Exploiting the effects of high hydrostatic pressure in ... - Science Direct

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Exploiting the effects of high hydrostatic pressure in biotechnological applications Vadim V. Mozhaev, Karel Heremans, Johannes Frank, Patrick Masson and Claude Balny Applying hydrostatic pressure to biological systems and processes can alter their characteristics. In addition to its use as a basic research tool for investigating the kinetics and thermodynamics of biological systems at the molecular level, the

application of pressure is also being used to modify the properties of biological materials to preserve or improve their qualities. This article reviews the principles underlying the observed effects of applied pressure on biological systems, and discusses current and potential application of pressure in biotechnological processes.

In recent years, there has been increasing interest in exploiting the effects of applied pressure on biological systems. During the past few years, progress in research into the use of high pressures in the food 1and pharmaceutical industries 2 has led to the manufacture of products at elevated pressures 3. In 1990, Meidi-ya Food Co. (Osaka, Japan) introduced into the market apple, strawberry and kiwi jams that had been sterilized using pressure alone. In addition, microbial culture at high pressure is now used to produce various compounds, including gases4, and the preparation of phenylalanine methyl ester (used in the synthesis of aspartame) in bioreactors operating under extreme conditions has also been describeds. Further progress in the use of high pressures in biotechnology will require the development of both processes and equipment, together with refinement of the theory of pressure effects on biomolecules and biosystems. Fortunately, many technical problems that previously hampered research and development into the use of high pressure in biosystems have been V. V. Mozhaev and C. Balny are at the Institut National de la Santd et de la Recherche Mddicale, I N S E R M U 128, Route de Mende, B.P. 5051, 34033 Montpellier cedex 1, France. V. V. Mozhaev is also at the Chemistry Department, Moscow State University, 119899 Moscow, Russia. IV. Heremans is at the Chemistry Department, Katholieke Universiteit Leuven, Celesijnenlaan 200 D, B-3001 Leuven, Belgium. J. Frank is at the Kluyver Laboratoryfor Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. P. Masson is at the Unitd de Biochimie, Service de Santd des Arm&s, Centre de Recherches Emile Bardd, 24 Avenue des Maquis du Grdvisaudan, B.P. 87, 38702 La Tronche, France. © 1994, Elsevier Science Ltd

solved, and apparatus for high-pressure treatment is now commercially available3. In this article, we evaluate the biotechnological potential of harnessing high pressure, focusing in particular on pressure-induced structural changes in proteins. Pressure effects on biological systems The behaviour ofbiosystems under high pressure is governed by Le Chatelier's principle, which predicts that the application of pressure shifts an equilibrium towards the state that occupies a smaller volume, and accelerates processes for which the transition state has a smaller volume than the ground state (see Box 1). In other words, pressure favours processes that are accompanied by negative volume changes. To understand further the principles of pressure effects on biochemical processes, it is helpful to consider the effects of changing volume on molecular processes. Effects on noncovalent molecular interactions

Noncovalent interactions constitute the main target for the modulation of molecular characteristics through pressure 6 (for quantitative data, see Box 2). Solvation of charged groups is accompanied by volume reduction resulting from electrostriction, i.e. the compact alignment of water dipoles owing to the coulombic field of the charged groups. Conversely, formation ofcoulombic interactions following dehydration of charged atoms is accompanied by positive changes in volume (A V), and is not favoured by pressure 7. Formation ofhydrophobic interactions between aliphatic groups is characterized by positive values of TIBTECHDECEMBER1994 (VOL12)

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reviews Box 1. Equations describing pressure effects on thermodynamics and kinetics For an elementary equilibrium process A *-, B, the changes in the free energy of the system (AG), thermal energy (AE), volume (AV) and entropy (AS) are expressed as a function of temperature (T) and pressure (p) by the equation: AG = AE + p A V - TAS

(Eqn 1)

where AV is equal to the difference in the volumes of the final (VB) and initial (VA) states, and at constant temperature is determined by the following expression: AV = VB-VA = (aAG/ap)r = -RT-(a InK/aP)r

