Caseins (αs1, β, αs2, e κ) represent about 80% of the whole protein content of ruminant milk. Each of these proteins is encoded by single copy genes (CSN1S1, CSN2, CSN1S2 and CSN3, respectively) clustered on a ∼200-kb segment of chromosome 6 (Ferretti et al. 1990; Gallagher et al. 1994) in the order: CSN1S1, CSN2, CSN1S2 and CSN3 (Mercier & Vilotte, 1993). Furthermore, in cattle and goat CSN1S1 and CSN2 are convergently transcribed (Leroux & Martin, 1996; Rijnkles et al. 1997) and are only 20 and 12 kb apart, respectively.

Cheddar cheeses were made from milks concentrated 3- to 6-fold by ultrafiltration. Modifications to the conventional cheesemaking process were designed to obtain the correct composition and pH value in the final curd, improve the fat and moisture retention and cheese texture, and increase the ripening rate. The compositions of all cheeses were similar to those of controls made with unconcentrated pasteurized milk. The curd and cheese structures and the textural properties of the cheeses were closer to those of the controls when made from milk concentrated 5-fold than when made from milk concentrated 3-fold. Light homogenization of the milk before concentration led to improved fat recovery and decreased graininess and crumbliness in the cheese. Lowering the coagulation temperature of concentrated milks gave improved curd structure and less grainy and more sticky cheeses. Addition of the bacterial proteinase, Neutrase, with the salt, stimulated proteolysis and flavour production in the cheeses and made them more sticky. Neither reduction of the mineral levels in the milk by acidification before concentration nor reduction of the lactose concentration by diafiltration had any significant effect on the composition or textural properties of the cheeses.

A dynamic mathematical model of blood flow regulation in the mammary glands of a lactating cow was constructed from a principle of local vasodilator release in response to changes in intracellular adenylate charge. An equation was derived to predict uptake of the milk precursors acetate, glucose, amino acids and fatty acids, as affected by mammary blood flow rate. Metabolism of the precursors to milk components and CO2 was simulated with a set of empirically derived equations. Relative rates of ATP production and utilization regulated both the number of perfused capillaries and the conductance of arteriolar segments in the mammary glands. The model simulated local control phenomena of functional and reactive hyperaemia, and simulation of autoregulation under changing arterial pressure suggested a predominance of precapillary sphincter regulation. It was predicted that an increase in blood flow without the mammary capacity to utilize blood metabolites efficiently would be detrimental to milk synthesis. Conversely, increased blood flow through changes in mammary activity resulted in predictions of higher milk production. It was proposed that the equation for uptake,

be used in analysis of mammary arteriovenous differences.

A total of 120 milk samples were collected from Comisana ewes throughout lactation. The ewes were ranked into two somatic cell count (SCC) categories: normal milk (N Milk) with SCC lower than 5·00×105/ml and high somatic cell milk (HSC Milk) with SCC higher than 1·00×106/ml. Milk samples were analysed in triplicate for pH, fat and protein contents, renneting parameters, and plasmin and plasminogen activities. The peptide profile due to total proteolytic activity (endogenous and exogenous enzymes) on α- and β-CNs were determined using urea-PAGE on sodium caseinate (pH 8·0 and pH 5·0) incubated at 37 °C for 4 d after sampling. The peptide profile due to non-plasmin enzyme activities at pH 5·0 was also determined using urea-PAGE. Plasmin activity was higher in the HSC milk than in the N milk throughout the study period. A decrease in plasmin activity was observed in the N milk during mid-lactation, which was probably related to decrease in pH, and in the HSC milk during late lactation, which may be ascribed to an enhanced influx of plasmin inhibitors from the blood stream. Proteolytic patterns in Comisana ewe milk were mainly affected by plasmin activity that increased with the SCC in milk. Also non-plasmin proteolytic activity was strongly enhanced by elevated SCC and resulted in a higher degradation of α-casein than of β-casein. In general, plasmin activity did not increase with the advancement of lactation and exhibited a different trend in HSC and N milk, suggesting that physiological factors did not play a key role in regulating the plasminogen-plasmin system in ewes' milk. Plasmin activity, detected with the colorimetric assay was consistent with proteolytic activity on sodium caseinate shown in urea-PAGE electrophoregram.

