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Recent advances in the microbiology of high pressure processing
Trends in Food Science & Technology 9 (1998) 152±158
Recent advances in the microbiology of high pressure processing J.P.P.M. Smelt Unilever Research Laboratorium, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands (tel: 31-10-4605578; fax: 31-10-4605188; e-mail: [email protected]) Ultrahigh Pressure (UHP) oers interesting possibilities for food processing ranging from extraction of plant compounds, restructuring foods and rapid formation of small ice crystals. Pressures between 300 and 600 MPa can inactivate yeasts, moulds and most vegetative bacteria including most infectious food-borne pathogens. Thus, pressure is a potential alternative to heat pasteurization as pressure leaves small molecules such as many ¯avour compounds and vitamins intact. In general, bacterial spores can only be killed by very high pressures (>1000 MPa). Bacterial spores, however, can often be stimulated to germinate by pressures of 50±300 MPa. Germinated spores can then be killed by relatively mild heat treatments or mild pressure treatments. However, in most cases a small fraction of spores can survive this treatment. Consequently, there is still no practical application of UHP treatment as a sterilization process, but it is still an interesting area for further exploration. # 1998 Elsevier Science Ltd. All rights reserved
This review deals with the eects of pressure on food microorganisms. For more general reviews on the eects of pressure on food, readers are referred to recent reviews [1, 2]. Increasing consumer demand for minimally processed additive-free, shelf-stable products prompted the exploration of physical treatments other than traditional heat treatments as potential alternatives. These alternatives include new ways of applying 0924-2244/98/$19.00. Copyright # 1998 Elsevier Science Ltd. All rights reserved PII: S 0 92 4 - 2 24 4 ( 9 8 ) 0 0 03 0 - 2
Review heat, such as Ohmic heating or microwave heating, and non-thermal methods, such as irradiation, application of high electric ®elds, high magnetic ®elds and high pressure. Ultrahigh Pressure (UHP) treatment has been known as a potential preservation technique for almost over a century, since Hite [3] demonstrated in 1899 that microbial spoilage of milk could be delayed by application of high pressure. High pressure has been applied for many years for production of ceramics, composite materials, carbon graphite and plastics. These technological developments increased the feasibility of commercial application in the food area. A range of pressure-treated products has been on the Japanese market for several years, including fruit preparations, fruit juices, rice cakes and raw squid. In France, pressure-treated fruit juices are available. Recently, a pressure-treated guacamole has been successfully launched on the US market. UHP causes inactivation of microorganisms and enzymes while leaving small molecules, such as ¯avours and many vitamins intact [4]. Emulsions, which are sensitive to heat can be pressuretreated without aecting the stability of the emulsion. Another application of UHP is to promote the formation of small ice crystals. Under a pressure of 207.5 MPa, water remains liquid at temperatures down to ÿ22 C. Sudden expansion at that temperature to atmospheric pressure is a means of formation of very small ice crystals provided the latent heat released by freezing can be removed. Pressure stimulates many chemical reactions and can release ¯avours from plant cells.
General principle of UHP
Two principles underlie the eect of high pressure. Firstly, the principle of le Chatelier, according to which any phenomenon (phase transition, chemical reactivity, change in molecular con®guration, chemical reaction) accompanied by a decrease in volume will be enhanced by pressure. One would expect that temperature would have an antagonistic eect because increasing temperature results in a volume increase. On the other hand, the reaction rate increases with increasing temperature according to Arrhenius' law. Secondly, pressure is instantaneously and uniformly transmitted independent of the size and the geometry of the food. This is known as isostatic pressure.
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The eects of pressure on microorganisms in food are determined by the eect of pressure on water, temperature during pressure treatment, the food constituents and the properties and the physiological state of the microorganism.
Mode of action of UHP on microorganisms
It can be expected that the mode of action of pressure on whole organisms is not necessary the same, but dependent on the pressure level. Elevated hydrostatic pressures between 30 and 50 MPa can in¯uence gene expression and protein synthesis. Hydrostatic pressure can induce tetraploidy in Saccharomyces cerevisiae [5], indicating that UHP can interfere with replication of DNA. At pressures of 100 MPa the nuclear membrane of yeasts was aected, and at more than 400±600 MPa further alteration occurred in the mitochondria and the cytoplasm. In particular, metal ions are released at pressures over 300 MPa [6]. Pressure-inducible proteins have been found in Methanococcus thermolyticus, Rhodothorula rubra and Escherichia coli, three organisms representing the three domains of life [7]. In the case of E. coli, a pressure of 53 MPa could induce proteins similar to those found at elevated temperature [7]. Although it is not yet known whether pressure can indeed enhance resistance to physical treatment, cells subjected to stress other than pressure (e.g. by sublethal heat) become more resistant to pressure [8]. The mechanism might be stabilization of the structures of membrane-bound enzymes. Exponentially growing cells are more sensitive to pressure than cells in the stationary phase [8, 9]. Stress might be induced during the stationary phase (e.g. through starvation or acidi®cation). A perturbation of the bacterial membrane is almost always involved during pressure treatment. Fatty acids of barophilic microorganisms become more polyunsaturated with increase in growth pressure [10]. A food spoilage organism, Lactobacillus plantarum in the exponential phase was more resistant to pressure when the cells were grown at suboptimal temperature [11]. Under these conditions, fatty acids were more unsaturated than in cells grown at optimum temperatures. When cholesterol is included, the ¯uidity of cell membranes of prokaryotes decreases and the cells become more sensitive to pressure [12]. The protective eect of dierent carbohydrates on the membrane during freezing is in the order glycerol
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Integral and peripheral membrane proteins become more detached from the plasma membrane when the membrane bilayer is suciently perturbed by pressure. UHP can also induce enzyme denaturation. There is an optimum temperature at which enzymes are most resistant to pressure [13]. As this is similar to that what has been found for microorganisms and bacteriophages, there is some circumstantial evidence that some microbial enzymes constitute the main target of pressure inactivation [14±16]. Enzymes of extreme thermophiles are not only more heat resistant but also more pressure resistant than mesophilic microorganisms stabilised by pressure [17]. Hydrostatic pressure can presumably directly aect enzymes and carriers of transport systems. Lactic dehydrogenase from rabbit muscle and glyceraldehyde-3-phosphate dehydrogenase from bakers yeast was deactivated by pressures of 200 and 100 MPa respectively [17]. Acidi®cation of the yeast vacuole might serve as a sequestrant of protons to prevent the cytosolic pH from acidifying [18]. A decrease intracellular pH after pressure treatment has also been found. The observations on membrane damage, protein denaturation, decrease of intracellular pH and the observations on yeasts suggest that membrane-bound enzymes associated with eux of protons may be at least one of the major targets in high-pressure inactivation. A clear candidate is membrane-bound F0F1 ATPase [9]. This enzyme might be inactivated or dislocated by pressure. Elevated hydrostatic pressure can in¯uence gene and protein expression