Journal of Applied Microbiology 2005, 98, 1400–1409
doi:10.1111/j.1365-2672.2005.02564.x
A REVIEW Microbiology of pressure-treated foods M.F. Patterson Department of Agriculture and Rural Development, Northern Ireland and Queen’s University, Belfast, UK 2004/0950: received 16 August 2004, revised and accepted 10 December 2004
1. 2. 3. 4.
Summary, 1400 Introduction, 1400 High-pressure processing equipment, 1401 Fundamental effects of pressure on microbial cells, 1401 4.1 Effect of pressure on cell membranes, 1401 4.2 Effect of pressure on cell morphology, 1401 4.3 Effect of pressure on biochemical reactions, 1402 4.4 Effect of pressure on genetic mechanisms, 1402 5. High pressure inactivation of micro-organisms, 1402 5.1 Inactivation kinetics, 1402 5.2 Pressure injury, 1402
1. SUMMARY High hydrostatic pressure has the potential to produce high quality foods that are microbiologically safe and with an extended shelf-life. Micro-organisms vary in their response to high pressure. Bacterial spores are the most resistant group and they cannot be significantly inactivated by pressure alone. Combination treatments using high pressure and heat have been proposed as a method of producing shelf-stable low acid foods. Viruses are less resistant than bacterial spores and their infectivity can be abolished without destroying their ability to elicit antibodies, leading to the possibility of vaccine production. Yeasts, moulds and vegetative bacteria vary in their response to pressure, depending on factors such as species, strain, processing temperature and substrate, and these are reviewed in the paper. A knowledge of how these factors interact is necessary in order to select the optimum processing conditions for foods. A number of pressuretreated foods are already commercially available and these are discussed in the paper. 2. INTRODUCTION Consumers in the 21st century are demanding high quality foods that are free from additives, fresh tasting, microbioCorrespondence to: Margaret Patterson, Agricultural, Food and Environmental Science Division (Food Microbiology Branch), Agriculture and Food Science Centre, Newforge Lane, Belfast BT9 5PX, Northern Ireland, UK (e-mail:
[email protected]).
5.3 Bacterial endospores, 1402 5.4 Viruses and prions, 1404 5.5 Vegetative bacteria, 1405 5.6 Yeasts and moulds, 1405 6. Extrinsic factors affecting the sensitivity of microorganisms to pressure, 1405 6.1 Effect of substrate, 1405 6.2 Effect of temperature, 1406 7. Pressure treatment to improve the microbiological quality of foods, 1406 8. References, 1407 logically safe and with an extended shelf-life. One food technology that has the potential to meet these demands is high pressure processing. High pressure processing, also known as high hydrostatic pressure or ultra-high pressure processing, uses pressures up to 900 MPa (c. 9000 atmospheres, c. 135 000 pounds per square inch) to kill many of the micro-organisms found in foods, even at room temperature. These pressures are immense. A mid-range food processing pressure of 500 MPa is equivalent to the weight of three elephants on a strawberry. The idea of using high pressure in food processing is not new. The first report of high pressure being used as a food preservation method was by Hite (1899). He reported that milk kept sweet for longer after a pressure treatment of c. 600 MPa for 1 h at room temperature. Hite et al. (1914) also reported that while pressure could be used to extend the shelf-life of fruits, it was less successful with vegetables. He concluded that fruits and fruit juices responded well to high pressure because the yeasts and other organisms having most to do with decomposition are very susceptible to pressure. Vegetables, however, he abandoned as hopeless due to the presence of spore-forming bacteria that survived the pressure treatment and could grow in the low acid environment. The problem of pressure resistant spores still remains one of the challenges for the technology today. Much experimental data has been produced over the last 100 years but it was not until the early 1990s that the first commercial food applications of the technology were seen. There are considerable ª 2005 The Society for Applied Microbiology
