January 26, 2007
Opportunities and Challenges in High Pressure Processing of Foods
By Rastogi, N K; Raghavarao, K S M S; Balasubramaniam, V M; Niranjan, K; Knorr, D
Consumers increasingly demand convenience foods of the highest quality in terms of natural flavor and taste, and which are free from additives and preservatives. This demand has triggered the need for the development of a number of nonthermal approaches to food processing, of which high-pressure technology has proven to be very valuable. A number of recent publications have demonstrated novel and diverse uses of this technology. Its novel features, which include destruction of microorganisms at room temperature or lower, have made the technology commercially attractive. Enzymes and even spore forming bacteria can be inactivated by the application of pressure-thermal combinations, This review aims to identify the opportunities and challenges associated with this technology. In addition to discussing the effects of high pressure on food components, this review covers the combined effects of high pressure processing with: gamma irradiation, alternating current, ultrasound, and carbon dioxide or anti-microbial treatment. Further, the applications of this technology in various sectors-fruits and vegetables, dairy, and meat processing-have been dealt with extensively. The integration of high-pressure with other matured processing operations such as blanching, dehydration, osmotic dehydration, rehydration, frying, freezing / thawing and solid- liquid extraction has been shown to open up new processing options. The key challenges identified include: heat transfer problems and resulting non-uniformity in processing, obtaining reliable and reproducible data for process validation, lack of detailed knowledge about the interaction between high pressure, and a number of food constituents, packaging and statutory issues.
Keywords high pressure, food processing, non-thermal processing
Consumers demand high quality and convenient products with natural flavor and taste, and greatly appreciate the fresh appearance of minimally processed food. Besides, they look for safe and natural products without additives such as preservatives and humectants. In order to harmonize or blend all these demands without compromising the safety of the products, it is necessary to implement newer preservation technologies in the food industry. Although the fact that "high pressure kills microorganisms and preserves food" was discovered way back in 1899 and has been used with success in chemical, ceramic, carbon allotropy, steel/alloy, composite materials and plastic industries for decades, it was only in late 1980's that its commercial benefits became available to the food processing industries. High pressure processing (HPP) is similar in concept to cold isostatic pressing of metals and ceramics, except that it demands much higher pressures, faster cycling, high capacity, and sanitation (Zimmerman and Bergman, 1993; Mertens and Deplace, 1993). Hite (1899) investigated the application of high pressure as a means of preserving milk, and later extended the study to preserve fruits and vegetables (Hite, Giddings, and Weakly, 1914). It then took almost eighty years for Japan to re- discover the application of high-pressure in food processing. The use of this technology has come about so quickly that it took only three years for two Japanese companies to launch products, which were processed using this technology. The ability of high pressure to inactivate microorganisms and spoilage catalyzing enzymes, whilst retaining other quality attributes, has encouraged Japanese and American food companies to introduce high pressure processed foods in the market (Mermelstein, 1997; Hendrickx, Ludikhuyze, Broeck, and Weemaes, 1998). The first high pressure processed foods were introduced to the Japanese market in 1990 by Meidi-ya, who have been marketing a line of jams, jellies, and sauces packaged and processed without application of heat (Thakur and Nelson, 1998). Other products include fruit preparations, fruit juices, rice cakes, and raw squid in Japan; fruit juices, especially apple and orange juice, in France and Portugal; and guacamole and oysters in the USA (Hugas, Garcia, and Monfort, 2002). In addition to food preservation, high- pressure treatment can result in food products acquiring novel structure and texture, and hence can be used to develop new products (Hayashi, 1990) or increase the functionality of certain ingredients. Depending on the operating parameters and the scale of operation, the cost of highpressure treatment is typically around US$ 0.05-0.5 per liter or kilogram, the lower value being comparable to the cost of thermal processing (Thakur and Nelson, 1998; Balasubramaniam, 2003).
The non-availability of suitable equipment encumbered early applications of high pressure. However, recent progress in equipment design has ensured worldwide recognition of the potential for such a technology in food processing (Could, 1995; Galazka and Ledward, 1995; Balci and Wilbey, 1999). Today, high-pressure technology is acknowledged to have the promise of producing a very wide range of products, whilst simultaneously showing potential for creating a new generation of value added foods. In general, high-pressure technology can supplement conventional thermal processing for reducing microbial load, or substitute the use of chemical preservatives (Rastogi, Subramanian, and Raghavarao, 1994).
Over the past two decades, this technology has attracted considerable research attention, mainly relating to: i) the extension of keeping quality (Cheftel, 1995; Farkas and Hoover, 2001), ii) changing the physical and functional properties of food systems (Cheftel, 1992), and iii) exploiting the anomalous phase transitions of water under extreme pressures, e.g. lowering of freezing point with increasing pressures (Kalichevsky, Knorr, and Lillford, 1995; Knorr, Schlueter, and Heinz, 1998). The key advantages of this technology can be summarized as follows:
1. it enables food processing at ambient temperature or even lower temperatures;
2. it enables instant transmittance of pressure throughout the system, irrespective of size and geometry, thereby making size reduction optional, which can be a great advantage;
3. it causes microbial death whilst virtually eliminating heat damage and the use of chemical preservatives/additives, thereby leading to improvements in the overall quality of foods; and
4. it can be used to create ingredients with novel functional properties.
The effect of high pressure on microorganisms and proteins/ enzymes was observed to be similar to that of high temperature. As mentioned above, high pressure processing enables transmittance of pressure rapidly and uniformly throughout the food. Consequently, the problems of spatial variations in preservation treatments associated with heat, microwave, or radiation penetration are not evident in pressure-processed products. The application of high pressure increases the temperature of the liquid component of the food by approximately 3C per 100 MPa. If the food contains a significant amount of fat, such as butter or cream, the temperature rise is greater (8-9C/100 MPa) (Rasanayagam, Balasubramaniam, Ting, Sizer, Bush, and Anderson, 2003). Foods cool down to their original temperature on decompression if no heat is lost to (or gained from) the walls of the pressure vessel during the holding stage. The temperature distribution during the pressure-holding period can change depending on heat transfer across the walls of the pressure vessel, which must be held at the desired temperature for achieving truly isothermal conditions. In the case of some proteins, a gel is formed when the rate of compression is slow, whereas a precipitate is formed when the rate is fast. High pressure can cause structural changes in structurally fragile foods containing entrapped air such as strawberries or lettuce. Cell deformation and cell damage can result in softening and cell serum loss. Compression may also shift the pH depending on the imposed pressure. Heremans (1995) indicated a lowering of pH in apple juice by 0.2 units per 100 MPa increase in pressure. In combined thermal and pressure treatment processes, Meyer (2000) proposed that the heat of compression could be used effectively, since the temperature of the product can be raised from 70-90C to 105-120C by a compression to 700 MPa, and brought back to the initial temperature by decompression.