in both 0.1 MPa adapted and high-pressure adapted microorganisms. A further important eect of pressure on membranes would be on ion movements mediated by ATPase enzymes [19]. Streptoccoccus faecalis could be adapted to grow at a pressure as high as 20 MPa. This strain had a regulatory defect and it produced large amounts of ammonia. Under pressure, non-adapted S. faecalis is hypersensitive to acid and the ammonia acted to neutralise metabolic acids. DNA and RNA are very resistant to pressure. However, an extreme condensation of the nuclear material was observed in Listeria monocytogenes and Salmonella typhimurium [20]. The hypothesis is that at elevated pressures, DNA comes in contact with endonucleases, which cleave DNA. This condensation has been found in many other instances and it is reversible and presumably also an enzyme responsible for renaturation is involved. If this enzyme is deactivated by UHP, the cell is no longer able to multiply. As in the case of heat, very severe pressure stress causes considerable damage to the cell and cells become more sensitive to adverse environmental conditions. The mode of action of pressure on bacterial spores is still a matter of speculation. Bacterial spores are killed directly by pressures higher than 1000 MPa. However, spores are sensitive to pressures between 50 and
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the nutrient-stimulated germination pathways. It is not known whether a preceding activation step is essential. Pressures of 300 MPa had hardly any eect on Bacillus subtilis spores, but a considerable decline was observed when alternating current was applied immediately after pressure treatment.
300 MPa [21, 22, 24]. It is generally agreed that at such pressures, spores germinate, followed by death of the germinated spore. Under atmospheric conditions `activation' of spores is often necessary prior to germination. Activation can be brought about by low pH or, in particular, heat. It seems to be a reversible event but it is often followed by germination. It is not known whether pressure induces `activation' similar to reversible heat activation or triggers germination irreversibly. It should be determined whether a similar mechanism is involved in pressure germination. It would be interesting to investigate whether the ®rst stage is the stage comparable to heat activation. Release of dipicolinic acid (DPA), which is not present in vegetative bacteria, is one of the ®rst events in germination. Bacillus subtilis in physiological saline looses 80% of its DPA after 60 MPa at 30 C [23]. At pressures over 1000 MPa [22], spores are killed more rapidly at low pH values. Germination caused by pressures between 50 and 300 MPa proceeds faster at neutral pH; the net eect is a faster kill, because the germinated spore is killed by pressure. Hauben et al. [25] concluded that high pressure activates
Classes of UHP resistance
In general heat-resistant microorganisms are also more resistant to pressure, but there are numerous exceptions. Comparisons are dicult because the test strains are mostly dierent and test conditions are often not comparable. Vegetative forms of eukaryotes, such as yeasts and moulds, are inactivated by pressures between 200 and 300 MPa [1]. Gram-positive bacteria are more resistant to heat and pressure than Gramnegative bacteria. Table 2 shows that the Gram-positive bacteria, Listeria and Staphylococcus aureus, are more resistant than the other organisms mentioned, which are Gram-negatives. It is remarkable that S. senftenberg which is notoriously resistant to heat is not particularly resistant to pressure. Finally, mutants were isolated,
Table 1. Approximate heat resistance and pressure resistance for some pathogenic bacteria Organism
D value at 60 C (minutes
Inactivation (log cycles) after 15 min pressure treatment. Pressure (MPa) 300
Aeromonas hydrophila Pseudomonas aeruginosa Campylobacter Salmonella spp. Yersinia entrocolitica Escherichia coli Escherichia coli 0157:H7 Salmonella senftenberg Staphyloccus aureus Listeria monocytogenes
0.1±0.2 0.1±0.2 0.1±0.2 0.1±2.5 2±3 4±6 2.5 6±10 1±10 3±8
400
>6 >6 >6 1±4.5 >6 1±2 3
500
600
2.5 0.1 1±3
1.9 >6
2.1
Data from Mossel et al. [43], Patterson et al. [35], Metrick et al. [44] and Styles et al. [45].
Table 2. De®ning targets for pressure inactivation Group of microorganisms Spoilage Yeasts Moulds Lactic acid bacteria Toxigenic/pathogenic Staphylococcus aureus Clostridium botulinum (mycotoxigenic moulds) Infectious pathogens Listeria monocytogenes Salmonella (Campylobacter) (Yersinia enterocolitica) (Aeromonas hydrophila)
Health implications No health risk
Requirements Inactivation or prevention of growth or delay of growth
Microorganisms not infectious, but they are able to form toxins in the food
Inactivation or prevention of growth
Microorganism itself is infectious
Inactivation
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that could withstand 15 min 750 MPa. These mutants were not particularly heat resistant. As shown in the Table 1 E. coli O157:H7 is very resistant to pressure, but the heat resistance of this strain is not known. Pressureresistant mutants of E. coli could be selected by repeatedly exposing subcultures of pressure-treated cells to increasing pressure levels [27]. Viruses are very heterogeneous and the pressure resistance of viruses varies considerably. The number of protein-DNA viruses such as bacteriophages, is considerably reduced at pressures of 300±400 MPa [26]. Lipid coated viruses such as the Sindbis virus retains complete infectivity at pressures from 300 to 700 MPa at temperatures down to ÿ20 C [27]. The number of infectious phage particles could be reduced by at least a factor 106. HIV viruses need a treatment of 10 min at 400±600 MPa to reduce the number of infectious particles by a factor 104±105 [28]. Whereas most spores from yeasts and moulds are easily inactivated by pressures of 400 MPa, ascospores of Byssochlamys are not only very resistant to heat, but also to pressure [29]. A pressure treatment of 15 min of 700 MPa at 70 C was not sucient to reduce the number of ascospores by a factor of >103. Bacterial spores can survive pressures over 1000 MPa. Pressures above 1000 MPa are needed to inactivate C. sporogenes spores in meat broth or carrot broth [30]. A reduction of maximum 1.5 log was obtained when treatments of up to 1500 MPa at room temperature were applied. Another study showed no inactivation of C. sporogenes using a pressure of 600 MPa for 60 min at 60 C. Heat treatments followed by milder pressures might be more promising. Indeed, temperatures above 75 C followed by treatment at 800 MPa were sucient to inactivate C. sporogenes by a factor of at least 105. These results should be con®rmed for the closely related C. botulinum to assess the safety of the process. A treatment of 680 MPa at ambient temperature for 60 min resulted in a ®ve-log reduction of C. sporogenes in chicken breast [31]. An inactivation ratio of four to six log cycles is obtained when spores of Bacillus strearothermophilus were subjected to four to six pressure cycle of 600 MPa at 70 C of ®ve minutes each [32]. A further reduction was not found when the pressure was increased to 820 MPa. A comparison between resistance of spores to pressure and heat shows that bacterial spores extremely resistant to pressure and are also resistant to heat, but there seems no relationship between pressure and heat resistance within the spore group of sporeformers. The pressure resistance and the heat resistance of six Bacillus strains was compared [33]. These strains represented almost the whole range of heat resistance of spores. Although the pressures applied were generally too low to inactivate the spores, one very heat resistant Bacillus strearothermophilus was most sensitive to pressure whilst a heat-sensitive strain like B. megaterium was not
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inactivated after a treatment of 40 min at 1000 MPa. As pointed out above, it is generally assumed that lower pressures stimulate germination followed by subsequent die o. Applying oscillatory pressure treatments alternating between 60 and 500 MPa with holding times of 1 min could reduce the number of B. subtilis spores by a factor >108 [34].
