Fresher Under Pressure Scientific Research

High Pressure Processing of Foods: An Overview

(FIRST PUBLISHED IN SCIENCE DES ALIMENTS, 19 (1999) P 619-661)

G. Tewari^*, ⁽¹⁾, D. S. Jayas⁽¹⁾, R. A. Holley⁽²⁾

Department of Biosystems Engineering, 438 Engineering Building, University of Manitoba, Winnipeg, MB, Canada R3T 5V6
Department of Food Science, University of Manitoba, Winnipeg, MB, Canada, R3T 2N2
* Correspondent's present address: Guelph Food Technology Centre, 88 McGilvray St., Guelph, ON, N1G 2W1, Canada. Phone: (519)-821-1246 (x 5019); Fax: (519) -836-1281;

Summary

High pressure processing (HPP) is gaining in popularity with the North American food industry because of its capacity to inactivate pathogenic microorganisms with minimal heat treatment, resulting in the almost complete retention of nutritional and sensory characteristics of fresh food without sacrificing shelf-life. Other advantages of HPP over traditional thermal processing include reduced process times; minimal heat damage problems; retention of freshness, flavor, texture, and color; no vitamin C loss; no undesirable changes in food during pressure-shift freezing due to reduced crystal size and multiple ice-phase forms; and minimal undesirable functionality alterations. Changes that may be made improve functional properties of food constituents resulting in value-added products. Minimization of damage during pressure-shift freezing and thawing using HPP; non-thermally-induced enzyme inactivation; and desirable changes in starch-gelatinisation properties are some other examples of potential benefits of HPP. However, spore-inactivation is a major challenge for HPP. Methods used to achieve full inactivation of spores using HPP are yet to be developed. In thermal processing, D (time required in min to reduce the microbial population 10-fold), Z (temperature in °C yielding a 10-fold change in D), and F_o (the integrated lethal value from all heat received by a treated food with a reference temperature of 121.1°C and assuming a Z-value of 10°C) values are standard processing parameters, however there is a need to develop and standardize HPP process parameters with respect to microbial inactivation, because none exist. This is essential before commercialization of this technology can take place. In this paper, basic principles of HPP are explained and the major research done on various HPP applications around the world is critically evaluated. Recommendations are made for major research areas in HPP for commercialization of this technology.

1. INTRODUCTION

Non-thermal food processing techniques are regarded with special interest by the food industry presently. Among non-thermal techniques (pulse-electric field pasteurization, high intensity pulsed lights, high intensity pulsed-magnetic field, ozone-treatment), high pressure processing (HPP) is also gaining in popularity with food processors not only because of its food preservation capability but also because of its potential to achieve interesting functional effects (LEADLEY and WILLIAMS, 1997). High pressure processing has had application for years in other industries which process or use ceramics, carbon graphite, diamond, steel/alloy, and plastics. HITE et al. (1914) were the first researchers who reported the effects of HPP on food microorganisms by subjecting milk to pressures of 650 MPa and obtaining a reduction in the viable numbers of microbes. For the last 15 years, the use of HPP has been explored extensively in food industry and related research institutions due to the increased demand by consumers for improved nutritional and sensory characteristics of food without loss of "fresh" taste. In recent years, HPP has been extensively used in Japan and a variety of food products like jams and fruit-juices have been processed (CHEFTEL, 1995). There have been 10 to 15 types of pressurized foods on the Japanese market, but several have disappeared, and those that remain are so specific that they would have little interest to European or American markets. Nevertheless, interest in HPP derives from its ability to deliver foods with fresh-like tastes without added preservatives. Examples of commercial pressurized products in Europe or US are: (1) orange juice by UltiFruit^®, Pernod Richard Company, France; (2) acidified avocado purèe (guacamole) by Avomex Company in US (Texas/Mexico); and (3) sliced ham (both cured-cooked and raw-cooked) by Espuna Company, Spain. Volumes produced are still very small. Pressurized fruit preparation from yoghurt should be coming soon on the market. The European "Novel Foods" Directive (May 1997) has introduced regulatory problems and slowed the introduction of new pressurized products. The capability and limitations of HPP have recently been reviewed and studied extensively by food scientists and food engineers (AUTIO, 1998; THAKUR and NELSON, 1998; CHEFTEL, 1995; LEADLEY and WILLIAMS, 1997; KNORR, 1995; FARR, 1990). The aim of this paper is to provide a current review of different aspects and potential applications of HPP, and to critically examine HPP-related studies.

2. MECHANISM of HPP

Any phenomenon in equilibrium (chemical reaction, phase transition, change in molecular configuration), accompanied by a decrease in volume, can be enhanced by pressure (Le Chatelier’s Principle: LEADLEY and WILLIAMS, 1997). Thus, HPP affects any phenomenon in food systems where a volume-change is involved and favors phenomena which result in a volume decrease. The HPP affects non-covalent bonds (hydrogen, ionic, and hydrophobic bonds) substantially as some non-covalent bonds are very sensitive to pressure, which means that low molecular weight food components (responsible for nutritional and sensory characteristics) are not affected, whereas high molecular weight components (whose tertiary structure is important for functionality-determination) are sensitive. Some specific covalent bonds are also modified by pressure. The other principles which govern HPP are; the Isostatic Principle which implies that the transmittance of pressure is uniform and instantaneous (independent of size and geometry of food), however, transmittance is not instantaneous when gas are present, and the Microscopic Ordering Principle, which implies that at constant temperature, an increase in pressure increases the degree of ordering of the molecules of a substance (HEREMANS, 1992). Another interesting rule concerns the small energy needed to compress a solid or liquid to 500 MPa as compared to heating to 100°C, because compressibility is small. HPP offers several advantages: reduced process times; minimal heat penetration/heat damage problems; freshness, flavor, texture, and color are well retained; there is no vitamin C loss; multiple changes in ice-phase forms resulting in pressure-shift freezing; and functionality-alterations are minimized compared with traditional thermal processing (CHEFTEL, 1995; LEADLEY and WILLIAMS, 1997; WILLIAMS, 1994; KNORR, 1995; FARR, 1990; MERTENS, 1992).

3. APPLICATIONS OF HPP

3.1 Sensory and Nutritional Characteristics - Retention, Shelf Life Extension, and Value-Added Products

As mentioned above, HPP does not depreciate the nutritional and sensory characteristics of food, and yet it maintains shelf life (table 1). ESHTIAGHI and KNORR (1993) compared the effect of HPP with water blanching on the microbial safety, quality (softness), and functionality [poly phenoloxidase (PPO) activity, leaching of potassium, and loss of ascorbic acid] of potato cubes. Total inactivation of microbes and PPO activity occurred at 20°C (using dilute citric acid solution at 0.5 or 1.0% as immersion medium). Water-blanched and high pressure-treated potato cubes had similar softness but potassium leaching was reduced by 20%, in addition, ascorbic acid was better retained (90% at 5°C to 35% at 50°C) in high-pressure-treated vacuum-packaged samples. However, they did not investigate use of different pressure-ranges, which may have resulted in different functional characteristics. ROVERE et al. (1997) studied the effects of HPP on chopped tomatoes. They pre-treated tomatoes at 25, 50, or 85°C, followed by cooling and vacuum packaging. After this, the tomatoes were subjected to pressures. They reported that color, sugar content, and pH were affected by pressure, and that viscosity decreased with increasing blanching temperature and increased with increasing pressures. They also found that pressure has a significant inactivating effect on polygalacturonase type pectic enzymes, but had only little effect on pectin esterases (PE). They concluded that HPP may control various quality parameters of chopped tomatoes.

KIMURA et al. (1994) compared the quality (volatile flavor components, anthocyanins, browning index, furfural, sucrose, and vitamin C content) of pressure-treated and heat-treated jams during storage at 5 and 25°C for 1-3 months. Immediately after processing, the pressure-treated jams had better fresh quality than heat-treated jams, and the quality was maintained in both at low temperature storage, but not at room temperature for pressure-treated jams. The presence of dissolved oxygen and enzymes was believed to have resulted in deterioration of pressure-treated jam held at ambient temperature. However, pressure-treated jam could be stored at refrigeration temperatures with minimal loss in sensory and nutritional characteristics for up to 3 months. In their study for microbiological stabilization of white and red grape must during HPP, MOIO et al. (1995) concluded that the rate of microbial inactivation was directly proportional to pressure used. Stabilization of white wine must occurred at 507 MPa for 3 min, whereas complete microbial inactivation was not achieved in red must (even at 811 MPa for 5 min), which could have been due to its high concentration of suspended solids. GOW and HSIN (1996) compared the quality and shelf life of pressure-treated with thermally pasteurized (88-90°C for 24s) "guava purée". A substantial inactivation of microbes (< 10 cfu/mL) was observed at 600 MPa, and the pressure-treated samples showed no color-change, no degradation of pectin, no cloud formation, and had the same ascorbic acid content as fresh samples. However, enzyme inactivation was more pronounced in thermally-treated samples. The pressure-treated "guava purée" (600 MPa) maintained good quality (similar to freshly extracted "guava purée") for 40 days when stored at 4°C. It is to be noted that "guava purée" is particularly sensitive to enzymatic browning reactions which are inhibited during HPP to some extent.

DONG et al. (1996) studied the effect of HPP on the shelf life and sensory characteristics of Angelica keiskei juice by subjecting it to a pressure of 558 MPa for 7 min and storing it at 4°C. Pseudomonas spp., E. coli, and coliform bacteria were totally inhibited by HPP. During storage, microbiological quality and sensory characteristics were monitored and it was found that HPP did not significantly influence freshness, sweetness, and bitterness; but after 8 days (at 4°C), pressure-treated juices had better freshness ratings than controls (non-pressurized juice). KLOCZKO and RADOMSKI (1996) studied preservation of fresh fruits, vegetables, fruit- and vegetable-juices by subjecting them to pressures and subsequently storing them at 6°C. They reported that HPP had no beneficial effects on keeping quality of fruits and vegetables, whereas immediately after pressurization, and after 55 days of refrigerated storage, pressure-treated juices had better aroma, flavor, and microbiological quality than untreated controls, and vitamin C content remained the same or declined slightly. PEHRSSON (1996) described experiments on HPP of microbially stable citrus juice, where they processed juices for 60-90 s at refrigeration or freezing temperature. The product was stored and distributed under refrigeration. The pressure-treated juice was stable for 6 months at 4°C, without losing any freshness (as compared to juices thermally treated at 98°C for 10s). DONSI et al. (1996) studied the high pressure stabilization of orange juice, by evaluating microbial activity and the chemical composition of orange juice treated at different pressure levels for various operating times. They obtained a 2 month shelf life for pressure-treated orange juice (at 350 MPa for 1 min at 30°C) stored under refrigeration. BUTZ et al. (1997) studied the effects of HPP on anti-mutagenic activities of fruits and vegetables juices. The anti-mutagenic activity was compared with raw and heated samples (100 or 50°C for 10 min). They reported that anti-mutagenicity of strawberry and grapefruit juices was not affected by heat and pressure. Also, vegetable juices exhibited moderate to strong anti-mutagenicity, whereas, the anti-mutagenic activity of carrot, leek, spinach, kohlrabi, and cauliflower juices was sensitive to heat treatment but remained unaffected by pressure treatment.

