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Interactions between phosvitin and milk proteins
Int. DairyJournal(1994)491 501 © 1994Elsevier Science Limited Printed in Great Britain. All rights reserved 0958-6946/'94/'$7.00
ELSEVIER
Interactions between Phosvitin and Milk Proteins
Athina Tziboula, A . W . M . Sweetsur & D . D . Muir Hannah Research Institute, Ayr, Scotland, UK KA6 5HL (Received 17 February 1993; revised version accepted 20 July 1993)
ABSTRACT Changes in heat stability and rennet coagulation induced by adding phosvitin to milk were investigated. Phosvitin was added to skim milk and concentrated skim milk (22% total solids ( T S ) ) as well as to whole milk, homogenized at different pressures (i.e. O, 6.8, 20.4 and 34.01 M P a ) and/ or concentrated to 31%o TS. Adding phosvitin to milk caused a decrease in pH, which resulted in destabilisation at the natural p H of the mixtures. The minimum in the heat coagulation time ( H C T ) - p H profile of unconcentrated milk was eliminated and the heat stability of concentrated milk improved. Increasing the homogenization pressure had a detrimental effect on heat stability, but unconcentrated milks containing phosvitin were more stable than controls. Phosvitin could not alleviate the detrimental effect of homogenization and concentration on heat stability, except when added after concentration in unhomogenized whole milk or milk homogenized at 6.8 MPa. Phosvitin delayed the enzymic coagulation of milk by inhibiting both steps o f the renneting reaction. These changes were the result of sequestering of calcium from milk by phosvitin. An additional effect arising from complexation of phosvitin with the micellar caseins should not be ruled out.
INTRODUCTION One of the problems most commonly encountered in the manufacture of dairy products is coagulation of the milk proteins during sterilization treatment. This is often compounded by seasonal variation in the inherent heat stability of fresh milk and processing operations (which have a detrimental effect on heat stability), such as concentration of the milk 491
492
A. Tziboula, ,4. W . M . Sweetsur, D.D. Muir
solids or homogenization of the milk fat. Methods of overcoming this problem include manipulation of the heat-treatment of milk and/or the addition of mineral salts, orthophosphates in particular. At present, there is increasing consumer pressure for the reduction of chemical additives in foods and an increasing demand for the use of alternative compounds that are both natural and safe. In response to these changing attitudes, we initiated a study on the effects of phosvitin, a phosphoprotein found in egg yolk, on the physicochemical properties of milk. Our initial findings demonstrated that adding phosvitin to milk caused dissociation of the casein micelles. This was most pronounced at concentrations of phosvitin exceeding 4mg/ml, whereas small amounts of phosvitin formed limited complexes with the remaining micellar caseins (Tziboula & Dalgleish, 1990). These findings prompted an investigation of the effect of adding low concentrations of phosvitin ( 0 4 mg/ml) on important technological aspects of milk, namely heat stability and rennet coagulation. At these concentrations of phosvitin, milk retained its organoleptic characteristics, and more than 75% of the total casein was in the micellar fraction.
MATERIALS AND METHODS Milk
Fresh milk from the Institute farm was skimmed by centrifuging at 1000g for 30 min at 20°C (MSE-Mistral type 2L centrifuge, Leicester, UK). Concentrated milk was prepared from whole or skim milk by evaporation (Rotary evaporator, Buchi Rotavapor-RE, Switzerland) to 31% and 22% TS, respectively. One-step homogenization was carried out on a laboratory homogenizer (Rannie, type 12.50, APV Rannie AS, Denmark 1001itres/h) at 60c:'C and at different homogenization pressures (i.e. 0, 6.8, 20.4 and 34.0 MPa). Phosvitin
Phosvitin was prepared from hen's egg yolks, according to the method of Joubert and Cook (1958). The major adjustment in the method was the treatment of phosvitin with EDTA to remove ionic material bound to the protein; the final preparation was dialysed against distilled water and freeze-dried. The purity of the preparation was assessed by anion exchange chromatography (FPLC on Mono-Q column) (Tziboula & Dalgleish, 1990).
Interactions between phosvitin and milk proteins
493
Heat stability assay Phosvitin ( 0 4 mg/ml) was added to 75ml portions of milk, and the samples were stirred for 2 h at 20°C. The heat stability of the mixtures was determined by the 'rocking tube' method (Davies & White, 1966) on 2.5 ml portions, over the pH range 6.0-7.2. The pH of the mixtures was adjusted by the addition of dilute NaOH or HC1 (50/~1 for unconcentrated milk and 10/tl for concentrated milk, 0.1, 0.2, 0.3, 0.4 and 0.5 M, respectively). The test was performed at 140°C for unconcentrated milk and at 120°C for concentrated milk.
Renneting of milk The rennet coagulation time (RCT) of milk was determined at 30°C at the unadjusted pH of milk as the time required for the visual flocculation of skim milk (10 ml) after the addition of l ml dilute rennet (chymosin purified from Kho,eromyces lactis, strength 1/15000, Gist-brocades, France, diluted with 0.01 M CaC12 to 1/2 1/500 fold dilutions) (IDF, 1982). When R C T was plotted as a function of the enzyme dilution factor, a straight line was obtained, the slope of which was related to the reaction rate of the first step of the renneting reaction (cleavage of K-casein by chymosin), the intercept being related to the second phase of the reaction (aggregation of renneted micelles).
