1st Meet Hpressure

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Process Optimisation andMinimal Processing of Foods

Proceedings of the first main meeting

European CommissionCOPERNICUS PROGRAMME

Concerted action CIPA-CT94-0195

Esco

laSupe

rior d

e Biot

ecnolo

gia,Port

o, Portugal

, December1995

Editor : Jorge C. OliveiraProject Coordinator : Fernanda A. R. Oliveira

Area leader : Dietrich Knorr

Volume 4: High Pressure


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The proceedings of the first workshop organised by the COPERNICUS concerted action ProcessOptimisation and Minimal Processing of Foods in December 1995 at Escola Superior deBiotecnologia, Porto, Portugal consist of five booklets, one for each project area:

Thermal Processing Freezing Drying

High Pressure Minimal and Combined Processes

Each booklet includes all communications that were presented at the meeting and laterforwarded by the authors as written text, plus the questions and answers that were recorded.

The editors found that the style of writing and correctness of language use was very varied, aswould be expected, and have tried to contribute to a greater harmonisation by taking libertieswith everybodys English. Not being native English speakers ourselves, it is evident that fullycorrect English has not resulted from this exercise, but we hope that in this way all texts are fullycomprehensible and more similar in style. However, the revision was not thorough and sometyping mistakes plus grammatical errors can certainly be found here and there. No review has

been made concerning the scientific content of the communications. The sole purpose of theedition of the texts was concerned with the language and style and if any change in meaning hasresulted, we sincerely apologise for the fact.

It is reminded that at the end of the project the communications that were orally presented inthe three project meetings as area overviews, plenary lectures and short communications will becollected for the publication of a book, through a professional scientific publisher. The contentswill then be scientifically reviewed by the area leaders and the publisher will make a professionalreview of the English.

We would like to thank all project participants and particularly those that have contributed withwritten versions of the presentations, thus allowing for the production of this set of booklets that

we consider to be most valuable for promoting the interchange of results among partners and forproviding a valuable project dissemination.

We look forward to receiving any suggestions regarding these booklets.

Finally, we would like to leave a warm word of appreciation to Mrs. Isabel Lino, who had to dealwith everything that had to do with typing, file converting, scanning, and all those very boringcomputing tasks that were required for the final editing and publishing and also for hercommitment and work towards this project.

Porto, November 12th, 1996

Fernanda A. R. OliveiraJorge C. Oliveira

i

Proceedings of the first project workshop

Forew ord


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Proceedings of the first project workshop High Pressure

Dietrich Knorr

Process Assessment of High Pressure Processing of Foods: an Overview 1

Jacek Arabas 6 Monika Fonberg-Broczek

The High Pressure Research Center of Warsaw, Poland 9

Suzanne de Cordt, L. Ludikhuyze, C. Weesmaes, Marc Hendrickx & Paul Tobback

Process Assessment in High Pressure / Thermal Processing of Foods: the Role of Kinetics 18

Horst Ludwig, Heidger Marx 6 Bernhard Tauscher

Behaviour of Organic Compounds in Food under High Pressure:

Diels-Adler Reactions of Food Components 31

Christian Schreck, Gunter van Almsick & Horst Ludwig

Influence of Culturing Conditions on the Pressure Sensitivity ofEscherichia Coli 38

Monika Fonberg-Broczek, Jacek Arabas, Sylwester Porowski, Stefan Podlasin, Janusz Szczepek,

Bozena Windyga, Halina Sciezynska, Krystyna Gorecka, Anna Grochowska, Kazimierz Karlowski,

Janusz Jurczak & Piotr Salanski

The Effect of Ultra High Hydrostatic Pressure on Vegetative Microorganisms

and Spores of Chosen Bacteria and Moulds 46

Jacek Szczawinski, Janina Peconek, Malgorzata Szczawinska, Monika Fonberg-Broczek & Jacek Arabas

High Pressure Inactivation ofListeria monocytogenes in Meat and Meat Products 51

ii

Table of Contents


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1

High Pressure

Key process advantages of high pressure applications to food systems are the independence

of size and geometry of the samples during processing, possibilities for low temperature

treatment and the availability of a waste free, environmentally friendly technology. Opportunities

for effective and relevant utilization of the potential of high hydrostatic pressure center around

preservation processes, product modifications, and processes based on phase transitions or

membrane permeabilization. Scientific challenges are the lack of kinetic data, little

understanding of mechanisms involved in high pressure effects on food systems, limited

knowledge regarding the role of food constituents, and storage related changes of pressure

treated products. Technical challenges of commercial applications of high pressure technology

include material handling, process optimization, sanitation, cleaning and disinfection as well aspackage design. Engineering aspects to be dealt with are heat transfer issues and temperature

distribution within pressure vessels.

Non-thermal processes are currently receiving considerable attention from consumers as well

as from producers and researchers. Processes that are under evaluation or development include

high hydrostatic pressure treatment (Balny et al. 1992, Hayashi et al. 1994, Ledward et al. 1995),

the utilization of high electric field pulses (Knorr et al. 1995; Quin et al. 1996), or high intensity

light pulses (Dunn et al. 1995), the application of supercritical carbon dioxide (Haas et al. 1988,

Lin et al. 1992), and the use of magnetic fields (Pothakamury et al. 1993).

In addition, treatments with biopolymers (Popper and Knorr 1990, 1993) or with natural

antimicrobials (Gould 1994) are being applied or attempted. Various combinations of the above

mentioned unit operations with thermal processes are also being evaluated. Excellent reviews on

these subjects have recently become available (ie. Leistner and Gorris 1994, Gould 1995).

It is the aim of this paper to provide an assessment - within the given framework -of the

advantages, opportunities and challenges of high pressure processing of food.

Knorr

Summary

1. Introduction

Process Assessment of High Pressure Processing of Foods:

an Overview

Dietrich Knorr

Department of Food Technology, Berlin University of Technology,Berlin, Germany


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In addition to advantages of the application of high pressure to foods or food constituents

provided in the scientific literature (Cheftel 1992, Hayashi et al. 1994, Tauscher 1995) which

include effects on reaction rates and reaction volumes, membrane permeabilization and

influences on phase transitions, the instant transmittance of high pressure throughout food

systems and the consequent independence of size and geometry of the sample represents a

major advantage over conventional thermal processing where size and geometry can be process

limiting factors. For example, size reduction required in conventional thermal processing to

improve heat and mass transfer during processing is often accompanied by elevated losses of

nutrients and subsequent environmental pollution (i.e. in hot water blanching processes). Such

independence of size and geometry of the samples could not only reduce process severity and

thus lead to higher product qualities, but also increase process flexibilities and ultimately

revolutionize food processing by making requirements for size reduction obsolete.

Another key advantage of high pressure application is the possibility to perform processing

at ambient or even lower temperatures. Indications exist that processing at subzero

temperatures can be more effective with regards to inactivation of microorganisms or enzymes

(Hayashi 1995, Knorr 1995). Low temperature processing can help to retain nutritional quality

and functionality of the raw materials treated and could allow maintenance of consistently low

temperatures during postharvest treatment, processing, storage, transportation and distribution

periods of the the life cycle of food systems.

Finally, the fact that high pressure processing is environmentally friendly, and a basically

waste free technology, needs attention. For example, Eshtiaghi and Knorr (1993) obtained

significantly less leaching of cell constituents after high pressure blanching of potato cubes as

compared to hot water blanching. In addition, potential for future omission of size reduction of

foods prior to processing could substantially reduce food processing wastes (i.e. resulting from

contents of ruptured plant or animal cells or tissues).

3.1.Preservation of foods and related substances

A vast amount of empirical information is available regarding the effects of high hydrostatic

pressure on a wide range of vegetative microbial cells (Gould, 1995; Hoover, 1993; Knorr, 1995).

Bottlenecks such as the baroresistance of microorganisms within environments of low water

activity (Oxen and Knorr, 1993) could be overcome by combinations with mild heat or by

pretreatment with ultrasound (Oxen-Bodenhausen, unpublished data). Work on pathogenic

microorganisms is still scarce in the published literature (Patterson et al., 1995) and needs

2

Process Optimisation and Minimal Processing of Foods Process Assessment

2. Advantages of high hydrostatic pressure

3. Opportunities for high hydrostatic pressure


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continued attention.

Increased pressure resistance of bacterial spores as compared to vegetative cells has been

demonstrated repeatedly (Gould, 1995). Temperature or pressure induced germination of

spores and subsequent inactivation of germinating or germinated cells by treatment with high

pressure or combination processes is one route that is currently being considered (Knorr,

1995). Methodologies have

been developed (Heinz and

Knorr, 1995) to study

germination processes via the

release of dipicolinic acid and

to monitor germination

processes during pressure

treatment via absorbance

measurements of spores in a

pressure cell with optical

windows (Figures 1 and 2).

Successfull preservation

operations often depend on the

effective reduction of

enzymatic activity during

processing. Consequently, one

of the requirements for high

pressure processing should

include the effective reduction

of undesirable enzymatic

activity (especially oxidases) to

ensure high quality, shelf stable

products. There is a vast

amount of publications dealing with the effects of high

pressure on food related

enzymes (Cheftel, 1992; Hara

et.al., 1990, Seyderhelm et al., 1996) indicating that certain food enzymes can be reduced by

high pressure to tolerable levels, but it also contains a wide range of sometimes conflicting

information (i.e. Anese et al., 1995).

