1. Proteolysis of β-casein by rennin and by pepsin was completely inhibited in the presence of 10% NaCl and was very significantly reduced by 5% NaCl. The rate of proteolysis of αs-casein was maximal in the presence of 5–10% NaCl. 2. The inhibitory effect of NaCl on the proteolysis of β-casein was independent of pH and incubation temperature. 3. The effectiveness of NaCl in controlling the development of bitter flavour in Cheddar cheese may be due to its inhibitory effect on the proteolysis of β-casein. 4. Rennin hydrolysates of β-casein were bitter in flavour whereas those of αs-casein were not.

The aim of this study was to investigate the effects of enzymatic hydrolysis with digestive enzymes of camel whole casein and beta-casein (β-CN) on their antioxidant and Angiotensin Converting Enzyme (ACE)-inhibitory properties. Peptides in each hydrolysate were fractionated with ultra-filtration membranes. The antioxidant activity was determined using a Trolox equivalent antioxidant capacity (TEAC) scale. After enzymatic hydrolysis, both antioxidant and ACE-inhibitory activities of camel whole casein and camel β-CN were enhanced. Camel whole casein and β-CN showed significant ACE-inhibitory activities after hydrolysis with pepsin alone and after pepsinolysis followed by trypsinolysis and chymotrypsinolysis. Camel β-CN showed high antioxidant activity after hydrolysis with chymotrypsin. The results of this study suggest that when camel milk is consumed and digested, the produced peptides start to act as natural antioxidants and ACE-inhibitors.

In a herd averaging forty-five to fifty cows in milk, large numbers of infections occurred during the dry period and over half of these developed during the first 3 weeks after the final milking-out of the udder. About 50% of the infections persisted and a like percentage of these produced clinical symptoms within the first 2 weeks of calving, half of them caused by staphylococci.

Aqueous solutions of reconstituted whey protein- concentrate (WPC) & isolate (WPI) powders were sonicated at 20 kHz in a batch process for 1–60 min. Sonication at 20 kHz increased the clarity of WPC solutions largely due to the reduction in the size of the suspended insoluble aggregates. The gel strength of these solutions when heated at 80°C for 20 min also increased with sonication, while gelation time and gel syneresis were reduced. These improvements in gel strength were observed across a range of initial pH values, suggesting that the mechanism for gel promotion is different from the well known effects of pH. Examining the microstructure of the whey protein gels indicated a compact network of densely packed whey protein aggregates arising from ultrasound treatment. Comparable changes were not observed with whey protein isolate solutions, which may reflect the absence of larger aggregates in the initial solution or differences in composition.

Proteomic analysis of bovine, caprine, buffalo, equine and camel milk highlighted significant interspecies differences. Camel milk was found to be devoid of β-lactoglobulin, whereas β-lactoglobulin was the major whey protein in bovine, buffalo, caprine, and equine milk. Five different isoforms of κ-casein were found in camel milk, analogous to the micro-heterogeneity observed for bovine κ-casein. Several spots observed in 2D-electrophoretograms of milk of all species could tentatively be identified as polypeptides arising from the enzymatic hydrolysis of caseins. The understanding gained from the proteomic comparison of these milks may be of relevance both in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products.

The development of typical flavour in Cheddar cheese was accelerated by the addition of bacterial neutral proteinase to the curds, but not by the addition of fungal acid proteinase which produced a bitter defect even at low concentrations. An optimum level of neutral proteinase addition was established at which general proteolysis was increased by about 20% in 2 months and treated cheeses of this age had flavour intensities typical of 4-month-old untreated cheeses. Enzyme-treated cheeses were softer-bodied and more brittle than untreated cheeses of the same age.

Skim milk was heated at 85 °C for different holding times. As a result of such heating, whey proteins, in particular β-lactoglobulin, denatured and associated with casein micelles. This led to an increase in size of the casein micelles but also to a different interaction between them. Both these changes could be described by using a quantitative model which was developed for the viscosity of so-called adhesive hard spheres. We applied the model successfully to skim milk and were able to describe on a quantitative basis the changes due to the heat treatment of milk. It was shown that after heating the casein micelles became larger and acquired a mutual attraction. The unfolding of the whey proteins and their subsequent association with the casein micelles appeared to be responsible for these changes. How this reaction influences the fouling of heat exchangers is discussed.