Statistical analysis Linear regression analysis was used to identify relationships between variables (Genstat 5, tel. 2.2).
RESULTS
Effect of phosvitin on heat stability
Skim milk Phosvitin had two main effects on the heat coagulation time (HCT)-pH profile of unconcentrated milk (Fig. l(a)) Firstly, with increasing concentration of phosvitin, there was a progressive improvement in heat stability in the region of the minimum of the profile, which, in a typical 'type A', milk occurs at pH 6-8-7.0 (control milk with no phosvitin added). Thus, the typical 'type A' H C T - p H profile of the control milk was converted to 'type B' (inflexioness curve) when the concentration of phosvitin in milk
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Fig. 1. Effect of phosvitin on the heat stability of skim milk (a) and concentrated milk (b). Phosvitin added at: (©) 0mg/ml; (I) 1.0 mg/ml; (~) 2mg/ml; (x) 3mg/ml (O) 4mg/ml. The unadjusted pH of milk is indicated by the arrows. exceeded 2 mg/ml. Secondly, phosvitin shifted the natural p H of milk to more acid values and, at the unadjusted pH, milks containing phosvitin were considerably less stable than the control milk (no phosvitin added). It seemed that there was an optimum concentration of phosvitin (2 mg/ ml) that fully eliminated the minimum from the H C T - p H profile but also stabilized milk over the entire p H range 6.4-7.0. Concentrations of phosvitin above 2 mg/ml had a further stabilizing effect at acid p H values (i.e. p H < 6.4), but there was no further improvement in heat stability at p H values above 6.5.
Concentrated skim milk A similar decrease in pH was also observed when phosvitin was added to concentrated milk. All samples were very unstable at the unadjusted p H
Interactions between phosvitin and milk proteins
495
and at all pH values below 6.4 (Fig. l(b)). However, with increasing concentration of added phosvitin, there was a progressive increase in H C T at all pH values above 6.4. For example, at 3 and 4 mg/ml of phosvitin, the maximum H C T was increased fourfold. Effect of storage on heat stability The effect of storage on the heat coagulation time of the milk-phosvitin mixtures (prepared form unconcentrated/or concentrated skim milk) was evaluated after 2, 24 and 48 h. With two exceptions, the stability of the mixtures was unaffected by storage. The first exception was with unconcentrated skim milk containing 1 mg/ml phosvitin, which showed a slight minimum ('type A' behaviour) after 2h, but was converted to 'type B' after storage for 24 and 48 h (Fig. 2(a)). The second exception was for concentrated milk containing 4 mg/ml phosvitin, the stability of which was reduced gradually with storage time (Fig. 2(b)). Effect of phosvitin on the heat stability of full-fat and homogenized milks Two aspects were studied: (a) the effect of phosvitin on the heat stability of whole milk homogenized at 0, 6.8, 20.4 or 34.1MPa, and (b) the importance of the order of addition of phosvitin in relation to homogenization/ concentration steps on heat stability. Unconcentrated whole milk The effect of phosvitin on the heat stability of whole milk was similar to that for skim milk. In general, increasing the homogenization pressure had a harmful effect on the heat stability of whole milk (Fig. 3), but, at each level of homogenization pressure, milks containing phosvitin were more stable than the corresponding controls (with no phosvitin added) (Fig. 3(b)). This effect was more noticeable at pH values above 6.5 and was partly attributed to the suppression of the minimum by phosvitin. For example, the H C T - p H profiles of the milk-phosvitin mixtures homogenized at 0 or 6.80 MPa were devoid of a minimum, but at more severe homogenization there was a slight minimum in the H C T - p H profile, albeit not as deep as in the control counterparts. No significant differences in heat stabilities were found in unconcentrated milks in which phosvitin was added before or after homogenization. Concentrated milk Three different schemes were used to prepare homogenized concentrated milk-phosvitin samples: (a) phosvitin (1 mg/ml) was added after homo-
496
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Fig. 2. Effect of storage period on the HCT pH profiles of phosvitin milk mixtures prepared with skim milk (a) and concentrated milk (b). Storage period: (©) 2h: (m) 24h: (~) 48 h. The unadjusted pH of milk is indicated by the arrows.
genization but before concentration of milk to 31% TS; (b) phosvitin (1 mg/ml) was added before homogenization and concentration, and (c) phosvitin (2.5 mg/ml) was added at the final step, after homogenization and concentration. (Note that in (a) and (b) the final concentration of phosvitin is 2.5 mg/ml.) Concentration of the milk solids resulted in a dramatic decrease in heat stability. The destabilization was further accentuated by homogenization (Fig. 4(a)). In the first two schemes, phosvitin failed to alleviate the detrimental effect of high homogenization pressures on heat stability. In scheme (c) (i.e. adding phosvitin after the homogenization and concentration steps), there was a spectacular improvement in heat stability at pH values above 6-4 when homogenization treatment was carried out at 0 and 6.80 MPa (Fig. 4(c)).
btteractions between phosvitin and milk proteins
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pH o f milk Fig. 3. Effect of homogemzation on the HCT-pH profiles of whole milk (a) and whole milk-phosvitin (1 mg/ml) mixtures (b); homogenization pressure: (©) 0 MPa; (ll) 6.8 MPa; (A) 20.4 MPa; (×) 34.0 MPa. The unadjusted pH of milk is indicated by the arrows.