As a thermodynamic parameter, pressure has far-reaching effects on the conformation of macromolecules, the transition temperature of lipids and water, and a number of chemical reactions (Cheftel, 1992; Tauscher, 1995). Phenomena that are accompanied by a decrease in volume are enhanced by pressure, and vice-versa (principle of Le Chatelier). Thus, under pressure, reaction equilibriums are shifted towards the most compact state, and the reaction rate constant is increased or decreased, depending on whether the "activation volume" of the reaction (i.e. volume of the activation complex less volume of reactants) is negative or positive. It is likely that pressure a\lso inhibits the availability of the activation energy required for some reactions, by affecting some other energy releasing enzymatic reactions (Farr, 1990). The compression energy of 1 litre of water at 400 MPa is 19.2 kJ, as compared to 20.9 kJ for heating 1 litre of water from 20 to 25C. The low energy levels involved in pressure processing may explain why covalent bonds of food constituents are usually less affected than weak interactions. Pressure can influence most biochemical reactions, since they often involve change in volume. High pressure controls certain enzymatic reactions. The effect of high pressure on protein/enzyme is reversible unlike temperature, in the range 100-400 MPa and is probably due to conformational changes and sub-unit dissociation and association process (Morild, 1981).
For both the pasteurization and sterilization processes, a combined treatment of high pressure and temperature are frequently considered to be most appropriate (Farr, 1990; Patterson, Quinn, Simpson, and Gilmour, 1995). Vegetative cells, including yeast and moulds, are pressure sensitive, i.e. they can be inactivated by pressures of ~300-600 MPa (Knorr, 1995; Patterson, Quinn, Simpson, and Gilmour, 1995). At high pressures, microbial death is considered to be due to permeabilization of cell membrane. For instance, it was observed that in the case of Saccharomyces cerevasia, at pressures of about 400 MPa, the structure and cytoplasmic organelles were grossly deformed and large quantities of intracellular material leaked out, while at 500 MPa, the nucleus could no longer be recognized, and a loss of intracellular material was almost complete (Farr, 1990). Changes that are induced in the cell morphology of the microorganisms are reversible at low pressures, but irreversible at higher pressures where microbial death occurs due to permeabilization of the cell membrane. An increase in process temperature above ambient temperature, and to a lesser extent, a decrease below ambient temperature, increases the inactivation rates of microorganisms during high pressure processing. Temperatures in the range 45 to 50C appear to increase the rate of inactivation of pathogens and spoilage microorganisms. Preservation of acid foods (pH ≤ 4.6) is, therefore, the most obvious application of HPP as such. Moreover, pasteurization can be performed even under chilled conditions for heat sensitive products. Low temperature processing can help to retain nutritional quality and functionality of raw materials treated and could allow maintenance of low temperature during post harvest treatment, processing, storage, transportation, and distribution periods of the life cycle of the food system (Knorr, 1995).
Bacterial spores are highly pressure resistant, since pressures exceeding 1200 MPa may be needed for their inactivation (Knorr, 1995). The initiation of germination or inhibition of germinated bacterial spores and inactivation of piezo-resistive microorganisms can be achieved in combination with moderate heating or other pretreatments such as ultrasound. Process temperature in the range 90-121C in conjunction with pressures of 500-800 MPa have been used to inactivate spores forming bacteria such as Clostridium botulinum. Thus, sterilization of low-acid foods (pH > 4.6), will most probably rely on a combination of high pressure and other forms of relatively mild treatments.
High-pressure application leads to the effective reduction of the activity of food quality related enzymes (oxidases), which ensures high quality and shelf stable products. Sometimes, food constituents offer piezo-resistance to enzymes. Further, high pressure affects only non-covalent bonds (hydrogen, ionic, and hydrophobic bonds), causes unfolding of protein chains, and has little effect on chemical constituents associated with desirable food qualities such as flavor, color, or nutritional content. Thus, in contrast to thermal processing, the application of high-pressure causes negligible impairment of nutritional values, taste, color flavor, or vitamin content (Hayashi, 1990). Small molecules such as amino acids, vitamins, and flavor compounds remain unaffected by high pressure, while the structure of the large molecules such as proteins, enzymes, polysaccharides, and nucleic acid may be altered (Balci and Wilbey, 1999).
High pressure reduces the rate of browning reaction (Maillard reaction). It consists of two reactions, condensation reaction of amino compounds with carbonyl compounds, and successive browning reactions including metanoidin formation and polymerization processes. The condensation reaction shows no acceleration by high pressure (5-50 MPa at 50C), because it suppresses the generation of stable free radicals derived from melanoidin, which are responsible for the browning reaction (Tamaoka, Itoh, and Hayashi, 1991). Gels induced by high pressure are found to be more glossy and transparent because of rearrangement of water molecules surrounding amino acid residues in a denatured state (Okamoto, Kawamura, and Hayashi, 1990).
The capability and limitations of HPP have been extensively reviewed (Thakur and Nelson, 1998; Smelt, 1998;Cheftal, 1995; Knorr, 1995; Fair, 1990; Tiwari, Jayas, and Holley, 1999; Cheftel, Levy, and Dumay, 2000; Messens, Van Camp, and Huyghebaert, 1997; Ontero and Sanz, 2000; Hugas, Garriga, and Monfort, 2002; Lakshmanan, Piggott,and Paterson, 2003; Balasubramaniam, 2003; Matser, Krebbers, Berg, and Bartels, 2004; Hogan, Kelly, and Sun, 2005; Mor-Mur and Yuste, 2005). Many of the early reviews primarily focused on the microbial efficacy of high-pressure processing. This review comprehensively covers the different types of products processed by highpressure technology alone or in combination with the other processes. It also discusses the effect of high pressure on food constituents such as enzymes and proteins. The applications of this technology in fruits and vegetable, dairy and animal product processing industries are covered. The effects of combining high- pressure treatment with other processing methods such as gamma- irradiation, alternating current, ultrasound, carbon dioxide, and anti microbial peptides have also been described. Special emphasis has been given to opportunities and challenges in high pressure processing of foods, which can potentially be explored and exploited.
EFFECT OF HIGH PRESSURE ON ENZYMES AND PROTEINS
Enzymes are a special class of proteins in which biological activity arises from active sites, brought together by a three- dimensional configuration of molecule. The changes in active site or protein denaturation can lead to loss of activity, or changes the functionality of the enzymes (Tsou, 1986). In addition to conformational changes, enzyme activity can be influenced by pressure-induced decompartmentalization (Butz, Koller, Tauscher, and Wolf, 1994; Gomes and Ledward, 1996). Pressure induced damage of membranes facilitates enzymesubstrate contact. The resulting reaction can either be accelerated or retarded by pressure (Butz, Koller, Tauscher, and Wolf, 1994; Gomes and Ledward, 1996; Morild, 1981). Hendrickx, Ludikhuy ze, Broeck, and Weemaes ( 1998) and Ludikhuyze, Van Loey, and Indrawati et al. (2003) reviewed the combined effect of pressure and temperature on enzymes related to the ity of fruits and vegetables, which comprises of kinetic information as well as process engineering aspects.
