μπι conversion decreased from 95% to 82%. The pressure drop decreased regularly with the increase in particle size (Fig. 2).

These results reflect the extent of internal diffusion limitations. The degree of conversion was relatively low, however, for the smallest particles (75-125 μνα). Within this range of small particle sizes the pressure drop was very large which might have originated channelling. The inefficient feed distribution probably led to suboptimal utilization of the enzyme load, and thus to a lower degree of conversion.

100

> i 90 '■£ o (0

a> 1 80 0)

0 5 10 15 20 Time (days)

Fig. 1. Operational stability of inulinase immobilized by adsorption (■) and adsorption/cross-linkage with glutaraldehyde (A) and tannic acid ( · ) .

l ^ ^ f

229

360

280

200 7 <

120

40 0 250 500 750 1000

Par t i c le s ize (μιη)

Fig. 2. Continuous hydrolysis of Jerusalem artichoke extract in packed-bed reactors by inulinase immobilized in activated carbon by the optimized tannic acid method: influence of particle size on degree of conversion (D) and pressure drop (■).

4 CONCLUSIONS AND FUTURE WORK An immobilization method based on adsorption/cross-linkage in granulated activated

carbon treated with a multifunctional reagent (glutaraldehyde or tannic acid) was developed for inulinase and resulted in highly active immobilized preparations with large operational stabilities, as compared with other methods. Under conditions optimized for enzyme coupling, the operational stability was higher with tannic acid. This immobilization method might become of industrial interest for the production, by enzymatic hydrolysis of fructan, of very-high-fructose syrups from fructan-rich crops.

Preliminary work carried out in continuous packed-bed reactors led to the selection of a particle size which results in maximal conversion. Current work is aimed at achieving enzyme reloading in situ.

Future work will concentrate on optimization of reactor operation: either in a packed-bed or a fluidized-bed mode. It is of interest to establish whether the latter mode of reactor operation, though more energy-consuming, performs better than the former with the small-size ( < 180 μπ\) carbon particles. In process optimization reactor productivity and energy consumption are among the most important variables. Upscaling of the method has not been

230

attempted so far. Since the transport parameters vary considerably with scale - in particular in the case of fluidized beds - special attention should be paid to dispersion effects.

5 ACKNOWLEDGEMENT This work is part of a research project sponsored by Junta Nacional de Investigagäo

Cientifica e Tecnologica, Lisboa.

6 REFERENCES

Cho, Y.K. and Bailey, J.E., 1978. Immobilization of enzymes on activated carbon: properties of immobilized glucoamylase, glucose oxidase, and gluconolactonase. Biotechnol. Bioeng., 20: 1651-1665.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275.

Somogyi, M., 1952. Notes on sugar determination. J. Biol. Chem., 195: 19-23.


PREPARATION AND CHARACTERIZATION OF AQUEOUS INULIN/EXO-INULINASE SYSTEMS

A. HUBER*, W. PRAZNIK**, T. SPIES**, S. KAMMERER** and C. KNABL* Institut für Physikalische Chemie der KF-Universität, Heinrichstrasse 28, 8010 Graz, Austria Institut für Chemie der Universität für Bodenkultur, Gregor-Mendel-Strasse 33, 1180 Wien, Austria

ABSTRACT

As a first step in the characterization of inulin/inulinase systems the specific mode and extent of the exo-inulinase-catalysed hydrolysis of Jerusalem artichoke inulin was studied. A non-Michaelis-Menten kinetic approach was applied for an adequate characterization of enzymatic activity. Since average molecular weight and molecular weight distribution of commercially available inulin samples vary greatly depending on their origin, as a matter of fact, the ß-2,l-glycosidic linkages rather than the badly defined polymer/ oligomer mixture should be taken as the real substrate. Consequently, the hydrolysed glycosidic linkages have to be considered as the reaction "products". For an exo-inulinase purified from a commercially available crude inulinase preparation which attacked Jerusalem artichoke inulin, activities were found to range from 3.3 to 10.9 / kat Γ1 and to be characterized by a mechanism which kept the molecular weight distribution of the inulin constant until the final stages of hydrolysis. This behaviour might be explained as well by a single-chain mechanism as by a multiple-chain mechanism. In fact, at present, it can not be decided yet which of these may be the right one.

1 INTRODUCTION

Biotechnological utilization of inulin requires explicit information about characteristic physicochemical properties of the inulin-attacking enzyme systems and of the available inulin at all stages of the biotechnological process. Among many others, Zittan (1981), Ettalibi and Baratti (1987, 1990) and Azhari et al. (1989) have presented extensive information about the action of exo- and endo-inulinases from the point of view of products formed, but little is known about the continuously changing polymer substrates during enzymatic attack. This is unfortunate because for the characterization of inulin/inulinase systems in fact only the continuously changing molecular weight distribution (MWD) of the mixture of initial inulin and reaction products indicates the mode and extent of the enzymatic action. To gain information about these products, adequate enzyme preparation

232

and sampling of reaction products as well as use of sophisticated analytical techniques, specific detection methods and data-processing are required. HPLC, calibrated size-exclusion chromatography (SEC) and chromatography/multidetection - in the actual case size-exclusion chromatography coupled with differential refractometer index and low-angle laser light scattering detection (SEC-DRI/LALLS) - have been utilized for the determination of the absolute MWD of the various inulin products formed during exo-inulinase attack.

In the study of inulin/exo-inulinase kinetics a non-Michaelis-Menten approach was chosen to characterize enzymatic activity. With this approach not the polymer inulin as such but the glycosidic linkages are the real enzyme substrate, and hydrolysed glycosidic linkages are considered as reaction "products". Further, inulin changes from being a substrate to a "substrate supplier", which carries glycosidic linkages that vary in their susceptibility to enzymatic hydrolysis. It means that, in the study of inulin/inulinase kinetics, characterization of inulin at increasing levels of degradation, as well as determination of numbers and positions - both terminal and in-chain - of hydrolysed glycosidic linkages in the G-Fn-chain are required.

It should be realized that this approach, in fact, should not be specific for inulin/exo-inulinase systems, but could - or, maybe, even should - be used for all polysaccharide/hydrolase systems. The integrated application of preparative and analytical techniques supplemented with advanced data-processing offers an effective way to gain comprehensive information needed for the proper analysis of such systems. As an example of this approach, the action of exo-inulinase against inulin will be presented in this paper.

2 MATERIALS AND METHODS 2.1 Samples

For the preparation of exo-inulinase the commercially available Novozym 230 inulinase from Novo Industri (Copenhagen, Denmark) - with a protein content of 2.210 mg ml"1 - was used. Inulin was isolated from Jerusalem artichoke tubers and purified by repeated precipitation from aqueous solution.

2.2 Preparation of exo-inulinase As illustrated in Fig. 1 Novozym 230 inulinase was fractionated first into a low-

retention volume (Fl) and a high-retention volume (F2) fraction utilizing Sephacryl SF 200 in a preparative SEC-system (column dimensions: 61 mm, 16 mm ID). Then, both Fl and F2 were fractionated further by DEAE ion exchange chromatography (Sepharose Fast Flow, column dimensions: 200 mm, 16 mm ID) with a discontinuous NaCl gradient with steps at 0.0, 0.1, 0.2, 0.3, 0.4 and 1.0 M.

The consecutive eluants were examined for their specific activity by means of thin-

233

Novozym 230

preparative SEC

F1 * ^ F2 DEAE (NaCI step gradient)

Ion Exchange Chromatography

Thin Layer Chromatography pre-check of fraction activity

F1-NaCI F2-NaCI 0.0,0.1,0.2 0.0,0.3 m

Exo Inulinase pool

Fig. 1. Flow chart for the preparation of exo-inulinase from Novozym 230 inulinase.

layer Chromatographie (TLC) analysis of the products formed from an aqueous solution of inulin (50 mg ml"1). Protein fractions eluted with 0.0, 0.1 and 0.2 M NaCI from Fl and with 0.0 and 0.3 M NaCI from F2 were identified as exo-inulinases by yielding fructose only. These fractions were pooled and subsequently utilized as exo-inulinase in further investigations.

2.3 Analytical techniques (a) HPLC with NH2-inatrix. F° r the characterization of inulin oligomers and

monomers a Spherisorb S5-column with aminated matrix, acetonitrile/H20 (75:25, v/v) as the eluant at a flow rate of 1.0 ml min"1 and mass detection by means of a differential refractive index (DRI) detector was utilized.

(b) Calibrated SEC. In accordance with Praznik and Beck (1985), a Fractogel guard column HW40 (100 mm, 10 mm ID) plus HW40 (300 mm, 10 mm ID) and Superdex 75 (300 mm, 10 mm ID) was utilized, with 0.05 M aqueous KCl (+ 0.02% NaN3) as the

234

eluant, at a flow rate of 0.6 ml min"1. Mass detection with a DRI-detector was used for MWD determination.

(c) Chromatography-Multidetection: SEC-DRI/LALLS. Absolute MWD was determined using a column system consisting of a TSK PW5000 column (600 mm, 7.5 mm ID) plus a TSK PW3000 column (600 mm, 7.5 mm ID) with 0.05 M NaCl as the eluant, at a flow rate of 0.8 ml min"1. Mass detection was performed with a DRI-detector, whereas scattering intensity was detected with a low-angle (5°) laser light scattering (LALLS) detector.

The software package PCLALLS from LDC-Analytical was used for data acquisition and data-processing. Details on theory and application of the specified SEC-DRI/LALLS equipment utilizing the software package PCL ALLS for flexible data acquisition, data-processing and documentation in polysaccharide characterization have been published by Eigner et al. (1988), Huber and Billiani (1990) and Huber (1991).

2.4 Kinetics Since the classical Michaelis-Menten approach for kinetic characterization of enzyme/

substrate systems is only valid for monomeric substrates, for the polymeric substrate inulin a non-Michaelis-Menten approach was introduced. When being hydrolysed by inulinase, inulin (G-Fn) generates one or two new non-monomeric molecules, G-Fn.m + Fm, with m = 1 for exo-inulinase, and m > 1 for endo-inulinase. During degradation, of each inulin molecule (G-Fn, with n > 1) the degree of polymerization (n + 1) continuously changes in a very characteristic way, depending on the mode of enzymatic attack and on the, possibly changing, susceptibility of the ß-2,1-glycosidic linkages to enzymatic breakdown. While being broken down each inulin molecule passes all stages from "initial-state" inulin, via intermediate inulins + oligomers + monomers to, finally, a monomer mixture of fructose (plus some glucose). To escape from the complexity of having to consider inulin as a "collection" of various substrates we suggest to look upon inulin as a "substrate supplier", which provides ß-2,1-glycosidic linkages as the actual substrate for inulinase. The susceptibility to hydrolysis of each of these linkages strongly depends on the molecular properties of the "substrate supplier": initial-state inulin versus intermediate inulins plus oligomers. Consequently, the corresponding enzymatic activity should be defined as the number of broken glycosidic linkages within a given time interval and for a given reaction volume, and should be expressed as kat Γ1. By definition, 1 kat is the "amount" (mol) of ß-2,1-glycosidic linkages hydrolysed within 1 sec. Thus, for a defined inulin concentration (mg ml"1) and enzyme concentration (mg protein ml"1), enzymatic activity can be expressed as kat per unit reaction volume (kat Γ1).

Additional information is provided by the continuously changing shape of the MWD,

235

which, at the same time, reflects the specific, viz. either exo or endo, mode of enzymatic attack.

3 RESULTS AND DISCUSSION 3.1 Inulin/exo-inulinase: fructose screening

HPLC analysis of the intermediates produced from inulin by the exo-inulinase-containing fractions supported the assumption of a pure and specifically active exo-inulinase being present: only monomers, no oligomers were detected. The reaction rate for the inulin/ exo-inulinase system was determined in a first approach by measuring the amount of fructose produced during hydrolysis (Fig. 2, Table 1). Conditions were selected so that complete hydrolysis took about 3 h. As a first result, during the initial and midrange part of the reaction fructose production appeared to be almost linear with time, suggesting a constant susceptibility of the glycosidic linkages to hydrolysis by the exo-inulinase present.

3.2 Inulin/exo-inulinase: MWD screening For the same stages of progressing hydrolysis, for which the production of fructose

was estimated, SEC-DRI/LALLS experiments were performed to gain information about the absolute MWD of the inulins present (Fig. 3). The gradual change in MWD during inulin degradation strongly indicates an exo-type of inulin hydrolysis by either a single-chain

100 90 80

? 70 CD CO 0 8 3 L 4-Ό

6 "5 60 90 120 150 180 210 24Ö" time of reaction [min]

Fig. 2. Fructose production by exo-inulinase during degradation of Jerusalem artichoke inulin. For experimental conditions, see Table 1.

236

Table 1. Weight-average molecular weight (Mw), polydispersity (Mw/Mn), fructose yield and enzymatic activity upon incubation of Jerusalem artichoke inulin with exo-inulinase, in relation to time of incubation8.

Reaction time (min)

0b

5 10 15 30 60

120 240

Mw (g mor1) + 3%

6500 6000 5600 5100 4100 2800

700 200

Mw/Mn

± 5%

1.5 2.5 3.7 6.4

10.3 14.0 7.0 2.0

Fructose yield (%) ± 1%

0 2 6

12 24 45 83

100

Enzymatic activity μ (kat Γ1) ± 5%

3.3 4.5

10.9 8.0 8.0 7.8

n.d.c

a Experimental conditions: inulin solution (5.03 mg ml"1); exo-inulinase solution containing 4.0 μg protein ml"1; reaction volume 9.4 ml; pH 5.6; ionic strength 0.05 M NaCl; room temperature (20° C); the reaction was stopped by boiling aliquots of 1 ml at 95 °C for 2 min at selected intervals. b Initial stage before addition of exo-inulinase. c n.d., not determined.

or a multiple-chain mechanism. As a matter of fact, the available experimental data showing

negligible changes in MWD of the polymer/oligomer fraction during the consecutive stages

of degradation, equally support both hypotheses. Assuming a single-chain mechanism, the

Fructose

!og(M) [g/mol]

Fig. 3. Absolute molecular weight distributions (MWD), as derived from SEC-DRI/LALLS-experiments, of Jerusalem artichoke inulin while being degraded by exo-inulinase, in relation to time of hydrolysis.

237

15 c Σ > 12

V c HS 9 I —I

0 E > 6

Σ ,

0 30 60 90 120 150 180 210 240 time of reaction [min]

Fig. 4. Weight-average molecular weight (Mw) and polydispersity (Mw/Mn) of Jerusalem artichoke inulin while being degraded by exo-inulinase, in relation to time of hydrolysis.

various G-Fn chains should be equally susceptible to hydrolysis, the exo-inulinase should have no preference for any specific chain length, and intermediate oligomers will not be detectable because they will remain bound to the enzyme. On the other hand, if a multiple-chain mechanism should be present, the activity of the exo-inulinase towards each attacked G-Fn chain should be identical, and independent of the chain length. In both cases, the primary product of hydrolysis will be fructose, in the first case arising by the successive removal of fructose units from one inulin chain, by the action of one exo-inulinase molecule, and in the second case by the removal of one fructose unit at a time, from different inulin chains, by one exo-inulinase which after each hydrolytic step is detached from the inulin molecule and then either rebound to the same chain or bound to another.

With respect to number-average molecular weight (Mn) and weight-average molecular weight (Mw) (Fig. 4, Table 1) the initially high-molecular (Mw = 6500 ±3%) and narrowly distributed (Mw/Mn = 1.5 + 5%) inulin constantly decreased in Mw and Mn, went through a maximum in polydispersity (Mw/Mn) and passed through three successive stages of molecular distribution,viz. monomodality (inulin) - bimodality (inulin, fructose/glucose) - monomodality (fructose/glucose), respectively.

GMw

• Mw/Mn

238

"0 30 60 90 120 150 180 210 240 time of reaction [min]

Fig. 5. Exo-inulinase activity hydrolysing Jerusalem artichoke inulin in kat Γ1, in relation to time of hydrolysis.

3.3 Inulin/'exo-inulinase: polymer-substrate kinetics When molecular weight (weight and number) distributions and molecular weight

averages of polymers like inulin at increasing levels of degradation are determined by SEC combined with specific data-processing, basic information is provided for a non-Michaelis-Menten kinetic characterization of such systems. Probably, the most important aspect of this approach is that it provides information about the number-average molecular weight (Mn) of the investigated polymer sample which is closely correlated to the number of particles in the reaction mixture. When the reaction proceeds, an increasing number of particles are accompanied with a decreasing number of glycosidic linkages still available for enzymatic attack. Simultaneously, these linkages might become less susceptible to hydrolysis. For exo-inulinase hydrolysing Jerusalem artichoke inulin, the increasing number of particles are correlated with the number of broken ß-2,1 -glycosidic linkages. Thus, the number of broken linkages produced within a specified time interval and for a given volume provide information on the average enzymatic activity for this interval in kat Γ1 (Fig. 5, Table 1).

4 CONCLUSIONS The characterization of inulin/inulinase systems requires an exact knowledge about

the various components of the inulinase preparation and their specific action against well-

239

defined substrates. As a first step towards a biochemical and physicochemical characterization of such a polymer substrate/enzyme system exo-inulinase was purified from a Novozym 230 inulinase preparation and subsequently utilized to investigate the hydrolysis of Jerusalem artichoke inulin. Powerful analytical techniques like HPLC, calibrated SEC and chromatography/multidetection were employed to obtain comprehensive basic information for the characterization of all degradation products of inulin during its hydrolysis by exo-inulinase. The rate of hydrolysis of the glycosidic linkages was then expressed as kat Γ1. In particular, analysis of molecular weight distribution at specified stages of the enzymatic reaction proved to be essential for a subsequent kinetic characterization of the inulin/exo-inulinase system.

In order to have a consistent terminology, which can also be applied to other polysaccharide/hydrolase systems, we suggest to slightly modify the existing nomenclature: glycosidic linkages, rather than the polysaccharide itself, should be considered as the real substrate. The polysaccharide, in its turn, changes from "substrate" to "substrate supplier", because it provides the glycosidic linkages for hydrolysis. Consequently, hydrolytic activity is expressed as the number of glycosidic linkages hydrolysed within a specified time interval and in a specified reaction volume, as kat Γ1.

From the molecular weight distribution of the inulin during hydrolysis the mode of action of the exo-inulinase towards Jerusalem artichoke inulin could be characterized as a mechanism which kept the MWD of the polymer/oligomer fraction constant until the final stages of hydrolysis. This finding can be explained as well by assuming the operation of a single-chain mechanism as of a multiple-chain one.

In conclusion, application of the techniques described here to inulin/inulinase systems will provide valuable information for the optimization of biotechnological processes used in the degradation or conversion of inulin.

5 ACKNOWLEDGEMENT This work was supported by the Austrian 'Fond zur Förderung wissenschaftlicher

Forschung' project number 7104 CHE.

6 REFERENCES

Azhari, R., Szlak, A.M., Ilan, E., Sideman, S. and Lotan, N., 1989. Purification and characterization of endo- and exo-inulinase. Biotechnol. Appl. Biochem., 11: 105-117.

Eigner, W.-D., Abuja, P., Beck, R.H.F. and Praznik, W., 1988. Physicochemical characterization of inulin and sinistrin. Carbohydr. Res., 180: 87-95.

Ettalibi, M. and Baratti, J.C., 1987. Purification, properties and comparison of invertase, exoinulinases and endoinulinases of Aspergillusficuwn. Appl. Microbiol. Biotechnol., 26: 13-20.

Ettalibi, M. and Baratti, J.C., 1990. Molecular and kinetic properties of Aspergillus ficuwn inulinases.

240

Agric. Biol. Chem., 54: 61-68. Huber, A., 1991. Characterization of branched and linear polysaccharides by size-exclusion chromatography

/ low-angle laser light scattering. J. Appl. Polym. Sei.: Appl. Polym. Symp., 48: 95-109. Huber, A. and Billiani, J., 1990. PC-unterstützte Molekulargewichtsbestimmung von Polymeren über

Ausschluß-Chromatographie. GIT Fachz. Lab., 34: 131-139. Praznik, W. and Beck, R.H.F., 1985. Application of gel permeation Chromatographie systems to the

determination of the molecular weight of inulin. J. Chromatogr., 348: 187-197. Zittan, L., 1981. Enzymatic hydrolysis of inulin - an alternative way to fructose production. Starch/Stärke,

33: 373-377.


PRODUCTION AND LOCALIZATION OF INULINASE IN KLUYVEROMYCES YEAST

Marco C M . HENSING, Robert J. ROUWENHORST, W. Alexander SCHEFFERS and Johannes P. van DIJKEN Department of Microbiology and Enzymology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands

ABSTRACT

The production of inulinase (EC 3.2.1.7) by Kluyveromyces marxianus was studied in continuous and fed-batch cultures.

In sucrose-limited chemostat cultures growing on mineral media the highest inulinase activity in these cultures was 52 U mg1 dwt. The inulinase activity in these cultures decreased from 52 U mg"1 dwt at low growth rates to 2 U mg"1 dwt at high growth rates. This suggests that the inulinase production is negatively controlled by the residual sugar concentration.

In high-cell-density (> 100 g dwt Γ1) fed-batch cultures up to 70,000,000 units of supernatant inulinase were produced at a 100-litre scale.

In K. marxianus part of the inulinase is secreted into the culture fluid and another part is retained in the cell wall. In order to explain the difference in localization both enzymes were purified to homogeneity. Denaturing gel electrophoresis of endo-H-treated supernatant and cell-wall inulinase showed both enzymes to consist of a 64-kDa polypeptide. The degree of glycosylation was 27-37% (w/w). Non-denaturing gel electrophoresis showed the supernatant and cell-wall inulinase to differ in size, due to a difference in subunit aggregation. The enzyme present in the culture fluid was a dimer and the enzyme retained in the cell wall a tetramer.

The amino-terminal end of inulinase was determined and compared with the amino terminus of the functionally related invertase from Saccharomyces cerevisiae. No homology was found in the first 20 residues except for the first one, serine, indicating that invertase and inulinase are different enzymes.

1 INTRODUCTION

Yeasts of the genus Kluyveromyces are well known for their ability to grow on fructans like inulin. From a screening of several Kluyveromyces strains, Kluyveromyces marxianus var. marxianus CBS 6556 was selected for a study of parameters relevant for the commercial production of inulinase.

The inulinase (2,1-ß-D-fructan fructanohydrolase, EC 3.2.1.7) of Kluyveromyces is an extracellular enzyme, partially associated with the cell wall and partially secreted into

242

the culture fluid. Yeast inulinases are responsible for the exo-wise degradation of fructans, of both the inulin- and the levan-type. The monosaccharides released upon hydrolysis of inulin are transported into the cell, where they are metabolized.

In yeasts, another enzyme may be involved in the hydrolysis of fructans, namely invertase. Invertase differs from inulinase in being only active on fructans with a low degree of polymerization, while inulinase is also active against fructans with a high degree of polymerization (DP > 10). Some authors consider this difference in activity towards fructans insufficient for a distinction between inulinase and invertase. In their view, inulinase is a special kind of invertase and should be classified as such (for references see Rouwenhorst et al., 1988). Whereas for the invertase of Saccharomyces cerevisiae extensive data on its molecular characteristics are available, the inulinase of K. marxianus had not been characterized in detail.

For the inulinase of Kluyveromyces three different locations can be distinguished: a) supernatant (enzyme secreted into the culture fluid) b) cell-wall (enzyme released by treatment of the cells with ß-mercaptoethanol) c) cell (cell-bound enzyme only released by disruption of the cells).

In order to explain the differences in localization, supernatant and cell-wall inulinases were purified to homogeneity and characterized. The characteristics of the purified inulinases will be compared with those of invertase.

2 PROTEIN SECRETION BY YEASTS In the industrial production of proteins, the down-stream processing of the product

is one of the main cost factors. If, however, an initial protein separation proceeds within the organism itself, resulting in the secretion of the desired protein, the costs of downstream processing will be reduced.

Another advantage of secretion in eukaryotes is that many proteins naturally secreted, such as interferon and calf prochymotrypsin, are not biologically active unless they are glycosylated and proteolytically processed through the secretory pathway of the cell.

The externalization of secretory proteins in yeasts appears to be associated with cell growth. By using fluorescein-labeled antibodies, it has been observed that newly synthesized invertase and acid phosphatase are mainly deposited around the developing bud (Tkacz and Lampen, 1973; Field and Schekman, 1980). Polypeptides destined to be secreted possess a leader sequence, consisting predominantly of hydrophobic amino acid residues at the N-terminal end. These leaders route the protein to the endoplasmic reticulum. There, the core oligosaccharides are linked to the proper asparagine residue in Asn-X-Ser(Thr) tripeptides, in which X may be any amino acid. The composition of the yeast oligosaccharides is equal to that of the mammalian type (Glc3Man9GlcNAc2). From the endoplasmic reticulum the

243

glycoprotein is then transported to the Golgi apparatus by specific vesicles. In the Golgi, the core oligosaccharides are elongated by attachment of further mannose residues. By the Golgi the secretory proteins are packaged in other vesicles, transported to the budding part of the cell, and delivered outside the plasma membrane by exocytosis.

All extracellular proteins in yeasts are glycosylated. The physiological significance of this glycosylation is still unclear. However, the following functions have been ascribed to glycosylation: influence on the rate of turnover of the protein; influence on the stability of the protein; prevention of irreversible protein aggregation; and a role in the retention of the protein in the cell wall.

3 THE YEAST CELL WALL After exocytosis over the cell membrane, part of the extracellular proteins is retained

in the cell wall. Models for the mode of retention of extracellular proteins in the yeast cell wall have been proposed by Lampen (1968) and by Kidby and Davies (1970); only the last-mentioned model is briefly described here. According to the model of Kidby and Davies (Fig. 1) the glycoprotein is not bound to the cell wall, but is soluble within the cell matrix.

The retention of invertase in the cell wall of S. fragilis (renamed K. marxianus) is dependent upon a cell-wall component which is reducible by thiol compounds like ß-mercaptoethanol. So, the enzyme is retained in the cell wall by a permeability barrier, the integrity of which is maintained by disulphide bonds. Retention of proteins is thus correlated with their molecular weight. An increase in molecular weight of a protein can be caused by multimerization or by a higher degree of glycosylation.

^Myjfv^

Cell membrane Disulphide

-linked protein

w

-Θ-Mannen

Fig. 1. Schematic structure of the yeast cell wall and the mode of enzyme retention according to Kidby and Davies (1970).

244

Table 1. Total inulinase activities and distribution in carbon-limited continuous cultures of K. marxianus CBS 6556 in mineral medium with 0.25% of various carbon substrates.

Carbon substrate

Inulin Inulin Sucrose Fructose Glucose Lactose Glycerol Ethanol

Dilution rate (h'1)

0.05 0.15 0.1 0.1 0.1 0.13 0.10 0.1

Total inulinase8

58 25 52 29 3.9 2.8 9.4

26

% Inulinase in

Supernatant

65 60 48 51 87 97 43 57

Cell wall

21 26 32 28 8 3

37 31

Cell

14 14 20 21 5 0

20 12

a Enzyme activities were measured with sucrose as the substrate.

4 PRODUCTION, LOCALIZATION AND PROPERTIES OF INULINASE IN KLUYVEROMYCES MARXIANUS Production and localization of inulinase in K. marxianus was studied in chemostat

cultures (Rouwenhorst et al., 1988). In carbon- and energy-limited continuous cultures of K. marxianus, the highest inulinase yields were obtained with inulin or sucrose as the limiting substrates (Table 1). Growth on glucose and lactose only supported a very low inulinase production, whereas on the non-fermentable substrates glycerol and ethanol considerable yields of inulinase were obtained.

The level of inulinase in sucrose-limited cultures was highly dependent on the dilution rate (which in continuous cultures per definition equals the growth rate). An increase in the dilution rate from 0.1 h"1 to 0.8 h"1 resulted in a decrease of total inulinase activity (sum of supernatant, cell-wall and cell-bound inulinase activities) from 52 to 2 U mg"1 cell dwt. The profile of inulinase activity (Fig. 2) in carbon-limited cultures suggested that the enzyme production is regulated by carbon catabolite repression. This hypothesis was confirmed by experiments with nitrogen-limited chemostat cultures. A higher concentration of residual carbon substrate resulted in a lower enzyme production.

Upon growth in sucrose-limited chemostat cultures, 45-50% of the inulinase was secreted, 30-35% was retained in the cell wall and the rest was cell-bound. This distribution was independent of the dilution rate.

5 PRODUCTION OF INULINASE DURING GROWTH OF KLUYVEROMYCES MARXIANUS AT HIGH CELL DENSITY The conditions supporting high inulinase yields in chemostat cultures were used to

develop a fed-batch process on a 100-1 scale. During the process, a dilution rate between

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Fig. 3. Production of supernatant inulinase during fed batch cultivation at a 100-1 scale. Inulinase activities were measured with sucrose as the substrate.

0.2 and 0.1 h"1 was maintained. The feed contained 500 g Γ1 sucrose as the carbon- and energy-limiting substrate. The maximum supernatant inulinase activity was obtained after 24 h of growth (Fig. 3). A disturbance in the pH-control caused a temporary drop from 4.5 to 2.7, and a drastic drop in inulinase activity.

246

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Fig. 4. Chromatography of inulinase preparations on DEAE Sephadex A-50. Crude preparations of supernatant (A) and cell-wall inulinase (B) were applied to a column equilibrated with 20 mM potassium phosphate buffer (pH 6.5) and eluted with a 0 to 0.5 M linear gradient of NaCl. Fractions were collected, scanned at 280 nm, and assayed for sucrose-hydrolysing activity ( · ) . Active peak fractions of supernatant and cell-wall inulinase were separated into six pools as indicated.

The supernatant inulinase production in this process, 70,000,000 units in 100 1, is the highest so far reported in the literature, and could even have been four to five times higher if no pH problems would have occurred.

6 PURIFICATION OF INULINASE Supernatant and cell-wall inulinases were concentrated by ultrafiltration. The

preparations were further purified by anion chromatography (Fig. 4). The supernatant inulinase was nearly free from contaminating proteins and eluted at

247

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0,015

I 0,010

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Fig. 5. FPLC gel filtration of the pooled DEAE Sephadex A-50 chromatography fractions of supernatant (A) and cell-wall inulinase (B). The pooled inulinase activities I, II, III (supernatant inulinase) and IV, V, VI (cell-wall inulinase) were applied to a Superose 12 HR 10/30 gel filtration column, equilibrated with 0.5 M potassium phosphate buffer (pH 6). Chromatography was performed at a flow rate of 24 ml h"1. Inulinase is given as sucrose-hydrolysing activity.

0.2 M NaCl. The cell-wall enzyme preparation contained a large fraction of contaminating proteins, which did not bind to the column and were eluted before the gradient elution was started. The cell-wall inulinase itself eluted at 0.15 M, and was relatively pure as indicated by the sharpness of the peak. The pooled active fractions I-VI (Fig. 4) were further purified by gel filtration.

The FPLC elution profiles of the supernatant inulinase all showed two peaks, of which only one had inulinase activity (Fig. 5). The cell-wall inulinase appeared to be almost pure; only one major protein peak was found which contained all the inulinase activity. When the elution times of the inulinases were compared with those of globular proteins of

III

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248

known molecular weight, molecular weights of 180 kDa and 450 kDa could be established for the supernatant and the cell-wall inulinase, respectively. However, in gel filtration glycoproteins tend to be eluted earlier than globular markers with the same weight, and hence the given molecular weights are likely to be an overestimation of the real values.

Another method to determine the molecular weight of proteins is native gel electrophoresis. With this technique, molecular weights of 165 kDa and 335 kDa were found for supernatant and cell-wall inulinase, respectively.

To establish the molecular weight of the subunits of inulinase, denaturating gel electrophoresis was used (Fig. 6). Supernatant and cell-wall inulinase showed a polydisperse band with molecular weights between 87 and 102 kDa. This polydispersity is probably caused by heterogeneity in the carbohydrate content of the proteins. After complete removal of the outer chain glycosylation with endo-N-acetyl-ß-glucosaminidase H (Endo-H), the supernatant inulinase and the cell-wall inulinase both migrated as one uniform band with a molecular weight of 64 kDa. Also silver staining, a more sensitive technique, revealed only one band on SDS-PAGE. The results of the denaturating gel electrophoresis with non-treated and Endo-H-treated inulinase showed that the intact inulinase monomer contains 27-37% of its weight as glycosyl units.

In order to clarify the relationship between the inulinase of K. marxianus and the invertase of S. cerevisiae, the amino acid sequence of the TV-terminal end of the inulinase was analysed (Fig. 7), and compared with the amino-terminal sequence of invertase as determined by Carlson et al. (1983). The first twenty TV-terminal amino acid residues were identical for purified supernatant and cell-wall inulinase. Apart from the first residue, serine, no homology was found between the amino acid sequence of inulinase and invertase.

Fig. 6. SDS-polyacrylamide gel electrophoresis of native and carbohydrate-depleted inulinases. Lanes 1, 2, 4: cell-wall inulinase; lanes 3, 5, 6: supernatant inulinase. Treatment with Endo-H was performed with native (lanes 2, 3) and denatured inulinases (lanes 4, 6).

249

I 10 Inulinase Ser-Gly-Asp-Ser-Lys-Ala-Ile-Thr- ? -Thr -Invertase Ser-Met-Thr-Asn-Glu-Thr-Ser-Asp-Arg-Pro

II 20 Inulinase Thr-Phe- ? -Leu-Asn-Arg-Pro-Ser-Val-Tyr Invertase Leu-Val-His-Phe-Thr-Pro-Asn-Lys-Gly-Trp

Fig. 7. Amino acid sequences of the amino-terminal ends of the inulinase polypeptide of K. marxianus CBS 6556 and the invertase polypeptide of S. cerevisiae. The amino-terminal sequence of invertase as determined by Carlson et al. (1983).

7 DISCUSSION With K. marxianus, the highest production of inulinase so far reported in the

literature was obtained. Depending upon the culture conditions, the inulinase produced is secreted up to 50% into the culture fluid. This high level of secretion facilitates the downstream processing of the enzyme. In combination with the possibility of high-cell-density cultivation, these properties make K. marxianus an attractive organism for the production of inulinase. The same characteristics also indicate K. marxianus as a favourable candidate for the production of heterologous proteins.

The inulinase of K. marxianus secreted into the culture fluid and the inulinase retained in the cell wall have identical subunits, consisting of a 64-kDa polypeptide that contains 26-37% of its molecular weight as carbohydrate. The two inulinase forms differ in degree of oligomerization: the supernatant inulinase consists of two subunits (165 kDa / 87 kDa) while the cell-wall inulinase is a tetramer (335 kDa / 87 kDa). Retention of glycoproteins in the periplasmic space of the yeast cell wall is thought to be caused by a permeability barrier in the outer regions of the cell wall. Oligomerization of proteins may then play a role in their retention in the cell wall. For invertase it is known that the secreted enzyme is a dimer and the form retained in the cell wall is an octamer (Esmon et al., 1987). Also in the case of inulinase, oligomerization appears to play a role in the retention of the protein in the cell wall.

Invertase and inulinase differ in their activities towards fructans. Further differences are encountered when the enzyme structures are compared: a 62-kDa polypeptide observed after treatment of the extracellular invertase of S. cerevisiae with Endo-H (Trimble and Maley, 1977) is only slightly smaller than the corresponding polypeptide of the inulinase of K. marxianus. The degree of glycosylation is different, around 30% of its molecular weight for the inulinase and 50% for the invertase. When the cell-wall enzymes are compared it is clear that the invertase with its molecular weight of 800 kDa is much larger than the inulinase of 335 kDa. The secreted enzymes also differ markedly in weight: 270

250

kDa versus 165 kDa for invertase and inulinase, respectively. The amino acid sequence of the amino-terminal end of invertase shows no homology

with that of inulinase except for the first residue serine, suggesting that inulinase and invertase are different polypeptides. However, the complete sequence of inulinase is unknown, so it can not be excluded that some internal homology between invertase and inulinase exists.

The difference in N-terminal amino acid sequence, together with the difference in molecular weight, in glycosylation and in the activity towards fructans support the conclusion that invertase of S. cerevisiae and inulinase of K. marxianus are different enzymes (Rouwenhorst et al., 1990).

8 REFERENCES

Carlson, M., Taussig, R., Kustu, S. and Botstein, D., 1983. The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence. Mol. Cell. Biol., 3: 439-447.

Esmon, P.C., Esmon, B.E., Schauer, I.E., Taylor, A. and Schekman, R., 1987. Structure, assembly, and secretion of octameric invertase. J. Biol. Chem., 262: 4387-4394.

Field, C. and Schekman, R., 1980. Localized secretion of acid phosphatase reflects the pattern of cell surface growth in Saccharomyces cerevisiae. J. Cell Biol., 86: 123-128.

Kidby, D.K. and Davies, R., 1970. Invertase and disulphide bridges in the yeast wall. J. Gen. Microbiol., 61: 327-333.

Lampen, J.O., 1968. External enzymes of yeast: their nature and formation. Antonie van Leeuwenhoek, 34: 1-18.

Rouwenhorst, R.J., Hensing, M., Verbakel, J., Scheffers, W.A. and Van Dijken, J.P., 1990. Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol., 56: 3337-3345.

Rouwenhorst, R.J., Visser, L.E., Van der Baan, A.A., Scheffers, W.A. and Van Dijken, J.P., 1988. Production, distribution, and kinetic properties of inulinase in continuous cultures of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol., 54: 1131-1137.

Tkacz, J.S. and Lampen, J.O., 1973. Surface distribution of invertase on growing Saccharomyces cells. J. Bacteriol., 113: 1073-1075.

Trimble, R.B. and Maley, F., 1977. Subunit structure of external invertase from Saccharomyces cerevisiae. J. Biol. Chem., 252: 4409-4412.


PRODUCTION OF fflGH-FRUCTOSE-CONTAINING SYRUPS FROM JERUSALEM ARTICHOKE EXTRACTS WITH FRUCTOSE ENRICHMENT THROUGH FERMENTATION

A. FONTANA, B. HERMANN and J.P. GUIRAUD Laboratoire de Microbiologie Industrielle, Centre de Gdnie et Technologie Alimentaires, Universitö Montpellier II (USTL), 34095 Montpellier Cedex 05, France

ABSTRACT

Jerusalem artichoke is a suitable raw material for fructose syrup production. The sugar content and the inul in/fructose ratio depend on various factors, in particular on the date of harvest. Incomplete fermentation of extracts by selected yeasts allows the production of syrups with increased fructose content. The yeast strains used (Saccharomyces cerevisiae, S. diastaticus, Schizosaccharomyces pombe and others) readily ferment sucrose and inulin oligomers, but not easily larger inulin polymers. The better strains bring about a distinct increase in the fructose/inulin ratio with a good yield in residual sugars. After fermentation of low-molecular-weight saccharides, followed by acid or enzymatic hydrolysis extracts of early and late harvested tubers led to syrups of good quality containing up to 95% and 90% fructose, respectively. This fermentative enrichment process is competitive with other ones (e.g. Chromatographie enrichment) employed in the technical production of fructose syrups: it can be applied in case of raw extracts, it simplifies purification and it permits a simultaneous valorization of by-products such as ethanol and yeast (in addition to the pulps).

1 INTRODUCTION

Fructose syrups are widely used in the food industry. Indeed, fructose is an interesting sweetener because of its high sweetening power (Pawan, 1973). Moreover, fructose is claimed to be less cariogenic than other sugars and to be more suitable for diabetics since its metabolism is insulin-non-dependent. Fructose has been found to stimulate the metabolism of ethanol and to increase the intestinal absorption of iron. Its physical properties make it well-suited to be utilized in various technological processes: it is more soluble in water and ethanol and has a lesser tendency to crystallize than sucrose. It is also useful for its hygroscopic and antioxidant properties, and for binding anions (Guiraud and Galzy, 1990).

Fructose can be obtained directly from fruits, but can also be prepared by oxidation of mannitol or sorbitol (Pilnik, 1973), by inversion of sucrose, by isomerization of glucose

252

or by acid or enzymatic hydrolysis of fructans like inulin (Willaman, 1920; Kierstan, 1980). Isomerose or high-fructose corn syrups (HFCS) are produced by isomerization of a corn starch hydrolysate to a 42% fructose/50% glucose mixture. Their fructose content can be increased up to 95 % (ultra-HFCS) by Chromatographie separation and subsequent recycling of the glucose component.

Because of their high content of fructose, fructans, like inulin, are interesting substrates from which to produce this kind of syrups. Inulin is especially found in some dicotyledonous plants. Several species within the Compositae are suitable sources, particularly dahlia (Haber et al., 1941), chicory (Rutherford and Weston, 1968), and Jerusalem artichoke (Kierstan, 1980). With a potential sugar production of 5-14 t ha"1, the latter species is one of the most valuable sources of fructose.

Inulin is composed of ß-2,1 -linked D-fructose residues, forming linear chains containing one terminal glucose residue. Their fructose content varies according to the mean degree of polymerization (MPD) (Bacon and Edelman, 1951) which was found to depend strongly on the Jerusalem artichoke cultivar used (Chabbert et al., 1985b), the growth and storage conditions, and the date of harvest (Chabbert et al., 1983). Indeed, late harvested tubers of Jerusalem artichoke contain inulin with an MPD ranging from 3 to 5, whereas those harvested early have inulin of MPD 10 to 15. That means that the fructose content of syrups obtained by hydrolysis of inulin also depends on the MPD of the raw material: 90-93% with the "early tuber" juices versus 67-80% with the "late tuber" extracts.

Chemical or enzymatic hydrolysis of inulin is easily performed. Enrichment of fructose from inulin can be realized in two ways: either after hydrolysis, by chromatography, like in the manufacture of isomerose or before hydrolysis, by precipitating the high-molecular-weight compounds (high MPD) or by eliminating the low-molecular-weight fractions (low MPD). The precipitation of high-MPD inulin can be performed using cold ethanol (Chabbert et al., 1985b). This method leads to a powder with a high fructose/glucose ratio (F/G) but also to a residue difficult to valorize.

The aim of the present work was to bring about fructose enrichment by the elimination of low-molecular-weight sugars (not containing fructose) through fermentation of extracts with selected yeast strains. This process will not only lead to the production of syrups with an increased fructose content but also enables the valorization of the eliminated sugars because of their concurrent conversion to ethanol.

2 MATERIALS AND METHODS 2.1 Yeast strains

Yeast strains from the Centraalbureau voor Schimmelcultures (CBS), from the Northern Regional Research Laboratory (NRRL) and from our own laboratory collection

253

(CGTA) were used: Candida salmanticensis CBS 5121; Kluyveromyces fragilis CBS 1555; Saccharomyces cerevisiae CBS 1200; Saccharomyces chevalieri CGTA L68; Saccharomyces diastaticus NRRL 2426; Schizosaccharomyces pombe CGTA L156.

2.2 Jerusalem artichoke extract preparation Tubers of Jerusalem artichoke cv. Violet de Rennes were used throughout. The

cultivar was grown in the Montpellier area and planted March 15, 1989. Tubers were harvested either October 20, 1989 ("early" tubers) or February 10, 1990 ("late" tubers). To prepare fermentable extracts, the tubers were washed, diced, crushed with a blender and pressed through a cloth with a hand press. After collecting the first juice, the pulp was mixed with water and pressed again. The two juices were combined and their concentration was adjusted by adding water to obtain a final concentration of about 130 g Γ1. The final juice was not purified before fermentation.

2.3 Yeast culture and fermentation techniques Cell cultures for inoculating fermentation media were grown in a yeast extract (5%)-

sucrose (5%) medium. The fermentation medium was a non-sterilized Jerusalem artichoke extract, adjusted to pH 3.5 with HC1. The fermentations were run at 28 °C for 4 days in static 100-ml erlenmeyer flasks, filled to 2/3 of their volume.

2.4 Analytical techniques Yeast cell density was determined by direct microscopic count using a counting

chamber. Dry matter content of the Jerusalem artichoke press juices was measured by weighing a lyophilized aliquot of the juice. The juices were purified by defecation (by heating them 5 min at 80 °C at a pH adjusted to 4.5 with H2S04) and concentrated under reduced pressure to 400 g sugar Γ1. The high-molecular-weight fructans were precipitated with ethanol (50%, v/v). Precipitation was performed during 48 h at 4 °C (Chabbert et al., 1983). The polymers were separated out by filtering, washed with an alcohol solution (80%) and dried in a microwave oven. A first approximation of the sugar concentration was obtained refractometrically. Reducing sugars were assayed using the dinitrosalicylate method according to Bernfeld (1955). Total sugar content was determined by the same method after hydrolysis (pH adjusted to 2 with H2S04; 30 min at 120 °C). Fructose and glucose were determined by the Boehringer enzymatic technique. Qualitative analysis of fructans was performed using HPLC (Conrad and Palmer, 1976). Ethanol was assayed using GLC (Chabbert et al., 1985a).

254

3 RESULTS 3.1 Direct production of fructose syrups from "early" and "late tuber" extracts 3.1.1 Characterization of the Jerusalem artichoke juices

The extraction yield for the "early tuber" juice was only about 50% whereas that of the "late tuber" extract was as much as 80%. The total sugar contents of the first and second press juices were 185 and 55 g Γ1 for the "early tuber" extract and 175 and 30 g Γ1

for the "late tuber" extract, respectively. The characteristics of the final juice are given in Table 1.

With the "early tuber" extract, the inulin yield, as obtained by alcohol precipitation, was 57.2%. The resulting powder contained 100% sugars with 94% fructose (MPD 16.4 or F/G ratio 15.4). In the case of the "late tuber" juice, the yield was only 7.5%; it was a powder containing 97% sugars with 91% fructose (MPD 15.2 or F/G ratio 14.2). Results of the HPLC analysis of the two extracts are shown in Fig. 1 (A, B). The "early tuber" extract contained less low-molecular-weight inulin polymers (peaks 1 to 10) and more high-molecular-weight fructans (peaks > 10) than the "late tuber" extract.

3.1.2 Chemical hydrolysis Fig. 1 (C) shows the HPLC chromatograms obtained after chemical hydrolysis of the

extracts. Hydrolysis was complete and led to extracts containing only glucose and fructose.

3.2 Fructose enrichment through fermentation 3.2.1 Choice of the used strains

The aim of the work was to preserve high-molecular-weight inulin polymers, eliminating oligo-, di- and monosaccharides. Then, on the basis of data by Barnett et al. (1983), we selected yeast strains unable to degrade high-polymer fructans (no or low inulinase activity) but capable of utilizing sucrose, glucose and fructose as carbon substrates. Selected strains were: C. salmanticensis, S. cerevisiae, S. chevalieri, S. diastaticus, and Sch. pombe. Of these species, C. salmanticensis and Sch. pombe are able to produce inulinase, though fermentation of inulin is either delayed or variable (Fuchs et

Table 1. Characteristics of the Jerusalem artichoke extracts.

Extracts Total sugars Reducing sugar MPD F/G (g I"1)

(g Γ1) % dry matter

"Early tuber" extract 131 80 10.5 9.5 8.5 "Late tuber" extract 137 71 11.5 3.4 2.4

255

Table 2. Composition of fermented "early tuber" juices.