(Eqn 2)

where R is the gas constant, equal to 0.082 mlatmK -lmo1-1 and K is the equilibrium constant. Pressure effects on the rate (v) of an elementary process A ---, B derive from the equation: AV" = V" - VA = (aAG'/aP)r = -RT(a Inv/aP)r

(Eqn 3)

where AG" and AV" are the changes in the activation free energy and volume, respectively, and AV" is equal to the difference in the volumes of the activated (V') and initial (VA) states. Le Chatelier's principle predicts that an increase in pressure favours the state that occupies the least volume in a system. For a process with AV of-16ml mo1-1, an increase in pressure from atmospheric pressure to lkbar leads to a nearly twofold increase in the value of K; for a process with AV" of -16 ml mo1-1, the same pressure increase results in a twofold acceleration of the rate of the reaction. In biochemical studies, the following pressure units are used most often: 1 atm = 1 kgcm -2 = 1.01325 bar = 0.101325 MPa = 14.6 Ibin-2 The difference between the atm, bar and kg cm-2 units is usually ignored if precise numerical values are not too important. The Pascal is the official SI unit.

A V, and is destabilized by increased pressure. Chargetransfer interactions and stacking of aromatic rings show small negative A V values and are favoured by increased pressure. Hydrogen bonds are formed and destroyed with nearly zero changes in volume, and are therefore almost pressure-insensitive 6,7. In general, it is not easy to interpret, at the molecular level, A V and A V* values observed in bioprocesses6 because too many interactions are disrupted or formed simultaneously. However, the data on volume changes that occur on the formation of major noncovalent interactions are helpful in predicting how pressure may change the structure of biomolecules.

Effects of pressure on

molecular structure intermolecular interactions

and

The covalent structure of low-molecular-weight biomolecules (peptides, lipids, saccharides), as well as the primary structure of macromolecules (proteins, nucleic acids and polysaccharides), is not perturbed by pressures -->10--20 kbar (1 bar = 1 × 105 Pa) because of the negligible compressibility of covalent bondsV; pressure ~icts predominantly on the spatial (secondary, tertiary, quaternary and supramolecular) structures of macromolecules. The double-helix structure of D N A is pressure-stable and does not unwind until the applied pressure exceeds 10kbar. Interactions between nucleic acids and proteins are mainly electrostatic and therefore disfavoured by high pressures. In the following sections, the effect of pressure on protein structure, protein-lipid interactions and biological membranes is discussed in more detail. TIBTECHDECEMBER1994(VOL12)

Pressure effects on protein structure Protein denaturation

Pressure is a valuable tool for studying two aspects of protein denaturation. The magnitude of volume changes upon protein denaturation (A Vd) and activation volumes for the denaturation-renaturation steps may be estimated from the pressure dependence of the equilibrium constant of protein denaturation, and rate constants for protein unfolding-refolding. In addition, pressure can be used to denature proteins by disturbing the delicate balance of stabilizing-destabilizing interactions8. At present, denaturation is usually studied at atmospheric pressure using high temperature, guanidinium chloride or urea as denaturants. Interpretation of the results obtained using such methods is then complicated by the facts that: (1) varying the temperature changes both the volume and the thermal energy of the system; and (2) the thermodynamic parameters of denaturation by guanidinium chloride or urea are influenced by the binding of these molecules to proteins 9. By contrast, if denaturation is induced at constant temperature in the absence of chemical denaturants, orie can try to describe the change in protein free energy as a function of the interatomic distances in the protein molecule which, in turn, are changed by variations in applied pressure. The use of pressure as a 'reagentless denaturant' is also advantageous from a methodological point of view: transition to standard conditions (renaturation) is achieved simply by releasing the pressure. In general, the effects of pressure on proteins are reversible, and only seldom are they accompanied by aggregation or changes in covalent structure7, s.

