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Process Optimisation and Minimal Processing of Foods

European Commission COPERNICUS PROGRAMME Concerted action CIPA-CT94-0195

Proceedings of the first main meeting

Volume 4: High Pressure

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December 1995 rtugal, to, Po Por gia, nolo tec

Editor : Jorge C. Oliveira Project Coordinator : Fernanda A. R. Oliveira Area leader : Dietrich Knorr

Proceedings of the first project workshop

Foreword

The proceedings of the first workshop organised by the COPERNICUS concerted action Process Optimisation and Minimal Processing of Foods in December 1995 at Escola Superior de Biotecnologia, 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 later forwarded 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, as would be expected, and have tried to contribute to a greater harmonisation by taking liberties with everybodys English. Not being native English speakers ourselves, it is evident that fully correct English has not resulted from this exercise, but we hope that in this way all texts are fully comprehensible and more similar in style. However, the revision was not thorough and some typing 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 the edition of the texts was concerned with the language and style and if any change in meaning has resulted, we sincerely apologise for the fact. It is reminded that at the end of the project the communications that were orally presented in the three project meetings as area overviews, plenary lectures and short communications will be collected for the publication of a book, through a professional scientific publisher. The contents will then be scientifically reviewed by the area leaders and the publisher will make a professional review of the English. We would like to thank all project participants and particularly those that have contributed with written 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 for providing 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 deal with everything that had to do with typing, file converting, scanning, and all those very boring computing tasks that were required for the final editing and publishing and also for her commitment and work towards this project.

Porto, November 12th, 1996 Fernanda A. R. Oliveira Jorge C. Oliveira i

Proceedings of the first project workshop

High Pressure

Table of Contents

Dietrich Knorr Process Assessment of High Pressure Processing of Foods: an Overview Jacek Arabas 6 Monika Fonberg-Broczek The High Pressure Research Center of Warsaw, Poland Suzanne de Cordt, L. Ludikhuyze, C. Weesmaes, Marc Hendrickx & Paul Tobback Process Assessment in High Pressure / Thermal Processing of Foods: the Role of Kinetics Horst Ludwig, Heidger Marx 6 Bernhard Tauscher Behaviour of Organic Compounds in Food under High Pressure: Diels-Adler Reactions of Food Components Christian Schreck, Gunter van Almsick & Horst Ludwig Influence of Culturing Conditions on the Pressure Sensitivity of Escherichia Coli 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 Jacek Szczawinski, Janina Peconek, Malgorzata Szczawinska, Monika Fonberg-Broczek & Jacek Arabas High Pressure Inactivation of Listeria monocytogenes in Meat and Meat Products

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Process Assessment of High Pressure Processing of Foods: an Overview


Dietrich Knorr

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

Summary
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 as package design. Engineering aspects to be dealt with are heat transfer issues and temperature distribution within pressure vessels.

1. Introduction
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. 1

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2. Advantages of high hydrostatic pressure


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. Opportunities for high hydrostatic pressure


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

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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 pressure absorbance

processes treatment

during via

measurements of spores in a pressure cell with optical

windows (Figures 1 and 2). Successfull preservation Figure 1 - Prototype of high pressure cell with optical windows

operations often depend on the effective enzymatic reduction activity of 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 on food of high 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)

pressure

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

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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 ElNour, 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 the requirement for studies on the kinetics of ice nucleation, or crystal size, distribution and growth, as well as on

recrystallization.

Figure 3 - Pressure and temperature conditions during pressure shift freezing of potato cubes (Koch et al. 1995) 4

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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.

50

sugar content (Brix)

40 30 20 10 0 20 40 60 A C D

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


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

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suspended in Ringers solution at 20C typically showed a sigmoid, non-symetric shape when plotted on logaritmic scale (Fig.5).
0 survivors log (N/No) -1

250MPa

-2

-3

-4

-5 0 400 800 1200 1600 UHP treatment time (s) 2000 2400

Figure 5 - Typical time-inactivation curve of Bacillus subtilis after high pressure treatment at 250 MPa and 20C (Heinz and Knorr, 1995) 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.

Acknowledgements

Parts of this work have been funded by grants from the European community (EC-AIR CT920296), 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

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References
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 (AIRCT92 - 0296) Hoover, D. G. (1993). Food Technology 47(6), 150 Kalichevsky, 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, 7 7

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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

Arabas & Fonberg-Brockzek

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The High Pressure Research Center of Warsaw, Poland


Jacek Arabas1, Monika Fonberg-Broczek1,2

1 High Pressure Research Center, Polish Academy of Sciences, Warsaw, Poland 2 Department of Food Research, National Institute of Hygiene, Warsaw, Poland

Summary
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.

1. Introduction
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.

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2. Outline of activities
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 (AIIIBV and 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 O2 10

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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. Food programme
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 stands and 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

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aureus), spores of Baccilius 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 - working pressure: 500 MPa, - 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)

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4. Food processing laboratory test stands for food samples 500 - 2000 cc - working pressure: 900 MPa, - 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 collaboration We have developed an international collaboration with: 1. Konan Womens University in Kobe, Japan (visiting professor of food technology 19951996), 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

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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 of Bacillus 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 of Listeria 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 MicroOrganisms 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 of Listeria 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.

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4. Conclusions
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.

Acknowledgements
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.

References
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 Science and 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 of Aspergillus 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

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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 on Listeria 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

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Process 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

Summary
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 Japanese market. However, Europe and the US are still awaiting the establishment of the reliability of HPtreatments. 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.

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


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

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High Pressure

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:

F=

D ref dt 0 D

(1)

where D is the decimal reduction time at temperature T, Dref the 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
t

F=

k
(2)

0 k ref

where k is the rate constant at temperature T, and kref the 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 Dref to 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 of a 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 19

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Process Assessment

Table I Survey of main kinetic models and parameters and their significance Thermal Death Time (TDT) terminology only appropriate for first-order! general terminology applicable for any value of order

t log (X) = log (X0 ) D


(at a specified constant temperature T and constant pressure P) where t is time, X (0) the system response (at time zero), and D the decimal reduction time D(T)(P) = decimal reduction time = time required, at a specified constant temperature T and constant pressure P, to reduce the system response (X) with one logarithmic unit (90%)

ln (X) = ln( X0 ) kt X=

(if n = 1)

1 n X0

+ ( n 1)kt

1 1 n

(if n 1)

(at a specified constant temperature T and constant pressure P) where n is the reaction order, t is time, X (0) the system response (at time zero), and k the rate constant k(T)(P) = rate constant at a specified temperature T and pressure P

log D(T ) = log DTref

T T + ref z

E 1 1 k( T ) = k Tref exp a R Tref T


(at a specified constant pressure P) where T ref is a chosen reference temperature, k (T) and k Tref the rate constants at T and at T ref, respectively, and E a the activation energy Ea = activation energy

(at a specified constant pressure P) where T ref is a chosen reference temperature, D (T) and D Tref the decimal reduction times at T and at T ref, respectively, and z the z-value z = temperature in- or decrement to respectively de- or increase D with one logarithmic unit D depends on P, but there is no generally accepted explicit equation

PV k( P) = k Pr ef exp RT
(at a specified constant temperature T) where P ref is a chosen reference pressure (usually atmospheric P), k (P) and k Pref the rate constants at P and at P ref, respectively, and V the activation volume

20

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High Pressure

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:

F=

1 k ref

X ln 0 Xt

n=1:

F=

1 k ref

X ln 0 Xt

F=

1 X1 n X1n t 0 k ref n 1

0 E 1 1 n1: dt F = exp A 1 X1 n X1n R Tref T t t 0 F= k ref n 1

advantage: simple

advantage: works with controlable variables

advantage: general applicability not yet available; being developed (e.g. References 8,9,14,40)

disadvantage: very limited applicability

disadvantage: limited applicability

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 physicalmathematical 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 indirectly involved, 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, the 21

Process Optimisation and Minimal Processing of Foods

Process Assessment

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 from Bacillus 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.

