Effects of high pressure on constituents and properties of milk

International Dairy Journal 12 (2002) 561–572

Review

Effects of high pressure on constituents and properties of milk Thom Huppertz, Alan L. Kelly*, Patrick F. Fox Department of Food Science, Food Technology and Nutrition, University College Cork, Cork, Ireland Received 9 March 2001; accepted 28 January 2002

Abstract High pressure (HP) treatment has significant and, in many cases, unique effects on many constituents of milk. The structure of casein micelles is disrupted and the whey proteins, a-lactalbumin and b-lactoglobulin, are denatured, with the former being more resistant to pressure than the latter. Pressure-induced shifts in the mineral balance in milk also occur and moderately high pressures (100–400 MPa) induce the crystallisation of milk fat. However, milk enzymes seem to be quite resistant to pressure. As a result of pressure-induced effects on individual milk constituents, many properties of milk are affected. HP treatment increases the pH of milk, reduces its turbidity, changes its appearance, and can reduce the rennet coagulation time of milk and increase cheese yield, thereby indicating potential applications in cheese technology. However, to fully understand the effects of HP treatment on milk and to evaluate the full potential of this process in dairy technology, further research is required in several areas, including the reversibility of pressure-induced changes in milk and the physical stability of HP-treated milk. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: High pressure; Milk

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

2.

High pressure equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

3.

Principles of high pressure processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

4.

Effects of high pressure on milk constituents . . . 4.1. Water . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Mineral balance in milk . . . . . . . . . . . . . 4.3. Whey proteins . . . . . . . . . . . . . . . . . . . . 4.4. Casein micelles . . . . . . . . . . . . . . . . . . . 4.5. Milk fat . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Milk enzymes . . . . . . . . . . . . . . . . . . . .

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Effects of high pressure on some properties of milk . 5.1. Appearance and pH of milk . . . . . . . . . . . . . 5.2. Rennet coagulation properties of milk . . . . . . 5.3. Cheesemaking properties of milk . . . . . . . . . 5.4. Yoghurt-making properties of milk . . . . . . . .

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Conclusions and future research needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570 *Corresponding author. Tel.: +353-21-4903405; fax: +353-21-4270213. E-mail address: [email protected] (A.L. Kelly). 0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 2 ) 0 0 0 4 5 - 6

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1. Introduction Although the first studies on the application of high pressure (HP) in food technology were carried out at the end of the 19th century (Hite, 1899), interest in HP processing of food developed only after 1970, principally due to a lack of suitable equipment before that time, and has increased considerably over the last decade or so (Balci & Wilbey, 1999). In 1990, the first HP-processed food, a fruit jam, was introduced onto the Japanese retail market (Mertens & Deplace, 1993); recently, a number of other HP-processed food products have been launched, including oysters in the USA, orange juice in France and guacamole in Mexico. So far, no HPprocessed dairy products are known to be available on the market. Processing of foods by HP offers unique advantages over traditional thermal treatments, as it exerts antimicrobial effects without changing the sensory and nutritional quality of foods. The major advantages of HP for the processing and preservation of foods are elimination or significant reduction of heating, thus avoiding thermal degradation of food components; high retention of flavour, colour and nutritional value; uniform and instant treatment of the product under pressure; reduced requirement for chemical additives; and potential for the design of new products due to the creation of new textures, tastes and functional properties (Datta & Deeth, 1999). In this survey, the current state of knowledge of the effects of HP on constituents and properties of milk and possible applications of HP treatment of milk prior to the production of yoghurt and cheese are reviewed. HP treatment of cheese, yoghurt or other dairy foods or effects of HP on the microflora of milk will not be discussed. Although effects of HP on isolated milk constituents have been studied often in model systems, this review will focus only on effects on constituents in milk. Whenever milk is mentioned in this review, this refers to bovine milk unless specified otherwise. Likewise, as most HP studies have examined effects of treatment at 20–251C, that treatment temperature may be assumed unless otherwise stated.

2. High pressure equipment A schematic diagram of basic equipment design used for HP processing is presented in Fig. 1. A typical HP system consists of four main parts: a high pressure vessel and its closure, a pressure-generating system, a temperature-control device and a material-handling system (Mertens & Deplace, 1993). The pressure vessel is usually a forged monolithic cylindrical vessel constructed of low-alloy steel of high tensile strength. The wall thickness is determined by the

Fig. 1. Schematic diagram of basic equipment design for high pressure processing of foods.

maximum working pressure, the vessel diameter and the number of cycles the vessel is designed to perform; this thickness can be reduced by using multi-layer, wirewound or other pre-stressed designs (Mertens & Deplace, 1993). Once loaded and closed, the vessel is filled with a pressure-transmitting medium; in food processing, potable water is generally used (Myllym.aki, 1996). Air must be removed from the vessel, by compressing or heating the medium, before pressure is generated (Deplace, 1995). In the food industry, vessels with a volume of several thousand litres are in use, with typical operating pressures in the range 100–500 MPa and holding times of about 5–10 min (Myllym.aki, 1996). Laboratory-scale HP equipment capable of reaching pressures up to 1000 MPa is also available.