UHP in real foods; eect of food constituents
In real food situations, two eects always determine microbiological safety and stability: the eect of the food during treatment and the eect after treatment during recovery of the microorganism. It should also be taken into account that results of studies in buers or laboratory media cannot be directly extrapolated to real food situations. For instance milk and cream protect microorganisms against pressure [35].
Acidity
In considering the eect of pH, one must consider that both temperature and pressure cause a pH shift and each buer has its own characteristics in this respect. When the activation volume of the buer is very large, the pH change due to pressure will be large [36]. For instance, the pH shift of phosphate buer caused by pressure is large and it is generally used as the reference buer in inactivation studies of microorganisms. In addition, buers have their speci®c eects on the physiology of the cell. Yeasts and moulds are quite resistant to low pH and a pH less than 4.0 hardly sensitizes these microorganisms against pressure. By comparison, vegetative bacteria are quite sensitive to pressure, to heat, and low pH. Bacterial spores are most resistant to the direct eects of pressure treatment at neutral pH. When spores are killed indirectly, following germination at pressures between 50 and 300 MPa, spores are most sensitive to pressure at neutral pH. Bacteria are more sensitive to suboptimal pH after heat or pressure treatment. Thus, pH not only enhances inactivation during treatment, but inhibits outgrowth of cells injured sublethally by heat or pressure. Apart from pH eects, no speci®c eect of organic acids have been observed. This might be due to the fact that pressure favours ionization and that organic acids are particularly inhibitory in the undissociated form. On the other hand, it is conceivable that the undissociated part might be more active under pressure.
Water activity
Although there is a general osmotic eect of water activity on the cell, there are also speci®c eects of factors that in¯uence water activity. Carbohydrates are generally more protective than salts. In general, low water activity protects cells against pressure [37], but microorganisms injured by pressure are generally more sensitive to low water activity. Recovery of pressure-
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treated cells was much lower when 2% salt was added to the medium [35]. The net eect of lower water activity is not always easy to predict.
Other antimicrobial compounds
Sorbic acid acts as an organic acid, but also interferes with the microbial membrane is more active in combination with pressure [38]. Microbes are particularly sensitive to nisin during or after pressure treatment. Bacillus coagulans was subjected to combinations of UHP (400 MPa), mild temperature and low pH and in the presence of nisin [39]. When nisin (0.8 IU mlÿ1) was added to the recovery medium, a reduction of at least six log cycles could be achieved [40]. Apparently Gram-negatives, such as E. coli and Salmonella which are normally resistant to nisin, can be sensitised to nisin when pressurized. This might be explained by the speci®c action of nisin. Nisin interacts with the cell membrane and it is possible that it could penetrate to the inner cell membrane. During pressure treatment E. coli was sensitized to lysozyme, nisin and ethylenediamine tetraacetic acid (EDTA) separately and even more to a combination of these compounds [41]. Propyl hexobenzoate and sodium ascorbate did not in¯uence pressure resistance of Listeria monocytogenes [38].
Design of safe process conditions
When considering microbiological safety and stability, the required pressure treatment is dependent on the target microorganisms to be eliminated. Infectious pathogenic bacteria must be completely killed. Toxigenic microorganisms, such as C. botulinum and S. aureus can form a toxin in the food, but they are harmless as long as they cannot grow. Sublethal injury of these microbes is sucient if combined with suboptimal growth conditions in the food or to extend shelf-life in a pack. The same holds for spoilage. In all cases, lethal inactivation to an acceptably low level guarantees
Box 1. Modelling inactivation of microorganisms by physical means The decimal reduction time (D value) is used to describe inactivation of microorganisms: Log Nt =N0 a1 ÿ D=t
1
where Nt number of microorganisms at time t. No initial number of microorganisms; a1 constant; D D value; and t time. An empirical equation describes the dependence of D on temperature: log D a ÿ T=z
2
where a is a constant; z is the so called z-value. T is mostly in C.