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reviews It is well-known that reversible denaturation of proteins usually obeys the two-state model, when only two protein forms, the native and denatured, are in equilibrium. Application of Eqn 2 in Box 1 enables the value ofA Vd, the difference between the volumes of a protein in the native and denatured states, to be determined. Values of A Va for denaturation by temperature or chemical agents, calculated in this way are usually negative (hundreds of mlmo1-1, i.e. 0), and above the line is the zone of thermodynamic instability of the native protein, where the denatured form is more stable (AGd < 0). Lines a and b correspond to the isothermal cross-sections, and line c to the isobaric cross-section of the phase diagram. At 20°C, an increase in pressure to values > 3 kbar (line a) promotes denaturation. More complex behaviour is observed at 43°C (line b): at atmospheric pressure most of the protein is in the denatured form and an increase in pressure to more than 0.5 kbar induces a transition back to the native form. This explains why proteins are stabilized against thermal denaturation at high pressures, and why heat-inactivated enzymes can sometimes be reactivated by applying high pressures. Finally, at pressures >2 kbar, the protein denatures again. Similar 'reactivation-inactivation' transitions may be obtained by increasing temperature at a fixed pressure (line c). Figure adapted from Ref. 22.

Modifying molecular structure Proteolysis Pressure within the range of 4-8 kbar can be used to promote two types ofproteolytic processes 2°. First, proteins can be unfolded, thus exposing new peptide bonds to proteotytic attack. If the protein is rigid, the number of potential cleavage sites exposed at high pressure is small, and limited selective proteolysis is 'possible. By contrast, if the protein is not too rigid, pressures of several kbar may accelerate nonspecific digestion by proteases. The extent of such digestion depends on the sequence and structure of the individual protein. Consequently, in a complex mixture, certain proteins can be hydrolysed preferentially at high pressures. Such pressure-induced selective proteolysis may find practical applications in food processing1; for example, selective degradation of lactoglobulins in whey reduces the allergenicity of milk 2o. TIBTECHDECEMBER1994 (VOL12)

One of the limitations of using chemical methods to alter protein structure is the inability to modify buried amino acid residues. This limitation can be circumvented by modifying the unfolded, rather than the folded, native protein 21. However, proteins modified in this way are often unable to refold into the catalytically active conformation. One possible explanation is that preliminary unfolding in concentrated solutions of guanidinium chloride or urea is so extensive that too many groups are modified, causing large changes in protein structure. Kunugi 2° has improved this modification strategy by using pressure, instead of denaturants, to limit protein unfolding. Glucose oxidase and albumin, partially unfolded at pressures of 4-5kbar, were reacted with ferrocenyl derivatives to attach several ferrocene groups per protein molecule. Once the applied pressure was released to atmospheric pressure, functionally active ferrocenylated proteins were refolded. Such modified proteins are potentially useful in biosensor systems as 'self-electron-mediating enzymes '2°, which can catalyse the enzymic reaction of interest and simultaneously communicate with an electrode without the need to couple the reaction to any external electron-transfer mediator. -

Exploiting pressure-temperature-inducedphase changes Both pressure and temperature can cause protein denaturation; consequently, the native conformation of a protein exists only over a rather limited range of both parameters. From Eqn 1 in Box 1, it follows that the region of stability of the native structure has a curved boundary when plotted on a temperature-pressure diagram (see Fig. 1) (P,,ef. 22). Such diagrams for denaturation transitions can be used to select useful biotechnological treatments for modulating the structure and stability of proteins 2°. In food biochemistry, it may be desirable, in some instances, to be able to denature protein products reversibly. At atmospheric pressure and high temperature (currently the conditions used most frequently in food processing), proteins tend to be irreversibly inactivated. However, by applying high pressures, it is possible to shift the region of denaturation to subzero temperatures (Fig. 1) and thus ensure that the irreversible denaturation of the protein product is negligible. Interest in pressure--temperature phase diagrams may be further stimulated by the recent observation that the stability of protein assemblies, bacteriophages, and even bacterial cells, may be described by diagrams similar to that shown in Fig. 1 (Ref. 23). There is also some evidence that the sol-gel transitions ofpolysaccharides show similar phase diagrams to those obtained for proteins 24.

