Alkylation of whole casein samples by reaction with cysteamine and cystamine in a bis-tris-propane–urea buffer (pH 7·0) followed by fast protein liquid chromatography (FPLC) at 20°C on a Mono Q HR5/5 column in the same buffer and using a NaCl gradient led to good resolution of the whole casein into fractions representing (i) γ2- plus γ3-caseins, (ii) κ-caseins, (iii) β-casein, (iv) αs2-caseins and (v) αsl-caseins, together with small amounts of unidentified materials. Quantitatively the FPLC values agreed well with those for αs1-, β-, αs2- and γ2- plus γ3-caseins obtained by ion-exchange chromatography on DEAE cellulose, Whatman DE52 and with those for º-caseins obtained by gel-permeation chromatography on Sephadex G–150.

The sizes of the casein particles in milk heated at 130°C were measured as functions of heating time and of initial pH. The effect of adding 10 mM-urea to the milks was also studied. Turbidities of the milks before and after dispersing the caseinate particles in 8 m-urea/EDTA/β-mercaptoethanol were measured, the latter measurement giving an indication of the extent of covalent cross-linking. Urea was found to modify the behaviour of the milk: diameters of the caseinate particles were considerably altered, and the extent of covalent cross-linking was diminished. These results agree with the observation that more protein is solubilized from the casein micelles when urea is present, indicating that a competitive reaction scheme involving cross-linking in opposition to micellar dissociation may be responsible for the ultimate precipitation of milk by heat.

On heating casein micelle systems containing β-lactoglobulin (β-lg) at 90°C for 10 min, β-lg complexed with casein micelles at pH < 6·9, probably as a result of interaction with κ-casein via sulphydryl-disulphide interchange, and co-sedimented with the micelles on ultracentrifugation. Complex formation with β-lg appeared to prevent the dissociation of micellar κ-casein on heating. However, at pH ≥ 6·9, κ-casein/β-lg complexes dissociated from the micelles on heating, thus enhancing the release of micellar κ-casein. High concentrations of β-lg (≥0·8%) induced coagulation at pH 7·3, essentially by promoting the dissociation of micellar κ-casein. It appeared that αs1-, αs2-, β- and κ-caseins dissociated from serum protein-free casein micelles to equal extents, but the presence of β-lg specifically enhanced the dissociation of κ-casein at pH values ≥ 6·9. Micelle hydration increased slightly when casein micelles were heated in the presence of β-lg at pH 6·7, while at pH 7·3 β-lg decreased the degree of hydration of casein micelles. Formation of a complex between β-lg and κ-casein appeared to stabilize the micelles in the pH range 6·5–6·7, possibly via increased micellar charge or degree of hydration or by preventing the dissociation of κ-casein.

A highly glycosylated phosphoprotein (HGPP) was isolated from a human casein fraction by reversed-phase high-performance liquid chromatography. This component contained carbohydrates to ∼ 38·2% (w/w) and phosphorus to ∼ 1·6% (w/w). The molecular weight of this HGPP as estimated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis ∼ 41000. Ultracentrifugal analysis revealed that the sedimentation coefficient of the HGPP was 2·6S in a 10 mM-imidazole-HCl buffer at pH 7·0 and 27 °C, but this component interacted with human κ-casein and formed a complex with s = 10·4S.

Heating milk at 140 °C caused an initial increase in the percentage of total casein sedimented at 10000 g for 1 h and in the relative viscosity, but as heating continued both parameters decreased before increasing again just before the onset of visible coagulation. This suggests that heating milk at high temperatures caused an initial aggregation of casein micelles with a concomitant increase in particle size and weight, followed by dissociation of these aggregates with reaggregation just before coagulation. Chromatography of heated milk on controlled pore glass confirmed the above suggestion. Calcium appeared to play a major role in the initial association of casein micelles which was also influenced by the initial pH and total solids concentration but whey protein had no effect.

