Formation of whey protein/κ-casein complexes in heated milk: Preferential reaction of whey protein with κ-casein in the casein micelles

Studies of the formation of soluble κ-casein/whey protein (WP) complexes in heated (90 °C 10 min⁻¹) milk and related mixtures of proteins have been made. The use of milk samples containing different genetic variants, and having different compositions, allowed the effects of changing the natural protein balance on the formation of particles to be investigated. In addition, studies were made of the effects of addition of WP or of purified κ-casein to the milk samples. The addition of WP caused an increase in the amount and the size of the complexes, but addition of κ-casein to the milk had little or no effect on the complex formation, nor did it seem that the added κ-casein could react with the WP in the milk. Conversely, in systems where the casein micelles were absent, the purified κ-casein reacted well with WP, suggesting that in milk heated at its normal pH the WP react preferentially with the κ-casein on the casein micelles.     

Rennet-induced coagulation of enzymatically cross-linked casein micelles

The enzymatic cross-linking of casein micelles with transglutaminase had an adverse influence on rennet-induced coagulation. Incubation with transglutaminase at 30 °C progressively reduced the levels of monomeric caseins and increased rennet flocculation time (RFT) in a Berridge test. For incubation up to 3 h at 30 °C, the reciprocal of the RFT was linearly correlated with the level of residual monomeric κ-casein, indicating that at complete cross-linking flocculation is absent. After treatment for 4–24 h at 30 °C, no residual monomeric κ-casein was detected and no rennet-induced flocculation of the casein micelle suspension was observed. Monitoring rennet-induced coagulation by diffusing wave spectroscopy revealed that transglutaminase-induced inhibition of rennet-induced coagulation of casein micelles is primarily due to an inhibition of the secondary phase of rennet coagulation, i.e., the gelation and gel-firming phase of the casein micelle coagulation. The gelation and fusion of κ-casein-depleted para-casein micelles as in normal milk appears to be absent if the casein macropeptide remains attached to the casein micelle.     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

Protein interactions in heat-treated milk and effect on rennet coagulation

Skim milk was heated at different pH values to cause differential association of whey proteins (WP) with the casein micelles. All of these milk treatments coagulated poorly with rennet. To understand this in more detail, the casein micelles from heated milk were redispersed in unheated serum or unheated micelles were suspended in the sera from heat-treated milk. Systems containing micelles from milk heated at pH 6.7 and 7.1 were marginally better than the heated milk, but that from milk heated at pH 6.3 was not. The sera from milk heated at pH 6.7 and 7.1 impaired the clotting of native micelles but that from the pH 6.3 milk did not. Native casein micelles were suspended in permeates or dialyzed (against unheated milk) sera from heat-treated milk. Permeate systems free of WP/κ-casein complexes produced significantly stronger rennet gels; as did dialyzed systems. The impaired rennet clotting of heat-treated milk was attributed to a synergistic effect of the casein micelles with their heat-modified surfaces, the soluble serum WP/κ-casein complexes, and other dialyzable serum components.     

Effect of NaCl on some physico-chemical properties of concentrated bovine milk

The influence of added NaCl (75–850 mmol L⁻¹) on some physicochemical properties of 2× or 3× concentrated milk (18 or 27%, w/v, total solids, respectively) was investigated. Adding NaCl did not influence average casein micelle size, but reduced the net-negative charge on the casein micelles and milk pH. The level of soluble and ionic calcium was increased by addition of NaCl, but the level of soluble inorganic phosphorus was not influenced. Addition of NaCl shifted the maximum in the pH–heat coagulation time (HCT) profile of 2× or 3× concentrated milk to a higher pH value and certain concentrations increased the maximum HCT, probably due to the fact that NaCl reduced the extent of heat-induced dissociation of κ-casein. Added NaCl reduced the ethanol stability, with the extent of this effect increasing with the concentration of NaCl. The key-mechanism though which added NaCl induces changes in the physico-chemical stability of casein micelles appears to be through changes in the charge on the casein micelles.     

Effect of pH at heat treatment on the hydrolysis of κ-casein and the gelation of skim milk by chymosin

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70-80% of the total after 30 min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para-κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para-κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness (G^′) of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.     

Casein Composition of Cow's Milk of Different Chymosin Coagulation Properties

Five good and four poor chymosincoagulating individual cow milk samples were analyzed for casein composition using hydroxyapatite chromatography and polyacrylamide gel electrophoresis to establish possible relationships between casein fractions and differences in coagulation properties.Samples exhibited wide variation in casein composition. Poor chymosincoagulating milk had higher content of γ- and degraded caseins and lower κ- and β-caseins than the good-coagulating milk. One milk sample that did not coagulate 30 min after chymosin addition had low α_S-casein concentration and an additional major casein fragment (not identified). A substantial peak representing unidentified minor caseins was apparent in a poorcoagulating milk sample, which coagulated early but whose coagulum did not become firm in 30 min. Excluding the nonclotting sample, less variability was observed in α_S-casein concentrations than in the other casein components.     

