The action of rennet on the stability of casein micelles has been described theoretically by combining Michaelis–Menten enzyme kinetics with von Smoluchow-ski's theory for slow coagulation. The casein micelle is considered to be stabilized by stcric interactions arising from a highly hydrated surface protein layer. The stability factor, in von Smoluchowski's theory, is then considered as a variable and determined by the concentration of unhydrolysed κ-casein.

The theoretical approach describes semi-quantitatively experimental observations on clotting time (CT) as a function of temperature, rennet concentration, and total protein concentration. The model also predicts that, under certain circumstances, the CT is inversely proportional to the enzyme concentration. Furthermore, the model agrees with other workers' experimental observations on the effect of κ-cascin concentration, the presence of a lag phase, and that the lag phase is commonly about 60% of the CT.

Thirty-nine British Friesian cows and 17 British Friesian heifers were used to measure the effects on milk yield, food intake and digestion, and N balance of increasing the protein concentration in the diet from 111 to 147 g crude protein/kg dry matter during the first 16 weeks of lactation. The comparison was made for animals given food either ad lib. or at a level which allowed them to gain 0·5 kg/d during the 8 weeks before parturition, and for animals given ad lib. diets of either 60:40 or 40:60 hay: concentrate ratio during early lactation. Milk yield was increased by a higher protein concentration in the diet (P < 0·01) and the increase was evident from the first week of lactation. Mean increases over the first 8 weeks of lactation were 3 kg/d for animals on the diet of high roughage content and 7 kg/d for animals on the diet of low roughage content. A higher crude protein content in the diet also increased food intake (P < 0·05) and digestion (P < 0·05), and the increases were greater for animals given the diet of low compared with high roughage content. Level of feeding before parturition did not influence food intake or milk yield and there were no differences between cows and heifers in their response to treatments. N balance over the first 10 weeks of lactation averaged –13 g/d for cows and 6 g/d for heifers (P < 0·025). and was not significantly altered by treatments. Mean N balance of all animals was estimated to increase from –37 g/d immediately after parturition to zero at 8 weeks of lactation. The results demonstrate that an increased protein concentration in the diet during early lactation is associated with increased food intake and digestibility of food, which are in turn largely responsible for higher levels of milk production.

The trypsin-inhibitor capacity of bovine milk was seen to increase in mastitits. This capacity showed good correlation with the California Mastitis Test score, somatic cell count (Coulter Counter method) and bovine serum albumin (BSA) content (radial immunodiffusion). The elevated antitrypsin level proved to be a safer indicator of mastitis than BSA alone. Using gel-filtration chromatography, the major trypsin-inhibitor activity of mastitic milk and plasma was eluted with BSA, indicating a similar mol. wt (70000). This fraction showed electrophoretic polymorphism. A large molecular weight fraction cross-reacted immunologically with human α2-macroglobulin. The antitrypsin activity in mastitic milk was clearly different from the principal activity in colostrum, which had a mol. wt of about 13000.

The leucocytes in sheep colostrum, milk and involution secretion were studied using the electron microscope. The predominant cell type in colostrum was the polymorphonuclear leucocyte (PMNL) (41–84%), followed by the macrophage (8–49%), and the lymphocyte (6–11 %). Plasma cells were present in low numbers (1–2%) while no secretory epithelial cells were observed. In mid-lactation the cell components changed so that the macrophage was the predominant cell (83–86%) followed by the lymphocyte (10–17%). In early involution secretion, PMNL reappeared but declined in secretion obtained 21 d after weaning, when the macrophage was again the predominant cell. Associated with the whole cells were membranous extracellular materials and ‘sunburst’ fragments of the secretory epithelial cells. These were phagocytosed by both PMNL and macrophages, which also engulfed fat droplets from the milk. The lymphocytes were examined for plasma membrane markers and the T-cell percentage (E+ rosettes) fluctuated within wide limits, (0–80 %). However, there was no discernible trend associated with stage of lactation. The B-cell markers (C1 and Fcγ) also fluctuated widely and it was evident that contaminating monocytes reduced the accuracy of the count. However, it appeared that more B-cells were present in milk than in blood.