Strains

C. salmanticensis K. fragilis S. cerevisiae S. chevalieri S. diastaticus Sch. pombe Initial juice Precipitated inulin

Ethanol (gr1)

2.2 42.4 15.5 17.8 5.8 9.1

--

Total sugars (g I"1)

107.4 27.7 76.3 69.3 98.2 86.1

130.4 -

Reducing sugars (gl1)

20.3 4.3 3.7

10.3 10.9 17.8 10.2 -

Sugar* yield (%)

77.6 12.8 53.7 49.2 73.4 63.3 -57.2*

F/Gb

ratio

11.4 17.8 15.2 11.9 13.9 13.9 8.5

15.4

Fructosec

enrichment

1.34 2.09 1.79 1.40 1.64 1.64 -1.80

a The sugar yield is the percentage of sugars recovered after fermentation or alcohol precipitation (*). b The F/G ratio as obtained after hydrolysis of the residual sugars. c The fructose enrichment is the ratio of final and initial F/G ratios.

al., 1985). Because of its ability to readily degrade inulin, K. fragilis was chosen as reference.

3.2.2 Fermentation of "early tuber" extracts The extracts were fermented under reduced oxygen pressure. Non-sterilized medium

was inoculated with 108 cells ml"1. After 4 days, yeast cell populations were about 2 to 2.5 109 cells ml1 in all assays. Table 2 summarizes the results of the analysis of fermented juices. Fig. 1 (D) shows the HPLC chromatogram obtained with an extract fermented by S. cerevisiae.

With the strains unable to use inulin, only 50%, or even less, of the sugars was consumed: C. salmanticensis and S. diastaticus fermented only about 25% of the initial sugar content. Consequently, the low level of fermentation led to a low ethanol production. The strain of K. fragilis, which can utilize inulin, fermented more but not all sugars, thus producing a large quantity of ethanol. The reducing-sugar content of the various fermented juices varied from 3.7 to 20.4 g Γ1 (5 to 20% of the total sugar content). This shows that sugar hydrolysis by the various yeasts was not the limiting factor of the fermentation. In all cases fructose was enriched. The best F/G ratio (17.8) was obtained with K. fragilis: the final fructose concentration was about twice the initial one. However, this result was not very interesting because only little sugar was left. The best yeast strains, S. cerevisiae, S. diastaticus and Sch. pombe, showed a good fructose enrichment and a low fermentation. The results obtained with these strains are nearly similar to those found upon alcohol precipitation (Table 2).

256

Table 3. Composition of fermented "late tuber" juices.

Strains Ethanol Total Reducing Sugar8 F/Gb Fructose0

(g Γ1) sugars sugars yield ratio enrichment -h (c 1-1 (g I'1) (g I"1) (%)

C. salmanticensis 5.5 104.5 10.0 68.3 3.1 1.27 S. cerevisiae 37.5 16.3 1.3 12.6 7.8 3.25 S. diastaticus 34.2 26.2 1.3 19.0 6.1 2.54 Sch. pombe 41.3 14.8 2.9 9.4 6.5 2.70 Initial juice - 136.9 11.2 - 2.4 Precipitated inulin - - - 7.5* 14.2 5.90

a The sugar yield is the percentage of sugars recovered after fermentation or alcohol precipitation (*). b The F/G ratio as obtained after hydrolysis of the residual sugars. c The fructose enrichment is the ratio of final and initial F/G ratios.

3.2.3 Fermentation of "late tuber" extracts The extracts were fermented under the same conditions as the "early" ones but only

with the best strains. After 4 days, the yeast cell populations were a little larger: 3 to 5 X 109 cells ml"1. Results of these fermentations are reported in Table 3. Fig 1 (E) shows the HPLC chromatogram obtained with an extract fermented by S. diastaticus.

The "late tuber" juice was fermented more readily than the "early tuber" extract. The poorest fermenting strain was always C. salmanticensis, whereas S. cerevisiae, S. diastaticus and Sch. pombe fermented most sugars and produced much more ethanol. The reducing-sugar content was low again. Compared with the initial juices all fermented juices had an increased fructose content. The best F/G ratio (7.8) was obtained with S. cerevisiae: here, the fructose concentration increased to about a threefold. However, in all cases, the increase in the F/G ratio was accompanied with a low sugar yield. In fact, S. diastaticus seems to be the most interesting strain. The increase in F/G ratio was always larger upon alcohol precipitation than after fermentation, but alcohol precipitation resulted in a lower sugar yield.

4 CONCLUSION Fructose enrichment of Jerusalem artichoke extracts by incomplete fermentation with

selected yeast strains seems to be an attractive technique. This process of fructose enrichment of the raw extract and the consecutive purification steps are much simpler than the usual technical processes employed in obtaining high-fructose syrups. Non-interesting sugars, like mono- or disaccharides and low-molecular-weight polymers, are directly valorized in the form of ethanol and yeast. The production of high-fructose syrups is easier with extracts from early harvested tubers which contain more high-molecular-weight

257

A) Crude "early" extract

10 11 12 13 1 4 1 5 1 6

0 2 4

I

1 R 7 8

6

9 10 11

8

12 13

10 12 14

D) Fermented "early" extract

^ ^ J f i e 17

16 Time (min)

18

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6

9 10

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Time (min)

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Fig. 1. HPLC chromatograms of carbohydrates in Jerusalem artichoke extracts. The "early tuber" extract was fermented by S. cerevisiae (D), the "late tuber" extract by S. diastaticus (E).

fructans. In every case, the fructose content of syrups is at least 9 0 % . Therefore, this

fermentative enrichment technique is competitive with other processes, for example, the

Chromatographie "enrichment".

258

5 REFERENCES

Bacon, J.S.D. and Edelman, J., 1951. The carbohydrates of the Jerusalem artichoke and other Compositae. Biochem. J., 48: 114-126.

Barnett, J.A., Payne, R.W. and Yarrow D., 1983. Yeast Characteristics and Identification. Cambridge University Press, Cambridge.

Bernfeld, P., 1955. Amylases, a and /?. Methods Enzymol., 1: 149-158. Chabbert, N., Braun, P., Guiraud, J.P., Arnoux, M. and Galzy, P., 1983. Productivity and fermentability

of Jerusalem artichoke according to harvesting date. Biomass, 3: 209-224. Chabbert, N., Guiraud, J.P., Arnoux, M. and Galzy, P., 1985a. Productivity and fermentability of different

Jerusalem artichoke {Helianthus tuberosus) cultivars. Biomass, 6: 271-284. Chabbert, N., Guiraud, J.P., Arnoux, M. and Galzy, P., 1985b. The advantageous use of an early

Jerusalem artichoke cultivar for the production of ethanol. Biomass, 8: 233-240. Conrad, E.C. and Palmer, J.K., 1976. Rapid analysis of carbohydrates by high-pressure liquid

chromatography. Food Technol., 30 (10): 84-92. Fuchs, A., De Bruijn, J.M. and Niedeveld, C.J., 1985. Bacteria and yeasts as possible candidates for the

production of inulinases and levanases. Antonie van Leeuwenhoek, 51: 333-343. Guiraud, J.-P. and Galzy, P., 1990. Inulin conversion by yeasts. In: H. Verachtert and R. De Mot (Eds.),

Yeast Biotechnology and Biocatalysis. Marcel Dekker, Inc., New York, pp. 255-296. Haber, E.S., Gaessler, W.G. and Hixon, R.M., 1941. Levulose form chicory, dahlia and artichokes.

Iowa State Coll. J. Sei., 16: 291-297. Kierstan, M., 1980. Production of fructose syrups from inulin. Process Biochem., 15 (4): 2,4,32. Pawan, G.L.S., 1973. Fructose. In: G.C. Birch and L.F. Green (Eds.), Molecular Structure and Function

of Food Carbohydrates. Applied Science, London, pp. 65-80. Pilnik, W., 1973. Food additives. Recent developments in the food industry. Gordian, 73: 208-214. Rutherford, P.P. and Weston, E.W., 1968. Carbohydrates changes during cold storage of some inulin-

containing roots and tubers. Phytochemistry, 7: 175-180. Willaman, J.J., 1920. Levulose syrup. Science, 52: 351-352.


MODIFICATION OF THE TRANSFRUCTOSYLATION ACTIVITY OF BACILLUS SUBTILIS LEVANSUCRASE BY SOLVENT EFFECT AND SITE-DIRECTED MUTAGENESIS

R6gis CHAMBERT and Marie-Frangoise PETIT-GLATRON Institut Jacques Monod, Centre National de la Recherche Scientifique, University Paris 7, Laboratoire Gdn&ique et Membranes, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05, France

ABSTRACT

A significant modification of the transfructosylation specificities of Bacillus subtilis levansucrase was obtained by two methods. Firstly, in concentrated solution of organic solvents this enzyme displays only its polymerase activity. Secondly, amino acid substitutions of the catalytically essential residue Arg331 generate levansucrase variants which have lost the ability of the wild-type enzyme to synthesize levan from sucrose, but have preserved the sucrose hydrolytic activity and the ability to synthesize the trisaccharide kestose. The latter reaction was investigated using a Lys331-variant.

The aim of the present study is to contribute to a better understanding of fructan synthesis in bacteria and to provide a way to investigate the hypothesis that the enzymes involved in fructan synthesis by bacteria and plants have a common ancestor.

1 INTRODUCTION The levansucrase of Bacillus subtilis (Dedonder, 1972) catalyses transfructosylation

from sucrose (or raffmose) to a variety of acceptors such as water, alcohols, monosaccharides, sucrose, oligosaccharides and levan, according to the reaction:

sucrose + acceptor > glucose + fructosyl acceptor This low level of fructosyl acceptor specificity contrasts with the high specificity of plant fructosyltransferases involved in fructan synthesis (Hendry, 1987). Moreover, it severely limits the use of this enzyme in the synthesis of defined fructosylated compounds.

The degree of polymerization of the final product and the relative rates of synthesis or hydrolysis of levansucrase depend upon the concentration of sucrose and acceptor and are also influenced by the pH, ionic strength and temperature of the assay medium (Chambert et al., 1974; Tanaka et al., 1979). Recently, we have undertaken two new approaches for the modification of transfructosylation specificity: i) by investigating the

260

catalytic behaviour of this enzyme in water-restricted environments, ii) by using site-directed mutagenesis to explore the role of the Arg331 residue. Isolation and characterization of such altered proteins has shown that the single substitution Arg331 > His strongly modifies the polymerase activity and to a lesser extent the hydrolase activity (Chambert and Petit-Glatron, 1991).

2 METHODS 2.1 Kinetic analytical methods

The transfructosylase activities of levansucrase occur by a simple catalytic pathway involving the formation of a transient fructosyl intermediate (Chambert et al., 1974)

ki k3 kA[A] E + AF E + S T ES . EF ^ C H [1]

k2 k4[G] kH^ E + F

where E, S, G, F and A are symbols for enzyme, sucrose, glucose, fructose and fructosyl acceptor, respectively. Measurements of vF and vG (which, respectively, denote the initial velocities of fructose and glucose liberation in the presence of various concentrations of a chosen fructosyl acceptor) allow the determination of the kinetic parameters and the proportion of the total transfer (Y), which utilizes the acceptor molecule A rather than water, both of which compete in the dissociation of the E-F intermediate. It is easy to demonstrate that

y=100(l-^) and ^ = 1 + __^_^] VG VF *H20

Measurements of vF and vG were carried out with [U-14C]-sucrose as the substrate. The [14C]-labelled sugar products were quantitatively analysed by paper chromatography (Chambert ei al., 1974).

2.2 Site-directed mutagenesis The different alleles of the structural gene for levansucrase were obtained by the

polymerase chain reaction method, details of which have been given in a recent paper (Chambert and Petit-Glatron, 1991). These alleles were cloned in the pBluescript plasmid and expressed in Escherichia coli. Production of 1-2 mg of pure levansucrase variants from 500 ml of E. coli culture allowed their complete kinetic characterization.

261

3 RESULTS AND DISCUSSION 3.1 Effects of organic solvents on enzyme behaviour

We studied the variation of the rates of hydrolase and synthetase activities of levansucrase by progressively replacing water with acetonitrile, acetone, 1,4-dioxane or dimethylsulphoxide in the reaction mixture at 22 °C.

The data presented in Table 1 indicate that the yield of levan synthesis is strongly enhanced by the presence of increasing concentrations of organic solvents. At concentrations higher than 50% the enzyme displays only its polymerase activity; Y then reaches a value of 100%, which means that the hydrolase activity of the levansucrase is completely inhibited under such conditions. From the data in Table 1, it can be concluded that the effect of a particular solvent on the behaviour of the enzyme does not correlate with the values, either of its dielectric constant or of its electric dipole moment.

The kinetic constants of levansucrase for sucrose and raffinose were evaluated in the presence of 60% acetonitrile and compared with the same constants determined for the aqueous medium. As shown in Table 2, in the presence of acetonitrile the Km values are nearly unchanged but the kcat values are increased for both substrates by a factor of 5.

It is noteworthy that the value of &cat for sucrose transformation is approximately the same as the value of the kinetic constant k3 (equation [1]) (Chambert and Gonzy-Trdboul, 1976). This may mean that, in the presence of solvent, the formation of fructosyl-enzyme is the rate-limiting step whereas the transfructosylation step from fructosyl-enzyme to water is the rate-limiting one in aqueous medium (Chambert et al., 1974).

Consideration of these data raises the question of whether the catalytic properties of levansucrase in water-restricted environments create new opportunities in the area of applied enzymology. For instance, would such properties allow the transfructosylation reaction of levansucrase to transfer fructose residues from sucrose to long-chain acyl alcohols solubilized in organic solvent-water mixtures?

Table 1. Percentage of total activity attributable to the polymerase activity of levansucrase with respect to the concentration of various solvents present in the reaction mixture.

Solvent e D Percentage polymerase activity

Solvent-water (v/v)

30:70 40:60 50:50 60:40 70:30

1,4-Dioxane 02.2 0.4 25 62 100 Acetone 20.7 2.9 29 95 100 Acetonitrile 38.8 3.4 23 86 100 100 100 Dimethylsulphoxide 45.0 4.3 40 73 080

262

Table 2. Kinetic constants of levansucrase for sucrose and raffinose.

Medium

Water 60% acetonitrile

Sucrose

*» (M)

2 10"2

3.2 ± 1 io-2

kcat min1

3 103

1.6 ± 0.5 104

Raffinose

Km(M)

1.5 IO"2

3 + 1 . 2 10"2

kc&l min"1

3 103

1.7 ± 0.6 104

3.2 Effects ofamino acid substitution at site 331 Based upon previous genetic analyses (Lepesant et al., 1974), various mutants of B.

subtilis producing altered levansucrase were isolated. In one of these, the QB252 strain, a

mutation - at the sacB allele - did not affect thermostability but modified the catalytic

properties of the enzyme in such a way that the polymerase activity was decreased relative

to the hydrolase activity.

We have cloned and sequenced this sacB allele. The mutation involves the

substitution of Arg 3 3 1 by a His residue. To restore the wild-type allele and to modify the

charge and polarity at this site, the following amino acid substitutions were carried out:

His 3 3 1 -> Arg, His 3 3 1 -> Lys , His 3 3 1 -*· Ser, His 3 3 1 -> Leu, and His 3 3 1 -* Gly.

The catalytic properties of the variants were studied by investigating the various

transfructosylation reactions catalysed by these modified levansucrase enzymes.

3 .2 .1 Effect on sucrose hydrolytic activity

In the presence of low concentrations of sucrose ( < 50 mM), levansucrase displays

only hydrolytic activity. The kinetic parameters Km and £cat for each variant are presented

in Table 3 . The amino acid substitutions at site 331 strongly modify the sucrose hydrolase

efficiency of levansucrase. The Ser3 3 1 and Gly3 3 1 variants displayed only a residual activity

(5% of the wild type). In contrast, the Lys331-variant had a two-fold higher catalytic activity

than the wild type.

Table 3. Michaelis constants8 for sucrose hydrolysis at 30 °C determined using a range of levansucrase variants.

Variant

*cat (a"1) 102 x Km (M)

H i s331

22 + 2 1.2 + 0.1

Lys33i

88 ± 5 1.1 ± 0.1

Leu331

7 ± 0.6 1.8 ± 0.2

Arg33i (wild-type)

35 ± 2 0.4 ± 0.04

Ser331

1.6 ± 0.2 1.8 ± 0.2

Gly331

1.8 ± 0.3 1.5 ± 0.2

a These values were calculated for each variant from the double reciprocal plot l/vF against 1/[S]. Numerical data were estimated by weighted regression analysis. The relative weights were assumed to be proportional to the square of the initial velocity. The results are means ± SD (n = 10).

263

Table 4. Percentage of total activity attributable to transfructosylase activity, with sucrose as fructosyl donor, and levan and inulin as fructosyl acceptors8.

Variant

Levan Inulin

H i s331

57 15

Lys 3 3 i

23 0

Leu331

23 0

Arg33i

82 37

Ser331

30 0

GIV331

25 0

a Transfructosylation from sucrose to fructan was measured in the presence of either 3.3 mM levan or 3.3 mM inulin. The initial concentration of sucrose was 50 mM.

3.2.2 Effect on chain elongation activity Two types of fructan were tested as fructosyl acceptor, a) B. subtilis levan of low

molecular weight (MW = 15,000 ± 3000), and b) dahlia inulin (MW = 5000). The percentage of fructosyltransferase activity from sucrose to both fructans at the same concentration of 3.3 mM in the reaction mixture was evaluated for each variant. As is evident from Table 4, amino acid substitution at site 331 strongly altered the efficiency of the chain elongation activity of levansucrase. The wild-type enzyme displayed highest efficiency for this reaction.

3.2.3 Effect on the polymerase activity with sucrose as the sole substrate In the presence of high sucrose concentrations, levansucrase catalyses the synthesis

Fig. 1. Transformation of sucrose catalysed by levansucrase variants. The reaction mixture contained 0.5 M [U-14C]-labelled sucrose in 0.05 M phosphate buffer (pH 6). The enzyme concentration was chosen in such a way that approximately the same rate of sucrose transformation was achieved for each variant. Products were analysed by paper chromatography after 30 min incubation at 30 °C.

264

of high-molecular-weight levan from sucrose, which plays a dual role as fructosyl donor and as the initial fructosyl acceptor. The polymerase activity of the variants in the presence of 0.5 M sucrose is illustrated in Fig. 1. As can be seen, the Arg331 and His331 variants synthesized [14C]-labelled levans which remained at the origin of the paper chromatogram. The other variants were unable to catalyse fructose polymerization. Only the trisaccharide kestose accumulated. With the Gly331-variant a similar result was obtained (results not shown).

3.3 Further kinetic characterization of the Lys33J-variant The Lys331-variant presents some interesting features since it displays a more efficient

sucrose hydrolysis activity than the wild type but is devoid of polymerase activity and accumulates kestose. Thus, in the presence of high sucrose concentrations, its activities may occur via the following reaction pathway

ki[S]

kestose

The competing activities of this variant release three products, G, F and kestose, from the sucrose substrate. This latter compound acts both as a fructosyl donor and as a fructosyl acceptor which competes for water. From the King and Altman procedure (1956) we can derive the following equation under the initial conditions of sucrose transformation:

v* W and v* CT [S]2

With φ0 = l/k3, φλ and φ2 are combinations of the individual rate constant, whereas VjK and VjF are the initial rates of kestose and fructose release, respectively. Fig. 2 shows the changes in the ratio v^/v^ in the presence of increasing concentrations of sucrose. The slope of the straight line is a direct measure of the competition between water and sucrose as a fructosyl acceptor.

-^-=0.3 *H20

265

0.2 A

0.1 A

vK

i—i—i—i—i—i—i—i » 0.2 0. 4 0. 6 0.8 (S) M 0,005 0.01 l/(S)mM

Fig. 2 (left). Plot of the ratio VjK/VjF versus sucrose concentration for the Lys331 variant.

Fig. 3 (right). Double reciprocal plot of the initial rate of kestose formation VjK versus sucrose concentration for the Lys331-variant.

kH 0 can be considered as a pseudo-first order rate constant containing the water concentration factor. The equation 1/VJK = f(l/[S]) results in a curve as shown in Fig. 3. Polynomial regression of this curve generated the equation:

— =2.6+0.9103— +0.6106—

Two conclusions may be drawn from this result, a) The sucrose concentration for which vA

K = vmK/2 is estimated to be 0.6 M. This value may be considered as a Km value

of the enzyme for sucrose as the substrate for kestose formation, b) From the values of φ0

and {E} the rate constant (k3) of fructosyl-enzyme formation for this variant was estimated to be 165 s"1. This is of the same order of magnitude as that calculated for the wild-type enzyme (Chambert et al., 1974).

4 CONCLUSIONS The nature of the amino acid at position 331 dramatically modifies the specificity and

the efficiency of the transfructosylation process catalysed by B. subtilis levansucrase. Do these observations help us to formulate a hypothesis concerning a possible common ancestor

266

for the fructosyltransferases involved in plant and bacterial fructan metabolism? In higher plants, the conventional view is that synthesis from sucrose involves the concerted action of two enzymes, sucrose: sucrose fructosyltransferase and fructan: fructan fructosyltransferase (Pollock, 1986). It has also been suggested, however, that synthesis may be mediated by phleinsucrase, an enzyme similar to the bacterial levansucrase (Suzuki and Pollock, 1986). The Lys331 variant of B. subtilis levansucrase partially mimics the activity of plant SST, since both enzymes produce only kestose and have approximately the same Km for sucrose.

In addition, the results described herein should facilitate both studies on the molecular mechanisms involved in the enzymatic processes of transfructosylation and potential new industrial applications of these enzymes.

5 REFERENCES

Chambert, R., Treboul, G. and Dedonder, R., 1974. Kinetic studies of levansucrase of Bacillus subtilis. Eur. J. Biochem., 41: 285-300.

Chambert, R. and Gonzy-Troboul, G., 1976. Levansucrase of Bacillus subtilis: kinetic and thermodynamic aspects of transfructosylation processes. Eur. J. Biochem., 62: 55-64.

Chambert, R. and Petit-Glatron, M.-F., 1989. Study of the effect of organic solvents on the synthesis of levan and the hydrolysis of sucrose by Bacillus subtilis levansucrase. Carbohydr. Res., 191: 117-123.

Chambert, R. and Petit-Glatron, M.F., 1991. Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis. Biochem. J., 279: 35-41.

Dedonder, R., 1972. Role and mechanisms of transglycosylation reactions. In: R. Piras and H.G. Pontis (Eds.), Biochemistry of the Glycosidic Linkage. Academic Press, New York, pp. 21-78.


King, E.L. and Altman, C , 1956. A schematic method of deriving the rate laws for enzyme-catalyzed reactions. J. Phys. Chem., 60: 1375-1378.

Lepesant, J.-A., Lepesant-Kejzlarovä, J., Pascal, M., Kunst, F., Billault, A. and Dedonder, R., 1974. Identification of the structural gene of levansucrase in Bacillus subtilis Marburg. Mol. Gen. Genet., 128: 213-221.

Pollock, C.J., 1986. Fructans and the metabolism of sucrose in vascular plants. New Phytol., 104: 1-24.

Suzuki, M. and Pollock, C.J., 1986. Extraction and characterization of the enzymes of fructan biosynthesis in timothy {Phleumpratense). Can. J. Bot., 64: 1884-1887.

Tanaka, T., Oi, S. and Yamamoto, T., 1979. Synthesis of levan by levansucrase. Some factors affecting the rate of synthesis and degree of polymerization of levan. J. Biochem. (Tokyo), 85: 287-293.


A BIOTECHNOLOGICAL AND ECOPHYSIOLOGICAL STUDY OF THERMOPHILIC INULIN-DEGRADING CLOSTRIDIA

W.J. DRENT, G.J. BOTH and J.C. GOTTSCHAL Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

ABSTRACT

In a study on the anaerobic degradation of inulin at 58 °C several new thermophilic clostridia were isolated. Two isolates were identified as new strains of Clostridiwn thermoautotrophicum, whereas a new species Clostridiwn thermosuccinogenes was proposed for four other closely related isolates to account for the unusual ability of these clostridia to produce significant quantities of succinate as a fermentation product.

C. thermoautotrophicum strain II and C. thermosuccinogenes strain IC were examined in greater detail. Product formation and inulinase synthesis were investigated in batch and continuous cultures. The temperature profiles of the inulin-hydrolysing enzymes were determined. In this paper the results of these investigations are presented, and their biotechnological and ecological implications are discussed.

1 INTRODUCTION Yeasts, fungi, mesophilic bacteria and thermophilic, aerobic bacilli have been studied

with respect to the potential industrial exploitation of inulin (Fuchs et al., 1985; Allais et al., 1987). Extensive research with these organisms has been carried out to improve the production of high-fructose syrups, alcohols and other solvents, and sugar oligomers of medical interest (Vandamme and Derycke, 1983; Yamashita et al., 1984; Marchai et al., 1985; McKellar and Modler, 1989). However, hardly any information is available on the potential of thermophilic, anaerobic species to produce useful compounds from inulin as a raw material.

In the past three years a number of new thermophilic clostridia have been isolated in our laboratory (Drent and Gottschal, 1991; Drent et al., 1991). The majority of the new strains were isolated from soils. In these habitats optimal temperatures for growth of thermophiles are probably never reached (Tansey and Jack, 1977). Therefore, studies on the physiology of thermophilic clostridia at optimal and suboptimal temperatures were considered to be helpful in understanding the possible role of our isolates in dutch soils.

268

Table 1. Major characteristics of Clostridium thermosuccinogenes strains IC and Clostridium thermoautotrophicum strain II.

Enrichment

Growth rate in batch culture with inulin

Optimum temperature for growth

Fermentation products

G + C content

C. thermosuccinogenes strain IC

• continuous culture • inulin as carbon

and energy source • inoculation with

mud from a tropical pond of a botanical garden

• , w 0.8 ± 0.05

• inulin 58 °C • fructose 72 °C

• formate, acetate, ethanol, lactate and succinate

• 35.9 mol%

C. thermoautotrophicum strain 11

• batch culture • pieces of dahlia

as carbon and energy source

• inoculation with pieces of dahlia

• M m a x : 0 . 8 ± 0 . 1

• inulin 58 °C • fructose 62 °C

• formate, acetate and ethanol

• 56.2 mol%

On account of these considerations the regulation of inulinase synthesis and the fermentation pattern of thermophilic anaerobes was investigated. Since the production of fermentation products and thermostable inulinases is commercially promising, ecophysiological research of this kind seems to be essential for the successful exploitation of thermophilic clostridia (Sonnleitner, 1985; Uhm et al., 1987). The present paper reports on the identification, ecology and biotechnology of the isolated thermophilic strains.

2 THERMOPHILIC INULIN-DEGRADING CLOSTRIDIA All anaerobic inulin-degrading thermophiles isolated in the present study can be

assigned to the genus Clostridium on the basis of the following characteristics: strictly anaerobic, sporeforming, rod-shaped and gram-positive.

The formation of succinate as a major fermentation product clearly distinguishes C. thermosuccinogenes from all other thermophilic clostridia described so far. In Table 1 the major characteristics of this new species have been summarized.

Strain II was assigned to the species C. thermoautotrophicum due to its ability to grow autotrophically with H2 + C0 2 . However, in contrast to the type strain, our isolate formed ethanol and formate as major fermentation products in addition to acetate (Table 1). Moreover, differences were observed in both the range of fermentable carbohydrates and

269

the temperature optimum for growth (Drent and Gottschal, 1991). Therefore, strain II represents a new strain of the species C. thermoautotrophicum.

3 PRODUCT FORMATION AND INULINASE SYNTHESIS Inulinase synthesis and product formation of both C. thermosuccinogenes strain IC

and C. thermoautotrophicum strain II were studied in batch and continuous culture (for preliminary results, see Drent et al., 1992).

Inulinase synthesis by the succinate-producing strain IC occurred only during growth in batch culture with inulin and a number of inulin oligomers (Table 2). When grown in an inulin-limited continuous culture C. thermosuccinogenes produced inulinase at all dilution rates tested, however, increasing the dilution rate resulted in a lower inulinase activity possibly due to some degree of repression by increasing concentrations of residual sugars. The major characteristics of the inulinase and its regulation have been summarized in Table 2.

Production of inulinase by C. thermoautotrophicum strain II was observed during growth on a variety of carbohydrates (Fig. 1, Table 2; see also Drent et al., 1992). Yet, the synthesis of the enzyme was not constitutive, since with some sugars and non-sugars no enzyme activity was present. In an inulin-limited chemostat, run at higher dilution rates, a drop in inulinase levels was observed as found with cultures of C. thermosuccinogenes (Fig. 2).

These results indicate that the synthesis of the inulin-degrading enzymes of both C.

<

GO O c

~5 c

110 j 100i 90-80-70-60-50-40-30-20i 1θ1 rii p-i n. il Ml J_

GLU FRU GF GF2 GF3 GF4

Growth Subst ra te GF.„

Fig. 1. Inulinase synthesis (U Γ1) by C. thermoautotrophicum strain II, grown in batch culture on fructose, glucose, inulin oligomers and inulin at 58 °C. In all cases the carbohydrate concentration was 0.15%. White bars represent cell-bound and black bars cell-free inulinase activity.

270

Table 2. Major characteristics of the inulinases and the regulation of the inulinase synthesis by Clostridium thermosuccinogenes strain IC and Clostridium thermoautotrophicum strain II. The inulinases were not purified and thus the enzyme activity was determined in crude extracts.

C. thermosuccinoges strain IC

C. thermoautotrophicum strain II

Substrates inducing inulinase synthesis

Substrates not inducing inulinase synthesis

Temperature characteristics of inulinase activity

Sugars formed during hydrolysis of inulin

fructosylnystose, inulin, kestose, nystose

fructose, galactose, glucose, lactose, maltose, raffinose, ribose, sucrose, xylose

Tmin: 30 °C Topt: 58 °C T · 72 °C 1max· '^ ^

fructose

fructosylnystose, inulin, kestose, nystose, sucrose

galactose, lactose, melibiose, starch, methanol, H2 + C0 2

Tmin: 30 °C Topt: 62 °C T · 70 °C

fructose

<

Ö c

500

400

300

200

l\ \ :L\

'il\ \ ■ + \ \

/ +\\ - + \ \ f \\ ■ ^:

7 \ ■ - v v-

Γ ^ ^+-—-

~~^+ . ■ ^

" ^ B

3 100 [ c

0.0 0.1 0.2 0.3 0.4 0.5 0.6

Dilution rate (hr1)

Fig. 2. Inulinase synthesis (U Γ1) by C. thermoautotrophicum strain II, grown in an inul in-limited continuous culture at 58 °C. (+) , ( ■ ) , (D): cell-bound, cell-free and total inulinase activity, respectively.

thermosuccinogenes and C. thermoautotrophicum is inducible and susceptible to catabolite repression. Therefore, the way in which inulinases are regulated by these two clostridia differs markedly from the repression/derepression regulation pattern, observed for the biotechnologically important yeast Kluyveromyces marxianus (Rouwenhorst et al., 1989).

271

In the continuous cultures, not only the production of inulinases was examined, but also the pattern of fermentation products. At high dilution rates C. thermoautotrophicum formed formate, H2, acetate and ethanol. At low growth rates the yield of acetate and H2

increased (data not shown). Also in C. thermosuccinogenes cultures the ratio of the different fermentation products formed, depended on the growth rate. Increasing the dilution rate resulted in higher yields of succinate (data not shown).

4 BIOTECHNOLOGY Thermophilic clostridia are biotechnologically promising microorganisms for the

production of fermentation products and thermostable enzymes (Sonnleitner, 1985; Uhm et al., 1987). In this respect, the formation of succinate by C. thermosuccinogenes is very interesting. This dicarboxylic acid is widely used as a specialty chemical for applications in foods, pharmaceuticals, and cosmetics but so far is produced almost exclusively by the petrochemical industry. Therefore, a patent application concerning the isolation and use of thermophilic succinate-producing clostridia has been filed (U.S. Patent 493.408).

Thermostable inulinases could find application in the production of high-fructose syrups. Elevated temperatures ensure higher solubility of inulin, which is a major limiting factor for the formation of sugar syrups at lower temperatures (Uhm et al., 1987). The temperature optima for the inulinases of the new isolates around 60 °C belong to the highest reported in the literature (Table 2).

However, the yeast K. marxianus exhibits still superior properties with respect to the quantity of inulinases produced. High levels were in particular observed when the yeast was cultivated in an inulin-limited continuous culture, run at low dilution rate (Rouwenhorst et al., 1988).

5 ECOLOGY An interesting aspect of the new isolates is their occurrence in soils, which probably

never reach the optimal temperature for growth (58 °C). In order to gain an understanding into the possible role of thermophilic bacteria living in soils, studies on the microbial physiology at suboptimal temperatures should be performed.

The effect of a down-shift in temperature was investigated during growth of C. thermosuccinogenes in an inulin-limited continuous culture. The temperature was shifted between optimum and near-minimum values. Most significantly, succinate increased relative to the other fermentation products. During the down-shift of temperature hardly any change in the production of the inulinases was observed. The underlying mechanisms of these changes in fermentation patterns are poorly understood. Therefore, studies are needed to elucidate the metabolic pathway and the regulatory factors controlling the product

272

formation. Such studies have recently been started in our laboratory.

6 CONCLUDING REMARKS The current study on the formation of fermentation products and the regulation of

inulinase synthesis by the newly isolated thermophilic clostridia offers interesting perspectives for the use of these organisms in biotechnological processes. However, more research is needed to fully understand the underlying mechanisms for inulinase synthesis and changes in fermentation patterns. In view of the relatively low inulinase activity of the above-described organisms, it is worth trying to isolate more productive thermophilic anaerobes.

7 REFERENCES

Allais, J.-J., Hoyos-Lopez, G., Kammoun, S. and Baratti, J.C., 1987. Isolation and characterization of thermophilic bacterial strains with inulinase activity. Appl. Environ. Microbiol., 53: 942-945.

Drent, W.J., Both, G.J. and Gottschal, J.C., 1992. Heterogeneity in colony formation of polysaccharide-degrading microorganisms (in preparation).

Drent, W.J. and Gottschal, J.C., 1991. Fermentation of inulin by a new strain of Clostridiwn thermoautotrophicum isolated from dahlia tubers. FEMS Microbiol. Lett., 78: 285-292.

Drent, W.J., Lahpor, G.A., Wiegant, W.M. and Gottschal, J.C., 1991. Fermentation of inulin by Clostridiwn thermosuccinogenes sp. nov., a thermophilic anaerobic bacterium isolated from various habitats. Appl. Environ. Microbiol., 57: 455-462.

Fuchs, A., De Bruijn, J.M. and Niedeveld, C.J., 1985. Bacteria and yeasts as possible candidates for the production of inulinases and levanases. Antonie van Leeuwenhoek, 51: 333-343.

Marchal, R., Blanchet, D. and Vandecasteele, J.P., 1985. Industrial optimization of acetone-butanol fermentation: a study of the utilization of Jerusalem artichokes. Appl. Microbiol. Biotechnol., 23: 92-98.

McKellar, R.C. and Modler, H.W., 1989. Metabolism of fructo-oligosaccharides by Bifidobacteriwn spp. Appl. Microbiol. Biotechnol., 31: 537-541.

Rouwenhorst, R.J., Visser, L.E., Van der Baan, A.A., Scheffers, W.A. and Van Dijken, J.P., 1988. Production, distribution, and kinetic properties of inulinase in continuous cultures of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol., 54: 1131-1137.

Sonnleitner, B., 1985. Biotechnology of thermophilic bacteria - growth, products, and application. Adv. Biochem. Eng. Biotechnol., 28: 69-138.

Tansey, M.R. and Jack, M.A., 1977. Growth of thermophilic and thermotolerant fungi in soil in situ and in vitro. Mycologia, 69: 563-578.

Uhm, T.-B., Byun, S.-M., Kwon, Y.-J., Han, S.-B. and Ryu, K.-S., 1987. Thermal stability of the multiple charge isoforms of inulase from Aspergillus niger. Biotechnol. Lett., 9: 287-290.


Yamashita, K., Kawai, K. and Itakura, M., 1984. Effects of fructooligosaccharides on blood glucose and serum lipids in diabetic subjects. Nutr. Res. 4: 961-966.


INULIN DEGRADATION BY PEDIOCOCCUS PENTOSACEUS

Wouter J. MIDDELHOVEN, Patrice F.L.A. van ADRICHEM, Martine W. REU and Monique KOOREVAAR Laboratory of Microbiology, Agricultural University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands

ABSTRACT

A lactic acid bacterium degrading inulin was isolated from ensiled Jerusalem artichoke herbage. It proved to be a homofermentative aerotolerant catalase-negative diplococcus, identified as Pediococcus pentosaceus. Inulin hydrolysis was catalysed by an exo-inulinase which released fructose from inulin with an average degree of polymerization (DP) of 25 or less. The inulinase strongly resembled that produced by yeasts: the pH optimum of the enzyme was 4.5-5.4. The apparent substrate constant for inulin (average DP 20) was about 10 mM. The enzyme was mainly bound to the cell wall but small activities were detected in the culture liquid.

Washed suspensions of inulin-grown cells hydrolysed inulin at pH 5.0 at a rate of 0.57 mg reducing sugars released mg"1 dry cells h"1 at 30 °C. Sucrose was hydrolysed under the same conditions at a rate of 2.3 mg reducing sugars mg"1 dry cells. Hence, the ratio of inulin hydrolysis and sucrose hydrolysis (S/I ratio) was 4.1, which is an exceptionally low value. Sucrose-grown cells showed a S/I ratio of 3.3. Specific sucrose-hydrolysing activities of these cells were about one third of those of inulin-grown cells.

1 INTRODUCTION

Inulin can be utilized in the food industry either as such or, after hydrolysis, as fructose syrup. In addition to these applications in foods and soft drinks, inulin and fructose may become interesting raw materials for the synthesis of miscellaneous chemical compounds (Fuchs, 1987).

In the present paper we report an attempt to the direct conversion of inulin into lactic acid, by bacterial fermentation. Lactic acid is widely applied in the food industry. It is also utilized for non-food applications (Benninga, 1990). The main topic of this'paper is the mechanism of inulin hydrolysis by a homofermentative lactic acid bacterium.

274

2 MATERIALS AND METHODS 2.1 Inulin

A commercial inulin preparation, Fibruline, was kindly provided by Dr. J. Fockedey (Warcoing S.A., Pecq-Warcoing, Belgium). It was prepared from chicory roots and contains inulin of variable chain length and small amounts of sucrose and fructose. The latter could be partly removed by dispersing 11 g Fibruline in 1 litre ice-cold water. After 2 h at 4 °C the supernatant was discarded. The undissolved fraction was dissolved in lukewarm water to yield a solution of about 100 g Γ1 (determined as fructose by the anthrone method). This fraction is designated as "inulin". Its average degree of polymerization (DP) proved to be about 20-25. The inulin solution was sterilized through 0.2-μπι filters before being added to growth media.

2.2 Isolation and cultivation The growth medium resembled MRS medium. It contained per litre demineralized

water: 5 g yeast extract, 5 g Lab Lemco meat extract, 1 g KH2P04, 0.5 g Na-acetate, 2 g Na-citrate, 1 g Tween 80, 10 mg MnCl2, 10 mg FeCl3, 10 ml trace element solution, and 1 ml vitamin solution (Middelhoven, 1970). The medium was adjusted to pH 6.0 and was sterilized at 120 °C for 20 min. Sugars were sterilized separately by filtration (0.2 μπι) and were added at 5 or 20 g Γ1. Solid media contained 20 g agar Γ1.

Lactic acid bacteria which utilized inulin were isolated from Jerusalem artichoke herbage that was harvested in the late autumn, chopped into 0.5-cm pieces, and ensiled at 30 °C on a laboratory scale, with addition of 20 g Fibruline kg"1. After 4 days a suspension was plated on solid growth medium (20 g Fibruline Γ1) and incubated at 30 °C. After 2 and 3 days colonies of different types were streaked pure. Only one type, represented by strain I30A4, turned out to grow at the expense of inulin and to be genetically stable. It was maintained in stab cultures (5 g inulin Γ1) in growth medium. The strain was grown in liquid medium as described above in screw-capped rubber-sealed bottles at 30 °C for 2 days.

2.3 Chemical analyses Fermentation products in culture supernatants were analysed by high-performance

liquid chromatography (HPLC) using a Chrompack Organic Acid Column provided with a Micro Guard Carbo P head column and an infrared detector. The eluant (0.60 ml min"1) was 10 mM sulphuric acid. This HPLC method allowed accurate measurement of fructose and other monosaccharides, lactic and acetic acids, and ethanol, but not of residual inulin. The latter was determined by the anthrone method (Trevelyan and Harrison, 1952). In enzyme preparations protein was quantified with Coomassie Blue B G250 reagent (Bradford,

275

1976). Reducing sugars were determined with Somogyi-Nelson reagent (Somogyi, 1952). This method was not used with culture supernatants since the latter contained substances which interfered with the analysis.

2.4 Enzyme determinations Since at least part of the inulinase was bound to the cell wall, a washed cell

suspension could be used as enzyme preparation. In most experiments, however, the cells were sonicated or the enzyme was solubilized. In the latter case the pellet of 100 ml 2-day-old culture (containing 10 g inulin Γ1) was taken up in 5 ml of 0.1 M phosphate buffer (pH 6.0) and treated with lysozyme (Sigma, 0.75 mg per ml) overnight at 30 °C. No cell lysis was seen after this treatment. To bring about cell lysis, subsequent sonication (15 times 30 sec with a Branson Sonifler B12, small tip) was necessary. Sonication without prior lysozyme treatment did not lyse the cells. The enzymes present in homogenate, sonicated cell suspensions or culture supernatants were partially purified with ammonium sulphate (80% saturation) at 0 °C. After centrifugation the precipitated and floating protein fractions were collected, dissolved in buffer and dialysed overnight in water to remove ammonium sulphate and substances which interfered with the determination of reducing sugars.

3 RESULTS 3.1 Isolation, properties and cultivation of strain I30A4

Among the lactic acid bacteria isolated from Jerusalem artichoke herbage one type appeared to be genetically stable with regard to inulin utilization. Strain I30A4 was selected as a representative of this group. It proved to be a Gram-positive aerotolerant catalase-negative diplococcus. Strain 130A4 was identified with the API 50 CHL system (API system S.A., Montalieu-Vercieu, France) as Pediococcus sp. Additional characteristics suggested it to be identical with Pediococcus pentosaceus. These were: arginine dihydrolase positive, growth at 45 °C positive, growth at 50 °C negative. The characteristic tetrads were not seen. The cells grew singly or in pairs, never in chains of more than two cells. The latter property excludes identification as a Streptococcus sp.

In Fig. 1 the results of a representative growth experiment are presented. The growth medium contained 3.5 g inulin Γ1. The culture was inoculated with a preculture (10 ml) in the same growth medium. In the course of time the fructose concentration (measured by the anthrone method) decreased and the amounts of lactic acid and biomass increased. A logarithmic plot of the latter with time showed that growth was exponential for most of the time. A growth rate of 0.55 h"1 and a generation time of 1.26 h were calculated by linear regression. From the data of Fig. 1 two moles of lactic acid appeared to be produced per mole of sugar consumed.

276

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3.2 Inulin degradation Suspensions of sonicated inulin-grown cells as well as supernatants of the culture

hydrolysed inulin. The amount of fructose (HPLC analysis) equalled the amount of reducing compounds liberated (Somogyi-Nelson method). Hence, the inulinase is of the exo-type like that of yeasts (Vandamme and Derycke, 1983; Fuchs et al., 1985; Guiraud and Galzy, 1990). Similar results were obtained with inulinase precipitated with ammonium sulphate from the culture filtrate. However, in the latter case the specific inulinase activity was only about one tenth of that in the sonicated cell suspension.

In Fig. 2 the pH activity curve of inulinase is shown. The enzyme preparation consisted of sonicated inulin-grown cells, partially purified by ammonium sulphate precipitation. A broad pH optimum of 4.5-5.4 was observed. A marked decrease in enzyme activity was observed at pH 6.5 and below pH 4.0. At the latter pH non-enzymatic inulin hydrolysis occurs which makes the data less reliable. Essentially the same pH activity curve was observed with inulinase precipitated from the culture supernatant.

Washed cells grown on inulin or on sucrose hydrolysed both of these carbohydrates (Table 1). In this experiment 10 mM sodium fluoride was added in order to inhibit fermentation of the released sugars. Inulin-grown cells displayed a higher activity than sucrose-grown cells. The ratios of the specific enzyme activities on sucrose and on inulin (the S/I ratio) for sucrose-grown cells and for inulin-grown cells were 3.3 and 4.1,

277

0.5 "I 1 1 1 1 ' ' r

3.5 3.8 4.2 4.6 5.0 5.4 5.8 6.2 6.6 PH

Fig. 2. pH activity curve of inulinase. The pellet of a sonicated suspension of inulin-grown cells of strain I30A4 was suspended in 50 mM sodium lactate buffer (pH 3.4-5.4) or 50 mM potassium phosphate buffer (pH 5.5 and 6.0). The inulin concentration was 4.0 g Γ1; 30 °C.

respectively. This suggests that hydrolysis of both inulin and sucrose are due to the activity of one enzyme.

Using an inulin preparation with an average DP of about 20, inulinase showed a high apparent substrate constant for inulin. By linear regression an apparent substrate constant of 37 g inulin Γ1 was calculated for solubilized inulinase. This value corresponds with a value of about 10 mM, as observed for yeast exo-inulinases (Vandamme and Derycke, 1983; Guiraud and Galzy, 1990). The maximum reaction rate observed in this experiment was 2.8 mg fructose released mg"1 protein min"1.

The production of inulinase appeared to be inducible. In Table 2 it can be seen that

Table 1. Specific activities of inulin and sucrose hydrolysis in washed suspensions of inulin-grown and sucrose-grown cells of strain I30A4. Conditions: 20 g inulin or 50 g sucrose Γ1 of 50 mM sodium acetate buffer (pH 5.0), supplied with 10 mM sodium fluoride; 30°C. Unit: mg reducing sugar (expressed as fructose released mg"1 dry cells h"1).

Growth substrate Reaction rate S/I ratio

Sucrose Inulin

Sucrose 0.78 0.24 3.3 Inulin 2.3 0.57 4.1

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Table 2. Production of inulinase by strain I30A4. Cells were grown on different substrates, solubilized and used to determine specific inulinase activities. Conditions in the enzyme assay: 50 mM sodium lactate buffer (pH4.8); 1.5 g inulin Γ1.

Growth substrate Specific inulinase activity (jig fructose mg1 protein min"1)

Inulin 43 Sucrose 15 Glucose 3

the enzyme is present after growth on inulin and sucrose, but is produced in much smaller amounts in glucose-fed cultures. The low specific inulinase activity present in sucrose-grown cells enables the cells to resume growth immediately after transfer to inulin medium. Cells grown in glucose medium, however, had to pass through a lag phase of about 3 h (unpublished results).