It seems clear that food constituents are affecting baroresistance of enzymes (Asaka and

Hayashi, 1991 Ogawa et al., 1990; Seyderhelm et al., 1996) and it also seems evident that when

evaluating pressure effects on given enzyme systems under given conditions, a case by case

approach is necessary.3

Knorr High Pressure

Figure 1 - Prototype of high pressure cell with optical windows

Figure 2 - Relative absorbance of Bacillus subtilis spores (ATCC

9327) during pressure treatment at 20C or 38.5C

measured at 580 nm (Heinz and Knorr 1995)


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3.2.Modifications of foods and related substances

An extensive set of data exists on gelling behaviour of proteins, polysaccharides and to some

extent also on protein/polysaccharide combinations under high hydrostatic pressure conditions

(Balny and Masson, 1993; Dumay et al., 1994; Ohshima et al., 1993). Because of the differences in

functionality experienced between pressure and temperature induced gels (Ohshima et al., 1993),

a wide field for product modifications via pressure or pressure/temperature treatments becomes

available. Changes in composition and functionality of plant tissues have also been identified. For

example hardening of vegetable tissues (Eshtiaghi, unpublished data; Kasai et al., 1995) and the

formation of solid gels during cold storage of kiwi or strawberry puree (Seyderhelm and Knorr,

unpublished data; Rovere, 1995) has been observed. The most likely explanation seems a

pressure induced change of pectins, which could also be caused by residual activity of pressure

tolerant enzymes such as pectin esterase and for the release of calcium ions.

Within this context it appears also highly interesting to indicate the effects of high pressure

on plant cell cultures as model systems for plant foods (Knorr 1994).

Current investigations in our laboratory on the stress response of cultured plant cells to high

pressure treatment indicate that treatment at 90 MPa and higher results in instant cell death

without subsequent stress reactions of the cell. Lower pressures lead to a time delayed stress

response, suggesting pectin degradation and an elicitor effect of such degradation products

(Drnenburg and Knorr, 1995).

3.3.Phase transition in food systems

Pressure induced phase transitions such as crystallization of lipids (Buchheim and Abou El-

Nour, 1992), or thawing or freezing of high moisture systems (Kalichevsky et al., 1995) offer

numerous opportunities for process or product development. However, some engineering

challenges still exist, such as the rapid removal of the heat of fusion (Fig.3) because of instant ice

crystal formation during

pressure shift freezing and therequirement for studies on the

kinetics of ice nucleation, or

crystal size, distribution and

growth, as well as on

recrystallization.

4


Figure 3 - Pressure and temperature conditions during pressure shift

freezing of potato cubes (Koch et al. 1995)


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3.4.Membrane permeabilization

The permeabilization of membranes of vegetative microbial cells as well as of plant

membranes has been demonstrated (Drnenburg and Knorr, 1994, Knorr, 1994, Osumi et al.,

1992). This has led to the inactivation of microbial cells and has opened new opportunities for

process development. For example, mass transfer during dehydration of plant tissues (Esthiaghi

et al., 1994), during processing of french fries, during pasteurization of strawbwerries (Fig.4), or

during high pressure blanching (Esthiaghi and Knorr, 1994) could be affected. Work is under way

in our laboratory to attempt to understand the mechanisms involved in the phenomena

observed.

4.1.Scientific challenges

Key areas where additional information is required, include the need for kinetic data on the

inactivation of microorganisms and enzymes as well as on the changes of food quality and

functionality; a better understanding of the mechanisms involved during high pressure treatment;

experiments clarifying the interactions between food constituents and high pressure effects on

food systems; the necessity to gain more knowledge regarding interactions between high

pressure and nutrients, toxins or allergens; and finally compilation of data during post pressure

treatment storage of food materials.

Attempts are under way to accumulate data on inactivation kinetics of microorganisms. Time-

inactivation curves of a statically pressurized test organism (Bacillus subtilis ATCC 9372)5

Knorr High Pressure

20 40 60 AB

CD

0

10

20

30

40

50

sugar

content

(Brix)

Immersion in sugar solution (min)

Figure 4 - Total solids of pressure thawed + pasteurized versus. athmospheric pressure thawed +

pasteurized strawberries (Esthiaghi and Knorr, unpublished data).

A=Freezing+Immersion in a sugar solution (25C, 60 min)

B=Freezing+Immersion in a sugar solution (92C, 20 min)

C=Freezing+HP(600 MPa, 50C, 15 min) +Immersion in a sugar solution (25C)

D=Freezing+HP(600 MPa, 50C, 15 min) +Immersion in a sugar solution (92C, 20 min)

4. Challenges for high pressure R&D in food science and technology


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suspended in Ringers solution at 20C typically showed a sigmoid, non-symetric shape when

plotted on logaritmic scale (Fig.5).

A mathematical description of experimental results was possible by fitting the accumulated

Weibull-distribution as a flexible two-parametric function. The resulting good agreement

between predicted and experimental number of survivors makes this approach a usefull tool for

comparison and development of high pressure processes.

4.2.Technical/engineering challenges

Technical challenges of commercial application of high pressure technology are, according to

Mertens (1995), material handling; package design; sanitation, cleaning and disinfection of high

pressure equipment; bulk or in-container processing; and high pressure short time processing or

low pressure long time processing. In addition, heat transfer within pressure transferring media;

temperature distribution within pressure vessels and pressure distribution within food materials

- due to differences in compressibilities because of the complex composition of foods (and other

biological systems such as microorganisms) - are engineering issues that require attention.

Parts of this work have been funded by grants from the European community (EC-AIR CT92-

0296), the German Research Foundation (DFG Kn 260/3-1,3-2,3-3) and the Research Foundation

of the German Food Industry (AIF-FV 8774, AIF-FV 9918). Major parts of this paper have also been

published in Hayashi, R. and Balny, C. 1996. High Pressure Bioscience & Biotechnology. Elsevier

Science Amsterdam.

6


-5

-4

-3

-2

-1

0

0 400 800 1200 1600 2000 2400

250MPa

UHP treatment time (s)

survivorslog (N/No)

Figure 5 - Typical time-inactivation curve of Bacillus subtilis after high pressure treatment at 250 MPa

and 20C (Heinz and Knorr, 1995)

Acknowledgements


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Anese, M., Nicoli, M. C., Dallglio D. & Lerici, C. R. (1995). J.Food Biochem. 18, 285.

Asaka, M. & Hayashi, R. (1991). Agric.Biol.Chem. 55, 2440

Balny, C., Hayashi, R., Heremans, K., Masson, P. (1992). High Pressure and Biotechnology, John

Libbey Eurotext, Montrouge

Balny, C. & Masson, P. (1993). Food Rev. 9 , 611

Buchheim, W. & Abou El-Nour, A. M. (1992). Fat Sci.Technol. 94 , 369

Cheftel, J. C. (1992), in: Balny, C., Hayashi, R., Heremans, K. & Masson, P. (eds), High Pressure and

Biotechnology, John Libbey and Co. Ltd., London, 195

Drnenburg, H. & Knorr, D. (1995). Enzyme Microb.Technol. 176, 74

Drnenburg, H. & Knorr, D. (1994). Food Biotechnol. 8, 57

Dumay, E., Kalichevsky, M. T. & Cheftel, J.-C. (1994). J.Agric.Chem. 42, 1861

Dunn, J., Ott, T., Clark, W. (1995). Food Technology 49(9), 95

Eshtiaghi, M. N. & Knorr, D. (1993). Journal of Food Science 58, 1371

Eshtiaghi, M. N., Stute, R. & Knorr, D. (1994). Journal of Food Science 59, 1168

Gould, G. W. (1995). New Methods of Food Preservation, Blackie Academic & Professional,

Glasgow

Haas, G. J., Prescott, H. E., Duddley, E., Dik, R., Hintlian, C., Keane, L. (1988). Journal of Food

Safety 9, 253

Hara, A., Nagahama., G., Ohbayashi, A., & Hayashi, R., (1990). Nippin Nogeikagaku Kaishi 64,

1025

(1994). High Pressure Bioscience, San-Ei Suppan Co., Kyoto

Kunugi, S., Shimada, S., Suzuki, A. (eds.) (1994), High Pressure Bioscience, San-Ei Suppan

Co.,Kyoto

Hayashi, R. (1996). Abstract for ISOPOW, 6th meeting St. Rosa, CA, 2-8 March

Heinz, V. & Knorr, D. (1995). Annual Report, EC project on High Pressure Processing of Foods (AIR-

CT92 - 0296)

Hoover, D. G. (1993). Food Technology 47(6), 150Kalichevsky, M. T., Knorr, D. and Lillford, P. J. (1995). Trends Food Sci.Technol. 6, 253

Kasai, M., Hatae, K., Shimada, A. & Iibuchi, S. (1995). Nippon Shokuhin Kagaku Kogaku Kaishi 42,

594

Knorr, D., Geulen, M., Grahl, T., Sitzmann, W. (1994). Trends Food Science & Technology 5, 71

Knorr, D. (1994). Trends Food Science & Technology 5, 328

Knorr, D. in: G. W. Gould (ed.), New Methods of Food Preservation, Blackie Academic &

Professional, 159, London 1995

Koch, H., Seyderhelm, I., Wille, P., Kalichevsky, M. T. & Knorr, D. Nahrung-Food (submitted)

Ledward, D. A., Johnston, D. E., Earnshaw, R. G. & Hasting, A. P. M. (1995) (eds.). High Pressure

Processing of Foods, Nottingham University Press, Nottingham, 77

Knorr High Pressure

References


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Leistner, L. & Gorris, L. G. M. (1994). Final Report, FLAIR Concerted Action no.7, Subgroup B, EC,

DG XII, Brussels

Mertens, B. in: G. W. Gould (ed.), New Methods of Food Preservation, Blackie Academic &

Professional, London 1995

Ogawa, H., Fukuhisa, K., Kubo, J. & Fukumoto, H. (1990). Agric. Biol. Chem. 54, 1219

Oshima, T., Ushiod, H.. Challenges of High Pressure Processing of Food

8



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This communication describes the food activities at the High Pressure Research Center

UNIPRESS of the Polish Academy of Sciences in Warsaw. A short description of Unipress history

and activities is given and the main fields of interest, current projects and high pressure

equipment available in the Center are described. Results of research projects involved in food

processing programs performed since 1992 are summarised. Specifications of the equipment

used for investigation of the process of high pressure treatment of foods are presented. Future

perspectives and basis of planned projects close the communication.

