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Dissociation of caseins in high pressure-treated bovine milk
ARTICLE IN PRESS
International Dairy Journal 14 (2004) 675–680
Dissociation of caseins in high pressure-treated bovine milk Thom Huppertz, Patrick F. Fox, Alan L. Kelly* Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland Received 10 July 2003; accepted 30 November 2003
Abstract In this study, reversibility of high pressure (HP)-induced solubilisation of as1 - and b-casein and HP-induced changes in micellar hydration and levels of ultracentrifugally-sedimentable solids in raw skim milk were examined. HP treatment of milk at 100– 600 MPa resulted in considerable solubilisation of as1 - and b-caseins, with the extent of solubilisation reaching a maximum around 250 MPa. HP-induced solubilisation of caseins was probably a result of solubilisation of colloidal calcium phosphate and disruption of hydrophobic interactions. On storage of HP-treated milk at 5 C, dissociation of caseins was largely irreversible but at 20 C, considerable reassociation of caseins was observed. Hydration of the casein micelles was increased by HP treatment at 100– 600 MPa, possibly due to HP-induced interactions between caseins and whey proteins; these changes were more extensive at higher pressures. The level of ultracentrifugally sedimentable solids was reduced by HP treatment, with a minimum occurring at 250 MPa; Changes in micellar hydration and the level of ultracentrifugally sedimentable solids were largely irreversible at 5 C, but partially reversible at 20 C. These results indicate that HP treatment increases levels of as1 - and b-caseins in the soluble phase of milk and produces casein micelles with different properties compared to those in untreated milk; therefore, HP treatment may have a considerable influence on the processing characteristics of milk. r 2004 Elsevier Ltd. All rights reserved. Keywords: High pressure; Milk; Casein micelles; Dissociation; Micellar hydration
1. Introduction Horne (1998) proposed a model of the casein micelle which is dependent on a balance between electrostatic repulsion and hydrophobic interaction; in this model, colloidal calcium phosphate (CCP) crosslinks the caseins and neutralises negatively charged phosphoserine groups, allowing the formation of hydrophobic interactions between caseins. In recent years, high pressure (HP) treatment has attracted considerable interest in dairy research. Under pressure, hydrophobic and electrostatic interactions between proteins are disrupted (Mozhaev, Heremans, Frank, Masson, & Balny, 1994, 1996) and CCP is solubilised (Buchheim, Schrader, Morr, Frede, & Schutt, . 1996; Schrader, Buchheim, & Morr, 1997; Lopez-Fandino, De la Fuente, Ramos, Lopez-Fandino & Olano, 1998; De la Fuente, Olano, Casal & Juarez, 1999); as a result, considerable changes
*Corresponding author. Tel.: +353-21-4903405; fax: +353-214270213. E-mail address: [email protected] (A.L. Kelly). 0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2003.11.009
in the size, structure and composition of the casein micelles occur. Casein micelle size is affected only slightly by HP treatment at pressures p200 MPa at 20 C (DesobryBanon, Richard, & Hardy, 1994; Needs, Stenning, Gill, Ferragut, & Rich, 2000b; Huppertz, Fox, & Kelly, 2004a). HP treatment at 250 MPa increases average micelle size by B30% (Huppertz et al., 2004a; Huppertz, Grosman, Fox, & Kelly, 2004b) and pressures X300 MPa reduce micelle size by B50% (DesobryBanon et al., 1994; Gaucheron, Famelart, Mariette, Raulot, Michel, & Le Graet, 1997; Needs et al., 2000b; Huppertz et al., 2003a, b). HP-induced changes in casein micelle size are generally irreversible on subsequent storage of the milk, except for the increase in micelle size at 250 MPa, which is partially reversible (Huppertz et al., 2004a). At pressures >100 MPa, denatured b-lactoglobulin (b-lg) interacts with casein micelles (Needs, Capellas, Bland, Manoj, McDougal, & Paul, 2000a; Scollard, Beresford, Needs, Murphy, & Kelly, 2000; Huppertz et al., 2004a). As a result of the disruptive effect of HP on casein micelles, increased levels of all caseins in the soluble phase of milk have been reported,
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with levels of soluble b- and k-caseins being the highest (Lopez-Fandino, et al., 1998; Arias, Lopez-Fandino, & Olano, 2000). This study investigated reversibility of HP-induced solubilisation of as1 - and b-caseins. The effect of HP treatment and subsequent storage on the weight of the ultracentrifugal pellet recovered from milk, hydration of casein micelles, and the level of sedimentable solids was also studied.
2. Materials and methods 2.1. Milk supply Raw whole bovine milk was obtained from a local dairy (CMP Dairies, Cork, Ireland) and skimmed by centrifugation at 2000 g for 20 min at 20 C, followed by filtration through glass wool to remove fat particles. Sodium azide (0.05% w/v) was added to the skimmed milk to prevent microbial growth. 2.2. High pressure treatment Milk samples (B250 mL) were placed in polyethylene bags and vacuum-sealed, placed in a second polyethylene bag containing B100 mL water, vacuum-sealed, and stored at 20 C for no longer than 4 h prior to HP treatment. Samples were HP-treated in a Stansted Fluid Power Iso-lab 900 HP food processor (Stansted Fluid Power, Stansted, Essex, UK), using a 90:10 mixture of ethanol and castor oil as pressurising fluid. This pressure vessel has a 2 L capacity and an internal diameter of 100 mm. Pressure was increased at a rate of 300 MPa min1 to 100, 250, 400 or 600 MPa, maintained at the desired pressure for 30 min and released at a rate of 300 MPa min1. The temperature of the vessel of the HP unit was thermostatically controlled at 20 C throughout treatment. An untreated control sample was maintained at atmospheric pressure at 20 C. Following HP treatment, samples were divided into two portions, transferred to sterile containers, and held at 5 C or 20 C for up to 48 h. The samples taken at 0 h were from the portion maintained at 20 C.