MOORMANS et al. (1996) investigated the use of high-pressure throttling (HPT) as an alternative to thermal processing of milk before acidification to yogurt. They reported that when HPT was used to increase milk temperature to 80°C (above minimum pasteurization temperature) followed by rapid cooling to 40°C, this resulted in a 4 log reduction of microbial numbers and increased viscosity, yielding a thick and creamy yogurt (requiring no further addition of polysaccharides).

SEVERINI et al. (1997) studied the effects of high pressure on the lipid oxidation of extra virgin olive and seed oils. Peroxidase-value, p-anisidine value, rancimat test, and volatile hydrocarbons were the analytical parameters measured to study the oxidative stability of the pressure-treated oils. They reported pressure treatment changed p-anisidine values, but not others (peroxidase values and volatile hydrocarbons). Other parameters affecting high pressure treatment were origin, composition, initial quality, and age of the oils, and it was found that olive oils were more resistant to oxidation which suggests need for replacement of seed oil with extra virgin olive oil during HPP to extend shelf life of foods. They conducted their study using only one pressure, temperature and time period. Further studies should be conducted using different combinations of pressure, temperature, and time intervals, which may result in other value-added products and opportunities.

3.2 Microbial Inactivation

Application of HPP as a method for microbial inactivation has stimulated considerable interest in the food industry. The effectiveness of HPP on microbial inactivation has to be studied in great detail to ensure the safety of food treated in this manner. Currently, research in this area has concentrated mainly on the effect of HPP on spores and vegetative cells of different pathogenic bacterial species. Detectable effects of HPP on microbial cells include an increase in the permeability of cell membranes and possible inhibition of enzymes vital for survival and reproduction of the bacterial cells (FARR, 1990). To design appropriate processing conditions for HPP of food materials, it is essential to know the precise tolerance levels of different microbial species to HPP and the mechanisms by which that tolerance level can be minimized. A knowledge of critical factors that affect the baroresistance of different bacterial species will help in the development of more effective and accurate high pressure processors. Inappropriate use of a variety of parameters like pressure range, processing temperature, initial temperature of sample, holding time, and packaging type may adversely affect the outcome of HPP. Thus a thorough understanding of the effect of a variation in critical factors on the intracellular changes undergone by pressure-treated microbial species is essential for documentation of a safe HPP. The physicochemical environment can adversely change the resistance of a bacterial species to pressure. In most cases, the effect of HPP on Gram-positive bacteria is less pronounced than on Gram-negative species. Factors such as the water activity and pH also influence the extent to which foods need to be treated to eliminate pathogenic microorganisms.

HOOVER et al. (1989) reported that most bacteria are baroduric, i.e., they are capable of enduring high pressures but grow well at atmospheric pressures. HAUBEN et al. (1997) studied HPP resistance development in Escherichia coli MG1655 mutants. Three barotolerant mutants (LMM1010, LMM1020, LMM1030) were isolated and pressure-treated. Mutants showed 40-85% survival at 200 MPa for 15 min (ambient temperature) and 0.5-1.5% survival at 800 MPa for 15 min (ambient temperature). In contrast, survival of the parent strain (MG1655) decreased from 15% at 220 MPa to 2x10^-8 % at 700 MPa. It should be noted that pressure sensitivity of the mutants increased from 10 to 50°C, as opposed to the parent strain which showed minimum sensitivity at 40°C. This research indicated that the development of high levels of barotolerance should be properly understood in order to predict the safety of HPP. Similar studies are needed to document the barotolerance of other potentially pathogenic bacterial species.

MAGGI et al. (1994) studied the effects of HPP on the heat resistance of fungi in apricot nectar and distilled water. They reported complete inactivation of T. flavus ascospores at 900 MPa for 20 min at 20°C; a two log cycle reduction in N. fischeri, but no effect was seen on B. fulva and B. nivea populations. In contrast, pre-heating apricot nectar (50°C) followed by pressure treatment of 800 MPa for 1-4 min resulted in complete inactivation of all four species. Also, lower pressure resistance was observed in distilled water samples.

3.2.1 Factors affecting microbial inactivation

Several theories on the effect of HPP on bacterial species have been proposed over the years to explain the mechanism behind microbial inactivation and to better optimize HPP of foods. The processing conditions (initial sample temperature, circulating water temperature, pressurizing medium, holding time) under which high pressures are applied significantly influence the level of inactivation as well as the overall effect on the nutritional and sensory characteristics of food. YE et al. (1996) studied the pressure tolerance of Saccharomyces cerevisiae, Escherichia coli, and Staphylococcus epidermidis in various media (agar, broth, apple jam, and juice), by subjecting the inocula to a pressure of 300 MPa at 5-25°C for 1-20 min. They reported that media pH played a very important role in the destruction of microbes; S. epidermidis was inhibited > 90% at 300 MPa in 11.2 min at pH 7.2 and in 4.8 min at pH 4.0. Variations in the pH and water activity of foods can result in different levels of lethality to a particular bacterium for the same high pressure processing parameters. Studies conducted by TIMSON and SHORT (1965) on B. subtilis showed that the pressure resistance of the bacterium (when subjected to 483 MPa for 30 min) was decreased as the pH in milk medium was lowered or raised from a pH value of 8. This value is not a constant for all microorganisms and the survival of B. subtilis at a specific pH can vary with the pressure and temperature of treatment. The type of culture medium used for growing the microbial species can also have a significant impact on the pressure and heat resistance of any microorganism. In general, the richer the growth medium, the better the baroresistance of the microorganisms. This is thought to be because of the increased availability of essential nutrients and amino acids to the stressed cell. It must be kept in mind that the parameters governing pressure tolerance are not constant for every bacterial species, they vary from one bacterium to another, and may also be different for a single species grown under different conditions or in different growth media. It is very likely that the application of pressure affects a multitude of functions in a cell, thus interacting to retard or even kill the cell (HOOVER et al. 1989). Therefore, studies related to the inactivation of bacterial species during HPP should specifically describe and document the processing conditions under which the inactivation took place. Also, specific information should be given about the variation in sample temperature during HPP.

HAYAKAWA et al. (1994) compared the pressure resistance of spores of six Bacillus strains. The spores were cultivated on nutrient agar and suspended in cold sterile distilled water with the filtrate being heated for 30 min at 80<C to destroy any vegetative cells. Spores of these six strains were then treated under pressures ranging from 196 to 981 MPa at 5-10<C for holding times of 20-120 min. It was found that B. stearothermophilus IAM 12043, B. subtilis IAM 12118 and B. licheniformis IAM13417 had the most resistance to pressure, but B. coagulans was actually activated when treated with high pressures. There was no actual correlation between pressure and heat resistance, although they chose rightly a number of spore-forming bacteria varying widely in heat resistance. This could be due to applications of very low pressure in most cases. However, variables including initial and final spore levels and dilution levels were not specified, which may help understanding the results better. It should also be noted that, in most cases, the reference heat treatment is much more inactivating that the pressure treatment to be compared, therefore, direct comparisons between heat resistance and pressure resistance is often not fair.

PATTERSON et al. (1995) studied the sensitivity of vegetative pathogens (Yersinia enterocolitica 11174, Salmonella typhimurium NCTC 74, Salmonella enteritidis, Staphylococcus aureus NCTC 10652, Listeria monocytogenes, Escherichia coli O157:H7) in buffer (pH 7.0), UHT milk, and poultry meat to high pressures up to 700 MPa at 20°C. A 10⁵ reduction in numbers was obtained in all cases when pressures in the range of 275-700 MPa for 15 min were applied at 20°C. Different strains of L. monocytogenes and Escherichia coli O157:H7 showed significant variation in pressure resistance, which were further used to examine the effect of substrates on pressure sensitivity, and indicated that substrate affected the baro-resistance of the microorganisms significantly.

The pH of a food material plays a very important role in determining the extent to which HPP affects the microorganisms under study. Several studies have documented and analyzed changes in heat resistance of organisms grown under different pH conditions, but there has not been very many studies on the pressure resistance of spores at different pH values. ROBERTS and HOOVER (1996) investigated the effects of changes in pH values combined with a variety of other factors on the pressure-resistance of B. coagulans ATCC 7050. They reported an increase in the effectiveness of pressurization as the pH of the buffer was lowered. A decrease of an additional 1.5 log was observed as the pH was decreased from 7.0 to 4.0. On the basis of these results, it is highly probable that, other factors remaining constant, a neutral pH value is most conducive to high pressure resistance in the cells. Earlier, TIMSON and SHORT (1965) observed that at high pressure the spores were most resistant at neutral pH and at low pressure, most sensitive to neutral pH. That is perfectly in line with other findings that a pressure between 50 and 200 MPa enhances germination followed by kill and direct killing at high pressures above 900 MPa. In contrast, SALE et al. (1970) reported that the inactivation of bacterial spores by pressurization was maximum when the buffer was at a pH near neutral and was lowest at extreme values of pH. The change in pH was thought to affect membrane ATPase and intracellular functions of the spore thereby destabilizing the microorganisms (MACDONALD, 1992). This effect of pH on the pressure resistance of any microorganism is accentuated by other factors like the addition of salts, temperature conditions, and general process parameters. A better understanding of the exact process by which the variations in the pH affects the stability of the spore is still awaited to give a better overall picture of the effect of high pressures. Therefore, studies to examine the effect of pH variation on the inactivation of different types of spores by HPP need to be done.

The water activity (a_w) of cells also affects the in pressure resistance. It is reported that the lower the a_w, the higher the pressure resistance of cells. PALOU et al. (1996) studied the combined effect of HPP and a_w on Zygosaccharomyces bailii inhibition. They reported complete inhibition of yeast at a_w > 0.98, and an increase in the surviving fraction with a decrease in a_w. They concluded that addition of sucrose (to decrease a_w) acts as a baroprotective layer, preventing inhibition of yeast even at high pressures. Such a mechanism can be utilized to prevent inhibition of favorable microbes during HPP. More work is also needed in this area.

3.2.2 Combined processes

3.2.2.1 Pressure and temperature

ALPAS et al. (1998) studied the interaction of pressure, time, and temperature on the viability of Listeria innocua strain CWD47 in peptone solution, by subjecting samples to pressures in the range of 138_345 MPa, temperature in the range of 25_50°C, and exposure times of 5_15 min. They showed that the combination of 345 MPa, 50°C and 9.1 min could reduce the microbial population by 7 logs, with a Z value (temperature change in °C causing a ten-fold reduction in D-value; here it stands for pressure in MPa needed to reduce D-value by ten-fold) of 173 MPa. Such studies resulting in description of Z values for a specific microbial species are aimed towards a standardization of HPP parameters, which will facilitate commercialization of HPP.

