Micellar calcium phosphate has the chemical composition and physicochemical properties that are consistent with it being a complex of the phosphate centres of casein with an acidic amorphous calcium phosphate. Similar acidic amorphous calcium phosphates have been prepared in the laboratory and for these, as well as for micellar calcium phosphate, the most appropriate crystalline model compound from which the short-range structure may be derived is brushite, CaHPO4.2H2O. The predicted secondary structures around sites of phosphorylation in the Ca2+-sensitive caseins often comprise an α-hclix-loop-α-helix motif with the sites of phosphorylation in the loop region. This motif may be important in linking the colloidal calcium phosphate with casein in native casein micelles.

Lamb rennet pastes were prepared by the procedure most commonly used by Idiazabal cheese manufacturers. We studied the effects on their coagulating and lipolytic activities of the state of the stomach at the time of death (full of milk or empty), the amount of NaCl added, the origin of the lambs and paste storage time. Coagulating activities were generally between 155 and 363 units/g tissue. Pastes prepared from stomachs of lambs from slaughterhouse flocks had significantly higher coagulating activities than those of lambs from separate flocks. No significant decrease in coagulating activity was observed after 1 year storage at 4 °C. Chymosin represented 75–80% of the total coagulating activity with the remainder being pepsin. Rennet paste extracts with pH < 4·7 did not have increased coagulating activities when their pH was lowered to 2·0, while those with pH > 5·2 had activities 1·5-fold those before treatment. Lipase activity was higher in extracts of rennet pastes prepared using the stomachs of lambs that arrived at the slaughterhouse in the morning just prior to slaughter than in those prepared with the stomachs of lambs that had arrived on the previous evening. However, the reverse was the case for esterase activity. Activating the coagulating activity by pH cycling completely destroyed both lipolytic activities. Storage at 4 °C for > 1 year did not affect esterase activity but lipase activity decreased substantially after 4–5 months. Lipase, but not esterase, activity was responsible for the liberation of short-chain free fatty acids from ovine milk fat.

Activity of lysosomal N-acetyl-β-d-glucosaminidase (NAGase) in milk has been used as an indicator of bovine mastitis. We studied NAGase activity of 808 milk samples from healthy quarters and quarters of cows with spontaneous subclinical and clinical mastitis. Associations between milk NAGase activity and milk somatic cell count (SCC), mastitis causing pathogen, quarter, parity, days in milk (DIM) and season were studied. In addition, the performance of NAGase activity in detecting clinical and subclinical mastitis and distinguishing infections caused by minor and major bacteria was investigated. Our results indicate that NAGase activity can be used to detect both subclinical and clinical mastitis with a high level of accuracy (0·85 and 0·99). Incomplete correlation between NAGase activity and SCC suggests that a substantial proportion of NAGase activity comes from damaged epithelial cells of the udder in addition to somatic cells. We therefore recommend determination of NAGase activity from quarter foremilk after at least six hours from the last milking using the method described. Samples should be frozen before analysis. NAGase activity should be interpreted according to DIM, at least during the first month of lactation. Based on the results of the present study, a reference value for normal milk NAGase activity of 0·1–1·04 pmoles 4-MU/min/μl for cows with ≥30 DIM (196 samples) could be proposed. We consider milk NAGase activity to be an accurate indicator of subclinical and clinical mastitis.

Acidity is an environmental condition commonly encountered by lactic acid bacteria and bifidobacteria in the gastrointestinal tract and fermented foods. In the present study, 22 strains of Bifidobacterium were screened for acid tolerance in artificial gastric juice (AGJ, pH 3·0) and fermented milk. AGJ tolerance was found to be strain-specific, with a pronounced variation among the strains. Several strains with a high survival rate in AGJ that belonged to Bifid. longum, Bifid. breve and Bifid. adolescentis were selected. Among them, only strain BL1 of Bifid longum was found to possess a high survival rate in fermented milk during refrigerated storage. Strain BL1 exhibited a survival rate of more than 25% in AGJ at pH 3·0 for 2 h and maintained a viable cfu level of more than 108 per gram of product in fermented milk (pH 4·6) under refrigerated conditions for 2 weeks. The acid tolerance of strain BL1 was found to depend on the final growth pH (<4·5). Rapid loss of acid tolerance was observed when the cells were shifted from acid to neutral conditions by addition of NaOH. Strain BL1 cells were able to maintain much higher intracellular pH under acid conditions, in comparison with those of AGJ sensitive mutant (BL1-S) or cells that lost acid tolerance following pH shifting from acid to neutral conditions. These results suggested that a cytoplasmic pH homeostasis system may function in the acid tolerance response in this strain.