As pointed out above, washed cell suspensions and to a much lesser extent filtrates of inulin-grown cultures were able to hydrolyse inulin. This suggests presence of the enzyme outside the cell membrane. Part of the inulinase activity could be released from the cells by treatment with lysozyme (20 mg ml"1 of 10 mM potassium phosphate buffer, pH 6.0) for 1 h at 37 °C. Cells did not lyse during this treatment. Complete solubilization of the inulinase was achieved by subsequent repeated sonication, which appeared to be necessary to break the tough cell walls. Specific inulinase activities in these homogenates were always considerably lower than those measured in suspensions of untreated cells. This loss in activity is probably due to the sonication treatment. Thus, most of the inulinase activity is firmly bound to the cell, outside the cell membrane and accessible to the substrate. Whether inulinase is also present in the cytoplasm is still unknown.

4 DISCUSSION From ensiled Jerusalem artichoke herbage a homofermentative lactic acid bacterium

was isolated which readily grew with inulin as source of energy, and which was identified as Pediococcus pentosaceus. Cells grown at the expense of inulin or sucrose readily hydrolysed inulin, but glucose-grown cells hardly did.

Inulin-grown cells when hydrolysing inulin yielded fructose as the only detectable reaction product. This was concluded from the observation that free fructose (as assayed by HPLC) and total reducing sugars were released from inulin at equal rates and to the same amounts. Hence, the inulinase is of the exo-type (2,1-0-D-fructan fructanohydrolase, EC 3.2.1.7) like that of yeasts (Vandamme and Derycke, 1983; Fuchs etal., 1985; Guiraud and Galzy, 1990) and many bacteria (for instance, Bacillus subtilis Marburg 168, sacL mutant, Kunst et al., 1977; and Actinomyces viscosus, Miller and Somers, 1978; for older data and

279

references see also Fuchs, 1959). The Pediococcus inulinase from the present study differs from that of yeasts by the

exceptionally low S/I ratio, viz. 3.3 for sucrose-grown cells and 4.1 for inulin-grown cells. S/I ratios of yeast and fungal inulinases (Vandamme and Derycke, 1983) are usually between 10 and 50. The Pediococcus inulinase resembles yeast inulinases with respect to the relatively low pH optimum, viz. 4.5-5.4, and the high substrate constant, viz. 10 mM for inulin with an average DP of about 20.

Inulin utilization by streptococci isolated from the dental plaque has been reported (cf. Fuchs et al., 1985). An unidentified Streptococcus hydrolyses inulin and levan to fructose at pH 5.0-7.0, probably by more than one enzyme (Da Costa and Gibbons, 1968). A related enzyme has been detected in S. mutans strain C67-1 (Kelstrup and Funder-Nielsen, 1972). In S. salivarius strain 51a ß-D-fructofuranosidase releasing fructose only from levan and from oligoinulins at pH 6.7 was demonstrated (Marshall and Weigel, 1980). Another strain of S. salivarius, viz. KTA-19, produces exolevanase and enzymes hydrolysing inulin and sucrose at pH 6.5 (Takahashi et al., 1983). Some of these inulinases from oral streptococci may bear some relationship to the inulinase in the present study. However, they all differ with respect to their relatively high pH optima.

5 ACKNOWLEDGEMENTS Thanks are due to Dr. Adriaan Fuchs for stimulating discussions, to Nees Slotboom

for the art work and to Ans Broersma-De Haan for preparing the typescript.

6 REFERENCES

Benninga, H., 1990. A History of Lactic Acid Making. Kluwer Academic Publishers, Dordrecht, 478 pp. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein

utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. DaCosta, T. and Gibbons, R.J., 1968. Hydrolysis of levan by human plaque streptococci. Arch. Oral Biol.,

13: 609-617. Fuchs, A., 1959. On the synthesis and breakdown of levan by bacteria. Ph.D. Thesis, State University

Leiden. Fuchs, A., 1987. Potentials for non-food utilization of fructose and inulin. Starch/Stärke, 39: 335-343. Fuchs, A., De Bruijn, J.M. and Niedeveld, C.J., 1985. Bacteria and yeasts as possible candidates for the

production of inulinases and levanases. Antonie van Leeuwenhoek, 51: 333-343. Guiraud, J.-P. and Galzy, P., 1990. Inulin conversion by yeasts. In: H. Verachten and R. De Mot (Eds.),

Yeast Biotechnology and Biocatalysis. Marcel Dekker, Inc., New York, pp. 255-296. Kelstrup, J.U. and Funder-Nielsen, T.D., 1972. Enzyme reactions in dental plaque matrix. Modification

of velocity by the presence of polymers. Acta Odontol. Scand., 30: 621-628. Kunst, F., Steinmetz, M., Lepesant, J.-A. and Dedonder, R., 1977. Presence of a third sucrose hydrolyzing

enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168. Biochimie, 59: 287-292.

Marshall, K. and Weigel, H., 1980. Extracellular 0-D-fructofuranosidase elaborated by Streptococcus

280

salivarius strain 51: preparation, and mode of action on a levan and on homologues of inulobiose. Carbohydr. Res., 83: 315-320.

Middelhoven, W.J., 1970. Induction and repression of arginase and ornithine transaminase in baker's yeast. Antonie van Leeuwenhoek, 36: 1-19.

Miller, C.H. and Somers, P.J.B., 1978. Degradation of levan by Actinomyces viscosus. Infect. Immun., 22: 266-274.

Somogyi, M., 1952. Notes on sugar determination. J. Biol. Chem., 195: 19-23. Takahashi, N., Mizuno, F. and Takamori, K., 1983. Isolation and properties of levanase from Streptococcus

salivarius KTA-19. Infect. Immun., 42: 231-236. Trevelyan, W.E. and Harrison, J.S., 1952. Studies on yeast metabolism 1. Fractionation and

microdetermination of cell carbohydrates. Biochem. J., 50: 298-303. Vandamme, E.J. and Derycke, D.G., 1983. Microbial inulinases: fermentation process, properties, and

applications. Adv. Appl. Microbiol., 29: 139-176.


IN VITRO SYNTHESIS OF INULIN BY THE INULOSUCRASE FROM STREPTOCOCCUS MUTANS

A.S. PONSTEIN* and M.B. van LEEUWEN ATO-Agrotechnologie, P.O. Box 17, 6700 AA Wageningen, The Netherlands

Present address: Mögen International N.V., Einsteinweg 97, 2333 CB Leiden, The Netherlands

ABSTRACT

An Escherichia coli clone containing the flf gene of Streptococcus mutatis encoding an inulosucrase was studied in in vitro experiments. The enzyme transferred fructose units donated by sucrose to water as well as to sucrose (and fructo-oligosaccharides). The apparent Km of the "self-transfer" reaction was 18 ± 2mM.

In the presence of inulin an enhanced fructosyltransferase activity was observed. Analysis of the reaction products demonstrated that inulin altered the molecular weight distribution of the reaction products formed. It is unclear at present whether the added inulin molecules function as acceptor or merely alter the specificity of the enzyme.

1 INTRODUCTION The chain length of enzymatically produced fructans depends on the origin of the

enzymes used. Short-chain fructo-oligosaccharides (with a degree of polymerization, DP, of 3 to 5) arise from incubation of concentrated sucrose solutions with fungal fructosyltransferases (Hidaka et al., 1988; Hayashi et aL, 1989). These saccharides are applied in the food industry since they improve food texture and mouthfeel.

Many plant species synthesize medium-sized fructan molecules, with average degrees of polymerization varying from < 10 in Jerusalem artichoke (Helianthus tuberosus L.) to about 200 in artichoke {Cynara scolymus L.) (Praznik and Beck, 1985), from 1-kestose (fructosylsucrose) as the fructosyl donor. Apart from being a carbohydrate reserve fructans are believed to play a role in frost tolerance in plants (Pontis, 1989). Therefore, the DP should remain low. Irrespective of DP, inulins may serve as a source of fructose, and may constitute a raw material in the manufacture of hydroxymethylfurfural, and ethanol and other fermentation products (cf. Fuchs, 1987). They may also be used as fructo-

282

oligosaccharides (see above), or transformed into cycloinulo-oligosaccharides (Kawamura et al., 1989), etc. Some applications for inulin have been developed based on these products. Other applications are conceivable for inulins of moderate DP (e.g., as occur in chicory, Cichorium intybus L.), and still others will arise after chemical or enzymatic modification of these molecules.

Our interest is aimed at the development of methods to modify inulin molecules enzymatically, and to find new applications for fructose oligomers and polymers. Since bacterial fructosyltransferases synthesize high-molecular-weight (HMW) fructose polymers we started to investigate these enzymes. The common donor molecule is sucrose and known acceptor molecules are water (hydrolysis), sucrose ("self-transfer") or reaction products of the "self-transfer" reaction (leading to HMW fructans). In addition, we investigated whether these enzymes are also able to elongate exogenously added inulin molecules. Here, we report on some of the characteristics of the fructosyltransferase from Streptococcus mutans. We used a gene of S. mutans cloned in Escherichia coli to prevent contamination with other glucosyl- and fructosyltransferases (Sato et al., 1984; Kametaka et al., 1987).

2 MATERIALS AND METHODS 2.1 Preparation of enzyme extracts

Cell cultures of E. coli were grown on TY medium (Maniatis et al., 1982) at 30 °C. T h e ^ 4 0 clone, encoding the fructosyltransferase from S. mutans (Aduse-Opoku et al., 1989), was kindly supplied by J. Aduse-Opoku. It was grown on TY medium supplemented with ampicillin (100 ^g ml"1). Cells were harvested in the exponential growth phase (OD450

= 0.4 to 0.6) by centrifugation (10 min at 10,000 g), and washed in 50 mM sodium phosphate buffer (pH 6.5). The final pellet was suspended in the same buffer containing 0.02% sodium azide. The cells were broken by sonication with a Branson sonifier 250 (six times 15 sec, 30% output) and the sonicate was used as the crude enzyme preparation.

Bacillus subtilis QB112 (Lepesant et al., 1972) was obtained by courtesy of M. Steinmetz. An enzyme extract was prepared by growing QB112 on a mineral medium supplemented with 8% sucrose. B. licheniformis NRC 9012 was supplied by K. Latta and grown according to Ramsay et al. (1989) on 8% sucrose. Bacilli were harvested in the stationary growth phase and the supernatant was used as the crude enzyme solution.

2.2 Assay ofinvertase activity, determination of glucose, fructose and sucrose Fructose, glucose and sucrose were quantified enzymatically. A kit provided by

Boehringer Mannheim was used according to the instructions. Assay conditions for invertase were as described for fructosyltransferase (see 2.3).

283

2.3 Assay offructosyltransferase activity Inulin was obtained from Suiker Unie, the Netherlands. Inulin was purified by

ethanol precipitation (80%, v/v) from an aqueous solution (1 %, w/v). The mean DP of the inulin preparation was 12 to 15. Hence, a 1% inulin solution corresponds to 4 to 5 mM.

Enzyme assays were performed in a total of 50 μΐ in 50 mM sodium phosphate buffer (pH 6.5), containing 0.01% sodium azide, 50 mM sucrose, radiolabelled sucrose ([U-14C-fructosyl]-glucose, New England Nuclear, specific activity 9.3 MBq mmol"1) and 1% (w/v) inulin (unless stated otherwise), at 37 °C for 7 h. The reaction was terminated by the addition of 0.4 ml ice-cold ethanol (96%). Reaction mixtures not containing inulin served as controls; upon termination of the enzyme assay they were also supplemented with inulin. Upon addition of ethanol precipitates formed immediately; they were collected by centrifugation. Supernatants were removed and pellets washed once with 1.0 ml ethanol, dried and suspended in 100 μΐ water.

Radioactivity was measured by liquid scintillation counting. To this end, 1.5 ml OptiPhase "HiSafe" 3 (Pharmacia) was added and the amount of radioactivity counted in a Wallac 1410 liquid scintillation counter (Pharmacia).

For the determination of the Km value for sucrose, sucrose concentrations between 5 and 50 mM were used. Kinetic parameters were determined by the method of Lineweaver and Burk.

2.4 Bio gel P-30 gel filtration Biogel P-30 was obtained from Bio-Rad, and used to pour a column of 50 ml (1.7

X 28 cm). The column was equilibrated with 50 mM sodium phosphate buffer (pH 6.5) containing 0.02% sodium azide at room temperature. Reaction products were solubilized in 200 to 300 μΐ buffer and applied to the column at room temperature. The flow rate during operation was 12 ml h"1. Fractions (1 ml) were collected and aliquots (0.5 ml) were taken for scintillation counting as described above.

2.5 Determination of carbohydrate content The carbohydrate content of the fractions obtained upon gel filtration was determined

according to Fairbairn (1953).

2.6 NMR analysis Part of the crude enzyme extract (25 ml) was incubated in 8% sucrose in the

presence of 0.05% sodium azide at 37 °C during 64 h. The reaction product was poorly soluble in water. After being collected by centrifugation (10 min at 10,000 g) the pellet was dried. The reaction product also appeared to be poorly soluble in DMSO.

284

A 13C-NMR spectrum was obtained using a Bruker AC 200 spectrometer operating at 50,323 MHz. About 1 mg inulin was solubilized in 0.6 ml D20, with acetone as the internal standard. Experiments were performed with complete proton-decoupling at 300 K; 14,348 scans were run and stored on a 32 Kb data base. The spectrum presented was obtained after 2 Hz line broadening.

3 RESULTS AND DISCUSSION 3.1 Demonstration of fructosyltransferase activity

Crude extracts from wild-type E. coli and E. coli containing the ftf gene from S. mutans were compared with respect to invertase and fructosyltransferase activity. Invertase activity was apparent at relatively low sucrose levels (Fig. 1), but only in case of the transformed E. coli.

Increasing the amount of sucrose resulted in an enhanced fructosyltransferase activity. This is visualized in Fig. 1 by the much larger amount of glucose formed as compared with that of free fructose formed. Fructosyltransferase activity was only present in extracts from the E. coli transformant. Thus, the ftf gene product is responsible for the cleavage of the glycosidic linkage in sucrose and the transfer of the fructose moiety, at low sucrose

[glucose] and [fructose] formed (in mM) % of label incorporated

W8& Ifruc] formed by ftf

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ABCC A B O D E F

■ m l

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5 15 1500 [sucrose] in assay mixture (in mM)

10 20 30 40 50 60 [sucrose] in mM

Fig. 1. (left) Histogram showing the production of fructose and glucose upon incubation of the inulosucrase from S. mutans with different amounts of sucrose. The formation of fructose and glucose is given for a protein-free control (A and D), the E. coli control (B and E) and the^f40 clone (C and F), respectively.

Fig. 2. (right) Diagram showing the amount of labelled, ethanol-insoluble product synthesized by the inulosucrase from S. mutans at various sucrose concentrations. The curves represent data of experiments carried out in the absence or presence of 5% inulin.

285

concentrations, to water and, at high sucrose concentrations, to sucrose or to growing inulin chains ("self-transfer"). No interference originating from the E. coli background was observed.

A radioactive assay was performed to study the fructosyltransferase activity of the enzyme in more detail. Upon incubation with sucrose, which was uniformly labelled in the fructose moiety, an ethanol-insoluble compound was formed only by the ftf enzyme preparation (data not shown). The incorporation of the labelled fructose units was linear with time for 12 h (data not shown). The formation of radiolabelled product increased with the enzyme concentration to at least 25 μg crude protein. The apparent Km value of the "self-transfer" reaction was 18 ± 2 mM sucrose. This value corresponds to data reported by Sato et al. (1984).

3.2 Effect of the addition of inulin on the activity ofdifferent fructosyltransferases The effect of the addition of inulin (1 %) on the transfructosylating activity of the

fructosyltransferases from B. subtilis, B. licheniformis and S. mutans was studied next. Only the latter enzyme preparation responded to the addition of inulin (data not shown).

The amount of fructose units transferred to the ethanol-insoluble product by the inulosucrase from S. mutans was increased in the presence of 1 % inulin. This increase was even more pronounced in the presence of 5% inulin (data not shown). An increase of incorporated label in the presence of 5 % inulin was only apparent at low but not at high sucrose concentrations (Fig. 2).

Two extremes (1 and 50 mM) were analysed further with respect to the reaction products formed (Fig. 3). The labelled reaction products were analysed by gel permeation chromatography. In the absence of exogenously added inulin mainly a high-molecular-weight product was formed. The HMW reaction product (containing 52.4% and 51.3%, respectively, of the total amount of label) was excluded from Biogel P-30. Literature data indicate that the molecular weight of this reaction product could be a few million Da (Ebisu et al., 1975). A LMW product was also formed (Fig. 3) but its nature is presently unclear.

In the presence of inulin a third reaction product was formed, eluting between the HMW and the LMW products. This reaction product (containing 59.4% and 39.0% of incorporated label, respectively) had about the same mol wt as the exogenously added inulin molecules (data not shown). The absolute amounts of label incorporated in the HMW and the LMW fractions in the presence of inulin, as measured by total carbohydrate estimation (anthrone), were distinctly lower than those incorporated in the same fractions in the absence of inulin. Thus, the addition of inulin to the reaction mixture resulted in the additional formation of a third reaction product (of moderate molecular weight, MMW), but also affected the synthesis of the HMW and LMW reaction products to some extent.

286

The question arises whether the exogenously added inulin molecules are modified by

the inulosucrase. Therefore, experiments were carried out in which labelled inulin was used

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Table 1. The incorporation pattern of radiolabelled fructose in fractions of different molecular weight.

Assay condition Absolute amount of incorporation in

HMW MMW LMW

Relative amount of incorporation in

HMW MMW LMW

01 mM sucrose / no inulin 01 mM sucrose / 5% inulin 50 mM sucrose / no inulin 50 mM sucrose / 5% inulin

4596 254 3921 3793 8506 2012 3928 1340 2389 3384 3615 2262

52.4% 02.9% 44.7% 26.5% 59.4% 14.1% 51.3% 17.5% 31.2% 36.5% 39.0% 24.4%

287

instead of labelled sucrose. Preliminary results indicate that the labelled inulin was converted neither to HMW nor to LMW fructans (data not shown). Thus, added inulin does not seem to serve as a fructosyl donor. Whether inulin can function as a fructosyl acceptor awaits further analysis.

3.3 Characterization of the fif 40 product The main type of fructosyl linkage formed was determined by N M R analysis and

appeared to be of the inulin type. This is consistent with data reported by Ebisu et al.

(1975). However, it should be noted that the reaction product of the enzymatic reaction was

poorly soluble in water. Thus, it is uncertain whether the N M R spectrum represents the

total product formed.

4 REFERENCES

Aduse-Opoku, J., Gilpin, M.L. and Russell, R.R.B., 1989. Genetic and antigenic comparison of Streptococcus mutans fructosyltransferase and glucan-binding protein. FEMS Microbiol. Lett., 59: 279-282.

Ebisu, S., Kato, K., Kotani, S. and Misaki, A., 1975. Structural differences in fructans elaborated by Streptococcus mutans and Strep, salivarius. J. Biochem. (Tokyo), 78: 879-887.

Fairbairn, N.J., 1953. A modified anthrone reagent. Chem. Ind., 1953: 86. Fuchs, A., 1987. Potentials for non-food utilization of fructose and inulin. Starch/Stärke, 39: 335-343. Hayashi, S., Imada, K., Kushima, Y. and Ueno, H., 1989. Observation of the chemical structure of

fructooligosaccharide produced by an enzyme from Aureobasidium sp. ATCC 20524. Curr. Microbiol., 19: 175-177.


Kametaka, S., Hayashi, S., Miyake, Y. and Suginaka, H., 1987. Electrophoretic studies of extracellular glucosyltransferases and fructosyltransferases from seventeen strains of Streptococcus mutans. Arch. Microbiol., 147: 207-212.

Kawamura, M., Uchiyama, T., Kuramoto, T., Tamura, Y. and Mizutani, K., 1989. Formation of a cycloinulo-oligosaccharide from inulin by an extracellular enzyme of Bacillus circulans OKUMZ 3IB. Carbohydr. Res., 192: 83-90.

Lepesant, J.-A., Kunst, F., Lepesant-Kejzlarovä, J. and Dedonder, R., 1972. Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg. Mol. Gen. Genet., 118: 135-160.

Maniatis, T., Fritsch, E.F. and Sambrook, F., 1982. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Pontis, H.G., 1989. Fructans and cold stress. J. Plant Physiol., 134: 148-150. Praznik, W. and Beck, R.H.F., 1985. Application of gel permeation Chromatographie systems to the

determination of the molecular weight of inulin. J. Chromatogr., 348: 187-197. Ramsay, J.A., Cooper, D.G. and Neufeld, R.J., 1989. Effects of oil reservoir conditions on the production

of water-insoluble levan produced by Bacillus licheniformis. Geomicrobiol. J., 7: 155-165. Sato, S., Koga, T. and Inoue, M., 1984. Isolation and some properties of extracellular D-

glucosyltransferases and D-fructosyltransferases from Streptococcus mutans serotypes c, ey and / . Carbohydr. Res., 134: 293-304. (Pon) OK!



THE CLONED BACILLUS SUBTILIS LEVANASE GENE AS A POTENT SYSTEM FOR THE EXPLOITATION OF INULIN IN BIOTECHNOLOGICAL PROCESSES

H. SCHWAB and E. WANKER Institut für Biotechnologie, Technische Universität Graz, Petersgasse 12, 8010 Graz, Austria

ABSTRACT

The levanase enzyme of Bacillus subtilis is able to hydrolyse sucrose as well as the fructans levan and inulin. The levanase gene was used to construct recombinant Escherichia coli strains for overexpression of levanase. From lysates of such clones showing high levanase activity, the levanase protein was purified to near-homogeneity in a few steps. Cloning the levanase gene into Saccharomyces cerevisiae resulted in yeast clones being able to grow on inulin as the sole carbon source. Levanase was shown to be secreted from yeast clones into the culture medium. Preliminary results indicate that the B. subtilis levanase gene can also be cloned and expressed in filamentous fungi {e.g. Penicillium chrysogenum).

1 INTRODUCTION Carbohydrates produced by agriculture are the basis of nearly all industrial

fermentation processes. Sucrose- and starch-containing products (e.g. molasses) represent the bulk of the various raw materials used as carbon sources in industrial bioprocesses. From many points of view there is a need to increase the spectrum of agricultural products which are potentially useful as carbon and energy sources to biotechnology-based industries. Fructans, especially inulin, aroused the interest of biotechnologists since various plants such as Jerusalem artichoke (topinambour) or chicory are capable of producing inulin at good yields (Guiraud and Galzy, 1981; Bajpai and Bajpai, 1991). Unfortunately, so far not much effort has been put into selecting or breeding high-yielding varieties of these potential agricultural crops.

In order to provide a better basis for the use of fructans in industrial processes, two targets can be considered. One of the possible strategies is to construct inulin-utilizing production strains by recombinant DNA techniques. A second strategy is to make available highly active inulin-hydrolysing enzymes in large quantities and at a reasonable price allowing the manufacturing of fructose syrups at low costs.

290

2 THE BACILLUS SUBTILIS LEVANASE GENE ENCODES A POTENT INULINASE Levanase is one of three B. subtilis enzymes capable of hydrolysing sucrose; in

addition, this enzyme also hydrolyses the fructans levan and inulin. A 2.5 kb DNA fragment from B. subtilis containing the levanase gene was cloned in Escherichia coli. The presence of this DNA fragment enables the E. coli clones to grow on sucrose as the sole carbon source. Cell lysates prepared from such clones were capable of hydrolysing the fructans inulin and levan (Friehs et al., 1986). The DNA sequence of the levanase gene was determined (Schörgendorfer et al., 1987). The encoded polypeptide (the levanase protein) has a size of about 75 kDa. The N-terminal end of the levanase polypeptide contains a signal sequence typical for secretory proteins. The amino acid sequence of the N-terminal part of levanase shows high homology to that of yeast invertase (Schörgendorfer et al., 1988).

Expression of the levanase gene in E. coli under the natural promoter was rather inefficient. Therefore, the levanase gene was cloned into an expression vector based on the strong inducible tac-promoter (Wanker et al., 1991). Two different constructs containing different parts of the 5'-region of the levanase gene were made (Fig. 1).

π,ΚΝΑ P'tl ATG mRNA B«11"1 ATG

Fig. 1. Expression plasmids for overexpression of the B. subtilis levanase gene in E. coli. The constructs are based on the inducible iflc-promoter expression vector pJF119HE (Fürste et al., 1986). LEV, levanase gene; SD, shine dalgarno sequence.

291

Table 1. Growth of E. coli clones on sucrose or inulin media.

Plasmid Carbon source Induction Growth after days

1

-

+ -(+)

+ --_

2

(+)

+ + ( + )

+ + + + _

3

+

+ + + (+)

+ + + + + + _

pKF3a

pKF3a

pES17HE pES17HE pES17HE pES17HE pJFEW66 pJFEW66 pJFEW66 pJFEW66

sucrose inulin sucrose sucrose inulin inulin sucrose sucrose inulin inulin

n.r. n.r. + -+ -+ -+ -

a pKF3 is a plasmid containing the levanase gene under the control of its natural promoter (Friehs et al.y 1986).

Analysing the growth behaviour of the E. coli HB101 clones which contained the levanase expression plasmids shown in Fig. 1, indicated good expression of the levanase gene. Both clones showed excellent growth on sucrose under induced conditions; the clone containing pJFEW66 also exhibited rather good growth on inulin, whereas the clone containing pES17HE grew only weakly. However, with a clone containing the levanase gene under the control of the natural promoter, no growth on inulin could be observed (Friehs et al.9 1986) (Table 1).

Since inulin is not transported into the cell or to the periplasm, growth on inulin is a good indicator for expression efficiency. Sufficient amounts of levanase allowing hydrolysis of inulin present in the culture medium seem to be only released from E. coli clones at high expression levels. Cell lysates from pES17HE clones were analysed by SDS-PAGE and a strong 75 kDa band could be seen which is not visible in lysates from non-induced cultures. Specific inulinase activities of about 20 U mg"1 protein were measured in such lysates (Wanker et al., 1991).

3 PURIFICATION OF THE LEVANASE ENZYME Efficient overexpression of the levanase gene in E. coli represents a good basis for

the preparation of pure enzyme. A procedure was set up to obtain pure levanase protein in a few and efficient purification steps. Important steps in this purification procedure are: 1. ammonium sulphate precipitation at 60-80% saturation, 2. ion exchange chromatography on DEAE-Sepharose CL-6B, 3. ion exchange chromatography on S-Sepharose.

292

These purification steps usually result in very pure preparations of levanase protein. To obtain highest purity a Chromatographie separation step on a Mono-Q column can be added. Interestingly, chromatography on DEAE-Sepharose CL-6B resulted in two different fractions containing inulinase-active material. However, in these two fractions the molecular weight of the levanase protein as determined by SDS-PAGE was the same.

4 EXPRESSION OF THE B. SUBTILIS LEVANASE GENE IN EUKARYOTIC MICROORGANISMS Two groups of eukaryotic microorganisms are most important in biotechnology:

yeasts and filamentous fungi. Laboratory strains of Saccharomyces cerevisiae and a strain of Penicillium chrysogenum once used as a penicillin production strain were chosen as model organisms for expression studies with the levanase gene.

After removal of most parts of the bacterial 5'-sequences of the levanase gene, the resulting fragment which was identical to that present in pJFEW66 (Fig. 1) was cloned into plasmid pMA91 (Mellor et al., 1983), an expression vector for S. cerevisiae based on the strong constitutive promoter of the yeast PGK gene. The resulting plasmid pMAEW66 is shown in Fig. 2.

DNA of plasmid pMAEW66 was transformed into different laboratory strains of S. cerevisiae. All transformants containing this plasmid were able to grow rapidly on media containing inulin as the sole source of carbon. Upon analysis of the levanase activity in different fractions of cultures of S. cerevisiae strains carrying the levanase expression

Fig. 2. Map of the levanase expression plasmid pMAEW66. The construction is described in Wanker et al. (1991).

293

Table 2. Levanase activity in culture fractions of yeast clones.

S. cerevisiae strain Levanase activity (U ml"1)

in lysate cell-bound in culture medium

W303a(pMAEW66) 0.11 0.11 0.49 DBY747a(pMAEW66) 0.28 0.15 0.47 W303a(pMAEW13)a n.d.b n.d. n.d. W303a(pMA93) n.d. n.d. n.d.

a This clone lacks the signal sequence and 22 amino acids of the N-terminus of levanase. b n.d., not detectable.

plasmid pMAEW66, it was evident that levanase is secreted from the yeast clones (Wanker et al.9 1991). Table 2 shows the distribution of levanase activity in culture fractions of S. cerevisiae clones.

The amounts of levanase secreted from yeast clones containing the expression plasmid pMAEW66 are very promising with regard to the development of strains for production of levanase enzyme preparations. For comparison, with one of the most potent extracellular inulinase producers, Kluyveromyces fragilis, levels of up to 1.6 U ml"1 have been reported (Negoro, 1978).

Hlndlll.Clal.Asull.Smal.Kpnl.EcoRI Hindlll.Sphl.Pstl

Fig. 3. Maps of levanase expression plasmids for filamentous fungi. Pips: promoter region of the isopenicillin N synthetase gene of P. chrysogenum (Kolar et al., 1991); Pgpd: promoter region of the glyceraldehyd-3-phosphate dehydrogenase gene of A. nidulans (Van Gorcom et al., 1986). The constructs are based on the E. coli plasmid pJFl 19HE (Fürste et al., 1986) and are designed for co-transformation with a plasmid selectable in fungi.

294

In order to express the B. subtilis levanase gene in the fungus P. chrysogenum several plasmids carrying the levanase gene under the control of a fungal promoter have been constructed. In Fig. 3 examples of fungal expression plasmids are shown.

Co-transformation experiments were performed with the plasmids shown in Fig. 3 and the plasmid p3SR2 (Hynes et al., 1983), which contains the acetamidase gene of Aspergillus nidulans. This system has already been proven to be suitable for selection in P. chrysogenum (Kolar et al., 1988). Preliminary results indicated that expression of the levanase gene could also be possible in P. chrysogenum.

5 CONCLUSIONS The levanase gene of B. subtilis represents a useful basis for the establishment of

industrial bioprocesses based on inulin as an agriculturally produced and thus renewable raw material. On the one hand, this gene can be used to construct microbial strains for efficient production of the levanase enzyme. First results with respect to overexpression of the levanase gene in E. coli and S. cerevisiae were quite promising. On the other hand, the levanase gene can be used to introduce the ability of direct utilization of inulin as a carbon and energy source into various microbial production strains employed in biotechnology-based industries. The results obtained with baker's yeast and with a penicillin production strain of P. chrysogenum also proved the utility of such a strategy.

6 ACKNOWLEDGEMENT This work was supported by grants from the FWF (P5384), the BMLF, the BMWF

and the Land Steiermark.

7 REFERENCES

Bajpai, P.K. and Bajpai, P., 1991. Cultivation and utilization of Jerusalem artichoke for ethanol, single cell protein, and high-fructose syrup production. Enzyme Microb. Technol., 13: 359-363.

Friehs, K., Schorgendorfer, K., Schwab, H. and Lafferty, R.M., 1986. Cloning and phenotypic expression in Escherichia coli of a Bacillus subtilis gene fragment coding for sucrose hydrolysis. J. BiotechnoL, 3: 333-341.

Fürste, J.P., Pansegrau, W., Frank, R., Blöcker, H., Scholz, P., Bagdasarian, M. and Lanka, E., 1986. Molecular cloning of the plasmid RP4 primase region in a multi-host range tacP expression vector. Gene, 48: 119-131.

Guiraud, J.-P. and Galzy, P., 1981. Production de fructose par hydrolyse chimique de rinuline. Ind. Aliment. Agric, 98: 45-52.

Hynes, M.J., Corrick, CM. and King, J.A., 1983. Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations. Mol. Cell. Biol., 3: 1430-1439.

Kolar, M., Punt, P.J., Van den Hondel, C.A.M.J.J. and Schwab, H., 1988. Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene. Gene, 62: 127-134.

Mellor, J., Dobson, M.J., Roberts, N.A., Tuite, M.F., Emtage, J.S., White, S., Lowe, P.A., Patel, T.,

295

Kingsman, A.J. and Kingsman, S.M., 1983. Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene, 24: 1-14.

Negoro, H., 1978. Inulase from Kluyveromyces fragilis. J. Ferment. Technol., 56: 102-107. Schörgendorfer, K., Schwab, H. and Lafferty, R.M., 1987. Nucleotide sequence of a cloned 2.5 kb Pstl-

EcoRl Bacillus subtilis DNA fragment coding for levanase. Nucleic Acids Res., 15: 9606. Schörgendorfer, K., Schwab, H. and Lafferty, R.M., 1988. Molecular characterization of Bacillus subtilis

levanase and a C-terminal deleted derivative. J. Biotechnol., 7: 247-258. Van Gorcom, R.F.M., Punt, P.J., Pouwels, P.H. and Van den Hondel, C.A.M.J.J., 1986. A system for

the analysis of expression signals in Aspergillus. Gene, 48: 211-217. Wanker, E., Schörgendorfer, K. and Schwab, H., 1991. Expression of the Bacillus subtilis levanase gene

in Escherichia coli and Saccharomyces cerevisiae. J. Biotechnol., 18: 243-254.



MODELLING INULIN METABOLISM

M.W. SHAW, K. LODGE and P. JOHN Department of Agricultural Botany, Plant Science Laboratories, University of Reading, Reading, RG6 2AS, U.K.

ABSTRACT

We have modelled the distribution of chain lengths of the inulin accumulated in the storage organs of species belonging to the Compositae. Our analysis reveals that the relative abundance of chain lengths can be shown to depend on the relative affinity for different chain lengths of the enzyme responsible for the reversible transfructosylation, fructan:fructan fructosyltransferase (FFT). When it is assumed that the affinity of FFT first decreases then increases with an increase in chain length, the predicted chain length distribution resembles that revealed by Chromatographie analysis of inulin.

1 INTRODUCTION Fructans are widely distributed as a carbohydrate reserve among a variety of higher

plant families, but it is in two species of Compositae - Jerusalem artichoke (Helianthus tuberosus L.) and chicory (Cichoriwn intybus L.) - that fructan accumulates to a sufficiently high level for commercial production. In the Compositae fructans are present as a homologous series of ß-2,1 -linked polymers, called inulins. For this inulin to be a practical crop product it is essential that the degree of polymerization (DP) be as high as possible. This is first because a high DP raises the relative amount of fructose compared to glucose, and second because longer chains are more easily purified by crystallization. Conventional chromatography reveals that inulin is present in tubers of Jerusalem artichoke with a limiting DP of about 35 (Edelman and Jefford, 1968). This value has recently been confirmed by the application of anion exchange chromatography to the inulin extracted from Jerusalem artichoke tubers (Chatterton et al., 1990) and from the storage organs of a variety of other Compositae (John, 1991).

The classic work of Edelman and colleagues (see Edelman and Jefford, 1968) established that the two enzymes sucrose .sucrose fructosyltransferase (SST) and fructan: fructan fructosyltransferase (FFT) were responsible for the synthesis of inulin from sucrose

298

r

I VACUOLE I

V / CYT050L

Fig. 1. Representation of the metabolism of fructan in the vacuole of the storage parenchyma of Jerusalem artichoke tubers. Yx denotes fructose or glucose, F2 sucrose, F3 isokestose = glucose-fructose-fructose, and Fj denotes a fructan of chain length i, that is, glucose-Cfructose); . These abbreviations will be used in the subsequent analysis.

in Jerusalem artichoke tubers. It was also shown (Edelman and Jefford, 1968) that the depolymerization of fructan was initiated by a fructan exohydrolase (FEH). None of these enzymes has yet been studied in a purified form from any member of the Compositae. In the absence of any concrete information at the biochemical level about the enzymes responsible for fructan synthesis, we have used differential equations to model the transfructosylation reactions likely to be taking place in the vacuole (Frehner et al., 1984; Darwen and John, 1989) of the storage parenchyma of an inulin-accumulating member of the Compositae such as Jerusalem artichoke and chicory. We have been able to attempt this because the large number of simply interrelated products are produced by the reversible transfructosylation action of a single enzyme, FFT. Our analysis makes it possible to relate the spectrum of products produced to the catalytic properties of the enzyme.

2 MODEL AND ANALYSIS 2.1 Metabolic basis

Fructan metabolism in the vacuole is assumed to be as illustrated in Fig. 1. The SST is active only during fructan polymerization, and the FEH only during fructan depolymerization, while FFT activity is always present (Edelman and Jefford, 1968).

2.2 Effects of FFT A model involving only FFT is developed first; elaboration will subsequently show

how SST and FEH can be incorporated. The strategy is to consider the rate of synthesis and

299

degradation of each possible fructan as the factors that determine its eventual concentration vary.

First, consider F2. In the following diagram the reactions that produce F2, the source reactions, are to the left, while the reactions which destroy F2 are to the right.

Sources F3 + F3 ^

fc(F4 + F3) >

V4(Fi + F3)

> F,

Sinks r F2 + F4

F2 + Fs

<

F2 + Fi

The overall rate of change is the sum of the rates of all these reactions:

d[F2l = Vi Σ [F3][F{\r3i + Vi[F3]2r33 - Σ [F2][Fi]r2i dt i=3 i=4

(1)

Here r is the rate constant for the reaction involving exchange between species i and j , and n is the maximum fructan length to be considered. The factor Vi enters because in general a reaction between Fj and Fj can have two outcomes, only one of which produces product i - 1 , thus:

Fi + Fj —-> F M + Fj+1

Fj + Fj > Fi+1 + Fj_!

In the absence of detailed information, it has to be assumed that these outcomes are equiprobable.

Next, consider F3 in the same way. The diagram is more complicated because F3 can be built up from F2 or degraded from F4. For the present, an input of F3 from the SST-catalysed conversion of imported sucrose will be ignored. This complication will be considered later.

300

So

Sources F4 + F

F5 + F2

F. + F ,

F2 + F4 ^ Fa + F,

Vi(Fi + F4) J

Sinks

Γ 2 ( F 3 + F 3 ) '/2(F3 + F4)

■+- <

F 3 + F i

dIE3l = Σ [F2][F;]r2i + V2 Σ [F4][Fi]r4i + [F4][F4]r44 + [F4][F2]r24 dt i=4 i=5

2[F3][F3]r33 + 'A[F3][F4]r34 + Σ [F3][FJr3i i=5

(2)

The factor of lA beside F3 + F4 comes because half the time this reaction simply regenerates F3 + F4; the factor of two beside F3 + F3 because this reaction always destroys two molecules of F3, whereas all the others only destroy one; and V2 beside F; + F4 because this will only generate an F3 molecule half of the time.

Last, consider a general fructan molecule with a chain length of i. The reactions to be considered are as follows:

301

Sources '/2(F3 + F M )

%(Fi.2 + Fi_!) > F i -1 + F i -1

'A(Fi+1 + Ρ Μ ) '/2(Fit2 + FM ) J

+ Fi+i ^

V4(FU2 +F i+1) ^ '/2(FU1 +F i+1)

F i+1 + F i+1 '/2(Fi+2 +F i+1)

Sinks

F; + F ,

F '/2(F

2(F '/2(F

F

+ F i -2

+ F i - l )

+ F i )

+ F i + l)

+ F i + 2

The factors of xh arise because a reaction only produces or destroys a molecule of Fj half the time, except in the case of FU 1 + F i+1, which half the time generates two molecules of FJ; but this reaction appears in two lists, which keeps the accounting straight. Fi+1 + Fi+1

and Fj_! + F M always produce a single molecule of F^ Fj + Fj always destroys two molecules. So the rate of change of concentration is given by

dIFjl = 1/2 Σ [Fi.JtFj]!-.! + V i t F i . j V u - i " ^[Fi-ilFdii dt j*i

i-1,2

+ 'Λ Σ [F^JfFjlrj^! + '/2[Fi+1]2ri+u+1 + '/2[Fi+1][F2]ri+1>2

- fe lFJIFj l ry - 'AIFJIFi.,]^., - '/i[Fi][Ful]rifU1 + [FJ 2 r ü | (3)

This gives us a complete set of equations, (1), (2) and (3), describing the dynamics of the various species in the absence of FEH activity or exogenous isokestose input.

To simplify the equations, consider the reaction

Fj + Fj - > Fi+1 + Fj.!

302

This is enzyme-mediated, and presumably proceeds by one of two general schemes. Either the enzyme binds both fructans at once:

Fi + Fj + E - > FiEFj - > FiÊFj.! - > Fi+1 + F j^ + E (4)

or sequentially:

Fj + E -> FjE -*■ FjÊ* -* Fi_l + E*

E + F j+1 - EFj+1 - E'Fj - E* + Fj (5)

In both cases the following argument holds, but it is easier to describe using route (4). Four factors determine the rate of this reaction: enzyme concentration [E], the enzyme affinities for Fj and Fj (given by Θ{ and 0j), and the rate at which FjEFj -> Fi+1 + Fj_x + E, r. So ry = r[E]9j0j. (If route 5 is correct Θ{ and 0j become average affinities of E and E* for Fj or Fj and r encompasses both the step FjE -> F ^ + E* and E*Fj -» E + Fj+1). Thus [E] has units mol dm"3, θ{ and 0j have units of mol'Mm3 and r has units of s"1.

Now applying this expression for ry to equations (1) - (3) allows a great simplification. We obtain

_ 1 _ d[F2l = 1/2[F3]03^ Σ [Fj]0j + [F3]03 U [F2]92 Σ [ F ^ (6) r[E] dt li=3 J i=4

J L d£F3l = [FJG, Σ [FjlGj + 1/2[F4]04J Σ [FjlGj + [F4]04 + [F2]02 r[E] dt i=4 IΊΦ3 \

- [F3]03 j Σ [Fj]0j + [F3]03 - V2[F4]04 V (7)

J_dIF j l = ViFêl Σ [Fj]0j + [ F M ] e M - [F2]02[ r[E] dt lj*i J

+ ^[Fj^jej^J^cFjiGj + [Fi+1]ei+1i

- [FilÖiJE [FjJOj - ^([Fj.jei.! + [Fi+1]0i+1) + [Έ-ίθΛ

303

(8)

(Here, Σ without upper or lower limits denotes summation over the whole possible range j

ofj)

Now [Fj]0j is a dimensionless measure of concentration: the effective concentration of species Fj from the viewpoint of one molecule of the enzyme E. It makes sense to ask about the total concentration of possible reactants. This is

T = Σ [FjlÖj j

Dividing (6), (7) and (8) by T and denoting [Fj]6j/T by aj we get (using the fact that Σ aj = 1 - aj etc):

1 d[F2] = 1/2a3(l+a3-a2) - a2(l-a2-a3) r[E]T dt

1 d[F3] = a2(l-a2-a3) + 1/2a4(l-a3+a4+a2) - a3(l-a2+a3-V2a4) r[E]T dt

1 dlEjl = Viai.jO-ai+ai^-a^ + teaâ-ai+a^+a^ - a ^ l - V ^ a ^ a ^ ) + %) r[E]T dt

which simplify somewhat to

_ l _ d [ F 2 l = V2a3(l+a3+a2) - a ^ l - a ^ (9) r[E]T dt

_J d[F3l = a2(l-a2) + 1/2a4(l+a4+a2) - a3(l+a3) (10) r[E]T dt

_ l _ d [ E J = ^ ^ ( l + V i - a ^ + ^ ( l + a ^ + a , ) - a^l+a,) (11) r[E]T dt

Given that Σ ^ = 1, this gives n equations in the n unknown a if the values of the i

304

lefthand side were known. At equilibrium the rates of change of all concentrations must be zero. Call the equilibrium values of ai? äj. With Σ äj = 1, setting (9) to (11) to zero gives n equations in the n unknown, äj. These equations cannot conveniently be solved explicitly because the solution for ai+1 in terms of 2L{ and a M is a quadratic, and the solution rapidly becomes very complicated. However, numerical solution by a simple bisection method is easy enough provided n is set. Results for n = 20, 30, 40, and 50 follow. In each case results are shown relative to a10. The essential points are that (i) äj = äj for i,j > 2, that is, the effective concentrations are all the same except for sucrose, and that (ii) a2 « 1/2a3, that is, the effective concentration of sucrose is half that of any other chain length. The sucrose concentration in Jerusalem artichoke tubers during synthesis is about 30 mM (Edelman and Jefford, 1968), but the vacuolar sucrose concentration in an equilibrium with the elongating fructan chains is not known.

Now, given an observed set of actual fructan concentrations, vanishing at some n, the relative affinities 0j/T can be deduced simply by

Θ/Τ = a/fFj] (12)

Because äj is effectively independent of j , 9j/T is simply proportional to the inverse of [FJ. Obviously the observed ultimately decreasing [Fj] is consistent with the theory only if Gj ultimately increases with j . That is, longer chains are more and more likely to be involved in the reaction, so their average lifetimes get shorter and shorter.

Table 1. Effective concentrations (ä) of fructans of different chain lengths deduced from equations (9) - (11), using various values for n, the maximum chain length. Values are relative to the effective concentration of Fio> ä10-

Chain length Maximum chain length

20 30 40 50

2 0.52 0.51 0.51 0.51 3 4 5

10 15 20 25 30 40 50

0.98 0.99 0.99 1 1.00 1.01 ---_

0.99 0.99 1.00 1 1.00 1.00 1.01 1.01 -_

1.00 1.00 1.00 1 1.00 1.00 1.01 1.01 1.01 _

1.00 1.00 1.00 1 1.00 1.00 1.00 1.01 1.01 1.01

305

2.3 Constant input of isokestose In this case equation (2) is modified by addition of a term, k, describing the rate of

input of isokestose by the activity of SST. But during growth the tuber grows rather than becoming indefinitely denser. So an increase in the concentration of fructans via kestose input must actually be offset by expansion and uptake of water, at least after the very early stages. This input of water to keep the osmolarity of the vacuole constant on average implies adding a term -[FJk/Σ [FJ to the equation for i the rate of change of [FJ. To see this consider a volume v which contains x moles of some molecule X. Its concentration is [X] = JC/V. Now suppose a little water enters, increasing the volume to v 4- dv. The new concentration is Jt/v+dv = Jt/v(l+dv/v) = [X]/(l+dv/v). Since dv/v is small (1+dv/v)-1 « (1-dv/v), so the new concentration [X] = [X](l-dv/v) and d[X] = -[X]dv/v. To keep osmotic pressure constant this dilution of all the molecular species present has to balance an exogenous input of any one of them, at least in the long term, k is the rate of input of isokestose in mol dm"V1; the rate of dilution of all the other species must match this input, so dJXIEJl = - k = L£IEJ1 dv dt v dt

Hence 1 dv = -k and, by linearity, v dt X[FJ

dffij = ^k[FJ dt I F J

The modified equations are then

J — d l F J = 1/2a3(l+a3+a2) - a2(l-a2) - kiF.l (13) r[E]T dt r[E]T £[FJ

1 drF3l = a2(l-a2) + 1/2a4(l+a2+a4) - a3(l+a3) + k Jl - [F31 i (14) r[E]T dt r[E]T[ E[F3]J

_ L _ dlFJ = V2ai_1(l-a2+ai_1) + Via^d+^+a^) - ^(1+^) - kfFJ (15) r[E]T dt r[E]T IfFJ

At equilibrium, as before, each equation is zero, by definition. If k/r[E]T and all the [FJ

306

are known, the equations can be solved for Θ{ as before. This is easiest to do if the scaling of 0j is such that T = 1 : choosing to scale Θ{ this way makes [E] not the true concentration but some multiple of it. In this case, if a single fructan Fj is incubated with FFT at physiological concentrations

dIFJ = ^rtEJfFJî2

dt

by reduction of (8), and

r[E] = -1 dIFJ (16) 2[Fi]20i

2 dt

Then using a set of concentrations of Fj at equilibrium, a measured k, and (16) the whole set of 0j could be deduced. The practical problem is to determine the physiological concentration of FFT.

