Muhidinov Zayniddin Kamarovich (992 37) 2257893 (сл.)

UDK 541.127:661.183.123.2 Effect of temperature on the intrinsic viscosity and conformation of

different pectins

©2009 г. Z.K. Muhidinov*, Kh.Kh. Avloev*, M.T. Norova*, A.S. Nasriddinov*, D.Kh.Khalikov*, M.L. Fishman**

Chemistry Institute of Tajikistan Academy of Sciences, 299/2 Ainy str., 734063

Dushanbe, Tajikistan

Eastern Regional Research Center ARS USDA 600 East Mermaind Lane, Wyndmoor 19038 PA, USA.

Received:

Accepted:

The effects of temperature on the intrinsic viscosity and on the

conformation of different pectins obtained from citrus, apple and sunflower in a 0,17M NaCl solution were studied. The intrinsic viscosity and the flow activation energy of the polymer (Ea) derived from slope of d In [η]/ d(l/T) as an index of stiffness of polymers have been evaluated at various temperature (20-60°C). These results showed: the intrinsic viscosities decreased linearly with increasing temperature, for all pectins expect apple pectin, therefore, a temperature-induced conformational transition did not occur in the temperature range studied. The results clearly indicate that only apple pectin has sufficient structural integrity to withstand the loss of neutral sugar side chains when subjected to temperatures as high as 60 degrees C. The value Ea were between 0.67 , 0.69 x 107 J/(kmol) for the commercial citrus and orange pectins and 1.34, 1.44 x 107 J/(kmol) for the sunflower and apple pectins respectively. Ea increased with decreasing molecular weight, indicating that pectins with low DE have aggregated species, while higher molecular weight pectins are more flexible. Data of the Ea indicate again that all studied pectins are stiff molecules. Key words: Pectin; Viscosity; Chain flexibility; Flow activation energy; destruction.

E-mail: zainy@mail.ru (Muhidinov Zayniddin Kamarovich)

1

Introduction

Pectin is structurally and functionally the most complex polysaccharide in

plant cell walls. Pectin has functions in plant growth, morphology, development,

and plant defense and also serves as a gelling and stabilizing polymer in diverse

food and specialty products and has positive effects on human health and has

multiple biomedical uses. Pectin is a family of galacturonic acid-rich

polysaccharides including homogalacturonan, rhamnogalacturonan I, and the

substituted galacturonans rhamnogalacturonan II (RG-II), and xylogalacturonan

(XGA)1. The main pectin chain is comprised of a (1-4)-linked D-galacturonic

acid residues. Many of the galacturonic acid residues have been esterified at C-6

to form methyl esters. Theoretically, the degree of esterification (DE) can range

from 0% to 100%. Pectins with a DE>50% are classified as high methoxyl (HM)

pectins, and consequently low methoxyl (LM) pectins have a DE <50%2.

Rhamnose residues are incorporated into the main chain at random intervals,

which results in a kink in the otherwise linear chain3. Side chains of arabinans

and galactans are also present, either randomly dispersed or in localised ‘‘hairy’’

regions. Besides the primary structure, the conformation and flexibility of a

pectin molecule is important to the functional properties in the plant cell wall

and also significantly affects their commercial use in the food and biomedical

industries4.

Knowledge of the viscosity is of primary importance to the pectin and

pectin containing fruit juice industry. The accurate viscosity data over wide

temperature and concentration regions are need for a various research and

engineering applications in any branch of the food industry, such as developing

food processes and processing equipment, the control of products, filters, mixers

and quality evaluation. Viscosity can become an important factor during the dia- 1 Debra Mohnen, Pectin structure and biosynthesis Current Opinion in Plant Biology 2008, 11:266–277. 2 Pilgrim, G. W., Walter, R. H., & Oakenfull, D. G. (1991). The chemistry of high-methoxyl pectins. In R. H. Walter (Ed.), The chemistry and technology of pectin (pp. 24–50). San Diego: Academic Press. 3 Axelos, M. A. V., & Thibault, J.-F. (1991). The chemistry of low-methoxyl pectin gelation. In R. H. Walter (Ed.), The chemistry and technology of p 4 Tombs, M. P., & Harding, S. E. (1998). An introduction to polysaccharide biotechnology. London: Taylor and Francis (pp. 14–20).Harding, 1998

2

ultrafiltration concentration of pectin solution, especially in the production of

high-density concentrates. Because pectin solution are subjected to different

temperatures and concentrations during processing, storage, transport, marketing

and consumption, their viscosity are studied as a function of temperature and

concentration. Thus, there is great practical interest in the study of the

combined effect of temperature and concentration on viscosity of pectin

solutions at operational conditions.