3. Principles of high pressure processing Pressure and temperature determine many properties of inorganic and organic substances. In food processing, thermal processing is commonplace. However, if a substance is exposed to increasing pressure, many changes also occur, especially at pressures of several hundred MPa. In general, changes associated with volume reduction are promoted under pressure (Buchheim & Prokopek, 1992). The behaviour of biological macromolecules under pressure is important for understanding the effects of HP on milk. Under pressure, biomolecules obey the Le Chatelier-Braun principle, i.e., whenever a stress is applied to a system in equilibrium, the system will react so as to counteract the applied stress; thus, reactions that result in reduced volume will be promoted under

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HP. Such reactions may result in inactivation of microorganisms or enzymes and textural changes in foods (Balci & Wilbey, 1999). Of particular interest in food processing are effects of HP on proteins. In their native state, proteins are stabilised by covalent bonds (including disulphide bridges) plus electrostatic interactions (ion pairs, polar groups), hydrogen bridges and hydrophobic interactions. Covalent bonds are almost unaffected by HP, at least at relatively low temperature (0–401C), and hence the primary structure of proteins remains intact during HP treatment (Mozhaev, Heremans, Frank, Masson, & Balny, 1994). On the other hand, changes in secondary structure occur at high pressures and lead to irreversible denaturation, because stabilising hydrogen bonds are enhanced at low pressures and ruptured only at very high pressures (Hendrickx, Ludikhuyze, Van den Broek, & Weemaes, 1998). Significant changes to the tertiary structure of proteins, which is maintained chiefly by hydrophobic and ionic interactions, are observed at >200 MPa (Hendrickx et al., 1998). Ionic bonds in aqueous solutions are strongly destabilised by pressure, due to the electrostrictive effect of separate charges: in the vicinity of each ion, water molecules are arranged more densely than in bulk water, thereby leading to a decrease in volume (Gross & Jaenicke, 1994). Likewise, hydrophobic interactions between aliphatic groups are characterised by increases in volume and thus are destabilised at increased pressures (Heremans, 1982; Mozhaev et al., 1994). Multimeric proteins, held together by non-covalent bonds, dissociate at relatively low pressures (o150 MPa), thereby disrupting quaternary structures. The exposure of protein surfaces which formerly interacted with each other to a solvent (hydrophobic solvation), results in the binding of water molecules, thereby reducing the volume of the system; thus, increasing pressure shifts the equilibrium between monomeric and multimeric states of proteins towards monomerisation (Gross & Jaenicke, 1994; Hendrickx et al., 1998). Overall, the structures of large molecules, such as proteins (including enzymes), may change under the influence of pressure. However, small molecules that have little secondary, tertiary and quaternary structure, such as amino acids, vitamins and flavour and aroma components contributing to the sensory and nutritional quality of food, remain unaffected (Balci & Wilbey, 1999).

–221C at 50, 100 or 210 MPa, respectively (Hinrichs, Rademacher, & Kessler, 1996a), due to the fact that ice formation is accompanied by an increase in volume (which is not favoured under pressure) and heat generation (2–31C per 100 MPa of applied pressure; Balci & Wilbey, 1999). Water, which is incompressible around atmospheric pressure, is compressed considerably at higher pressures, by about 4% at 100 MPa or 15% at 600 MPa (Hinrichs et al., 1996a). The pH of water is reduced under pressure, by about 1 unit at 1000 MPa, due to increased dissociation of water. A similar decrease in pH occurs on heating water from 251C to 1001C (Marshall & Frank, 1981). 4.2. Mineral balance in milk Although changes in the mineral balance in milk induced by heating, acidification or cold storage have been studied widely, changes due to HP treatment have been considered only recently. The mineral balance of milk refers to the solubility and state of ionisation of milk salts; as an example, there are a number of different forms of calcium in milk (Table 1). Effects of HP on minerals in milk can be divided into: (1) effects on the distribution between the colloidal and diffusible phases, and (2) effects on ionisation. Several authors have found either no (Johnston, Austin, & Murphy, 1992b; De la Fuente, Olano, Casal, & Juarez, 1999) or very small (Lopez-Fandino, De la Fuente, Ramos, & Olano, 1998) effects of a pressure o600 MPa on the concentration of ionic calcium in milk. Desobry-Banon, Richard, and Hardy (1994) reported that the level of diffusible calcium increased following treatment at 200 MPa, while a value similar to that of untreated milk was observed after treatment at 400–600 MPa. In comparison to thermal processes, where pasteurisation had little effect on the diffusible calcium content of skim milk but exposure to a higher temperature reduced it, HP treatment of previously pasteurised or high temperature-treated milk at 400 MPa increased

Table 1 Forms of calcium that can be distinguished in milk Total calcium Colloidal calcium

4. Effects of high pressure on milk constituents Diffusible calcium

4.1. Water Effects of HP on water, the major constituent of milk, include a decrease in its freezing point, to –4,
8 or

563

Ionic calcium

The total concentration of calcium in the milk Calcium associated directly with casein molecules in the absence of phosphates and calcium that forms an integral part of colloidal calcium phosphate (CCP) Also referred to as soluble calcium; calcium present in the soluble phase as free ionic calcium (Ca2+) or complexed predominantly by citrate or phosphate The ionic form of calcium (Ca2+)