microbiological safety and stability. In heat processing, inactivation is based on the assumption that death of microorganisms is log-linear with time. An outline of UHP modelling is given in Box 1. The approach has proven its value in heat processing and has been applied for many years. Although deviations from log-linearity have been found in heat inactivation, such deviations seem to be more common in pressure inactivation. Pressure inactivation is often characterised by pronounced `tailing'. The mechanism underlying inactivation kinetics is poorly understood and several possible explanations have been given. Deviation from log-linearity could be explained as a two step reaction passing through an intermediate stage [42]. A satisfactory description is possible by applying distribution models used in toxicology (Fig. 1). Whereas calculations with heat processing are complicated by the temperature pro®les in the container, calculations are simpler in this respect for pressure, because of the isostatic principle. On the other hand, pressure can be ignored in heat-resistance studies at atmospheric conditions or slightly higher. In pressure treatment, temperature change almost always plays a role due to adiabatic heating. In addition, there is much interest in the combined eect of temperature and pressure as an eective means of inactivation of microorganisms. Thus, the classical two-dimensional time-temperature models have to be replaced by three-dimensional timetemperature-pressure models. The eect of come-up time (ramp rate) and of depressurization rate has not yet been fully investigated. A slow ramp rate might induce a stress response and hence make the process less eective and it is often thought that a fast depressurization rate might contribute to the fast inactivation rate. Whereas it might be expected that organisms such as yeasts are sensitive to fast depressurization due to the vacuole in the cell, vegetative bacteria are probably quite insensitive under similar conditions. Bacterial spores are vulnerable to repeated pressure cycles probably due to a combination of germination and subsequent inactivation by pressure. Sublethal inactivation by pressure has been reported several timesÐi.e. sensitivity to bile salts and NaCl, and extension of the lag time. The same inactivation can be used for sublethal inactivation in the presence of sensitizing agents such as salt or low pH; models describing the relationship between pressure and subsequent lag times are being developed.
Outlook for UHP
Pressure treatment is still costly, mainly because of the initial capital expenditure, and this limits its application to high-value products. However, it can be expected that these costs will go down as a consequence of further progress in technology. An illustration is given by the presence of pressure-pasteurized milk on
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Fig. 1. Comparison of the traditional inactivation model (a) with distribution models (b, c and d). (b), Frequency distribution of resistance; (c), the cumulative distribution; and (d), a transformation of (c).
the British market. A clear advantage of pressure treatment is the low energy input and the stability of small molecules against UHP. Consequently, pressure will have less of an aect on the quality of vitamin-rich foods than heat. At present, the process can only be applied as an alternative to heat pasteurization. Alteration of the food structure is another application, but this can only be used if the microbiological safety is ensured. Although there are many reports showing inactivation of bacterial spores, this issue has not yet been fully solved. The physiology of spore germination has to be addressed to understand the mechanism and to know how pressure can be used most eectively. It means that much eort has still to be spent before pressure can be marketed as an alternative for sterilisation.
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12 MacDonald, A.G. (1993) `Eects of High Hydrostatic Pressure on Natural and Arti®cial Membranes.' in High pressure and Biotechnology. (C. Balny, R. Hayashi, K. Heremans and P. Masson eds), John Libby and Company Ltd London, pp. 67±75 13 Hawley, S.A. (1978) `High Pressure Techniques' in Methods in Enzymol. 49, 14±25 14 Hashizume, C., Kimura, K. and Hayashi, R. (1996) `Kinetic Analysis of Yeast Inactivation by High Pressure Treatment at Low Temperatures' in Biosci. Biotechnol. Biochem. 59, 1455± 1458 15 Gross, P. and Ludwig, H. (1992) `Pressure-temperature-phase Diagram for the Stability of Bacteriophage T' in High pressure and Biotechnology. (C. Balny, R. Hayashi, K. Heremans and P. Masson eds), John Libby and Company Ltd, London, pp. 57± 59 16 Sonoike, K., Setoyama, T., Kuma, Y. and Kobayashi, S. (1992) `The Eect of Pressure and Temperature on the Death Rates of Lactobacillus casei and Escherichia coli.' in High pressure and Biotechnology. (C. Balny, R. Hayashi, K. Heremans and P. Masson eds), John Libby and Company Ltd, London, pp. 297± 301 17 Jaenicke, R. (1991) `Protein Stability and Molecular Adaptation to Extreme Conditions' in Eur. J. Biochem. 202, 715±728 18 Abe, F. and Horikoshi, K. (1995) `Hydrostatic pressure promotes the Acidi®cation of Vacuoles in Saccharomyces Cerevisiae' in FEMS Microbiol. Lett. 130, 307±312 19 Marquis, R.E. and Bender, G.R. (1987) `Barophysiology of Prokaryotes and Proton Translocating ATPases' in Current Perspectives in High Pressure Biology. (H.W. Jannasch, R.E. Marquis and A.M. Zimmerman, eds), Academic Press Ltd, London, pp. 65±73 20 Mackey, B.M., Forestiere, K., Isaacs, N.S. and Brooker, B. (1994) `The Eect of High Hydrostatic Pressure on Salmonella thompson and Listeria monocytogenes examined by Electron Microscopy' in Lett. Appl. Bacteriol. 19, 429±432 21 Sale, A.J.H., Gould, G.W. and Hamilton, W.A. (1970) `Inactivation of Bacterial Spores by Hydrostatic Pressure' in J. Gen. Microbiol. 60, 323±334 22 Timson, W.J. and Short, A.J. (1965) `Resistance of Microorganisms to Hydrostatic Pressure' in Biotechnol. Bioeng. 7, 139± 159 23 HoÈlters, C., Sojka, B. and Ludwig, H. (1997) `Pressure-induced Germination of Bacterial Spores from Bacillus subtilis and Bacillus strearothermophilus' in High Pressure Research in the Bioscienses and Biotechnology (K. Heremans, ed.), Leuven University Press, pp. 257±260 24 Wuytack, E., Soons, J. and Michiels, C. (1997) `Rapid Measurement of Pressure-induced Germination of Bacillus subtilis Spores Expressing Green Fluorescent Protein' in High Pressure Research in the Bioscienses and Biotechnology (K. Heremans ed.), Leuven University Press, pp. 261±264 25 Hauben, K.J.A., Bartlett, D.H., Soontjes, C.C.F., Cornelis, K., Wuytack, E.Y. and Michiels, C.W. (1997) `Echerichia coli Mutants Resistant toInactivation by High Hydrostatic Pressure' in Appl. Env. Microbiol. 63, 945±930 26 Brauch, G., Haensler, U. and Ludwig, H. (1990) `The Eect of Pressure on Bacteriophages' in High Pressure Res. 5, 767± 769 27 Butz, P., Habison, G. and Ludwig, H. (1992) `In¯uence of High Pressure on a Lipid Coated Virus' in High Pressure and Biotechnology. (C. Balny, R. Hayashi, K., Heremans and P. Masson eds), John Libby and Company Ltd, London, pp. 61±64