Pectin methylesterase (PME) is an enzyme, which normally tends to lower the viscosity of fruits products and adversely affect their texture. Hence, its inactivation is a prerequisite for the preservation of such products. Commercially, fruit products containing PME (e.g. orange juice and tomato products) are heat pasteurized to inactivate PME and prolong shelf life. However, heating can deteriorate the sensory and nutritional quality of the products. Basak and Ramaswamy (1996) showed that the inactivation of PME in orange juice was dependent on pressure level, pressure-hold time, pH, and total soluble solids. An instantaneous pressure kill was dependent only on pressure level and a secondary inactivation effect dependent on holding time at each pressure level. Nienaber and Shellhammer (2001) studied the kinetics of PME inactivation in orange juice over a range of pressures (400-600 MPa) and temperatures (25-5O0C) for various process holding times. PME inactivation followed a firstorder kinetic model, with a residual activity of pressure-resistant enzyme. Calculated D-values ranged from 4.6 to 117.5 min at 600 MPa/50C and 400 MPa/25C, respectively. Pressures in excess of 500 MPa resulted in sufficiently faster inactivation rates for economic viability of the process. Binh, Van Loey, Fachin, Verlent, Indrawati, and Hendrickx (2002a, 2002b) studied the kinetics of inactivation of strawberry PME. The combined effect of pressure and temperature on inactivation kinetics followed a fractional-conversion model. Purified strawberry PME was more stable toward high-pressure treatments than PME from oranges and bananas. Ly-Nguyen, Van Loey, Fachin, Verlent, Hendrickx (2002) showed that the inactivation of the banana PME enzyme during heating at temperature between 65 and 72.5C followed first order kinetics and the effect of pressure treatment of 600-700 MPa at 10C could be described using a fractionalconversion model. Stoforos, Crelier, Robert, and Taoukis (2002) demonstrated that under ambient pressure, tomato PME inactivation rates increased with temperature, and the highest rate was obtained at 75C. The inactivation rates were dramatically reduced as soon as the essing pressure was raised beyond 75C. High inactivation rates were obtained at a pressure higher than 700 MPa. Riahi and Ramaswamy (2003) studied high- pressure inactivation kinetics of PME isolated from a variety of sources and showed that PME from a microbial source was more resistant \to pressure inactivation than from orange peel. Almost a full decimal reduction in activity of commercial PME was achieved at 400 MPa within 20 min.
Verlent, Van Loey, Smout, Duvetter, Nguyen, and Hendrickx (2004) indicated that the optimal temperature for tomato pectinmethylesterase was shifted to higher values at elevated pressure compared to atmospheric pressure, creating the possibilities for rheology improvements by the application of high pressure.
Castro, Van Loey, Saraiva, Smout, and Hendrickx (2006) accurately described the inactivation of the labile fraction under mild-heat and high-pressure conditions by a fractional conversion model, while a biphasic model was used to estimate the inactivation rate constant of both the fractions at more drastic conditions of temperature/ pressure (10-64C, 0.1-800 MPa). At pressures lower than 300 MPa and temperatures higher than 54C, an antagonistic effect of pressure and temperature was observed.
Balogh, Smout, Binh, Van Loey, and Hendrickx (2004) observed the inactivation kinetics of carrot PME to follow first order kinetics over a range of pressure and temperature (650800 MPa, 10-40C). Enzyme stability under heat and pressure was reported to be lower in carrot juice and purified PME preparations than in carrots.
The presence of pectinesterase (PE) reduces the quality of citrus juices by destabilization of clouds. Generally, the inactivation of the enzyme is accomplished by heat, resulting in a loss of fresh fruit flavor in the juice. High pressure processing can be used to bypass the use of extreme heat for the processing of fruit juices. Goodner, Braddock, and Parish (1998) showed that the higher pressures (>600 MPa) caused instantaneous inactivation of the heat labile form of the enzyme but did not inactivate the heat stable form of PE in case of orange and grapefruit juices. PE activity was totally lost in orange juice, whereas complete inactivation was not possible in case of grapefruit juices. Orange juice pressurized at 700 MPa for l min had no cloud loss for more than 50 days. Broeck, Ludikhuyze, Van Loey, and Hendrickx (2000) studied the combined pressure-temperature inactivation of the labile fraction of orange PE over a range of pressure (0.1 to 900 MPa) and temperature (15 to 65C). Pressure and temperature dependence of the inactivation rate constants of the labile fraction was quantified using the well- known Eyring and Arrhenius relations. The stable fraction was inactivated at a temperature higher than 75C. Acidification (pH 3.7) enhanced the thermal inactivation of the stable fraction, whereas the addition of Ca^sup ++^ ions (IM) suppressed inactivation. At elevated pressure (up to 900 MPa), an antagonistic effect of pressure and temperature on inactivation of the stable fraction was observed. Ly-Nguyen, Van Loey, Smout, Ozean, Fachin, Verlent, Vu- Truong, Duvetter, and Hendrickx (2003) investigated the combined heat and pressure treatments on the inactivation of purified carrot PE, which followed a fractional-conversion model. The thermally stable fraction of the enzyme could not be inactivated. At a lower pressure (50C), an antagonistic effect of pressure and heat was observed.
High pressures induced conformational changes in polygalacturonase (PG) causing reduced substrate binding affinity and enzyme inactivation. Eun, Seok, and Wan ( 1999) studied the effect of high-pressure treatment on PG from Chinese cabbage to prevent the softening and spoilage of plant-based foods such as kimchies without compromising quality. PG was inactivated by the application of pressure higher than 200 MPa for l min. Fachin, Van Loey, Indrawati, Ludikhuyze, and Hendrickx (2002) investigated the stability of tomato PG at different temperatures and pressures. The combined pressure temperature inactivation (300-600 MPa/50 -50C) of tomato PG was described by a fractional conversion model, which points to Ist-order inactivation kinetics of a pressure-sensitive enzyme fraction and to the occurrence of a pressure-stable PG fraction. Fachin, Smout, Verlent, Binh, Van Loey, and Hendrickx (2004) indicated that in the combination of pressure-temperature (5- 55C/100-600 MPa), the inactivation of the heat labile portion of purified tomato PG followed first order kinetics. The heat stable fraction of the enzyme showed pressure stability very similar to that of heat labile portion.
Peelers, Fachin, Smout, Van Loey, and Hendrickx (2004) demonstrated that effect of high-pressure was identical on heat stable and heat labile fractions of tomato PG. The isoenzyme of PG was detected in thermally treated (140C for 5 min) tomato pieces and tomato juice, whereas, no PG was found in pressure treated tomato juice or pieces.
Verlent, Van Loey, Smout, Duvetter, and Hendrickx (2004) investigated the effect of nigh pressure (0.1 and 500 MPa) and temperature (25-80C) on purified tomato PG. At atmospheric pressure, the optimum temperature for enzyme was found to be 55-60C and it decreased with an increase in pressure. The enzyme activity was reported to decrease with an increase in pressure at a constant temperature.
Shook, Shellhammer, and Schwartz (2001) studied the ability of high pressure to inactivate lipoxygenase, PE and PG in diced tomatoes. Processing conditions used were 400,600, and 800 MPa for 1, 3, and 5 min at 25 and 45C. The magnitude of the applied pressure had a significant effect in inactivating lipoxygenase and PG, with complete loss of activity occurring at 800 MPa. PE was very resistant to the pressure treatment.