Bovine milk procathepsin D: Presence and activity in heated milk and in extracts of rennet-free UF-Feta cheese

Milk samples were heat-treated at 72, 85 and 99°C for 15 or 60 s, and the effect on the stability of the milk acid proteinase zymogen procathepsin D was studied by combining immunoblotting using antibodies directed against bovine cathepsin D and its propeptide and by measuring residual procathepsin D-derived activity. Approximately half of the procathepsin D-derived activity detected in milk serum remained after heat treatment at 72°C/15 s or 72°C/60 s, while heat treatment at increased temperature further reduced the detectable activity. In accordance, immunoreactive procathepsin D was detected in serum from milk heated at 72°C/15 s and 72°C/60 s, while very low amounts of immunoreactivity were observed after treatment at higher temperatures. Contrary to the decrease in milk serum, the amount of procathepsin D antigen associated with casein micelles slightly increased with the temperature of the heat treatment, but still the measurable proteolytic activity derived from procathepsin D in the casein micelle samples decreased with temperature treatment. Moreover, the presence of procathepsin D and derived proteolytic activity was demonstrated in rennet free UF-Feta cheese. These results correlated with the finding of α_s1-I and para-κ-caseins in rennet free cheese. This is the first demonstration of procathepsin D in cheese, and of activity derived from indigenous procathepsin D in milk contributing to the proteolysis process in UF-products.     

Rheological properties and microstructure during rennet induced coagulation of UF concentrated skim milk

Rennet induced coagulation of ultrafiltrated (UF) skim milk (19.8%, w/w casein) at pH 5.8 was studied and compared with coagulation of unconcentrated skim milk of the same pH. At the same rennet concentration (0.010 International Milk Clotting Units g⁻¹), coagulation occurred at a slower rate in UF skim milk but started at a lower degree of κ-casein hydrolysis compared with the unconcentrated skim milk. Confocal laser scanning micrographs revealed that large aggregates developed in the unconcentrated skim milk during renneting. Following extensive microsyneresis the protein strands were shorter and thinner in gels from UF skim milk. Moreover, during storage up to 60 days (13 °C), the microstructure and the size of the protein strands of the UF gel changed only slightly. Hoelter–Foltmann plots suggested that the coagulation rate was reduced in the UF skim milk due to a high zero shear viscosity of the concentrate compared with the unconcentrated skim milk.     

Interactions Leading to Formation of Casein Submicelles

Gel chromatography was employed in studies of interactions of soluble whole casein that was prepared by dissociation of casein micelles with ethylenedia-minetetraacetate. With increasing protein concentration at pH 6.6 and 37°C, components of whole casein associate to polymers that approach molecular radii with apparent upper limit of 9.4 ± .4 nm. With decreasing protein concentration, κ-casein dissociates from the other casein components. This was shown by analysis of the eluted protein boundary by gel electrophoresis and radial immunodiffusion. The peak maximum elution volume and the broad, skewed character of the separated κ-casein peak indicate that in whole casein κ-casein exists in a size distribution of disulfide bonded polymers. This apparently suggests that SH-κ-casein monomers aggregate independently of the other casein components in the growth of casein submicelles, and additional studies with the purified casein components support this concept. However, after disulfide bond reduction with dithiothreitol, chromatography of whole casein over the same concentration range did not result in separation of SH-κ-casein polymers, because all of the casein was eluted under one peak. These findings show that, in vivo, casein submicelles could be formed by interaction of SH-κ-casein monomers with those of α_s- and β-casein, followed by S-S-κ-casein polymer formation through oxidation after milking.     

Adsorption of Positively-Charged β-Lactoglobulin Derivatives to Casein Micelles

Positively-charged β-lactoglobulin derivatives prepared by amidation or esterification of available carboxyl groups of the native protein were bound strongly by casein micelles. Increasing amounts of these additives progressively decreased rennet coagulation time of casein micelles. Higher concentrations of positively-charged proteins coagulated casein micelles without addition of rennet extract. Of the modified proteins tested, approximately 1.0 g amidated β-lactoglobulin, 2.0 g ethyl-esterified β-lactoglobulin, or 1.0 g methyl-esterified β-lactoglobulin would be required to coagulate 100 ml of casein micelles at concentrations in milk.     