2.4 Action of FEH and SST Exohydrolase activity will tend to alter all concentrations at a constant speed, so

dIEl = KFEHi[Fj] (17) dt

where KFEHi is the rate constant for FEH on species i. Then the resulting glucose is exported, converted to sucrose and then to isokestose and imported. The total rate at which extra isokestose appears is therefore

d[F3l = Σ ΚΡΕΗ][^] (18) dt j

If the activity is equal on all fructans, the right-hand-side of (18) becomes

KFEH Σ [Fj] j

Making the identification KFEH = k £ [F j ] j

307

produces a set of equations formally identical to (13), (14) and (15). That is, the joint action of FEH and SST is exactly equivalent to an extra isokestose input.

The affinities of FFT can then be deduced from (13) to (16), replacing k in (13) (14) and (15) by

k + KFEH Σ [Fj] j

and then solving iteratively as before. Provided k/r[E]T is small the solution deduced previously will still be approximately correct. That is, provided equilibration of the chain series is quick compared to the input of kestose, the input will hardly alter the equilibrium.

3 RESULTS The relative abundances of the fructan chains over a range of chain lengths, up to

a maximum at 40 are shown in Fig. 2. These curves owe their different shapes to the

(a) |~~

θ = θ~'

θ ί ~ θ 0 ' ( 3 +1 > ~ ' )

0 10 20 30 40 D P

Fig. 2. The relative abundances of inulin chains with a DP range up to DP 40 generated by the activity of a FFT enzyme in which the relationship between chain length and affinity is given by the accompanying equation. For explanation of terms, see text.

308

different assumptions that have been made for the catalytic properties of FFT. In Fig. 2a the affinity of FFT is assumed to be equal for all chain lengths, that is,

there is no preferential reactivity towards longer or shorter chains. This appears to be an unrealistic assumption as the resulting distribution is never found; there is always a decreasing abundance with a lengthening of the chain. In Figs. 2b and 2c it has been assumed that the affinity of FFT increases smoothly with the chain length. In Fig. 2b the affinity increases linearly with DP, and in Fig. 2c affinity increases exponentially with DP. The result is that there is a gradually decreasing abundance of chains as the DP increases from isokestose. Thus the tailing off that is apparent in actual analyses has been recreated. However in Jerusalem artichoke (Chatterton et al., 1990) and other Compositae (John, 1991) it is likely that oligomeric chains rather than isokestose, are the most abundant chain lengths. Distribution curves that show the most abundant polymers with chain lengths around DP 10 can be seen in Figs. 2d and 2e. Here it has been assumed that as the chain length is increased, the affinity of FFT initially decreases, then increases. A rationale for this relationship can be provided by assuming that with increasing chain length, the reaction rate drops, but the probability of encountering a chain increases, the latter eventually dominating.

4 CONCLUSIONS The present analysis provides a basis for the view that the size distribution of

fructans accumulated by the storage organs of the Compositae is determined in a predictable manner by the relative affinity of the transfructosylating enzyme for chains of different length. The significance of this factor relative to other factors that can determine size distribution of fructans (John, 1991) will become apparent only when future biochemical work reveals the actual catalytic properties of the enzymes responsible for fructan polymerization and depolymerization in the Compositae.

5 REFERENCES

Chatterton, N.J., Harrison, P.A., Thornley, W.R. and Draper, E.A., 1990. Oligosaccharides in foliage of Agropyron, Bromus, Dactylis, Festuca, Loliwn and Phlewn. New Phytol., 114: 167-171.

Darwen, C.W.E. and John, P., 1989. Localization of the enzymes of fructan metabolism in vacuoles isolated by a mechanical method from tubers of Jerusalem artichoke (Helianthus tuberosus L.). Plant Physiol., 89: 658-663.


Frehner, M., Keller, F. and Wiemken, A., 1984. Localization of fructan metabolism in the vacuoles isolated from protoplasts of Jerusalem artichoke tubers {Helianthus tuberosus L.). J. Plant Physiol., 116: 197-208.

John, P., 1991. Fructan quality and fructan synthesis. Biochem. Soc. Trans., 19: 569-572.

FOOD AND MEDICAL APPLICATIONS



THE OCCURRENCE OF FRUCTAN IN FOOD PLANTS

L.D. INCOLL* and G.D. BONNETT** Department of Pure and Applied Biology, University of Leeds, Leeds LS2 9JT, U.K. School of Agriculture and Forestry, The University of Melbourne, Parkville, Victoria 3052, Australia

ABSTRACT

The occurrence of fructan in food plants is reviewed in families with fructan-containing plants. Of these families, the Gramineae, Liliaceae, Iridaceae, Agavaceae, Asteraceae, Campanulaceae, Boraginaceae, and Menyanthaceae contain plants presently used for food or plants historically known to be sources of food. Of this set of food plants, evidence that the edible part contains fructan is often lacking. There are three other families with food plants, Cyperaceae, Portulacaceae and Zosteraceae where the presence of fructan-containing plants needs confirmation.

1 INTRODUCTION Fructan is a class of water-soluble oligomeric and polymeric carbohydrates that

occurs in food plants and of which inulin is just one example. Fructan is predominantly composed of fructose and usually contains one, but no more than one, glucose residue per molecule. In its role as a reserve carbohydrate, fructan is stored in the vacuoles of mesophyll, of the basal meristem of grass leaves, of swollen leaf bases in bulbs, and of tissues of underground storage organs such as tap-roots and tubers. Most of these tissues and organs are represented among the edible parts of plants.

Fructan is not digested by acid hydrolysis or by enzymes in the stomach or in the small intestine of man, so it passes undigested to the colon (Oku et al., 1984; Hosoya et al., 1988; Nilsson et al., 1988). In the colon it is almost completely metabolized by bacterial fermentation, in particular by the non-putrefying bifidobacteria (Modler et al., 1990). The benefits of fructan in human diets are two-fold. First, the small proportion of glucose in the molecule, at most 25% and often much less, makes it an alternative carbon source for diabetics. Secondly, the effect on the colonic bacterial flora tends to reduce digestive problems such as constipation (Hidaka et al., 1986). In this respect it has a similar effect to dietary fibre which is strictly plant cell wall material (Southgate et al., 1990).

310

Consequently, there is increasing interest in fructan-containing foods to the extent that mixtures of fructans of various degrees of polymerization are being prepared commercially by various methods in Europe and Japan for use as food additives e.g. Raftilose and Raftiline - Raffinerie Tirlemontoise S.A., Tienen, Belgium; Fibruline - Cosucra, Momalle, Belgium, and Neosugar - Meiji Seika Kaisha Ltd., Kawasaki, Japan. Despite this interest, the occurrence of fructan in food plants (and foods) is not well described especially when compared with that of starch. This is no doubt due to the lack of a reliable histochemical test for fructan, the positive identification of which has to be based on Chromatographie methods.

The purpose of this review is to examine and collate the available information on the occurrence of fructan in food plants both in relation to present-day diets and to ethnobotanical records.

2 OCCURRENCE OF FRUCTAN IN PLANTS IN GENERAL The most recent surveys of the occurrence of fructans in plants, all of which also list

plants without fructan, are those of Pollard and Amuti (1981), Pollard (1982) and Hendry (1987). The subject was reviewed by Meier and Reid (1982) and also by Hendry (1987). Hendry summarized "the reports of fructan at the family level where the evidence is modern, repeatedly confirmed and has employed techniques now held to be reliable". By excluding families with < 50 genera worldwide, he was left with the first 9 families of Table 1 plus the Boraginaceae, although in fact 4 of these families have < 50 genera. Menyanthaceae, Monotropaceae and Pyrolaceae can now be added to these (Hendry, pers. comm.). In the second part of Table 1 we have listed the families where the evidence that they have fructan-containing plants is restricted to single references. In some of these the evidence is restricted even more to one species. In the Cyanastraceae, Xanthorrhoeaceae and Zosteraceae (Pollard, 1982) only one species was assayed (Brunoniaceae has only one species). In the Lentibulariaceae, where only two species have been assayed, Pollard and Amuti (1981) found Utricularia vulgaris stems to be negative for fructan, whereas Pate and Dixon (1982) found U. menziesii tubers to be positive. Evidence of Pollard and Amuti (1981), Pollard (1982) and Pate and Dixon (1982) is based on colour reactions on paper chromatograms and on comparisons of RF values with those of sugars from known fructan storers. Doubt has been expressed, on taxonomic grounds (Hendry, pers. comm.) about some of these families purported to contain fructan. All the families in the second group therefore need independent confirmation. In his 1987 review, Hendry does not document the evidence for his 10 families nor does he state what techniques he considers to be reliable. There is a great need for a checklist of species where such evidence is collected and properly documented. First, fructan must be defined precisely. For some authors e.g.

311

Table 1.

Order

The occurrence of fructans

Family

in food plants in

Total

the families in which fructans occur.

genera8 Occurrence in each family of

food plants

fructan-containing food plants

1. Fructan shown to occur in a family by more than one investigator0"6

Monocotyledons

Cyperales Liliales

Dicotyledons

Asterales Campanulales

Ericales

Lamiales Solanales

Gramineae Agavaceae Haemodoraceae Iridaceae Liliaceae

Asteraceae Campanulaceae Goodeniaceae Stylidiaceae Monotropaceae Pyrolaceae Boraginaceae Menyanthaceae

635 18 16 92

294

1314 87 16 5

10 4

154 5

+ +

+ +

+ +

+ +

2. Fructan shown to occur in a family but in only one publication

Monocotyledons

Cyperales Liliales

Najadales

Dicotyledons

Calycerales Campanulales Caryophyllales Ericales Scrophulariales Solanales

Cyperaceaec

Cyanastraceaec

Xanthorrhoeaceaec

Zosteraceae0

Calyceraceaed

Brunoniaceaed

Portulacaceae6

Clethraceaed

Lentibulariaceae6

Polemoniaceaed

115 1 9 3

6 1

21 1 4

20

+ +

+ +

+ +

Key to sources: a Mabberley (1987) b Meier and Reid (1982) c Pollard (1982)

d Pollard and Amuti (1981) e Pate and Dixon (1982)

Pollock and Chatterton (1988), the simplest fructan is monofructosylsucrose, a trisaccharide

of which there are three isomers, 1-kestose and 6-kestose (basic structures glc-fru-fru) and

neokestose (basic structure fru-glc-fru). On the other hand, French (1989) defines a fructan

as a molecule "in which two or more fructosyl-fructose linkages constitute the majority of

312

linkages". Thus the presence of glucose is not part of the definition. For the shorter oligomers, tetrasaccharides derived from 1-kestose and 6-kestose (with structures glc-fru-fru-fru) fit the definition, that from neokestose (with structure fru-glc-fru-fru) does not, nor do the three trisaccharides.

Irrespective of the chosen definition, the likeliest source of misidentification is the presence of oligosaccharides of the raffinose series with a general structure of fru-glc-galn. For this reason identification requires separation by HPLC, followed by acid hydrolysis of separated carbohydrates and analysis of the resulting monosaccharides by TLC or HPLC. Linkage analysis i.e. methylation and GC-MS, is required to satisfy French's definition.

3 OCCURRENCE OF FRUCTANS IN FOOD PLANTS The families of Table 1 provide a starting point for assessing the occurrence of

fructan in food plants. Twelve families can be eliminated as our literature searches (e.g. in Heywood, 1985; Mabberley, 1987) show that they appear not to contain food plants (column 4, Table 1). The food plants in the remaining families are listed in Tables 2, 3 and 4. The major sources for these lists are Vilmorin-Andrieux (1885), Uphof (1959), Masefield et al. (1969), Purseglove (1968, 1972), Hedrick (1972), Anonymous (1974), Heywood (1985), Clayton and Renvoise (1986) and Mabberley (1987). We have tried to make the lists comprehensive, but hope that omissions will be pointed out to us. Some species have been omitted because we do not know enough about them e.g. putative cereal crops such as Sorghum sonorum, S. sumatrense, and Echinochloa utilis mentioned in Clayton and Renvoise (1986). The tables contain food plants that are major economic cereal and vegetable crops, important crops of limited distribution, and minor vegetables. Additionally, there are species that are recorded historically as food plants but are no longer cultivated or gathered. Finally, there are species that have been harvested in times of drought or famine. In this assemblage of food plants in families with fructan-containing plants, evidence that the edible parts of those food plants contain fructan is rarely available. Synonyms of common names are kept to a minimum for reasons of space with the English name usually given. To illustrate the problem; in Sorghum, possible species boundaries are indistinct, so, following Purseglove (1968), the cultivated sorghums are grouped under S. bicolor but the common names are given.

3.1 Gramineae The occurrence of fructan in grasses has been frequently demonstrated for over 50

years, but almost always in the vegetative parts viz. stems and leaves. The edible part of grasses is, however, with few exceptions (e.g. sugar cane, bamboo), the seed, and as its major storage carbohydrate is starch, the presence of fructan seems rarely to have been of

313

Table 2. Food plants in the Gramineae, a family with fructan-containing plants (excluding species solely used for fodder or for brewing).

Tribe: Bambuseae Bambusa beecheyana Dendrocalamus asper Gigantochloa verticillata Phyllostachys dulcis Tribe: Oryzeae Oryza glaberrima Oryza sativa Zizania aquatica Tizania caduciflora Tribe: Aveneae Avena abyssinica Avena byzantina Avena sativa Tribe: Triticeae Hordewn vulgäre Secale sativa Triticum aestivum Triticum dicoccum Triticum durum Triticum monococcum Tribe: Eragrostideae Eleusine coracana Eragrostis tef Tribe: Paniceae Brachiaria deflexa Digitaria exilis Digitaria iburua Echinochloa crus-galli Echinochloa frumentacea Panicum miliaceum Panicum sumatrensis Paspalum scrobiculatum Pennisetum cinereum Pennisetum nigritarum Pennisetum typhoides Setaria italica Tribe: Andropogoneae Coix lachryma-jobi Saccharum barberi Saccharum edule Saccharum officinarum

Common name

bamboo bamboo bamboo bamboo

African rice rice American wild rice gau sun

oat red oat oat

barley rye bread wheat emmer durum wheat einkorn

finger millet teff

hungry rice

barnyard millet Japanese millet common millet little millet Kobo millet bulrush millet bulrush millet pearl millet foxtail millet

Job's tears sugar cane

sugar cane

Edible part

shoot shoot shoot shoot

seed seed seed stem

seed seed seed

seed seed seed seed seed seed

seed seed

seed seed seed seed seed seed seed seed seed seed seed seed

seed stem inflorescence stem

Fructan in edible part

+a

+b

+ b

+b

+b

+ c flour

_ d

Fructan in other parts

+d stem

+ f stem

-1-f stem

- d stem

+* leaf

- d stem

314


Common name Edible part Fructan in Fructan in edible part other parts

Saccharum sinense sugar cane stem Sorghum bicolor sorghum, white durra, seed - e

feterita, Guinea corn, kafir corn, kaoliang milo, shallu, durra

Zea mays maize seed - b +g leaf

Key to sources: a Chan and Thrower (1980) e Colin and Belval (1934) b Belval (1924), MacLeod and Preece (1954) f Smouter and Simpson (1989) c Medcalf and Cheung (1971) « Chatterton et al. (1989) d Pollard (1982)

interest (Table 2). The recent papers of Chatterton et al. (1989) and Smouter and Simpson (1989) show that fructan is the major storage carbohydrate in the vegetative parts of species in the tribes Aveneae, Bromeae, Poeae and Triticeae. In contrast, they show that species in the tribes Ehrharteae, Stipeae, Eragrostideae, Cynodonteae, Andropogoneae, and Paniceae accumulate very little or no fructan (Smouter and Simpson (1989) actually include free as well as combined fructose in their fructan fraction). In agreement with these results, fructan has been shown to be present in the seeds of the temperate cereals, rye, barley, wheat and oats and absent from seeds of maize and sorghum (Table 2) but there are too few data to support the proposition that the seeds of all species in the two groups of tribes, "low fructan" and "high fructan", should be similarly low or high in fructan. Analyses of seeds of the "low fructan" group would be of great interest since it contains the cereals of warm-temperate, subtropical and tropical regions i.e. of a large proportion of the world's population. In such analyses it will be important to ensure that the pericarp is present in samples, if it is eaten, because, for example, in grains of wheat, fructan is found in the pericarp as well as the rest of the grain (Schnyder et al., 1988).

It should be noted that the surveys of Chatterton et al. (1989) and Smouter and Simpson (1989) did not cover all the tribes in the Gramineae and in particular two tribes containing food plants, Bambuseae and Oryzeae (Table 2). Of the food plants in Table 2, only wheat and barley were assayed by Smouter and Simpson and common millet and maize by Chatterton and his associates. With respect to the Oryzeae, Ojima and Isawa (1968) report that fructan is low in leaf sheaths and stems of rice, Oryza sativa, though we question the value of their data because they measured fructan in the residue after an extraction in 80% ethanol which will discriminate against fructan of low degree of polymerization. Of the Bambuseae, there are too many edible bamboos to list here (see for

315

example Mabberley, 1987); the four in Table 2 are important edible species representing hardy bamboos and tender tropical and subtropical types. None of these has been tested for fructan, however Pollard (1982) could not detect fructan in stems of the related edible species Phyllostachys aurea.

Another graminaceous stem vegetable, gau sun, consists of a remarkable host-parasite relationship between Zizania caduciflora and the smut fungus Ustilago esculenta (Thrower and Chan, 1980). Chan and Thrower (1980) show that low-molecular-mass fructans are at high concentrations in the edible non-sporulating culm but they qualify this parenthetically with the words "mainly fructose" - it is not clear how much of this fraction is fructan.

3.2 Other monocotyledonous families Of the plants listed in Table 3, four were described as food plants by the classical

Greek philosopher Theophrastus, Gladiolus italicus, Gyandriris sisyrinchium, Asphodelus aestivus, and Muscari comosus. M. comosus was cultivated but its cultivation ceased, probably because better cultivars were developed in other genera (Negbi, 1989). Pollock (1986) has shown that 98% of the total non-structural carbohydrate in M. atlanticus bulbs is fructan.

In many of this group of monocotyledons the presence of fructan in the edible part is unknown. In the genus Allium, two of the species investigated by Rubat du Merac (unpublished thesis cited in Meier and Reid, 1982), A. fistulosum and A. schoenoprasum, contained fructan but Meier and Reid do not name the assayed plant part. Asparagus officinalis is of particular interest because, although much research has been done on the biochemistry of the fructans of its roots by Shiomi (see Shiomi, 1989), the edible stem lacks fructan (Pressman et al., 1989). Camas (Camassia quamash and C. leichtlinii) and riceroot (Fritillaria camschatcensis and F. lanceolata) were important foods of the American Indians of the Pacific North-west, the use of which has died out following European conquest (Turner and Kuhnlein, 1983). Of the plants in the Cyperaceae, Pollard (1982) recorded fructan in the stems/leaf bases of all of the five species that he tested, including Eleocharis dulcis. The analyses recorded by Kay (1987) for tiger nut and Chinese water chestnut include starch, sucrose and reducing sugars in their dry matter but do not mention fructan. In his survey of the flora of the Sheffield (U.K.) region, Hendry (1987) recorded three species in the Cyperaceae as not containing fructan. Although the underground parts of the sedge family may be thought to be starch storers, starch and fructan are not mutually exclusive stored carbohydrates (e.g. they occur together in some rhizomes and tubers in the Iridaceae and some bulbs in the Liliaceae) so it is important that more analyses be done. The basis for including Zosteraceae among food plants is the descriptions by Mabberley (1987) of Zostera marina as a "promising grain (important bird food in winter)" - whether

316

Table 3. Food plants in monocotyledonous families with fructan-containing plants (excluding the Gramineae and species solely used as sources of aromatic compounds or for fermented liquors).

Iridaceae Gladiolus italicus Gyandriris sisyrinchium Liliaceae Alliwn ampeloprasum

Allium cepa

Allium chinense Alliwn fistulosum Allium sativum Allium schoenoprasum Allium tuberosum Asparagus officinalis Asparagus racemosus Asphodelus aestivus

Camassia (2 species) Fritillaria camschatcensis Fritillaria lanceolata Hemerocallis fulva Muscari comosus Agavaceae Cordyline terminalis Dracaena australis Yucca fllamentosa Cyperaceae Cyperus esculentus Cyperus papyrus Eleocharis dulcis

Zosteraceae Phyllospadix spp. Zostera marina

Key to sources:

Common name

corn flag Barbary nut

leek, kurrat, great-headed garlic onion, shallot, tree onion rakkyo,jao Welsh onion garlic chives Chinese chives asparagus

asphodel

camas riceroot riceroot jin zhen cai purse-tassels

palm lily

tiger nut papyrus Chinese water chestnut

eelgrass

a Darbyshire and Henry (1981) b Pollock (1986) c Pressman et cd. (1989) d Madan (1972) e Schlubach and Lendzian (1937), species of

Edible part

corm corm

leaf, bulb

bulb

bulb bulb bulb leaf leaf stem tuber leaf, stem, tuberous root bulb bulb bulb flower bulb

tuber tuber flower bud

tuber tuberous rhizome tuber or corm

root seed

Asphodelus not;


+ a leek

+ a , b onion

i a,b

_ c

+ d

+ e

+ f

+s + h

given f Yanovsky and Kingsbury (1938) g Boggs and Smith (1956) h Ekstrand and Johanson (1887) 1 Pollard (1982)


+ ' stem

+ ' stem

+ 1 stem

+ ' stem

this submerged halophyte gives a promising grain for humans is not stated - and of

317

Phyllospadix spp. as sources of "some locally eaten roots" on north Pacific coasts.

3.3 Dicotyledonous families It is generally believed that fructan is ubiquitous in the Asteraceae (Hegnauer, 1977).

In some, the occurrence of fructan in underground storage organs such as tap-roots and tubers (Table 4), is so well established as to almost not require confirming citations e.g. chicory, Jerusalem artichoke and elecampane. The latter, the roots of which were used as sweetmeats, was the plant from which inulin was first isolated by Rose in 1804. Tuberous roots of murnong are regarded by Gott (1983) as being a staple food of the aborigines of south-eastern Australia and she ascribes the collapse of the aboriginal population following European colonization to be, in part, a consequence of the loss of this staple. Yacon, a South American crop from the Andes of Venezuela to north-west Argentina (Zardini, 1991) is now being cultivated in Japan (Ohyama et al.9 1990). Table 4 shows that although the presence of fructan has often been demonstrated in tap-roots, there is almost no evidence for occurrence of fructan in vegetables where other organs are eaten e.g. the stem lettuce (woh sun) cultivar of China and the many leaf vegetables. Fructans have been shown to be synthesized in lettuce, chicory, dandelion and sow thistle leaves (all members of the tribe Lactuceae) by Chandorkar and Collins (1972) but only in disks on 5% sucrose i.e. in tissue from which translocation is not possible and accumulation of sucrose might thereby be induced to a level at which fructan is synthesized. Early reports of lack of fructan in Composite leaves e.g. from Helianthus tuberosus (Dean, 1904), were confirmed by Chandorkar and Collins (1972) for other members of the tribe Heliantheae, H. annuus and Dahlia variabilis. The Composite vegetables in Table 4 include species from the tribes Cynareae, Anthemideae and Senecioneae, the edible leaves of which need to be assayed by modern techniques.

Apart from Adenophora liliiflora, which is a cultivated root crop in Japan, the food plants in the other dicotyledonous families are less important economically. Mabberley (1987) records that the tap-rooted form of Campanula rapunculus was "much eaten but old clones with parsnip-like roots [were] apparently lost by [the] 1820s" though Vilmorin-Andrieux illustrates this form in 1885. Fructans have been shown to be present in the roots of other species of Adenophora and Phyteuma (see Hegnauer, 1964). Bogbean rhizome is powdered for bread by eskimos and the leaves are used for emergency food in Russia. Keegan's note (1916) that Bogbean rhizome contains "much inulin" is not supported by strong evidence. Pate and Dixon (1982) have shown that root tubers of Villarsia albiflora, a non-food species from the same family, contains fructan.

318

Table 4. Food plants in dicotyledonous families with fructan-containing plants (excluding species solely used as sources of aromatic compounds).

Asteraceae Arctium lappa Artemisia lactiflora Chrysanthemum moriflorum Chrysanthemum spatiosum Cichorium endivia Cichorium intybus

Cirsium oleraceum Cynara cardunculus Cynara scolymus Gynura bicolor Helianthus tuberosus lnula helenium Lactuca indica Lactuca sativa Microseris lanceolata Polymnia sonchifolia Scolymus hispanicus Scorzonera hispanica Sonchus oleraceus Taraxacum offlcinale Tragopogon porrifolius Campanulaceae Adenophora liliifolia Campanula rapunculus Phyteuma orbiculare Boraginaceae Cynoglossum offlcinale Symphytum offlcinale Menyanthaceae Menyanthes trifoliata Portulacaceae Claytonia perfoliata Portulaca oleracea

Key to sources: a Rundqvist (1909)

Common name

edible burdock juun jiu choi ju hua tong hao endive chicory

meadow cabbage cardoon globe artichoke guang cai Jerusalem artichoke elecampane

lettuce murnong yacon golden thistle black salsify ku mai, sow thistle dandelion salsify

rampion rampion

hounds tongue comfrey

bogbean, buckbean

winter purslane common purslane

b Rutherford and Phillips (1975) c Quillet and Bourdu (1952) d Edelman and Jefford (1968) e Rose (1804) f Incoll et al. (1989)

Edible part

tap-root, leaf leaf flower leaf leaf tap-root, leaf, chicon tap-root leaf stalk inflorescence leaf, shoot tuber root leaf leaf, stem tuberous root tuber tap-root tap-root leaf tap-root, leaf tap-root

root tap-root root

leaf leaf, shoot

leaf, rhizome

leaf leaf


+ a tap-root

+b tap-root +b

+ c

+ d

+ e

+ f

+*

+ a

+h tap-root

+ '

+h leaf

h Pollock (1986) 1 Bacon (1959) j Pollard and Amuti (1981) k Hendry (1987) 1 Bourdu (1957) m Bourdu (1954)


+j stem

+j stem + k

+ 1 root + mroot, +J stem

-H stem

—' stem

* Ohyama et al. (1990)

319

4 DISCUSSION It will be clear from the records of Tables 2 to 4 that the evidence for presence of

fructan in the edible parts of plants is not the result of systematic surveys of plants used for food. The data is derived coincidently from assays done for a variety of reasons e.g. to answer questions about taxonomic relationships (Pollard and Amuti, 1981; Pollard, 1982), for assessing occurrence of fructan-containing plants in a flora (Hendry, 1987), for assessing occurrence within a family (Smouter and Simpson, 1989) and in biochemical and physiological studies (Pollock, 1986). The only survey specifically to look at food plants was that of Yanovsky and Kingsbury (1938) where a subset of 66 plants was examined from a set of 1112 plants collected from the literature by Yanovsky (1936).

It will be clear also that some of the evidence for occurrence of fructan in the listed food plants does not meet the strict criteria set out in Section 2 above; some of the cited references predate the advent of chromatography. Much of the early evidence is based on optical rotation alone. We have preferred to record such evidence while emphasizing that there is a need for better confirming analytical work on fructan using modern techniques. Furthermore, it is important that negative results be published in the way that Pollard and Amuti have done in their surveys (1981, 1982).

The greatest interest in occurrence of fructan in food plants has been in species containing high concentrations like Jerusalem artichoke and chicory. This is unfortunate in our opinion because beneficial effects of fructan in human diets may be apparent at much lower concentrations. We plan to consider the subject of fructan in food, including its role in present and past diets, in greater detail in another paper.

Our survey may effectively be only "the tip of an iceberg" as far as fructan-containing plants are concerned in so far as it has mainly been concerned with economically important plants. Extending its scope to include more wild plants gathered for food would require much space. For example, in the "Useful Plants of Ghana", Abbiw (1990) lists 4 species of cormous Gladiolus, 11 Composite species used as semi-cultivated leaf vegetables and 34 wild edible graminaceous species, 19 of which he says are "worth developing into crops". Amongst ethnobotanical sources Turner (1981) under the title "A gift for the taking" listed 12 edible Allium species gathered by the Indians of the American North-west, Ebeling (1986) writes of the "onion valleys" of the Sierra Nevada and Cribb and Cribb (1987) describe 6 grasses used by the Australian aborigines.

5 ACKNOWLEDGEMENTS We thank Bing Liu and George Hendry for their advice.

320

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Bourdu, R., 1957. Contribution ä l'&ude du m&abolisme glucidique des Boraginacoes. Rev. Gen. Bot., 64: 153-192; 197-260.

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Chandorkar, K.R. and Collins, F.W., 1972. De novo synthesis of fructo-oligosaccharides in leaf disks of certain Asteraceae. Can. J. Bot., 50: 295-303.

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Cribb, A.B. and Cribb, J.W., 1987. Wild Food in Australia. Angus Robertson, North Ryde, 284 pp. Darbyshire, B. and Henry, R.J., 1981. Differences in fructan content and synthesis in some Allium species.

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as energy resources. J. Clin. Biochem. Nutr., 5: 67-74. Incoll, L.D., Bonnett, G.D. and Gott, B., 1989. Fructans in the underground storage organs of some

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on availability in rat and humans. J. Nutr., 118: 1325-1330. Ohyama, T., Ito, O., Yasuyoshi, S., Ikarashi, T., Minamisawa, K., Kubota , M., Tsukihashi, T. and

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Oku, T., Tokunaga, T. and Hosoya, N., 1984. Nondigestibility of a new sweetener, "Neosugar", in the rat. J. Nutr., 114: 1574-1581.

Pate, J.S. and Dixon, K.W., 1982. Tuberous, Cormous and Bulbous Plants, Biology of an Adaptive Strategy in Western Australia. University of Western Australia Press, Nedlands, 268 pp.

Pollard, C.J., 1982. Fructose oligosaccharides in monocotyledons: a possible delimitation of the order Liliales. Biochem. Syst. Ecol., 10: 245-249.

Pollard, C.J. and Amuti, K.S., 1981. Fructose oligosaccharides: possible markers of phylogenetic relationships among dicotyledonous plant families. Biochem. Syst. Ecol., 9: 69-78.

Pollock, C.J., 1986. Fructans and the metabolism of sucrose in vascular plants. New Phytol., 104: 1-24. Pollock, C.J. and Chatterton, N.J., 1988. Fructans. In: J. Preiss (Ed.), The Biochemistry of Plants, A

Comprehensive Treatise, Vol. 14, Carbohydrates. Academic Press, Inc., San Diego, pp. 109-140. Pressman, E., Schaffer, A.A., Compton, D. and Zamski, E., 1989. The effect of low temperature and

drought on the carbohydrate content of asparagus. J. Plant Physiol., 134: 209-213. Purseglove, J.W., 1968. Tropical Crops. Dicotyledons (2 vols.), Longmans, London, 719 pp. Purseglove, J.W., 1972. Tropical Crops. Monocotyledons (2 vols.), Longmans, London, 607 pp. Quillet, M. and Bourdu, R., 1952. Sur les glucides de reserve des organes souterrains des Composers au

dobut de la poriode de repos. C. R. Acad. Sei., 234: 1078-1081. Rose, V., 1804. A.F. Gehlen's Neues Allg. J. Chem., 3: 217. Rundqvist, L., 1909. Cited in: Czapek, F., 1913. Biochemie der Pflanzen, Vol. 1. Fischer, Jena, pp. 457-

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50: 463-473.

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72-85.


JERUSALEM ARTICHOKE AS A MULTIPURPOSE RAW MATERIAL FOR FOOD PRODUCTS OF HIGH FRUCTOSE OR INULIN CONTENT

J. BARTA Department of Canning Technology, University of Horticulture and Food Industry, M£nesi ut 45, 1118 Budapest, Hungary

ABSTRACT

In Hungary, research on inulin-containing plants is especially focussed on cultivation and processing of Jerusalem artichoke (Helianthus tuberosus L.). Product groups to be produced in Hungary by processing Jerusalem artichoke tubers are the following: a. sugar solutions, with a fructose content of 75-85% (w/v), b. juice concentrates of high mineral content, among others containing K and Mg, and the micro-elements Zn and Cr, both believed to be beneficial to human health, c. pure fructose syrup, containing 96-98% fructose (w/v), especially for diabetics, d. crystalline fructose, e. flour or granulate rich in both inulin and fibre, f. dried tuber cubes, to be used as an additive for dietetic products in the bakery and confectionery industry, g. quick-frozen tuber cubes, to be mixed with other vegetables to improve dietetic fibre content, h. heat-preserved hydrolysed tuber pulp, to increase the fruit content of fibre-rich soft drinks, i. pure inulin powder, to be used for nutritional and medical purposes.

1 INTRODUCTION Excessive consumption of carbohydrates, including sucrose, causes obesity and

diabetes in a significant part of the Hungarian population. One objective of the Hungarian long-term health program is the partial replacement of sucrose by fructose. The present demand for fructose in Hungary is estimated to be 150 to 200 tons per year which can only be satisfied by imports from hard-currency countries. Products with a high fructose content can be manufactured at low yield from sugar beet (sucrose) or maize (high-fructose corn syrup) or, at a much higher yield, from Jerusalem artichoke tubers (inulin) (Fig. 1).

Due to its lower energy content in relation to its sweetening ability, slow absorption and quick metabolic transformation, fructose has a beneficial physiological effect on people suffering from obesity or diabetes. Fructose is the sweetest natural sugar known; depending on the prevailing conditions it is about 20-30% sweeter than sucrose. Thus, foods sweetened with fructose bring considerably less energy into the body than those sweetened with sucrose, yet, at identical sweetness, having the same taste. Like fructose, a concentrate

324

Fig. 1. Fresh weight, total sugar and fructose yield (expressed as t ha"1) of some agricultural raw materials suitable for fructose production, from inulin (Jerusalem artichoke tuber), sucrose (sugar beet) and starch (maize corn), respectively. (Note: columns are not stacked, but consist each of three overlapping columns.)

made of Jerusalem artichoke can be used as a natural sweetener for a healthy diet. It has a good taste, sweetens pleasantly and tastily, is convenient in use, low in energy, and does not increase sugar, cholesterol and triglyceride levels of the blood. Consumed at a normal rate, it causes neither obesity nor the diseases related with it.

Cultivation of Jerusalem artichoke (Helianthus tuberosus L.) was already recommended in a book titled 'Posoni kert', by Jänos Lippay (1753). Since its publication several books on practical agronomy have praised the unfastidiousness of the plant regarding type of soil, nutritional and pesticide requirements, and climate.

In Hungary, in the 1950's, intensive research on the cultivation of Jerusalem artichoke has been performed at the Martonväsär Institute of the Hungarian Academy of Sciences, and an appropriate agrotechnology was worked out. Within the framework of this research, first the processing of Jerusalem artichoke tubers was developed for the distilling industry and, later on, laboratory and pilot-scale experiments were started for producing concentrates rich in fructose (Teri, 1955; Magyar, 1959). However, in 1959, Jerusalem artichoke growing and processing in Hungary was suspended to give way to the corn program.

In 1979, a new Jerusalem artichoke project comprising a more up-to-date growing and processing was launched supported by the Ministry of Agriculture and Food and the

325

Hungarian Committee for Technical Development. The Department of Canning Technology of the University of Horticulture and Food Industry, Budapest, joined the program by developing a new process technology based on scientific research.

2 CULTIVATION OF JERUSALEM ARTICHOKE IN HUNGARY The Jerusalem artichoke plant is similar to the sunflower, but unlike the latter species

it produces underground tubers. It stands extreme climatological conditions and can be grown in sandy soils where other cultivated plants hardly can grow. Thus, Jerusalem artichoke is one of the suitable plant species for being grown on the sandy soils in Hungary. It can preferably be grown in loose sandy soils, where a yield of 30 to 40 t ha"1 can be achieved in case of proper fertilization.

Planting techniques and treatment of Jerusalem artichoke tubers are similar to those of potato. Jerusalem artichoke is highly resistant to diseases and pests. It is a perennial plant and yields enough tubers for two successive years of cultivation. Growing Jerusalem artichoke for several years at the same site causes its tuber yield to drop and the small tubers to be inappropriate for processing. However, in that case the leaves, stalks and tubers can be used for feeding. For that, however, Jerusalem artichoke needs a two-stage harvest (stalk cutting and tuber lifting) (Molnär, 1985).

The tubers are strongly attached to the stolons which makes harvesting difficult, but this problem can be solved with a potato lifter of appropriate type. Among the known potato lifters the "Europa Super" of Grimme can properly be used for spring harvesting of tubers. Harvest losses and the level of contamination with adhering soil are influenced by the method of cultivation. In case of dam cultivation the loss caused by unlifted tubers is 1 to 2%, while in case of platband cultivation this value is as high as 4 to 7%. Contamination of the collected tubers is also less in case of dam cultivation. Because of the strong attachment of the tubers to the stolons, particularly in autumn, for autumn harvesting the combination of a lifter with revolving fork and manual lifting of tubers is required to obtain an acceptable result.

The tubers can stand the winter in the soil without lifting. Potential losses of their total sugar content significantly depend on the temperature conditions. If the tubers are left through the winter below +1 °C average temperature, the fructose content reduction is about 10% and this is proportional to that of the total sugar content.

The shape and composition of the tubers are variable. Thus, selection and improvement of cultivars is essential from the point of view of processing (Barta, 1984).

3 PROCESSING OF JERUSALEM ARTICHOKE TUBERS IN HUNGARY The major mass of edible carbohydrates in the Jerusalem artichoke consists of inulin

326

which can be hydrolysed to fructose. At the optimal time of harvest the inulin of the tubers contains from 75 to 85% fructose. As has been outlined in the Introduction all available data suggest that use of Jerusalem artichoke in the production of several foods is of practical importance taking into consideration the present-day results and current views of dietetics. In view of this the demand for Jerusalem artichoke concentrate of high fructose content in Hungary can be estimated to increase to 5000 to 60001 a'1. Understandably, this has greatly stimulated the development of new technologies for the manufacture of Jerusalem artichoke-based products rich in fructose.

3.1 Purified Jerusalem artichoke concentrate with high fructose content The production technology for purified Jerusalem artichoke concentrate of high

fructose content, which is practically a pure sugar solution, has been worked out based on data on the sugar content of tubers and on pilot-scale investigations.

A block diagram of the technology is shown in Fig. 2. The tubers can be delivered from the field in containers or in bulk.

3.1.1 Juice extraction Extraction of juice from the cleaned and chopped raw material can be carried out by

pressing or by diffusion. By one of the most up-to-date Bücher presses used in fruit juice production, an extract yield of up to 75% can be achieved, which can be increased up to 80% by a single aqueous extraction and repeated pressing of the press cake. Experiments on a single-screw inclined tray extractor showed that a significant increase in the diffusion temperature does not adversely affect the mechanic properties of the tuber slices.

Since the diffusion of the soluble substances is 30% higher at 90 °C than at 75 °C, an auxiliary device for recycling the heated liquor should be used (Fig. 3). The auxiliary system enables a diffusion temperature ranging from 90 to 92 °C in the heating zone to be reached ensuring increased permeability of the cells as well as uniform contact and increased mass transfer between the slices and the liquor. The temperature of the extracting water is 80 °C. Mass transfer is adequate over the whole length (8 m) of the device, thus enabling the extraction to result in an extract yield ranging from 95 to 98% (Fig. 4). Consequently, the process technology should preferably be based on extraction (Vukov and Barta, 1987).

3.1.2 Juice purification The main component of the juice is the inulin, which is chemically rather stable. In

addition, it contains mono- and disaccharides, salts and some free amino acids of the cell fluid as well as colloids and floating contaminants. The filtered juice contains the natural

327

TUBERS

Washing

I Mechanical cleaning

1

' ' Slicing

Chopping

1 f

' ' Diffusion L

Pressing

* ""

I Separating

Filtering

* Cation exchange

Hydrolysis

Anion exchange i

Evaporation j

Concentrate

Fig. 2. Flow-sheet of the Jerusalem artichoke process technology.

cations of the tubers, colouring agents and a major part of the added calcium ions. When using a clarification technology based on calcium hydroxide, the optimal temperature for clarification in case of pressed juice is between 85 and 90 °C, while in case of diffusion it is 60 to 65 °C (see Fig. 5).

The colloids and floating contaminants in the raw juice can be coagulated at pH 10 to 11.5 by means of heat and calcium hydroxide. Calcium hydroxide, amounting to 0.2% (for extracted juice) and to 0.4% (for pressed juice) calcium oxide equivalent of the juice (w/v), is satisfactory for this procedure. The so cleared juice can be readily filtered with

328

Juice

Drive» fl

Raw slices

■ ■ *

Heating (chambers

Condensate

-Steam pipe-

'LMm Γ IV V VI V MI VI 4= Condensate

Water heater

l l s i i

Spent sl ices

^ ^ i ice press |\

" *7 —'-da

Fig. 3. Diagram of the single-screw counterflow diffusion extractor.

Extract content (g/lOOg)

2 ( H 1 9 1 18 17-1 16 A 15-1 14J

1 3 I 12J 11H 1oJ

9 I 8 I 7A

6 I 5 I 4 I 3-I 2 I 1 H

Slices

Juice

Spent s l ices

0 0,5 1 1.5 2 2j5 3 3£ 4 4,5 5 T i m e ( h )

Fig. 4. Increase in extract contents during extraction of Jerusalem artichoke tubers.

filter aids. Beside the carbohydrates the clarified and filtered tuber juice contains organic and inorganic cations, anions and colouring agents, which can be removed by ion exchange.

Cation exchange can be carried out using the Hungarian Varion KSM resin. Utilizing 75% of the resin capacity, calcium, alkali ions and colouring agents can be removed from

329

Hydrated lime powder Juice

Filtering aid

i * Clarifying

60-85°C

τ

Filtering Juice

Fig. 5. Diagram of juice clarification.

Absorbance/juice 100% 1 [420 nm]

pH

10

8-j

64

4

2 1 oj

raw, untreated juice pH:10,1

colour: 3,2

0 1 2 3 4 5 6 7

Fig. 6. Effect of load of ion exchange resin on absorbance and pH of the juice produced.

the juice, and so its pH value can be stabilized at 1.9 to 2.5. Depending on the amount of calcium hydroxide used, a cation exchange volume of 1 m3 can be charged with 5 to 6 m3

juice. Practically, inorganic cations and amino acids can be totally removed, while the colour intensity of the juice can be diminished by 70 to 90% (Fig. 6) (Barta, 1986).

When removing the cations from the clarified and filtered tuber juice, hydrogen ions getting into the solution catalyse the hydrolysis of inulin. In practice, in the acidic juice which has a pH of 1.9 to 2.5 and a temperature of 90-95 °C, the inulin can be completely hydrolysed within 35 min (Fig. 7).

The fructose obtained by hydrolysis is highly heat-sensitive both in alkaline and in

330

8CH

70H

60H

Fructose content in the dry matter

%

- ^ 10 20 30 40 50 Time (min)

Fig. 7. Production of fructose during hydrolysis of inulin, at 90 to 95 °C.

acidic media. The heat stability of the fructose is maximal at about pH 4.5, thus the juice has to be set at this pH value by anion exchange with Varion ADM resin. This resin is strongly alkaline, thus, a good quality of the juice can be achieved only at a constant volume flow and careful pH control. In practice, the concentrated juice contains exclusively sugars, with a fructose content of 83 to 84% if processed in autumn and 71 to 75% if processed in spring. The absorbance of fructose-rich Jerusalem artichoke concentrates so prepared is only 1.5 cm"1 at 420 nm and at 100% soluble sugar content.

3.1.3 Concentration of the Jerusalem artichoke juice High-quality concentration of the purified juice with a fructose content of 75 to 85%

can be achieved by any quick vacuum evaporator at a temperature of 45 to 55 °C. Both FMC and Vogelbusch devices are appropriate for juice concentration.

The FMC Taste evaporator tower is a four-stage, six-unit, long-tubed rapid evaporator, in which the juice, under vacuum, stays for a short period and flows through each unit only once, without recycling. The input temperature of the juice is about 54 °C (preheated). Because of the proper heat transfer via the tube walls, the evaporation is so intensive that a concentrate of 70% Brix can be produced from a juice of 8 to 9% Brix within 5 to 10 min. The construction of the equipment ensures that the exhausted vapour

331

heats the pre-heaters and the tubes. Thus, only 1/3 kg of steam is required to evaporate 1 kg of water. The evaporator tower evaporates 3.8 t water h"1. In case of continuous operation the capacity is about 4.5 t juice h"1.

The Vogelbusch three-unit, falling-film evaporator is also suitable for producing Jerusalem artichoke concentrate. Its capacity is less than that of the FMC evaporator: 2500 1 of juice h"1. Its specific steam consumption q is 0.38 kg steam kg"1 evaporated water. The juice stays in the evaporator for about 15 min.

3.1.4 Features of the finished product The fructose content of the finished product varies from 70 to 85 %. The relative

fructose content depends on the variety of the Jerusalem artichoke used and on the time of harvest (autumn or spring). The pH of the juice is between 5.0 and 6.5 after desalting. After evaporation the pH drops to 4.3 to 5.2. This pH range equals that of pure sugar solutions. The high-fructose product manufactured with the above technology is a clear honey-like concentrate and has a good taste. During storage neither crystals nor other materials (proteins or saponins) precipitate. The product can be stored properly in tanks at room temperature and can be pumped without any difficulty. It can be used effectively in food-processing plants.

3.2 Concentrate of high fructose content containing all components of Jerusalem artichoke juice From the dietetic literature it is known that some minerals getting into the body

simultaneously with carbohydrates exert a beneficial effect on carbohydrate and fat metabolism. Among these minerals, the naturally occurring metals in plants are of special importance. Among the macro-elements, the relative amounts of potassium and magnesium are significant with respect to the cation balance of the human organism. As regards the micro-elements, proper levels of zinc and chromium are necessary for sugar metabolism (Angeli, 1983).

3.2.1 Enzymatic hydrolysis Since the mineral content of Jerusalem artichoke tuber juice is 3 to 5% and of this

potassium is about 2%, a mild technology without ion exchange had to be developed to obtain a purified, high-fructose Jerusalem artichoke concentrate containing all mineral components of the juice (Barta et al., 1989).