Polyphenoloxidase and Pemxidase
Polyphenoloxidase (PPO) and peroxidase (POD), the enzymes responsible for color and flavor loss, can be selectively inactivated by a combined treatment of pressure and temperature. Gomes and Ledward (1996) studied the effects of pressure treatment (100-800 MPa for 1-20 min) on commercial PPO enzyme available from mushrooms, potatoes, and apples. Castellari, Matricardi, Arfelli, Rovere, and Amati ( 1997) demonstrated that there was a limited inactivation of grape PPO using pressures between 300 and 600 MPa. At 900 MPa, a low level of PPO activity was apparent. In order to reach complete inactivation, it may be necessary to use high- pressure processing treatments in conjunction with a mild thermal treatment (40-50C). Weemaes, Ludikhuyze, Broeck, and Hendrickx (1998) studied the pressure stabilities of PPO from apple, avocados, grapes, pears, and plums at pH 6-7. These PPO differed in pressure stability. Inactivation of PPO from apple, grape, avocado, and pear at room temperature (25C) became noticeable at approximately 600, 700, 800 and 900 MPa, respectively, and followed first-order kinetics. Plum PPO was not inactivated at room temperature by pressures up to 900 MPa. Rastogi, Eshtiaghi, and Knorr (1999) studied the inactivation effects of high hydrostatic pressure treatment (100-600 MPa) combined with heat treatment (0-60C) on POD and PPO enzyme, in order to develop high pressure-processed red grape juice having stable shelf-life. The studies showed that the lowest POD (55.75%) and PPO (41.86%) activities were found at 60C, with pressure at 600 and 100 MPa, respectively. MacDonald and Schaschke (2000) showed that for PPO, both temperature and pressure individually appeared to have similar effects, whereas the holding time was not significant. On the other hand, in case of POD, temperature as well as interaction between temperature and holding time had the greatest effect on activity. Namkyu, Seunghwan, and Kyung (2002) showed that mushroom PPO was highly pressure stable. Exposure to 600 MPa for 10 min reduced PPO activity by 7%; further exposure had no denaturing effect. Compression for 10 and 20 min up to 800 MPa, reduced activity by 28 and 43%, respectively.
Rapeanu, Van Loey, Smout, and Hendrickx (2005) indicated that the thermal and/or high-pressure inactivation of grape PPO followed first order kinetics. A third degree polynomial described the temperature/pressure dependence of the inactivation rate constants. Pressure and temperature were reported to act synergistically, except in the high temperature (≥45C)-low pressure (≥300 MPa) region where an antagonistic effect was observed.
Gomes, Sumner, and Ledward (1997) showed that the application of increasing pressures led to a gradual reduction in papain enzyme activity. A decrease in activity of 39% was observed when the enzyme solution was initially activated with phosphate buffer (pH 6.8) and subjected to 800 MPa at ambient temperature for 10 min, while 13% of the original activity remained when the enzyme solution was treated at 800 MPa at 60C for 10 min. In Tris buffer at pH 6.8 after treatment at 800 MPa and 20C, papain activity loss was approximately 24%. The inactivation of the enzyme is because of induced change at the active site causing loss of activity without major conformational changes. This loss of activity was due to oxidation of the thiolate ion present at the active site.
Weemaes, Cordt, Goossens, Ludikhuyze, Hendrickx, Heremans, and Tobback (1996) studied the effects of pressure and temperature on activity of 3 different alpha-amylases from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis. The changes in conformation of Bacillus licheniformis, Bacillus subtilis, and Bacillus amyloliquefaciens amylases occurred at pressures of 110, 75, and 65 MPa, respectively. Bacillus licheniformis amylase was more stable than amylases from Bacillus subtilis and Bacillus amyloliquefaciens to the combined heat/pressure treatment.
Riahi and Ramaswamy (2004) demonstrated that pressure inactivation of amylase in apple juice was significantly (P
High pressure denatures protein depending on the protein type, processing conditions, and the applied pressure. During the process of denaturation, the proteins may dissolve or precipitate on the application of high pressure. These changes are generally reversible in the pressure range 100-300 MPa and irreversible for the pressures higher than 300 MPa. Denaturation may be due to the destruction of hydrophobic and ion pair bonds, and unfolding of molecules. At higher pressure, oligomeric proteins tend to dissociate into subunits becoming vulnerable to proteolysis. Monomeric proteins do not show any changes in proteolysis with increase in pressure (Thakur and Nelson, 1998).
High-pressure effects on proteins are related to the rupture on non-covalent interactions within protein molecules, and to the subsequent reformation of intra and inter molecular bonds within or between the molecules. Different types of interactions contribute to the secondary, tertiary, and quaternary structure of proteins. The quaternary structure is mainly held by hydrophobic interactions that are very sensitive to pressure. Significant changes in the tertiary structure are observed beyond 200 MPa. However, a reversible unfolding of small proteins such as ribonuclease A occurs at higher pressures (400 to 800 MPa), showing that the volume and compressibility changes during denaturation are not completely dominated by the hydrophobic effect. Denaturation is a complex process involving intermediate forms leading to multiple denatured products. secondary structure changes take place at a very high pressure above 700 MPa, leading to irreversible denaturation (Balny and Masson, 1993).
Figure 1 General scheme for pressure-temperature phase diagram of proteins, (from Messens, Van Camp, and Huyghebaert, 1997).
When the pressure increases to about 100 MPa, the denaturation temperature of the protein increases, whereas at higher pressures, the temperature of denaturation usually decreases. This results in the elliptical phase diagram of native denatured protein shown in Fig. 1. A practical consequence is that under elevated pressures, proteins denature usually at room temperature than at higher temperatures. The phase diagram also specifies the pressure- temperature range in which the protein maintains its native structure. Zone III specifies that at high temperatures, a rise in denaturation temperature is found with increasing pressure. Zone II indicates that below the maximum transition temperature, protein denaturation occurs at the lower temperatures under higher pressures. Zone III shows that below the temperature corresponding to the maximum transition pressure, protein denaturation occurs at lower pressures using lower temperatures (Messens, Van Camp, and Huyghebaert, 1997).
The application of high pressure has been shown to destabilize casein micelles in reconstituted skim milk and the size distribution of spherical casein micelles decrease from 200 to 120 nm; maximum changes have been reported to occur between 150-400 MPa at 20C. The pressure treatment results in reduced turbidity and increased lightness, which leads to the formation of a virtually transparent skim milk (Shibauchi, Yamamoto, and Sagara, 1992; Derobry, Richard, and Hardy, 1994). The gels produced from high-pressure treated skim milk showed improved rigidity and gel breaking strength (Johnston, Austin, and Murphy, 1992). Garcia, Olano, Ramos, and Lopez (2000) showed that the pressure treatment at 25C considerably reduced the micelle size, while pressurization at higher temperature progressively increased the micelle dimensions. Anema, Lowe, and Stockmann (2005) indicated that a small decrease in the size of casein micelles was observed at 100 MPa, with slightly greater effects at higher temperatures or longer pressure treatments. At pressure >400 MPa, the casein micelles disintegrated. The effect was more rapid at higher temperatures although the final size was similar in all samples regardless of the pressure or temperature. At 200 MPa and 1O0C, the casein micelle size decreased slightly on heating, whereas, at higher temperatures, the size increased as a result of aggregation. Huppertz, Fox, and Kelly (2004a) showed that the size of casein micelles increased by 30% upon high-pressure treatment of milk at 250 MPa and micelle size dropped by 50% at 400 or 600 MPa.
