In this study, the effects of the degree of thermal denaturation of whey protein (WP) added to milk on the dissociation of κ-casein from casein micelles were investigated, since they are related to the strength of acid milk gel and its factors. Acid milk gels were prepared by heating thermally denatured WP isolate (WPI) and undenatured milk mixtures and treating them with glucono-δ-lactone as a coagulant. The strength of these gels was negatively correlated with the WPI denaturation degree and strongly positively correlated with the extent of κ-casein dissociation from casein micelles. This behavior was ascribed to the fact that α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) contained in WPI denatured after heating and engaged in disulfide bond formation with each other. With an increase in the degree of denaturation and disulfide bond formation, the bonding between β-lactoglobulin and κ-casein was suppressed to decrease the amount of κ-casein–WPI complexes. When β-Lg forms SS bonds with α-La, the number of highly reactive, free SH groups decreases, which complicates the formation of SS bridges between β-Lg and κ-casein. Thus, the denaturation degree of WPI largely determined the degree of κ-casein dissociation from casein micelles and, consequently, the strength of acid milk gels. Adding WP to milk increases the strength of acid milk gel, and it can be controlled by changing the degree of thermal denaturation of the WP. Furthermore, it was clarified for the first time that the dissociation of κ-casein from casein micelles influences this effect. Further studies are needed to elucidate the structural features of κ-casein-dissociated micelles.

In this study, the relationship between the dissociation of κ-casein from casein micelles due to heat-induced denaturation and the strength of acid milk gel was investigated. The κ-casein-dissociated micelles were fractionated by gel filtration chromatography and two-dimensional polyacrylamide gel electrophoresis, and their zeta potential and surface hydrophobicity were measured. The negative charge of the κ-casein-dissociated micelles was lower than that of native micelles, and micellar surface hydrophobicity was higher. For confirmation, the isoelectric point of the casein micelles was measured. The κ-casein-dissociated micelles were found to cohere at an earlier stage of acidification than the native micelles. These results demonstrated that the heat-induced increase in the strength of acid milk gel was partly due to the decrease in micellar surface charge and partly to the increase in surface hydrophobicity caused by the dissociation of κ-casein.

This study investigated the antibacterial properties of plasmin, the plasmin hydrolysis of bovine κ-casein and the fractions (named κC1, κC2, κC3, κC4, and κC5) liberated from it using RP-HPLC. The target bacteria were Escherichia coli, Staphylococcus aureus (pathogenic), Lactobacillus casei and Lactobacillus acidophilus (probiotic). Three peptides (kC1, kC3, and kC4) were found to have antibacterial activity, with κC3 peptide being the most active. The plasmin digest of bovine κ-casein proved to be stronger than any of its fractions in terms of antibacterial potential. Measurement of the minimum inhibitory concentration (MIC) showed that Gram-positive bacteria are generally more sensitive to antibacterial activity than Gram-negative bacteria. The MIC of nisin, as a bacteriocin peptide, was also measured. The three antibacterial peptides were identified using LC-Mass. The molecular mass of kC1, kC3, and kC4 corresponded to the f(17–21), f(22–24), and f(1–3) of bovine κ-casein, respectively. It was also found that the positive charge and hydrophobicity of a peptide are not key factors in antibacterial activity. On the whole, the present study demonstrated that the plasmin digest of κ-casein has a high antibacterial potential and can be considered as a natural antibacterial agent in the food chain.

Caseinomacropeptide (CMP), a 7-kDa phosphoglycopolypeptide fragment released from κ-casein during milk renneting, is heterogeneous with respect to post-translational glycosylation. Several studies have reported that CMP has growth-promoting activity on lactic acid bacteria belonging to the genera Bifidobacterium. The aim of this study was to evaluate the effect of glycosylation and sequence variations between bovine and caprine CMP on the growth of two probiotics: Lactobacillus rhamnosus RW-9595-M and Bifidobacterium thermophilum RBL67. The growth-promoting activities of CMP (mixture of glycosylated (gCMP) and non-glycosylated (aCMP) fractions), aCMP and gCMP were measured in a basal minimal culture medium using turbidimetric microplate assay at 37 °C. Supplementation of the culture media at 2 mg/ml significantly improved maximum growth by 1·5 to 1·8 times depending on the strain, the additive (CMP, aCMP, gCMP), and the bovine or caprine origin (P < 0·05). CMP preparations also decreased the time needed to reach the inflexion point of the growth curve and increase the cell density at that time (P < 0·05). The effects of CMP preparations were dose dependent and significantly superior to the effect of bovine β-lactoglobulin added to the culture media. As gCMP and aCMP were as efficient as bovine and caprine CMP (P > 0·1), it was concluded that the presence of oligosaccharides linked to CMP was not essential for growth-promoting activity of CMP.