micellar phase, and the supernatant and pellet were weighed. 2.4. Determination of the level of as1 - and b-caseins in the soluble phase of milk Levels of as1 - and b-caseins in the soluble phase of milk were determined by urea polyacrylamide gel electrophoresis (Urea-PAGE) analysis (Andrews, 1983) of the ultracentrifugal supernatant of milk; gels were stained by the method of Blakesley and Boezi (1977). The level of as1 - or b-casein in the soluble phase of milk was quantified by densitometric analysis using Total Lab V1.10 software (Nonlinear Dynamics, Newcastleupon-Tyne, UK) and expressed as a percentage of the total level of as1 - or b-casein in untreated milk at 0 h. 2.5. Determination of the moisture content of the ultracentrifugal pellet The moisture content of the ultracentrifugal pellet was determined using a CEM smart system 5 moisture analyser (CEM Corporation, Matthews, NC, USA), equipped with an automated analytical balance. Pellet samples (2–4 g) were dried using microwave energy until a constant weight was obtained (generally after 3–5 min; the weight of a sample was assumed to be constant when changes in weight were o0.9 mg over 10 s); moisture content was determined in duplicate for all pellet samples. 2.6. Level of micellar hydration and sedimentable solids The level of micellar hydration (g H2O g1 solids) was calculated as follows: Micellar hydration ¼ Moisture content of pellet ð%; w=wÞ : ð100 Moisture content of pellet; %; w=wÞ The level of sedimentable solids in milk (g 100 g1 milk) was calculated as follows: Sedimentable solids ¼ Pellet weightðg 100 g1 milkÞð100 moisture content of pellet; %; w=wÞ 100
2.3. Separation of the soluble and micellar phases of milk
2.7. Statistical analysis
Immediately after HP treatment and after storage for 24 or 48 h, the soluble and micellar phases of milk were separated by ultracentrifugation at 100,000 g for 60 min at 20 C, using an Optimat LE-80 K preparative ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA, USA), equipped with a Beckman type 50.2 Ti rotor. After centrifugation, the supernatant, i.e., the soluble phase, was carefully separated from the pellet, i.e., the
All experiments were performed in triplicate on individual milk samples. Statistical analysis was performed using a randomised block design, using Minitab version 12 (Minitab Ltd., Coventry, UK). The effect of HP treatment and subsequent storage on the percentage of as1 - and b-caseins in the soluble phase of milk, the weight of the ultracentrifugal pellet, micellar hydration and the level of sedimentable solids was examined using
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the General Linear Model technique, with Tukey’s pairwise comparisons at a 95% confidence level.
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but had little effect on the percentage of soluble b-casein in milk treated at 100–600 MPa (Table 1). On storage at 20 C for 24 or 48 h, levels of soluble b-casein decreased in untreated milk and in milk treated at 100–600 MPa; greatest reductions were found for milk treated at 250– 600 MPa, and the extent of these reductions increased with storage time.
3. Results 3.1. Effect of HP treatment and subsequent storage on levels of as1 - and b-caseins in the soluble phase of milk
3.2. Effect of HP treatment on the sedimentable phase of milk
In untreated raw skim milk at 0 h, B3 or 6% of total as1 - or b-casein, respectively, was found to be in the soluble phase. On HP treatment of raw skim bovine milk at 100–600 MPa for 30 min, considerable increases in the levels of as1 - and b-caseins in the soluble phase of milk were observed (Table 1). Levels of both as1 - and b-caseins in the soluble phase increased with pressure up to 250 MPa, to B12 or 18% of total as1 - or b-casein in milk, respectively. After treatment at 400 or 600 MPa, levels of these caseins in the soluble phase of milk were slightly lower than those in milk treated at 250 MPa, but remained considerably higher than those in untreated milk or milk treated at 100 MPa. Although the absolute percentage of b-casein in the soluble phase of HPtreated milks was higher than that of as1 -casein in all samples, actual HP-induced increases in the percentage of soluble b-casein were less extensive than increases in the level of soluble as1 -casein (Table 1). On storage at 5 C for 24 or 48 h, the percentage of soluble as1 -casein in milk treated at 600 MPa at decreased; storage at 20 C for up to 48 h caused a reduction in the percentage of soluble as1 -casein in all samples (Table 1). Storage at 5 C for up to 48 h increased the level of soluble b-casein in untreated milk,
Compared to untreated milk, HP treatment at 100 or 250 MPa reduced the weight of ultracentrifugal pellet recovered from milk, whereas treatment at 400 or 600 MPa considerably increased the weight of the pellet (Table 2). Storage of milk samples at 5 or 20 C for up to 48 h did not affect the weight of the ultracentrifugal pellet from untreated milk or milk treated at 100 MPa; however, in milk treated at 250 MPa, storage at 20 C for 48 h increased the weight of the ultracentrifugal pellet. In milk treated at 400 or 600 MPa, storage at 5 C for 48 h increased the weight of the ultracentrifugal pellet (Table 2). Compared to untreated milk, treatment at 100 MPa had little effect on micellar hydration, but treatment at 250, 400 or 600 MPa increased this parameter significantly (Table 2). Storage at 5 C had no significant effect on micellar hydration in untreated milk and milk treated at 100–600 MPa. Storage at 20 C did not affect micellar hydration of untreated milk or milk treated at 100 MPa, but reduced hydration considerably for milk treated at 250–600 MPa, the extent of the reduction being larger in milk treated at higher pressures (Table 2).