Concentration of milk by reverse osmosis is severely hindered by the formation of deposits on the membrane. Electron micrographs of this deposit show a thin (11nm) continuous electron-dense layer adjacent to the membrane. An electronlucent zone (10–15nm thick) separates this layer from the main bulk of the deposit which is 30 μm thick and consists largely of granules with some vesicles in a diffuse matrix. The density of packing of the granules increases with proximity to the membrane. Their appearance, size and digestion with pronase suggest that these are milk protein granules. The order and nature of these various layers could explain the pattern of flux decline.

The type and relative amounts of plasminogen activator (PA) in different fractions of bovine milk obtained from 15 Holstein cows were examined. Raw milk was centrifuged to separate skim milk and a somatic cell pellet. PA was mainly localized within the casein fraction, being 42 times that in the serum, and in association with somatic cells. The predominant form of PA in milk casein was isolated from SDS-PAGE gel extracts and had a molecular mass of ∽75 kDa. Its activity was increased 41-fold (P < 0·01) in the presence of fibrin but was unaffected by the presence of amiloride, indicating that it was due to tissue-PA. The predominant forms of PA associated with milk somatic cells were isolated from SDS-PAGE gel extracts and had molecular masses of ∽ 30 and ∽ 50 kDa. The activity of both proteins was unaffected by the presence of fibrin but was dramatically reduced by the presence of amiloride, indicating that they represented urokinase-PA.

In this work the purification and the complete primary structure of κ-casein from equine milk are reported for the first time. Mares' milk casein was separated by RP-HPLC into four fractions. Complete primary sequence was obtained by sequence analysis of the protein in the fastest eluting peak isolated by chromatography. This sequence was 95% identical to that reported for the C-terminal portion of the zebras' κ-casein and showed high similarity with κ-caseins from sources other than Equidae, confirming that this protein was indeed κ-casein in equine milk. The presence of post-translational modifications in equine κ-casein was investigated by mass spectroscopy, after enzymic dephosphorylation. Two main components were found, the smaller component being more abundant. Equine κ-casein was recognized by a lectin specific for one of the glucosidic bonds in the saccharide moiety of bovine κ-casein. Sequence comparison with prevision studies showed that the distribution of charged and hydrophobic regions in equine κ-casein was similar, but not identical, to that found in the bovine protein; these regions are associated with the role of κ-casein in the formation and stabilization of the micellar structure of casein in milk.

A randomized, controlled field trial was performed in The Netherlands to determine the therapeutic efficacy of parenteral penethamate hydriodide (Leocillin®) against naturally occurring, chronic, streptococcal mastitis during lactation. Quarter milk samples were collected from subclinical cases of Streptococcus uberis or Streptococcus dysgalactiae mastitis to determine the effect of treatment on bacteriological cure and somatic cell count (SCC) at quarter level. A quarter was considered to be cured when the bacterial species, isolated prior to treatment, was not isolated from the quarter milk samples taken on days 10 and 20 post-treatment (bacteriological cure), or when a quarter milk SCC (QMSCC) was <250000 cells/ml on days 10 and 20 post-treatment (SCC cure). Longitudinal data analysis was performed to determine the effect of antibiotic therapy on SCC and milk yield at cow level. Bacteriological cure occurred in 59% of 29 treated quarters, while no cure was observed in any of the 21 untreated control quarters. Treatment resulted in a significant decrease in SCC at cow and quarter level in comparison with untreated controls. There was no significant effect of treatment on milk production. Antibacterial treatment of subclinical streptococcal infections during lactation also prevented clinical mastitis. Furthermore, the treatment may contribute to reduction of bulk milk SCC and to prevention of pathogen spread in dairy herds.