Effect of phosvitin on the renneting properties of milk
Phosvitin increased the RCT dramatically (Fig. 5). Despite the decrease in the pH of milk caused by phosvitin which was expected to accelerate the renneting reaction (Kowalchyk & Olson, 1977), there was an increase in renneting coagulation time by a factor of 4 at 0.4 mg/ml phosvitin and by a factor of 35 at 1 mg/ml phosvitin. From Fig. 5 and linear regression analysis of RCT against the chymosin dilution factor, it was evident that phosvitin retarded both the initial step of chymosin action (an increase in slope, Fig. 5 or Table 1) and the following aggregation stage of the renneted casein micelles (increasing value for constant in Table 1).
DISCUSSION The phenomena described in this paper arise mainly from the fact that phosvitin is a highly phosphorylated protein with a strong ion-binding capacity. Adding phosvitin to milk is equivalent to adding a considerable concentration of phosphate. From the known molecular weight and phosphoserine content of phosvitin, it can be calculated that adding 1 mg/ ml phosvitin is equivalent to adding 3.79mmol/dm 3 phosphate (Byrne et al., 1984). The total Pi content of milk is 22 mol/dm 3, whereas the casein phosphate content is approximately 7 mmol/dm 3. The binding constants
498
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6.1 6.2 6.3 6.4 6.5 6.6 6.7 pH of milk
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6.8
Fig. 4. Heat stability of whole homogenized concentrated milks: no phosvitin added (a), with phosvitin (1 mg/ml) added before the concentration step (b), and with phosvitin (2.5 mg/ml) added after the concentration step (c). Homogenization pressure: (O) 0 MPa; ( I ) 6.8 MPa; (A) 20-4 MPa; (x) 34.0 MPa. The unadjusted pH of milk is indicated by the arrows.
for C a 2+ by the serine phosphates of caseins and phosvitin are similar (Grizzuti & Perlmann, 1973). Thus, if the concentration of phosphate derived from phosvitin is higher than that derived from caseins, it can be expected that, in any competition between these phosphoproteins, calcium will bind to phosvitin. Indeed, it was shown that, when phosvitin was added to milk, phosvitin was bound to calcium and not to calcium phos. - . 40.0 ._c E
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Fig. 5. Effect of phosvitin on the rennet-coagulation time (RCT) of skim milk. Phosvitin added: (A) 0 mg/ml; ( i ) 0.4 mg/ml; (O) 1.0 mg/ml.
Interactions between phosvitin and milk proteins
499
TABLE 1 Linear-Regression Analysis: Rennet Coagulation Time against Dilution of Chymosin
Concentration of phosvitin (mg/ml)
Slope
0.0 0.4 1-0
0.067 0.378 6.250
Constant -41.60 - 12.68 +0.72
Correlation coefficient (r )
pH of sample
0.99*** 0.95"** 0.98***
6.80 6.76 6.67
***Statistical significance p < 0.001.
hate. Ultrafiltration of the milk-phosvitin mixtures using a membrane not permeable to any of the proteins showed an increase in the concentration of Pi in the permeate as the concentration of phosvitin increased, whereas most of the Ca remained with the protein fraction (Tziboula & Dalgleish, 1990). The binding of C a 2 + to phosphoserine residues in phosvitin is nonspecific (similar to demineralization by EDTA) and releases protons (Waugh et al., 1971), causing a decrease in pH. This effect is similar to that observed when an orthophosphate salt is added to milk (Sweetsur & Muir, 1982). In turn, the disturbance in the mineral equilibrium of milk affects heat stability (Rose, 1962) and a threshold level of ionic calcium is necessary for a 'type A' H C T - p H profile. Demineralization of milk eliminates the minimum, which results in 'type B' behaviour (Morrissey, 1969; Okonogi & Tamita, 1977; Fox & Hearn, 1978). Two theories have been proposed to explain the mechanism of heat-induced coagulation of concentrated milk within the minimum of 'type A' milk; Morrissey (1969), Fox and Hoynes (1975) and Snoeren and van der Speck (1977) suggested that the 'minimum' is the result of calcium-induced precipitation of a fllactoglobulin/~c-casein complex. However, other studies (Kudo, 1980; Singh & Fox, 1985, 1986; van Boekel et al., 1989) showed that the coagulation of milk within the 'minimum' is due indirectly to the heat-induced dissociation of micellar 1c-casein, the direct cause of the minimum being the sensitivity of the ~c-casein-depleted micelles to ionic calcium or heatprecipitated calcium phosphate. Phosvitin, acting as sequestrant of C a 2 + , prevented either the destabilization of the fl-lactoglobulin/tc-casein complex or the coagulation of micelles depleted in 1c-casein; hence there was the observed stabilization in the region of the minimum of the H C T pH profile and on maximum heat stability of concentrated milk. The binding of free C a 2+ to phosvitin was also responsible for the renneting behaviour of the milk-phosvitin mixtures, since the coagulation of the renneted micelles is highly dependent on the presence of C a 2 + (Dalgleish, 1982). However, this can only partly explain the phenomena described in this paper, and it is likely that phosvitin exerts a protective influence on
500
A. Tziboula, A.W.M. Sweetsur, D.D. Muir
the micelles perhaps forming a complex with the caseins in the micellar structure. Such a complex, for example, would be responsible for the suppression of the initial stage of the renneting reaction by inhibiting access of chymosin to the susceptible bond of ~c-casein.