Table 1 Effect of HP treatment at 100–600 MPa for 30 min at 20 C, followed by storage for 0, 24 or 48 h at 5 or 20 C, on the percentage of as1 - or b-caseins in the soluble phase of raw skim bovine milk Storage time (h)
Storage temp (C)
Untreated
Pressure (MPa) 100
250
400
600
% of as1 -casein in soluble phase 0 — 24 5 20 48 5 20
3.270.3aA 2.970.2abA 2.970.1abA 2.770.2abA 2.670.2bA
7.770.5aB 8.070.2aB 7.170.4bB 8.070.4aB 6.470.1cB
12.170.7aC 11.670.3aC 9.070.3bC 11.670.3aC 8.770.6bC
11.270.8aCD 10.170.8aCD 8.070.6bD 10.070.7aD 7.770.7bCD
10.670.6aD 9.670.1bD 7.470.9cBD 9.470.8abD 7.370.7cD
% of b-casein in soluble phase 0 — 24 5 20 48 5 20
6.370.4aA 9.270.3bA 5.670.5acA 9.070.4bA 5.270.6cA
10.270.3aB 10.470.2aB 8.670.9bB 10.370.9aA 7.670.9bB
17.870.9aC 17.270.2aC 12.870.7bC 17.570.6aB 12.870.7bC
15.970.8aD 15.670.3aD 12.070.8bC 15.970.7aC 11.070.5bD
15.870.7aD 15.270.6aD 11.470.8bC 14.871.2aC 10.770.7bD
Values are expressed as a percentage of the total concentration of as1 - or b-casein in untreated milk, 7standard deviation, and are the average of triplicate experiments on three individual milk samples. a,b,c Values without a common lower-case superscript in a column were significantly different (Po0:05). Comparisons were performed separately for as1 - and b-casein. A,B,C,D Values without a common upper-case superscript in a row were significantly different (Po0:05).
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Table 2 Effect of HP treatment of raw skim bovine milk at 100–600 MPa for 30 min at 20 C, followed by storage at 5 C or 20 C for 0, 24 or 48 h, on the weight of the ultracentrifugal pellet (g 100 g1 milk), micellar hydration (g H2O g1 solids), and the level of ultracentrifugally-sedimentable solids (g 100 g1 milk) Storage time (h)
Storage temp ( C)
Pressure (MPa) Untreated
100
250
400
600
12.470.5aA 12.570.7aA 12.470.8aA 12.570.7aA 12.570.8aA
11.270.5aB 11.070.6aB 11.170.5aB 11.170.5aB 11.070.4aB
10.770.5aB 10.870.8abB 11.770.6bAB 9.570.8aC 11.670.8bAB
13.970.6aC 14.770.7aC 14.170.2aC 15.070.5bD 14.170.6aC
15.770.5aD 16.771.3abC 15.170.6aD 16.770.6bD 14.571.0aC
Micellar hydration (g H2O g1 solids) 0 — 24 5 20 48 5 20
2.570.2aA 2.670.1aA 2.570.1aA 2.770.0aA 2.670.1aA
2.570.2aA 2.670.2aA 2.470.2aA 2.670.1aA 2.570.1aA
3.370.3aB 3.470.3aB 2.970.2bB 3.370.3aB 2.970.2bB
3.870.2aBC 3.970.1aC 3.270.1bC 3.970.1aC 3.170.1bB
3.870.2aC 3.970.1aC 3.170.1bBC 3.970.1aC 3.070.1bB
Sedimentable solids (g 100 g1 milk) 0 — 24 5 20 48 5 20
3.670.3aA 3.570.3aA 3.570.3aA 3.470.2aA 3.570.3aA
3.270.3aAC 3.170.3aAC 3.270.3aA 3.070.2aA 3.270.2aAB
2.570.1aB 2.570.2aB 3.070.2bB 2.270.3aB 3.070.1bB
2.970.2aBC 3.070.2aC 3.470.1bA 3.170.2abA 3.570.2bA
3.370.2aAC 3.470.3abAC 3.770.2abA 3.470.3abA 3.670.3abA
1
Ultracentrifugal pellet (g 100 g 0 — 24 5 20 48 5 20
milk)
Values are the average of triplicate experiments on three individual milk samples, 7standard deviation. a,b Values without a common lower-case superscript in a column were significantly different (Po0:05). Comparisons were performed separately for the weight of the ultracentrifugal pellet (g 100 g1 milk), micellar hydration (g H2O g1 solids), and the level of ultracentrifugally sedimentable solids. A,B,C,D Values without a common upper-case superscript in a row were significantly different (Po0:05).
Finally, as a result of HP-induced changes in the weight of the ultracentrifugal pellet and micellar hydration, the level of sedimentable solids in milk was also altered. Compared to untreated milk, HP treatment at 100–600 MPa reduced the level of sedimentable solids, with the maximum reduction in this parameter after treatment at 250 MPa. Storage at 5 C did not significantly affect the level of sedimentable solids of untreated milk or milk treated at 100–600 MPa. Storage at 20 C had little effect on the level of sedimentable solids in untreated milk or milk treated at 100 MPa, but in milk treated at 250, 400 or 600 MPa, significant increases in this parameter were observed on storage at 20 C. Compared to untreated milk, levels of sedimentable solids were lower in all HP-treated milk samples, except for milk treated at 400 or 600 MPa and subsequently stored for 24 or 48 h at 20 C, for which the level of sedimentable solids was comparable to that in untreated milk.