Sol-gel transitions in macromolecules The sensitivity to pressure of sol-gel transitions in protein and polysaccharide solutions may be explained by the crucial role that water plays in the mechanism

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reviews of gel formation. Gels are stabilized by interpolymer hydrogen bonds between, for example, the O H groups at positions 2 and 6 ofgalactose units in carrageenans, or the polar groups of amino acid residues in proteins 25. Such a process is accompanied by the liberation of water molecules that were previously involved in the hydration of the polar groups of biomolecules, and by an increase in the total volume of the system. Positive A V values have been determined for sol-gel transitions of K- and ~-carrageenans, ovalbumin and soy protein (Table 1). Conversely, negative A Vvalues have been found for sol-gel transitions of agarose and gelatin (Table 1). Intermolecular hydrogen bonds in these biopolymers may bridge water molecules involved in cross-linking junctions; such a mechanism has been demonstrated for gelatin and may also apply to agarose. This bridging process produces a large negative A Vvalue that is sufficient to compensate for the positive value of A V resulting from the decrease in hydration that occurs on gelling (Fig. 2). The parameters outlined in Table 1 are important considerations in choosing experimental conditions for the preparation of gels in the food industry. At high pressure, the temperature of the sol-gel transition in biopolymer solutions is often reduced, enabling technologists to produce food gels at ambient temperatures. These pressurized gels are often better in terms of strength and softness than the gels prepared at high temperature, and are therefore of potential interest for the food industry.

Modifying the catalytic behaviour of enzymes Reasons for pressure-induced changes in the rate of enzyme-catalysed reactions may be classified into three main groups: (1) changes in the structure of an enzyme; (2) changes in the reaction mechanism; for example, a change in the rate-limiting step; and (3) the effect of a particular rate-determining step on the overall rate. To our knowledge, there has been no definitive experimental proof of the first two mechanisms; for example, there is insufficient evidence that, under the action of pressure, an enzyme undergoes a transition from one catalytically active conformation to another. Thus, we prefer to limit our discussion to the possibility of modifying the rate of a particular rate-determining catalytic step with applied pressure. Reaction rates High pressure accelerates reactions that are accompanied by a decrease in volume on the formation of the transition state (see Eqn 3 in Box 1). For example, the hydrolysis of an anilide in reversed micelles, catalysed by ot-chymotrypsin, shows a negative value of A [7*; consequently, an increase in pressure from atmospheric pressure to 2 kbar results in a sevenfold increase in the reaction rate (curve 1 in Fig. 3). By contrast, hydrolysis of an ester by oL-chymotrypsin (curve 2 in Fig. 3) has a positive value ofA V~, and an increase in pressure up to 2 kbar leads to a more than tenfold deceleration in the rate of reaction 26.

Table 1. Thermodynamic parameters for the sol-gel transition of macromolecules at atmospheric pressure and 2 5 ° C a'b dTm/dpm) (Kkbar-1) c

Sample K-Carrageenan in 35 mM KCI ~-Carrageenan in 30 mM KCI Agarose at 1 kbar Gelatin at 1 kbar Ovalbumin at pH7 Soy protein at pH7.8

AVm {mlmol cross-links-I) c

-5.7

+36.3

- 4.0

+8.5

+2.3

-2.6

+3.9

-25.7

-12.1

+0.37

-17.7

+0.98

aAdapted from Ref. 25. bAbbreviations: Tin, transition temperature; Pro, transition pressure; and AVm, transition volume. CThesevalues are expressed as the quantities per mol of cross-linkingjunction. They were obtained by assuming a phase transition for sol-gel transition and using the Clapeyron-Clausiusequation.

The example in Fig. 3 also illustrates how pressure may be used to change enzyme selectivity. Owing to the difference in the values of A V* of > 60 ml mo1-1 for two substrates, an -100-fold increase in the relative anilide-hydrolysing activity (compared with the ester) is achieved in micelles at 2 kbar 26.

--.

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Sol

Activated

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-

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1 .

.

.

.