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engineering problems involved in repeatedly generating and containing the immense pressures in a vessel suitable for food products. However, within the last 25 years a range of specialist high pressure vessels, based on those used routinely in the production of polymers, ceramics and artificial diamonds, became available and reopened the possibility of commercial production of pressure-treated foods. This paper will review how high pressure can be used to improve the microbiological safety and quality of foods, including the problem of spore-forming bacteria and commercial food applications of the technology will also be discussed. 3. H I G H - P R E S S U R E P R O C E S S I N G EQ U I P M E N T A typical pressure treatment system consists of a pressure vessel, the pressure transmission fluid (usually water) and one or more pumps to generate the pressure. It is traditionally a batch process and pressure vessels used for commercial food production having capacities of 35– 350 l. Food packages are loaded into the vessel, the top is closed and the pressure transmission fluid is pumped into the vessel from the bottom. Once the desired pressure is reached, pumping is stopped, valves are closed and the pressure can be maintained without further need for energy input. The pressure is transmitted rapidly and uniformly throughout the pressure fluid and the food. The high pressure is applied in an isostatic manner so that all parts of the food are subjected to the same pressure at exactly the same time, unlike heat processing where temperature gradients are established. As it is equal from all sides, the pressure does not significantly affect the product shape. The pressure is released after the desired treatment time and the food packages can be unloaded. In the case of liquids, such as fruit juices, the whole vessel can be filled with the juice, which itself becomes the pressure transmission fluid. After treatment, the juice can be transferred to an aseptic filling line, similar to that used for UHT (ultra-high pressure treatments) liquids. A series of these vessels can work in sequence, with a vessel filling with juice, a vessel pressurizing and another emptying, all operating simultaneously, so the overall system can become semi-continuous. High pressure equipment suitable for food use is specialized and the capital equipment cost is relatively high, although running costs are relatively low. Typically a commercial vessel can cost £500 000 to over £1 million, depending on its size. It is likely that these costs would reduce if the use of the technology grows and more vessels are sold.
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4. FUNDAMENTAL EFFECTS OF PRESSURE ON MICROBIAL CELLS There is much published research on the changes induced by pressure treatment of microbial cells, including alterations in the cell membrane, cell morphology, effects on proteins, including enzymes and effects on genetic mechanisms of micro-organisms (for reviews see Hoover et al. 1989; Smelt et al. 2001). However, despite this effort, the mechanisms of microbial inactivation are still not fully understood (Paga´n and Mackey 2000). 4.1 Effect of pressure on cell membranes The cell membrane is generally acknowledged to be a primary site of pressure damage in micro-organisms (Morita 1975; Ulmer et al. 2000; Casadei et al. 2002). Evidence of physical damage to the cell membrane has been demonstrated as leakage of ATP or UV-absorbing material from bacterial cells subjected to pressure (Smelt et al. 1994) or increased uptake of fluorescent dyes such as propidium iodide that do not normally penetrate membranes of health cells (Benito et al. 1999). Stationary phase cells are normally more pressure resistant than exponential-phase cells. Man˜as and Mackey (2004) have proposed that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a more robust cytoplasmic membrane that can better withstand pressure treatment. This proposal was based on the fact that exponential-phase cells showed changes in their cell envelopes that were not seen in stationary-phase cells. These changes included physical perturbations of the cell envelope structure, a loss of osmotic responsiveness and a loss of protein and RNA to the extracellular medium. Loss of membrane functionality resulting from pressure treatment has also been described by Wouters et al. (1998), who reported that in Lactobacillus plantarum, pressure treatment at 250 MPa reduced F0F1 ATPase activity. The ability to maintain a DpH was also reduced and the acid reflux was impaired. 