A detailed kinetic study of hydroxymethylfurfural, lactulose and furosine formation was performed upon heating milk at temperatures between 90 °C and 140 °C. In case of prolonged heating, formation kinetics could be described by a fractional conversion model. Considering only the first phase of the model, kinetics could be simplified to a pseudo-zero order model. A first assessment of kinetic parameters was made by isothermal experiments. Data were analysed using both a 2-step linear and a 1-step non-linear regression method. Only for furosine, did the global 1-step regression approach seem to give better results than the individual 2-step regression approach. Next, the estimated parameters kref and Ea were re-evaluated under non-isothermal conditions by subjecting milk to a time variable temperature profile. Given the complexity of Maillard reaction, it seemed better to estimate kinetic parameters under non-isothermal conditions when using a simplified model. Formation of hydroxymethylfurfural, lactulose and furosine was characterized by an Ea value of 90·2 kJ/mol (k110 °C = 1·2 μmol/l, min), 99·1 kJ/mol (k110 °C = 51·5 mg/l, min) and 88·7 kJ/mol (k110 °C = 16·3 mg/100 g protein, min) respectively. Additionally, 90% joint confidence regions were constructed in order to obtain an accurate representation of the statistical confidence associated with the simultaneously estimated parameters.

In this study, the properties of casein particles reformed from alkaline disrupted casein micelles were studied. For this purpose, micelles were disrupted completely by increasing milk pH to 10·0, and subsequently reformed by decreasing milk pH to 6·6. Reformed casein particles were smaller than native micelles and had a slightly lower zeta-potential. Levels of ionic and serum calcium, as well as rennet coagulation time did not differ between milk containing native micelles or reformed casein particles. Ethanol stability and heat stability, >pH 7·0, were lower for reformed casein particles than native micelles. Differences in heat stability, ethanol stability and zeta-potential can be explained in terms of the influence of increased concentrations of sodium and chloride ions in milk containing reformed casein particles. Hence, these results indicate that, if performed in a controlled manner, casein particles with properties closely similar to those of native micelles can be reformed from alkaline disrupted casein micelles.

The goal of the study was to evaluate the composition of leucocyte populations in different milk fractions as a basis on which to judge their possible role in the immune response in different compartments of the udder. The milk of one healthy quarter of nine dairy cows (SCC/quarter [les ]125000/ml; bacteriologically negative) was removed separately and during the course of milking divided into: cisternal milk (C), alveolar milk 0–25%, 25–50%, 50–75%, 75–100% (A25; A50; A75; A100, respectively) and residual milk (R). Each fraction was analysed for the main constituents, SCC and distribution of leucocyte populations and their viability. The content of fat increased steadily during milking and reached highest values in R. Protein and lactose increased from C to A25, decreased from A25 to A100 and reached their minimum in R. Na and Cl ion levels diminished from C to A25 and thereafter increased from A50 to R. Electrical Conductivity (EC) also decreased from C to A25 but remained similar within the alveolar samples and reached a minimum in R. SCC decreased from C to a minimum in A25 and increased subsequently to a significant maximum in R. Somatic cell viability increased throughout consecutive fractions with a maximum value in R. The ratio of cell populations in the various milk fractions showed a reverse trend of macrophages (M) and polymorphonuclear neutrophils (PMN). M values were highest in C while PMN levels increased to their maximum in the R fraction. The lymphocyte (L) fraction remained similar in C, A25, A50, A75 and R but was higher in A100. Proportions of L, PMN and M were, respectively, 9·3%, 38·2%, 52·3% in C, 10·9%, 64%, 25·1% in A25–A100 and 10·2%, 64·9%, 24·8% in R. Numbers of L, PMN and M in milk showed a similar pattern for all three cell types: high levels in C decreased to a minimum at A25 and increased steadily thereafter to their maxima in R. It is concluded that, for healthy quarters, M, the predominant cell type in C, are located near the teat canal, the main entrance of pathogens. Obviously they are the first immunological barriers for invading pathogens. In contrast, PMN are the most important population in the alveolar compartment. However, each leucocyte fraction had a higher concentration in C than in early alveolar fractions, thus indicating the crucial role of immune defence in the cisternal compartment.

The effects of αs1-, β-, κ-casein and β-lactoglobulin genetic variants on rennet clotting time, rate of curd firming and coagulum strength, measured on the individual milks of 306 Rendena cows by a lactodynamographic apparatus, were evaluated statistically, with simultaneous adjustment for environmental and physicochemical factors. The importance of genetic polymorphism was confirmed, especially for the main effects of casein loci: interaction among them was not found to be significant. For κ-casein, BB genotype showed the best coagulation parameters (lower rennet clotting time, a higher rate of curd firming and higher coagulum strength), with AB in an intermediate position for clotting time and rate of curd firming. However, for αs1-casein and β-casein, dominance and overdominance effects between alleles were observed. Herd by genotype interaction was also found to be significant (herd by β-casein) or close to significance level (herd by κ-casein). From the results of the analyses, suggestions for selection programmes have been made.

A survey was made of the frequency of β-casein variants in the milk of cows of five major British breeds. Three variants A, B and C differing in electrophoretic mobility were found to occur either singly or in pairs, as expected if each were determined by a single allele. The A variant was the most common and found in all breeds. Ayrshires and Shorthorns produced no other variant, whilst the B variant occurred in Jerseys and, at low frequency, in Guernseys and Friesians. Production of the C variant was restricted to the Guernsey breed in which it occurred at low frequency. The technique of paper electrophoresis, used for β-casein ‘typing’ of some 600 cows, is described in detail.