Gel Figure 2 The changes in volume undergone by gelatin as it forms a gel, deduced from studying the formation of gelatin gels on the application of high pressure. AV is the volume change on gelation and AV~ is the activation volume of gelation. The positive value of AV~ is generated during the dehydration process, which occurs on the random association of gelatin polypeptidechains. Gelation is followed by the formation of interchain triple helix involvingthe bridging water molecules, and is accompanied by a large negativevolume change ( A V " = A V - A V ~ = -40 ml mol-Z). TIBTECHDECEMBER1994(VOL12)

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reviews critical state, and the properties of such systems). Carbon dioxide, currently the most widely used supercritical fluid, is nontoxic, nonflammable and dissolves many enzyme substrates that are poorly soluble in water. High pressure is necessary to generate the supercritical state (for carbon dioxide, the critical pressure and temperature are 73.8bar and 31°C, respectively), and can also be used in the control of such properties as specificity and enantioselectivity3°. In the near future, major developments in enzymology in supercritical fluids are expected for the synthesis of chiral compounds.

10

0.1

Pressure-assisted extraction processes Membrane systems 0 2.0 The main effect of high pressure on membranes is similar to that of low temperature: a stabilization of p (kbar) the more ordered crystalline (gel) state of the lipid matrix (see Ref. 31 for a comprehensive review). PressFigure 3 The dependence on pressure (p) of the rate of hydrolysis (v) of N-succinyl-L- ure-induced changes in the physical state of the memphenylalanine p-nitroanilide (11), and of N-carbobenzoxy-L4yrosine p-nitrophenyl ester brane usually lead to a weakening of protein-lipid (O) by ~-chymotrypsin in reversed micelles of Aerosol OT in octane. The values of v interactions and, at pressures of a few kbar, integral (relative to the rate of hydrolysis at atmospheric pressure) are plotted on a logarith- and peripheral proteins may be released from the mic scale. membranes. This phenomenon forms the basis of a new method for extracting and subfractionating proteins from membranes. Disaggregation of lipid-proAnother example is yeast carboxypeptidase2°. In tein assemblies by high pressure has the advantage of addition to its major carboxypeptidase activity, this avoiding the addition of surfactants, and thus favours enzyme catalyses acyl-transfer reactions and can thus the preservation of the native-like conformation of the be used in peptide synthesis. The low yield ofpeptide isolated proteins. By using high-pressure extraction, it synthesized by carboxypeptidase at atmospheric was possible, for example, to isolate protein kinase C pressure can be improved nearly sixfold at 2 kbar as a and other lipid-interacting proteins in complexes with result of a pressure-induced decrease in peptidase essential lipid molecules 32 - a difficult task using the activity, which is responsible for the wasteful substrate detergent-based procedure. Similarly, a high-pressure hydrolysis. method has been used to prepare blood-group antigens from human erythrocytes and immunogenic proteins from tumour cells31. Effects of mediura in rate modulation Volume changes accompanying enzyme catalysis may originate from changes in protein conformation, Application of raicro-emulsions as well as from changes in binding or dissociation of Another method for ffactionating membrane water molecules participating in the hydration of the enzymes, while preserving their native-like structure protein functional groups. Both processes depend sig- and environment, utilizes reversed micelles of surfactants in organic solvents as the extraction m e d i u m 33. nificantly on changes in the medium surrounding the enzyme and, for that reason, pressure effects in cataly- The problem encountered with this method is that the sis often correlate with some quantitative parameters ultimate isolation of extracted proteins from micelles of the medium and of the added solutes. often involves rather harsh conditions. An elegant apAdditional opportunities for manipulating reaction proach to solving this problem is based on the ability rates by altering pressure arise from the use of organic of certain gases dissolved in the aqueous pools of solvents as media for enzyme reactions. Catalysis in reversed micelles to Form clathrate hydrates - a proorganiq media is extremely sensitive to the hydration cess that can be controlled by pressure 34. These of enzymes27 which, among other factors, can be hydrates change the size of the mJcelles and aggreinfluenced by pressure. To date, there have been only gation number, thus changing the solubility of the a few studies on enzyme catalysis in organic media at encapsulated proteins. Proteins may be recovered From high pressure26.28; however, it appears that there is a reversed micelles by dissolving gases at pressures of clear tendency towards significantly larger volume 100 bar. changes in organic media compared with reactions in As an alternative to reversed micelles, it has been suggested that compressible-fluid-based microemulaqueous solutions. An exciting area of biotechnological research into sions, such as those formed by Tween-85 in subcritical propane, could be used For protein solubilization3s. the use of elevated pressure is biocatalysis in supercritiThe obvious advantage of supercritical systems For cal fluids (see Ref. 29 for a recent review of conditions where gases can be transformed into the superretaining the native properties of proteins is that 0.01