The High Pressure Research Center UNIPRESS of the Polish Academy of Sciences was created

in 1972. Unipress is entirely dedicated to high pressure research and it groups the largest

scientific and engineering staff working in the high pressure field in Poland - 55 scientists and

engineers. It is located in 4000 sq. m. in Warsaw, and there is also a second site, located in the

country village of Celestynw, close to Warsaw, with several laboratories and a conference center.

The Center was created and is headed since its opening by Professor Sylwester Porowski.

The main fields of interest of the researchers working in the Center are:

- high pressure physics of semiconductors, superconductors, metals, and high temperature

ceramics,

- materials science research related to high pressure technologies: crystallisation, plastic

deformation and sintering,

- high pressure research techniques and technologies, and

- food processing.

Recently, food projects have become one of the leading activities performed in the Center.

9

Arabas & Fonberg-Brockzek High Pressure

Summary

1. Introduction

The High Pressure Research Center of Warsaw, Poland

Jacek Arabas1

, Monika Fonberg-Broczek1,2

1 High Pressure Research Center, Polish Academy of Sciences, Warsaw, Poland2 Department of Food Research, National Institute of Hygiene, Warsaw, Poland


13/59

2.1 Departments of Unipress

The High Pressure Research Center consists of several research laboratories, among which the

last two are involved in food projects:

1. Laboratory of Semiconductor Research

(AIIIBVand AIIBVI semiconductors, impurity states, quantum wells, superlattices)

2. Crystallisation Laboratory

(nitrides AIIIN, AIIBVI, thermodynamics and crystal growth)

3. Superconductivity Laboratory

(MoN, NbN, Y-Ba-Cu-O, BSCCO)

4. Grain Boundaries and Sintering Laboratory

(bicrystals, diffusion and phase transitions in grain boundaries, sintering - SiC and Si3N4)

5. Hydroextrusion Laboratory

(copper, aluminium, gold melts, super conducting wires, hydroextrusion, mechanical

properties)

6. High Pressure Equipment Laboratory UNIPRESS EQUIPMENT

(development, manufacturing and commercialisation of high pressure equipment)

7. Laboratory of Food Processing

(processing of fruits and vegetables under HP, promotion of UHP food research in Poland)

2.2 High-pressure facilities

In our research laboratories, the high pressure facilities developed in the Center are used.

Among these are:

1. Crystallisation of single crystals and of epitaxial layers in high gas pressure and high

temperature-pressure up to 2.5 GPa (25 kbars); temperatures up to 2500 K.

2. Apparatus for the investigation of grain boundary diffusion.(Pmax = 1.5 GPa, Tmax = 1500 K)

3. Hydroextrusion of metals at elevated temperatures.

(Pmax = 1.5 GPa, Tmax = 800 K)

4. Electron transport and magnetotransport measurements in high gas and liquid pressures,

as well as in helium temperatures and magnetic fields up to 16T.

(Pmax = 1.7 GPa)

5. Measurements of magnetophotoconductivity in the far-infrared range.

(Pmax = 1.8 GPa, liquid helium temperatures, magnetic fields up to 9 T, far-infrared

wavelengths from 70 mm to 300 mm)

6. Investigation of chemical reactions, sintering and crystal growth processes in high O210


2. Outline of activities


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pressure.

(Pmax = 0.5 GPa, T = 1800 K).

7. Various designs of diamond anvil cells - available pressures up to 50 GPa, possibility of

working at low temperatures down to 4 K.

8. Food processing laboratory test stands:

* type 1 - for microbial cultures pressurised up to 2.0 GPa, sample volume 30-100 cc,

* type 2 - for food samples of larger volume (400 - 2000 cc).

The high pressure equipment made by Unipress is also used in several hundreds of research

laboratories of universities and scientific institutes all over the world.

2.3 High pressure technologies

Unipress possesses know-how in the field of many high pressures technologies, which already

have been commercially applied. For example:

2.3.1. Isostatic pressing - specially Cold Isostatic Pressing (CIP)

The technology of CIP is mainly applied as a shaping technology for powdered materials, for

example metal powder, ceramics, carbon/graphite and plastic powders. Depending on the type of

pressed materials, the pressures applied vary from 50 MPa up to 600 MPa. The parameters of CIP

technology are very similar to those applied in Ultra High Pressure (UHP) food technology.

2.3.2. Hydroextrusion

Hydrostatic extrusion is essentially the process in which a billet is extruded through a die

using liquid under high pressure, up to 1.5 GPa. The main application of hydroextrusion

technology is manufacturing wires (silver, gold, aluminium, copper), composites, pipes and

others.

2.3.3. Crystal growth and synthesis of materials under pressure

The newest technology on which the High Pressure Research Center focuses its activities is

crystal growth and synthesis of materials under pressure. This technology is applied to produce

semiconductors and superconductors with special properties. The parameters of this process are

extremely high, pressure up to 1.5 GPa and temperatures up to 2000 oC. The crystal growth

process lasts for several days.

Unipress successfully transferred high pressure technologies to several new companies

initiated and organised by the Center:

Cynel-Unipress, Warsaw, Poland - high pressure components for the metal and electrical

industries - solders, capillaries,

Hydron-Unipress, Lodz, Poland - technological extrusion equipment,11



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Izopress, Warsaw, Poland - Moscow, Russia - advanced ceramic materials: SiC, isostatic

pressing equipment,

Laboratory of Crystal Growth Physics, Warsaw, Poland - semiconductors,

KD-Unipress, Warsaw, Poland - devices for sample preparation: orientation, cutting, lapping,

especially superfine wire saws,

Wacer, Warsaw, Poland - high density alumina,

Small-Tube Poland, -hydroextrusion products: tubes, capillaries.

3.1 Background

The food programme is entirely new, combining the scientific basis of high pressure

techniques with food processing technology and food science. The laboratory of Food Processing

started the first project Application of ultra high pressure for processing of fruits in September

1992, in close collaboration with research groups from three other institutes:

- microbiologists from the Department of Food Research, National Institute of Hygiene,

Warsaw,

- chemists from the Institute of Organic Chemistry of the Polish Academy of Sciences, Warsaw,

- engineers from the Institute of Aeronautics and Applied Mechanics, Warsaw University of

Technology.

The activity of the interdisciplinary team co-ordinated by the High Pressure Research Center

led to a widespread programme with perspectives of taking laboratory experiments into

commercial application of UHP technology in Poland.

3.2 Project Evolution

Since 1992 we have performed over 1000 pressure tests in order to investigate samples of

model bacterial cultures and food products using our two food processing laboratory test standsand other high pressure installations. This activity was sponsored by the National Committee of

Sciences. The results of the study were accepted by the Committee and we obtained a new grant

for further investigation in this field in 1996-1997. Also, hundreds of high pressure tests for the

most active food research institutes in Poland, which we closely collaborate with, were

performed.

3.2.1. Scientific interests

The main lines of research are the following:

- Analysis of the effect of high hydrostatic pressure on Gram-negative bacteria (Salmonella,

Escherichia coli, Proteus mirabilis, Listeria monocytogenes), Gram-positive bacteria (Staphylococcus

12


3. Food programme


16/59

aureus), spores ofBaccilius cereus, yeasts (Sacharomyces cerevisiae, Candida albicans) and moulds

(Aspergillus flavus) in terms of pressure, temperature and time of exposure. The microorganisms

listed above often contaminate food and may cause foodborn diseases.

- Determination of mechanisms of killing bacteria with high pressure treatment - microscopic

studies of changes in microbial cell morphology.

- Evaluation of high hydrostatic pressure effect on the quality of foods: taste, flavour, colour.

3.2.2. Specification of equipment

In our investigations we have used the following high pressure equipment:

1. Laboratory test stands for microbial cultures equipped with U 101 set-up for research in

microbiology

- working pressure: 1300 MPa,

- temperature range: 278 - 393 K,

- pressure chamber volume: 30 - 100 cc,

- pressure chamber inner diameter: 16 mm,

- configuration: piston - cylinder,

- pressure transmitting medium: isopentane,

- source of pressure: hydraulic press 300 kN,

- pressure measurement: double measurement directly in the pressure chamber using

manganine gauge and Bourdon type manometer of the hydraulic press,

- heating or cooling: using an external thermostat coupled to the outer jacket of the chamber,

- temperature measurement: double measurement directly in the pressure chamber using a

thermocouple Cu+Constantan and a temperature gauge inserted in the cooling/heating jacket.