The effects of reducing the frequency of milking of cows in late lactation on milk somatic cell count (SCC), polymorphonuclear leucocyte (PMN) content, chemical composition and proteolytic activity were investigated. Intermittent milking is frequently practised by Irish farmers in late lactation, and the objective of this study was to determine whether this procedure could be linked to altered quality of milk. Seventeen Holstein Friesian cows in late lactation (>215 d in milk) were assigned to two treatment groups, and were either milked twice a day until drying-off (control group) or milked intermittently as the yield fell (test group). Milk composition and enzymic characteristics were measured on two occasions. At the first sampling, day 7, test cows were on once daily milking and at the second, day 15, the test cows were being milked every second day. Milk yields were significantly lower in test than control animals and decreased between days 7 and 15 in both groups. Milk SCC and PMN levels were increased on reducing milking frequency and, at day 15, the increase was not linked to decreased milk yield. Milk lactose levels were significantly decreased and pH, α-lactalbumin levels, plasmin activity and plasminogen activity significantly increased by reducing milking frequency. In conclusion, reduced frequency of milking in late lactation leads to the production of milk that is abnormal in character and this may be linked to reduced quality of dairy products manufactured from such milk.

The gastrointestinal tract is a complex and dynamic ecosystem. Commensal microorganisms (C), which proliferate in the intestine from birth, are crucial for gut homeostasis while non commensal (NC) microorganisms are transient and enter the organism from the environment and foods. We studied comparatively the influence of oral administration of C and NC Lactobacillus fermentum and Lactobacilus acidophilus on the gut-associated lymphoid tissue (GALT) of conventional mice. To determine the importance of the selection of probiotic host-specificity bacteria with immunomodulating capacity, we examined the interaction with the gut by transmission electron microscopy and FITC-labelled bacteria. We compared the immunomodulation capacities of C and NC strains by studying the number of IgA secreting cells and cytokine profile. No differences were found in the number of IgA+ cells; however, the pattern of cytokine response to C and NC bacteria was different. With regard to proinflammatory cytokine (IFNγ and TNFα), we found that TNFα was mainly produced by NC bacteria, while C bacteria were able to elicit mainly IFNγ. The regulatory cytokines (IL-10 and IL-4) were induced with different patterns for both C and NC strains. No differences in the pathway of internalization to the gut between C and NC were found. In summary, we determined that C and NC bacteria interact with the intestine in the same way; both C and NC bacteria were able to reinforce the surveillance of the gut mucosal immune system. The cytokine profile showed that C bacteria would be involved in the regulation of intestinal homeostasis rather than in the immune activation as the NC bacteria.

The principle of moving boundary electrophoresis has been employed for the measurement of the electrophoretic mobility and subsequent calculation of zeta potential of bovine casein micelles. The zeta potential of casein micelles was observed to increase with increase in temperature (from 10 to 50 °C) and to decrease with decrease in pH. Heat treatment of milk between 90 °C/30 min and 135 °C/50 min had no significant effect on zeta potential. The zeta potential of casein micelles during rennet action decreases until all the κ-casein has been cleaved by the enzyme.

The results of this study indicate that electrostatic interactions alone are not sufficient for an understanding of the absolute stability of casein micelles.

Rennin hydrolysed the phe-met bond in the peptide H-ser-leu-phe-met-ala-OMe (i.e. methyl ester), the amino acid sequence of which is similar to that around the phe-met bond attacked by rennin in κ-casein. Rennin did not attack other peptides from this sequence not containing serine, and it is suggested that, in both κ-casein and the pentapeptide, the enzymic attack is accelerated by the nearby serine side chain. Rennin also hydrolysed sulphite esters such as phenyl sulphite ester and some N-substituted imidazole compounds such as benzoyl imidazole. Phenyl sulphite esters may be suitable substrate for assaying the activity of preparations of rennin.

Changes in the distribution of minerals in skim goats' milk by high pressure (400 MPa) and/or heat (85°C for 30 min) treatment have been studied. Heat treatment caused reduced solubility of the calcium, magnesium and phosphorus, and this increased with the severity of heating. In contrast, high pressure released different levels of micellar elements into the soluble phase without causing appreciable changes in pH or ionic calcium concentration. The levels of soluble salts returned to their original values when the heated samples were subjected to high pressure. However, heating pressurized milk resulted in concentrations of soluble minerals that were lower than in control milks, and close to values found in heated milks. The salt balance in goats' milk was less affected by high pressure treatment at 75°C than was that of cows' milk. These results are discussed in relation to the effects of high pressure and heat treatment on mineral equilibrium and micellar structure.