4.2 Effect of pressure on cell morphology The cell wall is less affected by high pressure than the membrane and generally no morphological changes can be observed in prokaryotes and lower eukaryotes by observation under a light microscope. However, intracellular damage can be observed using electron microscopy. Ritz et al. (2001), using scanning electron microscopy (SEM), reported that bud scars appeared on the cell surface of Listeria monocytogenes after a 10 min pressure treatment at 400 MPa in citrate buffer. Similarly, Park et al. (2001) studied the effect of pressure on the ultrastucture of L. viridescens. Nodes on cell walls of organisms treated at 400 MPa and above for
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5 min at 25C were observed using SEM. Transmission electron micrographs indicated empty cavities between the cytoplasmic membrane and the cell wall after this treatment. 4.3 Effect of pressure on biochemical reactions High pressure treatment favours biochemical reactions that lead to a volume decrease while it can inhibit or retard reactions that lead to a volume increase. Most biochemical reactions result in a volume change and are therefore affected by pressure. Studies carried out on volume changes in proteins have shown that the main targets of pressure are hydrophobic and electrostatic interactions while hydrogen bonding, which stabilizes the a-helical and b-pleated sheet forms of proteins, is not significantly influenced by pressure (see Heremans 2001 for detailed review). Enzymes vary greatly in their ability to withstand pressure. Certain microbial enzymes, such as Bacillus subtilis a-amylase, can withstand pressures of 500 MPa (Suzuki and Kitamura 1963), while others, such as L. monocytogenes phosphoglucomutase and aconitase, are inactivated by 200 MPa (Simpson and Gilmour 1997a). However, in the latter case, L. monocytogenes was little affected at this pressure treatment, suggesting that the inactivation of these enzymes was not critical to survival. In addition, there was no relationship between pressure resistance of the 13 enzymes studies and the pressure resistance of the three L. monocytogenes strains included in the study. Covalent bonds are generally unaffected at the pressures used in food processing. This means that many of the components responsible for the sensory and nutritional quality of foods, such as flavour components and vitamins, are not destroyed by high pressure. This is an important benefit for the food industry. 4.4 Effect of pressure on genetic mechanisms Nucleic acids are relatively resistant to high pressures and as the structure of the DNA helix is largely the result of hydrogen bond formation, it is also stable under pressure. However, the enzyme-mediated steps involved in DNA replication and transcription are disrupted. It has been reported that pressure causes a condensation of nuclear material in L. monocytogenes, Salmonella Typhimurium (Mackey et al. 1994) and L. plantarum (Wouters et al. 1998). Chilton et al. (1997) postulated that at elevated pressures, DNA comes into contact with endonucleases that cleave the DNA.
5. HIGH PRESSURE INACTIVATION OF MICRO-ORGANISMS 5.1 Inactivation kinetics High pressure inactivation of micro-organisms is complex, and plotting the log of surviving numbers against time does
not always form a straight line relationship (first-order kinetics). Often there is an initial linear decrease in numbers followed by a decrease in the rate of kill leading to a pressure-resistant tail. Studies have shown that when this tail population is isolated, grown and again exposed to pressure, there is no significant difference in pressure resistance between it and the original culture (Metrick et al. 1989). Such tails are also found with heat processing but the phenomenon seems to be more pronounced with high pressure processing. The tailing effect is not fully understood. It may be because of inherent phenotypic variation in pressure resistance in some cells. Experimental conditions, such as the substrate and growth conditions, may also be a factor. Tailing phenomena can make calculation of pressure D-values difficult and have to be taken into account when doing challenge studies designed to optimize processing conditions for various foods. 