2. Food processing laboratory test stands for food samples of larger volume


- temperature range: 278 - 353 K,

- pressure chamber volume: 400 - 600 cc,

- pressure chamber inner diameter: 50 mm,- configuration: piston - cylinder,

- pressure transmitting medium: water,

- source of pressure: hydraulic press 2500 kN,

- pressure measurement: Bourdon type manometer of the hydraulic press,

- heating or cooling: using an external thermostat coupled to the outer jacket of the chamber,

- temperature measurement: temperature gauge inserted in cooling/heating jacket.

3. Gas compressor 1.5 GPa (commercialised under U11 name)

13



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4. Food processing laboratory test stands for food samples 500 - 2000 cc


- pressure transmitting medium: water,

5. Optical gas and liquid cells

- working pressure 1.2 GPa,

- temperature range 1 - 400 K,

- optical window diameter 2 - 4 mm.

3.2.3 National collaboration

Collaboration of national level includes:

1. Warsaw Agricultural University, Faculty of Veterinary Medicine, Department of Food

Hygiene (Listeria monocytogenes),

2. Warsaw Agricultural University, Department of Human Nutrition (meat and fruit juices),

3. Warsaw Agricultural University, Dept. of Technology of the Fruit and Vegetable Processing

Industry (modification of pea protein),

4. Olsztyn University of Agriculture and Technology, Institute of Food Biotechnology (cheese,

meat, plant oils),

5. Gdansk University, Department of Microbiology (high pressure shock in bacteria proteins),

6. National Food and Nutrition Institute (development of standards for UHP products on basis

of Polish legislation),

7. Institute of Agricultural and Food Biotechnology (fruit products),

8. Institute of Organic Chemistry of the Polish Academy of Sciences (natural food pigments),

9. Institute of Aeronautics and Applied Mechanics, Warsaw University of Technology (design

methodology).

It is worth mentioning that this collaboration has formed the basis for ten research works for

Master of Science degree.

3.2.4 International collaborationWe have developed an international collaboration with:

1. Konan Womens University in Kobe, Japan (visiting professor of food technology 1995-

1996),

2. Central Food Research Institute in Budapest, Hungary (combined processes - irradiation

and UHP)

3.5 Achievements

In the three years of activity of the Laboratory of Food Processing, several milestones have

been reached:

14



18/59

1. Collecting experimental data on high pressure inactivation of microorganisms - comparison

with data published in literature. Our study covered the following samples: Gram-positive

and Gram-negative bacteria, yeast, spores ofBacillus cereus and moulds as well as food

samples contaminated with bacteria: fruit, fruit juices, meat and meat processed products

(Fonberg-Broczek et al. 1995, Windyga at al. 1994).

2. Determination of the conditions of high pressure inactivation ofListeria monocytogenes

(Szczawinski at al. 1995).

3. Determination of the influence of high pressure on natural food pigments such as: carotene,

chlorophyll, annato, kurkuma (data not published).

4. Laboratory production of apple jam (the samples were used in sensory studies - evaluation

of discriminants: appearance, colour, flavour, taste).

5. Design of apparatus for high pressure research in microbiology. This system was

constructed in the Laboratory of Unipress Equipment and is now used in ours and fifteen

other centers.

6. Availability of our food processing laboratory test stands for other research centers.

Unipress is leading and co-ordinating center in high pressure food processing equipment

in Poland.

3.6 Perspectives

Interdisciplinary collaboration of many specialists contributing to our food programme

creates the possibility of designing new experimental research projects and technological

advancement.

1. Research project Investigation of Biologically Active Substances and Destruction of Micro-

Organisms and Enzymes in Gelled Fruit Products Preserved by High Pressure. (Sponsored

by the Polish Committee of Sciences for 1996 - 1997). Performers: High Pressure Research

Center, National Institute of Hygiene, Department of Food Research, Institute of

Agricultural and Food Biotechnology.

2. Comparison of two methods of inactivation ofListeria monocytogenes: irradiation and UHP.Application for a new research project Effect of UHP Treatment on Pathogens in Vacuum

Packed Sliced Meat Products. Collaborators: Warsaw Agricultural University, Faculty of

Veterinary Medicine, Department of Food Hygiene, Poland, National Institute of Hygiene,

Department of Food Research, Warsaw, Poland, Central Food Research Institute in

Budapest, Hungary.

3. Introduction of a new laboratory food processor (2.000 cc) as a commercial offer.

4. Construction of semi-industrial, medium capacity equipment (50.000 cc) in 1998. We are

seeking industrial partners for commercial processing of foods.

15



19/59

The Unipress programme of high pressure processing of food proceeded in our center since

1992 and provided basic experimental data on Ultra High Pressure effect on microorganisms. The

involvement and leading role of scientists and engineers of the High Pressure Research Center

has created an easy access to high pressure facilities and the possibility of developing new

laboratory and industrial food processes. A common action of interdisciplinary teams of

researchers may help overcome financial and psychological barriers against introducing high

pressure technology in commercial processing of food.

The High Pressure Research Center wishes to thank the co-ordinators of the Copernicus

Project Process Optimisation and Minimal Processing of Foods (CONTRACT CIPA - CT94-0195)

for the invitation to join the project.

Fonberg-Broczek, M., Windyga, B., Sciezynska, H., Grochowska, A., Gorecka, K., Salanski, P.,

Arabas, J., Szczepek ,J., Podlasin, S. & Porowski, S. (1995). Effect of High Hydrostatic Pressure on

Microorganisms. Symposium:High pressure effects on foods, 9th World Congress of Food Scienceand Technology, Budapest, Hungary, 07.30-8.04,1995, Abstracts Vol. II. P207, p. 74.

Fonberg-Broczek, M., Windyga, B., Sciezynska, H., Gorecka, K., Grochowska, A., Napiorkowska,

B., Karlowski, K., Arabas, J., Jurczak, J., Podlasin, S., Porowski, S., Salanski, P. & Szczepek, J.

(1995). The Effect of High Hydrostatic Pressure on Vegetative Bacteria and Spores ofAspergillus

flavus and Bacillus cereus. Joint XV AIRAPT & XXXIII EHPRG International Conference High

Pressure Science & Technology, Warsaw, Poland, September 11-15, 1995, Conference Proceedings

High Pressure Science & Technology by World Scientific, in press.

Fonberg-Broczek, M., Arabas, J. & Porowski, S. (1995). The Effect of High Hydrostatic Pressure in Vegetative Microorganisms and Spores of Chosen Moulds. 1st Main Meeting Process

Optimization and Minimal Processing of Foods COPERNICUS PROGRAMME, CONTRACT CIPA -

CT94-0195, Porto, December 6,7,8 1995, also published in this book.

Kolakowski, P., Reps, A., Babuchowski, A., Zmudzian, L, Podlasin, S. & Porowski, S. (1995).

Influence of High Pressure on Changes of Cheeses Characteristics. Joint XV AIRAPT & XXXIII

EHPRG International Conference High Pressure Science & Technology, Warsaw, Poland,

September 11-15, 1995, Conference Proceedings High Pressure Science & Technology by World

Scientific, in press

16


4. Conclusions

Acknowledgements

References


20/59

Szczawinski, J., Peconek, J., Fonberg-Broczek, M., Arabas, J. & Szczawinska, M., (1995).

Mozliwosci zastosowania wysokich cisnien do inaktywacji L. monocytogenes w miesie i

przetworach miesnych ( Listeria monocytogenes inactivation in minced meat and sliced ham).

Przeglad weterynaryjny, 4 (20), 516-519

Szczawinski, J, Peconek, J., Szczawinska, M., Porowski, S., Fonberg-Broczek, M., Arabas, J. (1995).

The Effect of High Hydrostatic Pressure onListeria monocytogenes in Minced Meat and Sliced Ham

in Ambient Temperature. 1st Main Meeting Process Optimization and Minimal Processing of

Foods COPERNICUS PROGRAMME, CONTRACT CIPA - CT94-0195, Porto, December 6,7,8 1995,

also published in this book

Windyga, B., Fonberg-Broczek, M., Sciezynska, H., Gorecka, K., Grochowska, A., Arabas, J.,

Szczepek, J., Podlasin, S., Porowski, S. (1994). Effect of High Hydrostatic Pressure on Yeast Candida

albicans. 7th International Congress of Bacteriology and Applied Microbiology Division, 7th

International Congress of Mycology Division, Prague, Czech Republic, July 3rd - 8th, 1994,

Abstract book, PC-6/52, p. 272

17



21/59

In the course of the last five years of research on high pressure (HP) effects (103 to 104 bar)

on bio-macromolecules and food, it has become established that HP has a potential as a new unit

operation in food processing and preservation. It has been shown clearly that HP can inactivate

microorganisms and enzymes while it only slightly affects nutritional and sensorial quality

aspects of food 5,20,32,37. This corresponds to the current consumer demand for fresh like

products and offers a major advantage in comparison with classical thermal (HT) preservation

technologies. Most authors believe that the most safe and economically feasible use of HP in

food preservation will be in combination processes, especially with moderate temperature

elevation (HP/T) 5,12,20,30,32,37.