Huppertz, Fox, and Kelly (2004b) demonstrated that the high- pressure treatment of milk at 100-600 MPa resulted in considerable solubilization of alphas 1- and beta-casein, which may be due to the solubilization of colloidal calcium phosphate and disruption of hydrophobic interactions. On storage of pressure, treated milk at 5C dissociation of casein was largely irreversible, but at 20C, considerable re-association of casein was observed. The hydration of the casein micelles increased on pressure treatment (100-600 MPa) due to induced interactions between caseins and whey proteins. Pressure treatment increased levels of alphas 1- and beta-casein in the soluble phase of milk and produced casein micelles with properties different to those in untreated milk. Huppertz, Fox, and Kelly (2004c) demonstrated that the casein micelle size was not influenced by pressures less than 200 MPa, but a pressure of 250 MPa increased the micelle size by 25%, while pressures of 300 MPa or greater, irreversibly reduced the size to 50% ofthat in untreated milk. Denaturation of alpha-lactalbumin did not occur at pressures less than or equal to 400 MPa, whereas beta-lactoglobulin was denatured at pressures greater than 100 MPa.
Galazka, Ledward, Sumner, and Dickinson (1997) reported loss of surface hydrophobicity due to application of 300 MPa in dilute solution. Pressurizing beta-lactoglobulin at 450 MPa for 15 minutes resulted in reduced solubility in water. High-pressure treatment induced extensive protein unfolding and aggregation when BSA was pressurized at 400 MPa. Beta-lactoglobulin appears to be more sensitive to pressure than alpha-lactalbumin. Olsen, Ipsen, Otte, and Skibsted (1999) monitored the state of aggregation and thermal gelation properties of pressure-treated beta-lactoglobulin immediately after depressurization and after storage for 24 h at 50C. A pressure of 150 MPa applied for 30 min, or pressures higher than 300 MPa applied for 0 or 30 min, led to formation of soluble aggregates. When continued for 30 min, a pressure of 450 MPa caused gelation of the 5% beta-lactoglobulin solution. Iametti, Tansidico, Bonomi, Vecchio, Pittia, Rovere, and DaIl'Aglio (1997) studied irreversible modifications in the tertiary structure, surface hydrophobicity, and association state of beta-lactoglobulin, when solutions of the protein at neutral pH and at different concentrations, were exposed to pressure. Only minor irreversible structural modifications were evident even for treatments as intense as 15 min at 900 MPa. The occurrence of irreversible modifications was time-dependent at 600 MPa but was complete within 2 min at 900 MPa. The irreversibly modified protein was soluble, but some covalent aggregates were formed. Subirade, Loupil, Allain, and Paquin (1998) showed the effect of dynamic high pressure on the secondary structure of betalactoglobulin. Thermal and pH sensitivity of pressure treated beta-lactoglobulin was different, suggesting that the two forms were stabilized by different electrostatic interactions. Walker, Farkas, Anderson, and Goddik (2004) used high- pressure processing (510 MPa for 10 min at 8 or 24C) to induce unfolding of beta-lactoglobulin and characterized the protein structure and surface-active properties. The secondary structure of the protein processed at 8C appeared to be unchanged, whereas at 24C alpha-helix structure was lost. Tertiary structures changed due to processing at either temperature. Model solutions containing the pressure-treated beta-lactoglobulin showed a significant decrease in surface tension. Izquierdo, Alli, Gmez, Ramaswamy, and Yaylayan (2005) demonstrated that under high-pressure treatments (100-300 MPa), the β-lactoglobulin AB was completely hydrolyzed by pronase and α-chymotrypsin. Hinrichs and Rademacher (2005) showed that the denaturation kinetics of beta-lactoglobulin followed second order kinetics while for alpha-lactalbumin it was 2.5. Alpha- lactalbumin was more resistant to denaturation than beta- lactoglobulin. The activation volume for denaturation of beta- lactoglobulin was reported to decrease with increasing temperature, and the activation energy increased with pressure up to 200 MPa, beyond which it decreased. This demonstrated the unfolding of the protein molecules.
Drake, Harison, Apslund, Barbosa-Canovas, and Swanson (1997) demonstrated that the percentage moisture and wet weight yield of cheese from pressure treated milk were higher than pasteurized or raw milk cheese. The microbial quality was comparable and some textural defects were reported due to the excess moisture content. Arias, Lopez, and Olano (2000) showed that high-pressure treatment at 200 MPa significantly reduced rennet coagulation times over control samples. Pressurization at 400 MPa led to coagulation times similar to those of control, except for milk treated at pH 7.0, with or without readjustment of pH to 6.7, which presented significantly longer coagulation times than their non-pressure treated counterparts.
Hinrichs and Rademacher (2004) demonstrated that the isobaric (200-800 MPa) and isothermal (-2 to 70C) denaturation of beta- lactoglobulin and alpha-lactalbumin of whey protein followed 3rd and 2nd order kinetics, respectively. Isothermal pressure denaturation of both beta-lactoglobulin A and B did not differ significantly and an increase in temperature resulted in an increase in thedenaturation rate. At pressures higher than 200 MPa, the denaturation rate was limited by the aggregation rate, while the pressure resulted in the unfolding of molecules. The kinetic parameters of denaturation were estimated using a single step non- linear regression method, which allowed a global fit of the entire data set. Huppertz, Fox, and Kelly (2004d) examined the high- pressure induced denaturation of alpha-lactalbumin and beta- lactoglobulin in dairy systems. The higher level of pressure- induced denaturation of both proteins in milk as compared to whey was due to the absence of casein micelles and colloidal calcium phosphate in the whey.
The conformation of BSA was reported to remain fairly stable at 400 MPa due to a high number of disulfide bonds which are known to stabilize its three dimensional structure (Hayakawa, Kajihara, Morikawa, Oda, and Fujio, 1992). Kieffer and Wieser (2004) indicated that the extension resistance and extensibility of wet gluten were markedly influenced by high pressure (up to 800 MPa), while the temperature and the duration of pressure treatment (30-80C for 2-20 min) had a relatively lesser effect. The application of high pressure resulted in a marked decrease in protein extractability due to the restructuring of disulfide bonds under high pressure leading to the incorporation of alpha- and gamma-gliadins in the glutenin aggregate. The change in secondary structure following high- pressure treatment was also reported.
The pressure treatment of myosin led to head-to-head interaction to form oligomers (clumps), which became more compact and larger in size during storage at constant pressure. Even after pressure treatment at 210 MPa for 5 minutes, monomieric myosin molecules increased and no gelation was observed for pressure treatment up to 210 MPa for 30 minutes. Pressure treatment did not also affect the original helical structure of the tail in the myosin monomers. Angsupanich, Edde, and Ledward (1999) showed that high pressure- induced denaturation of myosin led to formation of structures that contained hydrogen bonds and were additionally stabilized by disulphide bonds.