5.2 Pressure injury A high pressure treatment may not always completely inactivate micro-organisms but rather may injure a proportion of the population. Recovery of the injured cells will depend on the conditions after treatment and this has implications for microbiological enumeration. Compounds such as sodium chloride added to plating media can act as selective agents and inhibit growth of injured cells. Using selective agars can, therefore give inaccurate estimates of numbers of survivors (Patterson et al. 1995). 5.3 Bacterial endospores Micro-organisms vary in their response to pressure (Table 1). Bacterial endospores can be extremely resistant to high pressure, just as they are resistant to other physical treatments such as irradiation and heat, and can survive treatments of more than 1000 MPa (for a review see Smelt 1998). There can be significant variation between spores of different species and also between strains of the same species. Clostridium botulinum spores are among the most pressure resistant, especially nonproteolytic type B (Reddy et al. 2001). However, relatively low pressures (below 200 MPa) can trigger spore germination (Gould and Sale 1970). This has led to the suggestion that spores could be killed by applying pressure in two stages. The first pressure treatment would germinate the spores while the second treatment, at a higher pressure, would kill the germinated spores (for a review see Heinz and Knorr 2001). This process could be repeated several times leading to the idea that pressure cycling between relatively low and then high pressures could be one way of overcoming the problem of spore resistance. However, the extent of the inactivation can be highly variable (Heinz and Knorr 2001) and a small
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Table 1 Sensitivity of selected micro-organisms to high hydrostatic pressure
Micro-organism
Substrate
Treatment conditions
Spore-forming bacteria C. botulinum type Sorensen phosphate 827 MPa/5 min/ E spores (Alaska) buffer 50C (0Æ067 mol l)1, pH 7Æ0) C. sporogenes spores Chicken breast 680 MPa/80C/ 20 min
B. stearothermophilus
Water
C. sporogenes, Meat emulsion B. subtilis, B. stearothermophilus spores Viruses HIV-1 Culture medium
600 MPa/5 min/ 70C · 6 cycles 621 MPa/98C/ 5 min
Inactivation (log10 units of reduction)
5
2
Reddy et al. 1999
Level of inactivation increased if spores were subsequently irradiated to 3 kGy
Crawford et al. 1996
Hayakawa et al. 1994
>5 (C. sporogenes) Method relies on adiabatic >9 (B. subtilis) heating occurring. >10 (B. stearothermophilus) Product temperature peaked at 29Æ4C
Wilson and Baker 2001
Foot and Mouth Disease virus
Culture medium
Feline calicivirus
Tissue culture medium
275 MPa/21C/ 5 min
Hepatitis A
Tissue culture medium Sea water
Poliovirus
Tissue culture medium
450 MPa/21C/ 5 min 450 MPa/21C/ 5 min 450 MPa/21C/ 5 min
Prions Hamster-adapted scrapie agent
Hamster brain homogenate
700–1000 MPa/ 60C/2 h
Vegetative bacteria Campylobacter jejuni
Pork slurry Strained baby food
300 10 340 10
UHT milk Poultry meat
600 MPa/20C/ 15 min
Salmonella Senftenberg 775W Escherichia coli O157:H7 NCTC 12079
Reference
5
550 MPa/25C/ Infectivity titre reduced 10 min by 4log10 units Infectivity titre reduced 250 MPa/ )15C/1 mol l)1 by >4log10 units urea/60 min
MPa/25C/ min MPa/23C/ min
Comments
>6log10 reduction in plaque-forming units <2log10 inactivation
Otake et al. 1997 Treated virus, although no Ishimaru et al. 2004 longer infective, can still elicit neutralizing antibody production in rabbits. Gives the possibility of novel method of viral vaccine production 7 log 10 tissue culture Kingsley et al. 2002 infectious dose for 50% of the cultures was completely inactivated Kingsley et al. 2002
No reduction in plaque-forming units
Kingsley et al. 2002
Increase in survival rate Garcı´a et al. 2004 of hamsters following infection (47%) and delayed onset of disease in those that were infected (from 80 to 153 d) 6
Shigehisa et al. 1991
<2
Metrick et al. 1989
<2 3
Pressure-resistant strain
Patterson et al. 1995
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Table 1 (Continued)
Micro-organism Staphylococcus aureus Listeria monocytogenes Vibrio parahaemolyticus O3:K6 Lactobacillus helveticus Pseudomonas fluorescens Yeasts and moulds Saccharomyces cerevisiae Byssochlamys nivea ascospores