CONCLUSIONS The phenomena described in this paper were mainly the result of phosvitin acting as sequestrant of calcium. On this basis, one could explain the decrease in pH of the milk-phosvitin mixtures and the sequential decrease in heat stability (at the natural pH), the elimination of the minimum of the HCT-pH profile of unconcentrated milk, the improvement in the heat stability of concentrated milk, and the delay in the enzymic coagulation of milk. However, other aspects of phosvitin addition to milk, such as the improvement in the stability of unconcentrated milk outside of the minimum, the beneficial effect of phosvitin on homogenized, unconcentrated milks, or the delay in the initial stage of the renneting reaction, suggest that phosvitin exerts an additional protective role on the casein micelles, possibly via the formation of a complex between phosvitin and caseins in the micellar structure.
ACKNOWLEDGEMENT This research was funded by the Scottish Office Agriculture and Fisheries Department.
REFERENCES Byrne, B.M., van het Schop, A.D., van de Klundert, J.A.M.,Arnberg, A.C., Gruber,M. & Geert, A.B. (1984). Amino acid composition of phosvitin derived from the nucleotide sequence of part of the chicken vetellogenin gene. Biochemisto', 23, 4275-9 Dalgleish, D.G. (1982). The enzymatic coagulation of milk. In Developments in Dai O' Chemisto' 1: Proteins, ed P. F. Fox. Applied Science Publishers, London, pp. 157-87. Davies, D.T. & White, J.C.D. (1966). The stability of the milk protein to heat. I. Subjective measurement of heat stability of milk. J. Dait 3, Res., 33, 67-81 Fox, P.F. and Hearn, C.M. (1978). Heat stability of milk:Influence of dilution and dialysis against water. J. Dai~3' Res. 45, 149-57. Fox, P.F. and Hoynes, M.C.T. (1975) Heat stability of milk: influence of colloidal calcium phosphate and/J-lactoglobulin. J. Daio' Res., 42, 427-32.
Interactions between phosvitht attd milk proteins
501
Grizzuti,K. and Perlmann, G.E. (1973) Binding of magnesium and calcium ions to the phosphoglycoprotein, phosvitin. Biochemisto,, 12, 4399-403. International Dairy Federation (1982). Calf rennet and adult bovine pepsin. Determination of chymosin and bovine pepsin contents. IDF Standard 110. International Dairy Federation, Brussels, Belgium. Joubert, F.J. and Cook, W.H. (1958). Preparation and characterization of phosvitin from hen egg yolk. Can. J.Biochem. Physiol., 36, 399-408. Kowalchyk, A.W. and Olson, N.F. (1977). Effect of pH and temperature on the secondary phase of milk clotting by rennet. J. Daio' Sci., 60, 1256 9. Kudo, S. (1980). The heat stability of milk: Formation of soluble proteins and protein-depleted micelles at elevated temperatures. N Z J. Dairy Sci. Technol., 15, 255-63. Morrissey, P.A. (1969) The heat stability of milk as affected by varations in pH and milk salts. J. Dairy' Res., 36, 343 51. Okonogi S. and Tamita, M. (1977) Effect of pH on heat stability of skim-milk subjected to demineralization by means of electrodialysis with ion permselective membrane. Jpn J. Zootech. Sci., 48, 437 8. Rose D., (1962) Factors affecting the heat stability of milk. J. Dai O' Sci., 45, 1305 11. Singh, H. and Fox, P.F. (1985). Heat stability of milk:pH-dependent dissociation of micellar h-casein on heating milk at ultra high temperatures. J. Daio, Res., 52, 529 38. Singh, H. and Fox, P.F. (1986). Heat stability of milk: Further studies on the pHdependent dissociation of micellar K-casein. J. Dai O, Res., 53, 237-48. Snoeren, T.H.M. and van der Speck, C.A. (1977) The isolation of a heat-induced complex from UHTST milk. Neth. Milk Dai O' J., 31,352 5. Sweetsur, A.W.M. and Muir, D.D. (1982). Manipulation of the heat stability of homogenized concentrated milk. J. Soc. Dai O' Technol., 35, 126-32. Tziboula,A. and Dalgleish,D.G. (1990). Interaction of phosvitin with casein micelles in milk. Food Hydrocoll., 4, 149 59. van Boekel, M.A.J.S., Nieuwenhuijse, J.A. and Walstra, P. (1989). The heat coagulation of milk. 1: Mechanisms. Neth. Milk Dairy J., 43, 97-127. Waugh, D.F., Slattery, C.W. and Creamer, L.K. (1971). Binding of cations to caseins. Site binding, Donnan binding and system characteristics. Biochemistry, I0, 817-23.