28 Otake, T., Mori, H., Kawahata, T., Izumoto, Y., Nishimura, H., Oishi, I., Shigehisa, T. and Ohno, H. (1997) `Eects of High Hydrostatic Pressure Treatment on HIV Infectivity' in High Pressure Research in the Bioscienses and Biotechnology (K. Heremans ed.), Leuven University Press, pp. 233±236 29 Butz, P., Funtenberger, S., Haberditzl, T. and Tauscher, B. (1996) `High Pressure Inactivation of Byssochlamys nivea Ascospores and Other Heat Resistant Moulds' in Lebensm. Wiss. Technol. 29, 404±410 30 Rovere, P.P. (1996) `Prove di Sterilizzatione a 15000 bar per Ottenere la StabilataÁ Microbiologica ed Enzimatica' in Ind. Aliment. 35, 1062±1065 31 Crawford, Y.J., Murano, E.A., Olson, D.G. and Shenoy, K. (1996) `Use of High Hydrostatic Pressure and Irradiation to Eliminate Clostridium sporogenes Spores in Chicken Breast' in J. Food Prot. 59, 711±715 32 Hayakawa, K., Kanno, T., Tomito, M. and Fujio, Y. (1994) `Oscillatory Compared with Continuous High Pressure Sterilization' in J. Food Sci. 69, 164±167 33 Nakayama, A., Yano, Y., Kobayashi, S., Ishikawa, M. and Sakai, K. (1996) `Comparison of Pressure Resistances of Spores of Six Bacillus Strains with their Heat Resistances' in Appl. Environ. Microbiol. 62, 3897±3900 34 Sojka, B. and Ludwig, H. (1997) `Eects of Pressure Changes on the Inactivation of Bacillus subtilis Spores' in Pharm. Ind. 59, 436±438 35 Patterson, M.F., Quinn, M., Simpson, R. and Gilmour, A. (1995) `The Sensitivity of Vegetative Pathogens to High Hydrostatic Pressure Treatment in Phosphate Buered Saline and Foods' in J. Food Prot. 58, 542±529 36 Kitamura, Y. and Itoh, T. (1987) `Reaction Volume of Protonic Buering Agents. Prediction of Pressure Dependence of pH and pOH' in J. Solution Chem. 16, 715±725 37 Oxen, P. and Knorr, D. (1993) `Baroprotective Eects of High Solute Concentrations Against Inactivation of Rhodotorula rubra' in Lebensm. Wiss. Technol. 26, 220±223 38 Mackey, B.M., Forestiere, K. and Isaacs, N. (1995) `Factors Aecting the Resistance of Listeria monocytogenes to High Hydrostatic Pressure' in Food Biotechnol. 9, 1±11 39 Roberts, C.M. and Hoover, D.G. (1996) `Sensitivity of Bacillus Coagulans Spores to Combinations of High Hydrostatic Pressure, Heat Acidity and Nisin' in J. Appl. Bacteriol. 61, 363±368 40 Kalchchayanand, N., Sikes, T., Dunne, C.P. and Ray. (1994) `Hydrostatic Pressure and Electroporation have Increased Bactericidal Eciency in Combination with Bacteiocins' in Appl. Environ. Microbiol. 60, 4174±4177 41 Hauben, K.J.A., Wuytack, E.Y., Soontjes, C.C.F. and Michiels, C.W. (1996) `High-pressure Transient Sensitization of Eschericia coli to Lysozyme and Nisin by Disruption of Outer-membrane Permeability' in J. Food Prot. 59, 350±355 42 Heinz, V. and Knorr, D. (1996) `High Pressure Inactivation Kinetics of Bacillus subtilis Cells by a Three-state Model Considering Distributed Resistance Mechanisms' in Food Biotechnol. 10, 149±161 43 Mossel, D.A.A. , Corry, J.E.L., Struijk, C.B. and Baird, R.M. (1995) in Essentials of the microbiology of foods. (D.A.A. Mossel, J.E.L. Corry, C.B. Struijk and R.M. Baird, eds), John Wiley and Sons, Chicester, New York, pp. 88±89 44 Metrick, C., Hoover, D.G. and Farkas, D.F. (1989) `Eects of High Hydrostatic Pressure on Heat Resistant and Heat Sensitive Strains of Salmonella' in J. Food Sci. 54, 1547±1564 45 Styles, M.F., Hoover, D.G. and Farkas, D.F. (1991) `Response of Listeria Monocytogenes and Vibrio Paraheamolyticus to High Hydrostatic Pressure' in J. Food Sci. 56, 1404±1407
Recent advances in the microbiology of high pressure processing J.P.P.M. Smelt Unilever Research Laboratorium, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands (tel: 31-10-4605578; fax: 31-10-4605188; e-mail: [email protected]) Ultrahigh Pressure (UHP) oers interesting possibilities for food processing ranging from extraction of plant compounds, restructuring foods and rapid formation of small ice crystals. Pressures between 300 and 600 MPa can inactivate yeasts, moulds and most vegetative bacteria including most infectious food-borne pathogens. Thus, pressure is a potential alternative to heat pasteurization as pressure leaves small molecules such as many ¯avour compounds and vitamins intact. In general, bacterial spores can only be killed by very high pressures (>1000 MPa). Bacterial spores, however, can often be stimulated to germinate by pressures of 50±300 MPa. Germinated spores can then be killed by relatively mild heat treatments or mild pressure treatments. However, in most cases a small fraction of spores can survive this treatment. Consequently, there is still no practical application of UHP treatment as a sterilization process, but it is still an interesting area for further exploration. # 1998 Elsevier Science Ltd. All rights reserved
This review deals with the eects of pressure on food microorganisms. For more general reviews on the eects of pressure on food, readers are referred to recent reviews [1, 2]. Increasing consumer demand for minimally processed additive-free, shelf-stable products prompted the exploration of physical treatments other than traditional heat treatments as potential alternatives. These alternatives include new ways of applying 0924-2244/98/$19.00. Copyright # 1998 Elsevier Science Ltd. All rights reserved PII: S 0 92 4 - 2 24 4 ( 9 8 ) 0 0 03 0 - 2
Review heat, such as Ohmic heating or microwave heating, and non-thermal methods, such as irradiation, application of high electric ®elds, high magnetic ®elds and high pressure. Ultrahigh Pressure (UHP) treatment has been known as a potential preservation technique for almost over a century, since Hite [3] demonstrated in 1899 that microbial spoilage of milk could be delayed by application of high pressure. High pressure has been applied for many years for production of ceramics, composite materials, carbon graphite and plastics. These technological developments increased the feasibility of commercial application in the food area. A range of pressure-treated products has been on the Japanese market for several years, including fruit preparations, fruit juices, rice cakes and raw squid. In France, pressure-treated fruit juices are available. Recently, a pressure-treated guacamole has been successfully launched on the US market. UHP causes inactivation of microorganisms and enzymes while leaving small molecules, such as ¯avours and many vitamins intact [4]. Emulsions, which are sensitive to heat can be pressuretreated without aecting the stability of the emulsion. Another application of UHP is to promote the formation of small ice crystals. Under a pressure of 207.5 MPa, water remains liquid at temperatures down to ÿ22 C. Sudden expansion at that temperature to atmospheric pressure is a means of formation of very small ice crystals provided the latent heat released by freezing can be removed. Pressure stimulates many chemical reactions and can release ¯avours from plant cells.