2. Equipment performance in terms of process repeatability and uniformity


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

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Frame 1 State of the art on microorganisms number of papers:52

time period: 1899 - 1995

real kinetic studies:


18 8 Johnson & Lewin, 1946 : Escherichia coli 16 8 Johnson & co-workers, 1948 : Tobacco Mosaic Virus 33 8 Solomon & co-workers, 1966 : Coliphage T4 8 Clouston & Wills, 19706 : P-germination ofBacillus pumilis spores 8 Murrell & Wills, 197726: P-germination ofBacillus spores 4 8 Butz & co-workers, 1990 :

- non-sporeformers causing problems in pharmacy: Staphylococcus aureus E. coli, , Pseudomonas aeruginosa - ubiquitous germ:Bacillus subtilis spores - extremely heat resistant spores ofBacillus stearothermophilus - inactivation and optimal pre-treatments for P-germination
21 8 Ludwig & co-workers, 1994 : P. aeruginosa, S. aureus, E. coli, Bacteriophage T4,B. subtilis

spores

c 7 / 52
really food safety or quality related:
39 8 Timson & Short, 1965 : extremely P-resistant bacterial spores from milk (especially

Bacillus)
15 8 Hoover & co-workers, 1989 : review 27 8 Ogawa & co-workers, 1990 : yeasts and molds in satsuma mandarin juice 31 8 Shigehisa & co-workers, 1991 : m.o. associated with meat productsBacillus 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 35 8 Takahashi & co-workers, 1993 : B. subtilis, Microccus luteus, Candida albicans,

Saccharomyces cerevisiae, Aspergillus nigerand Penicillium citrinum in satsuma mandarin juice


3 8 Butz & co-workers, 1994 : microorganisms associated with onion 13 8 Hayakawa & co-workers, 1994 : B. stearothermophilusspores, causing flat sour spoilage of

canned liquid coffee


22 8 Mackey & co-workers, 1995 : Listeria monocytogenes

c 10 / 52
real kinetic studies of food safety or quality related microorganisms:

23

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Process Assessment

Frame 2 State of the art on enzymes number of papers:47

time period: 1914 - 1995

real kinetic studies:


17 8 Johnson & Campbell, 1946 : precipitation of human serum globulin 8 Miyagawa & Suzuki, 196323: inactivation of trypsin 34 8 Suzuki & Kitamura, 1963 : inactivation ofB. subtilis -amylase 8 Miyagawa & Suzuki, 196425: inactivation of Tak-amylase 24 8 Miyagawa & co-workers, 1964 : reactivation of Tak-amylase 8 Crelier & co-workers, 19957 : inactivation of pectin methyl esterase (PME) from tomato

c 6 / 47
really food quality related:
27 8 Ogawa & co-workers, 1990 : inactivation of pectin esterase in satsuma mandarin juice 2 8 Asaka & Hayashi, 1991 : activation of polyphenol oxidase (PPO) in pear fruit 8 Eshtiaghi & Knorr, 199311: inactivation of PPO in potato cubes 35 8 Takahashi & co-workers, 1993 : inactivation of PME in satsuma mandarin juice 3 8 Butz & co-workers, 1994 : inactivation and activation of onion PPO 1 8 Anese & co-workers, 1995 : inactivation, activation and reactivation of peroxidase from

carrot and PPO from apple


8 Crelier & co-workers, 19957 : inactivation of PME from tomato 29 8 Quaglia & Paoletti, 1995 : 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

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 Ptransferring 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

De Cordt, Ludikhuyze, Weemaes, Hendrickx & Tobback

High Pressure

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 zvalues 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.

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.

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. This installation 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

Process Optimisation and Minimal Processing of Foods

Process Assessment

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. 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 25C 40C 55C Table V Maximal temperature differences between the respective vessels for processes with fast P build up in the described multi-vessel HP-equipment Desired process pressure Desired process temperature 25C 40C 55C 2500 bar 0.9 C 1.2 C 1.3 C 5000 bar 2.0 C 2.0 C 2.6 C 7500 bar 3.0 C 2.7 C --2500 bar 4 min 4 min 4 min 5000 bar 5 min 5 min 4 min 7500 bar 5 min 5 min ---

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 25C 40C 55C 2500 bar 12.5 C 13.5 C 11.0 C 5000 bar 15.0 C 17.5 C 19.5 C 7500 bar 21.2 C 24.7 C ---

26

De Cordt, Ludikhuyze, Weemaes, Hendrickx & Tobback

High Pressure

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).

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.

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.

4. Conclusions
As schematically pictured in Frame 3, kinetics are the indispensable basis to enable the industrial application of HP(/T) as a novel preservation technology.

Acknowledgements

This research has been supported by the Flemish Institute for the promotion of scientifictechnological research in industry (IWT). 27

Process Optimisation and Minimal Processing of Foods

Process Assessment

Frame 3 Necessity of kinetics for enabling application of HP as a novel preservation technology application of HP(/T) for preservation

process impact quantification

performant equipment

concepts & methods

kinetics

References

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 of Bacillus 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 timetemperature 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. 28 (1995). Enzyme stability under high pressure and temperature. Oral presentation at the

De Cordt, Ludikhuyze, Weemaes, Hendrickx & Tobback

High Pressure

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. 12. Gould, G. W. and Sale, A. J. H. (1970). Inhibition of germination of bacterial spores by hydrostatic pressure. Journal of General Microbiology 60, 335-346. 13. Hayakawa, I., Kanno, T., Yoshiyama, K. and Fujio, Y. (1994). Oscillatory compared with 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 of Aspergillus species and Paecilomyces 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 of pressure 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). Gelatinisation temperature 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.

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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, Germany 2 Institute if Chemistry and Biology, Federal Research Centre for Nutrition, Karlsruhe, Germany * author to whom correspondence should be addressed

Summary
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 sixmember 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 K2 yields 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 to be studied whether the food matrix has a catalizing or inhibiting effect on this kind of reaction.

1. Introduction
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.

A + B [A B] AB V = AB A B V = A B describes the partial volumes of the reactants or of the products.


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

V is decisive; it is described by: d ln K V = RT dP T


(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:

d ln k V = RT dP T
Where k is the rate constant.

(2)

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

2. Materials and methods


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- K1 were 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 32

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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. EI70eV-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.

3. Results and discussion


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

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

Figure 2 - 6h-kinetic of the reaction between vitamin K3 and myrcene (1:3 in EtOH) 70C/650MPa Under pressure and increased temperature vitamin K1 reacts with myrcene to form two products as well (meta and para-isomers):

34

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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 K1 was 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.

Figure 3 - 60h-kinetic of the reaction between vitamin K1 and myrcene (1:3 in EtOH) 70C/650MPa

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 K2 vitamins of n=1-3 with myrcene was comparable to that of vitamin K1 with myrcene.

4. Conclusions

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.

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References
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 Diels-Alder reaction. Tetrahedron letters 35(8) 1189-1192 Adelhardt, R. & Spitelle, G. (1993). Products of the dimeriztion of unsaturated fatty acids. Part a kinetic studies on the dimerization of linolic acid. Fett Wiss. Technol 95(3) 85-90 Lawrence, B. M., Mookherjee, B. D. & Willis, B. J. (1986) (eds). Flavors and fragances: a world perspective. Proceedings of the 10th international Congress of Essent Oils, Fragances and Flavors. Washington DC, USA 16-20 Nov, 715-729 Burger, B. V. & Garbers, C. F. (1973). Diels-Alder-Reactions. Part III. Condensation of Methyl trans-Formylcrotonate with Retinol-Acetate, with a Note on the Structure and Stereochemestry of Kitol. J. Chem. Soc. Oerkin Trans I, 590-595 Ludwig, H., Marx, H. & Tauscher, B. (1994). Diels-Alder-Reactions of food-relevant compounds under high pressure I: myrcene and vitamin K3. Eur. High Pressure Groups. Proceedings of the XXXII Annual Meeting, Brno, Czech Republic, 203-206 Marx, H. (1996), Dissertation, Universitt Heidelberg, Germany

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Questions and Answers

Q A Q A

Does UHP have an accelerating effect on Maillard reactions? Petros Taoukis

Hayashi has found, that HP supresses the browning process rather than the initial condensation process in the total course of the Maillard reaction.

Could you comment on the possibility of unique UHP product-forming in food products? Petros Taoukis

A chemical reaction associated with a decrease in volume is favoured by pressure e.g.

ionization of water, acids, phenols and amines, formation of covalent bonds, quaternization of nitrogen ...see: Tauscher, Z Lebensm Unters Forsch (1995) 200, 3-13.