To this end, a pilot-plant scale process based on enzymatic hydrolysis of inulin is currently being developed. The hydrolysis of inulin is carried out with two enzyme preparations. One preparation is of fungal origin: Novo inulinase; the other one is of yeast

332

° RÖHM Rohalase I.10.X.

NOVO Inulinase

H 1 1 h 10 15 20 25 Time of reaction (h)

Fig. 8. Decrease of the inulin concentration in Jerusalem artichoke juice during enzymatic hydrolysis with two commercial enzymes.

origin: Röhm Rohalase I.10.X. The fungal inulinase is mainly an endo-enzyme which rapidly cleaves the long inulin

chain, while the enzyme from yeast is an exo-inulinase which sequentially splits off fructose units from the end of the inulin chain. The latter enzyme, thus, quickly hydrolyses the smaller oligosaccharides, but only slowly degrades longer inulin chains (Fig. 8). From this figure it is evident that equal quantities of inulin are hydrolysed during the first 5 h of the treatment, whereas later, e.g. after 15 h, the amount of non-decomposed inulin is still 10% in the case of Novo inulinase; in the case of Rohalase I.10.X it is even more.

3.2.2 Composition of the concentrates The composition of the concentrates obtained in laboratory test productions with the

two enzymes, as compared with that of the purified high-fructose concentrate, is shown in Table 1.

As is evident, total sugar comprises nearly 100% (98.8%) of the total extract content (dry matter) in the concentrate purified by ion exchange. In the enzymatically saccharified, non-purified concentrates this is much lower (79.0 and 86.8%). The level of hydrolysis is characterized by the reducing sugar content in relation to the total sugar content. In case of ion exchange and acid hydrolysis, the reducing sugar content is practically 100% (99%), in the juice hydrolysed by Novo inulinase it is 90.8% whilst in case of Röhm Rohalase I.10.X it is 86.9%. Of the total sugar content, 77.5, 82.7 and 75.8%, respectively, is

333

Table 1. Effect of ion exchange purification and enzymatic hydrolysis on the chemical composition of high-fructose concentrates of Jerusalem artichoke juice.

Total sugar Total fructose Total glucose Free reducing sugar Di- and oligosaccharides Colour0

Minerals pH

Mineral components

Macro-elements K Na Mg Ca P

Micro-elements Fe Cu Cr Mn Zn Ti

Purification by ion exchange ; hydrolysis

g/100 g dry matter

98.8

1.5 0.2 5.6

mg/100 g dry matter

27.3 88.4

210.1 167.0

7.6

1.16 0.70 0.02 0.33 0.20 0.01

and acidic

g/100 g total sugar

77.5a

22.5 99.0

1.0

Enzymatic

Novo inul

g/100 g dry matter

79.0

10.2 3.59 4.45

mg/100 g dry matter

1340 60 58 78

212

1.91 0.29 0.007 1.01 0.91 0.01

: hydrolysis without purification

inase

g/100 g total sugar

82.7b

17.3 90.8

9.2

Rohalasel.lO.X

g/100 g dry matter

86.8

12.3 4.77 4.0

mg/100 g dry matter

1780 14.3 45.3 90.8

225

5.28 0.24 0.03 2.54 0.57 0.05

g/100 g total sugar

75.8a

24.2 86.9 13.1

a Spring harvest and processing. b Autumn harvest and processing. c Absorbance per cm light path at 420 nm and at 100% soluble sugar content.

fructose, depending on the sort of the tubers processed and, mainly, on the time of harvest and processing. The colour of the juices in particular depends on juice clarification, but the presence of polyphenol oxidases in the tubers and their inactivation by acidification and heat also have a significant influence on the final colour obtained. The concentration of the macro-elements among the mineral components are significantly reduced by ion exchange. As compared to these macro-elements, the micro-element content is much less affected by

334

ion exchange. Organoleptic tests have shown that the taste of the purified high-fructose Jerusalem

artichoke concentrate is more pleasant than that of the enzymatically hydrolysed concentrates containing all components of the juice. This phenomenon may be due to the presence of amino acids and salts.

3.2.3 Area of application of the high-fructose Jerusalem artichoke concentrate An important area of application of the high-fructose Jerusalem artichoke concentrate

is the production of canned fruits and fruit juices of reduced energy content. Canned fruits for diabetics are produced by various Hungarian canning industries; often, in their manufacture the reciprocal taste-improving (synergistic) effect of fructose and cyclamate is being made use of. These canned fruits contain only 3 % added sugar (fructose) and their cyclamate content is also very low (0.04%). Their organoleptic characteristics, especially the taste, are very good.

The potential use of Jerusalem artichoke syrup instead of imported crystalline fructose in canned fruits for diabetics was tested by comparing both products on the basis of equal sugar content. To this end, the 3.0 kg crystalline fructose per 100 1 liquor prescribed by the factory was replaced by 5.6 kg Jerusalem artichoke concentrate (a concentrated extract with a dry matter content of 72%, of which 76% is sugar). In this way, 4.0 kg of sugar was put in 100 1 liquor, the fructose content of which was 3.0 kg. The quantity of the other components was unchanged as per the original technological prescription (cyclamate, fruit, citric acid).

Organoleptic tests of canned fruits made with Jerusalem artichoke concentrate showed that they could not be distinguished from those made with crystalline fructose, neither using a merit point system nor a triangle test. The most important components (fructose and glucose content) were estimated using HPLC. As shown in Table 2, only in the case of sour cherry the glucose/fructose ratio of the canned fruits made with crystalline fructose was slightly higher than that with Jerusalem artichoke concentrate. That means, that the variations in the original sugar composition of the fruits are of greater importance to the composition of the final product than the differences in sugar contents between the Jerusalem artichoke concentrate and pure fructose.

The use of Jerusalem artichoke concentrate (4.0 kg sugar/100 litres) in the liquors of the different canned fruits in every case reduced the total sugar and glucose content of the products and improved the fructose/glucose ratio (Table 3). From this Table it is evident that, based on their low glucose content, apple, pear and quince as well as strawberry and raspberry, and to some extent also sour cherry can be taken into consideration for producing canned fruits for diabetics with Jerusalem artichoke concentrates.

335

Table 2. Fructose and glucose content of canned fruits (g/100 g net weight).

Fruit

Sour cherry Cherry Pear

Produced with crystalline fructose

Fructose (F)

4.68 5.09 5.69

Glucose (G)

3.00 4.25 0.62

F/G

1.56 1.20 9.18

Produced with Jerusalem artichoke concentrate

Fructose (F)

5.09 4.95 6.28

Glucose (G) F/G

3.54 1.44 4.00 1.24 0.66 9.51

Table 3. Sugar composition of different fruits and that of the relevant canned fruits.

Fruit

Apple Pear Quince Plum Sour cherry Apricot Peach Raspberry Strawberry

Fresh fruit (g/100 g fwt)

Total sugar

11.8 ± 1.8 10.5 ± 1.2 9.1 ± 0.9

10.7 ± 2.9 9.1 ± 0.8 9.9 ± 1.7

11.6 ± 2.0 7.8 ± 2.0 7.1 ± 1.5

Total glucose

3.6 ± 1.3 2.4 ± 0.8 2.9 ± 0.4 6.0 ± 2.0 4.9 ± 0.6 5.2 ± 1.8 6.1 ± 1.4 3.8 ± 1.4 3.3 ± 0.7

Total fructose

8.2 ± 1.5 8.1 ± 0.9 6.2 ± 0.4 4.7 ± 2.0 4.2 ± 0.6 4.7 ± 1.9 5.5 ± 1.4 4.0 ± 1.4 3.8 ± 0.7

Canned fruit made with Jerusalem artichoke concentrate (g/100 g net

Total sugar

9.4 ± 1.3 8.5 ± 0.8 7.5 ± 0.6 8.6 ± 2.0 7.8 ± 0.6 8.0 ± 1.2 9.2 ± 1.4 6.6 ± 1.4 6.1 ± 1.0

weight)

Total glucose

2.7 ± 0.9 1.9 ± 0.6 2.3 ± 0.3 4.4 ± 1.4 3.9 ± 0.5 3.9 ± 1.3 4.5 ± 1.0 2.9 + 1.0 2.5 ± 0.5

Total fructose

6.6 ± 1.0 6.6 ± 0.6 5.2 ± 0.3 4.2 ± 1.4 3.9 ± 0.5 4.2 ± 1.3 4.7 ± 1.0 3.7 ± 1.0 3.6 ± 0.5

In view of a healthy nutrition, Jerusalem artichoke concentrates seem to be extremely suitable for replacing sucrose in the following products:

a. Canning industry - canned fruits - fibrous fruit nectars - filtered fruit juices - refreshments

b. Bakery and confectionery industry - sweet bakery products - flour-containing confectioneries - confectionery jellies

336

c. Dairy industry - cream curd - cocoa drink - puddings

In addition to Jerusalem artichoke concentrates suitable for diabetic purposes, also a fructose syrup (Fig. 9) has been produced having at least 96% fructose. From the fructose syrup also crystalline fructose has been produced using a technology elaborated at our department. Crystalline fructose is a product in great demand on the world market. Fructose enrichment was achieved usaing the calcium-fructosate method. Though this method is more labour-intensive, its investment demand is much less than that of the column Chromatographie methods recommended by Western companies. Starting from Jerusalem artichoke concentrate, the essence of the technology is the precipitation of fructose with a calcium hydroxide solution at a temperature of +2 to +8 °C. The calcium fructosate precipitate formed is separated from the calcium glucosate by filtration and washing. After

Jerusalem artichoke concentrate fructose content: 70-75% of DM

Ca-fructosate production (0-5 °C)

Chemical treatment and separation Filtration, neutralization, (sedimentation) filtration

Juice treatment Ion exchange

Evaporation Fructose syrup fructose content: 96% of DM

Crystallization

Crystalline fructose

Fig. 9. Flow-sheet of the production of fructose syrup and crystalline fructose.

337

the fructose has been set free by calcium phosphate or carbon dioxide, the insoluble calcium precipitate is filtered off as calcium phosphate or calcium carbonate, and the pure fructose solution concentrated under vacuum after ion exchange purification. Since its glucose content is 1 to 4% only, the fructose-rich concentrate obtained with this method is directly suitable for producing diabetic foods.

For the production of crystalline fructose a direct crystallization method (without alcohol) has been elaborated starting from a practically pure fructose concentrate. Under pilot-plant conditions well-grown crystals of even granulation were produced equivalent to those of imported fructose.

In addition to the products mentioned so far, others can be produced from Jerusalem artichoke. From Jerusalem artichoke tubers products can be made that contain the original inulin besides the natural fibre. Moreover, enrichment of both inulin and natural fibres can be accomplished by compressing the tubers to dry products, or the inulin can be partially hydrolysed to fructose as, for instance, in the production of pulp.

3.3 Jerusalem artichoke pulp The Jerusalem artichoke pulp produced by chopping the tubers contains all the

valuable materials of the tubers, viz. the components of the juice and the fibres. The production technology corresponds in every detail to that of fruit pulp manufacturing in the canning industry. The inulin in the pulp can be utilized as such or in a more or less hydrolysed - saccharified - from. The pulp can be used well as an additive to fibrous fruit juices, jams and bakery products. The pulp has very good nutritional qualities, since beside inulin it contains pectins and hemicelluloses (Table 4).

3.4 Dried Jerusalem artichoke products As starting material for dried products whole Jerusalem artichoke tubers or the by

product of juice extraction for the production of Jerusalem artichoke concentrate, the compressed pulp, can be utilized. The two raw materials constitute two types of products and require two different technologies. From the tubers dried dices of good quality and white colour can be made, whereas the compressed pulp can be made into a sized light-brown flour or groats with a high fibre content.

The dried semi-product can be used as a component of salads and vegetable products. They can be used in bakery and confectionery products as well, because the inulo-oligosaccharides (with a molecular mass of 700 to 3000 Da) help in keeping the bakery products fresh for a longer period. The pulp-derived dried groats or flour can directly be consumed in the form of granulates for increasing fibre intake.

The composition of the dried products is given in Table 4. Especially the data at the

338

Table 4. Composition (%, w/w) of dried Jerusalem artichoke tubers and pulp, and of dry fibre (%, w/w).

Dried flour Dried cubes Pulp

92.2 93.1 14.7 36.4 24.8 3.91 5.7 4.2 0.665

56.0 63.8 8.93 5.27 3.90 3.64 9.43 6.11 41.28

Dry fibreb

Cellulose 33 Pectic materials 25 Hemicelluloses 12 Lignin 1 Raw protein 26 Raw fat 3

a As a percentage of total sugar. b For comparison, dietetic fibre: 71%.

bottom of the Table clearly illustrate the good nutritional characteristics of dry fibre.

3.5 Inulin Inulin as such has a great potential importance both in the food and pharmaceutical

industry. Up to now, inulin has been mostly produced in small quantities from dahlia tubers or chicory roots.

In Hungary, inulin has not so far been produced from Jerusalem artichoke tubers. When considering Jerusalem artichoke as a potential source of industrially produced inulin, it should be realized that high-molecular-weight inulin is only present in tubers harvested in October and November and processed immediately.

Until recently, inulin was almost exclusively used for medical purposes, viz. as a test reagent to measure renal function. In the food industry, inulin of low molecular weight can be used in fine bakery and confectionery products. Both products have export potential to Western countries.

4 REFERENCES

Angeli, I., 1983. A cukorfogyasztäs korokozo hatäsa 6s az odesites problomäja. Az orvostudomäny aktuälis probl&näi. Medicina Kiado, Budapest, 45: 79-110.

Barta, J., 1984. Fruktoztartalmil levek 6$ süritmonyek technologiäja. Elelmez. Ipar, 38: 383-386. Barta, J., 1986. Tehnologijaproizvodsztvakrisztallicseszkojfruktozü. Pishch. Pererab. Promst', 12: 53-55.

(in Russian)

Dry matter Fibre Ash Total sugar Reducing sugar Reducing sugar8

339

Barta, J., Török, S., Vukov, K., Fodor, P. and Magyar-Pichler, 1989. Die technische Hydrolyse der Fructosane von Topinambur. Zuckerindustrie, 114: 397-400.

Lippay, J., 1753. 'Posoni kert\ Steibig Gergely J. Nyomda, Györ. Magyar, Κ-ηό, 1959. A csicsoka szonhidrattartalmänak kinyerese. Cukoripar, 12: 171, 189-194. Molnär, L., 1985. A csicsoka. Hosszuhegyi Mezögazdasägi Kombinat, Sükösd. Tori, T., 1955. A csicsokakutatäs eredmonyei 6s nopgazdasägi jelentösäge. METE publication, MTE

Kiskönyvtär. Vukov, K. and Barta, J., 1987. Csicsokaszörp 6s fruktoz gyärtäsa. Cukoripar, 40: 36-39.



PREPARATION OF PURE INULIN AND VARIOUS INULIN-CONTAINING PRODUCTS FROM JERUSALEM ARTICHOKE FOR HUMAN CONSUMPTION AND FOR DIAGNOSTIC USE

K. VUKOV*, M. ERDELYI and E. PICHLER-MAGYAR University of Horticulture and Food Industry, Faculty of Food Technology, Department of Canning Technology, Menesi ut 43, 1118 Budapest, Hungary * Deceased 17 October 1991.

ABSTRACT

Various technologies to obtain pure inulin for medical and diagnostic use, and different inulin preparations for human consumption are discussed. A product of extremely good quality could be obtained by microwave vacuum drying; however, in the latter case addition of foam structure-improving natural gums proved to be necessary. Ultrafiltration experiments - though unsuccessful so far - resulted in a possible alternative to ion exchange for the removal of high-molecular-weight impurities from inulin preparations.

1 INTRODUCTION Inulin is a reserve substance, among others in the roots and tubers of several

Campanulaceae and Compositae. Maximal concentrations are found in Jerusalem artichoke, chicory and dahlia, where it constitutes up to 72-80% of the dry weight. The degree of polymerization (DP) of inulin varies strongly, depending on the species, season, etc. In general, almost 90% of all the inulin in dahlia tubers has a DP > 10; this percentage is 70 and 50 for chicory roots and Jerusalem artichoke tubers, respectively.

For several reasons, there is a strong interest in Hungary in growing Jerusalem artichoke. Satisfactory tuber yields can be obtained even on sandy soils, where other crops hardly survive. Furthermore, Jerusalem artichoke has no common diseases, thus the costs of plant protection are remarkably low.

For diagnostic use, it is necessary to produce an extremely pure inulin with a high DP (> 20). For inulin to be used for human consumption in foodstuffs it is sufficient to remove toxic components and pathogenic organisms. Smaller inulins, with DP 6-10, could be utilized to improve the consistency of, for instance, cakes and other bakery products.

342

2 PREPARATION AND CLARIFICATION OF INULIN-CONTAINING JERUSALEM ARTICHOKE JUICE For the preparation and clarification of inulin-containing Jerusalem artichoke juice

the tubers are washed repeatedly, and then homogenized in an industrial apple crusher. During homogenization an edible acid is added to prevent enzymatic oxidation of the polyphenols present in the mash; if not, a quite dark juice would be produced. As is evident from Fig. 1 citric acid should be used preferably. The pressed juice is clarified by coagulation followed by heat treatment and acidification to pH 4, followed by filtration. Heating up and cooling down should proceed rapidly in order to prevent hydrolytic cleavage of the inulin chains. Subsequently, the clean juice is passed through a cation exchange resin where it becomes more acid (Fig. 2). This is done at low temperature again to prevent inulin breakdown. The liquid is then neutralized as soon as possible by passing it over an anion exchanger column. It is also recommendable to pass it through a decolorizer column filled with a regenerable adsorbent (Fig. 3). The effect of the above-mentioned processes on inulin breakdown was found to be only small: the reducing sugar content as a percentage of total sugar content increased with only 1.5% (Fig. 4).

The clarified carbohydrate solution which practically contains only inulin, and related oligo-, di- and monosaccharides is concentrated in a vacuum evaporator as quickly as possible. The concentrate is then dried at low temperature, which as edible inulin can then be favourably used in bakery products.

acid acid

Fig. 1. Effect of acid pretreatment on colour and turbidity of pressed Jerusalem artichoke tuber juice.

343

10

0

1 |

r i control

cation exchange

decolorization

anion exchange

modification of pH

0.00 1.25 2.50 375 5.00 6.25

Specific load (cm3 resin/cm3 juice)

Fig. 2. The effect of the specific load on the pH of the juice upon different treatments.

anion exchange

0.00 1.25 250 3.75 5.00

Specific load (cm3 resin/cm3 juice)

Fig. 3. The effect of the specific load on the absorbance of the juice, at 420 nm, upon different treatments.

For medical and diagnostic use, high-molecular-weight inulin (DP > 20) is separated from the oligo-, di- and monosaccharide fraction by ultrafiltration. The dried retentate is ready for use.

344

Fig. 4. The effect of ion exchange purification on the reducing sugar content (as % of total sugar) of the juice of three Jerusalem artichoke cultivars (a, b and c).

3 MICROWAVE VACUUM DRYING OF INULIN-CONTAINING JERUSALEM ARTICHOKE JUICE AND CLARIFIED INULIN SOLUTIONS An extremely high product quality could be obtained by microwave vacuum drying

of both natural, pressed Jerusalem artichoke juice concentrates and purified inulin solutions. The technological process of inulin powder production was achieved using a Labotron

600 laboratory microwave vacuum drier which enables water removal at quite low temperature (< 10 °C). However, depending on the material to be dried a specific drying technology has to be worked out. Thereby, two requirements should be met. Firstly, a foam structure should be created; in this case water removal takes place homogeneously through the whole volume of the material. Secondly, the foam structure should be dense enough to have a specifically large quantity of material on a specific drying area. In a series of experiments the following parameters were studied: a. the type of structure-modifying additives, b. the quantity of different additives (0.5-30%), c. the temperature of preheating (2-45 °C), d. the time interval before drying (0-15 min), e. the time of preliminary vacuum treatment (0-4 min), f. the microwave energy (300-600 W cm"2), g. the time of continuous drying (1-4 min), h. the time of periodic drying (2-20 min), and i. the time of cooling after drying (0-24 h).

With both natural, pressed Jerusalem artichoke juice concentrates and purified inulin solutions a good foam structure could be built up; only with the natural juice concentrate it proved to be necessary to apply structural additives. Of several foam structure-improving materials tested Guardian 178 and Carudan 147 (guar gum and St. Jones seed gum, respectively, both from Grinsted) proved to be satisfactory.

345

Fig. 5. The effect of a foam structure-improving additive on the reducing sugar content of dried inulin powder.

Powder samples were then dissolved in a quantity of 5 g per 100 ml distilled water. The following properties were measured (Fig. 5): turbidity at 720 nm, pH and sugar content. Reducing sugar and total sugar content of the powders made using different technologies did not differ, if the quantity of the additive used was less than 5 %. The glucose content of the various powders did not even differ at a 10% additive level. However, turbidity values were already different upon addition of 1% foam-structure improving material.

Preliminary ultrafiltration experiments with the natural Jerusalem artichoke concentrate to separate low- and high-molecular-weight inulin fractions have not been very successful, so far. However, they resulted in a possible alternative to the ion exchange method for the removal of high-molecular-weight impurities.



PRODUCTION AND CHARACTERISTICS OF FRUCTO-OLIGOSACCHARIDES

Masao HIRAYAMA, Koji NISHIZAWA and Hidemasa HIDAKA Bio Science Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1, Chiyoda, Sakado-shi, Saitama, 350-02 Japan

ABSTRACT

Fructo-oligosaccharides are short-chain fructans with a terminal or in-chain glucose moiety and are found in many plant species, such as onion, edible burdock, asparagus and others. A mixture of fructo-oligosaccharides, namely 1-kestose (GF2), nystose (GF3), and 1-fructofuranosylnystose (GF4), is commercially produced from sucrose through the transfructosylating action of an enzyme from Aspergillus niger.

Fructo-oligosaccharides possess useful physical and physiological properties and good sensory qualities which make them widely applicable in food- and feedstuffs. They are stable at neutral pH and at temperatures up to 140 °C, and have a sweetness of good quality. Because fructo-oligosaccharides are non-digestible, after ingestion they pass through the small intestine without being metabolized, and thus reach the large intestine unaltered, where they are selectively utilized by bifidobacteria of the intestinal microflora. Many studies have shown that fructo-oligosaccharides relieve constipation, improve the blood lipid composition in hyperlipidaemia, and suppress the production of intestinal putrefactive substances in both animals and humans. These results indicate the usefulness of fructo-oligosaccharides as a new sweetener, and as a healthy ingredient of foods and feeds.

1 INTRODUCTION In recent years, in Japan oligosaccharides have been extensively studied after the

discovery of the remarkable biological functions of fructo-oligosaccharides and their beneficial influence on human health by improving the intestinal microflora.

This paper describes the production and characteristics of fructo-oligosaccharides which have been developed in our laboratories and have been sold as food- and feedstuffs for the past ten years in Japan.

2 C H E M I C A L STRUCTURE AND PRODUCTION OF FRUCTO-OLIGOSACCHARIDES

2.1 Chemical structure The chemical structure of fructo-oligosaccharides is shown in Fig. 1. They consist

348

HO

CHtOH

OH

CH20H

H 0 l

HOCH

HO

H0CH20 Ö

~ < £ 1 H0C^ HO CH20H H 0 ?H*

HOCH ■''■2 Π I

CH20H

Ηθ4ν) HO

H0CH2n

Q HOCHt(

V

?& V^C HO

HOCH

CH2

HO/

HO Ο Η * ° Η Η0^^0 Η ι°Η

n=2 -kestose

n=3 nystose

n=4 1F-/Mructo-furanosyl nystose

1F (1 -ß-fructofuranosyO ^,—sucrose

Fig. 1. Structure of fructo-oligosaccharides (Neosugar).

of one glucose moiety and several ß-2,1 -linked fructose residues. Representative compounds are 1-kestose (GF2), nystose (GF3) and lF-fructofuranosylnystose (GF4). They occur widely in many kinds of edible plants, such as asparagus, garlic, onion, edible burdock, Jerusalem artichoke and so on (Table 1) (Whistler and Smart, 1953; Pollard and Amuti, 1981).

Table 1. Occurrence of fructo-oligosaccharides in plants.

Oligosaccharides Occurrence

GF2: 1-Kestose

6-Kestose Neokestose

asparagus, Chinese chive, onion, rye; edible burdock, Jerusalem artichoke Gramineae asparagus, banana, onion, Gramineae; sugar maple

GF3: Nystose Bifurcose Neobifurcose

asparagus, onion; edible burdock rye oat

GF4: Fructosylnystose asparagus, onion; edible burdock

349

2.2 Enzymatic preparation Fructo-oligosaccharides can be prepared from sucrose through the transfructosylating

action of two enzymes, namely ß-fructofuranosidase (= invertase, EC 3.2.1.26) and β-Ό-fructosyltransferase (EC 2.4.1.9), which occur in both microorganisms and plants (Hidaka et al., 1988). However, no method for their industrial production existed until we developed them as a new sweetener about 10 years ago. Fructo-oligosaccharides can be more efficiently prepared with a high concentration of sucrose by using an enzyme having high transfructosylating ability. Incubation of 50% sucrose with the Aspergillus niger (ATCC 20611) fructosyltransferase affords a mixture of fructo-oligosaccharides.

2.3 Manufacturing procedure The industrial production process developed is as follows. A 50-60% sucrose syrup

is passed through a column containing an immobilized enzyme with transfructosylating activity. Neosugar G is obtained from the effluent by a purification process involving decolorization over active carbon and desalination by ion exchange. Neosugar P is prepared from Neosugar G by removing the mono- and disaccharides.

The sugar composition of the two Neosugars used in the present study is shown in Table 2. Neosugar G consists of 35% glucose and fructose, 10% sucrose, and 55% fructo-oligosaccharides, of which 25% is GF2, 25 GF3 and 5% GF4. Neosugar P, on the other hand, consists of more than 95% fructo-oligosaccharides, of which 40% is GF2, 45% GF3

and 10% GF4. These products are commercially available in Japan.

3 PHYSIOLOGICAL FUNCTIONS 3.1 Non-digestibility

Fructo-oligosaccharides are not hydrolysed by rat or human digestive enzymes such as the disaccharidases of the intestinal mucosa or α-amylase of pancreatic homogenates (Oku et al., 1984). These properties are illustrated by the data of Fig. 2. It shows the result of an experiment on sugar tolerance which confirms that fructo-oligosaccharides are neither digestible nor absorbable unlike their monosaccharide components fructose and glucose. After ingestion of sucrose, plasma glucose and fructose concentrations increase and the insulin response is rapidly observed. However, fructo-oligosaccharide ingestion does not cause any increase in the plasma concentrations of these sugars (Yamada et al., 1990).

3.2 Available energy The utilization of fructo-oligosaccharides was investigated by a radiorespirometric

study and anaerobic incubation of [U-14C]-fructo-oligosaccharides (Hosoya et al., 1988). When [U-14C]-labelled fructo-oligosaccharides were ingested by healthy men, about 49%

350

Table 2. Composition of Neosugar G and P.

Trade name

Neosugar G Neosugar P

Monosaccharides Sucrose G + F GF

35 10 <5

Fructo-oligosaccharides GF2 GF3 GF4

25 25 5 40 45 10

Total FOS

55 >95

and 55 % of the administered radioactivity was detected as expired 14C02 after 24 and 48 h, respectively, with the maximum rate of 14C02 excretion occurring at 7 h after administration. Upon anaerobic incubation of the saccharide with the subjects' faeces, the saccharides were mainly catabolized to 14C02 (9.6%) and volatile fatty acids (VFAs). Together, the results of these two studies suggested the existence of a catabolic pathway in which the [14C]-fructo-oligosaccharides were fermented by intestinal bacteria into 14C02 and [14C]-VFAs, which were absorbed and utilized in man to give the respiratory 14C02. Therefore, caloric utilization of the saccharides could be quantified by estimating the amount of the [14C]-VFAs absorbed from the colon, and the available energy was estimated to be between 1.5 and 2.0 kcal g"1.

3.3 Selective utilization by intestinal bacteria Fructo-oligosaccharides are utilized by human intestinal bacteria. Table 3 shows the

utilization of several sugars by intestinal bacteria in vitro (Hidaka et al., 1986). Fructo-oligosaccharides were utilized by almost all Bifidobacterium species except B. bifidum, but were not utilized at all by undesirable putrefactive bacteria such as Clostridium perfringens, C. difficile and Escherichia coli.

51

^ *

2]

H

1 » Sucrose(o)

k FOS (. .;i-

30 60 90 120

•o j?150 a» o

X 5 0

I'' ■«■ %* Sucrose (o)

C irrte (m\ 1 FOS (·)

30 60 90 120 mm

Time after Oral Administration

Fig. 2. Plasma concentrations of glucose, fructose and inulin after oral administration of fructo-oligosaccharides (25 g) or sucrose (25 g) to healthy men.

351

Table 3. Utilization of several sugars by intestinal bacteria8.

Bacterial species

Bißffobaclerium adolescentis Bifidobactcrium longum Bifidobactcrium breve Bifidobactcrium infanlis Bifido bacterium bifidum Lactobacillus acidophilus Lactobacillus fermentum Lactobacillus salivarius Lactobacillus cosei Lactobacillus plantar urn Eubaderium aerofaciens Eubaeterium limosum Eubaderium lenlum Propionibacterium acnes Bacteroides fragilis Bacteroides thetaiolaom icron Bacteroides vulgatus Bacteroides dislasonis Bacteroides ovatus

c u

o d

4 3 3 2 2 3 4 2 1 1 1 1 1 1 4 3 2 1 1

o CO O

y

Ü 44 4f 41--14 41-44 41-44 41· 44 4f 4r -44 4r 44 44 44 44

ij

jo a υ rt

ΊΓ 44 4r 44 44 44 44--44 41-4+ 44 --— 44 -fl-44-4f 4f

0 ΙΛ

o *C O «3

IT 4f + 41---

- -+ -+ + --— -H-4f 44 +f 44-

Bacterial species

Bacteroides melaninogenicus Fusobacterium varium Megamonas hypermegas Mitsuokella mulliacidus Escheriehia coli Kleb Stella pneumoniae Enterococcus faecalis Enterococcus faecium Streptococcus intermedius Peptostreptococcus prevotii Peptostreptococcus parvulus Clostridium perfringens Clostridium difficile Clostridium parapulrißcum Clostridium clostridiforme Clostridium ramosum Clostridium butyricum Veillonella dispar Megasphaera elsdenii

1 o 6

1 2 2 2 2 1 1 1 2 1 1 4 2 2 2 2 1 2 1

o 8 υ 3

o 4f 44 44 44-44 4f 44· 44-4+ 44 44 4f 4+ 44-4f 44 +r --

3 ΰ

— -4f 4f 44 --f 44 41--— 41 — + 4f 4-f 44 --

0 <Λ bO 4>

U-. ΙΛ

~H~ -44 \ -44 + + 44 -44 --— + -f 44 — -

a Assessment of bacterial growth: 44, same level of growth compared to glucose; +, weaker growth compared to glucose; - , no growth; V, variable growth (strains may be either + or - ) .

Administration of fructo-oligosaccharides to senile in-patients ranging from 50 to 90 years old for two weeks (8 g day"1) resulted in a significant increase in the number of bifidobacteria in the faeces, without the counts of putrefactive bacteria being augmented. The average number of bifidobacteria per gram of stool increased about ten-fold from 108,8

to 109·7 after 14 days of administration of Neosugar. The total count of intestinal bacteria also increased, however, only from 10101 to 1010,3. These results suggest that bifidobacteria utilize fructo-oligosaccharides more rapidly than the species of the Bacteroides fragilis group, which are the dominant bacteria in the human intestine.

3.4 Cholesterol reduction Table 4 shows the results of measurements on serum total cholesterol, HDL-

cholesterol, triglycerides and apolipoproteins before and after administration of fructo-oligosaccharides (8 g day"1), to hypercholesterolemic subjects with Type Ha hyperlipoproteinaemia (Hidaka et al., 1991). Total cholesterol and triglycerides remained unchanged. Apoprotein E significantly increased. The daily intake of fructo-oligosaccharides lowered total serum cholesterol concentrations, mainly due to reduction of the LDL-cholesterol level.

352

Table 4. Effect of fructo-oligosaccharides (FOS) on serum lipids.

Serum component Initial8 After FOS

Total cholesterol 277.6 ± 15.1 260.0 ± 29.0 HDL-cholesterol 57.3 ±11.5 57.7 ± 13.4 Triglycerides 119.8 ± 41.8 119.6 ± 51.8 Apo A-l 138.3 ± 14.3 145.2 ± 22.1 Apo B 160.3 ± 20.3 152.2 ± 24.0 ApoE 3.6 ± 1.0 4.8 ± 1.6b

a Expressed as mean + SD of mgm ml"1 serum. b Significantly different from initial value at P < 0.05 (paired t-test).

3.5 Relief of constipation Fructo-oligosaccharides have a beneficial effect on constipation. To 15 constipated

subjects ranging from 20 to 80 years old fructo-oligosaccharides were administered for 28 days. After ingestion, 11 subjects (73%) improved in terms of constipation, and all the subjects were able to defecate more than once every 3 days (data not shown) (Kameoka et al., 1986). It might be considered that the alleviation of constipation by non-digestible saccharides is partly due to the high osmotic pressure of short-chain fatty acids produced by the intestinal bacteria, and consequently accelerated peristaltic movement.

4 CONCLUSION Fructo-oligosaccharides, produced by the transfructosylating action of a

fructosyltransferase from A. niger, have two characteristic properties, i.e. the non-digestibility for men and the selective utilization by intestinal bacteria. The beneficial effects on humans and animals are considered to be derived from these two properties. The non-digestible saccharides are utilized as nutrients by beneficial bacteria in the large intestine. This selective utilization results in an increase in bifidobacteria and the production of short-chain fatty acids. The concomitant lowered pH in the large intestine leads to suppression of putrefactive substances.

Because of their beneficial properties fructo-oligosaccharides are widely applied in foodstuffs and animal feeds.

5 REFERENCES

Hidaka, H., Eida, T., Takizawa, T., Tokunaga, T. and Tashiro, Y., 1986. Effects of fructooligosaccharides on intestinal flora and human health. Bifidobact. Microflora, 5: 37-50.


Hidaka, H., Tashiro, Y. and Eida, T., 1991. Proliferation of bifidobacteria by oligosaccharides and their

353

useful effect on human health. Bifidobact. Microflora 10: 65-79. Hosoya, N., Dhorranintra, B. and Hidaka, H., 1988. Utilization of [U-14C]fructooligosaccharides in man

as energy resources. J. Clin. Biochem. Nutr., 5: 67-74. Kameoka, S., Nogata, H. and Hamano, K., 1986. The effect of administration of fructo-oligosaccharides

on chronic constipation. Rinsho Eiyo, 68: 823-829. (in Japanese) Oku, T., Tokunaga, T. and Hosoya, N., 1984. Nondigestibility of a new sweetener, "Neosugar", in the rat.

J. Nutr., 114: 1574-1581. Pollard, C.J. and Amuti, K.S., 1981. Fructose oligosaccharides: possible markers of phylogenetic

relationships among dicotyledonous plant families. Biochem. Syst. Ecol., 9: 69-78. Whistler, R.L. and Smart, C.L., 1953. Fructans of higher plants. In: Polysaccharide Chemistry, Ch. 11.

Academic Press Inc., New York, pp. 276-291. Yamada, K., Hidaka, H., Inooka, G., Iwamoto, Y. and Kuzuya, T., 1990. Plasma fructosemic and

glucosemic responses to fructooligosaccharides in rats and healthy human subjects. Digestion and Absorption, 13: 88-91.



PRODUCTION OF FRUCTO-OLIGOSACCHARIDE-RICH FRUCTOSE SYRUP

H. YAMAZAKI and K. MATSUMOTO Department of Biology, Carleton University, Ottawa, Ontario, Canada K1S 5B6

ABSTRACT

Fructo-oligosaccharides (FOS) have been shown to provide a variety of health benefits for humans and animals. We have developed a simple and rapid process for producing FOS-rich fructose syrup from Jerusalem artichoke tubers. The tuber fructans are extracted by heating thin tuber slices in water. The extract is passed through a column of strong acid cation exchange resin, providing an effluent having a pH of 2.0 to 3.0. The effluent is then hydrolysed by heating at a temperature of 70-100 °C. The use of longer heating times and/or higher temperatures results in sweeter syrup of lower FOS content. Optionally, the hydrolysate is decolorized by contact with activated charcoal. The hydrolysate is passed through a column of weak base anion exchange resin, providing an effluent having a pH of 6.5 to 7.0. The effluent is then concentrated to a syrup of 40-70% solids by reverse osmosis followed by evaporation. The tuber pulp obtained after the fructan extraction is rich in nutrients and can be used as feed.

1 INTRODUCTION Recent Japanese studies (Hidaka et al., 1986) show that small fructo-oligosaccharides

(FOS) such as GF2 .4 (G = glucose; F = fructose) selectively stimulate the growth of "beneficial" intestinal bacteria (bifidobacteria in humans) and provide a variety of health benefits. Since humans can not utilize FOS, orally administered FOS is catabolized by bifidobacteria in the lower intestine. The resulting catabolites, organic acids, suppress the growth of "unfavourable" intestinal bacteria (e.g. Escherichia coli and Clostridium perfringens) which otherwise produce toxic substances (e.g. ammonia, amines, hydrogen sulphide, skatole and indole). The acids also retard the conversion of amines to nitrosamines (carcinogens) and the intestinal absorption of ammonia and amines (which contribute to high blood pressure), and stimulate bowel movement. Bifidobacteria also provide the hosts with vitamins, and stimulate intestinal immunity. A decline in the intestinal bifidobacteria population is commonly observed in unhealthy or elderly humans. FOS has been shown to reduce constipation, blood lipids, blood cholesterol, blood pressure and intestinal toxins in humans.

As a natural sweetener, fructose has several advantages over sucrose and glucose.

356

It is at least 1.3 times sweeter and less cariogenic than sucrose. The ingestion of normal amounts of fructose by humans does not depend on insulin. Thus, fructose is suitable for consumption by diabetics and calorie-conscious people who can enjoy the same sweetness with fewer calories. Furthermore, fructose crystallizes less readily than sucrose (thus giving a smoother texture in high-sugar or frozen foods); chelates metal ions (responsible for off-flavour); and enhances the inherent aroma of fruit and vegetable foods.

The present paper describes a simple and rapid process for producing FOS-rich fructose syrup from Jerusalem artichoke tubers.

2 MATERIALS AND METHODS The tubers of Jerusalem artichoke (cv. Challenger) were harvested late October in

Ottawa, stored at 4 °C and processed within two months. Ion exchange resins, Dowex 88 and Dowex 66, were obtained from Dow Chemical Co. Activated charcoals, Norit SX 2 and Norit ROX 0.8, were obtained from Van Waters and Rogers. Before use, the charcoals were washed to remove fines.

Inulofructosaccharides were analysed by HPLC using a Shimazu PNH2-10/S 2505 column, a mixture of acetonitrile and water (7:3, v/v) as a solvent, and Meioligo P (Meiji Seika Kaisha, Ltd.) as the standard.

Reverse osmosis was performed on a cellulose acetate membrane (7 Ä pore size) under 1000 psi of nitrogen gas.

3 RESULTS AND DISCUSSION The solids (representing about 20%) in the Jerusalem artichoke tubers consist of 68-

83% fructans; 1.5-1.6% proteins; 13% insoluble fibres; and 5% ash (Fleming and GrootWassink, 1979). The tubers are also rich in polyphenols and active polyphenol oxidases which catalyse the oxidation of the phenols to the quinones in air. The quinones in turn react with amino acids and proteins, causing brown discoloration and off-flavour. Therefore, production of fructose syrup should involve the control of discoloration and the removal of non-fructan components. Furthermore, the tubers should be processed as rapidly as possible to avoid the high cost of tuber storage.

Our process for producing FOS-rich fructose syrup is summarized as follows. The Jerusalem artichoke tubers were washed, mechanically sliced (to a thickness of 1-2 mm), and immediately mixed with an equal weight of hot water (> 80 °C). The mixture was heated at 80-100 °C for 20-30 min to extract fructans and inactivate the polyphenol oxidases. The juice was collected by hand-pressing the mixture in a filter cloth. The juice, while warm (> 40 °C), was passed through a column of strong acid cation exchange resin (H+), Dowex 88. The effluent (having a pH of 2 to 3) was hydrolysed by passing through

357

Table 1. Carbohydrate compositions of syrup preparations produced by hydrolysis at 100 °C and pH 2.5.

Heating (min)

2.5 5

10 15

time Composition (%)

Ga

4.6 7.2

13.5 22.1

F

16.4 26.2 45.1 63.8

DP 2

44.9 38.1 36.7 12.1

DP 3

11.9 8.1 3.3

~0

DP 4

8.6 6.6 1.6

~0

DP 5

6.0 4.6 0.6

~0

DP > 5

7.6 9.3

~0 ~0

a G, glucose; F, fructose; DP, degree of polymerization (DP = n contains Fn or GFn_!). ~0, negligibly small.

a 24-inch spirally coiled glass tubing (4 mm O.D., 2.5 mm I.D.) which was embedded in sand heated at 70-100 °C. The extent of hydrolysis was controlled by varying temperatures and flow rates (or heating times). If the effluent was coloured, it was decolorized by either mixing with 0.01 part of activated charcoal powder, Norit SX 2 or passing through a column of activated granular charcoal, Norit ROX 0.8. The charcoal was removed by filtering through a filter paper and a glass fibre filter (Whatman GF/F) or a membrane filter. The filter was then passed through a column of weak anion exchange resin (OH"), Dowex 66. The effluent (having a pH of 6.5 to 7) was concentrated to syrup of 40-70% weight solid either by evaporation at > 70 °C under vacuum (about 100 mm Hg) or by a combination of reverse osmosis followed by evaporation. The overall recovery of total sugars in the syrup (from the original juice) was at least 80%. The syrup contained no detectable amounts of proteins and ash. The syrup was colourless and transparent, and had a pleasant sweet taste. Table 1 illustrates examples of the syrup preparations which were produced by hydrolysis at 100 °C and pH 2.5. As predicted, longer heating times resulted in greater hydrolysis, which resulted in sweeter syrup of lower FOS content.

The process developed here is simple and rapid, and can be automated. This process should be applicable to production of similar syrups from the roots of chicory or dahlia. The sweet FOS-rich fructose syrup can be used as a "health" sweetener, particularly ideal for elderly people, calorie-conscious people and diabetics. The pulp obtained after the juice extraction is rich in protein and can be used as feed.

4 REFERENCES

Fleming, S.E. and GrootWassink, J.W.D., 1979. Preparation of high-fructose syrup from the tubers of the Jerusalem artichoke {Helianthus tuberosus L.). CRC Crit. Rev. Food Sei. Nutr., 12: 1-28.

Hidaka, H., Eida, T., Takizawa, T., Tokunaga, T. and Tashiro, Y., 1986. Effects of fructooligosaccharides on intestinal flora and human health. Bifidobact. Microflora, 5: 37-50.



POTENTIAL MEDICINAL AND NUTRITIONAL USES OF CHICORY ROOTS AND INULIN*

Anil K. GUPTA, Narinder KAUR, Maninder KAUR and Rangil SINGH Department of Biochemistry, Punjab Agricultural University, Ludhiana-141004, India

ABSTRACT

A significant increase in hepatic total lipid and triglyceride content was observed when rats were injected either with 2 ml ethanol kg"1 body weight for 10 days or with 0.5 mg dexamethasone kg"1 body weight for 13 days. Inclusion of 3 or 5% chicory roots in the feed of rats along with ethanol and dexamethasone injections, respectively, led to a significant reduction in the content of hepatic total lipids and triglycerides of these rats. Chicory roots also reduced the level of plasma triglycerides in ethanol-injected rats. As compared with normal feed the feed supplemented with chicory roots helped in the fast recovery of fatty liver caused by dexamethasone. Chicory roots given in normal feed for a period of 5 months lowered the content of hepatic total lipids, cholesterol and triglycerides.

Besides these potential medicinal uses of chicory roots, chicory root inulin could be used for the enzymatic production of fructose. To this end, synthesis of an extracellular inulinase was induced in Fusarium oxysporum by growing it on a medium containing chicory root extract as carbon source. Salt-soluble proteins from soybean and mungbean were made water-insoluble by heat treatment and inulinase, partially purified by ammonium sulphate fractionation, was immobilized by cross-linking it with these insoluble proteins. The immobilized inulinases had a higher temperature optimum (45 °C) than free inulinase (37 °C). Inulinase was also immobilized on DEAE-cellulose. Immobilized enzyme was used for the production of fructose from chicory root inulin.

1 INTRODUCTION

Chicory (Cichorium intybus L.) is known for its medicinal and nutritional qualities. In Indian medicine, this plant is used in the cure of fever, vomiting, diarrhoea and enlargement of spleen (Rao, 1960). Chicory roots are an excellent remedy for jaundice and all liver disorders (Levy, 1966). Besides for its medicinal values, the roots of this plant are used for blending with coffee and sometimes also as a substitute for coffee. Inulin, a polymer of ß-2,1 -linked fructose residues, is a reserve carbohydrate of chicory roots and is a potential material for the production of fructose. The conventional method for fructose

* This research was funded by the Indian Council of Medical Research, New Delhi, and the Department of Science and Technology, Government of India, New Delhi.

360

production from starch requires prior amylolysis with α-amylase and amyloglucosidase followed by glucose isomerase-catalysed conversion of glucose to fructose. The syrup thus obtained contains 42% fructose, 50% glucose and 8% oligosaccharides. The direct production of fructose from inulin using microbial inulinases can give a 90% yield of fructose.

In this paper, the effect of including chicory roots in feed in lowering the content of triglycerides in the liver and plasma of rats receiving ethanol and dexamethasone injections has been studied. In addition, some of the properties of inulinase, from Fusarium oxysporum, immobilized on various supports have been discussed in relation to fructose production.

2 MATERIALS AND METHODS C. intybus was sown in the first week of November 1988 and roots were harvested

in April of next year. The cut pieces of roots were dried at 70 °C and crushed to a fine powder. Male albino rats were kept on a diet being composed of (wt%): 75% wheat flour; 10% fat; 10% casein; 1% salt mixture; 3% NaCl; 0.5% choline chloride and 0.5% vitamin mixture. The composition of vitamin and salt mixture was as described by Hoy et al. (1983).

F. oxysporum (NCIM 1072) was grown on a medium prepared from an aqueous extract of chicory roots as described by Gupta et al. (1988).

After necessary feeding experiments (details of which are given in parentheses in Tables 1-4), rats were starved overnight, anaesthetized with ether and blood was collected by heart puncture using a heparinized syringe. Methodology described by Kaur et al. (1989) was used for the extraction of total lipids and for the estimation of triglycerides, cholesterol and phospholipids.

2.1 Isolation of inulinase Culture medium of F. oxysporum was collected after 9 days of incubation and

centrifuged at 5000 g. Precipitates obtained by addition of 40-60% ammonium sulphate were dissolved in 0.05 M sodium acetate buffer (pH 5.6) and dialysed overnight against an excess of this buffer before being immobilized on various supports.