MAGGI et al. (1995) studied the use of HPP for inactivation of Clostridium pasteurianum spores isolated from peeled tomato and inoculated into tomato serum. They reported that high pressure treatments of 900 MPa for 5 min at 60°C completely destroyed the spores (which was not obtained at temperatures < 60°C). Pressures of 700 or 800 MPa for 5 min at 60°C resulted in D-values of 2.4 and 3.4 min, respectively. They also studied spore inactivation using pressure-pretreatment followed by heat treatment and found that a pretreatment of 300 MPa or 500 MPa for 1 min at 60°C reduced heat resistance of spores by one-third and half, respectively. Their documented D-values for C. pasteurianum may be used for developing a data base for pressure-temperature-time requirements for complete destruction of different types of spores. ROBERTS and HOOVER (1996) studied the inactivation of Clostridium sporogenes PA3679 and Bacillus subtilis 168 using a combination of pressure, temperature, acidity, and nisin. They exposed the samples (buffer at pH 4-7, inoculated with spores) to 405 MPa at different temperatures (25-90°C) for 15 or 30 min. After pressure treatment, spores were pour plated into agar with or without nisin. They reported an increase in spore inactivation with a decrease in pH. Also, sterilization was achieved using higher temperatures and pressures, e.g. pressurization to 405 MPa at 45°C for 30 min resulted in complete inhibition of Clostridium sporogenes at pH 4, while 90°C yielded complete inhibition over a pH range of 4-6. Bacillus subtilis was completely inhibited by pressurization to 405 MPa at 70°C for 15 min at pH 4. Addition of nisin resulted in further reduction of microbial numbers. Unfortunately, this study was limited to only one pressure and D-value was not documented. The use of other combinations of pressures and temperatures may have given more promising results.

MAGGI et al. (1996) studied the combined effect of pressure and temperature on the inactivation of Clostridium sporogenes PA 3679 (ATCC 7953) spores in liquid media at pH 7.0 (beef or carrot broth medium, and phosphate buffer). They reported that 1500 MPa at 20°C for 5 min resulted in no spore-inactivation, whereas 1500 MPa at 60°C fully inactivated the spores, a pressure of just 800 MPa at 80-90°C resulted in sterilization of beef or carrot broth. ROVERE et al. (1996) studied the effect of high pressure (up to 1500 MPa) and temperature (20-88°C) on the destruction of Clostridium sporogenes strain PA 3679, ATCC 7955; and reported that total bacterial spore inactivation could be obtained using a combined pressure (1000 MPa) and temperature (50-60°C) treatment. BUTZ et al. (1996) also reported a combined pressure-temperature treatment for the inactivation of Byssochlamys nivea DSM 1824 ascopores. Researchers (AWAO and TAKI, 1990; MALLIDIS and DRIZOU, 1991; TAKI et al., 1991; SEYDERHELM and KNORR, 1992; CLOUSTON and WILLS, 1969; GOULD and SALE, 1970; NISHI et al., 1994) have also attempted to inactivate bacterial spores (Bacillus stearothermophilus, Bacillus licheniformis, Bacillus cereus, Bacillus coagulans, Clostridium botulinum) using combined high pressure (>0.7 MPa) and moderate temperatures (>50<C), and found satisfactory results. However, for complete spore inactivation, pressures > 100 MPa in combination with temperature 60-80°C are desired.

SONOIKE et al. (1992) examined the death rates of Lactobacillus casei Y1T9018 and Escherichia coli JCM1649 under various temperature (0-60°C) and pressure (0.1-400 MPa). They reported that death rates of both strains decreased with rising temperatures under a high pressure, and contours of constant death-rates of both strains on the pressure-temperature plane were elliptical and similar with that of free-energy difference for pressure-temperature-reversible denaturation of proteins.

HASHIZUME et al. (1995) studied the inactivation of yeast using HPP at low temperatures, by subjecting S. cerevisiae IFO 0234 to a pressure range of 120 to 300 MPa at -20 to 50°C. After performing regression analysis of 43 inactivation rates, they reported that the same degree of inactivation was achieved at higher pressures and higher temperatures as compared with low pressures and low (sub-zero) temperatures (e.g. the inactivation effect at 190 MPa and -20°C was similar at 320 MPa and room temperature). They concluded that high pressure treatment applied at sub-zero temperatures requires lower pressures than when conducted at high temperatures to achieve same degree of microbial inactivation. Since only a few studies have been performed using HPP at sub-zero temperatures, further studies are needed in this area to demonstrate the robustness of HPP at low temperatures, which may lead to interesting results.

3.2.2.2 Pressure and other processes

CRAWFORD et al. (1996) studied the combined use of pressure and irradiation to destroy Clostridium sporogenes spores in chicken breast. They reported a 5-log reduction at ambient temperature (25°C) with a pressure of 689 MPa applied for 60 min; heating samples at 80°C for 20 min resulted in the lowest number of survivors. They also reported that a 3.0 kGy irradiation treatment before and after pressurization at 80°C for 1, 10, and 20 min did not show any significant differences in spore numbers between samples that were pressurized and then irradiated or vice-versa. However, the irradiation D-value of Clostridium sporogenes decreased from 4.1 kGy to 2 kGy at high pressures (> 600 MPa at 80°C for 20 min); their research showed that high pressure reduced the irradiation dose required to produce chicken with an extended shelf life. They concluded that pre treatment with irradiation (prior to HPP) is a useful technique for inactivating Clostridium sporogenes spores, thereby reducing the radiation dose required to eliminate the spores by irradiation alone.

FORNARI et al. (1995) studied the inactivation of Bacillus spps. using a combination of pressure, time, and temperature. They studied 4 Bacillus spp. (B. cereus, SSICA/DA1 (from wheat flour), B. licheniformis SSICA/DA2 (from spices), B. coagulans SSICA 1881 (from tuna in tomato sauce), and B. stearothermophilus SSICA/T460 (from spoiled canned peas)) by subjecting prepared samples to a pressure range of 200-900 MPa for 1-10 min at 20, 50, 60, or 70°C. They also examined the effect of pressure-cycling on spore inactivation (pressure treatment of 200-500 MPa followed by 900 MPa). They found that B. cereus spp. were more sensitive to pressure treatment (inactivation of 4x10⁺⁵ endospores/mL was achieved at ambient temperature by treatment at 200 MPa for 1 min followed by 900 MPa for 1 min). However, for other species, a combination of higher pressure and moderate temperature was needed for significant reduction (B. licheniformis was inactivated at 800 MPa for 5 min at 60°C; B. coagulans was reduced to 10^-4 endospores/mL at 900 MPa for 5 min at 70°C; B. stearothermophilus was inactivated at 70 MPa for 5 min at 70°C).

ALEMAN et al. (1996) studied the effects of pulsed and static HPP on fruit preservation by inactivating Saccharomyces cerevisiae 2407-1a in unsweetened pineapple juice. They applied sinusoidal and step-pressure pulses and compared the inactivation effects with static pressure treatments. They reported that no inactivation was observed after the application of 40-4000 fast sinusoidal pulses (10 cycles/s) at 4-400 s over a pressure range of 235-270 MPa, whereas static pressure treatments of 270 MPa at 40 and 400 s gave 0.7 and 5.1 decimal reductions, respectively. Also, slower 0-270 MPa step pulses at 0.1 (10 pulses), 1 (100 pulses), and 2 (200 pulses) cycles /s with total time of 100 s resulted in 3.3, 3.5, and 3.3 decimal reductions, respectively. They also reported that the ratio of on-pressure time to off-pressure time also affected inactivation (e.g on-pressure time of 0.6 s and off-pressure time of 0.2 s resulted in a 4 decimal reduction in 100 s). They concluded that slower step pulses resulted in increased effectiveness of HPP as more reduction in microbial numbers was observed in less time in step-pressure processing. However, studies need to be done using step-pressure processing for inactivating other microorganisms and documenting their destruction kinetics. More studies are needed along the same lines for inactivation of other baroresistant microbes (e.g., Gram-positive bacteria, spores) using cycled pressure treatment. However, the high resistance of bacterial spores to HPP is still a major outstanding issue and is the subject of a variety of reports (SHIMADA, 1992; CRAWFORD et al., 1996; MAGGI et al., 1996; ROBERTS and HOOVER, 1996).

SHIMADA (1992) reported that the combined treatment of high pressure and alternating current yields lethal damage in E. coli and B. subtilis spores. Earlier, SHIMADA and SHIMAHARA (1985, 1987) found that the exposure of E. coli cells to an alternating current (a.c.) of 50 Hz caused the release of intracellular materials located in the nucleus region within the cells, causing a decrease in the resistance to basic dyes. This was believed to have resulted from loss and (or) denaturation of cellular components responsible for the normal function of the cell membrane, which suggested that the lethal damage to microorganisms may be enhanced when the organisms are exposed to a.c. before or after the pressure-treatment. SHIMADA (1992) subjected the E. coli cells to 300 MPa for 10 min immediately after a.c. exposure and B. subitlis suspension to 400 MPa for 30 min before a.c. exposure. A.C. exposure was carried out @ 0.6 A/cm² for E. coli cells at 35<C for 2 h and @ 1 A/cm² for spores at 50<C for 5 h. They found that the surviving fractions of E. coli cells and B. subtilis spores treated with a.c. and pressure were significantly reduced. It was also found that the susceptibility of E. coli cells and B. subtilis spores to some chemicals increased after the combination treatment, suggesting that the combined use of pressure and a.c. also lowers the tolerance-level of microorganisms to other challenges.

Response to pressure-cycling (KNORR, 1994; HONMA and HAGA, 1991), ultrasound with pressure (KNORR, 1994), and additives plus pressure (KNORR, 1994; PAPINEAU et al., 1991; POPPER and KNORR, 1990) have been studied, and significant interactive effects on microbial inactivation have been found. In addition, sensory and functional characteristics of foods were enhanced. KNORR (1994) reported that neither ultrasonic nor high pressure treatment alone was capable of inactivating Rhodoturola rubra, however, pretreatment of samples with ultrasonic waves (100 W/cm², 25°C, 25 min) followed by HPP (400 MPa, 25°C, 15°C) resulted in complete inactivation of R. rubra. POPPER and KNORR (1990) demonstrated the effectiveness of combinations of enzymes like lysozyme, lactoperoxidase, and glucose oxidase on the inactivation of microbes at atmospheric pressure. It is highly likely that the combination of enzyme pretreatment with HPP may result in significant inactivation of microbes even at low pressures.

The work done in combined pressure-temperature, pressure-a.c. exposure, pressure-cycling, pressure-ultrasound areas is at a preliminary level and the exact mechanisms of spore inactivation by such combined processes are not well known. There is no doubt that combined high pressure and moderate temperature (60-80<C) or high pressure and a.c exposure will have a beneficial impact on the sensory characteristics of heat-sensitive products, yet no such processes can be commercialized until microbial safety can be guaranteed. In most of the studies researchers did not evaluate the effect of pressure-induced "adiabatic" heating on sample temperatures nor did they examine spore inactivation during pressure "come-up" time, both of which may affect inactivation kinetics significantly. Also, the operational efficiency of a high pressure food processor is very important during combined treatments, therefore, proper equipment maintenance and effective training of personnel are prerequisite for such studies. A detailed evaluation of the database accumulated from studies which used a combination of pressure and other treatments (temperature, a.c. exposure, cycling) is needed prior to commercialization of HPP. Also, corresponding D-values for specific microorganisms need to be documented and validated to standardize HPP, before it can be commercialized.