Q A

Do you have any idea about the mechanism by which enzyme immobilization protects enzyme activity under HP? Harris Lazarides

Pressure affects the quaternary structure (e.g. through hydrophobic interactions), the tertiary structure (e.g. through reversible unfolding) and the secondary structure (through

irreversible unfolding). This may be supressed by covalently bonding the enzyme on a surface of a matrix.

Q A

What about the effect of HP on oxidation reactions? Harris Lazarides

The initial reaction, i.e. formation of radicals should be retarded by pressure, which propagation steps should be favoured. The effect of pressure on oxidative processes in

foods has been little investigated. We investigated the autoxidation of linolenic acid under pressure of up to 600MPa: the reaction is complex.

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Influence of Culturing Conditions on the Pressure Sensitivity of Escherichia Coli

Christian Schreck, Gnter van Almsick and Horst Ludwig

Institut fr Pharmazeutische Technologie und Biopharmazie, Gruppe Physikalische Chemie, Universitt Heidelberg, Germany

Summary
The kinetics of pressure inactivation was investigated for two strains of Escherichia coli at 25 C and 2.5 or 3 kbar. According to how the bacteria were grown, with or without oxygen, their sensitivity to pressure and the inactivation kinetics differed markedly. The characteristics of the kinetic curves also changed with pH. Bacteria from the exponential phase of growth were more sensitive to pressure than those from the stationary phase. This fact, and also the biphasic behaviour of inactivation curves, were shown to be caused very probably by various stages of the bacterial development in the cell cycle and by different amounts of protective proteins.

1. Introduction
Escherichia coli are facultatively aerobic bacteria. They grow very well in aerated media but can also develop and reproduce if oxygen is missing. Under anaerobic conditions the metabolism changes leading to alterations in the composition of the bacteria (Neidhard, 1987). Therefrom it might be expected that conditions of growth and availability of oxygen influence the sensitivity to pressure and the kinetics of inactivation.

2. Materials and methods


2.1. Materials

2.1.1. Microorganisms Escherichia coli ATCC 11303 were aerobically or anaerobically grown in Standard I nutrient broth. Escherichia coli ATCC 39403 were grown under aerobic conditions in M9 minimal medium. The bacteria were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany.

2.1.2. Substances Chloramphenicol (DAB 10, BP 93) was obtained from Eu-Rho-Pharma GmbH, Kamen-Heeren, 38

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Germany, and Trimethoprim (DAB 10) from Synopharm, Barsbttel / Hamburg, Germany. Nutrients were from Difco, Detroit, USA or from Merck, Darmstadt, Germany. All the other substances used were of p.a. or DAB 10 quality.

2.2. Methods

The bacteria were cultured just before each experimental run. Usually they were harvested in the early stationary phase and thus, suspended in nutrient broth, used in high pressure experiments. For these, they were enclosed in polyethylene tubes and then filled into the ten small pressure vessels of the high pressure device (figure 1). All vessels were thermostated at 25C and pressurized to 2.5 or 3 kbar. The single vessels were opened at different times to measure the kinetics of inactivation.

culture of bacteria

PE-tubes with silicon stoppers

incubation

petri dishes with suitable media

stepwise dilution

high pressure device

Figure 1 - Scheme of the experimental procedure The surviving organisms were then counted as colony forming units on agar plates (cfu = viable bacteria per ml). The culturing flask was either almost filled with the liquid medium to obtain nearly anaerobic conditions with restricted oxygen supply or contained a small amount of liquid and much of air for aerobic growth. In some cases of culturing E. coli ATCC 11303 additional oxygen was blown through the medium.

3. Results and Discussion


3.1.E. coli strain ATCC 11303

Figure 2 shows the inactivation of E. coli, strain ATCC 11303, at 2.5 kbar and two different temperatures. The bacteria had been grown aerobically, the culture being gased with additional oxygen. In figure 2 the logarithm of colony forming units is plotted as a function of time. The controls had been set to the same temperatures for the time indicated, but under normal pressure. The dashed line indicates the detection limit. The inactivation curves are very different depending on whether the temperature was low or 39

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high. At high temperature there was a simple first order reaction, a straight line in the semilogarithmic plot, but at a lower temperature of 30C or less biphasic inactivation was found. Deviations from simple first order reactions are also known for other inactivation methods (Moats, 1971; Rahn,1929; Wallhuer, 1995). All the subsequent results were at 25C and show this biphasic behaviour. Such curves can be described by three parameters: The D-values of the two linear parts, describing the slopes, and the ratio of sensitive to less sensitive specimens which is Figure 2 - Inactivation of E. coli at 2.5 kbar, w 30C; and s 50C; open symbols are controls

obtained from the intercept of the assymptote for high times. The D-value is the time in minutes needed to kill 90% of the bacteria, i.e to reduce them by one decade. The interpretation of biphasic behaviour is that the population contains a small fraction of less sensitive specimens in spite of the fact that it has been grown from one single clone. The initial pH value of the culture medium was 7.5 and usually it changed to 7 or somewhat smaller values in aerobic growth. However, in a growing culture of E. coli gased with additional oxygen, the pH reached a steady value of 9.3 in the stationary phase (figure 3). The reason for this strange evolution of pH in the growing culture is the fact that the oxygen gas expels the carbondioxide from the solution and thus changes the buffer system. Figure 4 gives the inactivation of the bacteria at pH 9.3. If the bacteria are transfered to the original medium with pH 7.5 they are much more resistant. This is also shown in figure 4, where the fast inactivation of the sensitive part of the bacteria is 18 times slower at pH 7.5 compared with that at pH 9.3. The inactivation curve changes

dramatically if the bacteria have been grown anaerobicaly, deficient in oxygen. A distinct lag time now appears before the inactivation of the insensitive fraction starts (figure 5). The description of such a curve needs 4 parameters, two slopes, less sensitive fraction, and duration of the lag time. In such an anaerobic culture the pH has become 6 because a lot of fatty acids have been 40 Figure 3 - Development of pH during aerobic growth (gased with oxygen) of E. coli at 37C in Standard I broth

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Figure 4 - Pressure inactivation of aerobically grown E. Figure 5 - Pressure inactivation of E. coli at 2.5 kbar coli (gased with oxygen) at 2.5 kbar and and 25C, grown for 6h with restricted 25C; s in the culture medium, pH 9.3; q oxygen supply, pH 6; open symbol is the centrifuged and suspended in a new culture control medium, pH 7.5; open symbols are controls produced in the fermentation process. If the pH is carefully adjusted to a value of 8 the lag time disappears and the insensitive fraction of the bacteria becomes smaller (figure 6). If the bacteria are resuspended in fresh medium adjusted to pH 6 the lag time comes back. Bacteria that had been grown with oxygen do not show a lag time, even if they are suspended in a medium used for anaerobic growth with pH 6 (figure 7).

Figure 6 - Pressure inactivation of E. coli at 2.5kbar and Figure 7 - Pressure inactivation of E. coli at 2.5 kbar 25C, grown for 6h with restricted oxygen and 25C, aerobically grown for 6h (gased supply, centrifuged, washed in 0.9% NaCI with oxygen), centrifuged and suspended solution, centrifuged and resuspended in the in sterile broth which had been used before same broth in which the bacteria had been to grow E. coli for 6h with restricted grown but sterilized before and adjusted to pH oxygen supply; pH 6; open symbol is the 8; open symbol is the control control

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3.2.E. coli strain ATCC 39403

The following results are for E. coli strain ATCC 39403. It was aerobically cultured but without additional supply of pure oxygen. It is able to grow in a well defined minimal medium consisting of salts, glucose, proline, thiamine and phosphate buffer. In this case the pH remains 7 during the exponential phase and reaches an end value of 6 in the stationary phase of the growth curve. These bacteria grow much slower than the strain given above. As can be seen from figures 9 or 11 it needs at least 10 hours to reach the stationary phase. Bacteria from the exponential phase have a much larger fraction of sensitive specimens than those of the stationary phase (figure 8). This fact, which is also well known in temperature
lg cfu
9

inactivation (Dabbah et al, 1969; Hansen and Rieman, 1963), gives the clue to reveal the underlying reasons for biphasic behaviour. For this purpose we added different amounts

of chloramphenicol between 2 and 8 g/ml in the exponential phase (figure 9) thus inhibiting competitively the synthesis of proteins in the bacteria. They were then harvested after 6 hours and pressurized. Figure 10 shows again the inactivation of stationary phase cells and exponential phase cells after 3.5 and 6 hours. With chloramphenicol the cells are even more sensitive than the exponential phase cells, but there is no difference between 2 and 8 g in spite of the fact that with 2 g a lot of new cells grow up (see figure 9). It seems that the biosynthesis of proteins is inhibited mainly for those proteins which are made during the stationary phase for the protection of the bacteria. Reeve et al (1984) have shown that carbon-starved E. coli K12 synthesize proteins which are needed to survive and that the synthesis of these proteins is inhibited by chloramphenicol. Another experiment gives additional insight. We stopped the growing culture in the exponential phase by adding the substance 42 Figure 9 - Growth of E. coli in minimal medium without oxygen aeration, arrow marks addition of chloramphenicol after 3.5h in concentrations of v 2; w 4; s 6; x 8 g/ml; q without chloramphenicol
-1 0 15 30 t / min 45