However, a survey of the literature reveals the scarcity of reliable

experimental viscosity data for pectin solutions in wide temperature and

concentration ranges. Only some experimental viscosity data sets are available

for other polysaccharides in the literature.

Materials and Methods

Pectin purification and characterization

Apple HM-pectin (red apples from Muminobod, Tajikistan, September

2007), citrus HM-pectin (mixture of albedo and peel of the orange fruits

exported from Turkey in 2007) and sunflower LM-pectin (local harvest in 2006,

Tajikistan) were obtained in laboratory condition. Citrus LM-pectin (GENUE ®

LM-12 CG) was kindly provided by CP Kelco Wilmington DE, USA. The

pectin samples were purified overnight by treatment with the mixture of acidic

ethanol (mixture of С2Н5ОН, concentrated НСI and water in ratio of

14.0:3.6:2.4). Pectin samples then were washed with 75% ethanol solution to

remove Cl- ions followed by 96% ethanol and dried under vacuum at 40-60о С.

The percentage of galacturonic acid and degree of methyl esterification,

were determined for selected pectin samples using method described in

references5,6.

Determination of intrinsic viscosity

5 Filisetti-cozzi T.M.C. C., Carpita N.C. Measurment of Uronic Acids without interference from Neutral Sugars. Anal. Biochem. – 1991 – p.197, p.157-162 6 CP Kelсo Control methods. Determination of pectin DE, 2001, p.3

3

Different concentration (0,1-0,08g/dL) solution of pectin in 0,17 M NaCl

(1%) were prepared. The intrinsic viscosity was measured using Ubellode

viscometer with solvent flow rate 54.97 second at 20.0°C. The temperature was

controlled with a thermostat (Masterline Model 70H, Forma Scientific, USA) to

maintain respective temperature at 20±0.1°C, 30±0.1°C, 40±0.1°C, 50±0.1°C,

60±0.1°C. Each sample was measured 3 times. The intrinsic viscosity was

determined from dilute solution viscosity data at zero concentration-limit of

specific viscosity (ηsp) divided by concentration (c):

C)/(0

limit][ spcηη

→= ,

where ηsp = [(η- ηs)/ηs], and η and ηs, are the viscosities of the solution and

solvent respectively.

Determination of pectin molar mass by SE chromatography

The methodology for the pectin HPSEC has been describe elsewhere7 and

modified according to requirements of the HPSEC systems8.

Depending on viscosity of pectin samples, the dried sample at a final

concentration of 1 or 2 mg\mL was dissolved on 0.05 M NaNO3 stirred until

dissolved, centrifuged at 20 000 g for 20 min at 20oC. Both solvent and pectin

solution were filtered with Nylon 0.45 mm (Millipore Millex-HN). The

injection volume was 100-200 µl. Samples were run in triplicate. The flow rate

was adjusted 0.80 ml\min. The solvent delivery system consisted of a 2-Channel

Vacuum Degasser, Waters 1515 Isocratic Pump, and 717+ Autoinjector

(Waters). Pectins were separated by one PL-Aquagel OH-60 and one PL-

Aquagel OH-40 size exclusion columns. The columns were connected in series,

enclosed in the column heater (Waters) and kept at 30oC. Column effluents

were detected by ViscoStar model differential pressure viscometer (Wyatt

Technology), and an Waters 2414 Refractive Index Detector in series. The

electronic outputs from the detectors were connected to separate serial ports in 7 Fishman, M.L., Chau, H.K., Kolpak, F., Brady, J. J. Agric. Food Chem. 2001, 49, 4494,-4501. 8 Z.K Muhidinov., M.L Fishman., R.M.Gorshkova, A.S.Nasriddinov, D.Kh Khalikov. Molar mass of Pectins obtained in autoclave. Kazakhstan Chemical Journal, special issue (21), 2008, p.60-66.

4

the same personal computer in a manner which permitted data to be collected

and processed by ASTRA 5.3.4.13 (Wyatt Technology) and Breez (Waters)

software simultaneously. The change in refractive index with polymer

concentration, dn/dc, in 0,05M NaNO3 for pectin was used, as reported from

reference [7] was 0,130 ml/g. The value of molar mass was obtained using

universal calibration curve. Columns are calibrated using a series of Pullulan

standard samples (Showa Denko K.K., Japan) with Mw 788KD; 667KD;

404KD; 112KD; 47.3KDand 22.8KD respectively.