' 0.5

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' 1.0

' 1.5

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reviews protein solubilization is achieved simply by a change in pressure, and does not involve any chemical changes in the aqueous microphase of the microemulsions, which may denature proteins. Immunoadsorption As a general rule, monomeric proteins do not denature at pressures below 4kbar. By contrast, protein-protein complexes can be dissociated at 1 kbar; consequently, at pressures of 1-4 kbar, dissociation of noncovalent antigen-antibody complexes occurs without denaturation. An example of the enzyme-linked immunoassay of albumin with immunoglobulin shows that dissociation of the complex below 2 kbar yields pure and active protein, without adversely affecting the binding ability of the immobilized antibodies 36. Immunoadsorption with recycling of ligands at high pressure has good potential for apphcation in bioseparation processes. High pressure in microbiology As 70% of the Earth is covered by sea water, a substantial proportion of all living organisms has to cope with a considerable hydrostatic pressure of hundreds of bar. Many surface-dwelling bacteria are able to grow, albeit slowly, at pressures of up to 600 bar. Above that pressure, however, growth will cease as a result of the complete inhibition of protein synthesis and the occurrence of abnormal phenomena, such as the formation of filaments and the disorganization of the cytoplasm. Although the occurrence of bacteria in the deep sea has been known for more than a century, the first barophihc bacterium (defined as an organism that grows well at pressures >400 bar) was isolated only in 1979 by Yayanos et al. 37 and, in contrast to other extremophiles, molecular characterization of the deep-sea bacteria has been started only recently. Growth at high pressure induces synthesis of a specific porin protein, indicating that the response to pressure has a genetic basis 38. The biotechnological potential of barophilic bacteria stems mainly from the fact that these are the only species that are likely to be found in the vicinity of hydrothermal vents in the deep sea where, as a result of the high hydrostatic pressure, water remains liquid at temperatures up to 300°C. Bacteria adapted to such an extreme environment and able to grow at 105°C and 200-400bar have been described recently39. These properties could prove valuable in the future, in biotechnological processes where a combination of high pressure and high temperature are required if the desired product(s) is to be obtained in sufficient yield.

High pressurefood processing The rationale behind the use of high pressure instead of high temperature in food processing is the improved preservation of food taste, flavour and colour 4°. This is based on the stability of the covalent structures of proteins, saccharides, vitamins, lipids and pigments to high-pressures, in contrast with their relative instability towards increased temperatures. The impact of

Box 3. Potential applications of high pressure in food technology a Pasteurization and sterilization at low or moderate temperature guarantees a better fresh taste, flavour, colour and longer shelf-life in a refrigerator, or at ambient temperature, in comparison with hightemperature sterilization. Protein modification via unfolding and aggregation results in restructuring and texturization (often tenderization) of meat, discolouration of haemoglobin (in animal blood) and inactivation of toxins. Changes in phase transitions: Reversible decrease in the melting point of ice permits food storage at -5 to -20-C without freezing. Reversible increase in the melting point of lipids may be used for tempering chocolate. Starch gelatinization at reduced temperatures softens the legume seeds and cereal grains. Gas removal or solubilization may be useful in deaeration by compression and saturation of aqueous solutions or moist foods with carbon dioxide. Extraction of constituents (such as pectin) from food, or dewatering of foods. Powder agglomeration may be used for the production of food bars, tablets, cubes, etc. Surface impregnation or coating of various foods: Adsorption of minerals, vitamins, flavours, pigments, antioxidants, etc. Encapsulation of liquids. Stabilization of emulsions. Protective layers and edible films. aAdaptedfrom Ref. 41.