General principle of UHP
Two principles underlie the eect of high pressure. Firstly, the principle of le Chatelier, according to which any phenomenon (phase transition, chemical reactivity, change in molecular con®guration, chemical reaction) accompanied by a decrease in volume will be enhanced by pressure. One would expect that temperature would have an antagonistic eect because increasing temperature results in a volume increase. On the other hand, the reaction rate increases with increasing temperature according to Arrhenius' law. Secondly, pressure is instantaneously and uniformly transmitted independent of the size and the geometry of the food. This is known as isostatic pressure.
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The eects of pressure on microorganisms in food are determined by the eect of pressure on water, temperature during pressure treatment, the food constituents and the properties and the physiological state of the microorganism.
Mode of action of UHP on microorganisms
It can be expected that the mode of action of pressure on whole organisms is not necessary the same, but dependent on the pressure level. Elevated hydrostatic pressures between 30 and 50 MPa can in¯uence gene expression and protein synthesis. Hydrostatic pressure can induce tetraploidy in Saccharomyces cerevisiae [5], indicating that UHP can interfere with replication of DNA. At pressures of 100 MPa the nuclear membrane of yeasts was aected, and at more than 400±600 MPa further alteration occurred in the mitochondria and the cytoplasm. In particular, metal ions are released at pressures over 300 MPa [6]. Pressure-inducible proteins have been found in Methanococcus thermolyticus, Rhodothorula rubra and Escherichia coli, three organisms representing the three domains of life [7]. In the case of E. coli, a pressure of 53 MPa could induce proteins similar to those found at elevated temperature [7]. Although it is not yet known whether pressure can indeed enhance resistance to physical treatment, cells subjected to stress other than pressure (e.g. by sublethal heat) become more resistant to pressure [8]. The mechanism might be stabilization of the structures of membrane-bound enzymes. Exponentially growing cells are more sensitive to pressure than cells in the stationary phase [8, 9]. Stress might be induced during the stationary phase (e.g. through starvation or acidi®cation). A perturbation of the bacterial membrane is almost always involved during pressure treatment. Fatty acids of barophilic microorganisms become more polyunsaturated with increase in growth pressure [10]. A food spoilage organism, Lactobacillus plantarum in the exponential phase was more resistant to pressure when the cells were grown at suboptimal temperature [11]. Under these conditions, fatty acids were more unsaturated than in cells grown at optimum temperatures. When cholesterol is included, the ¯uidity of cell membranes of prokaryotes decreases and the cells become more sensitive to pressure [12]. The protective eect of dierent carbohydrates on the membrane during freezing is in the order glycerol
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Integral and peripheral membrane proteins become more detached from the plasma membrane when the membrane bilayer is suciently perturbed by pressure. UHP can also induce enzyme denaturation. There is an optimum temperature at which enzymes are most resistant to pressure [13]. As this is similar to that what has been found for microorganisms and bacteriophages, there is some circumstantial evidence that some microbial enzymes constitute the main target of pressure inactivation [14±16]. Enzymes of extreme thermophiles are not only more heat resistant but also more pressure resistant than mesophilic microorganisms stabilised by pressure [17]. Hydrostatic pressure can presumably directly aect enzymes and carriers of transport systems. Lactic dehydrogenase from rabbit muscle and glyceraldehyde-3-phosphate dehydrogenase from bakers yeast was deactivated by pressures of 200 and 100 MPa respectively [17]. Acidi®cation of the yeast vacuole might serve as a sequestrant of protons to prevent the cytosolic pH from acidifying [18]. A decrease intracellular pH after pressure treatment has also been found. The observations on membrane damage, protein denaturation, decrease of intracellular pH and the observations on yeasts suggest that membrane-bound enzymes associated with eux of protons may be at least one of the major targets in high-pressure inactivation. A clear candidate is membrane-bound F0F1 ATPase [9]. This enzyme might be inactivated or dislocated by pressure. Elevated hydrostatic pressure can in¯uence gene and protein expression in both 0.1 MPa adapted and high-pressure adapted microorganisms. A further important eect of pressure on membranes would be on ion movements mediated by ATPase enzymes [19]. Streptoccoccus faecalis could be adapted to grow at a pressure as high as 20 MPa. This strain had a regulatory defect and it produced large amounts of ammonia. Under pressure, non-adapted S. faecalis is hypersensitive to acid and the ammonia acted to neutralise metabolic acids. DNA and RNA are very resistant to pressure. However, an extreme condensation of the nuclear material was observed in Listeria monocytogenes and Salmonella typhimurium [20]. The hypothesis is that at elevated pressures, DNA comes in contact with endonucleases, which cleave DNA. This condensation has been found in many other instances and it is reversible and presumably also an enzyme responsible for renaturation is involved. If this enzyme is deactivated by UHP, the cell is no longer able to multiply. As in the case of heat, very severe pressure stress causes considerable damage to the cell and cells become more sensitive to adverse environmental conditions. The mode of action of pressure on bacterial spores is still a matter of speculation. Bacterial spores are killed directly by pressures higher than 1000 MPa. However, spores are sensitive to pressures between 50 and
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the nutrient-stimulated germination pathways. It is not known whether a preceding activation step is essential. Pressures of 300 MPa had hardly any eect on Bacillus subtilis spores, but a considerable decline was observed when alternating current was applied immediately after pressure treatment.