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diffusible calcium levels. Thus, it appears that HP treatment solubilised both indigenous and heat-precipitated colloidal calcium phosphate (CCP) (Buchheim, Schrader, Morr, Frede, & Schutt, . 1996a; Schrader, Buchheim, & Morr, 1997). The opposite effects of heat and pressure treatments on levels of diffusible salts in milk were also reported for caprine milk (De la Fuente et al., 1999). Lopez-Fandino et al. (1998) reported that the concentrations of calcium, magnesium and phosphorus in the diffusible phase of bovine or caprine milk increased following treatment at up to 300 MPa, but were lower after treatment at 400 MPa than at 300 MPa. HP-induced changes in the level of diffusible salts were more pronounced in ovine milk, and increased with pressure up to 400 MPa (Lopez-Fandino et al., 1998). In contrast, Law, Leaver, Felipe, Ferragut, Pla, and Guamis (1998) noted little effect of treatment at 100–500 MPa at 201C or 451C on the levels of colloidal calcium and phosphorus in caprine milk. This disparity may be linked to the reversal of HP-induced effects on milk salts, due to storing the samples after HP treatment before analysis. Adjustment of pH alters the level of ionic calcium; for example, acidification results in dissolution of CCP. However, treatment of milk adjusted to pH values in the range 5.5–7.0 at 200 or 400 MPa had little additional effect on levels of ionic calcium (Arias, Lopez-Fandino, & Olano, 2000). 4.3. Whey proteins In one of the earliest investigations of the effects of HP on whey proteins, the amount of non-casein nitrogen in milk serum was shown to decrease with increasing pressure, suggesting denaturation and insolubilisation of whey proteins (Johnston et al., 1992b). Denaturation of individual whey proteins in milk is commonly determined by measuring their level in the pH 4.6-soluble fraction of milk and expressing the level of denaturation relative to control samples (LopezFandino, Carrascosa, & Olano, 1996; Scollard, Beresford, Needs, Murphy, & Kelly, 2000b). Treatment of raw milk at up to 100 MPa does not denature b-lactoglobulin (b-lg) (Lopez-Fandino et al., 1996; Lopez-Fandino & Olano, 1998a; Scollard et al., 2000b). Application of higher pressures results in considerable denaturation of b-lg, reaching 70–80% after treatment at 400 MPa (Lopez-Fandino et al., 1996; Lopez-Fandino & Olano, 1998a; Arias et al., 2000; Garcia-Risco, Olano, Ramos, & Lopez-Fandino, 2000; Scollard et al., 2000b). Relatively little further denaturation of b-lg occurs at 400–800 MPa (Scollard et al., 2000b). The level of denaturation caused by treatments before pressurisation may influence the amount of denatura-

tion measured afterwards, with studies reporting very different extents of denaturation of b-lg following HP treatment at 600 MPa of pasteurised milk (Needs, Capellas, Bland, Manoj, MacDougal, & Paul, 2000a) or reconstituted skim milk powder (Gaucheron et al., 1997). The reaction order of HP-induced denaturation of b-lg is 2.5 (Hinrichs, Rademacher, & Kessler, 1996b), indicating that the denaturation process is concentration-dependent and that a lower initial concentration of native b-lg should reduce the extent of denaturation of b-lg under pressure. A synergistic effect of temperature and pressure on the denaturation of b-lg has been reported, with similar levels of denaturation (almost 100%) after treatment of raw milk at 300 MPa at 50–601C or at 400 MPa at 40–601C (Lopez-Fandino & Olano, 1998a; Garcia-Risco et al., 2000). Denaturation was reduced by HP treatment at 41C relative to 201C (Gaucheron et al., 1997). HP-induced denaturation of b-lg was decreased when milk was acidified to pH 5.5 or 6.0 before treatment and was increased at pH 7.0, relative to milk at pH 6.7. Increased denaturation of b-lg at pH 7.0 may be due to enhanced reactivity of its free sulphydryl group at alkaline pH values (Arias et al., 2000). There may be differences in barostability of b-lg from milk of different species. Above 100 MPa, more extensive HP-induced denaturation of b-lg has been reported in caprine or ovine milk than in bovine milk (Felipe, Capellas, & Law, 1997; Lopez-Fandino & Olano, 1998b), possibly due to interspecies differences in the structure of b-lg but more likely due to other factors associated with the milk. Compared to b-lg, a-lactalbumin (a-la) is much more resistant to denaturation under pressure. Studies on raw milk (Lopez-Fandino et al., 1996; Lopez-Fandino & Olano, 1998a; Garcia-Risco et al., 2000), reconstituted skim milk (Gaucheron et al., 1997) and pasteurised skim milk (Needs et al., 2000a) have concluded that a-la is resistant to denaturation at pressures up to 500 MPa. Similar observations have been reported for ovine (Lopez-Fandino & Olano, 1998b) and caprine milk (Felipe et al., 1997; Lopez-Fandino & Olano, 1998b). However, treatment at higher temperatures (50–601C) greatly increases the extent of denaturation (LopezFandino & Olano, 1998a; Garcia-Risco et al., 2000). Differences in the barostability of a-la and b-lg may be linked to the more rigid molecular structure of the former (Lopez-Fandino et al., 1996; Gaucheron et al., 1997), caused partially by the numbers of intramolecular disulphide bonds in the two proteins (Hinrichs et al., 1996b; Gaucheron et al., 1997), and the lack of a free sulphydryl group in a-la (Lopez-Fandino et al., 1996). Few data are available on HP-induced denaturation of other whey proteins. Bovine serum albumin was resistant to pressures up to 400 MPa in raw milk