3.3 Protein Denaturation and Enzyme Inactivation

The four levels of protein structure are characterized as: primary (amino acids in a polypeptide chain joined by covalent bonding), secondary (coiling of peptide chains joined with hydrogen bonding), tertiary (arrangement of chains into globular shape by non-covalent bonding), and quaternary (various compact structures or sub-units joined by non-covalent bonding) (LEADLEY and WILLIAMS, 1997; HEREMANS, 1995). The secondary, tertiary, and quaternary structures can be significantly affected by HPP (because high pressure affects non-covalent bonds); therefore HPP can result in novel functional properties because tertiary structure is important in determining protein functionality. Pressures of 700 MPa have been shown to coagulate egg albumen completely (BRIDGMANN, 1914). The amount of pressure required depends upon the type and concentration of protein, pH and ionic strength of treated solutions (LEADLEY and WILLIAMS, 1997). Induction of coagulation and gelation in egg white, yolk, Alaskan pollack paste, rabbit meat paste, and soy protein (HAYASHI, 1989; HAYASHI et al., 1989; OKAMOTO et al., 1990); production of pressure-induced gels in whey protein, egg white, blood plasma, and haemoglobin protein concentrates (VAN CAMP and HUYGHEBAERT, 1995); induction of gelation in different Surimi types (SHOJI et al., 1990); increased elasticity, breaking strength, and syneresis resistance of pressure-treated skimmed milk due to the production of acid-induced gels (JOHNSTON et al., 1992); and increased rates of acid hydrolysis of proteins (HAYASHI et al., 1990) are some of the HPP applications used in the food industry and found to affect protein functionality.

LUDIKHUYZE et al. (1997) determined kinetic parameters of pressure-temperature inactivation of Bacillus subtilis alpha-amylase under dynamic conditions of pressure-cycling. They reported that multiple pressure treatments had a more pronounced effect on the inactivation of Bacillus subtilis alpha-amylase as compared to a single-cycle process. They attributed this result to more extensive and frequent temperature variation during multiple-pressure-cycling.

OHSHIMA et al. (1993) studied the effect of high pressures on fish muscle proteins. Myofibrils were first prepared by removing the water soluble glycolytic enzymes and proteins like collagen. Contractile, regulatory, and elastic structural proteins are the major components of muscle cells and their constituent myofibrils. When pressurized at 150 MPa for 30 min, the microscope revealed that destruction of the arrangement of myofibrils and cohesion of filaments occurred. Treatment at 38<C for 2 h did not revert the change. It was suggested that this denaturation of myofibrils was caused by depolymerization of myofibrillar proteins or aggregation of myosin (HAYASHI et al., 1990). When carp muscle was subjected to pressures of 200, 350 and 500 MPa and stored at 5<C, a delay in the natural decrease of inosine monophosphate (IMP) levels was observed at pressures of 350 and 500 MPa (OHSHIMA et al., 1993). This was important because IMP is responsible for the fresh taste of fish. Thus, it was suggested that the enzymes involved in ATP breakdown undergo denaturation and deactivation. Recently, HAUBEN et al. (1996) studied the effect of high pressure on the disruption of bacterial outer-membrane permeability under pressures of 220-320 MPa. In their experiments, E. coli was found to be sensitive to lysozyme and nisin, which are normally excluded by the outer cell membrane. Also, a periplasmic enzyme, $-galactosidase leaked into the extracellular medium under high pressure. This observed membrane damage was rectified after pressurization, thereby leading the authors to believe that the damage was either spontaneously reversible or repaired by enzymes. Another area that needs to be explored is the effect of high pressure on the constituent lipids and fatty acids of cells. Predictably, it has been reported that lipids in fish muscle are also affected by subjecting them to high pressures due to lipolysis (OSHIMA et al., 1993).

MACDONALD (1992) reviewed the effects of HPP on natural and artificial membranes and reported that pressure increases the order of model and natural lipid bi-layers which diminishes the void volumes between hydrocarbon chains, converts the liquid crystalline state and orthodox bi-layer structure to an ordered gel state and partial interdigitated bi-layer (phase-changes), respectively. Due to sufficient ordering of bi-layer by pressure, the integral and peripheral proteins get detached from the plasma membrane. Also, high pressure (>150 MPa) inactivates the ATP-ase activity, and increases the enzyme’s solvation. Due to membrane permeabilisation, HPP may facilitate the penetration of anti-microbials into vegetative cells.

TIMSON and SHORT (1965) suggested that a possible mechanism for protein denaturation under high pressures in the 345 - 2175 MPa range is ionization and subsequent precipitation of protein complexes. On the application of high pressure the solvation of ions and the ionization of weak electrolytes is increased. At these pressures the weakly charged molecules that are compressed together into the charged ionic groups, increase the total charge. This gives the oppositely charged groups enough energy to combine, which may not happen under normal conditions. Thus ionic bonds are formed decreasing the solubility of protein molecule due to a decrease in the number of free hydrophilic groups. As a result the proteins precipitate from solution. Also, electrostriction has been shown to occur under pressure, corresponding to charge separation and dissociation of ionic interactions. However, this may be reversible when pressure is released. CHEFTEL (1995) indicated that gel networks formed due to pressurization are weaker, if they are mainly comprised of hydrophobic interactions, than those in gels formed at atmospheric pressure. In contrast, gels formed mainly with hydrogen bonds are further reinforced due to pressurization (CHEFTEL, 1995). To fully understand the mechanisms of protein denaturation by high pressure, the effect of HPP on different protein types needs to be studied in depth. The extent of reversibility of denaturation for given pressure conditions also needs to be analyzed.

HPP affects enzymes in a variety of ways depending on other parameters of processing and also the type of enzyme (CHEFTEL, 1995). However, it should be noted that pressure-resistant enzymes can also affect HPP outcomes (ROVERE et al., 1994). Enhanced enzyme activity after HPP may be due from enzyme release from cellular compartments and closer contact with substrates. The latter workers first treated PPO (polyphenol oxidase, which is responsible for browning of fruits) at levels necessary to achieve complete inactivation of microbes. At these levels PPO was not inactivated. Other researchers (ASAKA and HAYASHI, 1991; GOMES and LEDWARD, 1996) also studied the effect of HPP on PPO activity and reported incomplete inhibition of PPO. GOMES and LEDWARD (1996) investigated the effects of pressure treatment (100-800 MPa for 1-20 min) on commercially available mushroom tyrosinase and tyrosinases from mushrooms, potatoes, and apples. They reported that the activity of a commercial PPO extract from mushrooms decreased at a constant rate with applied pressure and time in phosphate buffer at pH 6.5, with complete but reversible inactivation being obtained at 800 MPa for 5 min. The response of PPO extracted from potatoes and mushrooms was different than for commercial PPO. For potatoes, PPO activity was consistently lost with increasing pressure, but after 10 min at 800 MPa, 40% of PPO activity remained. For mushrooms, PPO activity was increased after treatment at 400 MPa for 10 min and even at 800 MPa for 10 min, 60% of PPO remained. They concluded that PPO-types affect the efficacy of HPP in their inactivation, and recommended that HPP should be coupled with other treatments to completely inhibit PPO activity. JAENICK (1991) found that enzyme inactivation under HPP was brought about by intra-molecular structural changes. They also reported that HPP increases reaction rates that involve enzymes. Thus the catalytic behavior of enzymes is affected by the application of pressure and this is probably due to volume changes resulting from changes in protein conformation. It has been found that pressures ranging from 102 to 304 MPa cause reversible denaturation and pressures over 304 MPa result in irreversible denaturation. JOLIBERT et al. (1995) studied the effects of HPP on PPO in fruits (plums, apricots, strawberries, and apples). They found that HPP increased apple-browning, but reduced PPO activity in plums; and suggested that a total inactivation of fruit PPO activity may be achieved at 600 MPa. They reported irreversible inactivation of PPO, which was also dependent on the substrate, fruit species, and pH (maximum inactivation occurred at low pH). They recommended addition of preservatives like sucrose, ethanol, or ascorbic acid to facilitate inhibition of fruit PPO by HPP.

A reduction in pectin esterase (PE) was observed by OGAWA (1992), SEYDERHELM and KNORR (1992), and BERG (1996) but results obtained were not promising as PE showed baroresistance even at high pressures (900 MPa). This resistance increased with an increase in soluble solids (OGAWA, 1992), and high sucrose concentration (SEYDERHELM and KNORR, 1992). PE is an enzyme that is found in citrus juices and can cause a loss of the cloudiness in fresh juices. GOODNER et al. (1998) studied the inactivation of PE in orange and grapefruit juices by high pressure. They reported that isostatic pressure $600 MPa caused substantial inactivation of the heat labile form of PE, whereas the heat stable form was not affected. Treatment times significantly affected total PE inactivation in orange juices (but not in grapefruit juices). They also reported that heat labile grapefruit PE was more sensitive to pressure than orange PE. BASAK and RAMASWAMY (1996) performed a kinetic study on the pectin methyl esterase (PME) during HPP of orange juice and evaluated the effects of pH and soluble solids concentration on PME inactivation. They subjected non-pasteurized and pasteurized orange juice to pressures 100-400 MPa for 0-720 min, and reported that PME inactivation was dependent on the pressure level, holding-time, pH, and total soluble solids. They found primary pressure inactivation to be dependent only on pressure level and secondary inactivation to be dependent on holding-time at each pressure level. ROVERE et al. (1996) also studied the inactivation of PME, peroxidase (POD), and PPO activities by combining pressures up to 100 MPa with temperatures of 20-88°C. They concluded that all of the enzymes could be inactivated using 100 MPa at 50-60°C. SEYDERHELM et al. (1996) studied the effects of HPP on PME, lipase, PPO, lipoxygenase, peroxidase, lactoperoxidase, phosphatase, and catalase, by subjecting samples to a pressure range of 0.1-900 MPa, at 25-60°C for 2-45 min (pH 3-7). They ranked these enzymes based on their pressure-induced inactivation, and reported that a combination of pressure and temperature increased the degree of enzyme inactivation. They also mentioned that actual foods provide a baroprotective layer preventing the inactivation of these enzymes during HPP, and concluded that inactivation of enzymes depends upon immersion medium, pH, temperature, and processing time.