processing on food quality is therefore reduced, and thus the quality of pressure-processed food depends more closely on the quality of the raw materials. Although the cost of pressure-treated food is currently higher than that of thermally processed food, the improved quality may find a niche market among a specific group of consumers who are willing to pay a premium. Pressure treatment consumes less energy than thermal processing and, in the future, this may lead to pressure-processed food being more comercially competitive. Physical processes exploited in food preparation, such as the formation of gels and granules of starch, protein coagulation, lipid phase transitions, powder agglomeration and others (see Box 3) are induced by pressurization just as effectively as by heating 41. One of the most important roles played by pressure is in sterilization1. At the turn of the century, studies of the effects of high hydrostatic pressure on microorganisms of food showed that short-term treatment with pressures of several kbar reduces the bacterial content in foods by several orders of magnitude 42. Microorganisms differ significantly in their ability to withstand pressure: bacterial spores and some viruses are among the most resistant and can survive pressures >10kbar. Spores are inactivated by pressure only after germination; therefore, repeated cychng between high and low pressures is recommended to remove spores. Inactivation of germinated spores and vegetative TIBTECHDECEMBER1994 (VOL 12)

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reviews 80

4O O

o

:e I ~ e V %40

Ice VI

/,oo,\ I

I

5

%1

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Figure 4 Solid-liquid phase diagram of water. Stable ice polymorphs (I to VI) exist in a well-defined region of pressure and temperature. Ice IV (not shown) is a metastable form of ice V. Solid lines show phase boundaries.

bacteria is often slowest at room temperature and is accelerated at both high and low temperature 43. The efficiency of cell destruction is increased by the combined action of pressure, temperature and other conditions, such as ultrasonic waves, shear, electromagnetic fields or high-voltage pulses. Ethanol, lysozyme, chitosan, sorbic and benzoic acids, and other additives enhance the effect of pressure on microorganisms, thus permitting lower pressures, temperatures, or shorter application time to achieve inactivation. By analogy to pasteurization, these sterilization procedures are termed 'pascalization'. Improved high-pressure food processing methods should result from the possibility of operating at temperatures below 0°C without freezing. According to the phase diagram of water (Fig. 4), at high pressures, a subzero temperature region exists where water remains liquid (for example, down to -20°C at 2 kbar). Application of such low-temperature, high-pressure conditions also allows either rapid freezing of biological materials, or rapid thawing of frozen ones, a factor that is often important for improved preservation of food quality. The industrial method of high-pressure processing of food is similar to the schemes already developed for conventional heat-based processes: after the necessary pretreatments, raw materials are put into plastic bags, sealed under vacuum, and pressurized to obtain the final products. Special high-pressure apparatus will be developed in the future that will meet the requirements of food technology. In order to gain fast depressurization and to ensure the safety of food, machines used in nonfood areas, for example, in the production of ceramics, steels, and superalloys for high-speed and carbide tools, are now being adjusted to meet the needs of the food i n d u s t r y 44.

Conclusion Over the past few years, R&D into the use of high pressure has been based on the principles of traditional

TIBTECHDECEMBER1994(VOL12)

physical chemistry and chemical technology. Integration of the knowledge gained in this way with the life sciences should enable research into high pressure to find many new applications as a clean and energysaving tool for processing biological materials, particularly in the food industry. Although, as yet, no sound pharmaceutical or biomedical applications of this technology have been reported, high pressure might be used in the preservation of pharmaceuticals, blood derivatives and transplant organs. More-ambitious goals, based on the rational modification of molecular structure-function relationships by pressure, await a more-detailed understanding of the effects of pressure at a molecular level.

Acknowledgements The authors are grateful to INSERM (Poste Vert, V. M.), I N S E R M / M V G (C. B. and K. H.), I N S E R M / N W O (C. B. and J. E) and D R E T (R M. and C. B.) for financial support. We acknowledge the thoughtful comments and linguistic advice of Peter Hailing and would like to thank Reinhardt Lange for critically reading the manuscript.

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