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(Lopez-Fandino et al., 1996), whereas immunoglobulins in caprine milk were resistant to pressures up to 300 MPa, although B35% denaturation occurred after treatment at 500 MPa (Felipe et al., 1997). 4.4. Casein micelles In one of the first studies of the effects of HP on milk (Schmidt & Buchheim, 1970), a decrease in the size of casein micelles after HP treatment of milk was observed by electron microscopy. In later studies, a variety of methods have been used to determine casein micelle size in HP-treated milk, such as laser granulometry, transmission electron microscopy (TEM) and photon correlation spectroscopy (PCS), as well as turbidimetry, an indirect method. Several investigators (Desobry-Banon et al., 1994; Gaucheron et al., 1997) have reported no apparent effect of treatment at 150–200 MPa on the average casein micelle size in reconstituted skim milk, but Needs, Stenning, Gill, Ferragut, and Rich (2000b) measured a slight increase (B9%) in size after treatment of raw skim milk at 200 MPa. Treatment at 250–600 MPa reduced micelle size by 40–50% in raw (Needs et al., 2000b) or reconstituted skim milk (Desobry-Banon et al., 1994; Gaucheron et al., 1997). TEM has indicated that treatment of raw skim milk at 400 or 600 MPa completely disrupted all large micelles into smaller fragments (Needs et al., 2000b). Measurement of the turbidity of HP-treated milk indicated slightly different effects from those estimated using laser granulometry, TEM or PCS. Treatment at 100 MPa had little effect on the turbidity of milk, but treatment at 200 or 300 MPa caused similar decreases in turbidity, which were greater after treatment at 400–500 MPa (Buchheim et al., 1996a; Schrader & Buchheim, 1998; Needs et al., 2000b). The effect of HP treatment of milk on micelle size is temperature-dependent. While treatment of reconstituted skim milk at 250 MPa at 201C had no significant effect on micelle size, treatment at 401C increased micelle size and treatment at 41C reduced it (Gaucheron et al., 1997). After treatment of milk at 250 MPa at 401C, two distinct micelle populations were observed by TEM, one with an average size similar to those in samples treated at 41C or 201C, the other being considerably larger, giving an increase in average size. There was a temperature-independent decrease in micelle size on treatment >450 MPa (Gaucheron et al., 1997). Temperature-related differences in turbidity of raw (Buchheim et al., 1996a) or pasteurised (Schrader & Buchheim, 1998) skim milk were more significant o300 MPa than at higher pressures. The effect of HP treatment at 51C or 101C on turbidity was probably related to solubilisation of CCP, while the increase in average micelle size or turbidity

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after HP treatment of milk at 401C may be due to interactions between fragments of casein micelles and HP-denatured whey proteins (Buchheim et al., 1996a; Gaucheron et al., 1997; Schrader & Buchheim, 1998). Pressure-induced whey protein-casein interactions, analogous to those caused by heat treatment, probably have a significant influence on the changes observed in casein micelles in HP-treated milk. When mixtures of k-casein and b-lg were HP treated at 400 MPa, the presence of b-lg reduced the susceptibility of k-casein to subsequent hydrolysis by chymosin, indicating interactions between the proteins (Lopez-Fandino, Ramos, & Olano, 1997). Heat treatment of milk before HP treatment influences the HP-induced effects on casein micelles. HP treatment (100–500 MPa) of ultra-high temperature (UHT)-treated skim milk reduced its turbidity, but to a lesser extent than in raw or pasteurised skim milk. This suggests that casein micelles in the latter types of milk are more sensitive to pressure than heat-induced caseinwhey protein complexes in UHT-treated milk (Buchheim et al., 1996a; Schrader & Buchheim, 1998). The increase in turbidity observed on HP treatment of raw or pasteurised milk at 401C was not observed by these authors for UHT milk, possibly because HP-induced interactions between casein micelle fragments and whey proteins are not significant in UHT milk, in which whey proteins are already denatured. In contrast to UHT milk, HP-induced shifts in the turbidity of pasteurised skim milk are not reversible on storage after treatment, which may be linked to the fact that in systems pre-treated at relatively low temperature, such as pasteurised milk, whey proteins interact with micelle fragments during HP treatment, thereby limiting the surface available for reaggregation of micelle fragments. However, in UHT milk denatured whey proteins are covalently bound to micellar casein before HP-induced disintegration of casein micelles occurs, preventing such interactions (Schrader & Buchheim, 1998). Lee, Anema, Schrader, and Buchheim (1996) and Anema, Lee, Schrader, and Buchheim (1997) observed that increasing the calcium concentration in a calcium caseinate suspension increased the resistance of micelles in the system to HP-induced disruption. This was suggested to be related to the fact that introduction of more calcium to the system shifts in the calcium equilibrium from the soluble to the colloidal phase. Schrader and Buchheim (1998) suggested that changes in the casein micelles in milk following HP treatment are due to three discrete processes: 1. Irreversible HP-induced dissolution of heat-precipitated CCP formed by severe heat treatment of milk, such as UHT-processing. 2. HP-induced partial dissolution of indigenous CCP, resulting in disintegration of casein micelles