TAMAGAWA et al. (1996) studied the effect of HPP on sterilization, viscosity, and browning of grated yam. They reported complete inactivation of most fungi and Gram-negative bacteria and a decrease in viscosity at pressures > 500 MPa (for 10 min at 20°C). At pressures > 500 MPa, a suppression of browning in yam was observed, suggesting inactivation of PPO by this treatment. Use of HPP for inhibition of proteolysis in milk whey (OKAMOTO and HAYASHI, 1990); protease activities in meat (OHMORI et al., 1991); activity of enzymes like polygalacturonase as well as pectin methylesterase, PPO, and POD (DÖRNENBURG et al., 1996) have also been studied further. Enhanced proteolysis of $-lactoglobin (e.g. trypsin) under pressure has been suggested to be used for preventing the allergenicity of dairy proteins. CASTELLARI et al. (1997) studied the effect of HPP on PPO activity of grape must, and reported that limited PPO inhibition was obtained between 300 and 600 MPa. They suggested that to totally inactivate PPO activity, very high pressures (> 900 MPa) along with mild thermal treatment (40-50°C) should be used. CANO et al. (1997) studied the pressure and temperature effects on inactivation of POD, PPO, and PME enzymes in strawberry and orange products. They subjected the samples to a pressure in the range of 50-400 MPa combined with heat treatment (20-60°C). They found that significant inactivation of strawberry PPO activity (60%) occurred up to 250 MPa and POD activity was reduced by 25% up to 230 MPa. In orange juice, POD and PME activities were reduced by 50% at 35°C. IBARZ et al. (1996) also investigated inhibition of PPO activity in apple slices during HPP. They immersed apple slices in water or ascorbic acid, citric acid, or 4-hexylresorcinol (4-HR) solutions as anti-browning agents at 50 ppm, prior to subjecting them to high pressures in the range of 138 - 690 MPa for 5 min. They did not find any PPO inactivation at 138 MPa (except in slices immersed in 4-HR), and suggested that to inhibit PPO activity, pressures > 276 MPa should be used or prior treatment with 4-HR should be done.

ASHIE and SIMPSON (1996) studied HPP to control enzyme-related fresh seafood texture by subjecting enzyme extracts (chymotrypsin, collagenase, cathepsin C, trypsin) from bluefish and sheephead fishes to pressures of 7-21 MPa. They found that fish enzymes were more sensitive to HPP as compared to their mammalian counterparts. They also concluded that HPP may serve as a tool to control the deterioration of sea food texture. Enzymes, like microorganisms are affected by HPP depending upon the amount of pressure applied, holding time, process temperature, and environmental/substrate conditions (LEADLEY and WILLIAMS, 1997). Nevertheless, enzymes may be inactivated reversibly or permanently depending on the amount and duration of pressure as well as the nature of associated processing conditions. However, more studies need to be done in the enzyme inactivation area using pre treatment with different combinations of processing conditions to optimize HPP and yield foods with excellent quality.

3.4 Food Packaging

The type of food packaging used also plays a very important role in HPP. Two basic requirements that the packaging material needs to possess are the ability to withstand the magnitude of pressure under operating conditions and good heat sealability. Currently, several different types of packaging are in use for HPP, like plastic stomacher bags, sterile tubes, polyester tubes, polyethylene pouches, nylon cast polypropylene pouches, and various other flexible pouch systems. The physical and mechanical properties of the material greatly influence the effectiveness of HPP on the food material. NACHAMANSON (1995) discussed various issues related to packaging materials used in HPP. He stated that the packages must have the ability to prevent any deterioration in the product quality during HPP, and excellent logistics should be applied to distribute the pressure-treated products. Foods to be treated by HPP may be either bulk or individually (consumer) packaged before or after (direct) processing. NACHAMANSON (1995) also stated that the presence of headspace must be kept as small as possible because air and other gases are compressed to ~ zero volume under high pressure, leaving deformation strains on the packages. Therefore, each package should be tested for permissible headspace because headspace cannot be avoided in practical situations. Film barrier properties and structural characteristics of polymer-based packaging material were unaffected when treated at 400 MPa for 30 min at 25°C was reported (NACHAMANSON, 1995).

MASUDA et al. (1992) also studied the effects of HPP on packaging materials for food (mainly gas barrier properties). They examined water vapor and oxygen permeability, tensile strength, and heat seal performance of pressurized gas barrier composite films, as affected by HPP. They reported that pressure treatment of #600 MPa at 40<C for 10 min or 5 pulses of intermittent treatment at 300 MPa, 20<C for 2 min did not affect the barrier properties. No change in the superior aromatic-proof properties of EVOH and PVOH films was observed (400 MPa, 20<C for 10 min). However, they found that voids appeared in composite films, including hydrophilic films (ON, EVOH, PVOH), which they suggested could be avoided by providing thick hydrophobic film-lamination, altering the ethylene content of EVOH, or reducing HPP time. They concluded that the properties of EVOH and PVOH are little affected by HPP and can be easily adapted for HPP to preserve freshness of food. Also, de-lamination of some multilayer composite films has been reported.

3.5 Starch

A few studies (THEVELEIN et al., 1981; MUHR et al., 1982; HAYASHI and HAYASHIDA, 1989; MUHR and BLANSHARD, 1982; EZAKI and HAYASHI, 1992) have been done to examine the effect of HPP on starch. A phenomenon similar to heat gelatinization has been observed with starches. EZAKI and HAYASHI (1992) studied the effect of pressure on 20 starches and compared effects with that of temperature for specific applications. They investigated the pressure effects of starches according to their A, B, or C-type classification (based on X-ray diffrectograms). A-type starches (corn, wheat, rice) were most susceptible to pressure, i.e., enzyme digestibility and swelling degree increased at about 200-400 MPa (>70% digestibility increase was obtained at 500 MPa). B-type starches (potato, marron, lily) were less susceptible to pressure than A-type starches, i.e., enzyme digestibility and swelling degree increased at $500 MPa. C-type starches (sweet potato, tapioca, mung bean) showed intermediate susceptibility between A and B- types. They concluded that the structure of pressure-treated starch was different from heat-gelatinized structure. Pressurization swelled starch granules allowing them to keep their granule like structure and improving enzyme-digestibility and gelatinized structure (without retrogradation).

THEVELEIN et al. (1981) examined the gelatinization temperature of starch under HPP. They reported that the application of high pressure results in an upward shift of gelatinization temperature (highly swollen stage of starch at a particular temperature or temperature at which an equal modulus of elasticity is obtained for gels formed at different pressures) which was

3-5<C/7 MPa. These studies were done on a laboratory scale and give limited insight to HPP of starches. A group of Finnish scientists have been reported to study HPP-induced changes in starch. Further work needed in this area should compare the effectiveness of HPP under different operating conditions, as it is highly probable that starch with unique functional properties can be obtained if treated with high pressures.

3.6 Pressure-Shift Freezing and Thawing

The rates of freezing and thawing influence the quality of food, because they cause changes in the texture and cooking properties believed to result from destruction of membrane structure and changes in the concentration of solutes (BEVILACQUA et al., 1979). Even cryogenic freezing may result in fracture of food products (as volume is decreased during cooling, followed by an increase in volume during freezing) (KALICHEVSKY et al., 1995). High pressure depresses the freezing temperature of water, which means that HPP (200 MPa) can result in unfrozen water existing even at -21<C. However, upon release of pressure, ice-crystals are formed uniformly as the pressure applied is isostatic. Thus, three potential applications of HPP can be envisioned, namely: pressure-shift freezing; super-cooling by storage at sub-freezing temperatures without freezing; and pressure-shift thawing ( LEADLEY and WILLIAMS, 1997). Only a few studies (HAAS et al., 1972; DEUCHI and HAYASHI, 1990, 1991, 1992; KNORR et al., 1998) have been done in this area.

HAAS et al. (1972) investigated the quality of pressure-frozen foods with that of freeze-dried products. They concluded that pressure-freezing followed by air-drying resulted in less textural damage, less shrivelling, more rapid dehydration and more uniform rehydration. DEUCHI and HAYASHI (1992) examined the application of HPP at sub-zero temperatures to preservation, rapid freezing, and rapid thawing of foods. They stored non-cooked foods, microorganisms, and freeze-sensitive microorganisms at temperatures from -5 to 20<C with pressure applied from 50 to 200 MPa for a few days or weeks. They found: strawberries retained fresh flavor, color, and texture; raw pork with no drippings; most microorganisms (coliforms, Enterobacteriaceae, Gram-negative and Gram-positive psychrophiles, enterococci, and LAB) were reduced in number; and freeze-inactivation of enzymes was partially prevented. They reported that storage of foods under moderate pressure at sub-zero temperatures preserved the natural characteristics of foods without microbial spoilage and damage. They also examined the rapid thawing of frozen foods by HPP. During their experiments, they pressurized ice (at -10, -15, -20, and -30<C) from 50 to 200 MPa, and held samples at 5<C for 30 min, and found that all ice was completely thawed. They recommended that rapid thawing of foods is a possible way to avoid qualitative changes, provided processing conditions (temperature, pressure, time) are carefully selected. They also studied the rapid freezing of food using HPP (pressurization up to 200 MPa followed by cooling to -20<C and rapid release of pressure) and found the procedure useful because of uniform formation of small ice-crystals throughout the food material, preserving the sensory characteristics of food.

KNORR et al. (1998) studied the effect of HPP on phase transitions of food, and performed a study of high pressure-freezing and thawing of potato. They designed a high pressure vessel which can withstand sub-zero temperatures, and suggested that due to transient nature of heat transfer, fast removal of latent heat is a major engineering challenge during pressure-freezing and thawing. They further stated that food research and development has until recently neglected taking advantage of the phase diagram of water; understanding phase changes during pressure-assisted freezing and thawing of foods can aid food process and product development.

KANDA and AOKI (1992) used this method to freeze tofu, and found that ice-crystals were uniform, granular, and very small in size. They thawed tofu at room temperature and found that the original shape, structure, taste, and texture of tofu were restored. FUCHIGAMI et al. (1998) studied high pressure-freezing effects on the textural quality and histological structures of Chinese cabbage, by subjecting the cabbage to high pressure freezing (100-700 MPa at -20°C). Freezing of samples at 100 MPa (ice I) and 700 MPa (ice VI) resulted in increased rupture strain, however, texture was comparatively intact at 200 MPa (liquid), 340 MPa (ice III), and 400 MPa (ice V). Also, pectin release and histological damage in samples frozen at 200 and 340 MPa were less than in those frozen at 100 and 700 MPa. They reported an increase in both textural and histological attributes of pressure-frozen samples as compared to results from traditional freezing. More studies are needed in high-pressure freezing and thawing, which may result in an excellent new processing technique for heat-sensitive food materials.