4. Discussion The results of this study indicate that both the colloidal and soluble phases of milk are affected considerably by HP treatment; solubilisation of caseins, increases in micellar hydration and reductions in the
level of sedimentable solids were observed. On subsequent storage under certain conditions, extensive reversibility in some of these parameters occurred in some HP-treated samples. 4.1. High pressure-induced solubilisation of caseins HP-induced increases in levels of as1 - and, in particular, b-casein in the soluble phase of milk are consistent with reports by Lopez-Fandino et al. (1998) and Arias et al. (2000). The maximum increase in the level of soluble caseins observed after treatment at 250 MPa (Table 1) is also consistent with results of Lopez-Fandino et al. (1998). HP-induced solubilisation of caseins is probably due to solubilisation of CCP, which is responsible for crosslinking caseins and neutralising the negatively charged phosphoserine groups (Horne, 1998), and disruption of hydrophobic bonds (Mozhaev et al., 1994, 1996), which are responsible for binding individual caseins within the casein micelles (Horne, 1998). The increase in the level of b-casein in the soluble phase on storage of untreated milk at 5 C for up to 48 h was consistent with previous reports (Rose, 1968; Davies & Law, 1983; Aoki, Yamada, & Kako, 1990; Law, 1996), and is probably a result of weakening of hydrophobic bonds (Law, 1996; De la Fuente, 1998),
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which occurs at low temperatures (Walstra, 2003). The reversibility of HP-induced increases in levels of soluble as1 - and b-caseins on storage of HP-treated milk at 20 C may be related to the reformation of micellar particles from fragments of HP-disrupted casein micelles (Needs et al., 2000a; Huppertz et al., 2004a); this process is probably due to the reformation of hydrophobic bonds (Needs et al., 2000a). The limited reversal of HP-induced solubilisation of caseins on storage at 5 C is probably related to the fact that hydrophobic bonds are considerably weaker at lower temperatures (Walstra, 2003); this is consistent with observations that HPinduced increases in exposed hydrophobic surface in milk were irreversible on subsequent storage at 5 C for up to 8 days (Johnston, Austin, & Murphy, 1992). 4.2. High pressure-induced changes in the micellar phase of milk HP-induced increases in the weight of the ultracentrifugal pellet from milk treated at 400 or 600 MPa (Table 2) were due to an increased moisture content in these pellets, as the levels of sedimentable solids were reduced by treatment at such pressures (Table 2). The reduction in the amount of sedimentable solids on HP treatment of milk is probably the result of HP-induced disruption of casein micelles, resulting in increased levels of soluble caseins (Table 1; Lopez-Fandino et al., 1998; Arias et al., 2000) and minerals (Buchheim et al., 1996; Schrader et al., 1997; Lopez-Fandino et al., 1998; De la Fuente et al., 1999). The level of sedimentable solids was lowest in milk treated at 250 MPa (Table 2); this is consistent with observations of a maximum level of solubilisation of caseins (Table 1; Lopez-Fandino et al., 1998) and minerals (Lopez-Fandino et al., 1998) at 200 MPa. After treatment at 400 or 600 MPa, the level of sedimentable solids was higher than after treatment at 250 MPa; this is possibly due to the interactions of denatured b-lg with casein micelles, as observed in previous studies (Needs et al., 2000a; Scollard et al., 2000; Huppertz et al., 2004a). HP-induced increases in micellar hydration (Table 2) were consistent with observations by Gaucheron et al. (1997), and are probably, at least partially, related to HP-induced interactions between denatured b-lg and casein micelles; Imafidon and Farkye (1996) reported that on heating milk, casein micelle hydration is increased as a result of the formation of casein–whey protein complexes, resulting in a larger net negative charge. HP-induced reductions in casein micelle size after treatment at 300–800 MPa (Desobry-Banon et al., 1994; Gaucheron et al., 1997; Needs et al., 2000b; Huppertz et al., 2004a, b) may also influence micelle hydration; Sood, Sidhu, and Dewan (1976) observed that the hydration of casein micelles, centrifugally separated into different size distribution classes, was
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higher for smaller casein micelles. Furthermore, changes in the shape of micellar particles, i.e., from roughly spherical to irregularly shaped particles, as occurs on HP treatment of milk (Gaucheron et al., 1997; Needs et al., 2000b; Scollard et al., 2000), may cause an increase in the voluminosity of casein micelles (Walstra, 1979), a parameter closely related to, and often used as, a measure of the hydration of casein micelles (Sood et al., 1976). Small increases in the level of micellar hydration on storage of untreated or HP-treated milk at 5 C (Table 2) are in consistent with increased micellar voluminosity on cold storage of milk (Walstra, 1979, 1990), which Walstra (1979) suggested is possibly due to the dissociation of b-casein from the micelles, as occurred in untreated milk (Table 1). Partial reversal of HPinduced increases in pellet hydration and decreases in sedimentable solids on storage at 20 C of milk treated at 250–600 MPa (Table 2) is possibly related to the reformation of micellar particles from HP-disrupted casein micelles; this process would increase sedimentable solids because monomeric caseins or caseins in small aggregates are not sedimentable, unlike reformed micellar particles. Furthermore, such reassociation may also partially reverse the increase in net negative charge on the micelles, induced by interactions with denatured b-lg; thereby reducing solvation hydration of the micelles. At 5 C, only little reassociation of materials of HP-disrupted casein micelles occurs, as a result of which only little reversal of HP-induced increases in pellet hydration or decreases in sedimentable solids occurred (Table 2). After storage for up to 48 h at 20 C, the amount of sedimentable solids in milk treated at 400 or 600 MPa was comparable to that in untreated milk (Table 2), despite a considerable amount of b-lg associated with the casein micelles (Huppertz et al., 2004a); this indicates that HP-induced solubilisation of caseins is only partially reversible, in agreement with results in Table 1. This provides further support for the suggestions by Johnston, Austin, and Murphy (1993) and Needs et al. (2000a) that casein micelles in HP-treated milk have a considerably altered composition, structure, and possibly surface properties, compared to those in untreated milk.
5. Conclusions HP treatment of milk resulted in significant changes in both the soluble and colloidal phase of milk; in the soluble phase, considerable increases in the level of soluble caseins were observed, whereas in the colloidal phase of milk, micellar hydration increased and sedimentable solids decreased. Many of these changes were virtually irreversible on subsequent storage at 5 C, but largely reversible on storage at 20 C. These results
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clearly indicate that casein micelles in HP-treated milk differ considerably from those in untreated milk; this may have implications for products made from HPtreated milk.
Acknowledgements This research was funded by the Food Institutional Research Measure (FIRM), which is administered by the Irish Government under the National Development Plan 2000–2006.