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or casein-whey protein aggregates, which is considered to be largely reversible. 3. HP-induced denaturation of whey proteins, to an extent dependant on pressure, temperature and the initial proportion of native whey protein. This process is superimposed on the disintegration of casein micelles under certain conditions and can increase the average particle size but cannot occur in severely heat-treated milk, in which whey proteins are already denatured. This model explains most of the results reported by Desobry-Banon et al. (1994), Buchheim et al. (1996a), Gaucheron et al. (1997) and Needs et al. (2000b). HP treatment (100–400 MPa) markedly increased the transfer of individual caseins from the colloidal to the soluble phase of milk from several species (LopezFandino et al., 1998). In bovine milk, dissociation of the caseins was in the order b > k > as1 > as2 ; whereas in caprine and ovine milk, the order was k > b > as1 > as2 : Extensive HP-induced dissociation of k-casein in caprine milk was also reported by Law et al. (1998). The order of dissociation of caseins largely corresponds to the serine phosphate content of the caseins, indicating that caseins which are more tightly bound to CCP dissociated to a lesser extent. In bovine milk, the greater dissociation of b-casein compared to k-casein, even though the former is more phosphosylated, may be related to the fact that some of the b-casein is not crosslinked by CCP in casein micelles (Aoki, Yamada, & Kako, 1990). Dissociation of individual caseins in milk under pressure (400 MPa) is affected by pH; relative increases in the levels of soluble caseins were higher in milk adjusted to pH 5.5 or 7.0 than in milk at pH 6.7 (Arias et al., 2000), probably due to the solubilisation of CCP or increased electrostatic repulsion, respectively. 4.5. Milk fat Relatively few studies have examined the effects of HP on milk fat. Crystallisation of milk fat in cream, as studied by TEM, is induced by HP treatment at 100–400 MPa, the effect being greatest at 200 MPa (Buchheim & Abou El-Nour, 1992; Buchheim, Schutt, . & Frede, 1996b). HP-treated cream had a higher solid fat content than untreated cream, also with a maximum effect at 200 MPa (Buchheim et al., 1996a, b). TEM showed no morphological differences between milk fat crystals formed during HP treatment or by conventional temperature-induced crystallisation at atmospheric pressure; pressures up to 400 MPa did not destabilise the cream. On comparing milk fat emulsions of identical fat composition and content but different average fat droplet sizes, Buchheim et al. (1996a, b) observed that reduced globule size delayed crystallisation of milk fat at both ambient and high pressures.

The induction of crystallisation of milk fat by HP is probably due to the fact that the solid/liquid transition temperature of milk fat is shifted to higher values under pressure. At up to 200 MPa, the crystallisation and melting temperatures of milk fat are increased by 16.31C and 15.51C/100 MPa, respectively (Frede & Buchheim, 2000). The lower extent of milk fat crystallisation at higher pressures (>350 MPa) may be due to reduced crystal growth because of reduced molecular mobility at higher pressures (Buchheim et al., 1996a, b). Kanno, Uchimura, Hagiwara, Ametani, and Azuma (1998) investigated the effects of HP on milk fat globules and the milk fat globule membrane (MFGM). Pressures o400 MPa did not affect the mean diameter or the size distribution of milk fat globules, but higher pressures (400–800 MPa) increased the former and broadened the latter. Since no increase in products of lipolysis was detected, no damage to the MFGM was thought to have occurred under HP, as suggested also by Buchheim et al. (1996b). 4.6. Milk enzymes Inactivation of indigenous enzymes in milk due to HP treatment is of interest due to their possible use as markers of the severity of treatment, analogous to the use of alkaline phosphatase as an index of pasteurisation of milk. Alkaline phosphatase appears quite pressureresistant, with no inactivation in raw milk after treatment at 400 MPa for 60 min at 201C (LopezFandino et al., 1996), 50% inactivation after 90 min at 500 or 10 min at 600 MPa and 100% inactivation after 8 min at 800 MPa (Rademacher, Pfeiffer, & Kessler, 1998). HP treatment of milk at higher temperatures generally increases inactivation of alkaline phosphatase (Seyderhelm, Boguslawski, Michaelis, & Knorr, 1996; Ludikhuyze, Claeys, & Hendrickx, 2000). Indigenous milk lactoperoxidase (Lopez-Fandino et al., 1996; Seyderhelm et al., 1996), phosphohexoseisomerase (Rademacher et al., 1998) and g-glutamyltransferase (Rademacher et al., 1998) are also resistant to pressures up to 400 MPa at 20–251C. The relatively high stability of these enzymes makes them unsuitable for use as markers for the severity of HP treatment of milk. Also of interest is the barostability of indigenous milk proteinases, such as plasmin. The barostability of plasmin may be monitored either directly, using specific enzyme assays, or indirectly, through incubation of milk or protein systems containing plasmin following HP treatment and analysis of proteolysis products, which yields information both on changes in enzyme activity and the susceptibility of substrate (casein) to proteolysis. Using the former approach, it has been shown that purified plasmin in phosphate buffer, in the presence or absence of sodium caseinate, was almost completely resistant to pressures up to 600 MPa (Scollard, Beres-

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ford, Murphy, & Kelly, 2000a); however, in the presence of b-lg, considerable inactivation occurred >400 MPa, indicating that the presence of b-lg destabilises the enzyme to pressure. Treatment of raw milk at 400 MPa reduced plasmin activity, with inactivation being far more significant at elevated temperature, e.g. 601C (Garcia-Risco et al., 2000; Scollard et al., 2000a). The level of proteolysis in HP milk treated at 100–400 MPa and then held for 48 h at 371C was identical to that in raw milk; the pattern of proteolysis was consistent with high residual plasmin activity (Lopez-Fandino et al., 1996). Proteolysis in milk during incubation at 371C was more extensive in milk treated at 300 MPa than in untreated milk, which may be the result of a combination of disruption of casein micelles, exposing surface area for access of proteolytic enzymes, with little inactivation of plasmin. After treatment at >300 MPa, the rate of proteolysis was lower than in untreated milk, probably due to increased inactivation of plasmin (Scollard et al., 2000b).