ELSEVIER
Interactions between Phosvitin and Milk Proteins
Athina Tziboula, A . W . M . Sweetsur & D . D . Muir Hannah Research Institute, Ayr, Scotland, UK KA6 5HL (Received 17 February 1993; revised version accepted 20 July 1993)
ABSTRACT Changes in heat stability and rennet coagulation induced by adding phosvitin to milk were investigated. Phosvitin was added to skim milk and concentrated skim milk (22% total solids ( T S ) ) as well as to whole milk, homogenized at different pressures (i.e. O, 6.8, 20.4 and 34.01 M P a ) and/ or concentrated to 31%o TS. Adding phosvitin to milk caused a decrease in pH, which resulted in destabilisation at the natural p H of the mixtures. The minimum in the heat coagulation time ( H C T ) - p H profile of unconcentrated milk was eliminated and the heat stability of concentrated milk improved. Increasing the homogenization pressure had a detrimental effect on heat stability, but unconcentrated milks containing phosvitin were more stable than controls. Phosvitin could not alleviate the detrimental effect of homogenization and concentration on heat stability, except when added after concentration in unhomogenized whole milk or milk homogenized at 6.8 MPa. Phosvitin delayed the enzymic coagulation of milk by inhibiting both steps o f the renneting reaction. These changes were the result of sequestering of calcium from milk by phosvitin. An additional effect arising from complexation of phosvitin with the micellar caseins should not be ruled out.
INTRODUCTION One of the problems most commonly encountered in the manufacture of dairy products is coagulation of the milk proteins during sterilization treatment. This is often compounded by seasonal variation in the inherent heat stability of fresh milk and processing operations (which have a detrimental effect on heat stability), such as concentration of the milk 491
492
A. Tziboula, ,4. W . M . Sweetsur, D.D. Muir
solids or homogenization of the milk fat. Methods of overcoming this problem include manipulation of the heat-treatment of milk and/or the addition of mineral salts, orthophosphates in particular. At present, there is increasing consumer pressure for the reduction of chemical additives in foods and an increasing demand for the use of alternative compounds that are both natural and safe. In response to these changing attitudes, we initiated a study on the effects of phosvitin, a phosphoprotein found in egg yolk, on the physicochemical properties of milk. Our initial findings demonstrated that adding phosvitin to milk caused dissociation of the casein micelles. This was most pronounced at concentrations of phosvitin exceeding 4mg/ml, whereas small amounts of phosvitin formed limited complexes with the remaining micellar caseins (Tziboula & Dalgleish, 1990). These findings prompted an investigation of the effect of adding low concentrations of phosvitin ( 0 4 mg/ml) on important technological aspects of milk, namely heat stability and rennet coagulation. At these concentrations of phosvitin, milk retained its organoleptic characteristics, and more than 75% of the total casein was in the micellar fraction.
MATERIALS AND METHODS Milk
Fresh milk from the Institute farm was skimmed by centrifuging at 1000g for 30 min at 20°C (MSE-Mistral type 2L centrifuge, Leicester, UK). Concentrated milk was prepared from whole or skim milk by evaporation (Rotary evaporator, Buchi Rotavapor-RE, Switzerland) to 31% and 22% TS, respectively. One-step homogenization was carried out on a laboratory homogenizer (Rannie, type 12.50, APV Rannie AS, Denmark 1001itres/h) at 60c:'C and at different homogenization pressures (i.e. 0, 6.8, 20.4 and 34.0 MPa). Phosvitin
Phosvitin was prepared from hen's egg yolks, according to the method of Joubert and Cook (1958). The major adjustment in the method was the treatment of phosvitin with EDTA to remove ionic material bound to the protein; the final preparation was dialysed against distilled water and freeze-dried. The purity of the preparation was assessed by anion exchange chromatography (FPLC on Mono-Q column) (Tziboula & Dalgleish, 1990).
Interactions between phosvitin and milk proteins
493
Heat stability assay Phosvitin ( 0 4 mg/ml) was added to 75ml portions of milk, and the samples were stirred for 2 h at 20°C. The heat stability of the mixtures was determined by the 'rocking tube' method (Davies & White, 1966) on 2.5 ml portions, over the pH range 6.0-7.2. The pH of the mixtures was adjusted by the addition of dilute NaOH or HC1 (50/~1 for unconcentrated milk and 10/tl for concentrated milk, 0.1, 0.2, 0.3, 0.4 and 0.5 M, respectively). The test was performed at 140°C for unconcentrated milk and at 120°C for concentrated milk.
Renneting of milk The rennet coagulation time (RCT) of milk was determined at 30°C at the unadjusted pH of milk as the time required for the visual flocculation of skim milk (10 ml) after the addition of l ml dilute rennet (chymosin purified from Kho,eromyces lactis, strength 1/15000, Gist-brocades, France, diluted with 0.01 M CaC12 to 1/2 1/500 fold dilutions) (IDF, 1982). When R C T was plotted as a function of the enzyme dilution factor, a straight line was obtained, the slope of which was related to the reaction rate of the first step of the renneting reaction (cleavage of K-casein by chymosin), the intercept being related to the second phase of the reaction (aggregation of renneted micelles).
