Explants from lactating bovine mammary gland were cultured in vitro in serum-free medium through 1–9 d. Casein synthesis was determined by [32P] incorporation into newly synthesized Ca rennin precipitable fraction. High correlation (γ = 0·98) was found between incorporation of [32P] and [3H]amino acids in explants cultured under different hormonal regimes, thus indicating that post-translational phosphorylation is not a rate-limiting step in casein synthesis.

Hormonal effects on casein synthesis were studied by supplementing the incubating medium with insulin (I), prolactin (PRL), cortisol (F), thyroxine (T4) and triiodothyronine (T3). It was found that both PRL and I were required absolutely for maximal synthesis and almost maximal effect was achieved with 50 ng/ml. The effect of F was less clear, but some increase was achieved at the 200–1000 ng/ml range. T4 and T3 did not affect casein synthesis at a range of 10-11–10-7M while a significant inhibition was observed at 2 × 10-5 M. A time-course study of casein synthesis further substantiated the dominant role of PRL in maintenance or even elevation of the initial rate of casein synthesis in the explants, through the first 4 d of incubation.

The heat stability–pH profile of milk is shifted to more acidic values by anionic compounds such as SDS, lysolecithin and β-lactoglobulin and to more alkaline values by cationic compounds such as quaternary ammonium compounds. Proline, which reduces the hydrophobicity of proteins, destabilized milk slightly. The results suggest that the heat stability of milk and the shape of the HCT–pH curve may be dependent on micellar charge effects.

The influence of mastitis and early lactation, and the effect of treating milk with heparin, blood serum and trypsin, on the proportion of lipoprotein lipase (LPL) activity in milk serum was investigated. The relative importance of milk serum LPL and LPL bound to micellar casein in promoting lipolysis was also examined. Colostrum contained LPL activity, 45% of which was found in the serum phase in samples obtained from the first milking post partum, but this value fell to 34% in samples taken 24 h later. The proportion of serum LPL was also increased in milks from quarters infected with Staphylococcus aureus, but not after overnight treatment of normal milk at 4 °C with 5% (w/v) blood serum or 2 µg/ml trypsin. The addition of 5 µg/ml heparin resulted in a consistent increase in serum LPL which varied between 14 and 50% of total milk LPL. Heparin did not release all the enzyme bound to casein micelles even after a second heparin treatment of resuspended micelles. Serum LPL was more effective in promoting lipolysis and was more responsive to blood serum activation than LPL bound to casein micelles. Lipolysis increased after heparin treatment but the increase was not related to serum LPL activity.

The 6 most widely used milk-clotting enzymes, i.e. chymosin, bovine and porcine pepsins and proteinases from Mucor miehei, M. pusillus and Endothia parasitica, have been purified and used to prepare rabbit antisera against each of them. The antisera were adsorbed with appropriate cross-linked antigens to remove non-specific antibodies. Monospecific antisera thus obtained were used to identify enzymes eventually added to calf or bovine rennets, using the double radial immunodiffusion technique. The threshold of sensitivity was c. 1%, expressed as clotting activity. This qualitative method complements the quantitative procedure recently proposed for determining chymosin and bovine pepsin A in commercial extracts of bovine veils.

Several dicarbonyl compounds (glyoxal, substituted glyoxals, diacetyl and 1, 2-cyclohexadione) had a marked stabilizing effect on the heat stability of milk, especially in the presence of urea. These reagents are believed to modify arginine more or less specifically suggesting an important role for arginine residues in heat stability. In contrast, modification of lysine residiles with dansyl chloride, acetic anhydride or cyanoborohydride had little effect on maximum heat stability although it did alter the HCT-pH profile. Since diacetyl is a natural constituent of fermented milks and cheese, it may be acceptable as an additive to increase the heat stability of ṃilk.

The effects of temperature, dilution, dialysis and the presence of heparin on the stability of lipoprotein lipase (LPL) in milk, skim-milk, milk serum and casein micelles were investigated. At 4 and 20 °C milk serum was the source of the least stable LPL and casein was that of the most stable. There was little difference between LPL stability in milk and skim-milk at these temperatures, or between serum and casein LPL at 50 °C. Heparin (5 µg/ml) increased stability although the effect was less for casein LPL than for serum LPL. A 40-fold dilution of serum LPL with either simulated milk ultrafiltrate (SMUF) or 0·01 M-Tris-Cl pH 8·3 increased the loss of serum LPL, but not of casein LPL. Dialysis of skim-milk against deionized water or SMUF increased stability at 4 or 20 °C but not at 37 °C. LPL activity was more stable in diluted samples of dialysed skim-milk than in diluted samples of the same milk which had not been dialysed. Dialysis against deionized water increased lipolysis but against SMUF it did not increase. Solutions prepared by dialysing water against some milks were found to inhibit lipolysis and this effect was overcome by heparin. The possibility that milk serum contains a factor which influences LPL stability is discussed.