Figure 8 - Inactivation of E. coli at 3kbar and 25C; (q culture in the stationary phase; v culture in the exponential phase); open symbols are controls

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?@@4V? H@@@'V ?Y(@@@@3 ?5@@@@@@N? H@@@@@@@3? 5@@@@@@@@N ?H@@@@@@@@@3 ?5@@@@@@@@@@N? ?@@@@@@@@@@@3? ?@@@@@@@@@@@@N Y0@@@@@@@@@@@@@'V? ?H5@@@@@@@@@@@@@@@3? Y(@@@@@@@@@@@@@@@@@? @@@@@@@@@@@@@@@@@? 5@@@@@@@@@@@@@@@@@@@@? ?H@@@@@@@@@@@@@@@@@@@@@N ?5@@@@@@@@@@@@@@@@@@@@@3 ?YH@@@@@@@@@@@@@@@@@@@@@@@N? ?5(@@@@@@@@@@@@@@@@@@@@@@@'V @@@@@@@@@@@@@@@@@@@@@@@@@3 ?@@@@@@@@@@@@@@@@@@@@@@@@@@@ ?YH@@@@@@@@@@@@@@@@@@@@@@@@@@@N? (@@@@@@@@@@@@@@@@@@@@@@@@@@@'V ?5@@@@@@@@@@@@@@@@@@@@@@@@@@@@@3N? H@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ 5@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@3? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?

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Therefore the cells accumulate in that stage of

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their cell cycle where the synthesis of nucleic

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acids is inhibited. The result is seen in the

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inactivation curves of figure 12. The more

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?M@? Y0@@ ?@@@@@@@@@@@@@@@@@@? 5@@@N?fh?1@@@@@@@@@@@@@@@@@? 1@@@@?fh?X62?6@2?6@@?@@@&O@? @@@@@@ L@@@J? ?@@@N? H@@@'V Y0?@@@J? @@@7 ?M(@@@@'IM04IM04I?fh5@@@2W @@@@@@@@@@@@@@@@@@fh@@ 1@@@@@@@@@@@@@@@@@h?M0@@&&O X)@@@@@@@@@@@@@@@@g?Y0@@@&WW? ?X)@@@&Of?@@@@@g?5@@@2W? L@@@J?fH@@@h?@@& H@@@f?Y(@@@gY0@@@JO? 5@@@fY(@@@7g5@@@7? 1@@@f5@@@2Wg@@@2W? ?@@@f@@@?h@@ 5@@@e?H@@@?g?H@@ @@@@eM(@@7?g?5@@ 1@@@?@@@@&W?g?@@@fh?M@? ?@@@?@@@&Wh?@@@fh@@7? 5@@@@@@&W?h?@@@N?eh@@J? @@@@@@&Weh?@@@'Ig?Y0@@7 1@@@@@J?eh?1@@@@4Vf?5@@@J L@@@@7fh?X)@@@@'I?M@V@@@@? H@@@@J X6@@@@@@@@@@2O )@@@ K)@@@@@@&O X)O?@? ?X62?6@2W?

M@I?fM@I? @@4VfhY0@@@@@@@@@@@@@@@@@? @@@3fh(@@@@@@@@@@@@@@@@@7? @@@@fh?K62?6@2?6@@@@@@@2W? @@@7 Y@@@ @@@? ?M(@@@ @@@3 @@@@ ?H@@@@@7 @@@@2W @@@7 M(@&O? ?H@@@J ?YY0@@@J ?@@@@N @@@@ Y((@@@@? @@ ?L@@@J ?Y(@@7 @@@? Y( @@@?fh?Y(@@@&W @@@?fh?5@@@2W? @@@?fh?@@@ @@@?fhH@@@ ?H@@@NfM+V?f5@@@ ?L@@@WM(@@@2W?f1@@?eh?@@@ ?@@@@@?Y0@@@*?f@@@7eh?@I? @@@@@@@@hL@@3ehH@@7 ?H@@@@@@@@h?@@@h?Y(@@J ?@@@@@@2O?h?1@@@4I?fM(@@7? ?L@@@&O?eh?XX6@@@@@@@@@@2WW? )@@@@4I?M0@@@& @@@J @@@? K)@@@@@@ ?X62OK6@

M@I? ?Y0@@@@@@@4Ifh?@@@ ?@M(@@@@@@@@@@4Ieh?@@@ @@@@@@@2?6@@@@@@h?@@@ ?@@@@&O?fK6@@@?h?@@@ H@@@@Jh?@@3hH@@@ 5@@@7?h?@@@h@@@@ 1@@@J?h?@@@hL@@@ ?@@@ehH@@@h?@@@ 5@@@N?g?Y(@@@h?@@@ 1@@@3?g?5@@@@h?@@@ X)@@@Ng?@@@@7h?@@@ ?XX)@@'?@IM0@@@&W?hH@@@ )@@'V?f?@@@&Wh?@@@ @@@@@@@@@@@Jeh@@@@ ?5@@@@@@@@@@@?ehL@@@ H@@@&OeK)@@@Neh?@@@ ?Y(@@&W?e?L@@@3ehH@@@ ?5@@@Jg1@@@N?h@@@@f?M@? ?@@@@?gL@@@@?hL@@@?Y0@@@@? ?1@@@?g?@@@eh?@@@Y(@@@@@? @@@NgH@@@3?hH@@@@@@@&O ?5@@@3g5@@@@?h@@@@@@@& ?@@@@@f?H@@@@@?hL@@@@@2WW? K)@@@4?@?(@@fh?@@@ ?X)@@@@@@@@7fh?@@7 X6@@@@@@@Jfh?@2W ?K62OK@?

?M04I? ?Y0@ ?M0@@@@@@4I?eh?5@@ ?Y0@@@@@@@@@@4I?h?@@@ ?5@@@@@2OK)@@@@7h?@@@ H@@&O?fX6@@@@h?@@@ ?Y(@@Jh?@@?h?@@@ Y@@@7?h?@@3h?@@@ ,(@@@?h?@@@h?@@@ L@@@J? ?@@@ ?@@@ ?@@@ ?@@@N? ?@@@ ?@@@3? ?@@@ ?1@@@?h?M+Vh?@@@ @@@? ?@@@ ?L1@@@4IfM0@@@3h?@@@ X)@@@@@4?0@@@@@7h?@@@ ?X6@@@@@@@@@@@@Jh?@@@ ?K6@K@O?=@@@@?h?@@@e?M@?+V @@@@@V@@@?h?@@@ ?@@@7?h?@@@?Y0@@@ ?@@@J?h?@@@Y(@@@@@* H@@@eh?@@@@@@@&O2W 5@@@eh?@@@@@@& ?H@@@@eh?@@@@@2WW? ?M04?0@4?(@@@7eh?@@@&O @@@@@@@@@@@@@Jeh?@@&W? @@@@@@@@@@@@7?eh?@2W ?K62O?K@O?K&W?

Y(IMY(I?@@@@@4?0@@ (@@@ 5@@@@@@@@@@@@@@@@@h?@4V 1@@@@@@@@@@@@@@@@@h?@@3 X)OK62OK62O?@@@@2Wh?@@@ @@@@@7h?@@@ @@@?ehH@@@ ?Y0@@@7?eh@@@@ Y(@@@2W?ehL@@@ ?Y(@@2O?fhH@@? ?@@7 Y0M(@@ @@@7 @@@3 5@@@2W L@@@ ?H@@&O ?@@7 Y(@@ H@@? ?Y(@@7J? 5@@3 ?5@@&W @@@@ H@@&W? @@@7 5@@J @@@?eM0@@@? @@@? @@@3 @@@?eh?Y0@h@@@@?@@@@@7? @@@Neh?5@7h1@@@?@@@@2 1@@'V?h?@@Jh?@@@@@@&O?W? L@@@'VgY0@@@?h5@@@@@&W ?@@@@'I?e?M(@@@7?h@@@@@2W? K)@@@@@@@@@@@2W?h@@@& ?L@@@@@@@@@&O?eh@@@JO? @@@@@@@@2Wfh@@@?