5. Effects of high pressure on some properties of milk 5.1. Appearance and pH of milk Effects of HP on milk mineral balance may alter its pH. Although Johnston et al. (1992b) reported no significant effect of pressure up to 600 MPa on the pH of skim bovine milk, several subsequent studies have observed increases, of varying magnitude, in the pH of HP treated caprine milk (De la Fuente et al., 1999) or raw (Buchheim et al., 1996a), pasteurised (Schrader et al., 1997; Schrader & Buchheim, 1998) or UHTtreated (Buchheim et al., 1996a; Schrader & Buchheim, 1998) bovine milk. Treatment at higher pressures or lower temperatures increased the extent of the pH-shift, which was less in raw or pasteurised milk than in UHTtreated milk and may thus be related to dissolution of CCP. HP-induced shifts in pH were completely reversed in pasteurised milk after 4 h at ambient pressure and temperature (Schrader & Buchheim, 1998); however, a shift of about 0.05 units persisted in UHT milk, probably due to the presence of heat-precipitated CCP, which dissociates irreversibly from the casein micelle on HP treatment (Schrader & Buchheim, 1998). The reversibility of HP-induced shifts in pH could explain the disparity in results from different studies. HP-induced changes in the Hunter L-value of milk, generally regarded as a measure of the whiteness, are due mainly to the disintegration of casein micelles. Treatment at 200 MPa at 201C had only a slight effect on the L-values, but treatment at 250–450 MPa significantly decreased the L-value of pasteurised or reconstituted skim milk. Treatment >450 MPa had little further effect on the L-values (Johnston et al.,

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1992b; Desobry-Banon et al., 1994; Gaucheron et al., 1997). 5.2. Rennet coagulation properties of milk Several investigators have studied the effects of HP on different parameters related to the rennet coagulation of milk, such as the rennet coagulation time (RCT), the time at which the coagulum is firm enough for cutting (or gelation time), and the firmness of the gel at cutting. In an early study, treatment at o150 MPa had no effect on the RCT of reconstituted skim milk, but the RCT was reduced markedly after treatment at 200–600 MPa (Desobry-Banon et al., 1994). The authors suggested that the decreased RCT was related to a reduction in casein micelle size, leading to increased specific surface area and increased probability of interparticle collision. However, these results were only partially confirmed by later studies, which showed that treatment of raw milk at up to 200 MPa for 30 min reduced the RCT, while higher pressures, up to 600 MPa, resulted in RCT values close to those for untreated milk (Lopez-Fandino et al., 1996, 1997; Johnston, Murphy, Rutherford, & McElhone, 1998; Needs et al., 2000b). The RCT of HP-treated milk is also affected by the duration of treatment at certain pressures. While treatment at 200 MPa for times in the range 10–60 min had similar effects on RCT, treatment at 400 MPa for 10 min reduced RCT but longer treatment times increased RCT considerably, with the value after 60 min being considerably higher than that for untreated milk (Lopez-Fandino et al., 1996, 1997). The RCT of HP-treated milk is affected by the temperature of treatment and the pH of the milk. Treatment at X200 MPa at 601C or X300 MPa at 501C hindered rennet coagulation of milk (Lopez-Fandino & Olano, 1998a). Acidification of milk (pH 5.5 or 6.0) prior to HP treatment decreased its RCT whereas alkalinisation (pH 7.0) had the opposite effect (Arias et al., 2000). HP treatment of pH-adjusted samples at 200 MPa decreased the RCT, whereas treatment at 400 MPa increased this parameter, compared to unpressurised milk. However, readjustment of the pH to 6.7 after HP treatment resulted in RCT values similar to those for milk maintained at this pH. This probably indicates an important role in shifts in the calcium equilibrium in milk on effects of HP on the RCT of milk. The effect of HP on the time required for renneted milk to become firm enough for cutting has been reported to be broadly parallel to changes in RCT. Treatment of raw milk at 200 MPa for 30 min reduced gelation time, but higher pressures, up to 400 MPa, resulted in a value close to that of untreated milk (Lopez-Fandino et al., 1996, 1997). As for the RCT,

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gelation time was dependent on the duration of treatment at certain pressures. Gel firmness was unaffected by HP treatment of milk at 100, 200 or 400 MPa for 30 min, but treatment at 300 MPa increased this parameter (Lopez-Fandino et al., 1996, 1997). An increase in gel firmness was also observed for milk treated for 10 min at 200 MPa, but longer treatments (up to 60 min) caused no further change. Treatment at 400 MPa for 10 min also increased gel firmness, but longer treatment times reduced this value, with the final value (after 60 min) being considerably lower than that for untreated milk. Effects of HP treatment on the rennet coagulation properties of milk vary with the species of origin. The RCT and gelation time of ovine milk decreased slightly after treatment at 100 MPa, increased significantly after treatment at 200–300 MPa, but decreased at 400 MPa to values slightly higher than those for untreated milk, whereas gel firmness was not affected by treatment at 100–400 MPa. The RCT, the gelation time and gel firmness of caprine milk were not significantly affected by pressures o200 MPa, but all three parameters increased after treatment at 300–400 MPa (LopezFandino & Olano, 1998b). In terms of understanding the influence of HP treatment on the rennet coagulation of milk, the effects of HP on the two phases of rennet coagulation, the enzymatic phase and the aggregation phase, have been studied separately. The primary stage, i.e., enzymatic hydrolysis of k-casein, may be followed by measurement of the release of the caseino-macropeptide (CMP). Less CMP was produced during renneting of milk treated at 400 MPa than from milk treated at 200 MPa or untreated milk (Lopez-Fandino et al., 1997). When considered in combination with the decreases in the RCT such treatments cause, this suggests acceleration of the aggregation phase of rennet coagulation in HPtreated milk. Temperature of treatment at 300 MPa did not influence the release of non-glycosylated CMP, but the amount of total CMP released after treatment at 40–601C was considerably smaller than after treatment at 251C, indicating a reduced release of glycosylated CMP from milk HP-treated at higher temperatures. The reduced release of CMP during the primary phase for samples treated at 400 MPa or at 300 MPa at >401C could be due to the interaction of HP-denatured b-lg with glycosylated k-casein, which would hinder the action of chymosin on k-casein (Lopez-Fandino et al., 1997; Lopez-Fandino & Olano, 1998a). In general support of this proposal, Needs et al. (2000b) reported that the RCT of milk HP-treated in the presence of the sulphydryl-blocking agent N-ethylmaleimide (NEM) was not affected, unlike samples without NEM, indicating that HP-induced effects on milk RCT involve –SH groups, presumably in interactions between b-lg and k-casein.