Since 1990, a number of HP-pasteurized fruit products have been introduced on the Japanesemarket. However, Europe and the US are still awaiting the establishment of the reliability of HP-

treatments. This requires specific technological research. Critical issues that should be studied

are the development of a terminology and methods for quantitative assessment of a HP(/T)

process impact, and equipment performance in terms of process repeatability and uniformity.

This communication discusses these two issues, and how they are closely related to kinetics.

Up to date, there is no mention of a concept, terminology or method for HP process impact

assessment in the open literature. However, this will be indispensable to fulfil legislative as well

as quality demands.

A straightforward approach to this challenge is to follow the lines of the well established

terminology and methods for quantitative evaluation of thermal (HT) preservation processes.

First, HP will probably be combined with moderate temperature elevation, hence the parameter

T will be involved again. Second, the concept used for quantitative evaluation of thermal

processes, i.e. equivalent time at a constant reference temperature, mostly denoted F , is very

robust. F represents the integral impact of time and temperature on a given system. The system

of interest may be a microorganism, an enzyme, a chemical substance (e.g. vitamin), colour, or

18


Summary

1. Development of a concept, terminology and methods for quantitative assessment

Process Assessment in High Pressure / Thermal processing of Foods: the Role

of Kinetics

De Cordt, S., Ludikhuyze, L., Weemaes, C., Hendrickx, M., and Tobback, P.

Universiteit te Leuven, Kardinaal Mercierlaan 92, 3001 Heverlee, BELGIUM


22/59

any other safety or quality determining factor.

In the Thermal Death Time terminology, commonly used in food technology but only

appropriate with first-order reactions, F is defined as:

(1)

where D is the decimal reduction time at temperature T, Drefthe decimal reduction time at a

chosen reference temperature Tref, and t time.

In a more general terminology working with rate constants, F can be written as

(2)

where k is the rate constant at temperature T, and krefthe rate constant at a chosen reference

temperature Tref.

In these definitions, the importance of kinetics is apparent. The D-values or rate constants are

explicitly present, and the z-value or activation energy Ea plays a role in the ratio of Drefto D,

and of k to kref, respectively. A survey of the main kinetic models and parameters and their

significance is presented in Table I.

Besides a concept and terminology for quantifying a process impact, one needs methods to

determine the exact process impact value. Again, it is straightforward to refer to the established

methodology in the area of thermal preservation. There, three approaches can be

distinguished.14 Definitions, formulas involved, advantages and disadvantages are summarized

in Table II.

In the in situ method, one is measuring the response of the actually monitored parameter

before (X0) and after (Xt) processing. This method is simple, but very limitedly applicable because

often it is a tedious job to measure X accurately, and the detection limits may be inconvenient.

A good example is the evaluation of the safety of low acid canned food, where the Probability ofa Non-Sterile Unit (PNSU) should be < 10-9.

The physical-mathematical method relies on integration of the actual (t,T) profile. The

necessary (t,T) data can be obtained by either direct registration of T during the process, or from

reconstruction of the (t,T) history, based on empirical formulas or theoretical heat transfer

models. The physical-mathematical method has a widespread use, but there are some limitations.

Direct registration of the time-temperature profile is often not appropriate and/or difficult, e.g.

the case of continuous processing of liquid foods containing solid particles. As to the use of

models for reconstruction of the profile, the most important problem is the lack of accurate

values of the model parameters (e.g. thermophysical and flow characteristics).

Time-temperature integrators (TTIs) are small, wireless devices that allow the calculation of

Fk

kref

t

= 0

FD

Ddtref

t

= 0

19

De Cordt, Ludikhuyze, Weemaes, Hendrickx & Tobback High Pressure


23/59

20


Table I

Survey of main kinetic models and parameters and their significance

Thermal Death Time (TDT) terminology general terminology

only appropriate for first-order! applicable for any value of order

(at a specified constant temperature T andconstant pressure P)

where t is time, X (0) the system response (attime zero), and D the decimal reduction time

(if n = 1)

(if n 1)

(at a specified constant temperature T andconstant pressure P)

where n is the reaction order, t is time, X (0)the system response (at time zero), and kthe rate constant

D(T)(P)= decimal reduction time = time

required, at a specified constant temperatureT and constant pressure P, to reduce thesystem response (X) with one logarithmic unit(90%)

k(T)(P)= rate constant at a specified

temperature T and pressure P

(at a specified constant pressure P)

where Trefis a chosen referencetemperature, D (T) and DTrefthe decimal

reduction times at T and at T ref, respectively,

and z the z-value

(at a specified constant pressure P)

where T refis a chosen referencetemperature, k (T) and kTrefthe rate constants

at T and at T ref, respectively, and E a the

activation energy

z = temperature in- or decrement torespectively de- or increase D with onelogarithmic unit

Ea = activation energy

D depends on P, but there is no generallyaccepted explicit equation

(at a specified constant temperature T)

where P refis a chosen reference pressure

(usually atmospheric P), k (P) and kPrefthe rate

constants at P and at P ref, respectively, and

V the activation volume

log X( ) = log X0( ) t

D

ln X( ) = ln X0( ) kt

X = X01 n + n 1( )kt[ ]

1

1 n

logD T( ) = logDTref +Tref T

z

kT( ) = kTref expEa

R

1

Tref

1

T

kP( ) = kPr efexpPV

RT


24/59

F from its response status. They are designed to display an easily and accurately measurable

response, thus avoiding the problems associated with the in situ method.

The problems associated with the respective approaches in the thermal area, will analogously

exist in the HP(/T) area. Then, since the applicability of the in situ method will also be very limited

in the HP(/T) area, one should rather focus on the development of the physical-mathematical

approach and design of integrators for evaluation of HP(/T) processes.

From the formulas in Table II, the importance of kinetics is obvious. With the physical-

mathematical method, the Ea-value is explicitly involved. With the in situ method and the use of

TTIs, the value of n determines which formula is to be used, and the values of kref(and of n if

n1) are explicitly involved. Furthermore, with the use of TTIs, the Ea- value is indirectlyinvolved, because the most important requirement of an integrator is that its Ea is equal to that

of the actually monitored parameter 9,14,40.

For quantification of a process impact on safety and quality, kinetic data are needed on

microorganisms, enzymes, quality and structural properties of food. We need kinetic data on

pathogenic and spoilage bacteria, yeasts, moulds and viruses, on their vegetative and (if

sporeforming) spore state, their inactivation, resuscitation, germination, and how all these states

and reaction kinetics are influenced by factors such as medium composition and growth. For food

quality related enzymes, the kinetics of inactivation, reactivation and activation should be

determined. In relation to quality, kinetic information on destruction of vitamins, changes in

appearance and flavour, and formation of new, possibly toxic compounds, is required. Also, the21


Table II

Methods for determination of thermal process impact

in situ physical-mathematical time-temperature -

integrators (TTIs)

= from response of

monitored parameter itself

= from (T,t) profile = small, wireless device

with representative

response9,14,40

n=1:

n1:

n=1:

n1:

advantage: simple

disadvantage: very

limited applicability

advantage: works with

controlable variables

disadvantage: limited

applicability

advantage: general

applicability

not yet available; being

developed (e.g. References

8,9,14,40)

F =1

krefln

X0

Xt

F =1

kref

X t1n X0

1n

n 1

F = expE A

R

1

Tref

1

T

t

0

dt

F =1

krefln

X0

Xt

F =1

kref

X t1 n X0

1n

n 1


25/59

kinetics of structural changes like gelification should be established.

In order to have a view on the state of the art in these fields, we compiled most papers that

appeared in journals and some contributions to conferences. For microorganisms and enzymes,

a survey is presented in Frame 1 and Frame 2, respectively.

Concerning the effects of HP on food quality, there is a recent review on chemical aspects37, a

study on chemical and sensory changes in onions3, and on how the Maillard reaction is affected36.

In relation to structural effects, there is a lot of interest in gelation of polysaccharides (e.g. 38) and

proteins (e.g. 28), and some authors examined the texture of HP-treated vegetables11.

Taking into consideration the time periods spanned by the literature surveys in Frame 1 and

Frame 2 (up to about one century), the numbers of references are small. Moreover, if only the

real kinetic and/or the real food related ones are considered, extremely few are retained. It is

clear that a tremendous amount of work remains to be done.

In the context of the above, the HP/T-induced inactivation of -amylase fromBacillus subtilis

and of polyphenol oxidase were recently studied in the Laboratory of Food Technology at

K.U.Leuven. The -amylase from Bacillus subtilis is a very thermostable enzyme, which was

formerly studied with a view to its possible application as a time-temperature-integrator for

quantitative evaluation of thermal preservation processes.40 The influence of enzyme

concentration, pH, Ca2+, ethanol, ethylene glycol, trehalose, glycerol, mannitol and sorbitol on

the inactivation under HT and HP/T was examined. Polyphenol oxidase is an enzyme causing

brown colouration in fruit and vegetable products. It is not readily inactivated by HP/T. The

influence of pH and the anti-browning agents EDTA, glutathione and benzoic acid on the

inactivation under HT and HP/T was examined. The results of these studies are summarized in10.

The question of repeatability is to what extent the same process, i.e. the same (t,T,P) profile,

can be reproduced in subsequent runs with the same settings of desired processing P and T.

Hence, it is especially a matter of control and accuracy of the equipment.The question of uniformity is to what extent there is a spread of process impact throughout

a vessel. Again, one can refer to an analogous problem in the thermal preservation area, i.e. of

thermal penetration and distribution. In principle, a spread in HP/T process impact can arise from

variations in P or in T with respect to time and position.