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International Dairy Journal 14 (2004) 675–680
Dissociation of caseins in high pressure-treated bovine milk Thom Huppertz, Patrick F. Fox, Alan L. Kelly* Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland Received 10 July 2003; accepted 30 November 2003
Abstract In this study, reversibility of high pressure (HP)-induced solubilisation of as1 - and b-casein and HP-induced changes in micellar hydration and levels of ultracentrifugally-sedimentable solids in raw skim milk were examined. HP treatment of milk at 100– 600 MPa resulted in considerable solubilisation of as1 - and b-caseins, with the extent of solubilisation reaching a maximum around 250 MPa. HP-induced solubilisation of caseins was probably a result of solubilisation of colloidal calcium phosphate and disruption of hydrophobic interactions. On storage of HP-treated milk at 5 C, dissociation of caseins was largely irreversible but at 20 C, considerable reassociation of caseins was observed. Hydration of the casein micelles was increased by HP treatment at 100– 600 MPa, possibly due to HP-induced interactions between caseins and whey proteins; these changes were more extensive at higher pressures. The level of ultracentrifugally sedimentable solids was reduced by HP treatment, with a minimum occurring at 250 MPa; Changes in micellar hydration and the level of ultracentrifugally sedimentable solids were largely irreversible at 5 C, but partially reversible at 20 C. These results indicate that HP treatment increases levels of as1 - and b-caseins in the soluble phase of milk and produces casein micelles with different properties compared to those in untreated milk; therefore, HP treatment may have a considerable influence on the processing characteristics of milk. r 2004 Elsevier Ltd. All rights reserved. Keywords: High pressure; Milk; Casein micelles; Dissociation; Micellar hydration
1. Introduction Horne (1998) proposed a model of the casein micelle which is dependent on a balance between electrostatic repulsion and hydrophobic interaction; in this model, colloidal calcium phosphate (CCP) crosslinks the caseins and neutralises negatively charged phosphoserine groups, allowing the formation of hydrophobic interactions between caseins. In recent years, high pressure (HP) treatment has attracted considerable interest in dairy research. Under pressure, hydrophobic and electrostatic interactions between proteins are disrupted (Mozhaev, Heremans, Frank, Masson, & Balny, 1994, 1996) and CCP is solubilised (Buchheim, Schrader, Morr, Frede, & Schutt, . 1996; Schrader, Buchheim, & Morr, 1997; Lopez-Fandino, De la Fuente, Ramos, Lopez-Fandino & Olano, 1998; De la Fuente, Olano, Casal & Juarez, 1999); as a result, considerable changes
*Corresponding author. Tel.: +353-21-4903405; fax: +353-214270213. E-mail address: [email protected] (A.L. Kelly). 0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2003.11.009
in the size, structure and composition of the casein micelles occur. Casein micelle size is affected only slightly by HP treatment at pressures p200 MPa at 20 C (DesobryBanon, Richard, & Hardy, 1994; Needs, Stenning, Gill, Ferragut, & Rich, 2000b; Huppertz, Fox, & Kelly, 2004a). HP treatment at 250 MPa increases average micelle size by B30% (Huppertz et al., 2004a; Huppertz, Grosman, Fox, & Kelly, 2004b) and pressures X300 MPa reduce micelle size by B50% (DesobryBanon et al., 1994; Gaucheron, Famelart, Mariette, Raulot, Michel, & Le Graet, 1997; Needs et al., 2000b; Huppertz et al., 2003a, b). HP-induced changes in casein micelle size are generally irreversible on subsequent storage of the milk, except for the increase in micelle size at 250 MPa, which is partially reversible (Huppertz et al., 2004a). At pressures >100 MPa, denatured b-lactoglobulin (b-lg) interacts with casein micelles (Needs, Capellas, Bland, Manoj, McDougal, & Paul, 2000a; Scollard, Beresford, Needs, Murphy, & Kelly, 2000; Huppertz et al., 2004a). As a result of the disruptive effect of HP on casein micelles, increased levels of all caseins in the soluble phase of milk have been reported,
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with levels of soluble b- and k-caseins being the highest (Lopez-Fandino, et al., 1998; Arias, Lopez-Fandino, & Olano, 2000). This study investigated reversibility of HP-induced solubilisation of as1 - and b-caseins. The effect of HP treatment and subsequent storage on the weight of the ultracentrifugal pellet recovered from milk, hydration of casein micelles, and the level of sedimentable solids was also studied.
2. Materials and methods 2.1. Milk supply Raw whole bovine milk was obtained from a local dairy (CMP Dairies, Cork, Ireland) and skimmed by centrifugation at 2000 g for 20 min at 20 C, followed by filtration through glass wool to remove fat particles. Sodium azide (0.05% w/v) was added to the skimmed milk to prevent microbial growth. 2.2. High pressure treatment Milk samples (B250 mL) were placed in polyethylene bags and vacuum-sealed, placed in a second polyethylene bag containing B100 mL water, vacuum-sealed, and stored at 20 C for no longer than 4 h prior to HP treatment. Samples were HP-treated in a Stansted Fluid Power Iso-lab 900 HP food processor (Stansted Fluid Power, Stansted, Essex, UK), using a 90:10 mixture of ethanol and castor oil as pressurising fluid. This pressure vessel has a 2 L capacity and an internal diameter of 100 mm. Pressure was increased at a rate of 300 MPa min1 to 100, 250, 400 or 600 MPa, maintained at the desired pressure for 30 min and released at a rate of 300 MPa min1. The temperature of the vessel of the HP unit was thermostatically controlled at 20 C throughout treatment. An untreated control sample was maintained at atmospheric pressure at 20 C. Following HP treatment, samples were divided into two portions, transferred to sterile containers, and held at 5 C or 20 C for up to 48 h. The samples taken at 0 h were from the portion maintained at 20 C.