The effects of the caprine αs1-casein (CSN1S1) polymorphisms on milk quality and cheese yield have been widely studied in French and Italian goat breeds. Much less is known about the consequences of κ-casein (CSN3) genotype on the technological and coagulation properties of goat milk. In the current study, we have performed an association analysis between polymorphisms at the goat CSN1S1 and CSN3 genes and milk coagulation (rennet coagulation time, curdling rate and curd firmness) and technological (time to cutting of curd and cheese yield) properties. In this analysis, we have included 193 records from 74 Murciano-Granadina goats (with genotypes constituted by different combinations of alleles B, E and F of the gene CSN1S1 and alleles A and B of the gene CSN3) distributed in three herds, which were collected bimonthly during a whole lactation. Data analysis, using a linear mixed model for repeated observations, revealed significant associations between CSN1S1 genotypes and the rate of the curdling process. In this way, milk from EE goats had a significantly higher curdling rate than milk from BB individuals (P<0·05). Contrary to previous experiments performed in French breeds, cheese yield was not significantly different in BB, EE and EF goats. Moreover, we have shown that CSN3 genotype has a significant effect on the rennet coagulation time (BB>AB, P<0·05) but not on cheese yield. No interaction between the CSN1S1 and CSN3 genotypes was observed.

Impact of milk protein composition on casein (CN) retention in curd during the milk coagulation process was studied using a model cheese making system. Individual milk samples from 110 cows in mid lactation of the Swedish Red and Swedish Holstein breeds with known genotypes of β-casein, κ-casein and β-lactoglobulin were defatted, coagulated with chymosin, subjected to syneresis and subsequent pressing simulated by centrifugation. The results indicated that κ-casein concentration of milk plays an important role in the curd formation process and initial syneresis (whey after cutting), whereas an increased CN ratio was associated with less casein in whey after simulated pressing. Increased κ-casein concentration of milk also characterized the milk samples with no measurable loss of casein in whey, compared with milk samples with casein lost in whey, both after cutting and after simulated pressing. Concentrations of αs1-casein, β-casein, and total casein in milk were positively associated with fresh curd yield, which showed a strong correlation with amount of casein retained in curd. No effect of protein genotype on fresh curd yield or casein in whey was found. The β-lactoglobulin BB genotype was associated with increased casein retention in curd, most likely due to the association of this genotype with CN ratio.

When κ-casein (κ-CN) was added to milk, and the milk was subsequently pH adjusted (pH 6·5−6·9) and heated (90°C/15 min), the serum contained considerably higher levels of denatured whey proteins than the milks without added κ-CN. When milk at pH 6·5−6·9 was heated at 90°C for different times, κ-CN was found in the serum in the early stages of heating and before significant levels of whey proteins were denatured. κ-CN reached its maximum level in the serum before the whey proteins were fully denatured. When milk at pH 6·5−6·9 was heated at 20−90°C for 15 min, κ-CN dissociated from the casein micelles at all temperatures, with the level in the serum increasing with the temperature and the pH at heating. κ-CN dissociated from the micelles at temperatures below those at which significant levels of the whey proteins were denatured. When taken together, the results from these experiments strongly indicate that the dissociation of κ-CN from the micelles can precede the interaction of the denatured whey proteins with κ-CN, and that there is a preferential interaction of the denatured whey proteins with serum-phase κ-CN.

We assessed polymorphisms in exon IV of the κ-casein gene (CSN3) in ten different breeds of domestic goat (Capra hircus) from three continents and in three related wild caprine taxa (Capra <ibex> ibex, Capra <ibex> sibirica and Capra aegagrus). Thirty-five DNA samples were sequenced within a 558 bp fragment of exon IV. Nine polymorphic sites were identified in domestic goat, including four new polymorphisms. In addition to four previously described polymorphic positions, a total of 13 polymorphisms allowed the identification of 13 DNA variants, corresponding to 10 protein variants. Because of conflicting nomenclature of these variants, we propose a standardized allele designation. CSN3*A, CSN3*B, and CSN3*D were found as widely distributed alleles in European goat breeds. Within Capra ibex we identified three variants and showed that the sequence of Capra aegagrus is identical to the most common Capra hircus variant, consistent with Capra aegagrus being the wild progenitor of domestic goats. A dendrogram was drawn to represent the molecular network between the caprine CSN3 variants.