Variations of P with position are avoided when working with hydrostatic pressure, this is

pressure transmitted by a liquid medium. Variations of P with time may occur, e.g. as a

consequence of a P-overshoot in the beginning of a process. However, it can be expected that

the amount of this overshoot, and what is more important, the effect of it on the treated

biological material in terms of (changes in) rate (constants), is negligible compared to that of the

temperature variations.

22


2. Equipment performance in terms of process repeatability and uniformity


26/59

23


Frame 1

State of the art on microorganisms

number of papers:52

time period: 1899 - 1995

real kinetic studies:

8 Johnson & Lewin, 194618: Escherichia coli

8 Johnson & co-workers, 194816: Tobacco Mosaic Virus

8 Solomon & co-workers, 196633: Coliphage T4

8 Clouston & Wills, 19706: P-germination ofBacillus pumilis spores

8 Murrell & Wills, 197726: P-germination ofBacillus spores

8 Butz & co-workers, 19904:

- non-sporeformers causing problems in pharmacy:Staphylococcus aureus, E. coli,

Pseudomonas aeruginosa- ubiquitous germ:Bacillus subtilisspores

- extremely heat resistant spores ofBacillus stearothermophilus

- inactivation and optimal pre-treatments for P-germination

8 Ludwig & co-workers, 199421: P. aeruginosa, S. aureus, E. coli, Bacteriophage T4,B. subtilis

spores

c 7 / 52

really food safety or quality related:

8 Timson & Short, 196539: extremely P-resistant bacterial spores from milk (especiallyBacillus)

8 Hoover & co-workers, 198915: review

8 Ogawa & co-workers, 199027: yeasts and molds in satsuma mandarin juice

8 Shigehisa & co-workers, 199131: m.o. associated with meat products (Bacillus spores,

Campylobacter, Salmonella, Yersinia, E. coli,

S. aureus, Streptococcus faecalis, ...)

8 Karatas & Ahi, 199219: Aspergillus sp. spoiling fruits and vegetables, andPaecilomyces

fulvus, spoiling canned, bottled

and carbonated fruit juices

8 Eshtiaghi & Knorr, 199311: microorganisms associated with potato cubes

8 Takahashi & co-workers, 199335: B. subtilis, Microccus luteus, Candida albicans,

Saccharomyces cerevisiae, Aspergillus nigerand Penicillium citrinum in satsuma mandarin

juice

8 Butz & co-workers, 19943: microorganisms associated with onion

8 Hayakawa & co-workers, 199413: B. stearothermophilusspores, causing flat sour spoilage of

canned liquid coffee

8 Mackey & co-workers, 199522: Listeria monocytogenes

c 10 / 52

real kinetic studies of food safety or quality related microorganisms:


27/59

A major cause of temperature variations with processing time will be adiabatic heating, the

extent of which is largely determined by the velocity of P build-up and the nature of the P-

transferring medium. The time it will take for T to decrease from its maximum value to the

desired T will depend on the heat dissipation rate, hence also on construction and dimensional

characteristics of the vessel, like the way of heating and thermostating, and surface to volume

ratio. In this context, studies of (t,T,P) profiles and how they are influenced by some factors, were

performed in two types of HP-equipment available in the Laboratory of Food Technology at

K.U.Leuven.

The first one is a single vessel system (National Forge, St.-Niklaas, Belgium) with a volume of

590 ml ( 5 cm; height 30 cm). The max. T is 100C and max. P is 6 kbar. The vessel has an

external electrical bandheater (5000 kW). The pressure-transferring medium is water with 3% oil.

We mounted 4 type-T thermocouples registering the temperature inside (dummy) samples at 4,

24


Frame 2

State of the art on enzymes

number of papers:47

time period: 1914 - 1995

real kinetic studies:

8 Johnson & Campbell, 194617: precipitation of human serum globulin

8 Miyagawa & Suzuki, 196323: inactivation of trypsin

8 Suzuki & Kitamura, 196334: inactivation ofB. subtilis -amylase

8 Miyagawa & Suzuki, 196425: inactivation of Tak-amylase

8 Miyagawa & co-workers, 196424: reactivation of Tak-amylase

8 Crelier & co-workers, 19957: inactivation of pectin methyl esterase (PME) from tomato

c 6 / 47

really food quality related:

8 Ogawa & co-workers, 199027: inactivation of pectin esterase in satsuma mandarin juice

8 Asaka & Hayashi, 19912: activation of polyphenol oxidase (PPO) in pear fruit

8 Eshtiaghi & Knorr, 199311: inactivation of PPO in potato cubes

8 Takahashi & co-workers, 199335: inactivation of PME in satsuma mandarin juice

8 Butz & co-workers, 19943: inactivation and activation of onion PPO

8 Anese & co-workers, 19951: inactivation, activation and reactivation of peroxidase from

carrot and PPO from apple

8 Crelier & co-workers, 19957: inactivation of PME from tomato

8 Quaglia & Paoletti, 199529: inactivation of peroxidase from green peas

c 8 / 47

real kinetic studies of food quality related enzymes:

8 Crelier & co-workers, 19957: inactivation of PME from tomato

c 1 / 47


28/59

10, 16, and 22 cm from the bottom. These sample temperatures were measured every 10 seconds

and registered by a data logger (Cobra 7-10, Mess+technik system GmbH), so that (T,t) profiles

could be filed. Similarly, the (P,t) profile was registered and filed. A typical (t,T) profile with fast P

build-up is shown in Figure 1. It shows that there are large temperature differences, especially

between the lowest measuring point (4 cm height) and the higher ones. Differences of this order

of magnitude cannot be neglected. They will drastically influence the (rates of) changes in

biological systems like microorganisms and enzymes being inactivated, which mostly have z-

values in the range 5-15 C. Also the T-overshoot in the beginning of the process is considerable,

and as a consequence it takes long to reach the set value.

The second type of equipment has 8 vessels (Resato, Roden, The Netherlands). Every vessel

has a volume of 8 ml, an individual water jacket for heating and thermostating, and can have a

thermocouple in its center. The pressure-transferring medium is a water/glycol mixture. Thisinstallation is particularly appropriate for kinetic experiments since one can start up a run with

up to 8 samples, which can be individually withdrawn at different times. Here, we examined the

temperature variations over the different vessels, and the effect of the P build-up velocity on

adiabatic heating. A typical (t,T,P) profile with fast P build-up (after pre-pressurization up to 1

kbar) is shown in Figure 2.

It is clear that whereas P is well under control and nearly constant, the T-overshoot due to

adiabatic heating is large, and it takes a considerably long time for T to come down to a constant

value. Compared to these adiabatic heating effects, the temperature differences between the

vessels are small. In tables III, IV and V, figures are given for a number of processes with fast P

build-up after pre-pressurization up to 1 kbar, similar to the process shown in Figure 2.25


Figure 1 - Typical (t,T,P) profile with fast P build-up in the single vessel equipment described in the text.

Desired processing P and T: 3.5 kbar and 45C. Dotted line: P. Full lines: T at the respective

positions in the vessel. The lowest line respresents the lowest measuring point (4 cm); the higher

lines represent measuring points at 10, 16 and 22 cm, resp., from the vessel bottom.


29/59

26


Table III

Adiabatic heating for processes with fast P

build-up in the described multi-vessel High

Pressure equipment

Desired process pressure

Desired process

temperature

2500

bar

5000

bar

7500

bar

25C 12.5 C 15.0 C 21.2 C

40C 13.5 C 17.5 C 24.7 C

55C 11.0 C 19.5 C ---

Table IV

Time between maximum and constant

temperature for processes with fast P build -

up in the described multi-vessel HP-equipment

Desired process

pressure

Desired process

temperature

2500

bar

5000

bar

7500

bar

25C 4 min 5 min 5 min

40C 4 min 5 min 5 min

55C 4 min 4 min ---

Table V

Maximal temperature differences between therespective vessels for processes with fast P build -

up in the described multi-vessel HP-equipment

Desired process pressure

Desired process

temperature

2500

bar

5000

bar

7500

bar

25C 0.9 C 2.0 C 3.0 C

40C 1.2 C 2.0 C 2.7 C

55C 1.3 C 2.6 C ---

Figure 2 - Typical (t,T,P) profile with fast P build-up after pre-pressurization up to 1 kbar in the described

multi-vessel equipment. Desired P and T: 7.5 kbar and 40C. Dotted line: P. Full lines: T in center

of vessels.


30/59

For determining the kinetics of a system, it is straightforward to proceed from data of

isobaric/isothermal experiments. Therefore, it can be concluded from Table IV that zero-time

references in kinetic experiments are best taken at 4 or 5 minutes after the maximum

temperature.

With a view to reducing the adiabatic heating, experiments with decreasing P build-up

velocities were carried out. In the extreme case where the P build-up was extended over 20

minutes, the temperature variations could be limited to the order of magnitude of a few degrees

C (Figure 3).

From this exercise, it can be concluded that in practice it is difficult to have isobaric and

isothermal HP/T processes. In such situation, an unequivocal quantification of process impact,

and any comparison of different processes, will require a concept of equivalent time at reference

conditions (of T and P), which at its turn relies on a kinetic basis.

As schematically pictured in Frame 3, kinetics are the indispensable basis to enable the

industrial application of HP(/T) as a novel preservation technology.