Application of 750 MPa for 20 minutes resulted in dimerization of metmyoglobin in the pH range of 6-10, whereas maximum pH was not at isoelectric pH (6.9). Under acidic pH conditions, no dimers were formed (Defaye and Ledward, 1995). Zipp and Kouzmann ( 1973) showed the formation of precipitate when pressurized (750 MPa for 20 minutes) near the isoelectric point, the precipitate redissolved slowly during storage. Pressure treatment had no effect on lipid oxidation in the case of minced meat packed in air at pressure less than 300 MPa, while the oxidation increased proportionally at higher pressures. However, on exposure to higher pressure, minced meat in contact with air oxidized rapidly. Pressures > 300-400 MPa caused marked denaturation of both myofibriller and sarcoplasmic proteins in washed pork muscle and pork mince (Ananth, Murano and Dickson, 1995). Chapleau and Lamballerie (2003) showed that high-pressure treatment induced a threefold increase in the surface hydrophobicity of myofibrillar proteins between O and 450 MPa. Chapleau, Mangavel, Compoint, and Lamballerie (2004) reported that high pressure modified the secondary structure of myofibrillar proteins extracted from cattle carcasses. Irreversible changes and aggregation were reported at a pressure higher than 300 MPa, which can potentially affect the functional properties of meat products. Lamballerie, Perron, Jung, and Cheret (2003) indicated that high pressure treatment increases cathepsin D activity, and that pressurized myofibrils are more susceptible to cathepsin D action than non- pressurized myofibrils. The highest cathepsin D activity was observed at 300 MPa. Cariez, Veciana, and Cheftel ( 1995) demonstrated that L color values increased significantly in meat treated at 200-350 MPa, the meat becoming pink, and a-value decreased in meat treated at 400-500 MPa to give a grey-brown color. The total extractable myoglobin decreased in meat treated at 200- 500 MPa, while the metmyoglobin content of meat increased and the oxymyoglobin decreased at 400500 MPa. Meat discoloration from pressure processing resulted in a whitening effect at 200-300 MPa due to globin denaturation, and/or haem displacement/release, or oxidation of ferrous myoglobin to ferric myoglobin at pressure higher than 400 MPa.
The conformation of the main protein component of egg white, ovalbumin, remains fairly stable when pressurized at 400 MPa, may be due to the four disulfide bonds and non-covalent interactions stabilizing the three dimensional structure of ovalbumin (Hayakawa, Kajihara, Morikawa, Oda, and Fujio, 1992). Hayashi, Kawamura, Nakasa and Okinada (1989) reported irreversible denaturation of egg albumin at 500-900 MPa with concomitant increase in susceptibility to subtilisin. Zhang, Li, and Tatsumi (2005) demonstrated that the pressure treatment (200-500 MPa) resulted in denaturation of ovalbumin. The surface hydrophobicity of ovalbumin was found to increase with increase in pressure treatment and the presence of polysaccharide protected the protein against denaturation. Iametti, Donnizzelli, Pittia, Rovere, Squarcina, and Bonomi (1999) showed that the addition of NaCl or sucrose to egg albumin prior to high- pressure treatment (up to 10 min at 800 MPa) prevented insolubulization or gel formation after pressure treatment. As a consequence of protein unfolding, the treated albumin had increased viscosity but retained its foaming and heat-gelling properties. Farr (1990) reported the modification of functionality of egg proteins. Egg yolk formed a gel when subjected to a pressure of 400 MPa for 30 minutes at 25C, kept its original color, and was soft and adhesive. The hardness of the pressure treated gel increased and adhesiveness decreased with an increase in pressure. Plancken, Van Loey, and Hendrickx (2005) showed that the application of high pressure (400- 700 MPa) to egg white solution resulted in an increase in turbidity, surface hydrophobicity, exposed sulfhydryl content, and susceptibility to enzymatic hydrolysis, while it resulted in a decrease in protein solubility, total sulfhydryl content, denaturation enthalpy, and trypsin inhibitory activity. The pressure- induced changes in these properties were shown to be dependent on the pressuretemperature and the pH of the solution. Speroni, Puppo, Chapleau, Lamballerie, Castellani, Aon, and Anton (2005) indicated that the application of high pressure (200-600 MPa) at 2OC to low- density lipoproteins did not change the solubility even if the pH is changed, whereas aggregation and protein denaturation were drastically enhanced at pH 8. Further, the application of high- pressure under alkaline pH conditions resulted in decreased droplet flocculation of low-density lipoproteins dispersions.
The minimum pressure required for the inducing gelation of soya proteins was reported to be 300 MPa for 10-30 minutes and the gels formed were softer with lower elastic modulus in comparison with heat-treated gels (Okamoto, Kawamura, and Hayashi, 1990). The treatment of soya milk at 500 MPa for 30 min changed it from a liquid state to a solid state, whereas at lower pressures and at 500 MPa for 10 minutes, the milk remained in a liquid state, but indicated improved emulsifying activity and stability (Kajiyama, Isobe, Uemura, and Noguchi, 1995). The hardness of tofu gels produced by high-pressure treatment at 300 MPa for 10 minutes was comparable to heat induced gels. Puppo, Chapleau, Speroni, Lamballerie, Michel, Anon, and Anton (2004) demonstrated that the application of high pressure (200-600 MPa) on soya protein isolate at pH 8.0 resulted in an increase in a protein hydorphobicity and aggregation, a reduction of free sulfhydryl content and a partial unfolding of the 7S and 11S fractions at pH 8. The change in the secondary structure leading to a more disordered structure was also reported. Whereas at pH 3.0, the protein was partially denatured and insoluble aggregates were formed, the major molecular unfolding resulted in decreased thermal stability, increased protein solubility, and hydorphobicity. Puppo, Speroni, Chapleau, Lamballerie, An, and Anton (2005) studied the effect of high- pressure (200, 400, and 600 MPa for 10 min at 10C) on the emulsifying properties of soybean protein isolates at pH 3 and 8 (e.g. oil droplet size, flocculation, interfacial protein concentration, and composition). The application of pressure higher than 200 MPa at pH 8 resulted in a smaller droplet size and an increase in the levels of depletion flocculation. However, a similar effect was not observed at pH 3. Due to the application of high pressure, bridging flocculation decreased and the percentage of adsorbed proteins increased irrespective of the pH conditions. Moreover, the ability of the protein to be adsorbed at the oil- water interface increased. Zhang, Li, Tatsumi, and Isobe (2005) showed that the application of high pressure treatment resulted in the formation of more hydrophobic regions in soy protein, which dissociated into subunits, which in some cases formed insoluble aggregates. High-pressure denaturation of beta-conglycinin (7S) and glycinin (11S) occurred at 300 and 400 MPa, respectively. The gels formed had the desirable strength and a cross-linked network microstructure.
Soybean whey is a by-product of tofu manufacture. It is a good source of peptides, proteins, oligosaccharides, and isoflavones, and can be used in special foods for the elderly persons, athletes, etc. Prestamo and Penas (2004) studied the antioxidative activity of soybean whey proteins and their pepsin and chymotrypsin hydrolysates. The chymotrypsin hydrolysate showed a higher antioxidative activity than the non-hydrolyzed protein, but the pepsin hydrolysate showed an opposite trend. High pressure processing at 100 MPa inc\reased the antioxidative activity of soy whey protein, but decreased the antioxidative activity of the hydrolysates. High pressure processing increased the pH of the protein hydrolysates. Penas, Prestamo, and Gomez (2004) demonstrated that the application of high pressure (100 and 200 MPa, 15 min, 37C) facilitated the hydrolysis of soya whey protein by pepsin, trypsin, and chymotrypsin. It was shown that the highest level of hydrolysis occurred at a treatment pressure of 100 MPa. After the hydrolysis, 5 peptides under 14 kDa with trypsin and chymotrypsin, and 11 peptides with pepsin were reported.