2.2 Preparation of support for immobilizing inulinase Hundred g flour from mungbean and soybean each was added to 500 ml of 0.1 M

NaCl and stirred for 30 min. The contents after passing through a double-layered muslin cloth were centrifuged at 5000 g for 15 min. The supernatant was kept at 100 °C for 20 min and denatured proteins obtained after centrifugation at 4000 g for 15 min were washed

361

with 0.1 M NaCl (three times) followed by water, chloroform: methanol (2:1, v/v) and finally again with water. These denatured proteins were dried at 65 °C and crushed to a fine powder. This material was used as support for immobilizing inulinase.

2.3 Immobilization of inulinase To 2 g of denatured proteins, 100 ml of 0.1 M acetate buffer (pH 5.4) and 8 ml of

25% glutaraldehyde were added. After stirring for 30 min at 25 °C, proteins were recovered by filtration and washed with water. To these denatured proteins, 25 ml of inulinase (2-5 units) and 100 ml of 0.1 M acetate buffer (pH 5.4) were added. The contents were stirred gently for 24 h at 4 °C. The immobilized inulinase, removed by centrifugation, was washed thoroughly with 1 M NaCl in 0.1 M acetate buffer (pH 5.4). The immobilized enzyme preparation was dried between two layers of Whatman No. 3 filter paper and stored at 4 °C. Inulinase was also adsorbed onto DEAE-cellulose. DEAE-cellulose (10 g) after washing with 0.5 N HC1, water, 0.5 N NaOH, water and 0.05 M acetate buffer (pH 5.6) was mixed with inulinase (4-10 units) and the mixture was stirred for 2 h. The adsorbed inulinase, obtained after centrifugation, was washed with 1 M NaCl (three times) and water (twice) before being dried between two layers of filter paper.

2.4 Assay of immobilized inulinase Fifty mg of immobilized enzyme and 1 ml inulin (0.1 %) in 0.1 M acetate buffer (pH

5.4) were incubated for a required time period at 37 °C. The reducing sugars resulting from the enzymatic reaction were estimated by the method of Nelson (1944).

3 RESULTS AND DISCUSSION 3.1 Effect of chicory roots on lipid profile of liver and plasma of ethanol-injected rats

On injecting ethanol (2 ml kg"1 body weight subcutaneously daily for 10 days), the contents of total lipids and triglycerides in the liver and cholesterol and triglycerides in the plasma increased significantly as compared with the control group in which ethanol was not injected. However, when chicory roots (3%) were included in the feed along with ethanol injections, the contents of total lipids and triglycerides in the liver decreased significantly (Table 1). A significant reduction in the content of cholesterol and triglycerides in plasma was also observed on including chicory roots in the feed of ethanol-injected rats (Table 1).

3.2 Effect of chicory roots on lipid profile of dexamethasone-injected rats Dexamethasone, a synthetic glucocorticoid, is used in the treatment of diseases

ranging from affections of the skin to malignancies. A side-effect of the administration of dexamethasone is the mobilization of fat from adipose tissue resulting in development of a

362

fatty liver (Kaur et al., 1989). Therefore, the effect of supplementing feed with chicory roots on the lipid profile of plasma and liver of dexamethasone-injected rats was studied.

The contents of total lipids, cholesterol and triglycerides in the livers of rats given dexamethasone injections daily for 13 days and kept on a feed supplemented with chicory roots were significantly less than those of rats given dexamethasone injections and kept on a chicory root-free feed. The content of these lipids in the former case was comparable to that of the control group receiving normal feed and no injections (Table 2).

In another experiment dexamethasone injections were given for 8 days and thereafter half of the rats were given feed supplemented with 5% roots and the other half was kept on a normal feed without chicory roots to ascertain the beneficial effect of these roots in the recovery of liver damaged by dexamethasone. The lipid profile of liver but not of plasma in the group in whicn roots were included in the feed was closer to the group in which rats received a normal feed (Table 3).

Table 1. Effect of diet containing chicory roots on the lipid composition of liver and plasma of ethanol-injected rats (mean + SD; n = 6).

Group

1 2 3

Total lipids

mg g"1 liver

35.5 ± 3.3 44.2 + 2.3" 34.3 + 3.8

Cholesterol

3.6 ± 0.3 4.4 ± 0.4 3.3 ± 0.6

Triglyceride

5.5 ± 2.0 9.6 ± 2.0" 5.1 ± 1.0

Cholesterol Triglyceride

mg/100 ml plasma

69.3 ± 10.4 52.5 ± 10.1 87.4 ± 16.3b 121.3 ± 22.0" 64.9 ± 5.8 75.2 ± 26.5

">b In comparison with group 1 and with group 3, P < 0.0Γ; 0.05b (Student's t test). Group 1 was given control feed ad libitum. Group 2 was given the normal feed and injected with 2 ml ethanol kg"1 body weight subcutaneously daily for 10 days. Group 3 was injected with ethanol as in group 2 along with 3% chicory roots in the normal feed and was pair-fed to group 2.

Table 2. Effect of diet containing chicory roots on the lipid composition of liver and plasma of dexamethasone-injected rats (mean + SD; n = 7).

Group

1 2 3

Total lipids

mg g"1 liver

37.2 ± 7.3 90.4 ± 25.5" 54.9 ± 9.4b

Cholesterol

5.1 ± 1.1 8.7 ± 2.4" 4.8 ± 1.4b

Triglyceride

6.2 ± 1.0 71.1 ± 22.1" 38.2 ± 7.1b


mg/100 ml plasma

88.6 ± 16.0 49.3 ± 12.8 108.9 ± 13.2 73.3 ± 16.4 122.1 ± 26.6 76.3 ± 9.9

" As compared to group 1, P < 0.01; b as compared to group 2, P < 0.01. Group 1 was given control feed ad libitum. Group 2 was given the normal feed and injected with 0.5 mg kg"1 body weight dexamethasone subcutaneously daily for 13 days. Group 3 was pair-fed to group 2 and given a feed containing 5% chicory roots.

363

Table 3. Effect of feeding diet containing 5% chicory roots for 3 days after injecting dexamethasone for 8 days on the lipid composition of liver and plasma (mean + SD; n = 7).

Group

1 2 3

Total lipids

mg g"1 liver

52.0 ± 9.0 43.0 ± 4.6 34.2 ± 7.8a

Cholesterol

4.2 ± 1.0 4.9 ± 0.6 4.8 ± 0.7

Triglyceride

23.2 ± 7.0 16.1 ± 3.6 11.8 + 2.9*


mg/100 ml plasma

111.8 ± 19.5 179.7 ± 61.2 65.6 ± 15.3 59.7 ± 22.7 77.5 ± 14.2 97.7 ± 28.2

a As compared to group 2, P < 0.05. All the three groups were injected daily with dexamethasone (0.5 mg kg"1 body weight subcutaneously) for 8 days. Rats in group 1 were sacrificed after 8 days. Group 2 and 3, after discontinuing dexamethasone injections, were given the normal feed and feed supplemented with 5% chicory roots, respectively, for 3 days and sacrificed.

Table 4. Effect of supplementing feed of rats with 5% chicory roots on the lipid composition of liver and plasma (means + SD; n = 8).

Group

1 2

Total lipids

mg g"1 liver

43.1 + 5.0 33.8 ± 2.8a

Cholesterol

4.4 + 0.2 2.5 ± 0.4a

Triglyceride

8.8 ± 1.7 5.1 ± 1.2a


mg/100 ml plasma

101.2 + 15.4 57.2 ± 12.4 104.9 ± 13.0 53.4 ± 14.3

a As compared with group 1, P < 0.01. Group 1 was given control feed and group 2 was given feed containing 5% chicory roots for a period of 5 months. Feed was given ad libitum as feed consumption was almost the same in both groups.

Feeding of chicory roots for 5 months led to a considerable lowering of hepatic total lipids, cholesterol and triglycerides as compared with rats fed on a normal feed for the same period. The plasma lipid profile was not much affected (Table 4).

These results clearly demonstrate the beneficial effect of chicory roots in keeping the lipid profile of liver, especially with respect to total lipids and triglycerides, to near a normal level if given in feed during ethanol and dexamethasone injections. The effect on blood plasma was not so prominent. Feeding chicory roots helped in the recovery of liver damaged subsequent to dexamethasone injections. Feeding these roots to rats for 5 months did not show any visual adverse side-effects to the animals. Efforts are in progress in our laboratory to pin-point a specific factor responsible for the hypotriglyceridemic effect of these roots.

3.3 Properties of immobilized inulinases F. oxysporum secretes 85 % of the inulinase in its medium when grown on a medium

364

Table 5. A comparison of properties of immobilized inulinases.

Source of support

Soybean Mungbean DEAE-cellulose

Table 6. Thermal

Source of support

Soybean Mungbean DEAE-cellulose

Immobilization method

cross-linked cross-linked adsorption

stability of immobilized inul

(mM)

0.44 0.25 0.30

linase.

% Activity after heating at 50

15

50.0 51.8 80.7

30

37.5 42.5 61.5

°Cfor

45

0 33.0 51.9

Temperature optimum (°C)

45 45 37

min

60

0 12.9 19.2

pH optimum

6.0 6.5 5.5

% Yield

23-30 20-25 30-40

prepared from chicory roots (Gupta et al., 1990). The pH optimum of the immobilized preparations remained in the acidic range (Table 5). Km values of the immobilized inulinases varied from 0.25 to 0.44 mM with inulin being used as a substrate (the molecular weight of the inulin used was considered to be 5000 Da) (Table 5) showing that the affinity of the immobilized inulinases towards inulin was not much different from each other. Maximal recovery of inulinase activity was obtained in case of inulinase immobilized on DEAE-cellulose. By cross-linking inulinase to the denatured protein isolate from mungbean and soybean, the temperature optimum of this enzyme could be raised from 37 °C for the free enzyme to 45 °C. The inulinase immobilized on DEAE-cellulose though does not have a higher temperature optimum than the free enzyme but has a much higher thermal stability than other immobilized preparations (Table 6).

In one typical experiment, inulinase (100 units) adsorbed on DEAE-cellulose (35 g wet) was packed in a column (2.0 cm X 30 cm) and was washed with distilled water. Hundred ml of 2% inulin was passed through this column at a flow rate of 10 ml h"1. After three cycles 90-95% of the inulin was converted to fructose. These data showed that these findings may be of use to produce fructose from inulin.

4 REFERENCES

Gupta, A.K., Nagpal, B., Kaur, N. and Singh, R., 1988. Mycelial and extracellular inulinases from Fusarium oxysporum grown on aqueous extract of Cichorium intybus roots. J. Chem. Technol. Biotechnol., 42: 69-76.

Gupta, A.K., Rathore, P., Kaur, N. and Singh, R., 1990. Production, thermal stability and immobilisation

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of inulinase from Fusarium oxysporum. J. Chem. Technol. Biotechnol., 47: 245-257. Hoy, C.-E., Holmer, G., Kaur, N., Byrjalsen, I. and Kirstein, D., 1983. Acyl group distributions in tissue

lipids of rats fed evening primrose oil (γ-linolenic plus linoleic acid) or soybean oil (a-linolenicplus linoleic acid). Lipids, 18: 760-771.

Kaur, N., Sharma, N. and Gupta, A.K., 1989. Effects of dexamethasone on lipid metabolism in rat organs. Indian J. Biochem. Biophys., 26: 371-376.

Levy, J.B., 1966. Herbal Handbook for Everyone. Faber and Faber Ltd., London, 42 pp. Nelson, N., 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J.

Biol. Chem., 153: 375-380. Rao, U.N., 1960. The Chicory in India. Indian Council of Agricultural Research, New Delhi, 16 pp.



VALORIZATION OF AN INULIN-RICH BY-PRODUCT OF CHICORY

Edith LECLERCQ* and Geja HAGEMAN** Department of Food Technology, Section Food Chemistry and Microbiology, Agricultural University, Wageningen, The Netherlands Present addresses: * Quest International, P.O. Box 2, 1400 CA Bussum, The Netherlands;

Department of Health Risk Analysis and Toxicology, State University Limburg, Maastricht, The Netherlands

ABSTRACT

The roots of chicory (Cichorium intybus L.) which remain after harvesting of the heads constitute a waste. Partly, these roots are used as cattle feed, but lots are destroyed. This waste material could be a source of inulin and of bitter compounds, both being interesting ingredients for the food industry.

In order to obtain the highest possible yield of inulin as well as of bitter compounds, chicory roots were liquefied with commercial enzyme preparations containing both pectinases and cellulases. The release of inulin and bitter compounds during liquefaction has been studied. The inulinase activity of the enzyme preparations has been investigated.

1 INTRODUCTION In the Netherlands, chicory is grown for the heads which are sold as a delicate

vegetable. In addition to bitter sesquiterpene lactones, the remaining roots contain another valuable material, namely inulin, which is a carbohydrate reserve. Inulin can be used, among others, as a raw material for the production of fructose (Zittan, 1981) and 5-hydroxymethylfurfural (Küster, 1990).

Inulin is usually extracted from the plant with hot water. The inulin yield of the aqueous extraction depends on various factors, such as the degree of polymerization of the inulin, and of the size of the chicory root slices from which it has to be extracted.

A patented process for the liquefaction of sugar beets by an enzymatic treatment using cellulases or pectinases is also suitable for chicory roots (De Baynast de Septfontaines et al., 1986). Simultaneous liquefaction of the roots and hydrolysis of the inulin can be achieved by the concurrent addition of inulinase to the commercial enzyme mixture. The resulting hydrolysate may be used for fermentation to produce alcohol. The advantage of

368

this process over conventional aqueous extraction is that no extra water is needed during incubation, and that it is suitable for various types of agricultural raw materials, among others Jerusalem artichoke tubers (Birtantie et al., 1988). However, there are no claims for a higher efficiency of the process as compared with hot water extraction.

The aim of the study reported here was to liquefy chicory roots with commercial pectinases and cellulases. The liquefaction process should not only release inulin but also the bitter constituents, and in addition, after simultaneous or subsequent treatment with inulinase, fructose. Thus a bitter, sweet syrup should be obtained, which might be used as a raw material in the production of beverages, e.g. tonic water.

Both release of inulin and inulinase activity of enzyme preparations used were investigated.

2 MATERIALS AND METHODS 2.1 Materials

Chicory roots were obtained from a grower and stored one week at 1 °C before being cut into small pieces (about 4 mm3) under liquid nitrogen, and then frozen at -25 °C.

Rapidase C80 (pectinases), Rapidase C600 (pectinases and cellulases), and Maxazym CL2000 (cellulases) were obtained from Gist-brocades, Delft, The Netherlands. An inulinase preparation, Novozym 230, was obtained from Novo Industri, Copenhagen, Denmark. According to the supplier the inulinase activity was 300 units g'1. Inulin was purchased from BDH Ltd., U.K.

2.2 Liquefaction of chicory roots Portions of frozen roots (10 g) were suspended in 10 ml of 0.1 M sodium acetate

buffer (pH 4) and incubated at 40 °C for 16 h under continuous stirring with 0.025 g Rapidase C80, 0.02 g Maxazym CL2000, 0.01 g Rapidase C600, and 0.02 g Rapidase C80 + 0.02 g Maxazym CL2000, respectively. The enzymes were added to the suspension as dry powders. Samples were centrifuged (30 min, 3000 g). The supernatant was analysed for reducing sugars using the Nelson-Somogyi test as modified by Spiro (1966). Total sugars were measured using the method according to Dubois et al. (1956), and glucose, fructose, sucrose and oligosaccharides were determined with HPLC.

Enzymatic liquefaction of fresh chicory roots was carried out as described by Leclercq and Netjes (1985) by incubating the roots with Rapidase C600 (0.01 or 0.1%). The release of inulin was followed as a function of time.

As a control, the same mixture was used, however, without enzymes added.

369

2.3 Inulinase activity Inulinase activity of Rapidase C600 was compared with that of Novozym 230 by

incubating pure inulin (100 ml of a 5% solution) at 40 °C for 4 h at pH 4.5 with 0.05 g Rapidase C600 or with 50 units of the commercial inulinase. The enzyme reaction was stopped by adding methanol (4 ml of methanol to 1 ml of inulin solution). The sample was filtered (0.45 μιτι filter), and analysed for fructose, glucose and sucrose as described by Leclercq and Hageman (1985).

2.4 Analysis Fructose, glucose and sucrose were determined with HPLC as described by Leclercq

and Hageman (1985). Chicory roots and the residues obtained after enzymatic liquefaction (both 1.5 g) were extracted twice with water (5 ml, 70 °C). Extracts were diluted with methanol (4 ml of methanol to 1 ml of suspension), filtered (0.45 μνη filter), and injected. The supernatants were likewise diluted with methanol (as described above) before analysis.

Inulin was measured indirectly using HPLC after incubation of the sample with inulinase. Before dilution with methanol, chicory root and residue extracts, and supernatants were incubated with inulinase. Inulinase (25 μΐ) was added to 1 ml of extract or supernatant, the pH adjusted to 4.5 with 0.1 N HC1 and incubated at 60 °C for 3 h. Addition of methanol (see above) was used to terminate the reaction.

3 RESULTS AND DISCUSSION 3.1 Enzymatic liquefaction of chicory roots

Several commercial enzyme preparations were evaluated for their ability to liquefy chicory roots and to release inulin by determining total sugar content and reducing sugars in the supernatant after liquefaction. A large amount of fructose was found in the supernatant upon liquefaction of chicory roots with Rapidase C80. Maxazym CL2000 and Rapidase C600, on the other hand, both gave large amounts of inulin. However, Maxazym CL2000 did not liquefy chicory roots very well, only 53 % of total sugars being recovered in the supernatant. Use of Rapidase C600 resulted in a 73 % recovery of total sugars, and produced a small amount of reducing sugars. This indicates that no inulin had been hydrolysed.

In order to confirm that Rapidase C600 showed no inulinase activity, pure inulin was tested as a substrate. Inulinase activity of Rapidase C600 was compared with that of commercial inulinase. Incubation of inulin with Rapidase C600 (4 h) showed no release of fructose, glucose or sucrose. Therefore, Rapidase C600 was used in all other experiments to investigate release of inulin from chicory roots.

370

3.2 Release ofinulin A study of the release of inulin with time, up to 24 h, was carried out with and

without addition of enzymes (Fig. 1). The glucose content increased with time due to hydrolysis of cellulose. Fructose, on

the other hand, remained constant up to 5 to 8 h of incubation with commercial enzymes (0.1 and 0.01%, respectively). This might be due to a low "over-all" inulinase activity of Rapidase C600, so that the final enzyme product (fructose) is seen only after prolonged incubation (> 4 h). Otherwise, Rapidase C600 might have mainly endo-inulinase activity, with a very low exo-inulinase activity. If so, then, first inulins with a lower degree of polymerization (DP) will be formed. No exo-inulinase activity of Rapidase C600 was

8 0/100 o chicory root

7

el· 6

4

3

2

1

0

i I

0

i I

fa ijfe i fe l·^ Wm mm 2 5 Time (h)

24

g/100 o chicory root

B

I P I i i

iSmM

i 1

-ffi 2 6 Time (h)

24

I fructose V/X oluoose H+H saccharose Ν\Ί Inulin

Fig. 1. Release of inulin and sugars during enzymatic liquefaction of chicory roots (A) and control mixture (B).

371

detected upon incubation for 4 h at 40 °C and pH 4.5. Hydrolysis of inulin might also be due to endogenous inulinase activity of the chicory

root cells. Singh and Bhatia (1971a, b) investigated fructose and inulin metabolism of chicory roots, and found that pH 5.6 and 37 °C were optimal for fructosyltransferase activity, that is the enzyme which catalyses the synthesis of inulin. In later stages of the growth of the chicory root, the enzyme fructan exo-hydrolase (inulinase) brings about the hydrolysis of inulin (Gupta et ed., 1985). No optimal conditions for the latter enzyme have been reported. Probably, fructan hydrolase has the same pH and temperature optima as has fructosyltransferase. If this is true, in our experiments the pH during enzymatic liquefaction has been too low for detectable endogenous inulinase activity. Though the temperature used was almost optimal for this endogenous enzyme, hydrolysis of inulin during enzymatic liquefaction of chicory roots by endogenous enzymes is therefore not very likely.

Maximal release of inulin was obtained after 2 h incubation with Rapidase C600 (Fig. 1). Incubation of the chicory roots with 0.1 or 0.01% Rapidase C600 gave no difference in release of inulin, however, the rate of production was different: hydrolysis of the cell wall components was more rapid when a larger amount of enzymes was added to the root suspension and maximal release of inulin was obtained after 2 h (Netjes, 1985). Recovery of inulin from enzyme-treated chicory roots was consistently higher than from non-treated, control roots. Apparently, without enzymes added inulin is only incompletely extracted from chicory roots.

Inulin was first determined with HPLC using the methods of Leclercq and Hageman (1985). However, this method appeared to be suitable only for inulin with a high DP, and not for the smaller oligosaccharides. The latter do not separate well, whereas other breakdown products, as for instance sucrose, give double peaks. Moreover, retention times of some sugars with the same DP are different. Inulin content in the samples was therefore measured indirectly by determining fructose, glucose and sucrose content before and after incubation with inulinase this procedure giving the best results.

4 CONCLUSIONS Inulin can be released by enzymatic liquefaction of chicory roots with a somewhat

higher recovery as compared with the non-enzyme treated control. The result of the liquefaction corresponds with that of a repeated batch-wise liquid-solid extraction of the roots with hot water. The increase in yield obtained during liquefaction may thus be ascribed to the inefficiency of the one-batch extraction of the untreated control, with which the enzymatic liquefaction was compared.

Incubation with Rapidase C600 (0.1%) has to be terminated within 5 h, because thereafter hydrolysis of inulin becomes apparent. No endogenous inulinase activity was

372

found during incubation of the roots.

Isolation of inulin using liquefaction of chicory roots seems commercially attractive

only in combination with the release of other interesting compounds, such as bitter

sesquiterpene lactones, or in the production of bulk chemicals such as ethanol from

unpurified root or tuber juices where the addition of hydrolytic enzymes such as found in

Rapidase C600 might result in the maximal release of fermentable sugars.

5 REFERENCES

Birtantie, I.R.K., Voragen, A.G.J., Pilnik, W. and Fuchs, A., 1988. Winning van inuline en fructose uit de aardpeer (Helianthus tuberosus L.) door enzymatische vervloeiing. In: A. Fuchs (Ed.), Versl. Tweede Themadag Inuline, 30 Oct. 1987, Wageningen. NRLO-rapport nr. 88/5, The Hague, pp. 50-58.

De Baynastde Septfontaines, R.J.M.P., Brouard, F.E.M.E., Baret, J.L.A.G., Gicquiaux, Y.G.A.J.M. and Olsen, H.S., 1986. Proce io de liquefaction de betteraves et de racines de chicore^ par hydrolyse enzymatique et hydrolysat liquide obtenu. EP0256899. Date of filing 7-7-86.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356.

Gupta, A.K., Mamta and Bhatia, I.S., 1985. Glucofructosan metabolism in Cichoriwn intybus roots. Phytochemistry, 24: 1423-1427.

Küster, B.F.M., 1990. 5-Hydroxymethylfurrural (HMF). In: A. Fuchs (Ed.), Proc. Third Seminar on Inulin, 1 March 1989, Wageningen. NRLO report 90/28, The Hague, pp. 28-37.

Leclercq, E. and Hageman, G.J., 1985. Release of inulin by enzymatic liquefaction of chicory roots. Food Chem., 18: 131-138.

Leclercq, E. and Netjes, J.J., 1985. Release of sesquiterpene lactones by enzymatic liquefaction of chicory roots. Z. Lebensm.-Unters. -Forsch., 181: 475-477.

Netjes, J.J., 1985. Isolatie van bittere sesquiterpeenlactonen uit witlofwortels. M.Sc. Thesis, Department of Food Technology, Agricultural University Wageningen.

Singh, R. and Bhatia, I.S., 1971a. Isolation and characterization of fructosyltransferase from chicory roots. Phytochemistry, 10: 495-502.

Singh, R. and Bhatia, I.S., 1971b. Substrate specificity of fructosyl transferase from chicory roots. Phytochemistry, 10: 2037-2039.

Spiro, R.G., 1966. Analysis of sugars found in glycoproteins. Methods Enzymol., 8: 3-26. Zittan, L., 1981. Enzymatic hydrolysis of inulin - an alternative way to fructose production. Staren/Stärke,

33: 373-377.


ACTIVITY OF INULINASE OF SOME STRAINS OF BIFIDOBACTERIUM AND THEIR EFFECTS ON THE CONSUMPTION OF FOODS CONTAINING INULIN OR OTHER FRUCTANS

PUDJONO*, G. BÄRWALD** and S. AMANU* IUC Biotechnology of the Gadjah Mada University, Yogyakarta, Indonesia Institute of Food Technology and Fermentation Technology, Technical University of Berlin, Seestrasse 13, 1000 Berlin 65, Germany

ABSTRACT

Commercial dahlia inulin and inulin extracted from Jerusalem artichoke were used as C-substrates in microbiological media employed as in-vitro test systems to simulate the intestinal tract. Selected strains from the intestinum, namely Bifidobacterium adolescentis, B. angulatum, B. bourn, B. breve, B. catenulatwn, B. globosum, B. infantis, B. pullorwn, B. sp. (subtile) and B. thermophilum were grown in these media. The main intestinal species, viz. B. adolescentis, showed the highest growth rate (μ = 0.337 h"1) in the dahlia inulin medium, at pH 6.8 and 37 °C under anaerobic conditions, whereas in the Jerusalem artichoke extract medium the growth rate was lower (μ = 0.273 h"1). Inulinase activity was found both in the cells and in the medium. The enzyme activity was maximal at pH 5.5 and 55 °C. It was stimulated by Mn2+ and was inhibited by Hg2+ and Mg2+. Besides inulinase activity, the crude enzyme displayed invertase activity.

1 INTRODUCTION

Bifidobacterium species are found in the faeces of human adults, in the human intestine and in that of some animals. Yazawa et al. (1978) reported about the utilization of oligo- and polysaccharides by B. infantis. In our experiments, growth in media containing pure dahlia inulin or Jerusalem artichoke tuber extracts showed no relationship with inulinase activity. Inulinases are produced by bacteria like Actinomyces longisporus (Ishibashi et aL, 1974), Arthrobacter ureafaciens (Uchiyama, 1975) and Lactobacillus plantarum (Takahasi and Soutome, 1975) and by yeasts like Candida kefyr (Negoro and Kito, 1973) and Kluyveromyces marxianus (Rouwenhorst et al., 1990) (for reviews see Vandamme and Derycke, 1983, and Fuchs et al., 1985). They are also produced by moulds like Aspergillus ficuum (Ettalibi and Baratti, 1987) and Penicillium purpurogenum (Odonera and Shiomi, 1988). According to Zittan (1981) Aspergillus inulinases show maximal activity

374

at pH 4.5 and 60 °C. In general, microbial inulinases have their pH optimum between 3.5 to 5.5, and their temperature optimum between 45 and 55 °C.

To study and simulate the digestion of fructans in the colon, microbiological tests were carried out in media containing either commercial dahlia inulin or partially modified inulin from Jerusalem artichoke, and inoculated with various Bifidobacterium species.

2 MATERIALS AND METHODS 2.1 Microorganisms

In this study some Bifidobacterium species were used from the collection of the DSM, Braunschweig: B. adolescentis (ATCC 15703), B. angulatum (ATCC 27535), B. bourn (ATCC 20432), B. breve (ATCC 15700), B. catenulatum (ATCC 27539), B. globosum (ATCC 25685), B. infantis (ATCC 15697), B. pullorum (ATCC 27685), B. sp. (subtile) (DSM 20209) and B. thermophilum (DSM 20209), and Saccharomyces cerevisiae (WET 136) from SIHA-Getränkeschutz, Langenlonsheim.

2.2 Chemicals Chemicals were obtained from Merck, Darmstadt, except enzymatic kits, which were

from Boehringer, Mannheim, and dahlia inulin from Sigma, Deisenhofen. Jerusalem artichoke (Helianthus tuberosus L.) inulin was extracted from tubers by pressing; the press juice was clarified and concentrated to 73% dry matter (Topina GmbH, Kassel), using the methods of Bärwald (1986).

2.3 Medium and cultivation The medium used to simulate the digestion in the colon contained the pressed and

concentrated Jerusalem artichoke juice mentioned before, in which the readily fermentable and metabolizable sugars (fructose, glucose and sucrose) were removed by fermentation with S. cerevisiae (Bärwald et al., 1989). The pH was adjusted to 6.8. After being inoculated with various Bifidobacterium species, the media were incubated anaerobically using a C02 atmosphere at 37 °C. For the preparation of the inoculum, cultures were grown for 24 h to a bacterial cell density of 106 ml"1 in a medium which contained 1 % dahlia inulin as the only carbohydrate.

The medium used for inulinase production was a modification of the usual Bifidobacterium medium (DSM, 1989), in which 1% glucose was replaced by 3% inulin. Precultivation took place in a 300-ml flask containing 50 ml of the medium at 37 °C for 3 days in an anaerobic (C02) atmosphere. Then, so much of the culture was used to inoculate a 2-1 fermenter containing 11 of the same medium as needed to obtain a initial concentration of 106 cells ml'1. The fermenter was then incubated at 37 °C, stirred at 200 rpm, and

375

anaerobic conditions were established by passing through 0.2 ml N2 min". The pH was adjusted and controlled at 6.8 with 5 N NaOH.

2.4 Preparation of inulinase from B. adolescentis Extracellular inulinase. During steady state cultivation, 11 of each of the fermented

media was collected, and the bacteria were separated off by centrifugation and washed with 0.9% NaCl. The culture fluid was concentrated to 47 ml, with a Filtron minisette stainless steel holder; concentration was followed by removal of contaminating low-molecular-weight proteins using a Filtron minisette ultrafiltration membrane, 0.16 μπι (30 kDa cut off). The concentrated preparation was used for the determination of inulinase activity.

Intracellular inulinase. The release of cell-wall associated inulinase was induced by autolysis of the cells with 1 M phosphate buffer (pH 8.0), pancreatine and toluene at 40 °C for various times (0, 4, 18 and 24 h). The suspensions were used for the determination of inulinase activity.

2.5 Assay of enzyme activity Inulinase activity. Inulinase was measured by determining the free sugars released.

The reaction mixture contained 2.0 ml of 2.5% inulin in 0.1 M phosphate buffer (pH 5.5) and 0.5 ml enzyme solution. The mixture was incubated at 55 °C in a water bath for 30 min. The free sugars were determined using enzymatic methods and TLC with 1-propanol/ethyl acetate/H20 (7:1:2, v/v/v) as the eluant. One unit (U) of inulinase activity was defined as 1 ^mole of fructose produced per min at 55 °C and pH 5.5.

Invertase activity. The ability of the enzyme preparation to hydrolyse sucrose was measured by determining the release of free sugars as described above. Two ml of a 1 % sucrose solution was used as substrate. One unit (U) of invertase activity was defined as 1 μπιοΐε of sucrose hydrolysed per min at 55 °C and pH 5.5.

2.6 Effect ofpH on enzyme activity The enzyme solution was incubated at various pH's at 55 °C for 30 min, and then

the activity was determined as described under Assay of enzyme activity. The effect of the pH on enzyme activity was investigated using acetate, phosphate and borate buffers in the range between 4.0 and 10.0 (Fig. 1).

2.7 The effect of temperature on enzyme activity The enzyme was incubated at various temperatures in 0.1 M phosphate buffer (pH

5.5) for 30 min. Then, the enzyme activity was determined as described under Assay of enzyme activity (Fig. 2).

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2.8 The effects of trace elements on enzyme activity Trace elements can influence enzyme activity. Therefore, the effects of some metal

ions were tested. To this end, to the reaction mixture consisting of 2.0 ml of 2% inulin and 0.5 ml enzyme solution in 0.1 M phosphate buffer (pH 5.5), 2.5 ml of CaCl2, FeCl3, HgCl2, MgCl2, MnCl2 or ZnCl2 was added to a final concentration of 1 mM. The reaction mixtures were incubated at 55 °C for 30 min. Then, the enzyme activity was determined as described under Assay of enzyme activity.

3 RESULTS AND DISCUSSION The various Bifidobacterium strains could be grown both in the media with Jerusalem

artichoke inulin and in those with dahlia inulin as C-substrate. As shown in Tables 1 and 2, B. adolescentis is the intestinal Bifidobacterium species, that showed a high growth rate in the dahlia inulin medium (μ = 0.337 h"1) as well as in the Jerusalem artichoke extract medium (μ = 0.273 h"1). Both Jerusalem artichoke and dahlia inulin were the substrates for model fermentations with typical bifidobacteria isolated from the human digestive tract: B. adolescentis, B. breve, B. catenulatum and B. infantis. The fermentation conditions are comparable with those prevailing in the human digestive tract. Thus, the experimental set-up can be considered as an in-vitro model, as already used in previous experiments (Bärwald et al., 1989). The lower growth rate of the bacteria in the Jerusalem artichoke juice (Table 2) as compared with that in the medium containing dahlia inulin as C-substrate (Table 1) may be due to the mineral content of the Jerusalem artichoke juice which might have resulted in some inhibition of the inulinase and therefore in a lower utilization of the

R E L A T I V E

A C T I V I T Y

4 ό 8 10 12 PH

BUFFER : Acetate —·— Phoiphat· Alkaline Borate

100

80

00

40

30

-

1 I

30 80 40 50 TEMPERATURE ( C)

Fig. 1. (left) The effect of pH on inulinase activity. Fig. 2. (right) The effect of temperature on inulinase activity.

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Table 1. Growth rate (μ) of some Bifidobacterium strains in a medium with 1% dahlia inulin as C-source.

Strain μ (h_1)

B. adolescentis 0.337 B. angulatwn 0.315 B. bourn 0.083 B. breve 0.180 B. catenulatum 0.137 B. globosum 0.337 5. infantis 0.219 B. pullorum 0.180 5. */?. fridtfifej 0.209 B. thermophilum 0.344

Table 2. Growth rate (μ) of selected Bifidobacterium strains cultivated in Jerusalem artichoke extract.

Strain μ (h1)

5. adolescentis 0.273 5. Z?reve 0.219 £. catenulatum 0.104 5. i/i/fl/iiw 0.245

Table 3. Effects of metal ions (as chloride salts) on inulinase activity.

Metal ions (1 mM) Remaining activity (%)

None 100 Ca2+ 99 Fe3+ 98 Hg2+ 3 Mg2+ 31 Mn2+ 114 Zn2+ 90

fructan. This assumption is substantiated by the inhibitory effect on the inulinase activity of various heavy metal ions, especially by Hg, and to a lesser extent by Mg; Mn, on the other hand, enhanced the enzyme activity (Table 3).

As shown in Figs. 1 and 2, the pH and temperature optima of the enzyme activity are at 5.5 and 55 °C, respectively. Release of enzyme from the cell wall by autolysis with 1 M phosphate buffer (pH 8.0) resulted in a maximal enzyme activity after 4 h (Table 4), however, after a longer autolysis time the activity decreased again. The apparent denaturation of the enzyme might be due to the phosphate concentration used, the relatively

378

Table 4. Effect of autolysis time on inulinase activity in 1 M phosphate (pH 8.0), at 40 °C.

Time (h) Inulinase activity (U) Invertase activity (U)

0 228 105 4 350 162

18 161 156 24 130 143

Table 5. Enzyme activities in a 1-1 fermentation with B. adolescentis and 3% dahlia inulin as C-source; fermentation time: 9 days.

Intracellular Extracellular, concentrated media

Total crude protein (mg) 1336 913 Inulinase activity (U)a 350 978 Specific inulinase activity (U mg"1 protein) 168 50 Invertase activity (U) 162 494 Specific invertase activity (U mg"1 protein) 162 25 Ratio inulinase/invertase 1 2

a Per 0.5 ml of enzyme solution; total inulinase activity in total crude protein was 224,000 U.

high pH and/or the use of - proteolytic - pancreatine in the release of cell-wall associated enzyme.

Of the inulinase activities in the fermentation fluid 17% was extracellular and 83 intracellular (and thus cell-bound). As shown in Table 5 the intracellular enzyme activities per mg protein were higher than those of the extracellular enzymes in the concentrated media (47 ml; > 30 kDa).

4 CONCLUSION Inulinase can be produced from B. adolescentis with inulin as C-substrate. Addition

of a trace of Mn to the common Bifidobacterium media was found to enhance enzyme production.

5 REFERENCES

Bärwald, G., 1986. Process for the preparation of a low-glucose digestion product from inulin-containing parts of plants. EP 0.201.676; US Patent 4.758.515; Can. Patent 1.274.483.

Bärwald, G., Amanu, S. and Pudjono, 1989. In-vitro-Test in der Magen-Darm-Passage von Helianthus iw^ro^MJ'-Kohlenhydraten. Erfahrungsheilkunde, 38: 298-304.

DSM, 1989. Catalogue of strains 1989, Braunschweig. Ettalibi, M. and Baratti, J.C., 1987. Purification, properties and comparison of invertase, exoinulinases and

endoinulinases of Aspergillus ficuwn. Appl. Microbiol. Biotechnol., 26: 13-20. Fuchs, A., De Bruijn, J.M. and Niedeveld, C.J., 1985. Bacteria and yeasts as possible candidates for the

production of inulinases and levanases. Antonie van Leeuwenhoek, 51: 333-343. Ishibashi, K., Onisi, M. and Amao, S.K., 1974. Cited from: Vandamme, E.J. and Derycke, D.G., 1983.

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Microbial inulinases: fermentation process, properties, and applications. Adv. Appl. Microbiol., 29: 139-176.

Negoro, H. and Kito, E., 1973. Purification and enzymatic properties of intracellular 0-fructofuranosidase from Candida kefyr. J. Ferment. Technol., 51: 103-110.

Onodera, S. and Shiomi, N., 1988. Purification and substrate specificity of endo-type inulinase from Penicillium purpurogenum. Agric. Biol. Chem., 52: 2569-2576.

Rouwenhorst, R.J., Hensing, M., Verbakel, J., Scheffers, W.A. and Van Dijken, J.P., 1990. Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol., 56: 3337-3345.

Takahasi, M. and Soutome, S., 1975. Cited from: Vandamme, E.J. and Derycke, D.G., 1983. Microbial inulinases: fermentation process, properties, and applications. Adv. Appl. Microbiol., 29: 139-176.

Uchiyama, T., 1975. Action of Arthrobacter ureafaciens inulinase II on several oligofructans and bacterial levans. Biochim. Biophys. Acta, 397: 153-163.


Yazawa, K., Imai, K. and Tamura, Z., 1978. Oligosaccharides and polysaccharides specifically utilizable by Bifidobacteria. Chem. Pharm. Bull., 26: 3306-3311.

Zittan, L., 1981. Enzymatic hydrolysis of inulin - an alternative way to fructose production. Starch/Stärke, 33: 373-377.



INULIN FERMENTATION IN GERM-FREE RATS ASSOCIATED WITH A HUMAN INTESTINAL FLORA FROM METHANE OR NON-METHANE PRODUCERS

C. ANDRIEUX, S. LORY, C. DUFOUR-LESCOAT, R. de BAYNAST and O. SZYLIT LEPSD, unito MBS, INRA, 78350, Jouy en Josas, France ARD, 27-29 rue Chateaubriand, 78383, Paris, France

ABSTRACT

Germ-free rats were inoculated with a human intestinal flora from either non-methane or methane producers. They were fed on a diet containing either 10% sucrose or 10% inulin. Gas excretion was measured in a respiratory chamber and other bacterial metabolites were determined in the caecum. In rats receiving inulin, inulin fermentation led to an increased production of hydrogen, lactic acid and butyric acid in both non-methane-producing and methane-producing rats. Furthermore, a decrease in methane production and an increase in total short-chain fatty acid production were observed in the methane-producing group. Inulin fermentation tended to convert methane-producing rats into non-methane producers.

1 INTRODUCTION Inulin does not seem to be digested by the endogenous enzymes in the gastrointestinal

tract, but is well fermented by the bacterial microflora in the hindgut (Nilsson and Björck, 1988). The nature of the fermentation products might depend on the origin of the microflora since 30 to 50% of western adult human subjects produce methane and hydrogen, whereas 50 to 70% only produce hydrogen, as shown by breath-test studies (Bond et al., 1971). To check whether the effect of inulin ingestion was similar in methane and non-methane producers, we studied inulin fermentation in initially germ-free rats associated with a human flora from either a non-methane producer or a methane producer. Gas production was measured in a respiratory chamber and caecal concentration of organic acids was estimated in the caecal contents of killed rats.

2 MATERIALS AND METHODS 2.1 Diet

Rats were fed on a "human-like" diet containing 46% cooked potatoes, 23% fish

382

meal, 10% lard, 5% cellulose, 4% maize oil, 2% vitamin and mineral mixture and either 10% sucrose (control diet) or 10% inulin extracted from chicory and provided by ARD (inulin diet). Diets were sterilized by irradiation at 40 kGy in vacuum-sealed plastic bags.

2.2 Animals Sixteen 3-month-old male germ-free rats of the Fischer strain 344 were used. They

were randomly divided into two groups of eight rats each. One group was fed on the control diet and the second group on the inulin diet. Faeces from a human non-methane producer were diluted in an anaerobic chamber and inoculated into four rats fed on control diet and into four rats fed on inulin diet. Faeces from a human methane producer were diluted and inoculated into the other rats. The heteroxenic rats received the diets for three weeks before hydrogen and methane production was measured in a respiratory chamber (Le Coz et al., 1989). Rats were then killed in fed state, the caecum was weighed and pH, short-chain fatty acids and lactic acids were measured in the caecal contents.

2.3 Analysis of fermentation products Hydrogen and methane were analysed using a Quintron apparatus (DP-Quintron

Instrument). Short-chain fatty acids (SCFA) were analysed using gas-liquid chromatography, whereas L- and D-lactic acids were determined enzymatically with lactate dehydrogenases (Boehringer-Mannheim).

2.4 Statistical analysis Results were expressed by mean and standard deviation (SD). Means were compared

by variance analysis and the Newman-Keuls test using STAT ITCF software (P < 0.05).

3 RESULTS 3.1 Gas production

In rats fed on the control diet, hydrogen production was similar whatever the bacterial status but methane production appeared only in rats associated with a methanogenic flora (Table 1). In the non-methane-producing rats, inulin ingestion strongly increased hydrogen production. In the methane producers, hydrogen production was increased whereas methane production was depressed.

3.2 Caecal metabolites In both methane- and non-methane-producing rats, caecal weight significantly

increased from 1.4 to 2.3 g per 100 g of body weight whereas caecal pH decreased from 6.4 to 5.4 by inulin intake.

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Table 1. Gas production (μηιοΐ per 10 g of food intake and per 100 g of body weight).

Hydrogen Methane

Non-methane producers

Control Inulin

2.2 ± 1.6 120a ± 22

Methane producers

Control

1.8 ± 0.8 10.6 ± 8.0

Inulin

40ab ± 26 0.8a ± 0.8

a Significantly different from control group. b Significantly different from non-methane producers.

Table 2. SCFA and lactic acids concentration (/xmol g"1 of caecal content).

Total SCFA L- and D-lactic acids

Non-methane producers

Control

69 ± 11 2.0 ± 0.6

Inulin

64 ± 16 5.7b ± 0.9

Methane producers

Control

37a ± 4 3.6 ± 0.6

Inulin

65 ± 5 6.0b ± 0.9

a Significantly different from other groups. b Significantly different from control group.

When rats were fed on the control diet, caecal SCFA concentration was 50% lower and lactic acids 80% higher in methane producers than in non-methane producers (Table 2).

Inulin intake did not modify caecal SCFA concentration in non-methane producers, but it led to a two-fold SCFA concentration in the caecum of methane producers. In the two groups of rats, the proportion of butyric acid increased from 14 to 30% whereas that of other SCFA was lowered. Lactic acid concentration increased two-fold in inulin-fed rats and the L/D ratio was reduced from 3:2 to 1:1 in non-methane producers and remained 1:1 in methane producers.

4 DISCUSSION The human microfloras kept their fermentative properties when inoculated into germ-

free rats fed on control diet. Bacterial floras from human methane producers only gave rise to methane production, whereas hydrogen production was similar in both methane and non-methane-producing rats. Furthermore, caecal SCFA concentrations were lower in methane-producing rats than in non-methane producers as was found in human faeces (Weaver et al., 1989) but the lactic acid concentration was higher as compared with that in non-methane-producing rats.

Some effects of inulin addition to the diet were similar in methane- and non-methane-

384

producing rats: decrease of caecal pH, increase of D- and L-lactic acid concentration, modification of SCFA profile and considerable increase of hydrogen production. In addition, some other specific effects of inulin were observed. In non-methane producers, the L/D lactic acids ratio decreased to reach the value obtained with methane producers. In methane producers, on the other hand, the caecal SCFA concentration increased to reach the value of non-methane producers and methane production was significantly reduced. The decrease in methane production might be related to the lowered caecal pH (Bryant, 1979). These results led us to conclude that inulin tends to convert methane-producing rats into non-methane producers and suggest that inulin might be able to reduce methane production in human methane producers.

5 REFERENCES

Bond, J.H., Engel, R.R. and Levitt, M.D., 1971. Factors influencing pulmonary methane excretion in man. An indirect method of studying the in situ metabolism of the methane-producing colonic bacteria. J. Exp. Med., 133: 572-588.

Bryant, M.P., 1979. Microbial methane production - theoretical aspects. J. Anim. Sei., 48: 193-201. Le Coz, Y., Morel, M.T., Bousseboua, H., Dufour, C. and Szylit, O., 1989. Mise au point d'une chambre

respiratoire connected sur isolateur pour la mesure in vivo des gaz de fermentation chez Γ animal gnotoxonique. Sei. Tech. Anim. Lab., 14: 35-59.

Nilsson, U. and Björck, I., 1988. Availability of cereal fructans and inulin in the rat intestinal tract. J. Nutr., 118: 1482-1486.

Weaver, G.A., Krause, J.A., Miller, T.L. and Wolin, M.J., 1989. Constancy of glucose and starch fermentations by two different human faecal microbial communities. Gut, 30: 19-25.


THE USE OF JERUSALEM ARTICHOKE FLOUR IN PIG AND CHICKEN DIETS

E.R. FARNWORTH*, J.D. JONES**, H.W. MODLER** and N. CAVE* Animal Research Centre, Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6 Food Research Centre, Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6

ABSTRACT

A cooked spray-dried flour produced from the tuber of Jerusalem artichoke, which contained 77.5% soluble carbohydrate (33.3% GF^, 46.4% GF3_4 and 20.3% GF^5) was incorporated into weaner pig diets and chick starter diets. Weaned pigs (15 pens of three pigs/diet group) were fed either a control diet, one containing 1.5% Jerusalem artichoke flour, or one containing 1.5% purified neosugars. There were no significant differences in the feed intake, body weight gain, or feed efficiency between groups. However, there was a very pronounced difference in the smell of the manure from the pigs, depending on the diet they received.