Milk ultrafiltrate and milks of varying protein, citrate and phosphate concentrations were heated in sealed containers. Protein was found not to be involved in the mechanism of formation of lactulose, but increasing the protein content of milk reduced the concentration of lactulose after heating. This was considered to be due to increased condensation of lactose and lactulose with amino groups of the protein, Less lactulose was formed in milk ultrafiltrate than in skimmed milk accorded the same heat treatment, which was attributed to the buffering capacity of the milk protein in skimmed milk. Activation energies for lactulose formation in skimmed milk and in ultrafiltrate were 128 and 131 kJ/mol respectively. Citrate and phosphate catalysed the formation of lactulose. It is proposed that the formation of free lactulose in heated milk and ultrafiltrate proceeds exclusively by the Lobry de Bruyn-Alberda van Ekenstein transformation with the naturally occurring phosphate and citrate acting as base catalysts.

Heat inactivation of a metalloproteinase, isolated from Pseudomonas fluorescens biotype I strain 112, was investigated in the temperature ranges 50–60 °C and 90–140 °C. At 90 °C the denaturation of the enzyme followed first-order kinetics with a decimal reduction time of 110 min and a velocity constant K of 3·5 × 10−4 s−1. Activation energy Ea was 100 kJ/mol for this temperature range. In the 50–60 °C region the proteinase was inactivated by autolysis, as shown by electrophoresis and gel filtration. At 55 °C the decimal reduction time was ∼ 22 s, at 57 °C it was 8 s. Rapid inactivation at 55 °C was only possible if the enzyme was heated from lower temperatures, but not if cooled down from 90 °C. This is due to a conformational change of the protein at this temperature. A model for the description of heat inactivation in the two temperature ranges is proposed.

Reducing the colloidal calcium phosphate (CCP) content of milk by 40% or increasing it by 20% did not significantly affect the heat-induced pH-dependent dissociation of micellar κ-casein. However, changes in soluble Ca and phosphate affected the dissociation of κ-casein markedly; decreasing the phosphate concentration or increasing the Ca concentration reduced the formation of non-sedimentable N (NSN) and non-sedimentable 12% TCA-insoluble N-acetyl-neuraminic acid (NANA). Dialysis of milk against water for short periods (∼ 5 h) reduced the formation of both NSN and non-sedimentable 12% TCA-insoluble NANA, as did NaCl at concentrations above 0·05 M. Modification of protein amino groups by succinylation promoted the release of κ-casein while amidation of carboxyl groups had the opposite effect. It appears that the pH-dependent dissociation of κ-casein produced on heating milk above 90°C is controlled by electrostatic interactions. The effects of soluble ions such as Ca2+ or Na+ appear to be due to shielding of such negatively-charged groups as seryl phosphate and carboxyl on the protein, thus reducing the release of κ-casein.

Skim milk samples from 126 Friesian and 147 Jersey cows in eight commercial herds were preheated at 85 °C for 30 min and concentrated to 200 g l−1 total solids. A heat coagulation time–pH curve was determined at 120 °C for each treated sample. Heat coagulation times ranged from 1 to 50 min at the non-adjusted pH and 1 to 60 min at the pH of maximum stability. The following statistically significant effects were found. Maximum heat stability was affected by genetic variants of κ-casein (B > AB > A; P < 0·001) and β-lactoglobulin (B, AB>A; P < 0·05) whereas natural heat stability was affected only by κ-casein genetic variants (B > AB > A; P < 0·001). Maximum and natural heat stability were corre-lated positively with β-casein and κ-casein concentrations and were negatively correlated with αs1-casein and β-lactoglobulin concentrations. Milk from Jersey cows had greater maximum and natural heat stability than milk from Friesian cows. Differences were found between herds within breed for natural heat stability, but not for maximum heat stability. Maximum heat stability declined with age of the cow. The heat stability of skim milk samples taken from 40 Jersey cows in one of the herds was determined at 140 °C. A considerable variation was found in the coagulation time–pH curves. There was a difference in natural heat stability between κ-casein variants (B > AB; P < 0°05). Natural and maximum heat stability were correlated positively with urea concentration. No relationship was found between the heat stability of preheated concentrated skim milk and the heat stability of the original skim milk. The pH of skim milk samples was associated with αs1-casein genetic variant, age of cow, stage of lactation and concentration of γ-casein.