This research has been supported by the Flemish Institute for the promotion of scientific-

technological research in industry (IWT).27


Figure 3 - (t,T,P) profile of a process with extremely slow P build-up to minimize adiabatic heating in the

described multi-vessel equipment. Desired P and T: 7.5 kbar and 40C. Dotted line: P. Full lines:

T in center of vessels.

4. Conclusions

Acknowledgements


31/59

1. Anese, M., Nicoli, M.C., DallAglio, G. and Lerici, C.R. (1995). Effect of high pressure treatments

on peroxidase and polyphenol oxidase activities. Journal of Food Biochemistry 18, 285-293.

2. Asaka, M. and Hayashi, R. (1991). Activation of polyphenoloxidase in pear fruits by high

pressure treatment. Agric. Biol. Chem. 55 (9), 2439-2440.

3. Butz, P., Koller, W.D., Tauscher, B. and Wolf, S. (1994). Ultrahigh pressure processing of onions:

chemical and sensory changes. Lebensmittel-Wissenschaft und -Technologie 27, 463-467.

4. Butz, P., Ries, J., Traugott, U., Weber, H. and Ludwig, H. (1990). Hochdruckinaktivierung von

Bakterien und Bakteriensporen. Die Pharmazeutische Industrie 52 (4), 487-491.

5. Cheftel, J-C. (1991). Applications des hautes pressions en technologie alimentaire. Actualits

des industries alimentaires et agro-alimentaires 108 (3), 141-153.

6. Clouston, J.G. and Wills, P.A. (1970). Kinetics of initiation of germination ofBacillus pumilis

spores by hydrostatic pressure. Journal of Bacteriology 103, 140-143.

7. Crelier, S., Tche, M.-C., Raemy, A., Renken, A. and Raetz, E. (1995). High pressure for the

inactivation of enzymes in food products. Poster presented at the 9th World Conference on

Food Science & Technology, July 30 - August 4, 1995, Budapest, Hungary.

8. De Cordt, S., Avila, I., Hendrickx, M. and Tobback, P. (1994). DSC and protein-based time-

temperature integrators: case study on -amylase stabilized by polyols and/or sugar.

Biotechnology and Bioengineering 44, 859-865.

9. De Cordt, S., Hendrickx, M., Maesmans, G. and Tobback, P. (1992). Immobilized -amylase from

Bacillus licheniformis: a potential enzymic time-temperature integrator for thermal processing.

International Journal of Food Science and Technology 27, 661-673.10. De Cordt, S., Ludikhuyze, L., Weemaes, C., Hendrickx, M., Heremans, K. and Tobback, P.

(1995). Enzyme stability under high pressure and temperature. Oral presentation at the28


Frame 3

Necessity of kinetics for enabling application

of HP as a novel preservation technology

application of HP(/T) for preservation

process impact

quantification

concepts &

methods

kinetics

performant

equipment

References


32/59

International Congress on High Pressure Bioscience & Biotechnology, November 5-9, 1995,

Kyoto, Japan.

11. Eshtiaghi, M.N. and Knorr, D. (1993). Potato cube response to water blanching and high

hydrostatic pressure. Journal of Food Science 58 (6), 1371-1374.

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hydrostatic pressure. Journal of General Microbiology 60, 335-346.

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continuous high pressure sterilization on Bacillus stearothermophilus spores. Journal of Food

Science 59 (1), 164-167.

14. Hendrickx, M., Maesmans, G., De Cordt, S., Noronha, J., Van Loey, A. and Tobback, P. (1995).

Evaluation of the integrated time-temperature effect in thermal processing of foods. Critical

Reviews in Food Science and Nutrition 35 (3), 231-262.

15. Hoover, D.G., Metrick, C., Papineau, A.M., Farkas, D.F. and Knorr, D. (1989). Biological effects

of high hydrostatic pressure on food microorganisms. Food Technology, March 1989, 99-107.

16. Johnson, F.H., Baylor, M.B. and Fraser, D. (1948). The thermal denaturation of Tobacco Mosaic

Virus in relation to hydrostatic pressure. Archives of Biochemistry 19, 237-245.

17. Johnson, F.H. and Campbell, D.H. (1946). Pressure and protein denaturation. Journal of

Biological Chemistry 163, 689-698.

18. Johnson, F.H. and Lewin, I. (1946). The disinfection of E. coli in relation to temperature,

hydrostatic pressure and quinine. Journal of Cellular Comparative Physiology 28, 23-45.

19. Karatas, S. and Ahi, E. (1992). Inactivation ofAspergillus species andPaecilomyces fulvus at high

hydrostatic pressure. Lebensmittel-Wissenschaft und -Technologie 25, 395-397.

20. Knorr, D. (1993). Effects of high-hydrostatic-pressure processes on food safety and quality.

Food Technology 47 (6), 156-161.

21. Ludwig, H., Gross, P., Scigalla, W. and Sojka, B. (1994). Pressure inactivation of

microorganisms. High Pressure Research 12, 193-197.

22. Mackey, B.M., Forestire, K. and Isaacs, N. (1995). Factors affecting the resistance of Listeria

monocytogenes to high hydrostatic pressure. Food Biotechnology 9 (1&2), 1-11.

23. Miyagawa, K. and Suzuki, K. (1963). Pressure inactivation of enzyme: some kinetic aspects ofpressure inactivation of trypsin. Review of Physical Chemistry of Japan 32, 43-50.

24. Miyagawa, K. Sannoe, K. and Suzuki, K. (1964). Studies on Taka-amylase A under high pressure

treatment. II. Recovery of enzymic activity of pressure inactivated Taka-amylase A and its

enhancement by retreatment at moderate pressure. Archives of Biochemistry and Biophysics

106, 467-474.

25. Miyagawa, K. and Suzuki, K. (1964). Studies on Taka-amylase A under high pressure. I. Some

kinetic aspects of pressure inactivation of Taka-amylase A. Archives of Biochemistry and

Biophysics 105, 297-302.

26. Murrell, W.G. and Wills, P.A. (1977). Initiation of Bacillus spore germination by high pressure:

effect of temperature. Journal of Bacteriology 129 (3), 1272-1280.29



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27. Ogawa, H., Fukuhisa, K., Kubo, Y. and Fukumoto, H. (1990). Pressure inactivation of yeasts,

molds and pectinesterase in satsuma mandarin juice: effects of juice concentration, pH, and

organic acids, and comparison with heat sanitation. Agricultural and Biological Chemistry 54

(5), 1219-1225.

28. Okamoto, M., Kawamura, Y. and Hayashi, R. (1990). Application of high pressure to food

processing: textural comparison of pressure- and heat-induced gels of food proteins.

Agricultural and Biological Chemistry 54 (1), 183-189.

29. Quaglia, G.B. and Paoletti, F. (1995). Study of the effects of high pressure treatments on

peroxidase activities in green peas. Poster presented at the 9th World Conference on Food

Science & Technology, July 30 - August 4, 1995, Budapest, Hungary.

30. Sale, A. J. H., Gould, G. W. and Hamilton, W. A. (1970). Inactivation of bacterial spores by

hydrostatic pressure. Journal of General Microbiology 60, 323-334.

31. Shigehisa, T., Ohmori, T., Saito, A., Taji, S. and Hayashi, R. (1991). Effects of high hydrostatic

pressure on characteristics of pork slurries and inactivation of microorganisms associated with

meat and meat products. International Journal of Food Microbiology 12, 207-216.

32. Smelt, J. P. P. M. and van Wely, E. J. M. (1993). Conservering van voedingsmiddelen met

ultrahoge druk. Voedingsmiddelentechnologie, 15 juli 1993, nr 14/15, 11-13.

33. Solomon, L., Zeegen, P. and Eiserling, F.A. (1966). The effects of high hydrostatic pressure on

coliphage-T4. Biochimica & Biophysica Acta 112, 102-109.

34. Suzuki, K. and Kitamura, K. (1963). Inactivation of enzyme under high pressure. The Journal

of Biochemistry 54 (3), 214-219.

35. Takahashi, Y., Ohta, H., Yonei, H. and Ifuku, Y. (1993). Microbicidal effect of hydrostatic

pressure on satsuma mandarin juice. International Journal of Food Science and Technology 28,

95-102.

36. Tamaoka, T., Itoh, N. and Hayashi, R. (1991). High pressure effect on Maillard reaction.

Agricultural and Biological Chemistry 55 (8), 2071-2074.

37. Tauscher, B. (1995). Pasteurization of food by hydrostatic pressure: chemical aspects.

Zeitschrift fr Lebensmittel-Untersuchung und -Forschung 200, 3-13.

38. Thevelein, J. M., Van Assche, J. A., Heremans, K. and Gerlsma, S. Y. (1981). Gelatinisationtemperature of starch, as influenced by high pressure. Carbohydrate Research. 93, 304-307.

39. Timson, W.J. and Short, A.J. (1965). Resistance of microorganisms to hydrostatic pressure.

Biotechnology & Bioengineering VII, 139-159.

40. Van Loey, A., Hendrickx, M., Ludikhuyze, L., Weemaes, C., Haentjens, T., De Cordt, S. and

Tobback, P. (1996). Potential Bacillus subtilis a-amylase based time temperature integrators to

evaluate pasteurization processes. Accepted for publication in Journal of Food Protection.