micellar phase, and the supernatant and pellet were weighed. 2.4. Determination of the level of as1 - and b-caseins in the soluble phase of milk Levels of as1 - and b-caseins in the soluble phase of milk were determined by urea polyacrylamide gel electrophoresis (Urea-PAGE) analysis (Andrews, 1983) of the ultracentrifugal supernatant of milk; gels were stained by the method of Blakesley and Boezi (1977). The level of as1 - or b-casein in the soluble phase of milk was quantified by densitometric analysis using Total Lab V1.10 software (Nonlinear Dynamics, Newcastleupon-Tyne, UK) and expressed as a percentage of the total level of as1 - or b-casein in untreated milk at 0 h. 2.5. Determination of the moisture content of the ultracentrifugal pellet The moisture content of the ultracentrifugal pellet was determined using a CEM smart system 5 moisture analyser (CEM Corporation, Matthews, NC, USA), equipped with an automated analytical balance. Pellet samples (2–4 g) were dried using microwave energy until a constant weight was obtained (generally after 3–5 min; the weight of a sample was assumed to be constant when changes in weight were o0.9 mg over 10 s); moisture content was determined in duplicate for all pellet samples. 2.6. Level of micellar hydration and sedimentable solids The level of micellar hydration (g H2O g1 solids) was calculated as follows: Micellar hydration ¼ Moisture content of pellet ð%; w=wÞ : ð100 Moisture content of pellet; %; w=wÞ The level of sedimentable solids in milk (g 100 g1 milk) was calculated as follows: Sedimentable solids ¼ Pellet weightðg 100 g1 milkÞð100 moisture content of pellet; %; w=wÞ 100
2.3. Separation of the soluble and micellar phases of milk
2.7. Statistical analysis
Immediately after HP treatment and after storage for 24 or 48 h, the soluble and micellar phases of milk were separated by ultracentrifugation at 100,000 g for 60 min at 20 C, using an Optimat LE-80 K preparative ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA, USA), equipped with a Beckman type 50.2 Ti rotor. After centrifugation, the supernatant, i.e., the soluble phase, was carefully separated from the pellet, i.e., the
All experiments were performed in triplicate on individual milk samples. Statistical analysis was performed using a randomised block design, using Minitab version 12 (Minitab Ltd., Coventry, UK). The effect of HP treatment and subsequent storage on the percentage of as1 - and b-caseins in the soluble phase of milk, the weight of the ultracentrifugal pellet, micellar hydration and the level of sedimentable solids was examined using
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the General Linear Model technique, with Tukey’s pairwise comparisons at a 95% confidence level.
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but had little effect on the percentage of soluble b-casein in milk treated at 100–600 MPa (Table 1). On storage at 20 C for 24 or 48 h, levels of soluble b-casein decreased in untreated milk and in milk treated at 100–600 MPa; greatest reductions were found for milk treated at 250– 600 MPa, and the extent of these reductions increased with storage time.
3. Results 3.1. Effect of HP treatment and subsequent storage on levels of as1 - and b-caseins in the soluble phase of milk
3.2. Effect of HP treatment on the sedimentable phase of milk
In untreated raw skim milk at 0 h, B3 or 6% of total as1 - or b-casein, respectively, was found to be in the soluble phase. On HP treatment of raw skim bovine milk at 100–600 MPa for 30 min, considerable increases in the levels of as1 - and b-caseins in the soluble phase of milk were observed (Table 1). Levels of both as1 - and b-caseins in the soluble phase increased with pressure up to 250 MPa, to B12 or 18% of total as1 - or b-casein in milk, respectively. After treatment at 400 or 600 MPa, levels of these caseins in the soluble phase of milk were slightly lower than those in milk treated at 250 MPa, but remained considerably higher than those in untreated milk or milk treated at 100 MPa. Although the absolute percentage of b-casein in the soluble phase of HPtreated milks was higher than that of as1 -casein in all samples, actual HP-induced increases in the percentage of soluble b-casein were less extensive than increases in the level of soluble as1 -casein (Table 1). On storage at 5 C for 24 or 48 h, the percentage of soluble as1 -casein in milk treated at 600 MPa at decreased; storage at 20 C for up to 48 h caused a reduction in the percentage of soluble as1 -casein in all samples (Table 1). Storage at 5 C for up to 48 h increased the level of soluble b-casein in untreated milk,
Compared to untreated milk, HP treatment at 100 or 250 MPa reduced the weight of ultracentrifugal pellet recovered from milk, whereas treatment at 400 or 600 MPa considerably increased the weight of the pellet (Table 2). Storage of milk samples at 5 or 20 C for up to 48 h did not affect the weight of the ultracentrifugal pellet from untreated milk or milk treated at 100 MPa; however, in milk treated at 250 MPa, storage at 20 C for 48 h increased the weight of the ultracentrifugal pellet. In milk treated at 400 or 600 MPa, storage at 5 C for 48 h increased the weight of the ultracentrifugal pellet (Table 2). Compared to untreated milk, treatment at 100 MPa had little effect on micellar hydration, but treatment at 250, 400 or 600 MPa increased this parameter significantly (Table 2). Storage at 5 C had no significant effect on micellar hydration in untreated milk and milk treated at 100–600 MPa. Storage at 20 C did not affect micellar hydration of untreated milk or milk treated at 100 MPa, but reduced hydration considerably for milk treated at 250–600 MPa, the extent of the reduction being larger in milk treated at higher pressures (Table 2).