Results and Discussion

The effect of temperature on the viscosity of the polymer is manifest. The

theological properties of a Newtonian fluid or polymer liquid are consistent with

the Arrhenius equation when the temperature is higher than the glass transition

temperature (Tg) or melting point9. The Arrhenius equation is as follows:

RTaEe

/Α=η (1)

where η is the apparent viscosity, A is the characteristic constant for polymers of

a specific shear rate and molecular weight, Ea is the activation energy for the

flow process, R is the gas constant and T is the absolute temperature (°K). The

slope (Ea/R) of the plot of natural logarithmic apparent viscosity (Ln η) and the

inverse of absolute temperature is the flow activation energy of the polymer

(Ea). Usually Ea, values are between 2.09 x 107 and 2.09 x 108 J/(kmol). Stiffer

polymers have larger Ea1,10. If the apparent viscosity is replaced with the

intrinsic viscosity or relative viscosity, the slope (d In [η]/ d(l/T) or (dln

ηrel/d(l/T)) can also be used as an index for stiffness of the polymer because it

relates to Ea. Stiffer polymers also have larger slopes11.

9 Nielsen LE. Polymer Rheology. New York: Marcel Dekker, 1977. 10 Chen RH, Lin WC. J Fish Soc Taiwan 1992; 19(4):299-309. 11Rinaudo M. Domard A. In: Skiak-Bræk G, Anthonsen T, Sandford P, editors. Chitin and Chitosan: Sources, Chemistry, Biochemistry, Physical Properties and Applications. London: Elsevier Applied Science, 1989:71-86.

5

The molecular flexibility of cellulose trinitrate, hydroxyethylcellulose, and

amylose increase with increasing temperature and cause the intrinsic viscosity to

decrease12. The effects of temperature on the Mark-Houwink exponent are very

significant when medium temperatures are close to the θ-temperature, and also

are very sensitive when the polymer is dissolved in a good solvent. However, if

the polymer is a stiff chain, the effects of temperature on the Mark-Houwink

exponent are limited13.

The effects of temperature on pectin conformation have rarely been

studied. Most of the studies focused on the effect of temperature on viscosity.

In this study we use apple pectin (red apple from Muminobod, Tajikistan,

fresh crop of September 2007) orange (mix albedo and peel, imported from

Turkey 2006) and sunflower (extracted from sunflower head residue crop of

2006 treated by NaCl)). The main characteristics of pectins are listed in the

table 1. Table 1. The main characteristics of pectin obtained from different sources

[η], dL/g Pectin sources GA,

% DE, %

Mw 10-3

MALLSRg, nm LS/VIS Ubbelode

a

Apple M (lab) 64,80 55,88 137,0 28,8 0,709 0,81 0,960 Orange (lab) 77,76 70,74 257,0 33,8 5,51 1,64 0,698 Sunflower (lab) 69,6 48,50 95,4 22,6 1,26 1,07 0,938 12LM-CG (CP Kelco) 69,0 31,0 212,7* - - 1,5 - * Mw obtained by SEC HPLC with RI and VIS detector using universal calibration curve

Galacturonic acid (GA) contain of pectins varied from 64 to 77 in series of

apple, commercial citrus, sunflower and orange pectins. Apple and orange

pectin have high degree of esterification (DE), which belong to HM pectins,

while sunflower and CP Kelco pectins are LM type. Both citrus pectins have

high viscosity and molar mass. The differences of [η] value obtained by on line

HPSEC and Ubbelode viscometer is to due polyelectrolyte effect of pectin

12 Launay B. Doublier JL, Cuvelier G. In: Mitchell JR, Ledward DA, editors. Functional Properties of Food Macromolecules.

London: Elsevier Applied Science,1986:1-78. 13 Bohdanecky M. Kovar J. Viscosity of Polymer Solutions.Amsterdam: Elsevier. 1982.

6

chains in the low 0.05M against 0.17M ionic strength of used solution. The

Mark-Houwink equations were obtained by MALLS method. Although the

resulted a value for orange pectin 0.698 in table 1 indicating random coil

conformation of polymer chains, but evidence from atomic force microscopy

(AFM)14 indicates orange pectin forms molecules with variety of shapes. Shapes

imaged included rods, segmented rods, kinked rods, rings, branched molecules,

and dense circular areas of pectin. Since no network structure is visible, it

appears that in the concentration range between 13.1 and 6.5 µg/mL a transition

occurs from fluid networks to individual molecules or aggregates of limited size.