Y(?0@4I? Y0?M(@@@@@@@4VV? 5@@@@@@@@@@@@'3? @@@@@2O?eK)@@@? @?g?X)@ @@@? ?H@@7?h?@7? ?5@@J?h@2W? ?@@@ ?@@@fM0@4I? ?@@@Y0@@@@@@@@4V ?@@@@@@2?6@@@@@3 ?@@@@&O?eK@W@@@N? H@@@&Wg?1@@ @@@@J?g?L@@3? L@@@eh@@@? ?@@7eh@@@? H@@?eh@@@? @@@3eh@@@N L@@@eh@@@@ ?1@@eh@@@J ?L@@N?h@@7? @@'If?Y0@@@ 1@@@@@eM(@@@7J? X6@@@@@@@@&O2W @@Y0@@@@ @@@@@2W?

@4IM0@ @@@@@@ ?M0@@@@@@@@@4V ?Y0@@@@@2?)@@@@3 ?5@@@@2OeX)?)@@ ?@@&O?g?L@@N? H@@Jeh@@3? ?Y(@@Neh@@@? ?,@@@@eh1@@N ?L@@@?ehH@@@ @@@JehL@@3 ?H@@@Neh5@@@ ?@@@@3eh@@@@ K)@@'IgM(@@@@ @@N?g?H@@@@ ?L@@@@4I?M0@@@@@@@ @@@@@@@@@@@@@@@7 K6@@@@@&O??@@J ?K&We5@@? H@@? @@7? ?@4Vh?H@@J? ?@@3hM(@@ ?@@@gY0@@@7 ?1@@@@@4IM(@@@2W ?X?K6@@@@@2O2O 6@@@@@@@@@ ?K62O?

?Y04IM+V M(@@@@@'V? ?YY0@@@@X)@@'I@? Y((@@@@@?L@@@@@? 5@@&O?f@@K)@? @Jh@@ ?H@@@?h?L@N ?5@@@?eh@3 ?@@@7?eh@@ ?@@@J? ?@@@N? ?@@@'V ?@@@@3 K)@@@@@@@? ?L@@@@@@7? @@ ?H@@@@@2W? ?5@&O? H@@J ?Y(@@N ?(?@@Jh?5@@ @@@@h?Y0@ ?@@Nh?@@@ ?@@'I?fM0@@@7 ?@@@@@@4?0@@@@2W K6K)@@@@2WO? @@@@@@@& ?X62O?

Y0?M04I?@4V? @@@@@@ ?Y(@@@@@@@@@'V ?5@@&?62?)@@@'V? ?@V@@&W?e?X)@@@'? ?1@@@JgX)@@ @@7?h@@3? ?5@@J?g?@@@@N ?@@7eh?@@3 H@@Jeh?@@7 5@@?ehH@@? @@@Neh@@@3 1@@@ehL@@@ ?@@Jeh?@@7 5@@?ehH@@? @@@?eh@@@3 1@@?ehL@@@ ?@@?eh?@@7 5@@NehH@@J 1@@'V?g?Y(@@? L@@@'?g?5@@7? ?1@@h?@@@J? ?L@@'VgH@@@ 1@@'V?e?Y(@ X)@@'?04?(@@@@ ?X6@K6@@@@2W @@@@@@@7

?Y04?0@@ M(@@@@@@ ?YY0@@@@@@@@@4V? ?5(@@@@2OKX6@@@3 @@g)@@@'V ?@@7h?@@7 ?@@Jh?@@? ?@@?h?@@3 ?@@@@?h?1@@ ?1@@@?eh@@N? @@@?h?L@@ ?5@@@?h?H@@@? ?@@@@?h?5@@ ?1@@@?h?1@@J? @@@?h?L@@N? ?5@@@?h?H@@@? ?@@@@?h?@@@J? ?1@@@?h?L@@ ?L@@@Neh@@ 1@@3h?H@@ L@@@h?5@@ ?@@@h?@@@ ?1@@N?g?@@@ ?L1@@@@4IM(@@@2W @@'IfY0@@@7 X6@@@@@@@&W? @@@@@@&O @O?K&W

inhibitor was added, the more the pressure

Y+V? M@ Y( M@I? 5@3?h@@@?f@@@?e?@@@h?@@@h?@@@ ?Y(@fhY0@@@@4V @@@?h@@@?f@@@?e?@@@h?@@@h?@@@ ?5@@fh(@@@@@@3 @@@?h@@@?f@@@?e?@@@h?@@@h?@@@N? ?@@7fh?K?K)@@@ 6@@@@@ @@@?h@@@?f@@@?e?@@@h?@@@N?g?@@@3? @@@?h@@@?f@@@?e?@@@h?@@@3?g?@@@7? ?YH@@J ?@@@ @@@?h@@@?f@@@?e?@@@h?@@@7?g?@@@ ?5(@@? @@7? 5@@@ @@@?h@@@?f@@@?e?@@@h?@@@J?g?@@@ ?@@@@? @@@@ @@@?h@@@?f@@@?e?@@@h?@@@h?@@@J? H@@&W? 1@@7 @@@?h@@@?f@@@?e?@@@h?@@@h?@@@ 5@@N ?@@? @@@?h@@@?f@@@?e?@@@h?@@@h?@@@ @@@J 5@@3 @@@?h@@@?f@@@?e?@@@h?@@@h?@@@ @@@@ 1@@@ @@@?h@@@?f@@@?e?@@@h?@@@h?@@@ L@@7 @@@?g?H@@@?f@@@?e?@@@hH@@@N?g?@@@ ?H@@ @@ H@@? @@@Ng?5@@@?f@@@?e?@@@N?g5@@@3?gH@@@ ?5@@ 5@@3 @@@3g?@@@@?f@@@?e?@@@'Vf?H@@@@@Nf?Y(@@@ ?@@@ @@@@ 1@@@N??Y0@@@@@@?f@@@?e?1@@@'V?eY(@@@@@'V?eM(@@@@ H@@@ @@@@ L@@@'IM(@@@@@@@?f@@@?e?L@@@@'I?M(@@@@@@@'IM0@@@@@@N?fh?Y(@@@ ?M0@@@@@@@@? ?@K6@@@@@2OK)@7?f1@7?fX6@@@@@@@2W?X6@@@@@@@@?@@2W?fh?@@@@7 @@@@@@@@@@@@@?f@@@?f1@@@@@@@@@&?)@@@@@@@@@?@@@*?fh?5@@@@ )@@@@@@@ X6@@@@@@@@@? K@O?fX-W?fX-W?hK@O?hK@O? ?@@@@J K)@@@@ H@@@@? ?L@@@@ @@@@ 5@@@7? @@@&W? 1@@@ @@@J L@@7 @4V? @@7? ?@2W @@*? @@J? @2W? @@

Figure 11 - The growth of E. coli was stopped within the exponential phase by adding Trimethoprim in different amounts of v 1; w 2; s 4 g/ml; q is the control; arrow marks addition of trimethoprim; culturing condition: M9medium; 37C

resistant fraction of the bacteria disappears. Figure 10 - Pressure inactivation of E. coli at 3 kbar and 25C, aerobically grown for different times Therefore, we concluded that the biphasic (without oxygen aeration) of s 3.5h; w 6h; behaviour of the inactivation curves reflects q 10h; v 6h but 2g/ml chloramphenicol after 3.5h; x 6 h but 8 g/ml chloramphenicol two stages of bacterial development in the cell after 3.5 h; open symbols are controls cycle.

synthesis of nucleic acids is slowed down.

activated carbon atoms. In consequence, the

purine and pyrimidine bases via the transfer of

the cell needs this molecule to synthesize

tetrahydrofolic acid cannot be produced, but

(Mutschler, 1991). If this enzyme is inhibited

important enzyme dihydrofolate reductase

trimethoprim in different amounts (figure 11).