Inactivation (log10 units of reduction)
Substrate
Treatment conditions
UHT milk Poultry meat UHT milk Poultry meat Oysters
600 MPa/20C/15 min
300 MPa/10C/3 min
2 3 <1 2 5
Ewe’s milk Ewe’s milk
500 MPa/10C/10 min 450 MPa/10C/10 min
3 4
Gervilla et al. 1997b Gervilla et al. 1997b
Pork slurry Grape juice, aw ¼ 0Æ97 Bilberry jam, aw ¼ 0Æ84
300 MPa/20C/10 min 700 MPa/70C/30 min
2 4 <1
Shigehisa et al. 1991 Butz et al. 1996
375 MPa/20C/15 min
proportion of each spore population seems to remain resistant to pressure-induced germination (Gould 1973). Another approach to the problem of the pressure resistance of bacterial spores is to combine high temperature along with pressure treatment. There have been many reports indicating that this can be very successful (Heinz and Knorr 2001). This approach is now being actively considered for the commercial production of shelf-stable foods and is the subject of a number of patents designed to achieve the commercial sterilization of foods that have a pH greater than 4Æ5. One such patent describes a process involving two or more cycles of high heat (>70C) and high pressure (>530 MPa) with a pause between the cycles (Mayer 2000). The temperature, pressure level, treatment time and time interval between the cycles can be varied depending on the product but are designed to give greater than the equivalent of a 12D process for C. botulinum. These treatments use an initial temperature of below 100C and rely on the fact that adiabatic heating occurs when the product is pressure treated. Adiabatic heating results from the work of compression during pressure treatment leading to an increase in the temperature of food. The extent of the temperature increase varies with the composition of the food but is normally 3–9C/100 MPa. The overall treatment conditions are less severe than conventional retorting. This results in shelf-stable products which are of higher quality in terms of texture, flavour and retention of nutrients than those obtained by conventional processing (Master et al. 2004). 5.4 Viruses and prions There is relatively little information on pressure inactivation of viruses compared with other micro-organisms but it
Comments
Reference Patterson et al. 1995
Most resistant of three strains studied Most resistant of 10 strains studied
Patterson et al. 1995 Cook 2003
does appear that viruses can vary significantly in their response to treatment (Table 1). Polio virus in tissue culture medium appears to be relatively resistant with 450 MPa for 5 min at 21C giving no reduction in plaqueforming units (PFU). The same treatment conditions with hepatitis A resulted in a 6log10 PFU ml)1 stock culture being reduced to undetectable levels (Kingsley et al. 2002). However, treatment in sea water increased the pressure resistance of hepatitis A virus, suggesting a protective effect of the salts. Feline calicivirus, a Norwalk virus surrogate, (Kingsley et al. 2002) and human rotavirus (Khadre and Yousef 2002) were more pressure sensitive than hepatitis A when treated in tissue culture medium. There are also reports that HIV-1 is relatively sensitive to pressure with 400–600 MPa for 10 min at 25C resulting in 4–5log10 reduction in viable particles when treated in tissue culture medium (Otake et al. 1997), although different strains varied in their pressure resistance. Foot and mouth disease virus (FMDV) is also relatively sensitive to high pressures. A treatment of 250 MPa at )15C and 1 mol l)1 urea for 1 h destroyed FMDV infectivity but maintained the integrity of capsid structure. The treated virus could also elicit neutralizing antibody production in rabbits. These results suggest that high pressure could be a safe, simple, cheap and reproducible method of producing viral vaccines (Ishimaru et al. 2004). Evidence is emerging that high pressure may have some effect on prions. Garcı´a et al. (2004) used hamster-adapted scrapie strain 263k to infect hamsters intracerebrally. Pressure treatment of the prions to >700 MPa at 60C for 2 h significantly increased survival rate of the animals. Further work still needs to be done but the early work suggests the possibility of producing safe products for specialized, high added-value markets, such as baby foods.