In the case of peach pectin15,16, microgel networks were observed at 10 µg/ mL,

so that for orange pectin, the transition range may start below 10 µg/mL but

above 6.5 µg/mL. Moreover, trends to aggregation of pectin chains rise with

reduction of DE, which promote intermolecular interaction induced by both

hydrogen bounds and crosslinking with bivalent metal ions. Thus in case of

pectin, the Mark-Houwink exponent measures compactness of macromolecule.

Furthermore, the a value for pectins is an average value of more then one shape

as indicated by the non-lineararity of the Mark-Houwink plot both by

fractionation method and SEC chromatography. The large value a for sunflower

(DE 48,5) and apple (DE 55.8) pectins indicate dissociation of aggregates.

Here we investigate influence of solution temperature on the intrinsic

viscosity for all pectins. With increasing solution temperature between 10 and

50°C, the intrinsic viscosity of pectins obtained from orange, sunflower and CP

Kelco decreases linearly, while apple pectin viscosity shows a non-linear

temperature dependency (Fig. 1). It is well known that17 in the same solvent θ-

temperature for branched polymers the less then liner one. This fact indeed

14 Fishman et al. Global Structure of High Methoxyl pectin from solution and gels. Biomacromolecules, 2007 15 Fishman, M. L.; Cooke, P.; Levaj, B.; Gillespie, D. T.; Sondey, S. M.; Scorza, R. Arch. Biochem. Biophys. 1992, 294, 253-260. 16 Fishman, M. L.; Cooke, P.; Hotchkiss, A.; Damert, W. Carbohydr. Res. 1993, 248, 303-316. 17 Rabek J.F. Experimaental Method in Polymer Chemistry. Translated into Russian. М: Мir, 1983.

7

confirms the presence of ramnogalacturonan fraction, which increases

branching number in the pectin molecules.

0

0,2

0,4

0,6

0,8

1

1,2

0 10 20 30 40 50 60 70

t,oC

[h],

dl/g

Apple

Sunflower

Orange

CP Kelco

Fig.1 Influence of temperature on the viscosity of pectins

Increasing the solution temperature usually results in a rapid decrease in the

ratio of radius of gyration to average molecular weight resulting in increased

chain flexibility and compactness of the molecule which, in turn, causes a

decrease in the intrinsic viscosity4. Noguchi18 reported that increasing the

temperature may result in decreasing hydrogen-bonded hydration water of

glucose, dextran, etc. Decrease in hydrogen-bonded hydration water may result

in a decrease in specific volume, and therefore the intrinsic viscosity decreases.

In case of sunflower and apple pectins we suggested the increase of temperature

promote aggregate dissociation, which in turn deceases molar mass and intrinsic

viscosity.

The hydrodynamic properties (intrinsic viscosity, [η]; infinite dilution

sedimentation coefficient, S20,w; weight average molecular weight, Mw and

translational frictional ratio, f/f0) of a high methoxy pectin have been evaluated

18 Noguchi H. In: Rockland LB. Stewart GF, editors. Water Activity: Influences on Food Quality. New York: Academic Press, p.281-293,1981.

8

at various temperatures (20–60°C)19. A reduction in the value of all four

hydrodynamic parameters is indicative of depolymerization due to β-

elimination, demethoxylation and loss of hairy side chain and is in agreement

with an other study using viscometry20. Hence the sharp decrease in case of

apple pectin results of aggregate dissociation and side chain breakdown, while

the decrease of [η] in case of orange pectin related to aggregate dissociation and

also β–elimination destruction.

Influences of temperature on the solvent nature are also investigated by

exploring the Huggins constant versus temperature. The Huggince constant (Kh,

equation 2) reflect solvent quality for the polymer solution and considered to be

an index of polymer-polymer interaction. ηsp/C=[η]+Kh[η]2C (2)

The extrapolation are usually done for relative viscosity values between

about 1.2 and 2.0 when the corresponding specific viscosities are between about

0,2 and 1,0. The Huggins constant Kh with a large number of the reported values

begin between 0,3-0,4 in the thermodynamics good solvents and 1,0 in theta

solvent21. In other solvent worth then θ-solvent, where occur chain aggregation

this value may reach 1-322.