Formation of soluble protein complexes and yoghurt properties influenced by the addition of whey protein concentrate

The effects of whey protein concentrate (WPC) on the formation of soluble protein complexes and yoghurt texture were evaluated. Skim milk (SM) and skim milk enriched with 1% WPC (SM + 1%WPC) or 2% WPC (SM + 2%WPC) were left unheated or heated and then made into yoghurt gels. Yoghurt prepared from heated SM + 2%WPC had significantly higher storage modulus, water holding capacity and firmness values and a denser microstructure than those prepared only from skim milk. Electrophoretic analysis of the milk showed that the level of β-lactoglobulin and κ-casein in the serum phase increased with increasing WPC concentration, indicating that the content of disulfide-linked β-lactoglobulin and κ-casein was higher in SM + 2%WPC than in SM, suggesting that more soluble protein complexes had been formed. Consequently, yoghurt prepared from heated SM enriched with WPC may have more bonds and more protein complexes in the protein network than yoghurt prepared only from SM, thus resulting in firmer gels.Practical applicationsYoghurt, one of the most popular fermented milk products, is of high economic importance to the dairy industry worldwide. In particular, high-protein yoghurt, such as Greek-style or set-type yoghurt, has been driving its ongoing popularity over recent years. In current industrial production of high-protein yoghurt, protein fortification and heat treatment of milk are two of the most important processing parameters affecting yoghurt texture. Whey protein concentrate has been added to milk to reduce whey separation and to increase the firmness of the yoghurt. From a technological point of view, the interaction of the denatured whey proteins with casein micelles or with κ-casein in the serum phases is regarded as responsible for obtaining a good yoghurt structure. The present research has shown that it is possible to produce yoghurt with a range of textural properties by precisely controlling the rate of whey protein fortification during its manufacture. Therefore, this study provides a better understanding of the effect of WPC fortification and aims to extend this insight for the production of good-quality yoghurt.     

Effect of hydrolysis of casein by plasmin on the heat stability of milk

This study examined the effect of hydrolysis of casein by added plasmin (6 mg L⁻¹) on the heat stability of raw, pre-heated, serum protein-free or concentrated skim milk. Plasmin activity markedly affected the heat stability–pH profile of skim milk and serum protein-free milk, apparently by altering the properties of the casein micelles. It is probable that changes in the surface charge of the micelles, as a result of the hydrolysis of caseins, contributed to this effect. Hydrolysis by plasmin reduced the zeta-potential of the casein micelles from ∼−19 to ∼−16 mV. The effect of hydrolysis of casein by plasmin on the heat stability of pre-heated milk was less pronounced, shifting the heat stability–pH profile to more alkaline values; the heat stability of concentrated milk was unaffected by plasmin. A very high (50 mg L⁻¹) level of added plasmin resulted in clearing of the skim milk; the L^* value decreased from ∼75 to ∼35 after 24 h incubation at 37 °C. Clearing was correlated with a change in casein micelle diameter from an initial value of ∼175 to ∼250 nm. It is suggested that plasmin-induced changes in zeta-potential may promote micellar aggregation or changes in micelle stucture.     

Heat-Induced Protein Changes in Milk Processed by Vat and Continuous Heating Systems

This study investigated and compared the effects of heating system and residence times on the physicochemical properties and interactions of casein and whey proteins in heated milk. Milk was processed by vat heating (85°C for 10 to 40 min), HTST heating (98°C for .5 to 1.87 min), UHT heating (140°C for 2 to 8 s), cooled, and fractionated into casein and whey by isoelectric precipitation.β-Lactoglobulin A and B variants were partially denatured by HTST and UHT heating and totally denatured by vat heating. Increasing residence time caused significant (P<.01) increases in denaturation of both β-lactoglobulin variants in UHT and HTST heating systems and of α-lactalbumin in the vat heating system. Surface hydrophobicity and sulfhydryl content were negatively correlated with whey protein denaturation. Sodium dodecyl sulfate electrophoresis of the casein fraction of heated milk indicated the presence of a high molecular weight component that would not enter the gel. Addition of 2-mercaptoethanol to heated casein samples dissociated this component, with the concurrent appearance of β-lactoglobulin and α-lactalbumin bands. In HTST and UHT heating systems, ratio of β-lactoglobulin to κ-casein increased linearly from the complex with increasing residence time.     