Table 1 Effect of HP treatment at 100–600 MPa for 30 min at 20 C, followed by storage for 0, 24 or 48 h at 5 or 20 C, on the percentage of as1 - or b-caseins in the soluble phase of raw skim bovine milk Storage time (h)
Storage temp (C)
Untreated
Pressure (MPa) 100
250
400
600
% of as1 -casein in soluble phase 0 — 24 5 20 48 5 20
3.270.3aA 2.970.2abA 2.970.1abA 2.770.2abA 2.670.2bA
7.770.5aB 8.070.2aB 7.170.4bB 8.070.4aB 6.470.1cB
12.170.7aC 11.670.3aC 9.070.3bC 11.670.3aC 8.770.6bC
11.270.8aCD 10.170.8aCD 8.070.6bD 10.070.7aD 7.770.7bCD
10.670.6aD 9.670.1bD 7.470.9cBD 9.470.8abD 7.370.7cD
% of b-casein in soluble phase 0 — 24 5 20 48 5 20
6.370.4aA 9.270.3bA 5.670.5acA 9.070.4bA 5.270.6cA
10.270.3aB 10.470.2aB 8.670.9bB 10.370.9aA 7.670.9bB
17.870.9aC 17.270.2aC 12.870.7bC 17.570.6aB 12.870.7bC
15.970.8aD 15.670.3aD 12.070.8bC 15.970.7aC 11.070.5bD
15.870.7aD 15.270.6aD 11.470.8bC 14.871.2aC 10.770.7bD
Values are expressed as a percentage of the total concentration of as1 - or b-casein in untreated milk, 7standard deviation, and are the average of triplicate experiments on three individual milk samples. a,b,c Values without a common lower-case superscript in a column were significantly different (Po0:05). Comparisons were performed separately for as1 - and b-casein. A,B,C,D Values without a common upper-case superscript in a row were significantly different (Po0:05).
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Table 2 Effect of HP treatment of raw skim bovine milk at 100–600 MPa for 30 min at 20 C, followed by storage at 5 C or 20 C for 0, 24 or 48 h, on the weight of the ultracentrifugal pellet (g 100 g1 milk), micellar hydration (g H2O g1 solids), and the level of ultracentrifugally-sedimentable solids (g 100 g1 milk) Storage time (h)
Storage temp ( C)
Pressure (MPa) Untreated
100
250
400
600
12.470.5aA 12.570.7aA 12.470.8aA 12.570.7aA 12.570.8aA
11.270.5aB 11.070.6aB 11.170.5aB 11.170.5aB 11.070.4aB
10.770.5aB 10.870.8abB 11.770.6bAB 9.570.8aC 11.670.8bAB
13.970.6aC 14.770.7aC 14.170.2aC 15.070.5bD 14.170.6aC
15.770.5aD 16.771.3abC 15.170.6aD 16.770.6bD 14.571.0aC
Micellar hydration (g H2O g1 solids) 0 — 24 5 20 48 5 20
2.570.2aA 2.670.1aA 2.570.1aA 2.770.0aA 2.670.1aA
2.570.2aA 2.670.2aA 2.470.2aA 2.670.1aA 2.570.1aA
3.370.3aB 3.470.3aB 2.970.2bB 3.370.3aB 2.970.2bB
3.870.2aBC 3.970.1aC 3.270.1bC 3.970.1aC 3.170.1bB
3.870.2aC 3.970.1aC 3.170.1bBC 3.970.1aC 3.070.1bB
Sedimentable solids (g 100 g1 milk) 0 — 24 5 20 48 5 20
3.670.3aA 3.570.3aA 3.570.3aA 3.470.2aA 3.570.3aA
3.270.3aAC 3.170.3aAC 3.270.3aA 3.070.2aA 3.270.2aAB
2.570.1aB 2.570.2aB 3.070.2bB 2.270.3aB 3.070.1bB
2.970.2aBC 3.070.2aC 3.470.1bA 3.170.2abA 3.570.2bA
3.370.2aAC 3.470.3abAC 3.770.2abA 3.470.3abA 3.670.3abA
1
Ultracentrifugal pellet (g 100 g 0 — 24 5 20 48 5 20
milk)
Values are the average of triplicate experiments on three individual milk samples, 7standard deviation. a,b Values without a common lower-case superscript in a column were significantly different (Po0:05). Comparisons were performed separately for the weight of the ultracentrifugal pellet (g 100 g1 milk), micellar hydration (g H2O g1 solids), and the level of ultracentrifugally sedimentable solids. A,B,C,D Values without a common upper-case superscript in a row were significantly different (Po0:05).
Finally, as a result of HP-induced changes in the weight of the ultracentrifugal pellet and micellar hydration, the level of sedimentable solids in milk was also altered. Compared to untreated milk, HP treatment at 100–600 MPa reduced the level of sedimentable solids, with the maximum reduction in this parameter after treatment at 250 MPa. Storage at 5 C did not significantly affect the level of sedimentable solids of untreated milk or milk treated at 100–600 MPa. Storage at 20 C had little effect on the level of sedimentable solids in untreated milk or milk treated at 100 MPa, but in milk treated at 250, 400 or 600 MPa, significant increases in this parameter were observed on storage at 20 C. Compared to untreated milk, levels of sedimentable solids were lower in all HP-treated milk samples, except for milk treated at 400 or 600 MPa and subsequently stored for 24 or 48 h at 20 C, for which the level of sedimentable solids was comparable to that in untreated milk.