30



34/59

Vitamin K, a dienophile with a naphthoquinone system, reacts, especially at higher

temperature and pressure, with a diene such as myrcene in a Diels-Alder reaction to form a six-

member ring system with a double bond (Diels-Alder-adduct). Isomeric compounds formed as

products were separated by HPLC, their structure explored by spectroscopic methods. Vitamin K,

and myrcene in ethanol at 70C and 650 MPa after 6 hours showed 100% yield compared to 3%

of blind. Vitamin K in ethanol at 70C and 650 MPa formed two isomeric products of 25% yield

each after 60 hours; the blind did not react under these conditions. At 40C vitamin K2yields

only traces of Diels-Alder products while at the 70C yield increased significantly. According,

Diels-Alder-products between food components may be expected at higher temperatures and

high pressure, in the case of vitamin K3 and myrcene as soon as after 15 minutes. It remains tobe studied whether the food matrix has a catalizing or inhibiting effect on this kind of reaction.

Pasteurization of food by ultrahigh hydrostatic pressure has attracted the attention of many

disciplines. Chemical reactions of low-molecular and oligomeric compounds in food under high

pressure have been little investigated. Generally any process and any reaction in food that follow

the principle of Le Chatelier are of interest. Under equilibrium conditions, a process associated

with a decrease in volume is favoured by pressure, and vice versa. Pressure influences rate and

equilibrium of reactions even in food (Tauscher, 1995).

For any reaction in solution (Matsumoto and Acheson, 1991) between reaction partners A and

B the reaction volume V and the activation volume V can be determined.

describes the partial volumes of the reactants or of the products.

A B A B AB

V

V

AB A B

A B

+ [ ]

=

=

31

Ludwig, Marx & Tauscher High Pressure

Summary

1. Introduction

Behaviour of Organic Compounds in Food under High Pressure: Diels-Alder

Reactions of Food Components

Horst Ludwig1, Heidger Marx1 and Bernhard Tauscher 2*

1 Institute of Pharmaceutical Technology, University of Heidelberg, Heidelberg, Germany2 Institute if Chemistry and Biology, Federal Research Centre for Nutrition, Karlsruhe, Germany

* author to whom correspondence should be addressed


35/59

For the location of the equilibrium of a chemical reaction in solution under pressure, the

reaction volume is decisive; it is described by:

(1)

where R is the general gas constant, T the absolute temperature, K the equilibrium constant,

and P the hydrostatic pressure.

Pressure influences not only the location of the equilibrium of a chemical reaction in solution,

but also its reaction rate. For the activation volume the following equation applies:

(2)

Where k is the rate constant.

Diels-Alder reactions have been studied extensively under pressure, as [2+4] cycloadditions.

The pressure induced acceleration of this reaction is one of the largest. The reaction between

electron-rich dienes and electron-deficient dienophiles is characterized by large negative

activation volumes. In some cases activation volumes exceed reaction volumes. The activated

complex hence has a very tight structure. The same applies also to inverse Diels-Alder reactions.

The selectivity of the reaction can be controlled by pressure; in this way the product whose

reaction volume is more negative is favoured. Optimal effects have been obtained by

simultaneous variation of pressure and temperature (Klrner, 1989). The chemo- and endo-

selectivity of Diels-Alder reactions in aqueous media is strongly affected by hydrophobic

interaction (Jenner, 1994).

In food, Diels-Alder products may form during thermal treatment. Unsaturated fatty acids may

turn into conjugated fatty acids which may react to Diels-Alder adducts (Adelhardt and Spitteller,

1993). Parsley -, lavender- and tagetes oil may also contain Diels-Alder products involving the

influence of terpenoidal compounds (Lawrence et al. 1980). Retinol may form dimer products as

well (Buger and Garbers, 1973).

Quinones may act as dienophiles and conjugated terpenoids as dimers (Ludwig et al, 1994).We therefore studied the reactions of the vitamin K group (quinones) with myrcene (diene) at

various pressures, temperatures and for different times.

Vitamin K3 and 2, 3- dimethylbutadiene were purchased from Aldrich, Steiheim. The cis/trans

isometric mixture of vitamin K1 and myrcene were from Roth, Karlsruhe. The homologes of

vitamin K2

and the pure trans- and cis- K1were grants of Hoffmann-La Roche, Basel, Switzerland.

All substances were used without further purification. Solvents for HPLC were from Serva,

Heidelberg whereas the solvent used for nuclear magnetic resonance experiments was supplied

V RTd k

dP T

ln=

V RTd K

dP T=

ln

V

32


2. Materials and methods


36/59

by Fluka, Neu-Ulm.

2ml of 0.058 molar solution of the dienophile in ethanol with a threefold excess of the diene

were pipetted into small bags of LDPE-aluminium-PET laminate and heat sealed.

The pressure apparatus used consisted of three separate microautoclaves (12ml volume each),

thermostated by water. Pressure up to 650 MPa was generated by an hydraulic pump.

After pressure treatment samples were fitered through 0.45m non-sterile syringe filters and

diluted two-hundred fold in the HPLC solvent. Normal-phase HPLC was performed on a 250x4mm

LiChrosorb Si60 analytical column (Knauer, Berlin). The products were eluted using a mixture of

n-hexane/ethylacetate at a ratio of 19:1 or 39:1 (v/v) depending on the polarity of the dienophiles.

The flow rate was 0.75 and 0.5ml/min, respectively. UV detection was performed at 254nm.

For a preperative separation of reaction products a 250x8mm LiChrosorb Si60 column

(Knauer, Berlin) was used, eluting with a mixture of n-hexane/ethylacetate in the range of 19:1 to

99:1 and a constant flow rate of 2.0 or 3.0ml/min depending on the products polarity and the

respective separation problem.

The Diels-Alder products and their possible isomers obtained were identified by 200/250

MHz-proton-and 50/62 MHz-carbon-NMR. To make a distinction between simultaneously forming

isomers, H-irradiation experiments at 400MHz and H-2D-COSY at 250MHz were carried out. EI-

70eV-mass spectroscopy was performed on a Finningan MAT90 by the direct inlet method. For

determination of the sum formula, a high-resolution MS was carried out in addition. The IR

spectra were recorded on a Bruker IFS 88 using the film technique, where applicable. UV spectra

were created on a Beckman DU-6 spectrophometer by measuring the wavelengths between 200

and 400nm.

The vitamin K-group includes vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone) present in

green plants, vitamin K2 (menaquinones Mk-n (n=1-7)) from bacteria, and the synthetic vitamin

K3 (2-methyl1-1,4-naphtoquinone).

A Diels-Alder reaction between vitamin K3 and myrcene is supposed to yield two isomers:Figure 1 shows the 60 h-kinetics, followed by HPLC, of the reaction of vitamin K3 and

myrcene. The reaction temperature was 40C, pressure was 650MPa. The peak appearing

between 10 and 13 minutes corresponds to vitamin K3, and the one appearing between 7 and 10

minutes corresponds to newly formed products. Product formation has clearly advanced already

after 30 minutes.

At 70C and 650MPa, however, the yield is considerable as early as after 15 minutes (fig. 2).

The group of peaks appearing between 7 and 10 minutes (fig. 2) shows that at least two products

were formed. There was no further separation. According to H-NMR, C-NMR, 2D-COSY, UV, IR and

MS the newly formed product was grouped with meta and para isomers.

33


3. Results and discussion


37/59

Under pressure and increased temperature vitamin K1 reacts with myrcene to form two

products as well (meta and para-isomers):

34


Figure 1 - 60h-kinetic of the reaction between vitamin K3 and myrcene (1:3 in EtOH) 40C/650MPa



38/59

Figure 3 shows the 60h-kinetics, followed by HPLC, of the reaction of vitamin K1 . The reaction

temperature was 70C and pressure was 650MPa. Vitamin K1was present as cis and trans isomer.

In the chromatogram shown in figure 3 cis-vitamin K1 appeared after 8.9 minutes, trans-vitamin

K1

after 9.8 minutes. The area ratio of cis: trans vitamin K1

was 12:88. The meta and para

isomeric Diels-Alder products appeared after 8.3 and 9.4 minutes. Kinetic measurements have

shown that small quantities of the Diels-Alder products have formed after three hours of pressure

exposure at 70C. No products were identified after 15 minutes. The structure of the new

products was explored by spectroscopic methods.

The pure cis- and trans-, isomers of vitamin K1 did not isomerize, as a response to pressure,

even after 60h at 70C and 650MPa.

Reactivity of the K2vitamins of n=1-3 with myrcene was comparable to that of vitamin K1with myrcene.

Diels-Alder reactions have been shown to occur between typical food components, also under

the conditions of high pressure sterilization of food. It remains to be studied whether the food

matrix has catalyzing or inhibiting effects on this kind of reaction.

35



4. Conclusions


39/59

Tauscher, B. (1995). Pasteurization of food by hydrostatic high pressire: chemical aspects. Z

Lebensm Unters Forsch 200, 3-13

Matsumoto, K. & Acheson, R. M. (1991) (eds.). Organic Synthesis at high pressures. Wiley, New

York, Chichester, Brisbane, Toronto, Singapore

Klrner, F. G. (1989). Chemie unter Hochdruck. Die Steuerung organisch-chemischer Reaktionen

nit hohem Druck. Chemie in unserer Zeit 23 (2) 53-63

Jenner, G. (1994). Effect of water on chemo- and endo-selectivity in high pressure D

Documents

1st Meet Hpressure