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1400–1409, doi:10.1111/j.1365-2672.2005.02564.x
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5.5 Vegetative bacteria
6.1 Effect of substrate
In general terms Gram-positive bacteria tend to be more resistant to pressure than Gram-negatives and cocci are more resistant than rod-shaped bacteria (Table 1). It has been suggested that the cell membrane structure is more complex in Gram-negative bacteria, making it more susceptible to environmental changes caused by pressure (Shigehisa et al. 1991). However, there are many exceptions to these general rules. Certain strains of Escherichia coli O157:H7, for example, can be exceptionally pressure resistant. Benito et al. (1999) reported that an E. coli O157:H7 strain isolated from a major hamburger patty outbreak in the US showed less than a 1log10 reduction when treated in laboratory medium at 500 MPa at <45C for 30 min. This strain was also more resistant to heat, acid, oxidative and osmotic stresses than a pressure-sensitive strain. However, other studies with pathogens such as Salmonella, have shown only a weak, or no correlation, between pressure resistance and resistance to other stresses (Sherry et al. 2004).
The chemical composition of the substrate during treatment can have a significant effect on the response of microorganisms to pressure. Certain food constituents such as proteins, carbohydrates and lipids can have a protective effect (Simpson and Gilmour 1997b). Inactivation data obtained using buffers or laboratory media, therefore, should not be extrapolated to real food situations where a more severe pressure treatment may be needed to achieve the same level of inactivation. For example, a treatment of 375 MPa for 30 min at 20C in phosphate buffer (pH 7Æ0) gave a 6log10 inactivation of a pressure-resistant strain of E. coli O157:H7. However, the same treatment gave a 2Æ5log10 reduction in poultry meat and only 1Æ75log10 reduction in milk (Patterson et al. 1995). Cations, such as Ca2+, can be baroprotective and this may explain why many micro-organisms appear more pressure resistant when treated in certain foods, such as milk (Hauben et al. 1998). A low water activity protects micro-organisms against the effects of pressure (Palou et al. 1997). Oxen and Knorr (1993) reported that reducing aw of the medium from 0Æ98– 1Æ0 down to 0Æ94–0Æ96 resulted in better survival of Rhodotorula rubra when it was subjected up 200–400 MPa for 15 min at 25C. However, the nature of the solute is important. At the same aw, cells were more pressure sensitive in glycerol than in monosaccharides and disaccharides. Trehalose is reported to confer most protection (see review by Smelt 1998). The pH of acidic solutions decreases as pressure increases and it has been estimated that in apple juice, there is a pH drop of 0Æ2 U per 100 MPa (Heremans 1995). To date, the pH change which occurs during pressure treatment cannot be measured directly in solid food, but methods have been developed for in situ pH measurement during pressure treatment of liquids (Hayert et al. 1999; Stippl et al. 2004). When the pressure is released, the pH reverts to its original value but it is not known whether these sudden changes in pH affect microbial survival in addition to the effect of pressure. It is known that pH and pressure can act synergistically leading to increased microbial inactivation. Linton et al. (1999) has shown that initial pH had a significant effect on inactivation rates of E. coli O157:H7 in orange juice. As pH was lowered, the cells were more susceptible to pressure inactivation and sublethally injured cells failed to repair and died more rapidly during subsequent storage of the juice. Food additives can have varying effects on microbial resistance to pressure. Pressure has been used to sensitize Gram-negative bacteria such as Salmonella, as well as Grampositive bacteria such as L. monocytogenes to nisin and lysozyme (Kalchayanand et al. 1998; Masschalk et al. 2001). The combination of high pressure and nisin was also used to
5.6 Yeasts and moulds Yeasts are generally not associated with food-borne disease but are important in spoilage, especially in acidic foods. They are relatively sensitive to pressure (Table 1) and this is one reason why pressure treatment of fruit products to extend shelf-life is particularly successful. There is relatively little information on the pressure sensitivity of moulds but it has been shown that vegetative forms are relatively sensitive, while ascospores are more resistant (Butz et al. 1996; Voldrich et al. 2004). The effect of pressure on preformed mycotoxins is thought to be limited as the treatment has little effect on covalent bonds. However, one study reported that patulin, a mycotoxin produced by several species of Aspergillus, Penicillium and Byssochlamys, was found to be degraded by pressure (Bru˚na et al. 1997). The patulin content in apple juice decreased by 42, 53 and 62% after 1 h treatment at 300, 500 and 800 MPa, respectively, at 20C. An explanation for this was not given. 6. EXTRINSIC FACTORS AFFECTING THE SENSITIVITY OF MICRO-ORGANISMS TO HIGH PRESSURE High pressure is no different from other physical preservation methods in that its effectiveness against micro-organisms is influenced by a number of factors. These all interact and contribute to the lethal effect and therefore have to be considered when designing process conditions to ensure the microbiological safety and quality of pressure-treated foods.