Effect of genetic polymorphism of milk proteins on rheology of chymosin-induced milk gels

Individual milk samples from 121 cows in mid lactation of the Swedish Red and Swedish Holstein breeds with known protein genotypes of β- and κ-casein and β-lactoglobulin were analysed. Chromatographically pure chymosin was added to skim milk and rheological properties of the gels were measured using a Bohlin VOR Rheometer. Coagulation time (CT) and curd firmness after 25 min (G′₂₅) were registered for each sample. The B allele of κ-casein was associated with improved coagulating properties of milk, whereas the E allele showed a negative effect on these traits. The β-casein A²A² genotype was associated with inferior milk coagulation characteristics. Total protein concentration of milk was positively associated with curd firmness, but showed no association with milk coagulation time.     

Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

Inactivation of an indigenous transglutaminase inhibitor in milk serum by means of UHT-treatment and membrane separation techniques

An indigenous inhibitor in raw milk inhibits cross-linking by transglutaminase (TG). The enzymatic cross-linking of micellar casein, compared with sodium caseinate, taking thermal inactivation of the TG inhibitor in the milk serum into consideration, was investigated. Inhibitor-free micellar casein was prepared by membrane separation combined with heat treatment of the UF permeate. The inhibitor permeated through MF (nominal pore size 0.1 μm) and UF (cutoff 25 kDa) membranes. TG-catalyzed cross-linking of casein micelles was clearly enhanced by UHT-treatment of UF permeate. Variation of the enzyme concentration showed that the inhibitory effect could not be compensated by higher enzyme concentrations when the casein micelles were suspended in unheated milk serum. Sodium caseinate, however, underwent high degrees of cross-linking even in unheated milk serum. By mixing an unheated milk serum and a UHT-treated milk serum at different ratios, the relative TG inhibitor activity was analysed. High inactivation (>80%) of the TG inhibitor is necessary to achieve high degrees of protein cross-linking.     

High-pressure-induced changes in the rennet coagulation properties of bovine milk

The mechanism of high-pressure (HP)-induced changes in rennet coagulation properties of milk, particularly the role of whey protein-casein micelle associations, was studied. Treatment at 100 or 250 MPa reduced the rennet coagulation time (RCT) of raw skimmed bovine milk, compared with untreated milk. Treatment at 400 MPa had little effect, but at 600 MPa, RCT increased considerably. HP-induced increases in RCT did not occur in serum protein-free milk or milk treated with the sulphydryl-oxidising agent KIO₃, which prevents association of denatured β-lactoglobulin with casein micelles. Treatment at 5 or 10 °C at 250–600 MPa resulted in shorter RCT than treatment at 20 °C. In milk without KIO₃, coagulum strength was highest after treatment at 250 or 400 MPa, whereas in milk with KIO₃ it was highest after treatment at 400 MPa. These results indicate the significance of HP-induced association of whey proteins with casein micelles for rennet coagulation properties of milk.     

Modifications in milk proteins induced by heat treatment and homogenization and their influence on susceptibility to proteolysis

The influence of the sequence of processing steps on protein integrity and susceptibility to proteolysis of skimmed and whole milks was examined. Laboratory-scale devices were used to homogenize and heat treat the milk. The results were confirmed in skimmed and whole milks processed in a direct ultrahigh temperature (UHT) pilot plant, with and without a homogenization step following heat treatment. The effects of homogenization varied depending on whether it preceded or followed heating. In general terms, homogenization slightly enhanced whey protein denaturation. But, although homogenization increased the concentration of micellar β-lactoglobulin (β-Lg) in whole milks, it led to high levels of soluble denatured β-Lg in skimmed milks. Homogenization after heating had a promoting effect towards proteolysis in skimmed milk, with κ-casein suffering the highest degradation. This was probably because these samples had high levels of soluble β-Lg and κ-casein that could have enhanced the susceptibility to proteolysis. Whole milk samples presented the lowest proteolysis levels, probably because of an increased complex formation between κ-casein and β-Lg.     

Comparison of rheological properties of acid gels made from heated casein combined with β-lactoglobulin or egg ovalbumin

Gelation of heat-treated milk protein solutions by acid fermentation was performed using a model system of micellar casein alone (systR), micellar casein and β-lactoglobulin (systB) or micellar casein and egg ovalbumin (systO) dissolved in a milk ultrafiltrate and heat-treated at 90°C for 24 min. Solubility of globular proteins at given pH values and their interactions with casein were determined. Particle size and ζ-potential of the protein systems were measured before and after heat treatment. Acid gelation of the heated systems was monitored using dynamic low-amplitude strain oscillation. Ovalbumin alone or with casein produced larger particles than casein alone or with β-lactoglobulin upon heat treatment. The systems gelled at different pH values, i.e. 4.88, 5.47 and 5.88 in systR, systB and systO, respectively. The latter two pH values were clearly related to the pH of the loss of solubility of the heated globular proteins. While the gelation pattern for systR resembled unheated milk, the pattern for systB and systO showed the usual maximum for tan δ of heated milk.