300 MPa [21, 22, 24]. It is generally agreed that at such pressures, spores germinate, followed by death of the germinated spore. Under atmospheric conditions `activation' of spores is often necessary prior to germination. Activation can be brought about by low pH or, in particular, heat. It seems to be a reversible event but it is often followed by germination. It is not known whether pressure induces `activation' similar to reversible heat activation or triggers germination irreversibly. It should be determined whether a similar mechanism is involved in pressure germination. It would be interesting to investigate whether the ®rst stage is the stage comparable to heat activation. Release of dipicolinic acid (DPA), which is not present in vegetative bacteria, is one of the ®rst events in germination. Bacillus subtilis in physiological saline looses 80% of its DPA after 60 MPa at 30 C [23]. At pressures over 1000 MPa [22], spores are killed more rapidly at low pH values. Germination caused by pressures between 50 and 300 MPa proceeds faster at neutral pH; the net eect is a faster kill, because the germinated spore is killed by pressure. Hauben et al. [25] concluded that high pressure activates
Classes of UHP resistance
In general heat-resistant microorganisms are also more resistant to pressure, but there are numerous exceptions. Comparisons are dicult because the test strains are mostly dierent and test conditions are often not comparable. Vegetative forms of eukaryotes, such as yeasts and moulds, are inactivated by pressures between 200 and 300 MPa [1]. Gram-positive bacteria are more resistant to heat and pressure than Gramnegative bacteria. Table 2 shows that the Gram-positive bacteria, Listeria and Staphylococcus aureus, are more resistant than the other organisms mentioned, which are Gram-negatives. It is remarkable that S. senftenberg which is notoriously resistant to heat is not particularly resistant to pressure. Finally, mutants were isolated,
Table 1. Approximate heat resistance and pressure resistance for some pathogenic bacteria Organism
D value at 60 C (minutes
Inactivation (log cycles) after 15 min pressure treatment. Pressure (MPa) 300
Aeromonas hydrophila Pseudomonas aeruginosa Campylobacter Salmonella spp. Yersinia entrocolitica Escherichia coli Escherichia coli 0157:H7 Salmonella senftenberg Staphyloccus aureus Listeria monocytogenes
0.1±0.2 0.1±0.2 0.1±0.2 0.1±2.5 2±3 4±6 2.5 6±10 1±10 3±8
400
>6 >6 >6 1±4.5 >6 1±2 3
500
600
2.5 0.1 1±3
1.9 >6
2.1
Data from Mossel et al. [43], Patterson et al. [35], Metrick et al. [44] and Styles et al. [45].
Table 2. De®ning targets for pressure inactivation Group of microorganisms Spoilage Yeasts Moulds Lactic acid bacteria Toxigenic/pathogenic Staphylococcus aureus Clostridium botulinum (mycotoxigenic moulds) Infectious pathogens Listeria monocytogenes Salmonella (Campylobacter) (Yersinia enterocolitica) (Aeromonas hydrophila)
Health implications No health risk
Requirements Inactivation or prevention of growth or delay of growth
Microorganisms not infectious, but they are able to form toxins in the food
Inactivation or prevention of growth
Microorganism itself is infectious
Inactivation
J.P.P.M. Smelt/Trends in Food Science & Technology 9 (1998) 152±158
that could withstand 15 min 750 MPa. These mutants were not particularly heat resistant. As shown in the Table 1 E. coli O157:H7 is very resistant to pressure, but the heat resistance of this strain is not known. Pressureresistant mutants of E. coli could be selected by repeatedly exposing subcultures of pressure-treated cells to increasing pressure levels [27]. Viruses are very heterogeneous and the pressure resistance of viruses varies considerably. The number of protein-DNA viruses such as bacteriophages, is considerably reduced at pressures of 300±400 MPa [26]. Lipid coated viruses such as the Sindbis virus retains complete infectivity at pressures from 300 to 700 MPa at temperatures down to ÿ20 C [27]. The number of infectious phage particles could be reduced by at least a factor 106. HIV viruses need a treatment of 10 min at 400±600 MPa to reduce the number of infectious particles by a factor 104±105 [28]. Whereas most spores from yeasts and moulds are easily inactivated by pressures of 400 MPa, ascospores of Byssochlamys are not only very resistant to heat, but also to pressure [29]. A pressure treatment of 15 min of 700 MPa at 70 C was not sucient to reduce the number of ascospores by a factor of >103. Bacterial spores can survive pressures over 1000 MPa. Pressures above 1000 MPa are needed to inactivate C. sporogenes spores in meat broth or carrot broth [30]. A reduction of maximum 1.5 log was obtained when treatments of up to 1500 MPa at room temperature were applied. Another study showed no inactivation of C. sporogenes using a pressure of 600 MPa for 60 min at 60 C. Heat treatments followed by milder pressures might be more promising. Indeed, temperatures above 75 C followed by treatment at 800 MPa were sucient to inactivate C. sporogenes by a factor of at least 105. These results should be con®rmed for the closely related C. botulinum to assess the safety of the process. A treatment of 680 MPa at ambient temperature for 60 min resulted in a ®ve-log reduction of C. sporogenes in chicken breast [31]. An inactivation ratio of four to six log cycles is obtained when spores of Bacillus strearothermophilus were subjected to four to six pressure cycle of 600 MPa at 70 C of ®ve minutes each [32]. A further reduction was not found when the pressure was increased to 820 MPa. A comparison between resistance of spores to pressure and heat shows that bacterial spores extremely resistant to pressure and are also resistant to heat, but there seems no relationship between pressure and heat resistance within the spore group of sporeformers. The pressure resistance and the heat resistance of six Bacillus strains was compared [33]. These strains represented almost the whole range of heat resistance of spores. Although the pressures applied were generally too low to inactivate the spores, one very heat resistant Bacillus strearothermophilus was most sensitive to pressure whilst a heat-sensitive strain like B. megaterium was not
155
inactivated after a treatment of 40 min at 1000 MPa. As pointed out above, it is generally assumed that lower pressures stimulate germination followed by subsequent die o. Applying oscillatory pressure treatments alternating between 60 and 500 MPa with holding times of 1 min could reduce the number of B. subtilis spores by a factor >108 [34].
UHP in real foods; eect of food constituents
In real food situations, two eects always determine microbiological safety and stability: the eect of the food during treatment and the eect after treatment during recovery of the microorganism. It should also be taken into account that results of studies in buers or laboratory media cannot be directly extrapolated to real food situations. For instance milk and cream protect microorganisms against pressure [35].