4. Discussion The results of this study indicate that both the colloidal and soluble phases of milk are affected considerably by HP treatment; solubilisation of caseins, increases in micellar hydration and reductions in the
level of sedimentable solids were observed. On subsequent storage under certain conditions, extensive reversibility in some of these parameters occurred in some HP-treated samples. 4.1. High pressure-induced solubilisation of caseins HP-induced increases in levels of as1 - and, in particular, b-casein in the soluble phase of milk are consistent with reports by Lopez-Fandino et al. (1998) and Arias et al. (2000). The maximum increase in the level of soluble caseins observed after treatment at 250 MPa (Table 1) is also consistent with results of Lopez-Fandino et al. (1998). HP-induced solubilisation of caseins is probably due to solubilisation of CCP, which is responsible for crosslinking caseins and neutralising the negatively charged phosphoserine groups (Horne, 1998), and disruption of hydrophobic bonds (Mozhaev et al., 1994, 1996), which are responsible for binding individual caseins within the casein micelles (Horne, 1998). The increase in the level of b-casein in the soluble phase on storage of untreated milk at 5 C for up to 48 h was consistent with previous reports (Rose, 1968; Davies & Law, 1983; Aoki, Yamada, & Kako, 1990; Law, 1996), and is probably a result of weakening of hydrophobic bonds (Law, 1996; De la Fuente, 1998),
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which occurs at low temperatures (Walstra, 2003). The reversibility of HP-induced increases in levels of soluble as1 - and b-caseins on storage of HP-treated milk at 20 C may be related to the reformation of micellar particles from fragments of HP-disrupted casein micelles (Needs et al., 2000a; Huppertz et al., 2004a); this process is probably due to the reformation of hydrophobic bonds (Needs et al., 2000a). The limited reversal of HP-induced solubilisation of caseins on storage at 5 C is probably related to the fact that hydrophobic bonds are considerably weaker at lower temperatures (Walstra, 2003); this is consistent with observations that HPinduced increases in exposed hydrophobic surface in milk were irreversible on subsequent storage at 5 C for up to 8 days (Johnston, Austin, & Murphy, 1992). 4.2. High pressure-induced changes in the micellar phase of milk HP-induced increases in the weight of the ultracentrifugal pellet from milk treated at 400 or 600 MPa (Table 2) were due to an increased moisture content in these pellets, as the levels of sedimentable solids were reduced by treatment at such pressures (Table 2). The reduction in the amount of sedimentable solids on HP treatment of milk is probably the result of HP-induced disruption of casein micelles, resulting in increased levels of soluble caseins (Table 1; Lopez-Fandino et al., 1998; Arias et al., 2000) and minerals (Buchheim et al., 1996; Schrader et al., 1997; Lopez-Fandino et al., 1998; De la Fuente et al., 1999). The level of sedimentable solids was lowest in milk treated at 250 MPa (Table 2); this is consistent with observations of a maximum level of solubilisation of caseins (Table 1; Lopez-Fandino et al., 1998) and minerals (Lopez-Fandino et al., 1998) at 200 MPa. After treatment at 400 or 600 MPa, the level of sedimentable solids was higher than after treatment at 250 MPa; this is possibly due to the interactions of denatured b-lg with casein micelles, as observed in previous studies (Needs et al., 2000a; Scollard et al., 2000; Huppertz et al., 2004a). HP-induced increases in micellar hydration (Table 2) were consistent with observations by Gaucheron et al. (1997), and are probably, at least partially, related to HP-induced interactions between denatured b-lg and casein micelles; Imafidon and Farkye (1996) reported that on heating milk, casein micelle hydration is increased as a result of the formation of casein–whey protein complexes, resulting in a larger net negative charge. HP-induced reductions in casein micelle size after treatment at 300–800 MPa (Desobry-Banon et al., 1994; Gaucheron et al., 1997; Needs et al., 2000b; Huppertz et al., 2004a, b) may also influence micelle hydration; Sood, Sidhu, and Dewan (1976) observed that the hydration of casein micelles, centrifugally separated into different size distribution classes, was
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higher for smaller casein micelles. Furthermore, changes in the shape of micellar particles, i.e., from roughly spherical to irregularly shaped particles, as occurs on HP treatment of milk (Gaucheron et al., 1997; Needs et al., 2000b; Scollard et al., 2000), may cause an increase in the voluminosity of casein micelles (Walstra, 1979), a parameter closely related to, and often used as, a measure of the hydration of casein micelles (Sood et al., 1976). Small increases in the level of micellar hydration on storage of untreated or HP-treated milk at 5 C (Table 2) are in consistent with increased micellar voluminosity on cold storage of milk (Walstra, 1979, 1990), which Walstra (1979) suggested is possibly due to the dissociation of b-casein from the micelles, as occurred in untreated milk (Table 1). Partial reversal of HPinduced increases in pellet hydration and decreases in sedimentable solids on storage at 20 C of milk treated at 250–600 MPa (Table 2) is possibly related to the reformation of micellar particles from HP-disrupted casein micelles; this process would increase sedimentable solids because monomeric caseins or caseins in small aggregates are not sedimentable, unlike reformed micellar particles. Furthermore, such reassociation may also partially reverse the increase in net negative charge on the micelles, induced by interactions with denatured b-lg; thereby reducing solvation hydration of the micelles. At 5 C, only little reassociation of materials of HP-disrupted casein micelles occurs, as a result of which only little reversal of HP-induced increases in pellet hydration or decreases in sedimentable solids occurred (Table 2). After storage for up to 48 h at 20 C, the amount of sedimentable solids in milk treated at 400 or 600 MPa was comparable to that in untreated milk (Table 2), despite a considerable amount of b-lg associated with the casein micelles (Huppertz et al., 2004a); this indicates that HP-induced solubilisation of caseins is only partially reversible, in agreement with results in Table 1. This provides further support for the suggestions by Johnston, Austin, and Murphy (1993) and Needs et al. (2000a) that casein micelles in HP-treated milk have a considerably altered composition, structure, and possibly surface properties, compared to those in untreated milk.
5. Conclusions HP treatment of milk resulted in significant changes in both the soluble and colloidal phase of milk; in the soluble phase, considerable increases in the level of soluble caseins were observed, whereas in the colloidal phase of milk, micellar hydration increased and sedimentable solids decreased. Many of these changes were virtually irreversible on subsequent storage at 5 C, but largely reversible on storage at 20 C. These results
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clearly indicate that casein micelles in HP-treated milk differ considerably from those in untreated milk; this may have implications for products made from HPtreated milk.
Acknowledgements This research was funded by the Food Institutional Research Measure (FIRM), which is administered by the Irish Government under the National Development Plan 2000–2006.
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