Milk is known to provide not only excellent nutrition but also protection against infection in mammalian neonates. Whey proteins, such as immunoglobulins, lactoferrin, lysozyme and lactoperoxidase, are well known antimicrobial components in milk. Håkansson et al. (1995) demonstrated that some multimeric human α-lactalbumin derivatives induced apoptosis in immature and tumour cells. Yoo et al. (1997) found that lactoferricin, which had been isolated from peptic lactoferrin as an antimicrobial peptide, induced apoptosis in tumour and immature mammalian cells. Apoptotic cell death is considered to be an important physiological function in the development and homeostasis of multicellular organisms and to be a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing tissue damage. Therefore, these findings suggest that whey proteins may have beneficial health effects by restricting specific cell population in the body.

Samples of bovine caseinomacropeptide (CMP) were isolated from κ-casein A and κ-casein B and fractionated to give aglycosylated CMP A and CMP B and monoglycosylated CMP A. The secondary structures of these three peptides were compared under neutral and acidic (pH 4·2) conditions, using two-dimensional (2D) 1H nuclear magnetic resonance (NMR) spectroscopy. The differences between the spectra at pH 4·2 and 7·0 and the spectra of the aglycosylated and glycosylated CMP A were subtle, indicating little change in backbone conformation with these changes. These results suggest that differences in the coagulation properties of milks containing either κ-casein A or κ-casein B are more likely to be related to factors, such as micelle size or charge, than to structural differences arising from altered backbone conformation of the macropeptide segments of the κ-caseins.

Polymorphism in the goat kappa casein gene was studied using the base excision sequence scanning (BESS) method and sequencing. Seven polymorphic sites, corresponding to single nucleotide transversions were detected. Three of these were silent mutations while the other four produced amino acid substitutions. The association between these polymorphic sites was investigated, which resulted in the identification of three goat kappa casein alleles, designated A, B, and C. Protocols for rapid genotyping of the C variant were developed by polymerase chain reaction–restriction fragment length polymorphism using Alw44I and BseNI restriction endonucleases. The occurrence of this allele was found to be very low in Spanish breeds but more frequent in the French Saanen goat. Further studies among different goat populations are necessary to establish the distribution of these alleles and their effects on the quality and functional properties of milk.

Two main genetic variants of κ-casein, A and B, are found in Holstein cows. A positive association between the κ-casein B genetic variant and milk characteristics of economic importance have been reported (reviews: Creamer & Harris, 1997; Ng-Kwai-Hang, 1998). This can be explained, at least in part, by a higher κ-casein content of the casein in milk of cows having the κ-casein B genetic variant (Donnelly et al. 1984; Law, 1993). We have recently reported the presence of a differential allele-specific expression of the κ-casein gene in Holstein cows by comparing the content of allele-A and allele-B specific mRNA in the mammocytes of lactating cows genotyped κ-casein AB (Robitaille & Petitclerc, 2000). For some cows, the A- and B-specific κ-casein gene alleles were expressed in a similar manner. These cows are named HH here to indicate that they were homozygous for κ-casein gene expression. The other group of cows, cows HL, presented a differential allele specific expression of the κ-casein gene allele B which was overexpressed compared to allele A. The genetic polymorphism governing this differential allele-specific expression of the κ-casein gene is likely to affect the physico-chemical properties of casein micelles and, hence, the technological properties of milk, as κ-casein is considered to be the stabilizing component of the milk micelles. The objective of the study was to evaluate the effect of this expression polymorphism of the κ-casein gene on milk ethanol stability.

In this work the purification and the complete primary structure of κ-casein from equine milk are reported for the first time. Mares' milk casein was separated by RP-HPLC into four fractions. Complete primary sequence was obtained by sequence analysis of the protein in the fastest eluting peak isolated by chromatography. This sequence was 95% identical to that reported for the C-terminal portion of the zebras' κ-casein and showed high similarity with κ-caseins from sources other than Equidae, confirming that this protein was indeed κ-casein in equine milk. The presence of post-translational modifications in equine κ-casein was investigated by mass spectroscopy, after enzymic dephosphorylation. Two main components were found, the smaller component being more abundant. Equine κ-casein was recognized by a lectin specific for one of the glucosidic bonds in the saccharide moiety of bovine κ-casein. Sequence comparison with prevision studies showed that the distribution of charged and hydrophobic regions in equine κ-casein was similar, but not identical, to that found in the bovine protein; these regions are associated with the role of κ-casein in the formation and stabilization of the micellar structure of casein in milk.