Schreck, van Almsick & Ludwig

-1.8

lg OD( =660 nm; d=1 cm)

-1.3

-0.8

-0.3

The bacteria were then harvested after 7 hours. Trimethoprim is a competitive inhibitor of the

t/h

Figure 12 - Inactivation of E. coli at 3 kbar and 25C in dependance on the concentration of added Trimethoprim; q 0; w 2; s 4; x 8 g/ml; q harvested in the stationary phase after 15h

-1

10

20

30 t/ min

40

50

43
lg cfu

High Pressure

Process Optimisation and Minimal Processing of Foods

Process Assessment

4. Conclusions
Aerobic and anaerobic growth of the same bacterial strain yield specimens with different behaviour. The pH value does not only influence the rate of inactivation but also the characteristics of the kinetic curves; the lag time found for anaerobicaly grown E. coli appears or disappears depending on pH. E. coli from the exponential phase are more sensitive to pressure than those from the stationary phase. One reason seems to be that stationary phase cells have additional stabilizing proteins. The biphasic behaviour of the inactivation curves of a single colony is caused by various stages of the bacterial development in the cell cycle.

Acknowledgements
This work was supported by the EU project AIR 1-CT92-0296

References
Dabbah, R., Moats, W. A. & Mattic, J. F. (1969). Factors Affecting Resistance to Heat and Recovery of Heat-Injured Bacteria. Journal of Dairy Science, 52, 608-614 Hansen, N. H. & Riemann, H. (1963). Factors Affecting Heat Resistance of Nonsporing Organisms. Journal of Applied Bacteriology, 26, 314-333 Moats, W. A. (1971). Kinetics of Thermal Death of Bacteria. Journal of Bacteriology, 105, 165-171 Mutschler, E. (1991). Arzneimittelwirkungen, 6. Auflage. Wissenschaftliche Verlagsgesellschaft, Stuttgart, 605 f Neidhard, F. C. (1987). Escherichia Coli and Salmonella Typhimurium, Cellular and Molecular Biology. Vol 1, Part 1, 3-127, American Soc. for Microbiology, Washington D.C. Rahn, O. (1929). The Non-Logarithmic Order of Death of Some Bacteria. The Journal of General Physiology, 13, 395-407 Reeve, C. A., Amy, P. S. & Matin, A. (1984). Role of Protein Synthesis in the Survival of CarbonStarved Escherichia coli K-12. Journal of Bacteriology, 160, 1041-1046 Wallhuer, K. H. (1995). Praxis der Sterilisation, 5 Auflage, Georg Thieme Verlag, Stuttgart

44

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Questions and Answers

When resistance in a population can arise to UHP, in a combined process, would this resistance render cells less sensitive to a subsequent treatment? It means the first strike

should be the best in order to have as little cells left at all. Leon Gorris

Yes, Microorganisms that survive the first treatment can produce factors to resist another second treatment, for instance a heat shock or pH shock can result in such resistance

factors.

45

Process Optimisation and Minimal Processing of Foods

Process Assessment

The Effect of Ultra High Hydrostatic Pressure on Vegetative Microorganisms and Spores of chosen Bacteria and Moulds
Monika Fonberg - Broczek1,2, Jacek Arabas1, Sylwester Porowski1, Stefan Podlasin1, Janusz Szczepek1, Bozena Windyga2,Halina Sciezynska2, Krystyna Gorecka2, Anna Grochowska2, Kazimierz Karlowski2, Janusz Jurczak3, Piotr Salanski3 1 High Pressure Research Center, Polish Academy of Sciences, Warsaw, Poland 2 Department of Food Research, National Institute of Hygiene, Warsaw, Poland 3 Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland

Summary
The experimental work presented in this communication analysed the effect of high hydrostatic pressure on Gram-negative and Gram-positive bacteria, moulds and spores of Bacillus cereus and Aspergillus flavus. The inactivation of microorganisms was found to be directly correlated to the level of high hydrostatic pressure, but significant differences between Grampositive and Gram-negative microorganisms were observed. It was stated that Bacillus cereus spores were the most resistant to high pressure and combined treatment with high pressure and temperature was needed for their inactivation, whereas conidia of Aspergillus flavus were inactivated in conditions similar to Gram-negative bacteria.

1. Introduction
Food products are an excellent environment for the growth of pathogens, which may cause foodborne diseases. The quality and shelf-life of food products depends greatly on the properties of the microorganisms that contaminate the food. Many methods of food preservation are used for providing microbiological safety, among which Ultra High Hydrostatic Pressure (UHP) seems very promising. The killing mechanism of microorganisms with UHP is thought to be based on the destruction of the microbial cell membrane, caused by changes of the cell volume and denaturation of proteins. Data known from literature differ in results and parameters of UHP used for inactivation of microorganisms. The aim of this study was to investigate the UHP effect on several microorganisms exposed to similar pressure and temperature conditions.

2. Materials and methods


2.1. Microorganisms studied

Investigations covered the following microorganisms: Gram-positive bacteria: 46

Fonberg - Broczek, Arabas, Porowski et al

High Pressure

- Staphylococcus aureus NCTC 4163, - Bacillus cereus ATCC 10876 Gram-negative bacteria: - Escherichia coli NCTC 8196, - Salmonella enteritidis, - Proteus mirabilis Moulds: - Aspergillus flavus Initial counts were 106 cells/ml for Staphylococcus aureus and Proteus mirabilis, 107 cells/ml for other bacteria and conidia Aspergillus flavus, 105 cells/ml for spores of Bacillus cereus.

2.2. Microbiological mediums

Bacteria were stored on the solid medium for strains maintaining and moulds - in the solid Czapek medium. Suspensions of specific density of bacteria in saline were prepared from 24h broth cultures at 37C for Salmonella, Escherichia coli and Staphylococcus aureus. Broth cultures of Bacillus cereus were performed for 24h at 30C and then the cultures were heated for 10 min. at 80C to kill vegetative forms. Suspensions of spores in saline were prepared after cooling. Aspergillus flavus strains were cultivated for 5 days at room temperature in solid Czapek medium and then spores were washed off from the surface with liquid medium. To detect the effect of UHP on mycelium, suspensions of hyphe were taken. Suspensions of vegetative forms and spores were collected in aseptic polypropylene tubes. Control samples and UHP treated samples were plated in nutrition agar and Czapek medium corresponding to the kind of microorganisms and the adequate ISO method was used for total number of bacteria or moulds.

2.3. High pressure treatment

The experiments were carried out in piston-cylinder type high pressure vessels with maximum pressure of 1500 MPa and volume of 30-50 cc, developed in the High Pressure Research Center. Distilled water was used as pressure transmitting medium. The samples were closed in Teflon or polypropylene ampoules. Working temperatures were stabilised by the heating/cooling jacket mounted on the vessel. Cultures of bacteria, moulds and suspensions of spores were treated with hydrostatic pressure ranging from 100 MPa to 1000 MPa for 5, 10 and 15 minutes at ambient temperature and at 10C to 50C for spores of Bacillus cereus.

47

Process Optimisation and Minimal Processing of Foods

Process Assessment

3. Results
Inactivation of microorganisms was found to be directly correlated to the level of high hydrostatic pressure, but significant differences between Gram-positive and Gram-negative bacteria were observed. Pressure of 500 MPa applied for 10 min. reduced the population of Gram-negative bacteria Salmonella, E. coli, Proteus mirabilis by 6 - 7 log. The population of 106 cells/ml of the Gram-positive bacteria Staphylococcus aureus was inactivated at 800 MPa, 20C (Table 1) and at 600 MPa, 10C. Table I The effect of pressure on survival of bacteria G(-) and G(+) - time 10 min., temperature 20C Bacteria Initial number (cells/ml) 100 200 300 Number of bacteria in suspension after UHP (cells/ml) 400 (MPa) Salmonella E. coli P. mirabilis S. aureus 3.0x10 7 2.5x10 7 1.8x10 6 3.3x10 6 2.5x10 7 1.1x10 7 1.2x10 6 2.7x10 6 2.7x10 6 2.6x10 6 1.2x10 5 2.1x10 6 1.4x10 6 1.0x10 6 9.0x10 2 1.7x10 6 1.5x10 2 1.5x10 2 absent 1.5x10 6 absent absent absent 4.8x10 5 absent absent absent 1.5x10 5 absent absent absent absent 500 600 800
(*) treatment