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increase the inactivation of B. cereus spores in cheese (Lo´pez-Pedemonte et al. 2003). However, in this case the pressure treatment conditions were relatively severe. The treatment (60 MPa at 30C for 210 min to germinate the spores followed by 400 MPa at 30C for 15 min to kill the vegetative cells) was carried out in the presence of 1Æ56 mg l)1 nisin in the cheese. This resulted in approx. 2Æ4log10 inactivation of the spores. Pediocin AcH also works synergistically with pressure. A combination of 345 MPa for 5 min at 50C in the presence of 3000 AU ml)1 pediocin AcH gave at least a 7log10 inactivation of a range of bacteria including L. monocytogenes, Salm. Typhimurium, Staphylococcus aureus, E. coli O157:H7 L. sake and Pseudomonas fluorescens. This level of inactivation could not be achieved by pressure alone (Kalchayanand et al. 1998). Similarly, the combination of high pressure with other antimicrobial agents such as lacticin 3147 (Ross et al. 2000), lactoperoxidase (GarciaGraellis et al. 2003) and carvacrol (Karatzas et al. 2001) can work synergistically to enhance the kill of micro-organisms, including pathogens. 6.2 Effect of temperature Temperature during pressure treatment can have a significant effect on microbial survival. Increased inactivation is usually observed at temperatures above or below 20C (Takahashi et al. 1992). High temperatures (>70C) can be particularly effective in helping to achieve high pressure sterilization, as discussed above. The combination of elevated temperatures (<50C) with pressure has also been suggested as a practical way to overcome the problem of pressure resistant strains of vegetative cells. Patterson and Kilpatrick (1998) reported approx. a 6log10 inactivation of a pressure-resistant E. coli O157:H7 in poultry mince and a 5log10 inactivation in milk using a treatment of 400 MPa at 50C for 15 min. Neither heat nor pressure alone could achieve this level of inactivation. Refrigeration temperatures can also enhance pressure inactivation. Gervilla et al. (1997a) reported that ewes milk pressurized at 450 MPa at 2C for 15 min was more effective at inactivating L. innocua than the same treatment at 25C but less effective than the pressure treatment at 50C. 7. PRESSURE TREATMENT TO IMPROVE T H E M I C R O B I O LO G I C A L Q U A L I T Y O F FOODS Pressure-treated fruit jams and sauces first became commercially available in Japan in the early 1990s. Treatment of fruit jams with around 400 MPa for up to 5 min at room temperature can significantly reduce the number of microorganisms, especially yeasts and moulds. Refrigeration of the
jam after processing is necessary due to browning and flavour changes caused by enzymatic activities. These products have a shelf-life of around 30 d and have superior sensory quality compared with those prepared in a conventional manner (Ludikhuyze and Hendrickx 2001). Fruit juices are normally processed at 400 MPa or greater for a few minutes at 20C or less. This can significantly reduce numbers of yeasts and moulds and so extend shelflife for up to 30 d. Pathogens, such as E. coli O157:H7 can also be destroyed by this treatment (Linton et al. 1999; Ramaswamy et al. 2003). Pressure-treated orange and grapefruit juices have been available in France since 1994, while pressure-treated apple juice is available in Italy. Pressure treatment of vegetable products is problematic because of their relatively high pH along with the possibility of survival and growth of pathogenic spore-forming organisms. However, one of the most successful pressure-treated foods in the USA is guacamole. Its market share continues to grow and is reportedly based on the consumer preference for the fresher taste of guacamole processed in this manner compared with heat-treated or frozen products. Treatment of around 500 MPa for 2 min is sufficient to extend shelflife from 7 to 30 d at refrigeration temperatures. Challenge studies with a variety of pathogens have shown that this treatment is sufficient to give a 5log10 reduction in numbers (Parnell 2003) and the process has been approved by the US Food and Drug Administration. Sliced cooked ham and other delicatessen meat products, in flexible pouches, can be successfully treated