Needs et al. (2000b) found that the release of glycosylated CMP was unaffected by HP treatment; rates of aggregation and gel formation of milk treated at 200 MPa were considerably higher than for untreated milk, as suggested by Lopez-Fandino et al. (1997), but these rates decreased at higher pressures. Samples treated at 400 or 600 MPa reached higher gel strengths than samples treated at 200 MPa or untreated samples (Needs et al., 2000b). Two opposing mechanisms may contribute to the effect of HP on the aggregation phase: the direct effect of pressure on micelles, which promotes aggregation (following hydrolysis of k-casein), and denaturation of b-lg, which reduces the rate of aggregation (Needs et al., 2000b). 5.3. Cheesemaking properties of milk The application of HP in cheese technology has been evaluated in several studies. While HP can be applied at several stages during the cheesemaking process, such as during brining or ripening, only the effects of HP treatment of milk prior to cheesemaking will be discussed here. Two approaches have been used to determine the effect of HP treatment of milk on cheese yield: determination of wet curd yield in model systems (milk is coagulated with rennet, the coagulum cut, and curd separated from whey by centrifugation and weighed; Lopez-Fandino et al., 1996), or estimation of the yield of cheese (Drake, Harrison, Asplund, Barbosa-Canovas, & Swanson, 1997; Trujillo, Royo, Guamis, & Ferragut, 1999b). While treatment of milk at p200 MPa had no effect on wet curd yield, although denaturation of b-lg was observed at 200 MPa, treatment at 300–400 MPa significantly increased wet curd yield, by up to 20% (Lopez-Fandino et al., 1996; Lopez-Fandino & Olano, 1998a; Arias et al., 2000), and reduced both the loss of protein in whey and the volume of whey. Increased treatment time, up to 60 min, at 400 MPa increased wet curd yield and reduced protein loss in whey; the changes were greatest during the first 20 min of treatment (Lopez-Fandino et al., 1996). Similar effects of pressure on wet curd yield have been reported for caprine milk while, for ovine milk, wet curd yield increased following treatment X200 MPa (Lopez-Fandino & Olano, 1998b). The wet curd yield was B15% higher for bovine milk treated at 400 MPa at 401C than at 251C (LopezFandino & Olano, 1998a), while Arias et al. (2000) reported that wet curd yield and moisture retention in the curd increased with increasing pH of milk, in the range 5.5–7.0, for milk treated at 400 MPa. Similarly, the yield of Cheddar cheese made from HPtreated milk (3 cycles of 1 min at 586 MPa) was B7% higher than that from raw or pasteurised milk (Drake et al., 1997), while the yield of fresh cheese from caprine

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milk treated at 500 MPa was increased by B5% relative to pasteurised milk (Trujillo et al., 1999b). Increased cheese yield reflects primarily greater moisture retention, but also incorporation of some denatured b-lg (LopezFandino et al., 1996). These factors are inter-related, since incorporation of denatured b-lg in the curd increases the net negative charge, which enhances solvation (Arias et al., 2000). HP may also increase solvation by disrupting casein micelles into smaller aggregates (Gaucheron et al., 1997). Furthermore, the casein micelles and fat globules in HP-treated milk may not aggregate as closely as in untreated milk, therefore allowing more moisture to be entrapped in the cheese (Drake et al., 1997). Except for a higher moisture content in Cheddar cheese made from HP-treated milk than from raw or pasteurised milk, no other compositional differences were observed by Drake et al. (1997). However, according to Trujillo, Royo, Ferragut, and Guamis (1999a) and Trujillo et al. (1999b), fresh cheese made from HP-treated (500 MPa) caprine milk had a higher pH throughout ripening, higher salt and moisture contents and a lower fat content than cheese made from pasteurised milk. The lower fat content of HP-treated milk cheese may be due to altered properties of the rennet-induced gel (Trujillo et al., 1999a), such as the tighter network of casein strands formed on rennet coagulation of HP-treated milk (Needs et al., 2000b). HP-induced effects on the salt content of fresh cheese may be due to differences in the moisture content, since the amount of salt absorbed by cheese during brinesalting is proportional to its moisture content (Geurts, Walstra, & Mulder, 1974). During ripening, the concentration of free fatty acids was higher in cheese made from HP-treated caprine milk than in that from pasteurised milk, probably due to a greater inactivation of milk lipase caused by pasteurisation than by HP treatment (Trujillo et al., 1999a). While no major differences in the electrophoretic pattern of pH 4.6-insoluble peptides were observed between these cheeses, levels of pH 4.6-soluble and TCA-soluble N increased during ripening at a slightly higher rate in cheese made from pasteurised milk, possibly due to differences in salt content, which influences water activity and hence proteolytic activity in cheese (Trujillo et al., 1999a). Irrespective of improvements in cheese yield and alterations to cheese composition resulting from HP treatment of milk, the quality of the resulting cheese is probably the major factor in determining the potential application of HP treatment of milk in cheesemaking. Drake et al. (1997) observed no significant differences in flavour between Cheddar cheeses made from pasteurised or HP-treated milk, but the latter had a pasty, weak texture, probably due to its higher moisture content. Sensory assessment of fresh cheese made from HP-