Statistical analysis Linear regression analysis was used to identify relationships between variables (Genstat 5, tel. 2.2).
RESULTS
Effect of phosvitin on heat stability
Skim milk Phosvitin had two main effects on the heat coagulation time (HCT)-pH profile of unconcentrated milk (Fig. l(a)) Firstly, with increasing concentration of phosvitin, there was a progressive improvement in heat stability in the region of the minimum of the profile, which, in a typical 'type A', milk occurs at pH 6-8-7.0 (control milk with no phosvitin added). Thus, the typical 'type A' H C T - p H profile of the control milk was converted to 'type B' (inflexioness curve) when the concentration of phosvitin in milk
A. Tziboula, A.W.M. Sweetsur, D.D. Muir
494
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Fig. 1. Effect of phosvitin on the heat stability of skim milk (a) and concentrated milk (b). Phosvitin added at: (©) 0mg/ml; (I) 1.0 mg/ml; (~) 2mg/ml; (x) 3mg/ml (O) 4mg/ml. The unadjusted pH of milk is indicated by the arrows. exceeded 2 mg/ml. Secondly, phosvitin shifted the natural p H of milk to more acid values and, at the unadjusted pH, milks containing phosvitin were considerably less stable than the control milk (no phosvitin added). It seemed that there was an optimum concentration of phosvitin (2 mg/ ml) that fully eliminated the minimum from the H C T - p H profile but also stabilized milk over the entire p H range 6.4-7.0. Concentrations of phosvitin above 2 mg/ml had a further stabilizing effect at acid p H values (i.e. p H < 6.4), but there was no further improvement in heat stability at p H values above 6.5.
Concentrated skim milk A similar decrease in pH was also observed when phosvitin was added to concentrated milk. All samples were very unstable at the unadjusted p H
Interactions between phosvitin and milk proteins
495
and at all pH values below 6.4 (Fig. l(b)). However, with increasing concentration of added phosvitin, there was a progressive increase in H C T at all pH values above 6.4. For example, at 3 and 4 mg/ml of phosvitin, the maximum H C T was increased fourfold. Effect of storage on heat stability The effect of storage on the heat coagulation time of the milk-phosvitin mixtures (prepared form unconcentrated/or concentrated skim milk) was evaluated after 2, 24 and 48 h. With two exceptions, the stability of the mixtures was unaffected by storage. The first exception was with unconcentrated skim milk containing 1 mg/ml phosvitin, which showed a slight minimum ('type A' behaviour) after 2h, but was converted to 'type B' after storage for 24 and 48 h (Fig. 2(a)). The second exception was for concentrated milk containing 4 mg/ml phosvitin, the stability of which was reduced gradually with storage time (Fig. 2(b)). Effect of phosvitin on the heat stability of full-fat and homogenized milks Two aspects were studied: (a) the effect of phosvitin on the heat stability of whole milk homogenized at 0, 6.8, 20.4 or 34.1MPa, and (b) the importance of the order of addition of phosvitin in relation to homogenization/ concentration steps on heat stability. Unconcentrated whole milk The effect of phosvitin on the heat stability of whole milk was similar to that for skim milk. In general, increasing the homogenization pressure had a harmful effect on the heat stability of whole milk (Fig. 3), but, at each level of homogenization pressure, milks containing phosvitin were more stable than the corresponding controls (with no phosvitin added) (Fig. 3(b)). This effect was more noticeable at pH values above 6.5 and was partly attributed to the suppression of the minimum by phosvitin. For example, the H C T - p H profiles of the milk-phosvitin mixtures homogenized at 0 or 6.80 MPa were devoid of a minimum, but at more severe homogenization there was a slight minimum in the H C T - p H profile, albeit not as deep as in the control counterparts. No significant differences in heat stabilities were found in unconcentrated milks in which phosvitin was added before or after homogenization. Concentrated milk Three different schemes were used to prepare homogenized concentrated milk-phosvitin samples: (a) phosvitin (1 mg/ml) was added after homo-
496
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Fig. 2. Effect of storage period on the HCT pH profiles of phosvitin milk mixtures prepared with skim milk (a) and concentrated milk (b). Storage period: (©) 2h: (m) 24h: (~) 48 h. The unadjusted pH of milk is indicated by the arrows.
genization but before concentration of milk to 31% TS; (b) phosvitin (1 mg/ml) was added before homogenization and concentration, and (c) phosvitin (2.5 mg/ml) was added at the final step, after homogenization and concentration. (Note that in (a) and (b) the final concentration of phosvitin is 2.5 mg/ml.) Concentration of the milk solids resulted in a dramatic decrease in heat stability. The destabilization was further accentuated by homogenization (Fig. 4(a)). In the first two schemes, phosvitin failed to alleviate the detrimental effect of high homogenization pressures on heat stability. In scheme (c) (i.e. adding phosvitin after the homogenization and concentration steps), there was a spectacular improvement in heat stability at pH values above 6-4 when homogenization treatment was carried out at 0 and 6.80 MPa (Fig. 4(c)).
btteractions between phosvitin and milk proteins
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pH o f milk Fig. 3. Effect of homogemzation on the HCT-pH profiles of whole milk (a) and whole milk-phosvitin (1 mg/ml) mixtures (b); homogenization pressure: (©) 0 MPa; (ll) 6.8 MPa; (A) 20.4 MPa; (×) 34.0 MPa. The unadjusted pH of milk is indicated by the arrows.