A rapid method for the direct enumeration of somatic cells in fresh and formalin-preserved raw milk is described. Heat treatment at 80°C for 10 mn fixed somatic cells sufficiently to prevent lysis and subsequent dilution allowed the equivalent of at least 0.5 ml milk to be filtered through a 1.0 μm pore size Nuclepore membrane filter. The somatic cells were concentrated on the membrane and, after staining with acridine orange, fluoresced green, yellow or orange under an epifluorescent microscope. Three different types of cell could be distinguished on the basis of nuclear structure and cell size. These were monocytes, polymorphonuclear leucocytes and the larger epithelial and/or secretory cells. For both fresh and preserved milks the count of somatic cells on the membrane correlated well (r ≽ 0·93) with the Coulter count. Differences between counts obtained by different operators were not significant. The technique is rapid, taking about 20min, and is suitable for milks containing between 2 × 104 and 1 × 107 somatic cells/ml.

The ability of total milk proteose peptone and individual proteose peptone components to inhibit lipolysis in milk was examined. Proteose peptone (1·5 mg/ml) when added to milk inhibited lipolysis, and component 3 was a more effective inhibitor than component 5 or component 8-fast. Inhibition by proteose peptone also occurred when the milk was treated with between 2 and 20% v/v bovine blood serum or with 5 μg/ml heparin. In vitro activity of milk lipoprotein lipase and its stability were not affected by proteose peptone. It was concluded that proteose peptone does not interact with lipase or its activator and that the mechanism of inhibition involves the substrate.

A large-scale survey of milks from healthy and mastitic bovine quarters was undertaken to establish the influence of mastitic infection on milk lipase activity and free fatty acid (FFA) level. Mastitic milks tended to have higher FFA levels, but lower lipoprotein lipase activities compared with milk from healthy quarters. These effects became significant at relatively severe levels of infection. The elevated FFA was attributable to higher FFA levels on secretion and to greater lipolysis during storage. Levels of carboxylesterase activity increased with severity of mastitis and showed high positive correlation with mastitis indices.

Marked increases in carboxylesterase, N-acetyl-β-D-glucosaminidase and phospholipase occurred following the induction of mastitis by intramammary infusion of Escherichia, coli endotoxin, in parallel with changes in somatic cell count and other mastitis indices. Relatively little change in lipoprotein lipase activity was observed.

Positively charged materials, polyethyleneimine, poly-L-lysine and partly estcrified proteins (i.e. methyl bovine serum albumin, methyl casein and ethyl casein), in aqueous solution at the natural pH of milk, caused milk coagulation. This occurred at pH 6–6·7 at ≤ 30 °C using a defined amount of coagulant, which was quantitatively recovered in the curd. The protein and fat recovery yields were higher than with acid and rennet coagulation; the (Ca + Mg) partition between whey and curd was very similar to rennet milk clotting: the curd obtained showed high syneresis rate.

By resuspending casein micelles in whey and dialysate it is shown that the role of whey proteins in the ethanol (EtOH)-induced coagulation of skim-milk is minimal. Experiments involving the interchange of milk sera indicated that the position of the EtOH stability/pH profile along the pH axis was governed by the diffusible constituents of the milk serum phase. The identities of those serum components governing the shape and position of the EtOH stability/pH profile were investigated. The addition of Ca2+ caused a shift in the entire profile to higher pH. Reduction of the available Ca2+ by addition of EDTA (up to 5 ran) shifted the profile to lower pH. The addition of phosphate (up to 5 mM) or citrate (up to 1 mM) had no effect on the profile, though higher concentrations of citrate (up to 5 mMi) caused slight shifts to lower pH. When equimolar amounts of Ca and phosphate were added, the system showed a shift in profile approximately equivalent to that of the free Ca introduced. Increasing the ionic strength of a milk by the addition of NaCl did not shift the profile, but decreased the maximum EtOH stability of the high pH arm of the sigmoidal profile. The EtOH stability/pH profile retained the same sigmoidal shape in all cases.

Dialysing milk against phosphate-free sera showed that the transition in the ethanol (EtOH) stability/pH profile was associated with the soluble phosphate component of the milk serum. Sigmoidal behaviour similar to that of milk was reproduced when the EtOH stability of artificial mixtures of casein, Ca and phosphate was measured as a function of pH. A mechanism for the s coagulation of skim-milk is discussed.