COMBINATION OF HIGHPRESSURE TREATMENT WITH OTHER NON-THERMAL PROCESSING METHODS
Many researchers have combined the use of high pressure with other non-thermal operations in order to explore the possibility of synergy between processes. Such attempts are reviewed in this section.
Crawford, Murano, Olson, and Shenoy (1996) studied the combined effect of high pressure and gamma-irradiation for inactivating Clostridium spmgenes spores in chicken breast. Application of high pressure reduced the radiation dose required to produce chicken meat with extended shelf life. The application of high pressure (600 MPa for 20 min at 8O0C) reduced the irradiation doses required for one log reduction of Clostridium spmgenes from 4.2 kGy to 2.0 kGy. Mainville, Montpetit, Durand, and Farnworth (2001) studied the combined effect of irradiation and high pressure on microflora and microorganisms of kefir. The irradiation treatment of kefir at 5 kGy and high-pressure treatment (400 MPa for 5 or 30 min) deactivated the bacteria and yeast in kefir, while leaving the proteins and lipids unchanged.
The exposure of microbial cells and spores to an alternating current (50 Hz) resulted in the release of intracellular materials causing loss or denaturation of cellular components responsible for the normal functioning of the cell. The lethal damage to the microorganisms enhanced when the organisms are exposed to an alternating current before and after the pressure treatment. High- pressure treatment at 300 MPa for 10 min for Escherichia coli cells and 400 MPa for 30 min for Bacillus subtalis spores, after the alternating current treatment, resulted in reduced surviving fractions of both the organisms. The combined effect was also shown to reduce the tolerant level of microorganisms to other challenges (Shimada and Shimahara, 1985, 1987; Shimada, 1992).
The pretreatment with ultrasonic waves (100 W/cm^sup 2^ for 25 min at 25C) followed by high pressure (400 MPa for 25 min at 15C) was shown to result in complete inactivation of Rhodoturola rubra. Neither ultrasonic nor high-pressure treatment alone was found to be effective (Knorr, 1995).
Carbon Dioxide and Argon
Heinz and Knorr (1995) reported a 3 log reduction of supercritical CO2 pretreated cultures. The effect of the pretreatment on germination of Bacillus subtilis endospores was monitored. The combination of high pressure and mild heat treatment was the most effective in reducing germination (95% reduction), but no spore inactivation was observed.
Park, Lee, and Park (2002) studied the combination of high- pressure carbon dioxide and high pressure as a nonthermal processing technique to enhance the safety and shelf life of carrot juice. The combined treatment of carbon dioxide (4.90 MPa) and high-pressure treatment (300 MPa) resulted in complete destruction of aerobes. The increase in high pressure to 600 MPa in the presence of carbon dioxide resulted in reduced activities of polyphenoloxidase (11.3%), lipoxygenase (8.8%), and pectin methylesterase (35.1%). Corwin and Shellhammer (2002) studied the combined effect of high-pressure treatment and CO2 on the inactivation of pectinmethylesterase, polyphenoloxidase, Lactobacillus plantarum, and Escherichia coli. An interaction was found between CO2 and pressure at 25 and 50C for pectinmethylesterase and polyphenoloxidase, respectively. The activity of polyphenoloxidase was decreased by CO2 at all pressure treatments. The interaction between CO2 and pressure was significant for Lactobacillus plantarum, with a significant decrease in survivors due to the addition of CO2 at all pressures studied. No significant effect on E. coli survivors was seen with CO2 addition. Truong, Boff, Min, and Shellhammer (2002) demonstrated that the addition of CO2 (0.18 MPa) during high pressure processing (600 MPa, 25C) of fresh orange juice increases the rate of PME inactivation in Valencia orange juice. The treatment time due to CO2 for achieving the equivalent reduction in PME activity was from 346 s to 111 s, but the overall degree of PME inactivation remained unaltered.
Fujii, Ohtani, Watanabe, Ohgoshi, Fujii, and Honma (2002) studied the high-pressure inactivation of Bacillus cereus spores in water containing argon. At the pressure of 600 MPa, the addition of argon reportedly accelerated the inactivation of spores at 20C, but had no effect on the inactivation at 40C.
The complex physicochemical environment of milk exerted a strong protective effect on Escherichia coli against high hydrostatic pressure inactivation, reducing inactivation from 7 logs at 400 MPa to only 3 logs at 700 MPa in 15 min at 20C. A substantial improvement in inactivation efficiency at ambient temperature was achieved by the application of consecutive, short pressure treatments interrupted by brief decompressions. The combined effect of high pressure (500 MPa) and natural antimicrobial peptides (lysozyme, 400 g/ml and nisin, 400 g/ml) resulted in increased lethality for Escherichia coli in milk (Garcia, Masschalck, and Michiels, 1999).
OPPORTUNITIES FOR HIGH PRESSURE ASSISTED PROCESSING
The inclusion of high-pressure treatment as a processing step within certain manufacturing flow sheets can lead to novel products as well as new process development opportunities. For instance, high pressure can precede a number of process operations such as blanching, dehydration, rehydration, frying, and solid-liquid extraction. Alternatively, processes such as gelation, freezing, and thawing, can be carried out under high pressure. This section reports on the use of high pressures in the context of selected processing operations.
Eshtiaghi and Knorr (1993) employed high pressure around ambient temperatures to develop a blanching process similar to hot water or steam blanching, but without thermal degradation; this also minimized problems associated with water disposal. The application of pressure (400 MPa, 15 min, 20C) to the potato sample not only caused blanching but also resulted in a four-log cycle reduction in microbial count whilst retaining 85% of ascorbic acid. Complete inactivation of polyphenoloxidase was achieved under the above conditions when 0.5% citric acid solution was used as the blanching medium. The addition of 1 % CaCl^sub 2^ solution to the medium also improved the texture and the density. The leaching of potassium from the high-pressure treated sample was comparable with a 3 min hot water blanching treatment (Eshtiaghi and Knorr, 1993). Thus, high- pressures can be used as a non-thermal blanching method.
Dehydration and Osmotic Dehydration
The application of high hydrostatic pressure affects cell wall structure, leaving the cell more permeable, which leads to significant changes in the tissue architecture (Fair, 1990; Dornenburg and Knorr, 1994, Rastogi, Subramanian, and Raghavarao, 1994; Rastogi and Niranjan, 1998; Rastogi, Raghavarao, and Niranjan, 2005). Eshtiaghi, Stute, and Knorr (1994) reported that the application of pressure (600 MPa, 15 min at 70C) resulted in no significant increase in the drying rate during fluidized bed drying of green beans and carrot. However, the drying rate significantly increased in the case of potato. This may be due to relatively limited permeabilization of carrot and beans cells as compared to potato. The effects of chemical pre-treatment (NaOH and HCl treatment) on the rates of dehydration of paprika were compared with products pre-treated by applying high pressure or high intensity electric field pulses (Fig. 2). High-pressure (400 MPa for 10 min at 25C) and high intensity electric field pulses (2.4 kV/cm, pulse width 300 s, 10 pulses, pulse frequency 1 Hz) were found to result in drying rates comparable with chemical pre-treatments. The latter pre-treatments, however, eliminated the use of chemicals (Ade- Omowaye, Rastogi, Angersbach, and Knorr, 2001).