3.7 Meat and Fish Industry

Researchers have studied the application of HPP in the meat industry using several combinations of pressure, time, and temperature (table 2). CARLEZ et al. (1992) studied the effect of high pressure and bacteriostatic agents on the destruction of Citrobacter freundii in minced beef muscles and also gave a pressure-destruction- kinetic equation for further comparison with thermal processing. By analogy with D-values (for thermal destruction), they calculated a decimal reduction time D_{230 MPa/20°C} of 14.7 min, i.e., the period of time at 230 MPa (pressure used in their study) and 20<C required to cause a decimal reduction in surviving microorganisms. They also studied the probable synergy between pressure and bacteriostatic substances (sorbic acid, benzoic acid, and CO₂ as a gas or in a supercritical state), and found little effect on the bacterial destruction. They suggested that essential oils or their constituents could be used as adjuncts for further study. SHIGEHISA et al. (1991) reported complete destruction of Salmonella typhimurium at 300 MPa after 10 min at 25<C. CARLEZ et al. (1993) reported that Citrobacter freundii, Pseudomonas fluorescens, and Listeria innocua were completely inactivated at pressures > 280, 200, and 400 M Pa, respectively at 20°C. They also noticed a paler color in samples of minced beef treated at pressures > 150 MPa, and greyish color in samples at pressures > 350 MPa. In addition, they also calculated decimal reduction times (D) values for all microorganisms tested. When studies generate D-values, then data can further be compared with other work, and eventually yield a data base of D-values (at a constant pressure and temperature) for different microorganisms which will be essential for standardization and commercialization of HPP.

MIYAO et al. (1993) investigated the effects of HPP on microorganisms in surimi paste. All of the microbes were destroyed at 300-400 MPa; fungi showed highest sensitivity to HPP followed by Gram-negative and Gram-positive bacteria. They also identified pressure-resistant bacteria, e.g., Moraxella spp. (viable at 200 MPa); Acinetobacter calcoaceticus (viable at 300 MPa); Streptococcus faecalis (viable at 400 MPa); and Corynebacterium spp. (viable at 600 MPa). They also reported a long lag phase in the growth curve of pressure-treated bacteria as compared to non-treated bacteria (e.g., Streptococcus faecalis subjected to 400 MPa showed a lag phase extended by 20 h). In their study with pressurized minced mackerel meat, FUJI et al. (1994) reported that in pressure-treated samples, Bacillus, Moraxella, Pseudomonas, Flavobacterium spp. were totally inactivated, whereas Staphylococcus and Micrococcus spp. dominated during storage after pressurization. The changes in freshness indicators (pH, amine content, histamine, TBA, lactic acid content) were related to bacterial growth, however, from sensory and freshness points of view, a 4 day increase in shelf life of pressure-treated samples was obtained.

CARLEZ et al. (1994) stored pressurized minced meat samples for 16-23 days at 3°C. Gram-negative bacteria were more sensitive to HPP than Gram-positive types. Total inhibition of microorganisms occurred at 400-450 MPa. However, Pseudomonas spp. were detected after 3-9 days at 3°C, which means that they were not fully inactivated but stressed during HPP. Therefore, HPP should be coupled with some other treatment (e.g., moderate temperature of 50°C) to eliminate viable Pseudomonas spp. In a subsequent study, CARLEZ et al. (1995) investigated the effects of HPP on color and myoglobin content of minced beef samples packaged under vacuum, air, or oxygen. They noticed a pink color of meat treated at 200-350 MPa (increase in L, lightness, color values), which turned grey-brown at 400-500 MPa (a decrease in L values). Also, a decrease in myoglobin content at 200-500 MPa, a decrease in oxymyoglobin, and an increase in metmyoglobin at 400-500 MPa were noticed. They also evaluated the effect of HPP in combination with ascorbic acid, cysteine, nicotinamide, nicotinic acid, sodium nitrite, sodium chloride, and an oxygen scavenger (Ageless FX-100, Mitsubishi France) in vacuum packaged meat to prevent oxidation and yield less discoloration of meat. Only sodium nitrite, sodium chloride, and the oxygen scavenger provided some protection against oxidation. They suggested that meat discoloration during HPP is due to a whitening effect at 200-300 MPa, caused by globin denaturation, haem displacement or release, or oxidation of ferrous myoglobin to ferric myoglobin at 400 MPa. However, there was no significant increase in haem iron extractable during HPP at different levels. They also mentioned that interaction between pressurized meat samples and chemicals from packaging was not taken into account, which may have had a significant effect on meat discoloration. They used only two types of packaging materials, and it could be possible that other types may prevent meat-discoloration. A separate study having packaging material as another variable is needed to examine this possibility. In this study, only one type of oxygen scavenger was tested, and there is significant variation in O₂ absorption rate among individual O₂ scavengers of the same type (TEWARI et al., 1999). Therefore, use of different commercially available oxygen scavengers in multiple numbers, with better O₂-absorbing kinetics might be useful in preventing meat discoloration. Further study should be conducted in this area.

CARPI et al. (1995) inoculated smoked creamed salmon samples with a variety of microorganisms: Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Saccharomyces cerevisiae, Pencillium expansum, Rhizopus oryzae, Clostridium sporogenes, Lactobacillus casei, and Enterobacteriaceae. They reported that an extended shelf-life from 60 to 180 days at 3 or 8°C (without significant chemical, microbiological, or sensory changes) was obtained for high pressure-treated samples. They also suggested that high pressure-treated samples must be stored at temperatures < 3°C to prevent outgrowth of surviving Clostridium botulinum spores. They did not mention the temperature used during HPP, which can have a significant effect on the lethality of any pressurized process. ANANTH et al. (1998) studied the shelf life extension, microbiological safety (Listeria monocytogenes Scott A and Salmonella typhimurium ATCC 13311), and quality of fresh pork loins using HPP. They reported the highest D_{414 MPa/25°C} value of 2.17 min for Listeria monocytogenes and highest D_{414 MPa/2°C} value of 1.48 min for Salmonella typhimurium. They did not find any significant difference in color (L^*, a^*, b^*), texture, and water-holding capacity between pressure-treated ( 414 MPa, 13 min, 25°C) samples and controls. However, pressure-treated samples at low temperature and processing time (414 MPa, 9 min, 2°C) resulted in improved texture as compared to the controls. They reported that the level of psychrotrophs was 5.7 log cfu/g for pressure-treated samples at 25°C as compared with 7.0 log cfu/g for controls. They recommended HPP for extending the shelf life of fresh pork loins, however, they did not report any significant difference in sensory characteristics between pressure-treated pork and controls. They performed a systematic study by calculating D-values and determining processing conditions for pork using HPP. More such studies are needed for other meat products.

EL MOUEFFAK et al. (1996) compared the effects of high pressure (300 and 400 MPa) at 50°C on microbial inactivation of duck foie gras with thermal pasteurization (80°C at coldest spot). They reported a reduction of vegetative mesophilic and psychrotrophic contaminants, destruction of coliforms, and Staphylococcus aureus (similar to pasteurization) in samples treated at 400 MPa for 10 min at 50°C. It is unfortunate that different processing times were not used and that D-values were not calculated, which would have been useful in developing a database for microbial inactivation. O’BRIEN and MARSHALL (1996) studied the microbiological quality of freshly ground raw chicken meat (initial microbial population of 10⁶ cfu/g) sealed in polyfilm pouches using HPP. They reported that application of 408, 616, and 818 MPa at ambient temperature for 10 min resulted in spoilage times of 27, 70, and > 98 days, respectively. They also concluded that facultative anaerobic psychrotrophs like Carnobacterium divergens and Serratia liquefaciens were highly resistant to applied pressures. Again, experiments were not performed at different holding times, preventing calculation of D-values to provide comparison with other similar studies. NISHIWAKI et al. (1996) studied the effects of HPP on Mg-enhanced ATPase activity of rabbit myofibrils, to examine the effect of high pressure on actin/myosin interactions in rabbit meat. They reported similar Mg-enhanced ATPase activity and its sensitivity to ionic strength in myofibrils from pressure treated muscles (up to 200 MPa) and from muscles conditioned for 7 days. A slight increase in ATPase activity was reported when isolated myofibrils were pressure-treated. These changes in ATPase activity may reduce time required for conditioning of muscles. Also, release of soluble material from pressure-treated myofibrils was increased significantly at pressures > 150 MPa, which may further lead to tenderization. BRUNN and SKIBSTED (1996) studied the effects of high pressure on the oxidation of nitrosylmyoglobin (an important pigment in cured meat products). They pressurized model cured meat systems (based on horse nitrosylmyoglobin) up to 300 MPa and reported decreased oxidation of nitrosylmyoglobin with increasing pressure. At 15°C in an air saturated solution (ionic strength 0.16 and pH 6.8), the first order rate constant for nitrosylmyoglobin oxidation was smaller by a factor of 5 at 300 MPa when compared with that at atmospheric pressure and ambient temperature. They explained that the pressure effect on oxidation was due to protein denaturation; and reduced oxidation rates favor the use of HPP with cured meats.

CHEAH and LEDWARD (1996) studied the effects of HPP on lipid oxidation in minced pork, by treating some samples at 800 MPa for 20 min at 20°C and cooking other samples at 80°C (controls were untreated). The samples were stored for 8 days at 4°C. They reported that pressure-treated samples showed faster oxidation than controls, however, a significant increase in the rate of oxidation in minced pork was observed only at pressures > 300 MPa. Also, a significant denaturation of myofibrillar and sarcoplasmic proteins and conversion of reduced myoglobin/oxymyoglobin to the oxidized ferric form were also observed at pressures > 400 MPa. In a subsequent study, CHEAH and LEDWARD (1997) also studied the effect of HPP on inhibition of metmyoglobin formation in fresh beef longissimus dorsi and psoas major muscles. The samples were treated at 80-100 MPa for 20 min at 2 or 20 days post mortem, and it was found that during early post mortem, color stability was improved, (a greater effect was observed on longissimus dorsi than psoas major muscles), whereas little effect on color stability was found during latter post mortem period. This is an important study which may lead to the solution of transient discoloration problem in centrally prepared retail beef cuts packaged under controlled atmosphere of 100% CO₂/N₂, due to the presence of residual oxygen. However, a study using modified atmosphere packaging (high anoxic packaging) coupled with pressure treatment to reduce discoloration of beef has not been documented.

PAUL et al. (1997) studied the effect of HPP, gamma-irradiation, and combined treatments on the microbiological quality of lamb meat during chilled storage. Gamma-irradiation (1.0 kGy) or pressure (200 MPa for 30 min at 30°C) or a combination of both was used to examine the shelf life of minced lamb meat after subsequent storage at 0-3°C. All coliforms were inactivated by either of these two treatments, whereas Staphylococcus spp. showed a reduction of only 1 log cycle when treated with irradation or pressure alone. In contrast, a combination treatment resulted in complete inactivation of Staphylococcus spp. (initial number in the untreated sample was 10⁴ cfu/g) in samples immediately after the treatment. Also, low number of injured cells were recovered only after 3 weeks of storage following the combined treatment which were not present in the samples treated with irradiation or pressure alone. This suggests an interactive effect against Staphylococcus spp. However, it should be noted that Staphylococcus spp. do not grow at temperatures < 9°C, therefore the non-permissive storage temperature used following treatment could have provided opportunity for organisms to recover from injury sustained. Additionally, their study was restricted to one level of irradiation and pressure treatment. It is likely that with some other levels of treatments total inhibition of Staphylococcus spp. may be obtained, and calculation of D-values for these organisms would have been possible. CHEFTEL and CULIOLI (1997) reviewed the effects of HPP (100-800 MPa) on different components and quality attributes of meat. A detailed explanation and review of research done on various aspects (meat enzymes, meat structures, isolated myofibrillar proteins, meat texture, pressure-induced gelation, meat myoglobin, meat lipids, meat microorganisms, as well as sub-zero processing of meat) of pressurized meat is given. However, they did not discuss the effect of combined HPP and gamma-irradiation on the microbiological quality of meat. There was also no attempt to generate D-values for HPP treatment of meat.