The thermal behaviour of β-lactoglobulin was studied by differential scanning calorimetry (DSC) in the temperature range 40–160 °C. The DSC curves revealed, in addition to the usually observed denaturation peak near 80 °C, a distinct endothermal peak between 130 and 150 °C. When the pH was increased from 6·5, the area under the peak near 80 °C (denaturation heat) decreased significantly, whereas the peak area near 140 °C increased. The temperature of maximum heat absorption in the peaks near both 80 and 140 °C gradually increased as the pH decreased. Addition of sugars and variation of the heating rate both caused a temperature shift of the endothermal heat effect at 140 °C, similar to that at 80 °C. No peak near 140 °C was observed when β-mercapto-ethanol was added to the β-lactoglobulin solution before scanning. The origin and nature of the high temperature denaturation peak is discussed in terms of conformational changes of the protein.

The main components of bovine whole casein were characterized by electrofocusing; pi values of αs1-, β-, κ-, γ1-, γ2- and γ3-caseins were determined. A further identification of casein components was achieved by a 2-dimensional electrophoresis study. 2-Dimensional patterns of γ-caseins obtained from a hydrolysate of β-cascin by bovine plasmin are in good agreement with those of γ-caseins naturally present in whole casein.

Using polyacrylamide gel electrophoresis, 2-dimensional gel electrophoresis and electrofocusing, it has been shown that 4 basic fragments were produced by action of plasmin on buffalo β-casein. These fragments were present in whole buffalo casein prepared from bulk milk. Their mol. wts, 20000, 16000 and 11000 (2 components) were determined by SDS-gradient polyacrylamide gel electrophoresis. It is suggested that 3 of them are homologous to bovine γ1-, γ2- and γ3-caseins respectively, while the last one (mol. wt 16000), which has no counterpart in plasmin digest of bovine β-casein, could arise from a cleavage at bond 68–69.

The action of gastric proteinases on bovine caseins was studied in vivo on rats fed skim-milk, or whole casein in water or whole casein in mineral solution, by analysing the gastric content after 30 min digestion. Clotting of the diet in the stomach greatly reduced the rate of gastric emptying. The proteolysis of caseins observed by gel electrophoresis appeared to follow a different pathway for the 3 different diets. The amino acid compositions of the trichloracetic acid sediments of the stomach contents did not differ between the 3 diets. The presence of free amino acids in the stomachs at significant levels (0·8–5 % of the total amino acid content) was observed.

Pickled soft cheese was made from recombined milk (30 % total solids, 8 % fat) containing buttermilk powder (0–4%) and stored in the pickle at room temperature (25±5 °C) for 135 d. The yield, quality and composition of cheese were followed periodically during storage. The addition of buttermilk slightly increased the yield and improved the flavour intensity of the cheese, whilst only slightly affecting its chemical composition.

Milks were prepared at 1·7- to 4-fold the initial concentration by combining skim-milk concentrated by ultrafiltration with cream, and used for Cheddar cheese-making. Starter growth was unaffected, but the increased buffering capacity in the more concentrated milks resulted in a slower decline in pH and a higher pH value in the cheese. Curd formation was faster despite the use of reduced amounts of rennet. With milk concentrated more than 2-fold, large amounts of fat were lost in the whey, so that the cheeses had less fat than normally. Fat losses may be partly related to the lower degree of aggregation of the casein micelles when the curd was cut. As the concentration factor of the milk increased, the rate of casein breakdown, the intensity of Cheddar flavour, and the levels of H2S and methanethiol in the cheese decreased. These factors may relate to the reduced concentration of active rennet retained in the curd at pressing.

Samples of curd and cheese, taken throughout cheese-making with milks concentrated to different extents, were examined by light microscopy and by scanning and transmission electron microscopy. The mature cheeses were analysed for textural parameters by instrumental and sensory means. The protein was packed in larger, more compact areas and the fat was more segregated in the curds and cheeses from the more concentrated milks. The protein network increased in coarseness in proportion to the concentration factor of the milk, the relative differences being the same throughout cheese-making into the mature cheese. Thus, the basic structure of the protein network was laid down during the curd firming process and was not fundamentally altered later in cheese-making. The fat/protein interfaeial area in the cheese and the changes in structure during maturation decreased as the concentration factor of the milk increased. The instrumental firmness, cohesiveness and force and compression required to cause fracture, and the sensory firmness, crumbliness, granularity and dryness of the cheese all increased as the concentration factor of the milk increased. The adhesiveness declined and there was little change in elasticity, with increase in milk concentration factor. The textural differences were related to the smaller fat content, lower level of proteolysis, stronger links in the protein matrix and reduced capacity of the fat and protein phases to move relative to each other in the cheeses from the more concentrated milks.