The chicks (six pens of eight birds/diet group) received either a control diet, or one containing 1%, 5% or 10% Jerusalem artichoke flour, or purified neosugars. The birds receiving the diets with either the Jerusalem artichoke flour or the neosugars had increased feed consumption and body weight gain, but poorer feed efficiency.

The incorporation of Jerusalem artichoke flour in the diets of pigs or chicken may have benefits in production efficiency, as well as barn environment improvement.

1 INTRODUCTION The addition of fructo-oligosaccharides to the diet of monogastric animals has been

shown to bring about several metabolic/physiologic changes that have been attributed to a change in the make-up of the intestinal microflora population. Changes in the intestinal microflora of domestic animals might in turn lead to improvements in feed efficiency, reduced diarrhoea and reduction of smell in faeces. Such changes would have obvious benefits to domestic animal producers.

Recently, a process was patented (U.S. Patent # 4.871.574) that describes how the inulin in tubers of Jerusalem artichoke (Helianthus tuberosus L.) can be hydrolysed to short-chain fructo-oligosaccharides.

It was the purpose of this study to determine the effects of adding Jerusalem artichoke tuber flour rich in fructo-oligosaccharides to swine and poultry rations.

386

Table 1. Composition of Jerusalem artichoke flour.

Constituent % Composition (% of dry matter)

Nitrogen* 2.1 Insoluble fibrea 16.2 Asha 4.2 Soluble carbohydrate 77.5

Constituents5

GF!.2 33.3 GF3_4 46.4 GF^5 20.3

a Determined using AOAC methods. b Fructose polymers (fructans) consisting of a terminal glucose and 1, 2, 3, 4, 5, or > 5 fructose units.

2 MATERIALS AND METHODS 2.1 Jerusalem artichoke flour

Jerusalem artichoke flour was obtained by a patented process (U.S. Patent # 4.871.574). The process involves macerating the tubers, heating the macerate to 66 °C, and then spray-drying the mixture. The resulting flour has a composition as described in Table 1. The free-flowing flour was then incorporated into either the swine or the chick rations.

2.2 Swine experiment Swine from the Animal Research Centre SPF herd were used in the pig trials.

Groups of three (two of one sex) were then assigned randomly to one of the weaning diets, a control diet, one containing 1.5% Jerusalem artichoke flour (+JA), or one containing 1.5% purified neosugars (+NS). The three pigs were housed together in raised flat-deck cages. This was repeated until 15 groups had been placed on test. Pigs were weighed initially and then weekly for 4 weeks. Feed consumption per pen of three pigs was measured weekly. Daily, pigs were checked for signs of diarrhoea; any diarrhoea was treated. At the end of 4 weeks, faecal samples were taken from pigs and evaluated by a panel of judges. Samples were rated on a scale of 1 (strongest smell), 2 or 3 (least smell). This was repeated for five groups of pigs. Panelists were also asked to describe the smell of the faeces.

2.3 Poultry experiment One-day-old meat strain chicks from the Animal Research Centre poultry facilities

were group-housed in brooder cages (13/pen) and fed a standard starter ration ad libitum until age 7-days. Birds were then weighed, allocated to a diet and housed in groups of 8 in

387

Petersime battery cages. The experimental diets were either a control diet, or one containing 1 %, 5% or 10% JA flour, or 1 %, 5% or 10% NS. Each diet group contained three groups of females and three groups of males. Birds consumed the experimental diets for 2 weeks. Total body weight and total feed consumption was obtained on a per pen basis.

3 RESULTS AND DISCUSSION The data in Table 2 show that the feed intake in the first week of the experiment of

the +JA groups was greater than the others, but there were no significant differences attributed to diet, either in the first week or over the whole experiment.

The growth of the +JA groups tended to lag behind the others (Table 3), but again, there were no significant diet effects. There was large variability in the feed efficiency data in the first week of the experiment, but neither in the first week nor over the whole 4-week trial were feed efficiencies different between the three diet groups (Table 4).

There were no differences in the incidence of diarrhoea between the three diet groups. The faeces of the +NS and -f JA groups tended to be a different colour from those of the control group. When the faecal samples were evaluated by the panel, both the +NS and the +JA samples scored higher on average than the control (1.5 for control, 2.4 for

Table 2. Feed intake (kg) of weaned pigs (per pen of three pigs).

Diet na 0-7 days 0-28 days

Control 15 4.89 ± 1.66b 55.56 ± 9.89 + NS 15 4.83 ± 1.79 55.78 ± 7.76 + JA 15 5.09 ± 1.96 53.67 ± 9.48 Effect of diet n.s.c n.s. a 15 repetitions of three pigs per repetition. b Mean ± standard deviation. c n.s., not significant (P > 0.05).

Table 3. Weaned pig body weight gains (kg).

Diet na 0-7 days 0-28 days

Control 45 0.62 ± 0.52b 11.01 ± 1.93 + NS 45 0.66 ± 0.52 11.18 ± 1.79 -I- JA 45 0.53 ± 0.64 10.54c+ 1.81 Effect of diet n.s.d n.s. a 15 repetitions of three pigs per repetition. b Mean ± standard deviation. c 44 pigs. d n.s., not significant (P > 0.05).

388

Table 4. Feed efficiency8 of weaned pigs.

Diet 0-7 days 0-28 days

Control + NS + JA Effect of diet

15 15 15

0.35 + 0.21b

0.38 ± 0.18 0.23 + 0.05 n.s.c

0.60 ± 0.04 0.61 ± 0.06 0.59 ± 0.05 n.s.

a Total pen weight gain (three pigs)/total feed consumed per pen. b Mean + standard deviation. c n.s., not significant (P > 0.05).

Table 5. Chicken feeding trial results.

Initial body weight (g) Final body weight (g)a

Weight gain/bird/day (g)b

Feed consumed/bird/day (g)

Feed/gainc

Control

120 483

25.4 41.2

1.63

JA

1%

122 493

26.3 42.9

1.63

5%

125 520

27.0 44.6

1.65

10%

119 491

25.6 45.2

1.77

NS

1%

122 505

27.3 43.8

1.60

5%

120 496

26.3 44.5

1.70

10%

123 496

26.3 46.8

1.78

a At end of 21 days. b Mean total pen weight gain (14 days)/mean bird days. c Per bird per day.

+JA and 2.1 for +NS). These scores were not significantly different, however, due to a large week-to-week variation. Panelists did indicate that they could smell a difference, but when asked what the samples smelled like, there was no clear pattern of descriptor words.

Adding either Jerusalem artichoke flour or neosugars to the chick diet resulted in increased (but not significantly) body weight gains (Table 5). These diets also increased the amount of feed consumed per bird per day, and as a result, the feed to gain ratios generally were increased compared with those in the control birds. Both feed consumption and feed to gain ratio were significantly affected by diet (P < 0.05 and P < 0.01, respectively). The feed was fed to the birds as a crumble, as it was found that when fed as a mash, stickiness of the feed hampered proper feeding of the birds. Mortalities of birds during the feeding trial were not attributable to diet.

CONCLUSIONS

This was a first attempt at feeding Jerusalem artichoke flour to pigs and chickens.

389

It is evident that both species accepted the additive. At a level of 1.5% in the diet, changes were evident in the smell of pig faeces. Higher levels of inclusion may produce more significant results. When added to chicken diets, weight gain and feed consumption increased. In neither the pig nor the chicken experiment were the diets formulated to be isocaloric with the control diet. Therefore, the effects of the added Jerusalem artichoke may have been reduced.



THE USE OF INULIN FOR THE DETERMINATION OF RENAL FUNCTION: APPLICABILITY AND PROBLEMS

N. GRETZ*, M. KIRSCHFINK** and M. STRAUCH* Clinic of Nephrology, University of Heidelberg, Klinikum Mannheim, 6800 Mannheim, Germany Department of Immunology and Serology, University of Heidelberg, 6900 Heidelberg, Germany

ABSTRACT

® Inulin, though difficult to handle, is the ideal marker of renal function. With Inutest an inulin preparation has been made available, which is easy to handle. As distinct from usually applied inulin preparations Inutest does not cause complement activation. In this paper the different methods to determine renal function will be discussed. Furthermore, a short survey of the methods used in the chemical analysis of inulin will be given.

1 INTRODUCTION In 1935, Shannon pointed out that inulin, a starch-like polymer of fructose, can be

employed to measure renal function. During the following years it was shown that inulin is filtered freely, exhibits no plasma binding, and does not result in toxic side-effects or allergic adverse reactions. Furthermore, inulin is neither secreted nor reabsorbed by the tubules. Thus, inulin is the ideal substance to quantify glomerular filtration rate (GFR). Tubular function, however, can not be assessed by this approach. Despite of the fact that inulin is the ideal marker for GFR, its use decreased over the subsequent years for several reasons: inulin is insoluble and therefore difficult to prepare for injection; the conventional procedure of performing GFR measurements by using inulin is tedious; and finally, the chemical determination of inulin is cumbersome and fairly imprecise.

As an alternative to inulin, methods using radioactive isotopes were introduced. It is, however, not quite clear whether all isotopes are really only filtered and not additionally secreted or reabsorbed by the tubuli (Mulhern and Perrone, 1990). Furthermore, use of radio-isotopes may not always be permitted. The latter problem might be fairly important in the near future, when the corresponding laws of the different member states of the

392

European Community will be adjusted and the use of radioactive isotopes restricted. Besides, patients are more and more unwilling to be exposed to any unnecessary radiation.

The aim of this paper is to present a survey on the applicability of inulin for the measurement of renal function in humans and to demonstrate that to-day the problems mentioned above have virtually been overcome.

2 PREPARATIONS OF INULIN AS USED FOR INJECTION Inulin as used normally is insoluble and has to be heated to be solubilized. This

sometimes results in heat decomposition of inulin. Furthermore, if the inulin is solubilized incompletely, blood vessels may be obstructed, especially in the lung. It should be noted that immunologists use inulin, though in ten times higher concentrations than used in humans, for the in vitro activation of the complement system. This activation, however, is probably due to a different inulin configuration or due to impurities, as activation does not

® ®

occur with Inutest . The fructan Inutest (sinistrin), which has a molecular weight of about 3500 and thus a degree of polymerization of about 21 (range: 12-28), is available as an aqueous solution, ready for injection. Thus, it does not suffer from the disadvantage of having to be heated. Furthermore, even with high plasma concentrations of Inutest no adverse reactions due to a complement activation are observed, as has been shown by us

® recently. In 43 patients we injected 5 g of Inutest as a bolus in order to perform a GFR determination. Blood was sampled before, and 15 and 60 min after injection. No changes in the complement concentration could be detected with time indicating that no activation had occurred (Fig. 1).

C1 C3b C3d

8 12 120 10 = 1 0 0 — — -

80-i — i — = — = — —i—■—i— EBBE ;^ f f S i 0 15 60 u o 15 60 0 15 60

time (min) time (min) time (min)

Fig. 1. Results of a time-dependent GFR measurement in 43 patients injected with Inutest showing that no activation of the different components (Cl, C3b and C3d) of the complement system occurred, as there is no decrease of the concentrations with time (mg dl"1). No difference between the three times of sampling could be detected by ANOVA analysis of variance (P > 0.05).

393

3 METHODS OF PERFORMING A GFR MEASUREMENT In principle, there are two methods to perform a GFR measurement, either by

continuous infusion of inulin or by bolus injection (also called single-shot method).

3.1 Continuous infusion of inulin This approach is regarded as the gold standard of GFR measurement. The GFR is

defined as the ratio between the total amount (mg) of inulin excreted by the kidney per unit of time (min) and the plasma concentration (mg ml"1) of inulin. GFR is thus expressed as ml min"1. Continuous infusion of inulin implies that a steady state plasma concentration can be achieved. If due to "over"- or "under"-infusion the plasma concentration changes (Fig. 2) during the clearance period, the data concerned should not be included in the calculations. Before starting GFR measurement the patient has to be hydrated by giving water or tea orally. During the clearance periods the urinary output should be at least 500 ml h"1. After the hydration a bolus injection of 1.5 g m"2 inulin is given followed by a constant infusion of 10 mg m"2 min"1 in order to attain a steady state plasma concentration of inulin for the whole clearance period. This is the most essential and also the most difficult part of the procedure, especially in patients having renal failure, since then inulin excretion is reduced and thus the inulin concentration rises, or in diabetic or hypertensive patients exhibiting hyperfiltration, as a result of which the inulin concentration decreases (Fig. 2). The blood and urine sampling should not be started before 90 to 120 min after injection in order to obtain a steady state in the plasma. Thereafter, three to four times blood and urine samples (30 to 40-min collecting periods) are obtained and the GFR is calculated: GFR (ml min"1) = Cinulinurine * Vurine / Cinulinplasma (C: concentration [mg ml"1]; V: volume per time [ml min"1]). The calculation is done for each of the collecting periods. Then, the mean value of these calculated GFRs is taken. In its original form the procedure is fairly inconvenient as patients have to be catheterized. Nowadays, one tries to overcome

Inulin concentration (mg/ml)

1.4-j 1.2-j

11 0.8i 0.61 0.41 0.2J

0 30 60 90 120 180 240 time (min)

Fig. 2. Diagram showing the effects of "over"- and "under"-infusion of inulin with increasing and decreasing inulin concentrations. Such a finding invalidates the clearance measurement.

394

this problem by checking the emptying of the bladder via ultrasound. However, there is still the serious problem that, if small urine volumes are passed, inulin is left in the bladder.

3.2 Single-shot inulin clearance In this type of clearance the disappearance of inulin from the circulation is

determined by frequent blood sampling (Fig. 3). The clearance is then calculated as the area under the curve as exemplified in Fig. 3. Usually a two-compartment model is used, one (0 to 90 min) determined by the rapid distribution of inulin in the vascular and extracellular space, and the other (90 to 300 min) by the renal excretion of inulin resulting in a linear decrease of the concentration. Usually, the patient is given 5 g of inulin, then blood is sampled at 0, 30, 60, 120, 180, 240, and 300 min. With this method, exact timing of the sampling is not necessary, but an exact documentation of data is mandatory so that these can later be included in the regression analysis (Gretz et al., 1989). The decline of the inulin concentration in one person exhibiting a normal GFR is depicted in Fig. 3. In patients with poor renal function the linear decay is much slower. Therefore, it is recommended to sample blood also after 5 hours. Especially in patients with a minute decrease in the inulin concentration with time, an adequate method for determining inulin is imperative.

Unlike patients with poor renal function, diabetic or hypertensive patients exhibiting hyperfiltration have a much more rapid decline of the inulin concentration. Therefore, a fairly frequent sampling is necessary so that also this decline can be assessed correctly. Furthermore, methods for the chemical determination of inulin are needed, which are not interfered with by plasma glucose levels. Usually, there are no other problems in performing such a clearance.

H

N 2 3

I - 2 - :

δ 1 N ί

0 1 2 3 4 5 6

TIME (HOURS)

Fig. 3. The plasma concentration of inulin during a single-shot clearance in a normal person.

395

Any clearance is standardized to a body surface of 1.73 m2. With both methods, however, erroneous results are obtained if there is a flux of inulin into the third space, e.g. ascites or edema, since then redistribution takes place after a certain period of time.

4 METHODS FOR THE CHEMICAL DETERMINATION OF INULIN Another major obstacle for a more frequent use of an inulin clearance is its

cumbersome chemical determination. In addition, the precision of these determinations is often not as high as is needed. This is especially troublesome with the single-shot clearance in patients with a slow decrease in inulin concentration where minute differences have to be detected, and also in patients showing a very rapid decline of inulin concentration so that it comes close to the background measured.

Currently, various methods are available for chemical determination of inulin: resorcin (Roe et al., 1949), anthrone (Führ et al., 1955), thiobarbiturate (Zender and Falbriard, 1966), cysteine-tryptophane (Waugh, 1977), and ß-fructosidase (Kühnle et al., 1993). Micromethods are available for the anthrone method (0.01 ml), the cysteine-tryptophane method (0.01 ml), and the ß-fructosidase method (0.05 ml). A word of caution is due here with regard to substances, like lipids or bilirubin, which might interfere with the photometric determination; besides, there is the usual problem with high plasma glucose levels, which has been solved by the ß-fructosidase method.

Recently, Kühnle et al. (1993) described a modified ß-fructosidase method allowing a more precise determination of inulin. In a first step, the glucose is oxidized by glucose oxidase and H202 as both the glucose oxidation and the enzymatic hydrolysis of inulin can be run at an identical pH and in the same buffer solution. This has also the advantage that the glucose moiety of the inulin after hydrolysis is also oxidized and does not disturb any of the subsequent determinations. In the next step, fructose is converted via fructose-6-phosphate into glucose-6-phosphate, which is then determined as glucose by adding NADP. This is probably the most sensitive method currently available.

5 CONCLUSIONS At present, a sufficient number of approaches exist which simplify GFR

determination with inulin. Furthermore, a more precise chemical determination of inulin is available allowing the use of the so-called single-shot clearance, which is more convenient

® than an infusion clearance. As Inutest is an easy-to-handle inulin preparation no further development in this area is needed. With respect to the chemical determination of inulin, however, a still more convenient method is required.

396

6 REFERENCES

Führ, J., Kaczmarczyk, J. and Kriittgen, C.-D., 1955. Eine einfache colorimetrische Methode zur Inulinbestimmung für Nieren-Clearance-Untersuchungen bei Stoffwechselgesunden und Diabetikern. Klin. Wochenschr., 33: 729-730.

Gretz, N., Kühnle, H.F. and Strauch, M., 1989. A SAS procedure for evaluating inulin clearance data. Proc. SEUGr89, 9-12 May 1989, Cologne, pp. 564-573.

Kühnle, H.F., Linzmeier, P. and Doerge, L., 1993. Determination of glomerular filtration rate in rats. In: N. Gretz and M. Strauch (Eds.), Experimental and Genetic Rat Models of Chronic Renal Failure. Karger, Basel, pp. 331-336.

Mulhern, J.G. and Perrone, R.D., 1990. Accurate measurement of glomerular filtration rate. In: V.E. Andreucci and L.G. Fine (Eds.), International Yearbook of Nephrology. Kluwer Academic Publishers, Boston, pp. 277-290.

Roe, J.H., Epstein, J.H. and Goldstein, N.P., 1949. A photometric method for the determination of inulin in plasma and urine. J. Biol. Chem., 178: 839-845.

Shannon, J.A., 1935. The excretion of inulin by the dog. Am. J. Physiol., 112: 405-413. Waugh, W.H., 1977. Photometry of inulin and polyfructosan by use of a cysteine/tryptophan

reaction. Clin. Chem., 23: 639-645. Zender, R. and Falbriard, A., 1966. Analyse colorim&rique des c&o-hexoses et de I'inuline par

la reaction de l'acide thiobarbiturique. Clin. Chim. Acta, 13: 246-250.

MISCELLANEOUS



SOME ASPECTS OF RESEARCH ON INULIN AND INULIN-CONTAINING CROPS IN THE UKRAINE

G. LEZENKO*, L. BOBROVNIK*, I. GRINENKO*, R. GRUSHETSKY*, I. GULY*, J. TSOKUR** and O. VDOVENKO***

Kiev Technological Institute of Food Industry, Kiev, Ukraine Farm "Ioisk", Odessa region, Ukraine Ukrainian Agricultural Academy, Kiev, Ukraine

ABSTRACT

Some aspects of research carried out in the Ukraine on the composition of Jerusalem artichoke tubers, and on the isolation, purification and properties of inulin are presented in the form of three extended abstracts.

1 THE INFLUENCE OF CLIMATIC FACTORS AND GROWING CONDITIONS ON THE CARBOHYDRATE COMPOSITION OF JERUSALEM ARTICHOKE TUBERS (J. Tsokur, O. Vdovenko and G. Lezenko) Inulin is one of the most valuable components of Jerusalem artichoke. So far,

however, the dependence of inulin content on environmental factors, like climatic and soil conditions, and quality and quantity of fertilizers used, is poorly understood. Therefore, the objective of our investigations was to examine the influence of different climatic and agrochemical factors on carbohydrate accumulation in Jerusalem artichoke tubers. For this purpose, we compared and analysed both available literature data and the results of our own research on the chemical composition of Jerusalem artichoke, grown in different regions of the Ukraine and in climatic regions markedly differing from them.

Carbohydrate accumulation in tubers proved to depend on the cultivar used. There are particular groups of cultivars, mainly selected for special local climatic conditions, which yield maximal quantities of inulin only under these conditions. For example, cv. Interes, which is well-known for its high yields of inulin and therefore widespread in the former USSR, yields not more than 15% carbohydrates (on a fwt basis) in the western part of the Ukraine, where local cultivars accumulate more than 23 %.

398

Carbohydrate accumulation also appeared to depend on soil fertility and fertilizer usage. Thus, P-fertilizers enhanced carbohydrate content. Local cultivars seemed to be better adapted to special local soils; in other areas, even on better soils, they showed a lower carbohydrate content. For instance, local West-Ukrainian cultivars grown on dried peat contained more inulin in their tubers than when grown on the black earth of central Ukraine.

The inulin content of the tubers proved to increase from North to South. For instance, cv. Dietic Pink, which has been selected in the Central Botanical Garden of the Ukraine, contained only 7-8% carbohydrates in the Northern regions of Russia (Ural), however, 12-18% in central Ukraine, and even up to 19-20% in Armenia and in Usbekistan. It seems justified to assume that there is some relationship between the inulin content and the dose of ultraviolet radiation received by the plant: in South-East Asia and in the mountains of Armenia and in the Carpathians the inulin content was nearly identical.

2 SOME PHYSICOCHEMICAL CHARACTERISTICS OF JERUSALEM ARTICHOKE INULIN (G. Lezenko, L. Bobrovnik, R. Grushetsky and I. Grinenko) In the processing of Jerusalem artichoke tubers for the isolation of inulin various

problems are commonly encountered. For instance, drying of tubers and heating of aqueous extracts or press juices may cause conversion of inulin to fructose. Boiling of juice may lead to formation of precipitates which may interfere with fractionation. Clarification of juices or extracts using lead salts is impossible if the inulin has to be used in foodstuffs or medicines. In view of these problems, a - relatively inexpensive - method of isolation of inulin from Jerusalem artichoke has been worked out. The inulin prepared by our method is of a purity fully suitable for food or medicinal uses. Because of its lack of toxicity, it may be applied intravenously.

In our studies, different modifications of inulin were obtained, and their conformation and physicochemical behaviour were studied, because of the known dependence of biological activity on the molecular configuration and properties of inulin. The behaviour of inulin was examined in various solvents and their mixtures. Investigations on swelling of inulin in generally used solvents enabled us to select the best solvents, such as ethanol, dioxane, and acetone, and also to establish the optimal solvent/extract ratio to precipitate inulin from aqueous extracts. In addition, measurements were made on the viscosity of inulin solutions, and on its ability to absorb and desorb water depending on the environmental humidity.

Packing of fructofuranose residues was experimentally investigated by estimating the isotopic H/D and D/H interchange from steam and liquid phase. A complete D/H and H/D interchange was found only for inulin in the -form. Both - and -forms apparently have

399

a globular shape. Furthermore, the interaction of inulin with cations of some metals, including

strontium, was studied, in view of the potential removal of metal ions, especially radionuclides, from the human body. In this respect, it was of special interest that in vitro a given quantity of inulin bound 2.5-3 times more strontium than other elements under otherwise identical experimental conditions. Currently, research work is in progress on the synthesis and biological activity of inulin derivatives.

3 SOME METHODS OF ACIDIC AND NON-ACIDIC, COMPLETE AND PARTIAL HYDROLYSIS OF INULIN (R. Grushetsky, G. Lezenko, L. Bobrovnik, I. Grinenko and I. Guly) The demands of the food industry and medicine for fructose exceed the output and

possibilities of its production by currently used methods. Hydrolysis of natural fructans is one of the alternative methods of fructose production. With prior purification of these fructans, it is possible to obtain fructose of high purity and quality. Acid hydrolysis at elevated temperatures as a rule is accompanied with formation of considerable amounts of by-products, arising by oxidation, breakdown, and condensation, and all leading to the formation of coloured multifunctional compounds.

Therefore, we studied the possibility of inulin hydrolysis at reduced temperatures, namely in cooled and frozen solutions of hydrochloric acid. As a result, even at ambient (room) temperature the rate of formation of coloured compounds proved to be significantly reduced in comparison with the rate of hydrolysis, colour formation being virtually absent in frozen solutions. Liquid chromatography showed fructan oligomers (with degrees of polymerization of 2 to 6) to be formed as intermediates, with fructose as the final product.

In a study on the reaction kinetics it was established that this heterogeneous reaction was even possible at sub-zero temperatures. The order of reaction - 0.6 - was apparently due to an intensified reversion process at increasing fructose concentrations. In accordance with the kinetic properties of monomolecular reactions the reaction rate at sub-zero temperatures in frozen suspension was reduced significantly. However, near 0 °C because of solvent crystallization the reaction mixture became more concentrated, resulting in increased reaction rates. Hence, the rates of hydrolysis at 4-10 and -5 °C were practically equal.

A significant acceleration of inulin hydrolysis was reached using another heterogeneous reaction, namely hydrolysis as a continuous process on columns packed with cation exchangers in H+-form. The cation exchangers served here as heterogeneous catalysts and as adsorbents at the same time. As a result, after additional purification fructose syrups of high purity and quality were obtained which could be concentrated by

400

freeze drying. At present, other methods of inulin hydrolysis at low temperature are being worked

out using ultrasound and electrochemical methods.

Full papers of these communications have unfortunately never been received. However, some interesting aspects of the research carried out by these Ukrainian workers, in my opinion, made it worth-while to publish the extended abstracts in their original form, even though some data had to remain unexplained. The Editor.

401

INDEX

Page numbers followed by a + sign always refer to the first page of a paper. They are used for items given in the title or mentioned on 4 or more pages of the paper concerned.

a value ([A:cat/A:m]I/[A:cat/^m]S), 214 a-amylase, 349, 360 α-cyclodextrin (= cyclomaltohexaose), 145 a-glucosidase, 71, 72, 220 (ß-2,1-)glycosidic linkages - as "substrate", 231+ (ß-2,1 -)glycosidic linkages - hydrolysis, 231+ ß-2,1-linked fructan/fructo- oligosaccharides, 65, 168,

202, 203, 348 ß-2,1-linked fructose polymers, 65, 205, 224, 252, 297,

359 ß-D-fructo(furano)sidase (see: invertase) ß-fructosidase method (for determination of inulin),

395 1-FFT, 168 1-kestose, 65, 93+, 107+, 121+, 129, 130, 133, 147,

168, 173+, 191+, 281, 311, 312, 348, 349 -accumulation/synthesis, 178, 182 - assignment, 130, 132, 133 - chemical shifts, 132, 133 - coupling constants, 124, 132, 133 - crystal structure, 124 - extraction, 107+ - molecular structure, 130 - NMR spectrum, 124, 132 -NOE data, 124 - purification, 107+ - structural analysis, 129 1D-NMR (spectra/spectroscopy), 129+ lF-(ß-)fructofuranosylnystose (see: fructosylnystose) 2,1-linked cyclofructo-oligosaccharide, 144 - 13C-NMR spectrum, 144 2,5-bis-(hydroxymethyl)furan, 153 2,5-furandicarboxaldehyde, 153 (2,5-)furandicarboxylic acid, 149+ 2-(4-methyl-2,6-dinitroanilino)-^V-methyl

propionamide (L-MDMP) - as an inducer of fructan accumulation, 182

2D-NMR (spectra/spectroscopy), 129+ 5-hydroxymethylfurfural (see:

hydroxymethylfurfural [dehyde]) 6-G FT, 169 6-kestose, 94, 97, 107+, 173+, 196, 311, 312, 348 -accumulation/synthesis, 178, 182 - extraction, 107+ - purification, 107+ acetate - as fermentation product, 268, 271 acetone-butanol - as biofuel, 211 acid phosphatase, 242

Actinomyces longisporus, 373 Actinomyces viscosus, 278 activated carbon - as immobilization matrix, 223+ active charcoal chromatography, 143 acyl donor, 162, 163, 166 Adenophora liliiflora, 317, 318 Adenophora spp., 317 adhesives (from HMF-based resins), 156 adipic acid, 138 adipic acid anhydride, 136, 137 adipose tissue, 361 adsorption - as immobilization method, 224, 227, 228 adsorption/cross-linkage - as immobilization method,

223+ Aerobacter levanicum, 202 affinity chromatography, 176, 200, 201 African rice (see: Oryza glaberrima) Agavaceae, 311, 316 Agropyron sp. - leaf extract, 170 Agrostis avenacea Gmel., 108 alcohol - as fermentation product, 367 Allium ampeloprasum (= great-headed garlic, kurrat,

leek), 316 Allium cepa L. (= onion, shallot, tree onion), 107+,

174, 181, 182, 316, 348 -bulbs, 108,110, 112, 173+ - seeds, 173+ Allium chinense (= jao, rakkyo), 316 Allium fistulosum (= Welsh onion), 315, 316 Allium sativum (= garlic), 316, 348 Allium schoenoprasum (= chives), 315, 316 Allium spp., 315, 319 Allium tuberosum (= Chinese chive[s]), 316, 348 American wild rice (see: Zizania aquatica) amyloglucosidase, 220, 360 amylolysis, 360 Andropogoneae, 313, 314 anion/cation/ion exchange (chromatography/resin), 70,

71, 93+, 103, 108, 116, 121, 168, 175, 178, 200-202, 218-220, 232, 233, 246, 291, 292, 297, 323+, 328-330, 342, 344, 345, 349, 356, 357, 399

annual ryegrass (see: Lolium rigidum Gaud.) Anthemideae, 317 antibacterial compounds, 151 apolipoproteins - in serum, 350, 352 Arctium lappa (= edible burdock), 318, 348 Artemisia lactiflora (=juun jiu choi), 318 Arthrobacter ureafaciens, 373

402

ascites, 395 asparagus (see: Asparagus officinalis) Asparagus officinalis L. (= asparagus), 107, 168,169,

315,316,348 -roots, 168,315 Asparagus racemosus, 316 Aspergillus ficuum, 68, 211+, 373 Aspergillus nidulans, 293, 294 Aspergillus niger, 144, 349, 352 Aspergillus sp., 224 - inulinase, 373 asphodel (see: Asphodelus aestivus) Asphodelus aestivus (= asphodel), 315, 316 Asteraceae, 311, 317, 318 Asterales, 167, 173, 311 autoradiographic detection (of 14C-labelled

carbohydrates), 193, 195 Avena abyssinica (= oat), 313 Avena byzantina (= red oat), 313 Avena sativa (= oat), 313, 348 Aveneae, 313, 314 Bacillus circulans, 143, 146 Bacillus licheniformis, 282, 285 Bacillus macerans, 146 Bacillus subtilis, 278, 282, 285, 290 - levanase gene, 289+ - levansucrase, 259+ - subtilisin, 162, 163 bacteria, human intestinal, 350-352, 355 Bacteroides distasonis, 351 Bacteroides fragilis, 351 Bacteroides melaninogenicus, 351 Bacteroides ovatus, 351 Bacteroides thetaiotaomicron, 351 Bacteroides vulgatus, 351 bakery/confectionery industry, 335 bakery/confectionery products, 335, 337, 338, 341,

342 bamboo (see also: Bambusa beecheyana,

Dendrocalamus asper, Gigantochloa verticillata and Phyllostachys dulcis), 309+

Bambusa beecheyana (= bamboo), 313 Bambuseae, 313, 314 banana, 348 Barbary nut (see: Gyandriris sisyrinchium) barley (see: Hordeum vulgäre L.) barnyard millet (see: Echinochloa crus-galli) basic chemicals (industrial), 149+ beverages, bitter sweet fructose syrup-containing, 368 bifidobacteria, 66, 67, 350, 352 - effect on bowel movement, 355 - effect on intestinal immunity, 355 - in the colon of man, 309 bifidobacteria, intestinal, 355 Bifidobacterium adolescentis, 351, 373+

Bifidobacterium angulatum, 374, 377 Bifidobacterium bifidum, 350, 351 Bifidobacterium bourn, 374, 377 Bifidobacterium breve, 351, 374, 376, 377 Bifidobacterium catenulatum, 374, 376, 377 Bifidobacterium globosum, 374, 377 Bifidobacterium infantis, 351, 373+ Bifidobacterium longum, 351 Bifidobacterium pullorum, 374, 377 Bifidobacterium sp. (subtile), 374, 377 Bifidobacterium spp., 350, 373+ Bifidobacterium thermophilum, 374, 377 bifidogenic factor, 57, 63 bifurcose, 348 binders (from HMF-based resins), 156 bioenergy, 86 biofuel,211 biomass, 31, 32, 86, 118, 149, 150, 158,193, 275, 276 biomass crop, 21 black salsify (see: Scorzonera hispanica) blood sampling - for inulin measurement, 393, 394 body weight gain, 387, 388 bogbean (see: Menyanthes trifoliata) - rhizome as "bread" for eskimos, 317 Boraginaceae, 310, 311, 318 Brachiaria deflexa, 313 bread wheat (see: Triticum aestivum) breath-test studies, 381 Bromeae, 314 Brunoniaceae, 310, 311 buckbean (see: Menyanthes trifoliata) bulrush millet (see: Pennisetum cinereum and P.

nigritarum) butyl acetate - as acyl donor in transesterification of

HMF, 162, 163 butyric acid, 383 C-3 cereals, 185 Calyceraceae, 311 Calycerales, 311 camas (see: Camassia leichtlinii and C. quamash) Camassia leichtlinii (= camas), 315 Camassia quamash (= camas), 315 Camassia spp. (= camas), 316 Campanula rapunculus (= rampion), 317, 318 Campanulaceae, 311, 318, 341 Campanulales, 311 Candida kefyr, 373 Candida salmanticensis, 251+ canned fruits - for diabetics, 334 canned fruits - with Jerusalem artichoke (tuber juice)

concentrate, 334, 335 canning industry, 335, 337 carbodiimide - as used in enzyme immobilization,

223+ carbohydrate metabolism - effect of metal ions, 102

403

carbohydrate metabolism - effect of titanium, 105 carbon-celite chromatography, 107 cardoon (see: Cynara cardunculus) Caryophyllales, 311 catabolite repression, 270 cellulase, 367, 368 cereals/cereal crops, 171, 312, 314 CFT-ase (see: cycloinulo-oligosaccharide

fructanotransferase) chemical shifts, 130 CHI (see: cycloheximide) chicken - weight gain, 389 chicken/poultry, 386, 388, 389 chicory (see: Cichorium intybus L.) Chinese chive(s) (see: Allium tuberosum) Chinese water chestnut (see: Eleocharis dulcis) chives (see: Allium schoenoprasum) cholesterol - estimation, 360 cholesterol - reduction in serum, 351 cholesterol level, in blood, 324 cholesterol, HDL- - in serum, 350, 352 cholesterol, LDL- - in serum, 351 cholesterol, total - in serum, 350, 352 Chrysanthemum moriflorum (= ju hua), 318 Chrysanthemum spatiosum (= tong hao), 318 Cichorium endivia (= endive), 318 Cichorium intybus L. (= chicory), 29+, 191+, 200,

202, 203, 206, 208, 224, 252, 282, 289, 297, 298, 317-319, 341, 359, 360, 382

- biomass distribution, 35 - biomass production/yield, 33, 35, 37 - breeding, 29, 37 -chicon,29, 191, 196 - crop characteristics, 29+ - dry matter distribution, 29+ - dry matter production/yield, 29+ - (effect of) nutrient supply, 31, 36 - effect of plant density, 35, 37 - effect of row width, 30 - effect of sowing date, 30, 33, 35 - effect of sowing density, 33, 35 - (effect of) water supply (natural, artificial), 31, 36 - heads - as vegetable, 367 - inulin content (of stems and roots), 30 - inulin production/yield, 29+ - leaf (area) expansion/development, 31, 35 - leaf area (index), 29+ - light interception, 29+ - light use efficiency, 33, 35 - plant growth, 191+ -productivity, 30, 31 - root(s), 29+, 65+, 77+, 135, 150, 191+, 205+, 224,

274, 357, 359+,367+ - - as a raw material, 150 - - as sink, 191

- - as source, 191 - - bitter constituents, 368 - - cortex, 205+ - - effect on cholesterol in liver/plasma of rats, 361-

363 - - effect on hypotriglyceridemia, 363 - - effect on lipid composition/profile in liver/plasma

of rats, 361-363 - - effect on total lipids in liver/plasma of rats, 361-363 - - effect on triglycerides in liver/plasma of rats, 359+ - - (enzymatic) liquefaction, 367+ - - forcing in hydroponic system, 191 - - (hot water) extraction, 367, 368, 371 - -maturation, 191 - - potential medicinal and nutritional uses, 359+ - - sesquiterpene lactones, 367, 372 - - sink-source transition, 191, 192, 196 - - vascular bundles, 205+ - - yield, 29+ - root extracts, 224 - - fresh weight, 193 - root fructan (content), 193 - (root) inulin, 121, 135+, 199+, 338, 341 - - chemical modification, 135+ - - potential medicinal and nutritional uses, 359+ -root raw juice, 77+ - use in Indian medicine, 359 - valorization of inulin-rich by-product, 367+ - yield formation, 30 Cirsium oleraceum (= meadow cabbage), 318 Claytonia perfoliata (= winter purslane), 318 Clethraceae, 311 Clostridium butyricum, 351 Clostridium clostridiforme, 351 Clostridium difficile, 350, 351 Clostridium paraputrificum, 351 Clostridium perfringens, 350, 351, 355 Clostridium ramosum, 351 Clostridium spp., 67 Clostridium thermoautotrophicum, 267+ - batch culture, 269 - continuous culture, 269, 271 - continuous culture, inulin-limited, 269, 270 Clostridium thermosuccinogenes, 267+ - batch culture, 269 - continuous culture, 269, 271 - continuous culture, inulin-limited, 269 coating materials (from HMF-based resins), 156 coffee additive, 29, 359 coffee substitute, 29, 359 Coix lachryma-jobi (= Job's tears), 313 colon, human, 309, 350, 374 comfrey (see: Symphytum officinale) common millet (see: Panicum miliaceum) common purslane (see: Portulaca oleracea)

404

complement system - activation by inulin, 392 complement system - components, 392 Compositae, 224, 252, 297, 298, 308, 319, 341 configurational disorder, 123 conformational energy map, 122, 123 conformational wheel, 122, 123 consumer goods, 135 Cordyline terminalis (= palm lily), 316 corn, 52, 53 corn cobs - as a raw material for furfural, 154 corn flag (see: Gladiolus italicus) cosmetics, 271 covalent binding/linkage - as immobilization method,

224, 226 crested wheatgrass, 179 (crop growth) model, 30, 36,45 cross-linking agent, 223+ cryoprotection, 120 crystallography, 122 Cyanastraceae, 310, 311 cycloamylose, 125 cyclodextrin glucanotransferase, 146 cycloheximide (= CHI), 177, 178, 182 cycloinulin, 125, 126 cycloinulo-octaose, 125, 145 cycloinulo-oligosaccharide fructanotransferase (=

CFT-ase), 143+ - coupling (by intermolecular transfructosylation), 147 - cyclization, 146 - disproportionation, 147 - hydrolysis, 147 cycloinulo-oligosaccharides, 143, 147, 282 - enzymatic synthesis, 143+ - structure, 143+ cycloinuloheptaose, 125, 145-147 cycloinulohexaose, 125, 126,143+ - 18-crown-6 skeleton, 145 - crystal structure, 121, 125, 144 - ORTEP drawing, 145 cyclopentenone derivatives - as industrial end-products

from HMF, 155 Cynara cardunculus (= cardoon), 318 Cynara scolymus L. (= [globe] artichoke), 281, 318 Cynareae, 317 Cynodonteae, 314 Cynoglossum officinale, 318 Cyperaceae, 311,315, 316 Cyperales, 311 Cyperus esculentus (= tiger nut), 315, 316 Cyperus papyrus (= papyrus), 316 cytosol, 179 D-fructohexaose anhydride, 144 D-lactic acid, 382-384 Dactylis glomerata L., 187 - fructan (exo)hydrolase, 203

dahlia (see: Dahlia variabilis) Dahlia variabilis (= dahlia), 29, 30, 211, 252, 268,

317, 341 - tuber(s), 30, 357 - (tuber) inulin, 102, 103, 121, 218, 220, 263, 338, 341,

373+ dairy industry, 336 dandelion (see: Taraxacum officinale Weber) data acquisition, 234 data-processing, 232, 234, 238 Dendrocalamus asper (= bamboo), 313 dexamethasone - effect on lipid metabolism in rats,

359+ di-D-fructofuranose dianhydride III, 143 diabetes - contra-indication for use of inulin to

measure GFR, 393, 394 diarrhoea (in man), 359 diarrhoea (in pigs/swine), 63, 385-387 dicotyledons, 252, 311, 317, 318 Diels-Alder products (HMF-based), 157 diet, chicken, 385+ diet, human, 57, 309, 310, 319, 324 diet, monogastric animals, 385 diet, pig, 385+ diet, rat, 360, 362, 363, 381-383 dietary fibre, 66, 67, 309 differential refractometer index (= DRI) detection,

231+ differential refractometry, 192 difructose anhydride I producing enzyme, 143 difructose anhydride II - molecular structure, 143 difructose anhydride III - molecular structure, 143 difructose anhydride III hydrolysing enzyme, 143 difructose anhydride IV producing enzyme, 143 digestive tract, human, 376 Digitaria exilis (= hungry rice), 313 Digitaria iburua, 313 distilling industry, 324 Dracaena australis, 316 DRI (see: differential refractometer index) durra (see: Sorghum bicolof) durum wheat (see: Triticum durum) dyes - as industrial end-products, 149 EC 2.4.1.9 (see: inulosucrase) EC 2.4.1.10 (see: levansucrase) EC 2.4.1.93 (see: inulin fructotransferase

[depolymerizing]) EC 2.4.1.99 (see: sucrose:sucrose fructosyltransferase

[SST]) EC 2.4.1.100 (see: fructan:fructan fructosyltransferase

[FFT]) EC 3.2.1.7 (see: inulinase [= 2,1-ß-D-fructan

fructanohydrolase]) EC 3.2.1.26 (see: invertase [= ß-D-fructofuranosidase]) EC 3.2.1.65 (see: levanase)

405

EC 3.2.1.80 (see: fructan [exo]hydrolase, F[E]H) Echinochloa crus-galli (= barnyard millet), 313 Echinochloa frumentacea (= Japanese millet), 313 Echinochloa utilis, 312 edema, 395 edible burdock (see: Arctium lappa) eelgrass (see: Zostera marina) efficient temperature (definition), 48 Ehrharteae, 314 einkorn (see: Triticum monococcum) elecampane (see: Inula helenium) Eleocharis dulcis (= Chinese water chestnut), 315, 316 Eleusine coracana (= finger millet), 313 emergency food, 317 emmer (see: Triticum dicoccum) end-products (industrial), 149+ endive (see: Cichorium endivia) endo-inulinase, 65+, 102, 144, 212-214, 218-220, 231,

234, 370 - amino acid composition, 213 - catalytic constant (kcat), 213, 214 - catalytic power (kcat/Km), 214 - sedimentation coefficient, 213 endoplasmic reticulum, 242 Enterococcus faecalis, 351 Enterococcus faecium, 351 enzyme immobilization/immobilized enzymes, 224 Eragrostideae, 313, 314 Eragrostis tef(= teff), 313 Ericales, 311 Erysiphe cichoracearum (see: Helianthus tuberosus L.