The use of a commercial laser Doppler electrophoresis apparatus to measure electrophoretic mobilities of milk constituents is demonstrated, and calculations of apparent ζ-potentials from these mobilities are given. Systems studied were: native and renneted casein micelles suspended in milk ultrafiltrate and different buffers containing Ca2+, particles from homogenized milk before and after renneting, fat globules, and homogenized fat/caseinate systems. The results confirmed previous estimates of the ζ-potentials of these materials, but also extended to cover the effect of changing the concentrations of Ca2+ and other salts on the electrokinetic properties of the particles. Increasing concentration of Ca2+ caused considerable decrease in the ζ-potentials of native and renneted casein micelles, and temperature increase caused the apparent ζ-potential to increase in both cases. Particles from homogenized milk showed ζ-potentials very similar to those of native casein micelles, and renneting of the homogenized milk gave behaviour similar to renneted casein micelles. The ζ-potential of the homogenized materials could be distinguished clearly from those of native fat globules. Likewise, milk fat which had been homogenized using whey protein showed distinct differences in its surface properties from particles based on caseinate or casein micelles.

Casein phosphopeptides (GPP) were produced by tryptic hydrolysis of sodium caseinate and further purified by precipitation and chromatography on QAE-Sephadex A-25. Their physico-chemical properties were compared with the properties of an enzymically dephosphorylated equivalent preparation (DPP). Binding of Ca2+ to the peptides was measured using a Ca selective electrode and was found to increase with pH and to show 1/1 stoicheiometry Ca/Porg in CPP at pH 6·5 a.nd 7·6. Klotz plots indicated equivalent binding sites at these two pH values, but some heterogeneity was seen at pH 3·5. In contrast, DPP did not bind significant amounts of Ca2+.

CPP effectively inhibited the formation of insoluble calcium phosphates at different Ca/P ratios. The effective CPP concentration was 10 mg/1 and complete stability of calcium phosphate solutions was obtained at about 100 mg/1. This stabilizing effect was dependent on the presence of organic P.

The content and composition of protein in milk samples from creameries in south-west Scotland were determined over a period of 12 months. The composition of the whole casein was expressed in terms of αsl-, β-, κ-, αs2- and γ-caseins, and that of the total milk serum protein in terms of β-lactoglobulins (β-lg), α-lactalbumins, bovine serum albumin, and a mixture of immunoglobulins, proteose-peptone component 3 and lactoferrin (IPL). Concentrations of the individual caseins varied appreciably and for most, concentration was closely correlated with and showed the same seasonal pattern as total casein concentration. Concentrations of the milk serum proteins also varied but only those of β-lg and the IPL fraction were closely correlated with that of total milk serum protein and seasonal trends were not marked. Relative amounts of the individual proteins, on the other hand, showed smaller variations and so throughout the experimental period the milks contained a protein complex of comparatively constant composition. Because of this comparative constancy it would appear that seasonal variations in milk properties in south-west Scotland are unlikely to be determined to a major extent by milk protein composition, but could be more affected by protein concentration.

Quantitative polyacrylamide gel electrophoresis has been used to measure residual native whey proteins remaining after heat treatment of skim-milk and cheese whey in a kinetic study. The denaturation of α-lactalbumin (α-la) appeared to be first order, but was probably a second-order reaction displaying pseudo first-order kinetics. The denaturation of both β-lactoglobulin A and B (β-lgA and β-lgB) followed second-order kinetics while that of serum albumin was more complex, and could equally well be described as first or second order. Equations are given relating log k1 (in s-1) to temperature for α-la denaturation in skim-milk between 70 and 95 °C and between 100 and 150 °C. Similarly, equations relating log k2 (in lg-1s-1) to temperature are given for ²-lgA in skim-milk between 100 and 150 °C, and for ²-lgB between 95 and 150 °C. The relative heat stability of ²-lgA and ²-lgB was found to vary.

Below 95 °C ²-lgA appeared slightly more thermostable than ²-lgB in skim-milk, and the same was observed in cheese whey below 100 °C. Above these temperatures ²-lgB appeared more stable than ²-lgA.

Denaturation of ²-lgB was only slightly more rapid in skim-milk than in whey at temperatures below 95 °C, but was significantly slower at higher temperatures.