The effects of bulk milk cell count (BMCC) and stage of lactation on the quality and storage characteristics of UHT milk were investigated. The UHT milk was manufactured in a pilot plant using milk of low BMCC from early and late lactation, and milk of high BMCC from early and late lactation. Upon storage at 20°C, early lactation UHT milk gelled far ahead of late lactation milk. Within each stage of lactation, high BMCC milk tended to gel first. Few differences in the organoleptic properties of the UHT milks were observed. It was apparent that the onset of age gelation may not always be related directly to the level of proteolysis, and that other factors influencing milk composition and the reactions between milk components may play more important roles. At a particular stage of lactation, proteolysis induced by mastitis may hasten the onset of gelation.

A new method is described for the determination of the free and masked sulphydryl (-SH) groups in heated milk and milk products. The procedure is based on the use of the specific -SH reagent p-chloromercuribenzoate, and utilizes another specific -SH reagent, 5,5' -dithiobis(2-nitrobenzoate), as an indicator producing yellow colours which are matched with the aid of standard glasses in the Lovibond Comparator.

The method was applied to heated skim-milks and a series of commercially prepared milk powders. Measurements were made in particular of the -SH content of UHT-treated milk. The changes in the -SH content of such milks on storage were studied; these changes could not be explained by a reversal of the denaturation of the whey proteins, although evidence was obtained that small amounts of heatdenatured whey protein can revert to the native state.

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.

The effect of varying the casein composition of artificial micelle milk on rennet coagulation time and syneresis was examined in order to determine whether either of these processes is dependent on the concentration of particular casein components. It was found that the levels of κ-and β-caseins had a significant effect on coagulation, whereas syneresis was only affected by the level of β-casein. Partial dephosphorylation of preformed micelles or the incorporation of dephosphorylated or partly dephosphorylated β-casein into artificial micelle milk was found to have an adverse effect on both coagulation and syneresis. It was concluded that the phosphate groups of casein, particularly those of β-casein, are directly involved in the micelle-micelle interactions which occur during coagulation and syneresis.

Two monoclonal antibodies (MAb) raised against bovine κ-casein were developed and applied in an automated optical biosensor (Biacore 3000) to create easy and fast direct and inhibition biosensor immunoassays (BIA) for the detection of cows' milk in the milk of ewes and goats. With both assay formats, low limits of detection (<0·1%) and fast run times (around 5 min) were obtained. For sample preparation, milk was diluted in buffer (direct assay) or in an antibody-containing buffer (inhibition assay) only. For quantitative analysis, calibrants of cows' milk in ewes' or goats' milk were used. Advantages of the direct BIA are: the single reagent format (biosensor chip immobilized antibodies only); the use of small amounts of antibodies (2 μg for >350 tests); and the wide measurement range (0·1 to 10% cows' milk). Despite these advantages, the inhibition BIA (using κ-casein immobilized on the chip) was preferred because of the possible application of non-purified Mab, the higher responses, the higher sensitivity at relevant low percentages of cows' milk and its robustness (>800 cycles per chip).

Six batches of Casar de Cáceres raw ewes' milk cheeses were examined throughout their ripening. Three were made in winter, the others in spring. The microbial counts in the interior of the cheeses were ∼1·5 log units higher in the winter cheeses for total viable counts, lactic streptococci and leuconostocs, and >2 log units higher for total enterobacteria, coliforms and faecal coliforms in the 60 d samples. Staphylococci were not detected after day 15 of maturation in spring cheeses, but reached 1 log unit at day 60 in the interior of winter cheeses. In the surface samples, differences in microbial counts were more marked. Values of some physicochemical indices, such as pH, % moisture content and % NaCl were also determined.

Of the lactic acid bacteria isolated, 720 strains from thirty-six typical Cheddar cheeses have been classified. The dominant organism in New Zealand cheese has been shown to be Sbm. plantarum. Sbm. casei occurs much less frequently, while betabacteria and betacocci are found in still smaller numbers. Betabacteria tend to replace Sbm. plantarum in the final stage of cheeseripening. The flora of good quality cheese has been found to consist mainly of one or two varieties of Sbm. plantarum, often associated with Sbm. casei or a small proportion of betabacteria. In poor-quality cheese a larger number of types of lactic acid bacteria frequently occur. A simple routine method is described for distinguishing the different groups present in cheese.

The effects which different strains of lactobacilli and betacocci produce in cheese have been correlated with their biochemical characteristics. Sbm. casei in general appears to be beneficial to cheese quality. The strains of Sbm. plantarum isolated have been grouped in four varieties, mainly on the basis of sugar reactions; one variety has been shown to be beneficial to cheese quality, another variety has very little apparent effect, while the remaining two varieties produce serious defects—bad flavours, discoloration and, occasionally, open texture. The same defects are produced by most strains of betabacteria and betacocci when present in large numbers.

The biochemical characteristics of the lactobacilli studied have shown a more regular gradation from Sbm. casei through Sbm. plantarum to Bbm. breve (the only species of Betabacterium isolated) than is indicated from the generally accepted systems of classification.

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.