A method capable of determining the interfacial tension (γ) of individual emulsified liquid drops has been investigated and applied to a study of the surface properties of milk fat globules. Measurements on several organic liquids in bulk against water were made to validate and standardize the technique. With one exception, results agreed well with literature values, but the reproducibility of measurements was not as good as that of other, standard, methods. Measurements performed on small drops were affected by several factors and precision was not high.

The influence of different milk treatments upon the interfacial tension of milk fat globules was studied. Agitation of milk had the effect of increasing γ while the effect was reversed upon allowing agitated milk to stand. Dilution of normal milk with water resulted in an increase in γ while acidification and alkalization produced a decrease. Pasteurization of raw milk caused a slight, non-significant decrease. Values of γ were low, 1–2 mN m-1, irrespective of the nature of the milk treatment and were an order of magnitude smaller than those obtained for interfaces of butter oil and milk protein solutions. The results support the concept of a 2-layer membrane and suggest that changes in γ follow mainly the adsorption/desorption of surface active components in the outer hydrophilic layer of the globule membrane. An inner, firmly bound lipophilic layer, resistant to many different milk treatments, appears to determine the overall low γ value under most conditions.

Changes in the concentrations of various metabolites in mammary secretions (organic acids, UDP-hexoses and nucleotides) have been observed at the onset and cessation of lactation. All metabolites, with the exception of galactose, increased dramatically in concentration just before and immediately after parturition. Galactose concentrations decreased with the onset of lactation. After regular milking ceased, the concentrations of these metabolites in milk contained within the udder fell, but the rates of decline varied.

Oxidized flavour developed in whole milk only through the catalytic effect of either Cu or light. The O2 requirement for the 2 processes differed as did the characteristics of the off-flavours produced. Cu-induced oxidized flavour was described as ‘cardboardy’ and light-induced oxidized flavour was ‘painty’. Light-induced oxidized flavour increased in intensity with O2 loss, and could be prevented in stored milk by restricting access of O2. In UHT milk with a dissolved O2 content of 6·6 mg/1, and in the absence of access of further O2, light-induced oxidized flavour did not develop; similarly, O2 uptake of 7·5 mg/1 in in-bottle sterilized milk exposed to fluorescent light did not result in flavour formation. When light-induced oxidized flavour developed consistently in whole milk none developed in skim-milk, indicating the lipid source of the flavour. In contrast Cu-induced oxidized flavour development was not associated with high O2 uptake. Although nearly complete deoxygenation of whole pasteurized milk contaminated with Cu prevented the formation of the flavour, moderate deoxygenation resulted in even greater flavour intensity than non-deoxygenation. The 2 oxidized flavours also differed in relation to ascorbic acid (AA) oxidation. Light-induced oxidized flavour developed only after AA oxidation was complete, whereas Cu-induced flavour developed with AA still present. AA oxidation was greatly accelerated through the effects of both Cu and light. In milk free from Cu contamination and protected from light, after AA oxidation (plus SH group oxidation in the case of UHT milk) was complete, no further loss of O2 occurred, even during prolonged storage at 5°C, despite the presence of large O2 concentrations. However, at 20°C, a small consumption of O2 was measured, and this was associated with stale flavour.

The keeping quality of commercial HTST-pasteurized milk and laboratory pasteurized milk from a common bulk raw supply has been investigated for 5 dairies. Spoilage occurred at levels of total bacterial counts around 107 colony forming units/ml, but with a slightly higher off-flavour threshold for the commercial milks than the laboratory pasteurized milks. The predominant microflora at spoilage and the type of off-flavour produced differed between the 2 types of milk. Raising the storage temperature from 5 to 11 °C caused a slight shift in the spoilage microflora and led to an average reduction in the shelf life of the laboratory pasteurized milk from 28 to 6 d and of the commercial pasteurized milk from 13 to 5 d. Changes in the level of post-pasteurization contamination (PPC) were reflected in changes in keeping quality, particularly at 5 °C. However, the greatest improvements were found in the absence of PPC.

A sample of milk fat was fractionated into high, medium and low molecular weight triglycerides by silicic acid column chromatography. Each fraction was further separated by silver ion adsorption thin-layer chromatography into triglyceride classes which were subjected to stereospecific analysis. Trans-monoene fatty acids were incorporated into triglycerides in the same manner as their cis-isomer. The specific positional distribution for fatty acids in intact milk fat triglycerides was not always maintained in the molecular weight fractions or in the various classes of unsaturation. The significance of these findings is discussed in relation to biosynthetic mechanisms for milk fat synthesis.