Acidity
In considering the eect of pH, one must consider that both temperature and pressure cause a pH shift and each buer has its own characteristics in this respect. When the activation volume of the buer is very large, the pH change due to pressure will be large [36]. For instance, the pH shift of phosphate buer caused by pressure is large and it is generally used as the reference buer in inactivation studies of microorganisms. In addition, buers have their speci®c eects on the physiology of the cell. Yeasts and moulds are quite resistant to low pH and a pH less than 4.0 hardly sensitizes these microorganisms against pressure. By comparison, vegetative bacteria are quite sensitive to pressure, to heat, and low pH. Bacterial spores are most resistant to the direct eects of pressure treatment at neutral pH. When spores are killed indirectly, following germination at pressures between 50 and 300 MPa, spores are most sensitive to pressure at neutral pH. Bacteria are more sensitive to suboptimal pH after heat or pressure treatment. Thus, pH not only enhances inactivation during treatment, but inhibits outgrowth of cells injured sublethally by heat or pressure. Apart from pH eects, no speci®c eect of organic acids have been observed. This might be due to the fact that pressure favours ionization and that organic acids are particularly inhibitory in the undissociated form. On the other hand, it is conceivable that the undissociated part might be more active under pressure.
Water activity
Although there is a general osmotic eect of water activity on the cell, there are also speci®c eects of factors that in¯uence water activity. Carbohydrates are generally more protective than salts. In general, low water activity protects cells against pressure [37], but microorganisms injured by pressure are generally more sensitive to low water activity. Recovery of pressure-
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treated cells was much lower when 2% salt was added to the medium [35]. The net eect of lower water activity is not always easy to predict.
Other antimicrobial compounds
Sorbic acid acts as an organic acid, but also interferes with the microbial membrane is more active in combination with pressure [38]. Microbes are particularly sensitive to nisin during or after pressure treatment. Bacillus coagulans was subjected to combinations of UHP (400 MPa), mild temperature and low pH and in the presence of nisin [39]. When nisin (0.8 IU mlÿ1) was added to the recovery medium, a reduction of at least six log cycles could be achieved [40]. Apparently Gram-negatives, such as E. coli and Salmonella which are normally resistant to nisin, can be sensitised to nisin when pressurized. This might be explained by the speci®c action of nisin. Nisin interacts with the cell membrane and it is possible that it could penetrate to the inner cell membrane. During pressure treatment E. coli was sensitized to lysozyme, nisin and ethylenediamine tetraacetic acid (EDTA) separately and even more to a combination of these compounds [41]. Propyl hexobenzoate and sodium ascorbate did not in¯uence pressure resistance of Listeria monocytogenes [38].
Design of safe process conditions
When considering microbiological safety and stability, the required pressure treatment is dependent on the target microorganisms to be eliminated. Infectious pathogenic bacteria must be completely killed. Toxigenic microorganisms, such as C. botulinum and S. aureus can form a toxin in the food, but they are harmless as long as they cannot grow. Sublethal injury of these microbes is sucient if combined with suboptimal growth conditions in the food or to extend shelf-life in a pack. The same holds for spoilage. In all cases, lethal inactivation to an acceptably low level guarantees
Box 1. Modelling inactivation of microorganisms by physical means The decimal reduction time (D value) is used to describe inactivation of microorganisms: Log Nt =N0 a1 ÿ D=t
1
where Nt number of microorganisms at time t. No initial number of microorganisms; a1 constant; D D value; and t time. An empirical equation describes the dependence of D on temperature: log D a ÿ T=z
2
where a is a constant; z is the so called z-value. T is mostly in C.
microbiological safety and stability. In heat processing, inactivation is based on the assumption that death of microorganisms is log-linear with time. An outline of UHP modelling is given in Box 1. The approach has proven its value in heat processing and has been applied for many years. Although deviations from log-linearity have been found in heat inactivation, such deviations seem to be more common in pressure inactivation. Pressure inactivation is often characterised by pronounced `tailing'. The mechanism underlying inactivation kinetics is poorly understood and several possible explanations have been given. Deviation from log-linearity could be explained as a two step reaction passing through an intermediate stage [42]. A satisfactory description is possible by applying distribution models used in toxicology (Fig. 1). Whereas calculations with heat processing are complicated by the temperature pro®les in the container, calculations are simpler in this respect for pressure, because of the isostatic principle. On the other hand, pressure can be ignored in heat-resistance studies at atmospheric conditions or slightly higher. In pressure treatment, temperature change almost always plays a role due to adiabatic heating. In addition, there is much interest in the combined eect of temperature and pressure as an eective means of inactivation of microorganisms. Thus, the classical two-dimensional time-temperature models have to be replaced by three-dimensional timetemperature-pressure models. The eect of come-up time (ramp rate) and of depressurization rate has not yet been fully investigated. A slow ramp rate might induce a stress response and hence make the process less eective and it is often thought that a fast depressurization rate might contribute to the fast inactivation rate. Whereas it might be expected that organisms such as yeasts are sensitive to fast depressurization due to the vacuole in the cell, vegetative bacteria are probably quite insensitive under similar conditions. Bacterial spores are vulnerable to repeated pressure cycles probably due to a combination of germination and subsequent inactivation by pressure. Sublethal inactivation by pressure has been reported several timesÐi.e. sensitivity to bile salts and NaCl, and extension of the lag time. The same inactivation can be used for sublethal inactivation in the presence of sensitizing agents such as salt or low pH; models describing the relationship between pressure and subsequent lag times are being developed.
Outlook for UHP
Pressure treatment is still costly, mainly because of the initial capital expenditure, and this limits its application to high-value products. However, it can be expected that these costs will go down as a consequence of further progress in technology. An illustration is given by the presence of pressure-pasteurized milk on
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157
Fig. 1. Comparison of the traditional inactivation model (a) with distribution models (b, c and d). (b), Frequency distribution of resistance; (c), the cumulative distribution; and (d), a transformation of (c).
the British market. A clear advantage of pressure treatment is the low energy input and the stability of small molecules against UHP. Consequently, pressure will have less of an aect on the quality of vitamin-rich foods than heat. At present, the process can only be applied as an alternative to heat pasteurization. Alteration of the food structure is another application, but this can only be used if the microbiological safety is ensured. Although there are many reports showing inactivation of bacterial spores, this issue has not yet been fully solved. The physiology of spore germination has to be addressed to understand the mechanism and to know how pressure can be used most eectively. It means that much eort has still to be spent before pressure can be marketed as an alternative for sterilisation.
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