- pathogens) Escherichia coli, 260, 282, 284, 285, 289+, 350, 351,

355 ethanol - as biofuel, 211 ethanol - as fermentation product, 52, 55, 56, 251+,

268, 271, 281, 372 ethanol - as fuel additive, 85+ ethanol - effect on lipid metabolism in rats, 359+ ethanol blends, 89,90 - for use as gasoline extender, 90 - for use as octane enhancer, 90 ethanol feedstock, 87, 89, 90 ethnobotany, 310, 319 Eubacterium aerofaciens, 351 Eubacterium lentum, 351 Eubacterium limosum, 351 exo-inulinase, 66, 68, 72, 102, 212-214, 218-220,

231+, 276, 278, 332, 370 - activity, 237, 238 - amino acid composition, 213 - catalytic constant (itcat), 213, 214 - catalytic power (kcat/Km), 214 - TV-terminal amino acid sequence, 213 - sedimentation coefficient, 213

exo-inulinase, yeast, 277 exolevanase, 279 expression vector, 290 externalization (of secretory proteins), 242 extracellular space, 394 faeces - smell, 385+ faeces, human, 373, 382, 383 faeces, pig, 387 fast-performance liquid chromatography (= FPLC),

175, 180, 181, 212 fatty acids, volatile, 350 fatty liver, 362 FD-MS analysis, 144 (feed) additive, 389 feed consumption/intake, 385+ feed crop, 52 feed efficiency, 385, 387, 388 feed to (weight) gain ratio, 388 feed(stuff)s, 347, 352, 357, 359+, 388 FEH (see: fructan [exo]hydrolase) fermentation (general), 374 - as industrial process, 289 fermentation pattern, 271, 272 fermentation products (general), 272 Festuca arundinacea Schreb., 118 feterita (see: Sorghum bicolor) FF-inulo-oligosaccharide, 66, 71 FFT (see: fructan:fructan fructosyltransferase) FH (see: fructan [exo]hydrolase) Fibruline, 274, 310 finger millet (see: Eleusine coracana) fluorescein-labeled antibodies, 242 fodder, 313 (food) additive, 135, 310, 337 food crop, 52 food industry, 63, 150, 205, 211, 251, 273, 281, 338,

399 food plants, 309+ food plants, fructan-containing, 311 food texture, 281 foods, fructan-containing, 310 foods, fructan/inulin-containing - consumption,

373+ food(stuff)(s), 77, 80, 82, 135, 151, 271, 273, 310, 319,

323+, 341,347, 352,356, 398 formate - as fermentation product, 268, 271 FOS (see: fructo-oligosaccharides), 355 foundry products (from HMF-based resins), 156 foxtail millet (see: Setaria italica) FPD (see: freezing-point depression) FPLC (see: fast-performance liquid chromatography) freezing-point depression (= FPD), 115+ Fritillaria camschatcensis (= riceroot), 315, 316 Fritillaria lanceolata (= riceroot), 315, 316 frost tolerance, 281

406

fructan(s), 93, 97, 107+, 115+, 167, 169, 170, 173+, 185+, 191+, 199+, 217, 218, 229, 241, 249, 250, 251+, 263, 281, 282, 287, 289, 290, 297+, 309+, 356, 377, 386, 392, 399

- accumulation/(bio)synthesis, 53, 107, 115,118, 167+,173+,185+, 191+, 259, 298

- as a raw material, 289 - chain elongation, 174, 181, 304 - colligative properties, 115+ -definition, 310-312 - degradation, 55, 143, 168, 189, 242, 299 - depolymerization, 191+, 298, 308 - digestion, 374 - enzymatic methods for quantitative analysis, 101+ -extraction, 52, 116 - hydrolysis, 108, 170, 191, 196, 199, 217, 218, 229,

242, 252, 290, 399 - metabolism, 107, 167+, 183, 185, 187, 189, 266, 298 - - vacuolar localization, 298 - metabolism by fermentation, 309 - non-digestibility, 309 - occurrence, 309+ - physics and chemistry, 121 - polymerization, 191+, 298, 308 - production, 187 -purification, 116 - sources, 107+ - synthesis in microvesicles, 171 - vacuolar localization, 207 fructan(s), branched (with both ß-2,1- and ß-2,6-

linkages), 202 fructan(s), cyclic, 147 fructan-metabolizing enzymes, 168 - purification, 168 fructan (exo)hydrolase (F[E]H, EC 3.2.1.80), 108, 112,

168, 199+, 207, 209, 298, 306, 307, 371 -activity, 301,306 - extraction, 200 - Michaelis-Menten kinetics, 203 - purification, 200, 201 - vacuolar localization, 207 (fructan) oligomers (see also: [fructan]

oligosaccharides), 93+, 116,118, 120, 312, 399 - separation, 93+ (fructan) oligosaccharides, 93+, 109,112, 115+, 123,

193, 200 (fructan) pentasaccharide, 110, 112, 177, 193 (fructan) polymers, 253 (fructan) tetrasaccharide, 110, 112, 177, 193, 312 (fructan) trisaccharide, 94, 107+, 168, 173+, 188, 189,

193,196,203,264,311,312 - synthesis, 168-170, 195 fructan:fructan fructosyltransferase (FFT, EC

2.4.1.100), 107, 112,168, 169, 174, 181, 182, 196, 266, 297+

- relationship between fructan chain length and enzyme affinity, 307, 308

fructofuranose - anomeric effects, 125 fructofuranose - conformations, 121+ fructofuranose - molecular models, 121+ fructofuranose - NMR, 121 fructofuranose ring - envelope (E) conformations/

forms/shapes, 122, 123 fructofuranose ring - northern conformations/shapes,

124, 125 fructofuranose ring - pseudorotation, 122, 123 fructofuranose ring - puckering, 122, 123, 125 fructofuranose ring - - amplitude, 123 fructofuranose ring - - phase angle, 123 fructofuranose ring - southern conformations/shapes,

124, 125 fructofuranose ring - twist (T) conformations/forms/

shapes, 122, 123, 145 fructo-oligomers (see: [fructo-]oligosaccharides) fructo-oligosaccharide-rich fructose syrup, 356 - production, 355+ (fructo-)oligosaccharides (= FOS), 53, 55, 57+, 65-67,

108, 109, 144,146, 147, 281, 282, 347+, 355, 385 - as an ingredient of milk-replacers, 63 - characteristics, 347+ - chemical structure, 347 - effect on blood pressure, 355 - effect on constipation, 352, 355 - effect on human health, 347, 352, 355 - effect on intestinal toxins, 355 - effect on serum lipids, 350, 352, 355 - enzymatic preparation, 349 - hydrolysis, 62 - metabolism by fermentation, 66 - non-digestibility, 349, 352 - production, 65, 347+ - selective utilization by intestinal bacteria, 350-352 - synthesis, 65 fructo-oligosaccharides, cyclic, 143,144 fructo-pentasaccharide, 108 fructose enrichment (through fermentation), 251+ fructose oligomers - applications, 282 fructose polymers - applications, 282 fructose syrup (see also: high- and very-high-fructose

syrup), 72, 205, 251, 254, 257, 273, 289, 334, 336, 356, 360, 399

- bitter, sweet syrup from chicory, 368 fructosyl acceptor, 168, 174, 196, 259+, 282, 287 fructosyl donor, 168, 174, 181, 196, 263, 264, 281,

282, 287 fructosyl intermediate, 260 fructosyl transfer, 167+, 173+, 193, 196 fructosyl-fructose linkage, 311 fructosylation, 65 fructosylnystose (= GF4), 65, 147, 270, 348, 349

407

fructosylnystose (continued) - structural analysis, 129 fructosylraffinose, 94, 97 fructosylstachyose, 94, 97 fructosylsucrose, 187, 281 fructosyltransferase (activity) (general), microbial, 65,

263, 266, 281+, 349, 352 fructosyltransferase (activity) (general), plant, 168,

169, 171, 173+, 195, 259, 266, 371 fructotransferases, 143 fruit juices, 334 y?/40 gene (product) (from Streptococcus mutatis),

281+ (fuel) additive, 85, 90 fuel ethanol, 85+ fungi (filamentous) - importance in biotechnology, 292 furan aramide, 156 furfural, 154, 158 furfurylalcohol, 154, 158 Fusarium avenaceum (see: Helianthus tuberosus L. -

pathogens) Fusarium culmorum (see: Helianthus tuberosus L. -

pathogens) Fusarium oxysporum, 360, 363 Fusarium sporotrichoides (see: Helianthus tuberosus

L. - pathogens) Fusobacterium varium, 351 (gallo)tannic acid - as cross-linking agent, 223+ garlic (see: Allium sativum) gas(-liquid) chromatography (= G[L]C), 52, 55, 94,

253,382 gas(-liquid) chromatography/mass spectrometry (=

G[L]C-MS), 144, 312 gasohol - as ethanol blend, 89, 90 gastrointestinal tract, human, 381 gau sun (see: Zizania caduciflord) GC (see: gas [-liquid] chromatography) GC/MS (see: gas [-liquid] chromatography/mass

spectrometry) gel electrophoresis, denaturing, 248 gel electrophoresis, native, 248 gel filtration/permeation (chromatography), 67, 72, 94,

109, 111, 112, 116, 144, 176, 180, 200, 212, 218, 247, 248, 283, 285

GF-inulo-oligosaccharide, 66, 71 GFR (see: glomerular filtration rate) Gigantochloa verticillata (= bamboo), 313 Gladiolus italicus (= corn flag), 315, 316 Gladiolus spp., 319 GLC (see: gas[-liquid] chromatography) GLC-MS (see: gas[-liquid] chromatography/mass

spectrometry) (globe) artichoke (see: Cynara scolymus L.) glomerular filtration rate (= GFR), 391, 394 - definition, 393

- determination/measurement, 391+ glucose isomerase, 360 glucose oxidase, 395 glucosyltransferase, 282 glutaraldehyde - as cross-linking agent, 223+ glycosylation (of proteins), 241+ golden thistle (see: Scolymus hispanicus) Golgi apparatus, 243 Goodeniaceae, 311 grain - as an agricultural feedstock, 86, 87 grain sorghum, 52, 53 Gramineae (= grasses), 108, 167+, 173,181, 185,187,

196, 309+, 348 - "high fructan" tribes, 314 - "low fructan" tribes, 314 Gramineae cell suspension cultures - fructan-

producing, 187 grasses (see: Gramineae) great-headed garlic (see: Allium ampeloprasum) growth pattern determinism, 46 guang cai (see: Gynura bicolor) Guinea corn (see: Sorghum bicolor) Gyandriris sisyrinchium (= Barbary nut), 315, 316 Gynura bicolor (= guang cai), 318 Haemodoraceae, 311 halophyte, 316 Heliantheae, 317 Helianthus annuus L. (= sunflower), 1, 39, 317, 325 - productivity, 39 Helianthus tuberosus L. (= Jerusalem artichoke,

topinambour), 1+, 11+, 21+, 29+, 39+, 45+, 51+, 57+, 86, 87, 90, 94, 102, 111, 167+, 196, 202, 203, 209, 211, 224, 229, 252, 281, 289, 297, 298, 308, 317-319, 323+, 341, 348, 356, 374, 385, 389, 397, 398

- accession, 2, 7, 8 - agronomic characteristics, 1+, 11+, 87 - (agronomic) performance, 15, 53 - (animal) feed, 51,57,63, 89 - application, 51+ - as an agricultural feedstock, 89, 90 - biomass, 36, 37, 52 - biomass distribution, 35 - biomass yield, 35, 37 - breeding, 29, 37, 42, 43, 52 - carbohydrate content/yield, 52, 53, 56 - carbon assimilation, 39, 40, 48 - compositional characteristics, 1+, 11+ - costs of production, 85+ - crop characteristics, 29+ - (crop) productivity, 30, 31, 39+, 45 - cultivar selection, 61 - cultivation, 51+, 324, 325 - developmental stage, 47, 48 - diseases, 25

408

Helianthus tuberosus L. (continued) - dry matter (content), 1+, 11+ - dry matter distribution, 31, 33, 34 - dry matter production/yield, 1+, 11, 16, 33-35, 41,

46, 47, 53 - effect of light saturation, 48 - effect of (long-term) storage of tubers, 57+ - effect of nitrogen nutrition/supply, 39+ - (effect of) nitrogen partitioning, 40, 43 - (effect of) nutrient supply/nutrition, 11+, 25, 31, 36,

38,45 - effect of photoperiodism, 48 - effect of plant density, 2, 11, 17, 37, 49, 52, 54 - (effect of )plant distance/row width/spacing, 12, 40,

52 - effect of planting date of tubers (autumn/spring), 21+ - (effect of) respiration (rate), 48, 60 - (effect of) soil pH, 25 - effect of soil type, 52, 53 - effect of temperature regime, 48 - (effect of) water supply (natural, artificial), 11+, 26,

31,36,45 - evapotranspiration, 11,12, 15 - flower induction, 29, 48 - flower initiation, 31, 34 - germplasm (variability), 2, 8 - growth characteristics, 11+ - herbage, 274, 275, 278 - inulin content (of stems and tubers), 30, 37 - inulin production/yield, 29+ - inulin-containing products, 341+ - - food use, 341+ - leaf (area) expansion/development, 31, 35 - leaf area (index) (= LAI), 11+, 30, 32, 35, 40, 41, 45,

47,48 - leaf area duration, 34 - leaf nitrogen (level/concentration), 39+ - light interception, 29+, 47, 48 - light (use) efficiency, 33, 35, 47, 48 - maturity (groups) (early, medium, late), 1+ - meristem culture, 22 - microplants, 22, 24 - micropropagation, 22, 26 - microtubers, 24 - minitubers, 24, 25 - pathogens, 26 - photosynthesis, 39+ - photosynthetic capacity/rate, 39+ - plant height, 3, 7, 8, 12,13 - potential feed and non-food uses, 26 - potential for the production of ethanol, 2 - potential for the production of foods, feed and fiber,

2 - powdery mildew, 25 - productivity modelling, 45+

-propagules, 21, 26 - raw material (industrial), 51 - reasons for non-competitiveness as a crop, 1 - relative sink effects, 49 - reserve (sugar) distribution, 48, 49 - seed tuber diseases, 25 - seed tuber size, 25, 26 - shoot dry matter (yield), 45 - shoot/top, 58, 88, 89 - shoot/top growth, 1, 11+, 45+ - shoot/top yield, 1, 13, 14,16, 52, 88, 89 - sink activity, 33, 34 - (sink-driven) assimilate balance/distribution, 45+ - stem (feed and fibre quality), 35 - stem as sink, 48 - tissue differentiation, 29 - to offset soil degradation problems, 87, 88 - tuber(s), 1, 2, 11+, 22, 25, 26, 29+, 40,42, 48, 53,

57+, 68, 72, 87, 88, 94, 95, 102, 105, 107, 115, 116, 167, 173+, 191,192, 195, 200, 207, 224, 232, 252, 253, 297, 298, 304, 323+, 342, 356, 368, 385, 397, 398

- - analysis, 52 - - application/utilization, 57, 63 - - as a raw material, 323+, 368 - - blanching, 62 - - carbohydrate analysis, 13 - - composition, 2, 13, 17, 18, 325 - - disease resistance, 2, 8 - - dormancy, 29 - - dried products, 337, 338 - - dry fibre, 338 - - dry matter (yield), 45-47 - - effect of climatic factors on carbohydrate

composition, 397 - - effect of fertilizers on carbohydrate composition,

397,398 - - effect of growing conditions on carbohydrate

composition, 397 --filling, 31,34,42 - - formation, 15, 16 - - frost tolerance, 23 - - fructo-oligosaccharide content, 57+ - - growth (rate), 33, 36,42, 46-48 - - harvesting, 325 - - histology/histogenesis, 13, 15 - - initiation, 15, 34, 37 - - (long-term) storage, 53, 56 - - process technology, 326, 327 - - processing, 51+, 57+, 324 - - production, 57 - - rots, 25 - - shape, 2, 17, 18, 325 - - sink activity/capacity, 42 - - storage quality, 2

409

Helianthus tuberosus L. - tuber(s) (continued) - - trisaccharide content, 17 - - use as/in feed and food (products), 51, 57, 63 - - yield, 1+, 11+, 21+, 29+, 39+, 49, 52, 54, 58, 59,

61,325,341 - (tuber) extract, 223+, 373+ - (tuber) extracts/(press/raw) juice - as fermentation

medium, 251+ - (tuber) flour, 58, 62, 63, 337, 338, 385+ - - as feed for/effect on poultry, 63, 385, 386 - - as feed for/effect on swine, 63, 385, 386 - (tuber) inulin, 129, 202, 231+, 326, 338, 341+, 374,

376 - - content, 17, 18, 398 - - food, and medical and diagnostic use, 341+ - - physicochemical characteristics, 398 - (tuber) juice, 323+, 342-344, 374, 376, 398 - - clarification, 327, 329, 333, 342 - - concentration, 330 - - effect of ion exchange resin, 329 - - extraction, 326, 328, 337, 357 - - microwave vacuum drying, 344 - - preparation, 342 - - purification, 326 - (tuber juice) concentrate, 323+, 344, 345 - - application, 334 - tuber macerate, 58, 62, 63, 386 - - measures to reduce browning, 62 - - spray-drying, 58, 62, 386 - (tuber) pulp, 337, 338, 357 - tuber raw juice, 53, 54 - - hydrolysis, 53 - - metabolism by fermentation, 52, 55, 56 - tuberization, 48 - use of tops, 16 - yield (formation), 30, 38, 40, 56 Hemerocallis fulva (= jin zhen cai), 316 hetero-oligomers, 65 HFCS (see: high-fructose corn syrup) high-fructose corn syrup (= HFCS), 252, 323 high-fructose product, 331 high-fructose syrup, 211, 256, 267, 271 -production, 251+ high-performance anion exchange (= HPAE)

chromatography, 71, 73, 74, 94, 95, 98 high-performance liquid chromatography (= HPLC),

53, 54, 56, 94, 106, 109, 111, 186, 192, 231+, 251+, 274, 276, 278, 312, 334, 356, 368, 369, 371

hindgut, human, 381 HMF (see: hydroxymethylfurfural[dehyde]) HMF-acetate, 163 HMF-stearate, 164, 166 - NMR spectra, 164,165 homo-oligomers, 65 homofermentative lactic acid bacteria, 273, 278

homology (between levanase and yeast invertase), 290 Hordeum spp., 108 Hordeum vulgäre L., 173+, 313, 314 - fructan (bio)synthesis in leaves, 173+ - fructan (exo)hydrolase, 203 - invertase, 179, 180, 203 - (primary) leaves, 169, 170, 173+, 207 - protoplast extracts, 182 - seedlings, 175, 176 - stem, 107+ host-parasite relationship, 315 hounds tongue (see: Cynoglossum officinale) HPAE (see: high-performance anion exchange

chromatography) HPLC (see: high-performance liquid chromatography) humic acids - as by-product of HMF production, 159 hungry rice (see: Digitaria exilis) hydrogen (also: H2) - as fermentation product, 271,

381+ hydrophobic interaction chromatography, 176 hydroxymethylfurfural(dehyde) (= HMF), 149+, 161+,

281,367 - amination, 152 - chemical instability, 157 - decarbonylation, 154 -dehydration, 151 - Diels-Alder cycloaddition reactions, 154 - dimerization, 152 - esterification, 151 - esterification with long-chain fatty acids, 161 - etherification, 152 - Friedel-Crafts reactions, 152, 154 - functionalities, 151 - halogenation, 152, 154 - high reactivity, 157 -nitration, 154 - oxidation, 153, 159 - polymerization, 159 - reduction, 152, 153 - reductive amination, 153, 158 - self-condensation, 159 - sulphonation, 154 - transesterification, 161, 162, 166 hydroxymethylfurfural derivatives, 152 - in corrosion inhibitors, 153 - in shampoos, 153 hydroxymethylfurfural dimer, 151 hydroxymethylfurfural esters - enzymatic synthesis,

161+ hypercholesterolaemia, 351 hyperfiltration - contra-indication for use of inulin to

measure GFR, 393, 394 hyperlipoproteinaemia, 351 hypertension - contra-indication for use of inulin to

measure GFR, 393, 394

410

infusion clearance, 395 intermediates (industrial), 149+ intestinal putrefactive substances, 67 intestine, human (lower), 122, 351, 355, 373 intestine, human small, 309 intestine, large, 66,67, 352 Inula helenium (= elecampane), 317, 318 inulin(s), 29+, 53, 55, 57, 63, 65+, 77+, 93+, 101+,

121+, 129, 135+, 143+, 150, 158, 161, 181, 205+, 211+, 217+, 224, 226, 231+, 241, 242, 244, 252, 254, 255, 263, 267+, 273+, 281+, 289+, 297, 298, 307, 309, 317, 323+, 341-343, 359+, 367+, 374, 376, 378, 382-385, 391+, 397+

- 13C-NMR spectrum, 284, 287 - adsorption, 82 - adsorption isotherm, 82, 83 - appearance, 80 - application, 129, 135, 282 - as a filling agent, 135 - as a raw material, 150,158, 267, 273, 281, 289, 294,

324 - as a thickener, 135 - behaviour of aqueous solutions like Newtonian

liquids, 82 - behaviour of aqueous suspensions, 82 - behaviour of inulin vs. corn starch suspensions, 82 - bolus injection (see also: single-shot method), 393 - characterization, 232 - chemical modification/derivatization (chemical),

135+,398 - clearance, 395 - composition, 80 - conformations, 121 - continuous infusion, 393 - conversion, 239 - (conversion by) fermentation/as fermentation

substrate, 211, 254, 273, 376, 378, 381+ - crystallization, 78, 79 - degradation, 53, 68, 129,146, 231+, 242, 273+, 342 - derivatization/esterification with (mixed acetic/adipic

acid anhydride), 135+ - derivatization/esterification with Na-

trimetaphosphate, 135+ - digestion, 381 - distribution in the human body, 394 - enzymatic estimation, 103 - extraction (pilot-scale), 135 - functional properties, 135 - heat decomposition, 392 - helix in model, 126 - hydrolysis, 55, 56, 65, 66, 78, 102, 103, 105, 205,

211+, 217, 221, 227, 231+, 242, 252, 270, 273+, 289-291, 323+, 342, 367,369, 371, 395, 399, 400

- - at low temperature, 399, 400 - - by ultrasound and electrochemical methods, 400

- - multiple-chain mechanism, 236, 237, 239 - - single-chain mechanism, 235, 236, 239 - hydrolysis products, 65+ - hysteresis effects of inulin suspensions, 82 - in chocolate creams, 82 - in foods, as compared to maltodextrins, 82 - in low-calorie sandwich spreads, 82 - in mayonnaise, 82 - inability to measure tubular function, 391 - (industrial) exploitation (in biotechnology), 267,

289+ - ingestion/intake, 381-383 - isolation by aqueous extraction of sliced chicory

roots, 77+ - isolation by grinding and wet milling of whole

chicory roots, 79 - linkage, 124 - metabolism, 371 - (methods for) chemical determination, 391, 394, 395 - microwave vacuum drying of clarified solutions, 344 - model, 123, 126 - modelling metabolism of inulin, 297+ - molecular distribution, 237 - molecular size, 121 - molecular structure, 129 - molecular weight distribution (= MWD), 103,106 - - effect of titanium, 106 - mouthfeel of inulin-containing foodstuffs, 82 -non-digestibility, 121, 135 - non-toxicity, 398 - number-average molecular weight, 237, 238 - physicochemical properties, 129 - physiological characteristics, 391 - pilot-scale production, 77+ - polydispersity, 236, 237 - problems with injection, 391, 392 - production, 65+ - purification by ultrafiltration, 343, 345 - renal excretion, 394 - solid separation, 79 - spray-dried/spray-drying, 77+ - (steady state) concentration in plasma, 393-395 - structural chemistry, 121+ - synthesis, 281+, 371 - (temperature-dependent) solubility, 80-82 - ultrafiltration, 77+ - use for medical purposes, 338, 398 - use in foodstuffs, 77+ - use in measuring GFR, 391 - use in removal of metal ions/radionuclides, 399 - use in the determination of renal function, 391+ - utilization, 231, 275, 279, 294 - viscosity of aqueous solutions/suspensions, 78, 80,

82, 398 - weight-average molecular weight, 236-238

411

inulin(s) (continued) - X-ray diffraction, 80, 81 - yield, 289, 397 inulin, native, 77, 83, 135,142 - chemical, physical and/or functional properties, 77,

80,82 - flow curve of inulin vs. corn starch, 83 - (thermoreversible) gel, 140 - viscosity (characteristics), 140 - viscosity/temperature curve, 138,139 inulin-containing crops, 86, 87, 397+ - as an (alternative) agricultural feedstock, 86, 87 - breeding, 289 inulin adipate derivatives - viscosity, 140 inulin adipate derivatives - viscosity/temperature

curve, 138, 139 inulin adipic acid ester (see also: inulin adipate

derivatives), 136, 138 inulin derivatives, 136, 142, 161 - biological activity, 399 - gel (formation), 137, 140 - gel firmness, 136, 140 - gelation, 136 -rheology, 136 - solubility, 142 - viscosity (characteristics)/properties, 136, 138, 142 inulin fructotransferase (depolymerizing) (EC

2.4.1.93), 143 inulin gels - gel strength, 140 inulin homologues, 129 (inulin) oligomers (see also: inulin oligosaccharides),

93+, 121+, 231+, 269, 308 - conformations, 121+ - molecular models, 121+ - NMR spectra, 121 - separation, 93+ - torsion angles, 121+ inulin oligomers, cyclic, 121, 125,126 inulin oligosaccharides, 112, 131 - structural analysis, 129+ inulin pentasaccharide, 107+, 192,194 - extraction, 107+ - purification, 107+ (inulin) polymers, 231+, 254, 297, 308 inulin powder - structure-improving additives, 344,

345 inulin tetrasaccharide, 194 inulin trimetaphosphate derivative, 140 - solubility (characteristics), 140 - (thermoreversible) gel, 140 - viscosity (characteristics), 140 - viscosity/temperature curve, 141 inulin/exo-inulinase kinetics, 232, 239 inulin/exo-inulinase systems - characterization, 231+ inulinase (= 2,1-ß-D-fructan fructanohydrolase, EC

3.2.1.7), 55, 56, 62, 68, 74, 78, 79, 102, 105, 205+, 211+, 217+, 223+, 231+, 241+, 254, 267+, 273+, 290, 292, 293, 331, 332, 360, 361, 363, 367+, 373+

- activity, 196, 225, 269, 272, 278, 291, 369, 373+ - amino acid sequence of amino-terminal end, 248-250 - antiserum against, 212 - biochemical characterization, 211+ - biotechnological applications, 211+ - catabolite repression-regulated production, 244 - dilution rate-dependent production, 244, 245 - effects of (heavy) metal ions/trace elements on

activity/production, 376-378 - extraction from chicory roots, 206 - fed-batch process for production, 244, 245 - immobilization (matrix/method), 212, 214, 215, 218,

223+, 361, 364 - inhibitors, 209 - kinetic properties, 212-214, 220 - Lineweaver-Burk plot, 209 - localization, 241+ - metabolites affecting activity, 208 - Michaelis-Menten kinetics, 213, 215 - molecular properties, 212 - monomer, 248 - oligomerization, 249 - physicochemical properties, 231 - polydispersity, 248 - production, 241+, 268, 269, 271, 277, 278 - production in chemostat culture, 244, 245 - production in continuous culture, 244 - properties, 205+, 244 -purification, 212, 246 - regulation of activity, 205+ - synthesis, 269, 270, 272 - yield, 244 inulinase, cell-free/extracellular/supernatant, 241+,

269, 270, 375, 378 inulinase, cell-wall - tetramer, 249 inulinase, cell-(wall)(bound)/intracellular, 205+, 241+,

269, 270, 275, 278, 375, 378 inulinase, covalently bound, 207, 208 inulinase, fungal, 279 inulinase, immobilized, 214-216, 217+, 223+, 359+ - operational stability, 223+ -properties, 215, 220 - thermal stability, 364 inulinase, ionically bound, 206, 207 inulinase, microbial, 211, 217 inulinase, plant, 211, 217 inulinase, soluble, 205+, 217+ - Michaelis-Menten kinetics, 208 inulinase, supernatant - dimer, 249 inulinase, yeast, 242, 279 inulobiose, 66, 71, 122, 124, 126, 144, 147 inulofructosaccharides, 356

412

inulohexaose, 144 (inulo-)oligosaccharides, 65+, 332, 337 - production, 65+ - separation, 70 inulosucrase (EC 2.4.1.9), 281+ inulotetraose, 144,147 inulotriose, 147 Inutest ®, 392, 395 - physiological characteristics, 392 invertase (= ß-D-fructofuranosidase, EC 3.2.1.26),

102, 103, 105, 144, 147, 169, 170, 173+, 187, 199+, 211+,241+,279,282, 349

- activity, 108, 282, 284, 375, 378 - amino acid sequence of amino-terminal end, 248-250 - catalytic constant (fcCat)> 214 - catalytic power (kcat/Km), 214 - vacuolar localization, 179 invertase, acid, 173+ invertase, acid - properties, 178 invertase, acid - purification, 178 invertase, cell-wall - octamer, 249 invertase, fungal, 178, 200 invertase, plant, 178, 182 invertase, supernatant - dimer, 249 invertase, yeast, 94, 108,110, 111, 170, 178, 218, 290 Iridaceae, 311,315, 316 I/S ratio, 211, 212 isokestose, 297+ isomerose, 252 isophthalic acid, 157 jao (see: Allium chinense) Japanese millet (see: Echinochloa frumentacea) Jerusalem artichoke (see: Helianthus tuberosus L.) jin zhen cai (see: Hemerocallis fulva) Job's tears (see: Coix lachryma-jobi) ju hua (see: Chrysanthemum moriflorum) juun jiu choi (see: Artemisia lactiflora) kafir corn (see: Sorghum bicolor) kaoliang (see: Sorghum bicolor) kestose, 259+, 270, 305, 307 Kevlar®, 156 Klebsiella pneumoniae, 351 Kluyveromycesfragilis, 253, 255, 293 Kluyveromyces marxianus, 52, 55, 56, 241+, 270, 271,

373 - continuous culture, inulin-limited, 271 Kluyveromyces marxianus var. marxianus, 241 Kluyveromyces sp., 241+ Km - Agropyron sp. (hydrolase activity) (for sucrose),

170 ^m - Agropyron sp. (transferase activity) (for sucrose),

170 Km - Allium cepa sucrose:sucrose fructosyltransferase

(for sucrose), 173+ Km - Aspergillus ficuum endo-inulinase ("Novozym

230") (for sucrose), 103, 104, 213, 214, 220 Km - Aspergillus ficuum endo-inulinase ("Novozym

230") (for [dahlia] inulin), 103,104, 213, 214 Km - Aspergillus ficuum exo-inulinase ("Novozym

230") (for sucrose), 103, 104, 213, 214, 220 Km - Aspergillus ficuum exo-inulinase ("Novozym

230") (for [dahlia] inulin), 103,104, 213, 214, 220 Km - Aspergillus ficuum inulinase ("Novozym 230")

(for dahlia inulin; sucrose), 103, 104 Km - Aspergillus ficuum invertase ("Novozym 230")

(for inulin), 213,214 Km - Aspergillus ficuum invertase ("Novozym 230")

(for sucrose), 213, 214 Km - Bacillus subtilis levansucrase (hydrolase activity)

(for sucrose and raffinose), 259+ Km - Fusarium oxysporum inulinase (for inulin), 364 Km - Helianthus tuberosus sucrose:sucrose

fructosyltransferase (for sucrose), 168 Km - Hordeum vulgäre sucrose:sucrose

fructosyltransferase (for sucrose), 173+ Km - Lolium rigidum fructan exohydrolase (for fructan

of DP 3-7, and 30), 201-203 Km - Saccharomyces cerevisiae invertase (hydrolase

activity) (for sucrose), 170 Km - Saccharomyces cerevisiae invertase (transferase

activity) (for sucrose), 170 Km - Streptococcus mutans inulosucrase (=

fructosyltransferase) (for sucrose), 283, 285 Kobo millet (see: Paspalum scrobiculatum) ku mai (see: Sonchus oleraceus) kurrat (see: Allium ampeloprasum) L-lactic acid, 382-384 lactate dehydrogenase, 382 lactate/lactic acid - as fermentation product, 268, 273+,

382, 383 lactic acid bacteria, 274, 275 Lactobacillus acidophilus, 351 Lactobacillus casei, 351 Lactobacillus fermentum, 351 Lactobacillus plantarum, 351, 373 Lactobacillus salivarius, 351 Lactuca indica, 318 Lactuca sativa (= lettuce), 317, 318 Lactuceae, 317 lactulose, 351 lactulose - crystal structure, 123 LAI (see: leaf area index) LAI phase - decreasing, 46 LAI phase - increasing, 46 LALLS (see: low-angle laser light scattering) Lamiales, 311 laminate resins (from HMF-based resins), 156 leaf area development, 36 leaf area duration, 31 leek (see: Allium ampeloprasum)

413

Lentibulariaceae, 310, 311 lettuce (see: Lactuca sativa) levan, 259, 263, 264, 79, 290 - degradation, 242 - hydrolysis, 290 - synthesis, 261 levanase (EC 3.2.1.65), 207, 289+ - (amino acid sequence of) N-terminal end, 290 - activity, 292, 293 - purification, 291 levanase gene - 5'-region, 290 levanase gene - DNA sequence, 290 levanase gene - expression in Penicillium

chrysogenum, 292-294 levanase gene - expression plasmids, 289+ levanase gene - expression vector, 290 levanase gene - (over)expression in Escherichia coli,

290, 291, 294 levanase gene - (over)expression in Saccharomyces

cerevisiae, 292-294 levansucrase (EC 2.4.1.10), 259+ - catalytic constant (fccat), 261, 262 - chain elongation activity, 263 - effect of organic solvents on activity, 259+ - hydrolase activity, 260-262 - kinetic constants, 261, 262 - modification of transfructosylation activity, 259+ - polymerase activity, 259+ - synthetase activity, 261 - transfructosylase activity, 260, 263 levansucrase, bacterial, 170 light interception, 31, 36 light interception (definition), 46 light use efficiency, 31, 32, 36 light (use) efficiency (definition), 46 Liliaceae, 107, 311, 315,316 Liliales, 173,311 linkage analysis, 312 LINTUL ("sink-limited") model, 46, 48,49 lipase, 162, 163,166 lipids (total) - extraction, 360 (liquid) cell suspension culture, 185+ little millet (see: Panicum sumatrensis) loliose, 94,97 Lolium perenne L. (= ryegrass), 94, 115+ - leaf elongation, 117-119 Lolium rigidum Gaud. (= annual ryegrass), 108, 199+ - fructan (exo)hydrolase, 203 - invertase, 203 - leaves, 201 Lolium temulentum L., 174 -leaf extracts, 169, 180 - leaves, 181 long-chain alkyl-HMF-esters, 162 low-angle laser light scattering (= LALLS), 231+

(low-temperature) X-ray diffraction, 121 Lycopersicon esculentum Mill (= tomato), 94 M5E3 - as ethanol blend, 89, 90 maize (see: Zea mays) malto-oligosaccharide, 96 Mannich(-type) addition reaction, 158 manure odour (of swine), 63 meadow cabbage (see: Cirsium oleraceum) Megamonas hypermegas, 351 Megasphaera elsdenii, 351 melezitose, 97 Menyanthaceae, 310, 311, 318 Menyanthes trifoliata (= bogbean/buckbean), 318 methane - as fermentation product, 66, 381+ methane producers, 381+ methylation, 312 methylation analysis, 144 Michaelis-Menten kinetics, 234 (micro)flora, human bacterial/intestinal, 347, 381+ (micro)flora, intestinal - domestic/monogastric

animals, 385 (micro)flora, methanogenic, 382 Microseris lanceolata (= murnong), 317, 318 - as staple food of Australian aborigines, 317 microtitre plate method, 176 milo (see: Sorghum bicolor) Ntitsuokella multiacidus, 351 molecular dynamics, 132 molecular weight distribution (= MWD), 231+ monocotyledons, 311, 315, 316 monofructosylsucrose (see also: fructan trisaccharide),

174,311 Monotropaceae, 310, 311 mucosa, intestinal, 349 multimerization, 243 murnong (see: Microseris lanceolata) Muscari atlanticus, 315 Muscari comosus (= purse-tassels), 315, 316 MWD (see: molecular weight distribution) Na-trimetaphosphate, 137 Najadales, 311 neobifurcose, 348 neokestose, 94, 96, 97, 107+, 168, 173+, 196, 311,

312, 348 - extraction, 107+ - purification, 107+ - synthesis, 178 neosugar - as product, 57, 386, 388 Neosugar - as trade-name, 65,66, 94, 31(X 34$, 351 Neosugar G, 349, 350 Neosugar P, 107+, 203, 349, 350 NMR analysis, 283, 287 NMR spectroscopy, 147, 162, 163, 166 NOE, 132,133 Nomex®, 156

414

non-ionic detergents, 161 non-methane producers, 381+ non-Michaelis-Menten kinetics, 232, 234, 238 nystose, 65, 93+, 107+, 122, 124, 126, 129, 130, 133,

147, 192, 194, 270, 348, 349 - assignment, 129+ - chemical shifts, 132, 133 - coupling constants, 132, 133 - crystal structure, 121, 125 - extraction, 107+ - HMBC spectrum, 131 - molecular structure, 130 -NMR spectra, 132 - purification, 107+ - structural analysis, 129 oat (see: Avena abyssinica and A. sativa) oats, 314 oligofructan, 177, 194-196 oligofructoside, 213 oligoinulin, 103, 279 oligomers (general), 267 oligosaccharides (see also: [fructan], [fructo-], inulin

and [inulo-]oligosaccharides), 65+, 167-169, 242, 243, 254, 259, 312, 347, 360, 368, 371, 373

- in (animal) feed, 67 onion (see: Allium cepa L.) OP (see: osmotic potential) organic acids - as fermentation products, 381 Oryza glaberrima (= African rice), 313 Oryza sativa (= rice), 313, 314 Oryzeae, 313,314 osmotic potential (= OP), 116-118 PAD (see: pulsed amperometric detection) PAGE (see: polyacrylamide gel electrophoresis) palm lily (see: Cordyline terminalis) Paniceae, 313, 314 Panicum miliaceum (= common millet), 313, 314 Panicum sumatrensis (= little millet), 313 paper chromatography, 93, 260, 263, 264, 310 papyrus (see: Cyperus papyrus) PAR (see: photosynthetically active radiation) Paspalum scrobiculatum (= Kobo millet), 313 pBluescript plasmid, 260 pearl millet (see: Pennisetum typhoides) pectinase, 367, 368 pectolyase, 182 Pediococcus pentosaceus, 273+ Pediococcus sp., 275, 279 Penicillium chrysogenum, 292-294 Penicillium purpurogenum, 373 Pennisetum cinereum (= bulrush millet), 313 Pennisetum nigritarum (= bulrush millet), 313 Pennisetum typhoides (= pearl millet), 313 Peptostreptococcus parvulus, 351 Peptostreptococcus prevotii, 351

periplasmic space, 249 permethylation, 94 pharmaceutical industry, 63, 338 pharmaceuticals, 271 pharmaceuticals - as industrial end-products from

HMF, 149+ phleinase (see: levanase) phleinsucrase, 266 Phleum pratense L., 185+ phospholipids - estimation, 360 photosynthates, 13, 18, 174,176, 182,185, 191 photosynthetically active light/radiation (= PAR), 12,

15,18,31,32,46 Phyllospadix spp.f 316, 317 Phyllostachys aurea, 315 Phyllostachys dulcis (= bamboo), 313 Phyteuma orbiculare (= rampion), 318 Phyteuma spp., 317 pig (= porcine) pancreas lipase (type II), 162, 163 pig/swine, 385+ plant protection agents - as industrial end-products

from HMF, 152,155 planteose, 94, 97 plants, fructan-containing, 309+ plasticizers - as industrial end-products, 149 Poeae, 314 Polemoniaceae, 311 polyacrylamide disc gel electrophoresis, 146 polyacrylamide gel electrophoresis (= PAGE), 168 polyaddition products - as industrial end-products from

HMF, 155 polyamides, 150, 156 - decomposition temperature, 156 - glass temperature, 156 - isophthalic acid-based, 156 - terephthalic acid-based, 156 polycondensates - as industrial end-products from

HMF, 155, 156 polyesters, 150, 156 polyfructosylsucrose (see: fructan) polymers (general), 57, 256 - as industrial end-products from HMF, 149+ polymers, conducting (HMF-based), 157 polymers, conducting (HMF-based) - in batteries,

sensors, switches, 157 polymers, special (HMF-based), 155, 157 Polymnia sonchifolia (= yacon), 317, 318 polyphenol oxidase, 356 polysaccharide(s), 82, 94, 98, 121, 135, 167, 188, 232,

239, 373 - characterization, 234 Portulaca oleracea (= common purslane), 318 Portulacaceae, 311, 318 potato, 52, 53 premium plus gasoline - as ethanol blend, 89, 90

415

pressure atomization, 62 Propionobacterium acnes, 351 Pseudomonas fluorescens - lipase, 162, 163,166 Pseudomonas syringae pv. tagetis (see: Helianthus

tuberosus L. - pathogens) pulsed amperometric detection (= PAD), 71, 93+ purse-tassels (see: Muscari comosus) pyridine derivatives - as industrial end-products from

HMF, 155 pyridoxal - as inhibitor of invertase and

sucrose:sucrose fructosyltransferase, 179, 180 pyridoxin - as inhibitor of invertase and

sucrose:sucrose fructosyltransferase, 179, 180 Pyrolaceae, 310, 311 pyrrole derivatives - as industrial end-products from

HMF, 155 radioactive isotopes - use in measuring renal function,

391, 392 radiorespirometry - to study utilization of fructo-

oligosaccharides by man, 349 raffinose, 97, 180, 181, 202, 259, 261, 270, 312 Raftiline, 310 Raftilose, 310 rakkyo (see: Allium chinense) rampion (see: Campanula rapunculus and Phyteuma

orbiculare) rat, 349, 359+ - caecal content(s), 381-383 - caecal metabolites, 382 - caecal pH, 382, 384 - caecal SCFA, 383, 384 - caecal weight, 382 rat, germ-free, 381+ raw materials - agricultural/industrial/multipurpose(s),

149+,273, 281, 289, 294, 323+, 368 reactor performance (for inulin hydrolysis), 216, 220 - operational stability, 216 - space velocity, 216 - specific productivity (of sugar from inulin), 216 - volumetric productivity (of sugar from inulin), 216 recombinant DNA techniques, 289 red oat (see: Avena byzantina) refractive index (= RI) (detection/measurement), 109,

111, 116,117,129 (renal) clearance, 393 renal failure - contra-indication for use of inulin to

measure GFR, 393 resins - as industrial end-products from HMF, 154-156 resistance to cold stress, 115 reverse osmosis, 356, 357 reverse-phase system, 94 reversed-phase high-performance liquid

chromatography (= RP-HPLC), 107+, 129 rheograms (of rheopex-plastic substances), 82 Rhizopus sp. (see: Helianthus tuberosus L. -

pathogens) RI (see: refractive index) ribulose bisphosphate carboxylase, 43 rice (see: Oryza sativa) riceroot (see: Fritillaria camschatcensis and F.

lanceolata) Rohalase (yeast exo-inulinase), 332 root crops, 317 rotary disc atomization, 62 RP-HPLC (see: reversed-phase high-performance

liquid chromatography) rye (see: Secale sativa) ryegrass (see: Lolium perenne L.) Saccharomyces cerevisiae, 52, 55, 56, 108, 241+,

251+, 292-294, 374 Saccharomyces cerevisiae transformant - growth on

inulin, 292 Saccharomyces chevalieri, 253-255 Saccharomyces diastaticus, 251+ Saccharomyces fragilis, 243 Saccharum barberi (= sugar cane), 313 Saccharum edule, 313 Saccharum officinarum (= sugar cane), 313 Saccharum sinense (= sugar cane), 314 salsify (see: Tragopogon porrifolius) SCFA (see: short-chain fatty acids) Schizosaccharomycespom.be, 251+ Sclerotinia sclerotiorum (see: Helianthus tuberosus L.

- pathogens) Sclerotinia sp., 59 Scolymus hispanicus (= golden thistle), 318 Scorzonera hispanica (= black salsify), 318 Scrophulariales, 311 SDS-PAGE (see: sodium dodecyl sulphate -

polyacrylamide gel electrophoresis) SEC (see: size-exclusion chromatography) Secale sativa (= rye), 313, 314, 348 sedges, 315 Senecioneae, 317 sensory evaluation/test (of inulin-containing

foodstuffs), 82 Setaria italica (= foxtail millet), 313 shallot (see: Allium cepa) shallu (see: Sorghum bicolor) short-chain alkyl-HMF-esters, 162 short-chain fatty acids (= SCFA), 352, 382-384 S/I ratio (see also: I/S ratio), 276, 277, 279 single crystal X-ray diffraction, 143 single-screw (counterflow diffusion) extractor, 326,

328 single-shot (inulin) clearance/method, 393-395 sinistrin, 392 sink strength, 49 sink tissue, 173 sink-source relations, 36

416

site-directed mutagenesis, 259+ size-exclusion chromatography (= SEC), 78, 231+ smut fungus, 315 sodium dodecyl sulphate-polyacrylamide gel electrophoresis (= SDS-PAGE), 176,180,181, 212,

248,291,292 soft drinks, 273 Solanales, 311 solvents - as industrial end-products, 149+ Sonchus oleraceus (= ku mai, sow thistle), 317, 318 sorghum (see: Sorghum bicolof) Sorghum bicolor (= durra, feterita, Guinea corn, kaflr

corn, kaoliang, milo, shallu, sorghum, white durra), 312, 314

Sorghum sonor urn, 312 Sorghum sumatrense, 312 source tissue, 173 sow thistle (see: Sonchus oleraceus) SST (see: sucrose:sucrose fructosyltransferase) stachyose, 97,181 stachyose - crystal structure, 123 Staphylococcus spp., 67 starch, 135-137, 173,185, 224, 310, 312, 315, 324,

360 - chemical modification/derivatization, 135-137 - derivatives, 138 - functional properties, 135 starch hydrolysis products, gel-forming, 140 starch, corn, 252 stem lettuce, 317 Stipeae, 314 streptococci, oral, 279 Streptococcus intermedius, 351 Streptococcus mutans, 279, 281+ Streptococcus salivarius, 202, 279 Streptococcus sp., 275, 279 Stylidiaceae, 311 succinate - as fermentation product, 268, 269, 271 succinate production - effect of temperature

down-shift, 271 sucrose:sucrose fructosyltransferase (SST, EC

2.4.1.99), 107, 168-170, 173+, 192, 195, 196, 266, 297+

- activity, 107,196 - properties, 173+ - purification, 173+ - vacuolar localization, 174, 179 sugar beet, 52, 53, 78, 79, 323, 324 - (enzymatic) liquefaction, 367 - productivity, 30 sugar cane (see also: Saccharum barberi, S.

officinarum and S. sinense), 312-314 sugar maple, 348 sunflower (see: Helianthus annuus L.) surfactants - as industrial end-products, 149+

sweeteners, 205, 211, 217, 251, 323, 324, 349, 355, 357

sweetmeats, 317 Symphytum officinale (= comfrey), 318 iac-promoter, 290 Taraxacum officinale Weber (= dandelion), 211, 317,

318 - fructan (exo)hydrolase, 209 teff (see: Eragrostis tef) temperate forage grasses, 185 temperature sum, 34, 36,46,49 terephthalic acid, 150,157 thermophilic anaerobes - fermentation pattern, 268 thermophilic clostridia, 267+ - inulinase synthesis, 269, 270, 272 - product formation, 269 thermophilic clostridia, inulin-degrading, 267+ thermoreversible (maltodextrin) gels, 82 thermostable enzymes, 271 thin-layer chromatography (= TLC), 70, 93,109,116-

118, 146, 162,163, 166,173+, 192, 193,195, 202, 213, 218, 221, 232, 233, 312, 375

thiophene derivatives - as industrial end-products from HMF, 155

third space, 395 tiger nut (see: Cyperus esculentus) timothy, haplocorm - levanase, 207 titanium (ascorbate), 102, 105,106 TLC (see: thin-layer chromatography) tomato (see: Lycopersicon esculentum Mill) tong hao (see: Chrysanthemum spatiosum) tonic water, 368 topinambour (see: Helianthus tuberosus L.) Tragopogon porrifolius (= salsify), 318 transfructosylation, 259+, 298, 308, 349, 352 transportation fuel, 85+ tree onion (see: Allium cepa) Trichoderma sp. (see: Helianthus tuberosus L. -

pathogens) triglyceride level, in blood, 324 triglycerides - estimation, 360 triglycerides - in serum, 350, 352 Triticeae, 313, 314 Triticum aestivum L. (= bread wheat), 108, 313 Triticum dicoccum (= emmer), 313 Triticum durum (= durum wheat), 313 Triticum monococcum (= einkorn), 313 two-compartment model (of plasma concentration of

inulin), 394 ultra-high-fructose corn syrup (= ultra-HFCS), 252 Ultrafiltration, 78, 80, 82, 246, 375 - retentate, 80 - - spray-drying, 78, 80 urethanes (HMF-based), 157 Urginea maritima Baker, 202

417

urinary output - during inulin clearance, 393 urine sampling - for inulin measurement, 393 Ustilago esculenta, 315 Utricularia menziesii, 310 Utricularia vulgaris, 310 vascular space, 394 vegetable crops, 312 Veillonella dispar, 351 very-high-fructose syrup, 223+ Villarsia albiflora, 317 vinyl acetate, 161-163 vinyl esters - as acyl donors in transesterification of

HMF, 161+ vinyl stearate, 162,166 volatile fatty acids - as fermentation products, 66 Welsh onion (see: Alliumfistulosum)

wheat, 314 white durra (see: Sorghum bicolor) winter purslane (see: Claytonia perfoliata) woh sun, 317 X-ray analysis, 147 Xanthorrhoeaceae, 310,311 yacon (see: Polymnia sonchifolia) yeasts (general), 242, 256, 267, 276, 278, 279, 292,

373 Yucca filamentosa, 316 Zantac® (an HMF-based anti-ulcer drug), 158 Zea mays (= maize), 314, 323, 324 Zizania aquatica (= American wild rice), 313 Zizania caduciflora (= gau sun), 313, 315 Zoster a marina (= eelgrass), 315, 316 Zosteraceae, 309+

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[a Fuchs] Inulin and Inulin-containing Crops(BookZZ.org)