19 G. A. Morris, T. J. Foster and S. E. Harding. Carbohydrate Polymers, Vol. 48, (4) , p. 361-367, 2002. 20 Axelos, M.A.V., & Branger, M. Food Hydrocolloids, 7, p. 91–102, 1993 21 Rao, M.A. Rheology of fluid and semisolid foods: Principal and application. A Chapman & Hall food science book 12-14, 1999. 22 Berger, R. Macromol. Chem..102, p.24, 1967.

9

-0,500

0,000

0,500

1,000

1,500

2,000

2,500

3,000

0 20 40 60 80

toC

Kh

SunflowerPectin

OrangePectin

Citruse CPCelco

Apple Pectin

Fig. 2 Influence of temperature on Huggins constant

Fig. 2 shows the influence of temperature on the Huggins constant (Kh) for

all pectins studied. Kh goes through a maximum in the range of temperatures

between 20 to 60 degree for all pectins but apple. From the shape of its curve, it

appears that the apple curve could go through a maximum at a temperate higher

than 60 degrees provided it is not degraded. It appears that the various pectins

reach a maximum value of Kh at different temperatures. In other words each

pectin had an optimum temperature for polymer-polymer interactions. In the

temperature range studied, initially Kh decreases for all pectins studied but

orange pectin. The initial decrease in Kh may indicate polymer dissociation

induced by an increase in the dissociation of hydrogen ions from carboxyl

groups. The maximum in Kh for orange, sunflower and CP Kelco citrus pectin

may indicate maximum intermolecular association for these polymers at the

temperature of the maximum. At temperatures beyond the maximum, the

polymers may undergo dissociation due to increased Brownian motion with

increasing temperature. Presumably, if a wider temperature range were studied,

Kh for all the polymers would undergo a minimum followed by a maximum

with increasing temperature provided no other event interfered with the

processes discussed. The displacement of the curves along the temperature axis

may be due to structural differences among the polymers studied. The Huggin's

10

constant for sunflower pectin increased from 1.50 to 2.63 with increasing

temperature from 25 to 50°C. This was attributed to an increase in chain-chain

interactions of homogalacturonans in sunflower pectin with increasing

temperature.

The chain flexibility was investigated from this data by plotting Ln[η]

against the inverse of absolute temperature (1/T). The slope dLn[η]/d(1/T) can

be used as chain flexibility index9 (fig.3)

-1,40

-1,20

-1,00

-0,80

-0,60

-0,40

-0,20

0,002,90 3,00 3,10 3,20 3,30 3,40 3,50

1/T.103

Ln[h

] SP

OP

CPK

AP

Fig. 3 Plot of the natural logarithmic intrinsic viscosity (Ln[η]) versus inverse absolute temperature (1/T, К) of different pectins in 0,1 M NaCl. Each curve indicates different pectin sources: (SP) sunflower; (OP) orange. (CPK) CP Kelco; (AP) Apple. Table 2 shows the slope of dln[η]/d(l/T), Ea, higher for sunflower and apple

pectin and smaller for low methoxyl CP Kelco pectin (CPK) and HM orange

pectin (OP). This indicates that the polymer chains of larger molecular weight

pectins are more flexible than the smaller molecular weight ones. The results are

in accordance with the Wang and Xu23 reported Ea, of 75 and 91% DD chitosan

in 0.2 M acetic acid to be 1.5 x 107 and 2.5 x 107 J /(kmoll), respectively. Data of

the Ea indicate that among all studied pectins sunflower and apple pectin are

stiff, whereas citrus pectins are less stiff molecules. Table 2 The slope of dln[η]/d(l/T), Ea for studied pectins.

23 Wang W, Xu D. Int J Biol Macromol. 16, p.149-52, 1994.

11

Pectins dln[η]/d(l/T R2 Ea, J/(Kg mol) 10-7

Orange Pectin, lab. 834.7 0,9458 0,69 Citrus Pectin, CP Kelco 809.4 0,8822 0,67 Sunflower Pectin, lab 1610.9 0,9278 1,34 Apple pectin, lab 1731.0 0,9384 1,44

The results obtained clearly indicate that among with pectins the apple

pectin is strongly susceptible to a structural integrity at elevated temperature (up

to 600C) due to NS side chain breakdown. Data in table 2 indicate that all

pectins studied with the exception of apple undergo association-dissociation in

the temperature range studied. This may be of significances as many HM-

pectins are exposed to high temperatures during in both the food and

pharmaceutical industries. Acknowledgements The authors would like to thank the USDA and ISTC (T-1420 project) for support of this research. Cooperation with H.K. Chau at the ERRC ARS USDA on SEC/MALS experiments and data analysis is gratefully acknowledged.

12