A milk-related model system, 2 mM-trilinolein in 0·1 M-piperazine-N, N′-bis-2-ethanesulphonic acid buffer, pH 6·8, stabilized by lysophosphatidylcholine was used to assess the effect of milk proteins on lipid oxidation in emulsion, catalysed by Cu2+ ions. In some experiments the trilinolein was replaced by sunflower oil (2 g 1-1). Oxidation at 25°C was assessed by measurement of the rate of O2-utilization and by the thiobarbituric acid assay. The features, relevance and reliability of the model are described in terms of the observed effects in the absence and presence of added protein. Casein was antioxidative at concentrations relevant to those in bovine milk, human milk, infant milk formulations and ‘high linoleic acid’ milk. Whole whey protein was a less effective inhibitor of lipid oxidation at similar concentrations. Purified α-lactalbumin was also marginally antioxidative, and its effect was independent of the molar Cu2+: protein ratio between 1:1 and 3:1.

The rates of milk clotting and of formation of para-κ-casein in milk, colloidal phosphate-free milk and isolated κ-casein by pepsin and by its soluble size-fractionated conjugates with dextran were determined. Milk clotting with all the enzyme derivatives was dependent on the rate of the enzymic phase and required essentially complete κ-casein hydrolysis at 30 °C and throughout the range of pH 5·6–6·7. κ-Casein hydrolysis by pepsin at pH 6·6 was fastest in milk and slowest in isolated κ-casein, but the rate decreased as the enzyme size increased, especially with milk. When corrected for the changes in pepsin activity, the rates of κ-casein hydrolysis in all substrates were identical at 30 and 5 °C, but increased with decrease in pH, especially with the larger enzyme conjugates. Hydrolysis of the C-terminal bonds of β-and κ-casein in native and disrupted casein micelles by carboxypeptidase A and soluble conjugates of it were also investigated. κ-Casein was hydrolysed much faster, and β-casein slightly faster, in native than in disrupted micelles by the native enzyme. Increase in the size of carboxypeptidase A increased the rate of hydrolysis of κ-casein in disrupted micelles and also induced lag periods before hydrolysis commenced, especially with disrupted micelles. The results are compatible with a model for the casein micelle in which the κcasein is on the outside and the casein components are in a more ordered arrangement than in the casein complexes formed on micelle disruption. They also indicate that immobilized coagulants would be unable to clot milk.

The effect of limiting the amount of O2 present in commercially pasteurized milk on bacterial growth and on the keeping quality of the milk was investigated. No increase in bacterial count of laboratory pasteurized milk during storage for 9 d at 5 °C was observed and O2 consumption due to bacterial activity was low. In commercial pasteurized milk containing post-pasteurization contamination (PPC) growth of strict aerobes was encouraged by high O2 content while that of coliforms was less dependent on O2 level. The amounts of headspace (HS) O2 consumed in milk with PPC growth were large. Reducing the dissolved O2 content of commercial pasteurized milk stored in O2-impermeable containers without HS had little effect on bacterial activity and shelf-life.

The milk-related model system described by Allen & Wrieden (1982) was used to assess the effects of a number of purified milk metalloproteins, at concentrations relevant to those in milk and dairy products, on triglyceride oxidation in emulsions. Oxidations were monitored by the rate of O2 utilization (ROU) and by the thiobarbituricacid (TBA) assay. The proteins lactoferrin, lactoperoxidase, superoxide dismutase and xanthine oxidase were used either on their own or in conjunction with 10 μM-Cu2+ or Fe2+. Lactoferrin alone had little effect on oxidation, although both the iron-free and iron-saturated protein were able to protect the lipid to some extent from Fe2+-catalysed oxidation. Cu2+-catalysed oxidation was slightly promoted. Lactoperoxidase was pro-oxidative in the absence and presence of added Cu2+ or Fe2+. Heat treatment at 72°C/20 s had little effect, but oxidation was greatly reduced by treatment at 80°C/20 s. Superoxide dismutase strongly reduced the rate of lipid oxidation in the absence of added metals, but had no effect in the presence of 10 μM-Cu2+. xanthine oxidase had little effect on lipid oxidation in the absence of added metals, but was strongly pro-oxidative in the presence of 10 μ-Cu2+. Similar effects were observed down to 1 μM-Cu2+. The enzymic activity of xanthine oxidase was rapidly eliminated by 10 μM-Cu2+, but not by 10 μM-Fe2+. A moderate pro-oxidative effect was observed in the presence of 10 μM-Fe2+. Heat treatment of xanthine oxidase at 80°C/20 s reduced its lipid pro-oxidative effect in the presence of Cu2+ more effectively than treatment at 72°C/20 s.