(*) UHP - Ultra High Pressure

Even more resistant were the spores of Bacillus cereus. The treatment of 1000 MPa, 20C, 10 min. reduced the count of spores by about 1 log. The same effect was observed for 800 MPa applied for 10 min. The pressure of 800 MPa, 10 min. was sufficient to destroy all spores when the temperature was increase to 50C. These results are in agreement with other studies indicating that it is necessary to combine high pressure and temperature treatment for inactivation of all bacteria spores (Table 2). Conidia of Aspergillus flavus were destroyed at 400MPa, 20C, 10 min. (Table 3). The inactivation of bacterial cells was irreversible. The ability of bacteria to grow was also investigated in three time conditions: immediately after the UHP Table II The effect of pressure and temperature on survival of spores of solution - time 10 min. Initial number (cells/ml) 2.8x10 4 7.8x10 4 5.0x10 4 (C) 10 20 50 200 MPa 1.6x10 4 ----400 MPa 7.7x10 3 3.3x10 3 --Temperature Number of spores in solution after UHP (*) treatment (cells/ml) 600 MPa 3.1x10 3 2.1x10 3 --800 MPa 1.9x10 3 1.9x10 4 absent 1000 MPa --3.4x10 3 absent Bacillus cereus saline in

(*) UHP - Ultra High Pressure

48

Fonberg - Broczek, Arabas, Porowski et al

High Pressure

Table III The effect of pressure and temperature on survival of spores of Aspergillus flavus in saline solution - time 10 min. Initial number (cells/ml) 7.0x10 4 4.8x10 5 (C) 20 10 300 MPa 2.7x10 2 1.6x10 2 400 MPa absent absent Temperature Number of spores in solution after UHP (*) treatment (cells/ml) 500 MPa absent absent 800 MPa absent 1000 MPa absent -

(*) UHP - Ultra High Pressure

treatment, 24 hours and 48 hours after the treatment. No growth was observed in these conditions. A combined treatment: 800MPa, 50C, 10 minutes kills all Bacillus cereus spores - 106 cells/ml. For inactivation of the conidia of Aspergillus flavus 400MPa, 20C, 10 min suffice. Inactivation is slowest at room temperature, higher and lower temperatures give better results.

4. Conclusions
The study confirmed significant variation in pressure sensitivity between Gram-positive and Gram-negative bacteria. A combined treatment with UHP and temperature is required for inactivation of Bacillus cereus spores. Conidia of Aspergillus flavus were inactivated in conditions similar to Gram-negative bacteria.

Acknowledgements
This work was funded by the National Committee of Scientific Research, Poland (Grant N 5S 307 005 05 1993/1994) and the High Pressure Research Center of the Polish Academy of Sciences, Warsaw, Poland.

References
Balny, C., Hayashi R., Heremans, K., Masson, P. Eds. (1992). Proceedings of First European Seminar on High Pressure Biotechnology, High Pressure and Biotechnology. La Grande Motte, France, September 1992. John Libbey and Company Ltd Butz, P. & Ludwig, H. (1986). Pressure Inactivation of Microorganisms at Moderate Temperatures. Physica 139 and 140 B, 875-877 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 Science and Technology, Budapest, Hungary, 07.30-8.04, 1995, Abstracts Vol. II. P207, p. 74 49

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Gould, G. W. (1995). The Microbe as a High Pressure Target. in High Pressure Processing of Foods. Ed. Ledward D. A., Johnson D. E., Earnshaw R. G., Hasting A. P. M.. Nottingham University Press, 27-35 Knorr, D. (1993). Effects of High Hydrostatic Pressure Processes on Food Safety and Quality. Food Technology, 47 (6), 156-161 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, 272

50

Szczawinski, Peconek, Szczawinska, Broczeck & Arabas

High Pressure

High Pressure Inactivation of Listeria monocytogenes in Meat and Meat Products


Jacek Szczawinski1, Janina Peconek1, Malgorzata Szczawinska1, Monika Fonberg-Broczek2,3, Jacek Arabas2 1 Department of Food Hygiene, Faculty of Veterinary Medicine, Warsaw Ag. University, Poland 2 High Pressure Research Center, Polish Academy of Sciences, Warsaw, Poland 3 Department of Food Research, National Institute of Hygiene, Warsaw, Poland

Summary
High pressure technology raises a lot of interest among scientists from all over the world and in the nearest future a dynamic progress in this field is expected. This study confirmed the usefulness of high pressure technology for inactivation of Listeria monocytogenes in cured, pasteurized, sliced, vacuum-packaged meat products.

1. Introduction
The phenomenon of damage of bacterial cells resulting from exposure to high hydrostatic pressure has been known since 1895. High pressure technology, however, has been used in food processing since just a few years mostly for preservation of jams, yoghurts and other food products. High pressure technology raises a lot of interest among scientists from all over the world and in the nearest future a dynamic progress in this field is expected (Johnston, 1995, Lewicki, 1992 and Kolakowski et al, 1994). The aim of this study was to define the usefulness of high pressure treatment for inactivation of Listeria monocytogenes in raw meat and in cured, pasteurized, sliced, vacuum-packaged pork ham. In our previous studies it has been found that L. monocytogenes is comparatively sensitive to high temperature. Properly conducted heat treatment during preparation of food at home (boiling, frying, stewing, baking), as well as in industrial food processing, should cause complete inactivation of L. monocytogenes in pork-butchers meat products. The problem is caused by secondary contamination, which occurs after heat treatment. It is practically unavoidable during industrial processing of food: slicing and packaging of cured, pasteurized food products. As a result of contamination there is an increase in the microbial load in ham and other sliced and vacuum-packaged meat products. Microbial contamination consists of various microorganisms, commonly present in animal derived food products (Kwiatek, 1991, 1993) and in meat processing plants (Berends et al. 1995), including pathogenic bacteria: Staphylococcus aureus, Salmonella, Listeria. Contamination with L. monocytogenes appears to be very hazardous. In contrast to the majority of pathogenic microorganisms L. monocytogenes (classified as a psychotrophic microorganism) has the ability to multiply in vacuum-packaged food products, stored in the 51

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refrigerator. Listeria monocytogens is responsible for severe food poisoning and may cause death among sensitive persons (Zareba and Borowski, 1994)

2. Materials and methods


Lean beef meat was minced, divided into 10-gram portions and subsequently placed in polyamide-polyethylene bags (Multiseven 78 TOP, Wipak, Finland). In order to restrict the growth of saprophytic microflora (which might have affect the determination of L. monocytogenes), the samples were decontaminated by gamma irradiation at a dose of 10kGy. Cured, sliced, pasteurized pork ham (bought in a butcher store) was divided into 10g portions. Every portion, made of two adjoining slices of ham, was packaged in PA/PE bag in the same way as meat. These samples were not exposed to ionizing radiation. Both types of samples (meat and ham) were inoculated with a mixture of five strains of L. monocytogenes, (incubated on BHI at 37C for 24 h) obtained from the Polish National Veterinary Institute. After inoculation all samples were vacuum-packaged (Henkovac 1000). The samples were exposed to high pressure treatment in the High Pressure Research Center of the Polish Academy of Sciences, where a special stand for food testing was built. The process of high pressure treatment requires the following steps: a high pressure chamber, closed from the bottom with a special cover, is filled with distilled water; after putting vacuum - packaged food products in the chamber, the top cover is closed; pressure is generated in 10-60 seconds by a special plunger, descending into the chamber. The measurement of hydrostatic pressure inside the chamber is done indirectly with the help of a manometer, indicating the pressure under the piston rod. After high pressure treatment is finished, the pressure in the chamber is lowered in about a twice longer time than it has been generated in. In our study samples were exposed to 100, 150, 200, 300 and 400 MPa (1000, 1500, 2000, 3000, 4000 bars). The samples were subsequently stored at 4C for 18 hours. The number in of Listeria pressure Pressure Table I Effect of high pressure on sensory parameters of cured ham Mean score of 12 samples (points 1 - 9) (MPa) 0 100 150 200 300 Appearance 6.8 a 6.3 a 6.0 a 6.3 a 6.8 a 6.4 a Colour 7.0 a 6.4 ab 6.1 ab 6.2 ab 6.7 ab 5.8 b Smell 6.5 a 6.1 a 5.8 a 6.2 a 6.2 a 6.2 a Taste 6.2 a 5.8 a 5.8 a 6.2 a 6.2 a 5.9 a

monocytogenes

high

treated and in control samples was determined using a dilution

technique. The samples were plated onto Oxford medium (Oxoid) and incubated for 72 hours at 30C. The experiment was repeated three times. The results were

transformed into logarithms and statistically analyzed, using analysis of variance and linear regression. The 52

400 a,b Means within the same column having different superscripts are different at 95% confidence level (Tukey's test)

Szczawinski, Peconek, Szczawinska, Broczeck & Arabas

High Pressure

statistical package Statgraphics was used. Additionally, some control (uninoculated) samples were treated with high pressure. These samples were used in sensory studies using 9-point quality scores for appearance, colour, smell and taste (Table I). The evaluation was conducted by a trained panel (4 persons). Results were statistically transformed using two-factor analysis of variance. Mean values were compared using Tukeys test.