Effect of phosvitin on the renneting properties of milk
Phosvitin increased the RCT dramatically (Fig. 5). Despite the decrease in the pH of milk caused by phosvitin which was expected to accelerate the renneting reaction (Kowalchyk & Olson, 1977), there was an increase in renneting coagulation time by a factor of 4 at 0.4 mg/ml phosvitin and by a factor of 35 at 1 mg/ml phosvitin. From Fig. 5 and linear regression analysis of RCT against the chymosin dilution factor, it was evident that phosvitin retarded both the initial step of chymosin action (an increase in slope, Fig. 5 or Table 1) and the following aggregation stage of the renneted casein micelles (increasing value for constant in Table 1).
DISCUSSION The phenomena described in this paper arise mainly from the fact that phosvitin is a highly phosphorylated protein with a strong ion-binding capacity. Adding phosvitin to milk is equivalent to adding a considerable concentration of phosphate. From the known molecular weight and phosphoserine content of phosvitin, it can be calculated that adding 1 mg/ ml phosvitin is equivalent to adding 3.79mmol/dm 3 phosphate (Byrne et al., 1984). The total Pi content of milk is 22 mol/dm 3, whereas the casein phosphate content is approximately 7 mmol/dm 3. The binding constants
498
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6.1 6.2 6.3 6.4 6.5 6.6 6.7 pH of milk
6.2
6.4
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6.8
Fig. 4. Heat stability of whole homogenized concentrated milks: no phosvitin added (a), with phosvitin (1 mg/ml) added before the concentration step (b), and with phosvitin (2.5 mg/ml) added after the concentration step (c). Homogenization pressure: (O) 0 MPa; ( I ) 6.8 MPa; (A) 20-4 MPa; (x) 34.0 MPa. The unadjusted pH of milk is indicated by the arrows.
for C a 2+ by the serine phosphates of caseins and phosvitin are similar (Grizzuti & Perlmann, 1973). Thus, if the concentration of phosphate derived from phosvitin is higher than that derived from caseins, it can be expected that, in any competition between these phosphoproteins, calcium will bind to phosvitin. Indeed, it was shown that, when phosvitin was added to milk, phosvitin was bound to calcium and not to calcium phos. - . 40.0 ._c E
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8 20.0 /
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I 100
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200
300
I 400
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Dilution Factor
Fig. 5. Effect of phosvitin on the rennet-coagulation time (RCT) of skim milk. Phosvitin added: (A) 0 mg/ml; ( i ) 0.4 mg/ml; (O) 1.0 mg/ml.
Interactions between phosvitin and milk proteins
499
TABLE 1 Linear-Regression Analysis: Rennet Coagulation Time against Dilution of Chymosin
Concentration of phosvitin (mg/ml)
Slope
0.0 0.4 1-0
0.067 0.378 6.250
Constant -41.60 - 12.68 +0.72
Correlation coefficient (r )
pH of sample
0.99*** 0.95"** 0.98***
6.80 6.76 6.67
***Statistical significance p < 0.001.
hate. Ultrafiltration of the milk-phosvitin mixtures using a membrane not permeable to any of the proteins showed an increase in the concentration of Pi in the permeate as the concentration of phosvitin increased, whereas most of the Ca remained with the protein fraction (Tziboula & Dalgleish, 1990). The binding of C a 2 + to phosphoserine residues in phosvitin is nonspecific (similar to demineralization by EDTA) and releases protons (Waugh et al., 1971), causing a decrease in pH. This effect is similar to that observed when an orthophosphate salt is added to milk (Sweetsur & Muir, 1982). In turn, the disturbance in the mineral equilibrium of milk affects heat stability (Rose, 1962) and a threshold level of ionic calcium is necessary for a 'type A' H C T - p H profile. Demineralization of milk eliminates the minimum, which results in 'type B' behaviour (Morrissey, 1969; Okonogi & Tamita, 1977; Fox & Hearn, 1978). Two theories have been proposed to explain the mechanism of heat-induced coagulation of concentrated milk within the minimum of 'type A' milk; Morrissey (1969), Fox and Hoynes (1975) and Snoeren and van der Speck (1977) suggested that the 'minimum' is the result of calcium-induced precipitation of a fllactoglobulin/~c-casein complex. However, other studies (Kudo, 1980; Singh & Fox, 1985, 1986; van Boekel et al., 1989) showed that the coagulation of milk within the 'minimum' is due indirectly to the heat-induced dissociation of micellar 1c-casein, the direct cause of the minimum being the sensitivity of the ~c-casein-depleted micelles to ionic calcium or heatprecipitated calcium phosphate. Phosvitin, acting as sequestrant of C a 2 + , prevented either the destabilization of the fl-lactoglobulin/tc-casein complex or the coagulation of micelles depleted in 1c-casein; hence there was the observed stabilization in the region of the minimum of the H C T pH profile and on maximum heat stability of concentrated milk. The binding of free C a 2+ to phosvitin was also responsible for the renneting behaviour of the milk-phosvitin mixtures, since the coagulation of the renneted micelles is highly dependent on the presence of C a 2 + (Dalgleish, 1982). However, this can only partly explain the phenomena described in this paper, and it is likely that phosvitin exerts a protective influence on
500
A. Tziboula, A.W.M. Sweetsur, D.D. Muir
the micelles perhaps forming a complex with the caseins in the micellar structure. Such a complex, for example, would be responsible for the suppression of the initial stage of the renneting reaction by inhibiting access of chymosin to the susceptible bond of ~c-casein.