Figure 2 (a) Effects of various pre-treatments such as hot water blanching, high pressure and high intensity electric field pulse treatment on dehydration characteristics of red paprika (b) comparison of drying time (from Ade-Omowaye, Rastogi, Angersbach, and Knorr, 2001).
Figure 3 (a) Variation of moisture and (b) solid content (based on initial dry matter content) with time during osmotic dehydration (from Rastogi and Niranjan, 1998).
Generally, osmotic dehydration is a slow process. Application of high pressures causes permeabilization of the cell structure (Dornenburg and Knorr, 1993; Eshtiaghi, Stute, and Knorr, 1994; Fair, 1990; Rastogi, Subramanian, and Raghavarao, 1994). This phenomenon has been exploited by Rastogi and Niranjan (1998) to enhance mass transfer rates during the osmotic dehydration of pineapple (Ananas comsus). High-pressure pre-treatments (100-800 MPa) were found to enhance both water removal as well as solid gain (Fig. 3). Measured diffusivity values for water were found to be four-fold greater, whilst solute (sugar) diffusivity values were found to be two-fold greater. Compression and decompression occurring during high pressure pre-treatment itself caused the removal of a significant amount of water, which was attributed to the cell wall rupture (Rastogi and Niranjan, 1998). Differential interference contrast microscopic examination showed the ext\ent of cell wall break-up with applied pressure (Fig. 4). Sopanangkul, Ledward, and Niranjan (2002) demonstrated that the application of high pressure (100 to 400 MPa) could be used to accelerate mass transfer during ingredient infusion into foods. Application of pressure opened up the tissue structure and facilitated diffusion. However, higher pressures above 400 MPa induced starch gelatinization also and hindered diffusion. The values of the diffusion coefficient were dependent on cell permeabilization and starch gelatinization. The maximum value of diffusion coefficient observed represented an eight-fold increase over the values at ambient pressure.
The synergistic effect of cell permeabilization due to high pressure and osmotic stress as the dehydration proceeds was demonstrated more clearly in the case of potato (Rastogi, Angersbach, and Knorr, 2000a, 2000b, 2003). The moisture content was reduced and the solid content increased in the case of samples treated at 400 MPa. The distribution of relative moisture (M/M^sub o^) and solid (S/S^sub o^) content as well as the cell permeabilization index (Zp) (shown in Fig. 5) indicate that the rate of change of moisture and solid content was very high at the interface and decreased towards the center (Rastogi, Angersbach, and Knorr, 2000a, 2000b, 2003).
Most dehydrated foods are rehydrated before consumption. Loss of solids during rehydration is a major problem associated with the use of dehydrated foods. Rastogi, Angersbach, Niranjan, and Knorr (2000c) have studied the transient variation of moisture and solid content during rehydration of dried pineapples, which were subjected to high pressure treatment prior to a two-stage drying process consisting of osmotic dehydration and finish-drying at 25C (Fig. 6). The diffusion coefficients for water infusion as well as for solute diffusion were found to be significantly lower in high-pressure pre- treated samples. The observed decrease in water diffusion coefficient was attributed to the permeabilization of cell membranes, which reduces the rehydration capacity (Rastogi and Niranjan, 1998). The solid infusion coefficient was also lower, and so was the release of the cellular components, which form a gel- network with divalent ions binding to de-esterified pectin (Basak and Ramaswamy, 1998; Eshtiaghi, Stute, and Knorr, 1994; Rastogi Angersbach, Niranjan, and Knorr, 2000c). Eshtiaghi, Stute, and Knorr (1994) reported that high-pressure treatment in conjunction with subsequent freezing could improve mass transfer during rehydration of dried plant products and enhance product quality.
Figure 4 Microstructures of control and pressure treated pineapple (a) control; (b) 300 MPa; (c) 700 MPa. ( 1 cm = 41.83 m) (from Rastogi and Niranjan, 1998).
Ahromrit, Ledward, and Niranjan (2006) explored the use of high pressures (up to 600 MPa) to accelerate water uptake kinetics during soaking of glutinous rice. The results showed that the length and the diameter the of the rice were positively correlated with soaking time, pressure and temperature. The water uptake kinetics was shown to follow the well-known Fickian model. The overall rates of water uptake and the equilibrium moisture content were found to increase with pressure and temperature.
Zhang, Ishida, and Isobe (2004) studied the effect of highpressure treatment (300-500 MPa for 0-380 min at 20C) on the water uptake of soybeans and resulting changes in their microstructure. The NMR analysis indicated that water mobility in high-pressure soaked soybean was more restricted and its distribution was much more uniform than in controls. The SEM analysis revealed that high pressure changed the microstructures of the seed coat and hilum, which improved water absorption and disrupted the individual spherical protein body structures. Additionally, the DSC and SDS-PAGE analysis revealed that proteins were partially denatured during the high pressure soaking. Ibarz, Gonzalez, Barbosa-Canovas (2004) developed the kinetic models for water absorption and cooking time of chickpeas with and without prior high-pressure treatment (275-690 MPa). Soaking was carried out at 25C for up to 23 h and cooking was achieved by immersion in boiling water until they became tender. As the soaking time increased, the cooking time decreased. High-pressure treatment for 5 min led to reductions in cooking times equivalent to those achieved by soaking for 60-90 min.
Ramaswamy, Balasubramaniam, and Sastry (2005) studied the effects of high pressure (33, 400 and 700 MPa for 3 min at 24 and 55C) and irradiation (2 and 5 kGy) pre-treatments on hydration behavior of navy beans by soaking the treated beans in water at 24 and 55C. Treating beans under moderate pressure (33 MPa) resulted in a high initial moisture uptake (0.59 to 1.02 kg/kg dry mass) and a reduced loss of soluble materials. The final moisture content after three hours of soaking was the highest in irradiated beans (5 kGy) followed by high-pressure treatment (33 MPa, 3 min at 55C). Within the experimental range of the study, Peleg's model was found to satisfactorily describe the rate of water absorption of navy beans.
A reduction of 40% in oil uptake during frying was observed, when thermally blanched frozen potatoes were replaced by high pressure blanched frozen potatoes. This may be due to a reduction in moisture content caused by compression and decompression (Rastogi and Niranjan, 1998), as well as the prevalence of different oil mass transfer mechanisms (Knorr, 1999).
Solid Liquid Extraction
The application of high pressure leads to rearrangement in tissue architecture, which results in increased extractability even at ambient temperature. Extraction of caffeine from coffee using water could be increased by the application of high pressure as well as increase in temperature (Knorr, 1999). The effect of high pressure and temperature on caffeine extraction was compared to extraction at 100C as well as atmospheric pressure (Fig. 7). The caffeine yield was found to increase with temperature at a given pressure. The combination of very high pressures and lower temperatures could become a viable alternative to current industrial practice.
Figure 5 Distribution of (a, b) relative moisture and (c, d) solid content as well as (e, f) cell disi