3. Results and discussion


Results indicate (Figs 1, 2) that in various products (beef meat, cured pork ham) the number of L. monocytogenes decreases proportionally to the increase in high pressure and time of treatment.
4 10 8 6

log cfu

On the basis of analysis of variance it was observed that the effect of these two factors (level and time of high was
Log no. of listeria
0 0 5 2

Time (min) 15

10

0 MPa 100 MPa 150 MPa 200 MPa 300 MPa 400 MPa

pressure statistically

treatment)

significant

Figure 1 - Survival of L. monocytogenes in minced beef at various pressure levels and treatment time

(P<0,01). After 15 minutes of high pressure treatment (400 MPa) a decrease of the
log cfu
7 6 5 4 3 2 0 MPa 100 MPa 150 Mpa 0 5 10 15 200 MPa 300 MPa 400 MPa

L.monocytogenes number in raw meat was observed: 5,81 logarithmic units in

comparison to reduction of 6,55 logarithmic units in cured ham, treated with the same parameters of high pressure. Therefore it can be assumed that in the environment of raw meat L. monocytogenes has a higher resistance to high pressure than in cured, sliced ham.

Log no. of listeria 1


0

Time (min)

Figure 2 - Survival of L. monocytogenes in cured ham at various pressure levels and treatment time

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Process Assessment

Similar range of reduction of L. monocytogenes as a result of high pressure treatment was observed in previous studies (Rnner, 1994). In a recently conducted study in Poland (FonbergBroczek et al. 1995), evaluating the impact of high pressure treatment on bacterial cultures, a reduction of 6 - 7 logarithmic cycles of Gram-negative bacteria after exposure to 500 MPa for ten minutes was shown. Similar effect on Gram-positive bacteria was observed after treatment with 800 MPa. Still higher pressure was necessary for inactivation of bacterial spores. (FonbergBroczek et al. 1995; Johnston, 1995, Rnner, 1994). The direct comparison of the results is complicated because of differences in experimental schemes applied by the authors (especially different combinations of time of treatment and high pressure level). It seems that proper comparison of different results as well as accurate prediction of the effects of high pressure on microflora would be possible if mathematical methods were applied, similarly to thermobacteriology (Szczawinski and Stanczak, 1994, Ziemba, 1980). Table II shows D values (calculated using linear regression) for L. monocytogenes in various pressure parameters. D value indicates time (in minutes) necessary for a decimal reduction of L. monocytogenes number (that means a reduction of 90% of L. monocytogenes or one logarithmic unit) for a given pressure level. The D value, known for different pressure levels, enables the calculation (regression analysis) of the coefficient z (Table II), which states by how many MPa to increase or decrease pressure in order to obtain a ten times decrease or increase of the D value. It seems that in the range of pressure levels and treatment times applied in our experiment, the calculated D values might be useful to predict the extent of L.monocytogenes inactivation. It has to be stressed that the experiments described include comparatively small numbers of samples and must be treated as preliminary results. Further investigations are needed to define the conditions in which concepts of thermobacteriology might be applied to high pressure technology. Table II D-values (time required for decimal reduction of and z-values (coefficient of resistance of Raw meat -1/b* D100 MPa D150 MPa D200 MPa D300 MPa D400 MPa z-value 124.3 min 81.8 min 25.6 min 9.8 min 2.6 min 177.6 MPa Correlation 0.7869 0.8820 0.9547 0.8610 0.9852 0.9930 L. monocytogenesat given pressure) L. monocytogenesto high pressure) Cured ham -1/b 31.8 min 31.7 min 28.3 min 5.8 min 2.4 min 240.3 MPa Correlation 0.8586 0.7398 0.7502 0.9810 0.9911 0.9642

(*)b - linear regression coefficient

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Raw meat samples which underwent high pressure treatment revealed undesirable organoleptic changes, specially visible when pressures of 300 and 400 MPa were employed. Even before opening the experimental bags, changes in colour and consistency of samples and also leakage were observed. The usefulness of high pressure in meat processing should not be questioned on the basis of these findings, however, because high pressure effects on raw meat depends on many factors (Lewicki, 1992), among which the degree of post-mortem changes plays a major role. In the experiment described negative effects on organoleptic changes in meat might result from the previous radiation tratment and mincing. It seems that appropriate selection of technological parameters will enable good microbiological results while keeping the organoleptic and functional properties of meat. Results shown in Table I explicitly indicate a positive opinion on the application of high pressure technology in decontamination of cured, sliced and pasteurized pork ham. On the basis of the analysis of variance it was stated that the level of high pressure had a statistically significant impact on the colour of samples only, and time did not have any influence on any of the sensory discriminants. Table I contains mean arithmetic results for all times of treatment used. Comparison of these data indicate that only samples which underwent a 400 MPa treatment differ statistically (P < 0,05) in colour from the control samples.

4. Conclusions
The application of high pressure technology to the processing of cured, sliced, pasteurized pork ham cause a reduction in L. monocytogenes contamination by 6 logarithmic cycles, with practically unchanged organoleptic qualities. The results obtained seem very promising and confirm the need for further investigations in this field.

References

Berends, B., Burt, S., Snijders, J. (1995). Critical Points in Relation to Breaking Salmonella and Listeria Cycles in Pork Production. New Challenges in Meat Hygiene: Specific Problems in Cleaning and Disinfection. Ed. Sara, A. Burt and Friedrich Bauer. Utrecht ECCEAMST Foundation, The Netherlands, 9-20 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. Abstracts of 9th World Congress of Food Science and Technology, July 30 August 4, 1995; vol. 2: 74 Johnston, D. E. (1995). High Pressure Effects on Milk and Meat. High Pressure Processing of Foods. Ed. Ledward, D. A., Johnston, D. E., Earnshaw, R. G., Hasting A.P.M., Nottingham University Press, 99-121 55

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Kolakowski, P., Reps, A., Babuchowski, A. (1994). Przydatnosc Wysokich Cisnien w Utrwalaniu i Przetwarzaniu Zywnosci. Przemysl Spozywczy, 48, (4), 108-111 Kwiatek, K. (1991). Wystepowanie Listeria monocytogenes w Wolowinie, Wieprzowinie i Miesie Drobiowym. Medycyna Weterynaryjna, 42, (12), 69-71 Kwiatek, K. (1993). Wystepowanie Listeria monocytogenes w Miesie Oraz w Produktach Miesnych. Zycie Weterynaryjne, 68, (12), 304-306 Lewicki, P. P. (1992). Zastosowanie Wysokich Cisnien w Technologii Zywnosci. Przemysl Spozywczy, 46 (11), 280-282 Rnner, U. (1994). Food Preservation by Ultrahigh Pressure. Food Preservation by Combined Processes. Ed. Leistner, L., Gorris, L.G.M., Final Report FLAIR Concerted Action No. 7, Subgroup B, EUR 15776 EN, 1994, 31-35 Szczawinski, J., Stanczak, B. (1994). Nowoczesne Narzedzie Pracy Inspektora WIS - Elektroniczny Przyrzad do Pomiaru Temperatury i Wartosci Pasteryzacyjnej. Magazyn Weterynaryjny , 3, (3), 5457 Zareba, M. L., Borowski J. (1994). Podstawy Mikrobiologii Lekarskiej. Ed. Wydawnictwo Lekarskie P.Z.W.L., Warszawa. Ziemba, Z. (1980). Podstawy Cieplnego Utrwalania Zywnosci. Ed. Wydawnictwo NaukowoTechniczne, Warszawa.

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