CONCLUSIONS The phenomena described in this paper were mainly the result of phosvitin acting as sequestrant of calcium. On this basis, one could explain the decrease in pH of the milk-phosvitin mixtures and the sequential decrease in heat stability (at the natural pH), the elimination of the minimum of the HCT-pH profile of unconcentrated milk, the improvement in the heat stability of concentrated milk, and the delay in the enzymic coagulation of milk. However, other aspects of phosvitin addition to milk, such as the improvement in the stability of unconcentrated milk outside of the minimum, the beneficial effect of phosvitin on homogenized, unconcentrated milks, or the delay in the initial stage of the renneting reaction, suggest that phosvitin exerts an additional protective role on the casein micelles, possibly via the formation of a complex between phosvitin and caseins in the micellar structure.
ACKNOWLEDGEMENT This research was funded by the Scottish Office Agriculture and Fisheries Department.
REFERENCES Byrne, B.M., van het Schop, A.D., van de Klundert, J.A.M.,Arnberg, A.C., Gruber,M. & Geert, A.B. (1984). Amino acid composition of phosvitin derived from the nucleotide sequence of part of the chicken vetellogenin gene. Biochemisto', 23, 4275-9 Dalgleish, D.G. (1982). The enzymatic coagulation of milk. In Developments in Dai O' Chemisto' 1: Proteins, ed P. F. Fox. Applied Science Publishers, London, pp. 157-87. Davies, D.T. & White, J.C.D. (1966). The stability of the milk protein to heat. I. Subjective measurement of heat stability of milk. J. Dait 3, Res., 33, 67-81 Fox, P.F. and Hearn, C.M. (1978). Heat stability of milk:Influence of dilution and dialysis against water. J. Dai~3' Res. 45, 149-57. Fox, P.F. and Hoynes, M.C.T. (1975) Heat stability of milk: influence of colloidal calcium phosphate and/J-lactoglobulin. J. Daio' Res., 42, 427-32.
Interactions between phosvitht attd milk proteins
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Grizzuti,K. and Perlmann, G.E. (1973) Binding of magnesium and calcium ions to the phosphoglycoprotein, phosvitin. Biochemisto,, 12, 4399-403. International Dairy Federation (1982). Calf rennet and adult bovine pepsin. Determination of chymosin and bovine pepsin contents. IDF Standard 110. International Dairy Federation, Brussels, Belgium. Joubert, F.J. and Cook, W.H. (1958). Preparation and characterization of phosvitin from hen egg yolk. Can. J.Biochem. Physiol., 36, 399-408. Kowalchyk, A.W. and Olson, N.F. (1977). Effect of pH and temperature on the secondary phase of milk clotting by rennet. J. Daio' Sci., 60, 1256 9. Kudo, S. (1980). The heat stability of milk: Formation of soluble proteins and protein-depleted micelles at elevated temperatures. N Z J. Dairy Sci. Technol., 15, 255-63. Morrissey, P.A. (1969) The heat stability of milk as affected by varations in pH and milk salts. J. Dairy' Res., 36, 343 51. Okonogi S. and Tamita, M. (1977) Effect of pH on heat stability of skim-milk subjected to demineralization by means of electrodialysis with ion permselective membrane. Jpn J. Zootech. Sci., 48, 437 8. Rose D., (1962) Factors affecting the heat stability of milk. J. Dai O' Sci., 45, 1305 11. Singh, H. and Fox, P.F. (1985). Heat stability of milk:pH-dependent dissociation of micellar h-casein on heating milk at ultra high temperatures. J. Daio, Res., 52, 529 38. Singh, H. and Fox, P.F. (1986). Heat stability of milk: Further studies on the pHdependent dissociation of micellar K-casein. J. Dai O, Res., 53, 237-48. Snoeren, T.H.M. and van der Speck, C.A. (1977) The isolation of a heat-induced complex from UHTST milk. Neth. Milk Dai O' J., 31,352 5. Sweetsur, A.W.M. and Muir, D.D. (1982). Manipulation of the heat stability of homogenized concentrated milk. J. Soc. Dai O' Technol., 35, 126-32. Tziboula,A. and Dalgleish,D.G. (1990). Interaction of phosvitin with casein micelles in milk. Food Hydrocoll., 4, 149 59. van Boekel, M.A.J.S., Nieuwenhuijse, J.A. and Walstra, P. (1989). The heat coagulation of milk. 1: Mechanisms. Neth. Milk Dairy J., 43, 97-127. Waugh, D.F., Slattery, C.W. and Creamer, L.K. (1971). Binding of cations to caseins. Site binding, Donnan binding and system characteristics. Biochemistry, I0, 817-23.