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treated or pasteurised caprine milk indicated that the cheeses were of similar quality (Trujillo et al., 1999a). Therefore, HP treatment of milk does not appear to significantly adversely affect its overall quality, although there are relatively few published studies in this area. 5.4. Yoghurt-making properties of milk Several studies on the effects of HP on the acid coagulation properties of milk have been reported. These fall into two categories: acid coagulation after HP treatment, or acid coagulation under HP. Rigidity and force at the breaking point of acidinduced gels formed from HP-treated milk increased with increasing pressure and of treatment time (Johnston, Austin, & Murphy, 1992a, 1993). The volume of whey released after cutting the gel decreased with increasing pressure, indicating greater resistance to syneresis, consistent with microscopic observations of a greater density of network strands in gels from milk treated at 600 MPa (Johnston et al., 1993). Acidification of HP-treated reconstituted skim milk with glucono-dlactone (GDL) to pH 4.5 showed that HP-treated milk coagulated at a higher pH and yielded a stronger gel than untreated milk (Desobry-Banon et al., 1994). Similarly, when yoghurt was made from HP-treated milk (350 or 500 MPa at 251C or 551C) by fermentation, coagulation occurred at a higher pH and yoghurt firmness was higher compared to yoghurt made from pasteurised or heat-treated (5 min at 951C) milk (Ferragut, Martinez, Trujillo, & Guamis, 2000). In yoghurt made from milk treated at 600 MPa, casein micelles appeared as smooth-surfaced particles and formed densely packed strands (Needs et al., 2000a). Despite intimate intermicellar connections, the micelles appeared not to have coalesced, and interspersed within the matrix were clumps of dense amorphous material. In contrast, in yoghurt made from heat-treated (20 min at 851C) milk, the micelles were separated by dense filamentous projections from their surface. Yoghurt made from HP-treated milk had a higher storage modulus (G0 ) than yoghurt made from heat-treated milk, which may reflect a greater number of strong bonds, such as short-range micelle–micelle bonds or interactions, in the network in the former case. The firmness of yoghurt made from HP-treated milk (Needs et al., 2000a) increased with increasing treatment pressure and was higher for yoghurt made from milk HP-treated at 551C than that treated at 101C or 251C (Ferragut et al., 2000). This increased firmness may be linked to disruption of casein micelles, resulting in a greater effective area for surface interaction. However, HP treatment of milk at 551C resulted in little disruption of casein micelles, indicating that another important factor was probably whey protein denaturation. The level of syneresis in yoghurt made from milk HP-treated

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at 101C at 200–500 MPa or at 10–551C at 200 MPa was comparable to that made from pasteurised milk, whereas HP treatment of milk at 350 or 500 MPa at 251C or 551C gave similar syneretic properties as heattreatment (5 min at 951C). The level of syneresis in yoghurt made from HP-treated milk did not change during storage for 20 d at 41C, whereas syneresis of pasteurised or heat-treated milk yoghurt increased during storage (Ferragut et al., 2000). In a second group of studies, acid coagulation of milk under pressure was examined. Milk to which sufficient GDL was added to reduce the pH to 5.5 after 60 min at atmospheric pressure coagulated during HP treatment for >40 or >10 min at 100 or 200 MPa, respectively, and increasing pressure or temperature reduced the coagulation time (Schwertfeger & Buchheim, 1998, 1999). A large difference in the structure of the coagula was evident. Treatment of milk at 100 MPa for 40 min resulted in coarse floccules, whereas milk treated at 200 MPa for 10, 20 or 40 min at 200 MPa formed fine floccules, a fine-stranded coherent coagulum or a compact, fine-stranded sediment, respectively. Schwertfeger and Buchheim (1998) suggested that several factors influenced the type of acid coagulum formed during HP treatment of milk: disintegration of casein micelles, loss of the electrostatic stabilisation of casein micelles at around pH 5.3 (Banon & Hardy, 1992), destruction of the hydration sphere around micelles at >150 MPa, and increased frequency of particle collisions during HP treatment.

6. Conclusions and future research needs The main effects of HP treatment appear to include casein micelles, salts and whey proteins in milk, resulting in increased pH and reduced Hunter L-value and turbidity of milk following HP treatment. HP treatment of milk before cheese manufacture may have potential for commercial application since it appears to reduce RCT and increase cheese yield. Another possible application is in yoghurt production, due to improvement of the texture of products made from HP-treated milk. However, further research is required to evaluate the full commercial potential of HP treatment of milk through complete understanding of the effects of pressure. Several aspects have received only little attention to date, such as the reversibility of HP-induced changes in milk, the stability of HP-treated milk during subsequent storage, the heat and alcohol stabilities and age-gelation behaviour of HP-treated milk. Regarding HP-induced inactivation of enzymes, two points remain undefined: indigenous milk enzymes as markers for the effectiveness of HP treatment, and effects on milk enzymes of significance for the quality of

milk and dairy products (lipases, psychrotrophic enzymes). The effects of HP on lactose have not been studied thus far, although HP treatment of milk may affect the Maillard reaction or the mutarotation equilibrium of lactose. Most research has focussed on the effects of HP on skim milk, thus the influence of milk fat on the effects of HP on other constituents and the properties of milk is another aspect of interest as are creaming and lipid oxidation in HP-treated milk, not investigated to date. Effects of specific HP-processing conditions such as HP treatment at high (>501C) or sub-zero temperatures, cycled HP treatment and repeated HP treatment with holding periods between treatments at atmospheric pressure also remain to be studied for milk.

Acknowledgements Funding for the preparation of this review was provided by the Irish Government under the National Development Plan 2000–2006.

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