Buffalo milk fat was first separated by thin layer chromatography (t.l.c.) into high (HMT), medium (MMT) and low (LMT) molecular weight triacylglycerols. The triacylglycerol (TG) fractions thus obtained were further resolved by argentation t.l.c. into saturated, trans-monoene, cis-monoene, diene and polyene TG species. Whereas 18:1 was concentrated in HMT, 6:0, 8:0 and 10:0 tended to be located in MMT. LMT was unique in containing almost all 4:0. HMT, MMT and LMT showed great variations in their levels of different TG species. The distribution of fatty acids among the TG species was found to be characteristic of each TG species.

The effect on the composition and properties of casein micelles of the binding of ionic materials which accelerate the coagulation of milk on rennet treatment, was investigated. When considered in terms of their relative charge concentration, all the materials tested caused similar effects. The casein, inorganic phosphate and Ca contents of the micelles increased slightly. Micelle hydration decreased as additive binding increased. Casein and Ca dissociation on cooling increased at low concentrations of bound material, then progressively decreased at higher concentrations. The mean size of micelles and their electrophoretic mobility was little affected by bound ionic materials. The aggregation of the casein complexes in colloidal calcium phosphate-free milk was markedly increased by adding ionic materials, the efficiencies of these additives paralleling their efficiencies in accelerating the coagulation of milk by rennet. The results suggested that the ionic materials were bound in the interior of the casein micelles and promoted aggregation after rennet treatment by shielding charged groups, thus increasing the micellar hydrophobicity.

The effects of concentrations of 10–30 mM/1 of CO2 on the growth of psychrotrophic milk spoilage organisms were examined, both in untreated whole milk and in pure cultures of Pseudomonas fluorescens growing in skim-milk and broth. Spoilage of refrigerated untreated whole milk was found to be inhibited by the addition of CO2 and the effect was enhanced with increasing concentrations of CO2 and decreasing storage temperature. Experiments with pure cultures of Ps. fluorescens showed that the inhibitory effects of CO2 were not due to increased acidity or to displacement of dissolved O2, but to the presence of CO2per se which induced an increase in the duration of the lag phase of growth and had only a small effect on the logarithmic phase.

With either Na caseinate or skim-milk powder, 1% (w/v) additions of pepsin, chymotrypsin or Alcalase and an incubation time of 24 h at 37°C gave the best hydrolysates for subsequent plastein production. In spite of the fact that peptide species which were produced by treatment with the 3 enzymes were entirely different, they were all capable of producing qualitatively similar amounts of plastein when concentrated and further incubated with proteinase, suggesting that the identity of peptide components was relatively unimportant. αs1-, β- and κ- caseins, Na-caseinate and skim-milk powder all led to plastein products with broadly similar properties, confirming the comparative unimportance of peptide composition. This also indicated that for most practical applications fractionation of initial protein mixtures (as in many food products) would not be justified. Optimum peptide concentration for plastein formation with pepsin and caseinate hydrolysates was 20–35% (w/v) with an optimum pH range of 4–5; the best peptide molecular weight varied between 400 and 800 and the optimum temperature was either 37°C for 24 h or 50°C for 4–6 h. Higher temperatures (70°C) gave more rapid plastein formation but poorer yields. Lower temperatures (e.g. 20°C) gave similar yields, but incubation times required to be extended to at least 48 h. The same proteinase should be used for plastein synthesis as that used in the initial hydrolysis stage, or else further hydrolysis (over hydrolysis) could occur during the plastein formation stage causing lower yields due to differences in the specificity of the 2 enzymes. Even under ideal conditions plastein yields never exceeded 27% of the weight of peptide taken and at the present time it seems unlikely that the plastein reaction will have anything more than curiosity value. Economics must preclude its application in low-cost, high volume situations such as generally exist within the food industry.

A scintillation counter was used to measure chemiluminescence (CL) from milk powder and reconstituted milk. Only a small amount of CL, just above the background level, was observed from powders, but reconstituted milks emitted easily measurable CL. CL from milk was stimulated by exposure to light and found to decline gradually but non-exponentially after the exposure ceased. CL was at a maximum immediately after the milk was reconstituted from milk powder. Without light exposure, CL then declined gradually but non-exponentially over several hours to reach a constant value related to the level of oxidation of the powder. When determined under standard conditions 60 min after reconstitution, CL of milk reconstituted from stored milk powders correlated well with peroxide values and oxidized flavour as assessed by a taste panel.