Assay procedures were developed for a number of enzymes in milk which apparently originate from leucocytes. The enzymes studied were acid phosphatase, N-acetyl-β-D-glucosaminidase, β-glucuronidase, arylsulphatase, α-mannosidase, and catalase. Quarter-milk samples were analysed for enzyme activity and results compared with the electronic cell count and the Wisconsin Mastitis Test. All enzymes measured except acid phosphatase and α-mannosidase showed good correlation with the electronic cell count. Of the other 4 enzymes tested, β-glucuronidase and arylsulphatase were unsuitable as diagnostic aids owing to the lengthy incubation periods required in their assay procedures. The assay of catalase, which involved the measurement of the initial rate of release of O2 using an O2 analyser apparatus, was rapid, sensitive and reasonably reliable, if fresh milk samples were used. The assay procedure for N-acetyl-β-D-glucosaminidase was considered to be the most reliable, simple and rapid enzymic method for estimating the number of somatic cells in milk.

The cheesemaking properties of recombinant chymosin from Kluyveromyces lactis (Gist-Brocades Chymosin 610) were Compared with those of standard rennet in parallel trials with the same milk and mixed culture starter. For each pair of cheeses the cheesemaking characteristics, mass balance results, composition of the cheeses at 6 weeks and maturation rates were similar. A taste panel was not able to differentiate between the cheeses at 3, 6 or 12 months.

A method has been developed, using the fluorogenic substrate 4-methylumbelliferyl oleate, to measure the activity of the extracellular lipase of the psychrotroph Pseudomonas fluorescens AR11 in milk. Inhibition of lipolysis by constituents of skim-milk was prevented by including both sodium taurocholate and cetyltrimethylammonium bromide in the reaction mixture. Cream also interfered with the reaction and skim-milk was used for high sensitivity. Natural milk lipase was readily inactivated by heat treatment to which AR11 lipase was relatively resistant.

The occurrence and isolation of bacteriophages from cultures of lactic streptococci (“starter cultures”) is described. Aeration of the milk in which the phage-infected cultures are grown is shown to have a stimulating action on the development of the phage. The characteristics of phages for lactic streptococci are described.

Although casein micelles are disrupted by removal of Ca, individual caseins remain aggregated in sub-micellar casein aggregates or sub-units. These sub-units have been studied by: (1) the use of gel filtration on Sepharose 4B at 6, 20 and 37°C at pH 6·7 and 0·1 ionic strength, (2) ultracentrifugation and (3) electron microscopy. At 37°C the protein composition of the sub-units varied across the gel-filtration peak, with κ-casein being eluted towards the leading edge and the ratio of αs1- to β-casein being almost constant across the peak. Re-chromatography of the protein from the leading edge of this peak gave a new wide peak with the κ-casein again being eluted towards the leading edge. However, αs1-casein was eluted before β-casein in the leading edge of the new peak. Prior treatment of the casein micelles by dispersion with 6 m-urea solution, precipitation with acid or reduction with 2-mercaptoethanol did not alter the gel-filtration pattern. An examination of the purified casein components and their mixtures showed that a 1:1 ratio mixture of αs1- and β-casein had the same peak maximum elution volume as casein micelle sub-units. κ-Casein by itself eluted at the void volume of the gel-filtration column, but after admixture with a sample of small micelles it eluted at the leading edge of the sub-unit peak and was indistinguishable from the κ-casein normally present. These results suggest that the sub-units are in equilibrium with their component caseins and that their size distribution is determined by only those factors (such as protein concentration, pH, temperature and ionic strength) which determine the strength of association between the casein components. The results from electron microscopy and ultracentrifugation support these conclusions.

Analyses of the neutral volatile components from 3 Cheddar cheese are presented. Two were made aseptically in an aseptic vat, with and without starter culture; the third was made with starter culture in an open vat. Gas–liquid chromatography and mass spectrometry detected the same volatiles in starterless cheese having little or no Cheddar flavour as in cheese made with starter and having a characteristic Cheddar flavour. Methyl disulphide and dimethyl sulphide were the only compounds consistently detected in higher concentrations in the cheese made with starter than in the cheese made without starter. However, using a total trapping technique, it was found that the combination of components recovered from the effluents of the chromatographic columns did not have the cheese-like aroma of the distillate vapours that were injected. Reasons for this are discussed.