TAKAHASHI and HAGA (1997) studied the HPP of uncooked pickle-cured and fermented hams by monitoring changes in microbial growth, color, and composition of hams. They injected porcine longissimus thoracis muscle with Lactobacillus SK-1001 (10⁶ cells/g) which was cured (3% salt) and fermented for 1 week at 5°C and then samples were subjected to a pressure treatment. They reported reduction in the survival of staphylococci and coliforms in pressure-treated fermented hams after pressure-treatment as compared to untreated ones, however, there was no significant change in the number of lactic acid bacteria within 25 min after pressurization and 1 week later. Also, a significant increase in pH and a color change was noticed in the pressure-treated fermented hams (L* and H-0 values increased and a*, b*, and c* values decreased).

Acceleration of changes which take place during ageing, better meat tenderization (SUZUKI et al., 1992; MACFARLENE, 1985); coagulation of pork slurries (SHIGEHISA et al., 1991); increase in the tensile strength of beef patties (MACFARLENE et al., 1984); changes in color and myoglobin content of minced beef (CHEFTEL, 1996) are other effects induced by HPP treatment that may have further application in the meat processing industry.

3.8 Dairy and Egg Industry

High pressure processing may also have application in the dairy and egg industries due to changes induced in the functional properties of whey protein, as well as in other milk components and native constituents (table 3). DUMAY et al. (1994) investigated the effects of HPP on the unfolding and aggregation of an industrial beta-lactoglobulin ($-LG) protein isolate prepared from sweet or mixed whey. For processing, $-LG solutions having pH of 7.5 with 0-5.0 % sucrose (a baroprotectant) were prepared. Significant unfolding of the proteins occurred as )H (residual enthalpy of denaturation) was decreased by 44 or 54 % when the 2.5 or 5.0 % protein solutions were pressure-processed at 0 % sucrose, respectively. However, $-LG remained soluble. The solubility of $-LG in 2M ammonium sulphate decreased due to pressure-induced protein aggregation. A partial reversibility of pressure-induced unfolding and aggregation were obtained on storage for 7 days at 4°C. It was also found that the presence of 2.5 or 5.0 % sucrose reduced $-LG unfolding and slightly increased the recovery of protein solubility in 2M ammonium sulphate. GALAZKA et al. (1995) studied the effect of HPP on the emulsifying behavior of whey protein concentrate (WPC). The pressure was applied to the protein before homogenization or to the emulsion prepared with native WPC. Functional properties of WPC were examined along with the relationship between stability of WPC emulsions and degree of adsorption of the protein emulsifier. They found that oil-in-water emulsions (0.4 wt.% protein, 20 vol.% n-tetradecane, pH 7) prepared with pressure-treated WPC solutions gave a broader droplet size distribution than emulsions made with native untreated protein. An inverse relationship was obtained between emulsifying efficiency and applied pressure plus treatment time. Also, HPP had little effect on the stability of WPC emulsions made with native protein.

LOPEZ et al. (1996) studied the effects of HPP on whey protein denaturation and cheese-making properties of raw milk. They reported that high pressure slightly improved the microbiological quality of milk without modifying lactoperoxidase activity (a native milk enzyme). $-lactoglobulin was denatured by pressures > 100 MPa, whereas, "-lactalbumin and bovine serum albumin were pressure-resistant (400 MPa for 60 min). An increase in cheese yield was found (at 300 and 400 MPa) in conjunction with additional $-lactoglobulin and moisture retention. They concluded that HPP can improve the coagulation properties of milk and can increase moisture retention of fresh cheese. BUCHHEIM and FREDE (1996) investigated the effect of HPP on the crystallization of emulsified fats, using model emulsions (ultra high temperature, UHT-treated, whipped cream and coffee cream, having 30 and 10% fats, respectively). They found that pressure-treatment resulted in higher content of solid fats, and this result was also affected by pretreatment tempering at 37 or 60°C and the average fat globule size. They also demonstrated combination of pressure and temperature used during HPP of dairy products, which may be used for the optimization of HPP control unit (pressure-temperature-time) parameters. Their study was geared towards standardization of HPP for improvement in the functional characteristics of dairy fats and they were able to describe a relationship between pressure-temperature-time, which may lead to a better design of high pressure food processors exclusively used for accelerating crystallization processes for emulsified fats, and permit development of other value-added dairy products. PITTIA et al. (1996) investigated changes in structure and surface properties of $-lactoglobulin by pressurizing 0.25% (w/v) $-lactoglobulin to 300 MPa for 10 or 30 min; 600 MPa for 10 or 15 min; and 900 MPa for 5 or 10 min. Pressure-treated $-lactoglobulin showed reduced emulsifying capacity and foamability compared to untreated controls, whereas the capacity for protein-protein interaction in the adsorbed layers at interfaces increased (due to an increased surface-dilational modulus and resistance to displacement by a surfactant in foams). They reported that there was a pressure-induced structural change in $-lactoglubulin which increased its hydrophobic characteristics and aggregate formation potential (this accounted for reduced emulsifying capacity and foamability).

CAPELLAS et al. (1996) investigated the effects of HPP on the populations of aerobic mesophiles and inoculated E. coli during storage of fresh goat milk cheese. They manufactured pasteurized goat-milk cheese containing an added E. coli (strain 405 CECT) population of 10⁸ cfu/g. After pressure treatment, samples were stored at 2 to 4°C. Microbial numbers were determined at 1, 15, 30, and 60 days after treatment. No colonies of E. coli were detected throughout storage and low numbers (2-3 log cfu/g) of aerobic mesophilic bacteria were consistently present. In their experiments, only three holding-time periods were used and unfortunately none of these was < 5 min. This information would have been valuable from an industry perspective where processing that require only a couple of minutes for complete destruction of microorganisms is attractive. GERVILLA et al. (1997a) studied the effect of HPP on Listeria innocua 910 CECT inoculated into ewe’s milk. Interestingly enough, at a temperature of 2°C, higher inactivation of Listeria innocua occurred than at room temperature when pressures between 450 and 500 MPa for 10 to 15 min were used. They reported first order destruction kinetics for Listeria innocua, and calculated D-values of 3.12 min at 2°C (400 MPa) and 4 min at 25°C (400 MPa). They commented that D-values using thermal processing at temperatures between 64 and 71.7°C are in the range of 0.95-0.09 s, which are much lower than D-values calculated using HPP. They noted that HPP generates reduced physicochemical modifications in milk compared with thermal processing, and that HPP at low temperatures can be used for significant inactivation of L. innocua. They also concluded that although fat has a thermoprotective effect, which is more dominant in ewe’s milk than cow’s or goat’s milk, a significant reduction (7.7 log units at 400 MPa for 20 min at 2°C) in L. innocua was obtained using HPP. They recommended further studies be done to examine the baroprotective effects of ewe’s milk on other microorganisms. This study was also aimed at determining D-values for inactivation of Listeria innocua using HPP, which is a requirement if HPP is to be adopted by the food industry.

WALKENSTROM and HERMANSSON (1997) studied the microstructural (using light and transmission electron microscopy) and rheological (using dynamic oscillatory measurements and tensile tests) properties of high pressure-treated mixed and pure gels of gelatin and WPC at pH 7.5 and 5.4. They reported that pressure-treated pure WPC gels had a higher degree of aggregation than conventionally heat-treated WPC gels but gelatin gels remained unaffected by HPP. Further, the rheological properties of pressure-treated mixed gels indicated a higher degree of gelatin continuity than heat-treated mixed gels at pH 7.5. In contrast, at pH 5.4 the high pressure-treated mixed gels formed a phase-separated network with a gelatin continuous phase and a discontinuous WPC phase. Not surprisingly, the rheological properties of mixed gels were the same as that of pure gelatin (independent of WPC). DRAKE et al. (1997) studied the effect of cycled high pressure treatment of milk on the microbiological and sensory characteristics of Cheddar cheese. They reported the same flavor scores for pressurized and pasteurized milk cheeses, but higher moisture and wet weight yields for pressurized milk cheese were associated with some texture defects. Nevertheless, they recommended HPP as an alternative to thermal pasteurization before cheese-making, but they did not examine functional changes (protein denaturation) in pressurized milk cheese which might affect its market value. Also, a comparison between the quality of static pressure-treated and cycled pressure-treated milk was not done. GAUCHERON et al. (1997) investigated the combined effects of HPP on the physicochemical characteristics (lightness, Ca and P content, casein micelles, exposure of hydrophobic regions of milk proteins, and particle size) of skim milk processed with a holding-time of 10 min. They reported that HPP produced an irreversible disintegration of casein molecules into smaller particles and caused an increase in protein hydrophobicity, casein micelle hydration, P and Ca solubilization, and $-LG denaturation.

FELIPE et al. (1997) compared the effects of HPP and thermal pasteurization on whey protein in goat’s milk. They examined denaturation of the individual whey proteins using gel permeation FPLC (fast protein liquid chromatography). They found rapid aggregation of $-LG and precipitation of disulphide- linked immunoglobulins and proteins upon pressure treatment at 25°C. The denaturation of WPC was affected differently by pressure and thermal pasteurization, while alkaline phosphatase activity in goat’s milk was reduced or remained unaffected by thermal pasteurization and pressure, respectively. ADAPA et al. (1997) determined the effect of HPP on the functional properties of skim milk (9% solids) due to HPP, by subjecting concentrated (18% solids, using ultra-filtration) and unconcentrated milk to a pressure of 310 MPa for 0.3 s. This was followed by chilled storage of samples at 4°C. Stable foams and emulsions were produced only by pressurized milk, which was also more viscous. Also, L^* (lightness), a^* (red/green), and b^* (blue/yellow) values were lower in pressurized unconcentrated milk samples than controls. Creamers were made with pressurized milk and samples were reported to be stable but, their study was limited to only one level of pressure treatment. ERKMEN and KARATAS (1997) studied the effect of HPP on Staphylococcus aureus (ATCC 27690) in milk at pressures in the range of 50-350 MPa for up to 12 min at constant temperature (20±2°C). They found no survival at pressure treatments of 350 MPa for 6 min and 300 MPa for 8 min. Also, D-values were reported as 211.8, 15.0, 3.7, and 2.6 min at 200, 250, 300, and 350 MPa, respectively. While these data are valuable it was unfortunate that the study was limited to use of only one temperature. Other temperatures (< 20 or > 20°C) may have resulted in total inhibition at lower pressures. GERVILLA et al. (1997b) investigated the effects of HPP on Escherichia coli 405 CECTand Pseudomonas fluorescens 378 CECT strains in ovine milk by subjecting inoculated milk samples to different combinations of pressure, temperature, and time. Temperature played a very important role in inactivation of microbes as > 6 log cfu/ml reduction in microbial populations was observed at 50°C for all pressure-time combinations. They reported that the test strain of Escherichia coli was more pressure-resistant than the Pseudomonas fluorescens strain. However, they did not calculate D-values for HPP of microbial inactivation in ovine milk. Also, if shorter holding-time levels (< 5 min) had been used, these would have been useful in determining the effects of "instant" treatment at high isostatic pressure. It is possible that most microbial inactivation occurs during the "come-up" time of pressurization. Their research also did not determine the influence of adiabatic heating (resulting from pressure-application), which may have affected their results.