The isotherms for Ca2+ binding to bovine β-casein have been measured at 5 temperatures in the range 4–40 °C and at 4 different ionic strengths. The results are interpreted by an interactive-site binding model, and are compared with results previously obtained on αs1-casein. The affinity of β-cesein for the first Ca2+ to bind is similar to the affinity of αsl-casein for the same binding event: however, binding of subsequent Ca2+ to β-casein is weaker than the binding to αs1-casein. The results are discussed in terms of precipitability of the 2 caseins caused by the binding of Ca2+.

In this study, high pressure (HP)-induced denaturation of α-lactalbumin (α-la) and β-lactoglobulin (β-lg) in dairy systems was examined. In both milk and whey, β-lg was less baroresistant than α-la; both proteins were considerably more resistant to HP-induced denaturation in whey than in milk. HP-induced denaturation of α-la and β-lg increased with increasing proportion of milk in mixtures of milk and whey. Addition of a sulphydryl-oxidising agent, KIO3, to milk or whey increased HP-induced denaturation of β-lg, but reduced the denaturation of α-la. Denaturation of both α-la and β-lg was prevented by adding a sulphydryl-blocking agent, N-ethylmaleimide, to milk or whey prior to HP treatment, highlighting the crucial role of sulphydryl-disulphide interchange reactions in HP-induced denaturation of α-la and β-lg. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of α-la and β-lg significantly. The higher level of HP-induced denaturation of α-la and β-lg in milk than in whey may be the result of the abscence of the casein micelles and colloidal calcium phosphate from whey, which facilitate HP-induced denaturation of α-la and β-lg in milk.

Fourier transform infrared spectroscopy and differential scanning calorimetry were used as complementary techniques to study changes in the secondary structure of β-lactoglobulin under various physicochemical conditions. The effects of pH (3–9), NaCl (0–2 M), and lactose, glucose and sucrose (100–500 g/l) in the temperature range 25–100 °C on the conformation sensitive amide I band in the i.r. spectrum of β-lactoglobulin in D2O solution were examined. The 1692 cm−1 band in the amide I band profile had not been definitively assigned in previous studies of the i.r. spectrum of β-lactoglobulin. The decrease in this band at ambient temperature with time or upon mild heating was attributed to slow H-D exchange, indicating that it was due to a structure buried deep within the protein. The disappearance of the 1692 cm−1 band on heating was accompanied by the appearance of two bands at 1684 and 1629 cm–1, assigned to β-sheets. The 1692 cm−1 band was therefore attributed to a β-type structure. β-Lactoglobulin showed maximum thermal stability at pH 3 and was easily denatured at pH 9. On denaturation, the protein unfolded into more extensive random coil structures at pH 9 than at pH 3. After 10 h at pH 9 (25 °C), β-lactoglobulin was partly denatured. Heating to 60–80 °C generally resulted in the loss of secondary structure. At all pH values studied, two new bands at 1618 and 1684 cm−1, characteristic of intermolecular β-sheet structure and associated with aggregation, were observed after the initial denaturation. Differential scanning calorimetry studies indicated that the thermal stability of β-lactoglobulin was enhanced in the presence of sugars. The Fourier transform i.r. results obtained provide evidence that sugars promoted the unfolding of β-lactoglobulin via multiple transition pathways leading to a transition state resisting aggregation.

The temperature-dependent dissociation of β-casein from the casein micelles of milk and from the soluble casein complexes of colloidal phosphate-free (CPF) milk was investigated by high-speed centrifugation and gel-filtration. The percentage of the total casein in supernatants prepared by high-speed centrifugation of mid-lactation milks increased from approximately 6 to 15% on cooling the milks from 30 to 5 °C; β-casein accounted for about 46% of this increase, while αs-and κ-casein constituted 30 and 23%, respectively. On gel-filtration both of skim-milk and CPF milk on Sepharose 2B at 0, 2, 5, 10 and 25 °C, maximum amounts of free β-casein (c. 60% of total) were obtained at 5 °C. The remainder of the β-casein appeared to be more strongly bound to the αs- and κ-casein and may be involved in the internal cohesion of casein micelles. The free β-casein of both milk preparations appeared to be in equilibrium with the bound β-casein. On Sephadex G-200 columns at 5 °C, approximately 5 and 60% of the β-casein of skim-milk and CPF milk, respectively, was eluted in the free form in the expected position for a globular protein of molecular weight about 200000. At low temperatures, particularly at 5 °C, colloidal phosphate appeared to play an integrating role in the association of over half the total β-casein with the other casein components of native micelles. However, when the equilibrium between micellar and free β-casein was disturbed by gel-filtration on Sepharose 2B, the presence of colloidal phosphate did not prevent the release of most of the β-casein from casein micelles. Some problems encountered in the use of densitometry for the estimation of individual caseins on electropherograms are described.