The ethanol (EtOH) stability of skim milk and the stability towards aggregation of casein micelles diluted into ethanolic buffer solutions were compared using data obtained from previously published experiments. Differences in absolute stability and in relative response were observed when Ca2+ level and pH were adjusted, the buffer system results lying below those from skim milk in both cases. Increasing the ionic strength of skim milk adjusted to pH 7·0 lowered its EtOH stability whereas increasing the ionic strength of the diluting buffer increased the stability of the casein micelles. The hypothesis is put forward that the differences are due to the simultaneous precipitation of Ca phosphate when EtOH is added to skim milk. This draws calcium from the caseinate sites of the micelle, counteracting the destabilizing effects of the EtOH towards the micelle. Such removal and the consequent restructuring are kinetically controlled and micellar precipitation in skim milk finally occurs when the micellar coagulation time falls within the time scale of the restructuring reactions.

Three strains of psychrotrophic Pseudomonas spp., each with different lipolytic and proteolytic phenotypes (including a proteinase-deficient mutant), were cultured separately in whole milk at 7°C. Growth rates were the same during logarithmic growth phase, but during early stationary phase the cell densities were related to the activities of lipase and proteinase in the cultures. Only one strain underwent pronounced death phase. Proteinase activity was not detected in the culture of the proteinase-deficient mutant, but in those of the other strains it increased to a plateau, or continued to increase linearly. Lipase activity of the culture of each strain reached a peak in early stationary phase; in late stationary phase activity was highest for the proteinase-deficient mutant strain where degradation of lipase by bacterial proteinase would have been least. The ability of psychrotrophic bacteria both to survive for long periods and to produce high levels of proteinases and lipases on prolonged incubation in milk emphasizes the spoilage potential arising from psychrotrophic bacteria in inadequately cleaned dairy equipment.

The combined effect of ultrasonic waves (20 kHz, 150 W) and heat treatment applied simultaneously (thermoultrasonication) on the survival of a strain of Staphylococcus aureus was studied in 0·05 m-phosphate buffer pH 6·8 and ultra-heat-treated whole milk. This combined process decreased by 63% the decimal reduction times for the heat treatment when the organism was suspended in buffer and by 43% when suspended in milk. These effects were much greater than the additive effect of the two agents considered independently.

Kareish cheese was made from ultrafiltered milk (UF) and standard milk and the chemical composition, organoleptic properties and microstructure of the cheese compared. Cheese made with the UF had higher moisture, protein, fat, pH, soluble N, non-protein N, amino acid N, tryptophan and free amino acids than the standard cheese. The microstructure of both types of cheese was very similar, while the organoleptic properties were more acceptable in the UF cheese.

Heat stability profiles of a number of indigenous bovine milk enzymes were examined with the object of being able to monitor heat treatments slightly more severe than typical pasteurization conditions by measurements of residual enzyme activity after heating. Assay procedures were limited to simple fluorimetric, or preferably colorimetric, methods that would be most likely to form the basis of a quick, simple and inexpensive test. Both lipoprotein lipase and α-fucosidase were relatively sensitive to heat and were totally inactivated at temperature/time combinations below those of pasteurization, but the latter may be satisfactory for studying temperatures in the range 55–65°C. Rather more heat stable were N-acetyl-β-glucosaminidase and γ-glutamyl transpeptidase, which may be most appropriate for 65–75°C and 70–80°C respectively. Higher temperature treatments between 80 and 90°C could best be investigated by following α-mannosidase or xanthine oxidase activity.