PONCE et al. (1998) studied the inactivation of Listeria innocua inoculated in liquid whole egg using HPP by subjecting the food to different combinations of pressures, temperature, and time. Total inhibition of Listeria innocua was not reported, however reduction was > 5 log at 2°C for 15 min (450 MPa). D-values for Listeria innocua were reported as 7.35 min at 2°C (400 MPa) and 8.23 min at 20°C (400 MPa). The upper temperature limit used was 20°C, and it is possible that combinations of higher temperature and pressure (e.g., 40°C and 450 MPa) might have resulted in total inhibition of the organism. However, their approach is valuable because it documents D-values during HPP of liquid whole eggs. They also reported that the reduction of Listeria innocua was a little greater at 2 and -15°C than at room temperature (at pressures of 300 and 350 MPa). They explained this behavior as being due to the greater susceptibility of some proteins to denaturation at low temperatures. This study is one of the very few that used HPP at sub-zero temperatures. They also recommended work be done to pressure-treat Salmonella-inoculated egg, since these organisms frequently contaminate egg products. The effects of different components of liquid eggs which might act as baroprotective agents for microorganisms need to be studied in greater depth.

3.9 Cell Sensitiser

Results indicate that high pressure treatment can be an effective antimicrobial process, enhancing the lethality of parallel physical and chemical treatments, thereby increasing the overall efficacy of preservation processes. EARNSHAW (1992) reviewed the potential applications of HPP as a cell sensitiser and mentioned that high pressure damages cell membrane structure and results in cytoplasmic leakage even at 101 MPa. NACHMANSON (1995) discussed synergistic effects of HPP and modified atmospheres as well as HPP and packaging material containing zeolite (which can be used in packaging material to inhibit the growth of microorganisms). He reported a large reduction in viable numbers of yeasts (Saccharomyces cerevisae and Candida tropicalis) following treatment at 3-400 MPa when the headspace was filled with CO₂. When treatment was done using packaging material containing zeolite there was a significant reduction of the microbial population. When the two procedures were used separately the results were relatively poor. Application of pressure with natural antimicrobials (food acids, herbs and essential oils, bacteriocins), synthetic chemicals, or modified gas atmospheres may yield another dimension for fresh food preservation, by erecting a series of hurdles preventing growth by spoilage and pathogenic bacteria at levels of individual agent intervention that leave the food essentially unaffected (EARNSHAW, 1992).

4. SUMMARY AND FUTURE RESEARCH NEEDS

There is no doubt that HPP represents another interesting and promising dimension for food processing not only because of it inactivates microorganisms but also because it provides opportunities for development of new "value-added" food products. The need for an alternative to thermal processing as the primary means of eliminating pathogenic and spoilage microorganisms is substantial. High pressure processing holds promise since food materials treated by this method retain their natural flavor, color, and texture without loss in vitamin or nutrient content. Furthermore, predictable changes in functional characteristics of proteins and complex carbohydrates (where little work has been done), mean that there are some exciting avenues of work in HPP treatment of foods that remain to be explored. Although a lot of research has been conducted in the area of high pressure processing, a lot remains to be done in terms of understanding the critical limits of the process and the extent to which this might ensure appropriate treatment of food materials. Research has been done in various research institutions, universities, and food industry R&D laboratories, yet a direct comparison of the data obtained is not possible. There can be no possibility of extensive implementation of this technology unless specific processing parameters are established for each food material treated by HPP. The possibility of commercialization of HPP also depends on its economic viability. Therefore, a detailed economic analysis of HPP needs to be done by comparing the process costs with the present costs of processing by conventional means and at the same time factoring in the added value of the improved sensory characteristics of HPP-treated foods. Some indications on investment and operational costs of HPP are available from the machine manufacturers, although perhaps not fully reliable.

Thus far no concerted effort has been made to validate HPP procedures for complete elimination of pathogenic bacteria. It is important that standard operating criteria and processing conditions be established that will ensure the reliability of HPP as an alternative to thermal processing. For example, thermal processing has had standards like D- and Z- values, holding- times, and minimum temperatures developed for different foods; likewise, standards need to be set for HPP. Very few studies on the HPP inactivation of microorganisms discuss HPP effects in terms of the D-value associated with each particular strain of organism (table 4). More research is needed along these lines so that a database of HPP D-values for different microorganisms can be prepared. Without a well-documented database of D-values, there is no means to effectively compare the results of experiments performed with different microorganisms under different processing conditions. There is a need for comparable standards that might indicate the resistance of various microorganisms to pressure and temperature combinations used in HPP. Different D values at constant values of temperature and pressure may be used as an indication of resistance of a particular bacterial species with respect to another species. Thus, an effort should be made to understand and derive both D-values and Z-values so that minimum processing conditions may be developed for contaminants in target foods. Since the general norm followed in the food industry is to subject food products to treatments that ensure a 12D reduction of microorganisms, to ensure that HPP is effective in inactivating microorganisms, treatments that result in 12D reduction must be another goal. Perhaps it is not surprising that no database of D and Z values for microbial species has been developed for HPP. It must be kept in mind that these parameters have been derived for temperature and time effects together without regard to pressures. This, therefore does not mean that even if we have these values for various microorganisms, we can predict the precise parameters of pressure, temperature, and time required to eliminate the bacteria completely from the food by HPP. To fully validate HPP, a relationship has to be developed between the pressure of processing and the reduction in microbial population under treatment over a fixed time and temperature. The methods adopted to derive this parameter may be different but the end result must provide an effective means of predicting bacterial reductions due to HPP.

During high isostatic pressure processing, the sample is not immediately subjected to target temperature, time or processing pressure. When a process is started, there is usually a finite period of time before the pressure in the chamber reaches the set point from the initial value. The sample is therefore subjected to a preliminary rise in pressure for a finite period (come-up time), in addition to the treatment at the set point. Temperature is another factor that is influenced by this rise in pressure and which influences the extent of treatment received by the sample. Whenever the pressure within the high pressure chamber rises it is accompanied by a corresponding rise in the temperature of the chamber. This rise in temperature is known as adiabatic heating, which can result in the sample being subjected to higher temperatures than originally set for during a process. In most of the research reviewed, adiabatic heating has not been taken into consideration, which generates uncertainty and complicates standardization of the associated thermal treatment effects. Research is being done at The National Center for Food Safety and Technology, Summit-Argo, IL (USA), to incorporate adiabatic heating and pressure come-up time during experimental execution. Recently, WEEMAES et al. (1998) were able to avoid problems due to adiabatic heating during pressure build-up by taking a blank measurement (enzymatic activity when entering a constant pressure phase, A₀, at t=0 min) 3 min after desired pressure was achieved, to exclude the non-isobaric/non-isothermal phase from their experiments. They also reported the temperature profiles in the pressure vessel during pressure build-up and subsequent holding phase. In a similar manner, adiabatic heating should be addressed while performing microbial inactivation studies using HPP.

The effects of come-up time on the viability of microorganisms may be significant. Therefore, during studies related to HPP, it should be imperative that overall destruction of microorganisms during total processing time (holding time+come-up time) and the proportion of microorganisms destroyed during come-up time should be calculated separately, by proper experimental execution. To quantify microbial inactivation during pressure come-up time, experiments could be performed while holding-time is still at zero. Recently, PALOU et al. (1998) studied the effect of come-up time at pressures in the range of 50-689 MPa on Saccharomyces cerevisiae and Zygosaccharomyces bailii grown at 21°C, and reported significant effects of pressure come-up times on the fraction of yeast surviving. They also mentioned that the pressure build-up velocity (or come-up time), which is usually neglected during HPP, substantially reduces the microbial population. Therefore, it is imperative that rate of pressure compression and decompression should be reported in the studies as well as microbial inactivation prior to pressure stabilization. They also suggested using the Fermi equation (Eq.1) to model pressure as the lethal agent, which can allow comparison between pressure responses at different growth phases, a_w, and among different bacterial species. It is to be noted that the concept is not completely consistent with the D-value concept as in thermal processing, due to the dependence of D on time.

(1) where
S(P)= fraction of surviving microorganisms,
P = pressure applied (MPa),
Pc= critical P level, where surviving fraction is 0.5, and
k= constant (MPa) representing the steepness of the survival curve around Pc.

As adiabatic heating also affects the performance of HPP, the following points should always be taken into account while planning experiments.

(i) Initial temperature of the sample: different baseline control experiments should be performed to monitor the change in temperature of the sample from the desired temperature at that point where the test pressure is reached initially,

(ii) Temperature of the circulating water: in most high pressure processors, water is circulated around the pressure chamber to control the chamber temperature, and this affects the sample temperature during processing. This should also be carefully monitored and documented for different experiments,

(iii) Holding time: a series of baseline control experiments should always be done to allow for equipment calibration and facilitate use of proper combination of initial temperature and holding- time, so that the desired operating conditions are met and come-up times are calculable separately,

(iv) Sample temperature during processing: sample temperature should be carefully monitored and documented during the high pressure holding-time, and proper care should be taken to avoid significant variation in temperatures during HPP.

Remaining questions are variability of pressure resistance between different strains of same microbial species; possible protecting effects of food constituents, making it important to study microbial inactivation in given foods; stressed cells, and their possible recovery during chilled or ambient storage; effects of high pressure and sub-zero temperature processing, effects of pressure-cycling and pressure-a.c. exposure treatments to reduce the microbial-viability. An exciting aspect of this technology is its effect on different functional characteristics of foods (starch gelatinization, protein denaturation, enzyme inactivation). Not only do these areas require further study but also HPP as a method of storage by freezing foods (pressure-shift freezing, rapid thawing, rapid cooling) requires further work. From the methodological standpoint, it would be necessary to monitor temperatures in the food sample, and pressure at different positions in the HP vessel, and to use well-calibrated two pressure gauges. Until today, high pressure food processors are available for batch and/or semi-continuous processes. Developing a continuous high pressure food processor still remains an engineering challenge.

Although HPP shows promise in its ultimate usefulness for food processing, limitations with respect to difficulty in data comparison and complexity associated with understanding interactive components of the process currently limit full acceptance of the practice.

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