The relationship between extent of κ-casein proteolysis and aggregatability of milk casein micelles has been studied using chymosin either bound to porous glass supports or free in solution. Both enzyme preparations demonstrated that, overall, 86–90 % of the κ-casein had to be destroyed before any aggregation could occur. Based on these results, a mathematical model of chymosin action on casein is described involving (i) the attack on κ-casein by chymosin, via a Michaelis–Menten mechanism (ii) the probability that sufficient κ-casein on any micelle is destroyed to allow aggregation, and (iii) the aggregation of para-casein micelles by a von Smoluchowski mechanism.

The decline in milk yield after peak lactation in dairy animals has long been a biological conundrum for the mammary biologist, as well as a cause of considerable lost income for the dairy farmer. Recent advances in understanding the control of the mammary cell population now offer new insights on the former, and a potential means of alleviating the latter. The weight of evidence now indicates that a change in mammary cell number, the result of an imbalance between cell proliferation and cell removal, is a principal cause of declining production. Further, it suggests that the persistency of lactation, the rate of decline in milk yield with stage of lactation, is strongly influenced by the rate of cell death by apoptosis in the lactating gland. Mammary apoptosis was first demonstrated during tissue involution after lactation, but has now been detected during lactation, in mammary tissue of lactating mice, goats and cattle. Those factors that determine the rate of cell death by apoptosis are as yet poorly characterized, but include the frequency of milking in lactating goats. Initial evidence suggests that nutrition also is likely to influence cell survival after peak lactation, an important factor being the degree of oxidative stress imposed by feed and the tissue's ability to deal with, and prevent damage by, reactive oxygen species. Comparison of cows in calf or not pregnant during declining lactation also indicates a likely influence of reproductive hormones, with oestradiol and progesterone acting to preserve mammary ductal and alveolar integrity during the dry period, while allowing a degree of apoptosis and cell replacement. In each case, the molecular mechanisms controlling mammary cell survival (or otherwise) are as yet poorly defined. On the other hand, more persistent lactations are likely to benefit animal welfare through fewer calvings and by placing less emphasis on maximal production at peak lactation, and modelling of persistent lactation with longer calving intervals indicates their likely economic benefits. In these circumstances, there is considerable incentive to elucidate the determinants of mammary apoptosis, and the factors controlling the dynamic balance between cell proliferation and cell death in the lactating mammary gland.

In development of fermented dairy products and protein hydrolysates with high inhibitory activity towards angiotensin-converting enzyme (ACE), it is crucial to have a reliable assay for measuring the ACE activity. In the present study, the performance of two commonly used assays based on synthetic N-derivates of tripeptides as substrates were tested with respect to reliability in determination of ACE activity per se and to the inhibitory effect of a tryptic whey protein digest and captopril. In one test, the ACE activity was calculated from the amount of hippuric acid liberated from hippuryl-His-Leu (HHL) during 30 min of incubation with ACE, as quantified after HPLC separation of reaction products. In the other assay, the ACE activity was measured directly as the rate of decrease in the absorbance at 340 nm during the first 30 min of ACE catalysed hydrolysis of furanacroloyl-Phe-Glu-Glu (FA-PGG). Both assays, in the absence of inhibitor, showed a good performance with relative standard deviation between replicate samples around 7%. In the presence of inhibitor solutions, relative standard deviations for both assays varied between 1 and 18% for the variously diluted inhibitors. Both assays gave values for the concentration of inhibitor needed to inhibit ACE by 50% similar to those previously reported for whey protein digests and captopril. Different results from the two assays, however, emphasize the importance of controlling the actual ACE-activity for comparison between assays. The limitations of each assay are discussed. Considering the fewer steps in the assay using FA-PGG as substrate, and thus less time and chemicals consumed per sample, and the simpler equipment needed, this assay is recommended for the screening of clear peptide samples.