Changes in the composition of the microflora in Cheddar cheese ripened at elevated temperatures were examined. Cheeses were stored under the following ripening conditions: (i) control, 8°C for 32 weeks; (ii) 15°C, (iii) 17·5°C or (iv) 20°C, all for 8 weeks, then followed by 8°C for 24 weeks; (v) 15°C, (vi) 17·5°C or (vii) 20°C, all for 32 weeks. Generally, the total bacteria, lactic acid bacteria, lactobacilli and ‘undesirable lactobacilli’ (producing off flavours and CO2) counts in cheeses stored above 8°C were higher than in the control cheese. This effect seemed to be dependent on continued storage at the elevated temperature. Streptococcal counts rose slightly during ripening at elevated temperatures and fell slightly during ripening at the control temperature. Numbers of non-lactic acid bacteria were unaffected by elevated storage temperatures. In the only treatments where off flavours developed (17·5°C for 32 weeks, 20°C for 16 weeks or more) there was no apparent relationship between any bacterial group and the off flavours.

Lactobacillus bulgaricus strains LT1 and LT4 previously isolated from a yogurt factory were shown to be lysogenic and inducible by mitomycin C or u.v. irradiation, induction being optimal at the beginning of exponential growth. Five indicator strains (four of L. lactis, one of L. bulgaricus) were found in which the two phage lysates propagated well in a liquid medium, but formed plaques on bacterial lawns less readily. Ca2+ was required for the phage infection of the indicator strains to reach termination. Phage particles from each strain exhibited a comparable ultrastructural morphology under the electron microscope, having an isometric head, a triple collar and a noncontractile fibre-containing tail.

A proteinase was purified from a cell wall extract of a culture of Streptococcus lactis NCDO 763 grown in skim milk. Being active at a low pH (at pH 4·8 on haemoglobin and pH 6·–6·5 on casein) and completely inhibited by diisopropylfluorophosphate, it was considered to be a serine proteinase partly inhibited by EDTA; the mol. wt was ∼ 80000.

A mechanistic model of dairy cow metabolism is described. The model was constructed as part of a programme directed toward quantitative and dynamic analysis of concepts and data regarding factors which influence the partition of nutrients in lactating dairy cows. Sensitivity and behavioural analyses undertaken using the model indicate that concepts and data arising from tissue level experiments conducted in vitro can be used to structure and parameterize whole-animal models since the quantitative and dynamic behaviour of such a model is acceptable. These analyses indicate further that such models can be used to evaluate factors which influence patterns of nutrient utilization. By way of illustrating the model's utility regarding evaluation of concepts relating to the interpretation of energy balance experiments, results of a simulated energy balance experiment are presented. Apparent costs of milk synthesis are partitioned among biosynthetic costs, physiological costs, ion transport costs and the reductions in energy expenditures in synthesis of body components which accompany increasingly negative energy balances due to feed restriction.

Milk clotting activity of Mucor miehei proteinase (commercial name Fromase) was determined in combined rennets as the difference between the total activity of combined rennet and the residual activity after inhibition of Fromase by the purified immunoglobulin fraction of a Fromase antiserum. The proposed method is simple, rapid, and makes possible the use of any arbitrary method for assaying milk clotting activity. The application of the immunoglobulin fraction, instead of the whole antiserum, is necessary owing to the presence of nonspecific proteinase inhibitors in blood serum.

Thirty six samples of Manchego and 36 of Burgos cheese from retail outlets were examined. The mean log microbiological counts/g in Manchego cheese were 2·6 coliforms, 3·76 enterococci, 7·07 lactic acid bacteria, 8·02 mesophiles, 3·84 psychrotrophs 4·47 yeasts and moulds and 4·11 staphylococci, and in Burgos cheese were 7·17, 3·45, 7·25, 8·89, 8·18, 5·09 and 5·12 respectively. Coagulase-positive staphylococci were detected only in three samples of Manchego cheese, one of these isolates producing enterotoxin A. Staphylococcal thermonuclease was detected in eight samples of Manchego and 12 of Burgos cheese, the mean values of enzyme detected being 0·29 and 0·32 ng/g cheese respectively. Staphylococcal enterotoxins were not detected in any of the cheeses tested. Possible relationships between microbial counts and physicochemical characteristics were investigated by regression analysis.