using 500 MPa for a few minutes. The sensory properties of ham are preserved, and shelf-life can be extended to 60 d under chilled storage. Cooked delicatessen products have a risk of postprocessing contamination from pathogens such as L. monocytogenes. High pressure treatment as a final preservation step, after packaging, can give additional microbiological safety assurance. During challenge studies a treatment of 500 MPa can cause a 5log10 reduction in L. monocytogenes in dry-cured ham (Minerich and Krug 2003). Pressure-treated cooked and vacuum-packaged ham is available in Spain and in the USA. Another example of commercially successful pressuretreated foods available in the USA are oysters. The initial aim of the pressure treatment was to eliminate Vibrio spp. from oysters, which are often eaten raw or only lightly cooked. Vibrio spp. are relatively sensitive to high pressure, although there can be species variation (Cook 2003). Typical treatments of 250–350 MPa for 1–3 min at ambient temperature are used commercially without significantly affecting sensory quality. An additional benefit of pressure-treated oysters is the mechanical shucking effect it causes, releasing the adductor muscle from the shell (He et al. 2002). For this reason, a heat shrink plastic band is placed around each oyster prior to processing so that the shell is kept shut until
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the meat is required. The main processor of pressure-treated oysters in the USA uses a trademark plastic gold band and these products have achieved several national awards for quality products. Pressure will successfully shuck other shellfish, such as mussels, Nephrops and crabs as well as improving their microbiological quality and it is likely that the technology will be more widely used for this purpose. There is also increasing interest in pressure-treating fin fish to improve microbiological safety and quality as well as using the technology to produce a range of novel food products (Lakshmanan and Dalgaard 2004), including surmi gels (Ohshima et al. 1993; Ashie and Simpson 1996). Pressure treatment of milk and dairy products to improve microbial safety and quality has been of interest since the early work of Hite (1899). However, it is likely that the technology will only be used commercially for niche applications, where it can provide a commercial advantage over existing, usually heat-treated, products. For example, pressure treatment may be of value in treating milk that is to be used in the manufacture of raw milk cheese, where it could reduce the numbers of pathogens such as L. monocytogenes. However, some pathogens, such as certain strains of E. coli O157:H7, are known to be extremely pressure resistant in milk (Patterson et al. 1995). Therefore, this approach will not solve all the microbiological safety problems associated with raw milk cheese. High pressure treatment of yoghurt has also been investigated. Tanaka and Hatanaka (1992) investigated the effectiveness of using high pressure to prevent after-acidification of yoghurt during storage. They concluded that a treatment of 200–300 MPa for 10 min at room temperature prevented the continued growth of lactic acid bacteria during storage and so maintained the yoghurt quality. The range of products now being considered for high pressure treatment continues to grow year-on-year. A range of complete meal kits have recently been launched in the USA. The kits consist of pressure-treated cooked meat or chicken, salsa, guacamole, peppers and onion are now also available. Only the flour tortillas are not pressure treated. The products have a chilled shelf-life of at least 30 d and only required to be reheated in a microwave before consumption. It is likely that the range of added-value, high quality pressure-treated foods will increase within the next 5 years. 8. REFERENCES Ashie, I.N.A. and Simpson, B.K. (1996) Application of high hydrostatic pressure to control enzyme related fresh seafood texture deterioration. Food Res Int 29, 569–575. Benito, A., Ventoura, G., Casadei, M., Robinson, T. and Mackey, B. (1999) Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses. Appl Environ Microbiol 65, 1564–1569.
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