A new method for determining the composition of the colloidal phosphate of milk has been developed, based on analysis of milk free from colloidal phosphate. Preparation of this material is described.

The results suggest that the so-called colloidal calcium phosphate of milk should be more properly described as a colloidal phosphate—citrate.

This colloidal phosphate—citrate shows considerable analytical resemblance to the precipitate formed on neutralizing acidified milk serum. Both can be represented approximately by the empirical formula of a hypothetical citrate apatite.

Heat-induced association of β-lactoglobulin (β-lg) and casein micelles, mainly at 85–90 °C, was studied by means of preparative ultracentrifugation, using [3H]-labelled β-lg. Qualitative aspects were studied by gel electrophoresis and electron microscopy. In this association, the formation of intermolecular S–S bonds between β-lg and κ-casein plays a role, but hydrophobic bonds are also involved.

After heating mixtures containing 25 g/kg casein and 4 g/kg β-lg at 90 °C for 20 min, the amount of β-lg sedimented with the casein micelles by ultracentrifugation decreased by approximately 30% when the milk salt buffer system was reduced to 0·25 of its normal concentration; it decreased by about 20% when the pH was increased from 6·8 to 7·3 and increased by 15% when the pH was reduced from 6·8 to 5·8. When the β-lg concentration was decreased from 4 to 2 g/kg, the amount of sedimented β-lg decreased by 25% after heating at 90 °C for 6 min. After heating in milk salt buffer, the amount of sedimented β-lg was 15% higher than after heating in a salt solution which contained neither Ca nor citrate ions.

Although α-lactalbumin (α-la) is involved in the heat-association of β-lg and casein micelles, no influence of α-la on the amount of associated β-lg was found. In addition to the heat-association product of β-lg and whole casein micelles, an association product consisting mainly of β-lg and κ-casein was also formed. This product was observed by electron microscopy as noodle-like particles. The size of these particles depended on the β-lg/κ-casein ratio.

The main objective was to determine the prevalence of intramammary infections (IMI) in dairy cows in Flanders, Belgium. Data were obtained from quarter milk samples of dairy herds subjected to a mandatory yearly screening of all lactating cows. A total of 178 668 quarter milk samples were collected at 1087 cross-sectional dairy herd screenings performed in three consecutive years. Of the dairy cows, 40% had at least one culture-positive quarter. More than 50% of all IMI were caused by non-aureus staphylococci. Streptococcus agalactiae is almost eradicated in Flanders, whereas Staphylococcus aureus was isolated from 18% of the culture-positive quarters. In addition, the distribution of mastitis pathogens in quarter milk samples from selected dairy cows with an elevated somatic cell count (SCC) is described. From 6390 cows with a geometric mean composite SCC ⩾250 000 cells/ml, nearly 65% had at least one culture-positive quarter. The majority of the IMI were caused by non-aureus staphylococci (41·1%), whereas Staph. aureus and aesculin-positive cocci were found in respectively 25% and 18% of the culture-positive milk samples. We conclude that more efforts are needed in the prevention and control of subclinical mastitis in Flanders. Non-aureus staphylococci are the predominant cause of IMI, warranting more research regarding the epidemiology and pathogenicity of those species.

The detailed chemical composition of 25 milks from different breeds of cow, sheep and goat were related to their properties of rennet clotting, coagulum development and syneresis at pH 6·4. Experiments in which concentrations of fat and whey proteins in milk were manipulated, and in which milks were homogenized at different pressures, were also carried out and the effects observed were related to the above processes.

The composition of milks varied widely and many relations between concentrations of components could be related to their known modes of secretion from the alveolus or to their structural functions in the colloidal phase of milk. Rennet clotting was related to total Ca concentration and also to the proportions of αs- and β-caseins present. Coagulum strength was strongly related to total casein concentration in milk but was unaffected by total fat content or by casein/fat ratios. Syneresis was inversely related to fat content of milk and little affected by coagulum strength. Homogenization retarded syneresis, probably by altering coagulum structure and modifying entrapment of fat globules within the coagulum.

The rates of coagulation of completely renneted casein micelles have been measured as functions of ionic strength, temperature, and concentration of Ca2+. At 25 °C and below, the rate constants for the coagulation were found to be low, but increased with temperature so that at 60 °C the particles were coagulating at almost maximum rate permitted by diffusion. This maximal rate at 60 °C was achieved at nearly all of the ionic strengths and concentrations of Ca2+ used. At lower temperatures the rate constant decreased with increasing ionic strength, the dependence being more marked at lower temperatures. Increasing concentration of Ca2+ also increased the rate at low and moderate temperatures. The implications of these results are discussed in terms of specific and non-specific ionic interactions and of hydrophobic bond formation.

Effects of genetic variants of the milk proteins, αs1-casein, β-casein, κ-casein and β-lactoglobulin (βlg), on milk yield and composition, particularly the protein composition, were investigated in milk samples from 289 Jersey and 249 Friesian cows in eight commercial herds.

Milk protein genotypes had no significant effect on yields over a complete lactation of milk and fat, but significant differences in fat content were detected for β-casein (B, A1B, A2 > A1A2) and βlg (B, AB > A) variants. Significant differences between β-lg variants were also found with total solids (B, AB > A), casein (B, AB > A), whey protein (A > AB > B) and βlg (A > AB, AC > B > BC) concentrations. Casein genotypes were not significantly different in total protein and casein concentrations but many differences were found in casein composition. αs1-Casein variants significantly affected αs1-casein (BC > B) and κ-casein (B > BC) concentrations. β-Casein variants affected concentration and proportion of β-casein (A1B, A2B > A1, A1A2, A2, B), αs1-casein (A1, A2 > B) and κ-casein (B > A2) and concentration of whey protein (A1 > most other β-casein variants). κ-Casein variants affected concentration and proportion of κ-casein (B > AB > A), proportion of αs1-casein (A > AB > B) and concentration of βlg (A > AB, B) and α-lactalbumin (A, AB > B). Differences in milk composition were found between breeds, herds and ages, and with stage of lactation. The potential use of milk protein genotypes as an aid in dairy cattle breeding is discussed.

A new spectrofluorimetric assay procedure for bovine milk N-acetyl-β-D-glucosaminidase (NAGase) is described for use as a routine screening test for the detection of abnormal uddersecretions. This procedure uses 4-methylumbel-liferyl-N-acetyl-β-D-glucosaminide as substrate. On the basis of the greater sample throughput, increased product sensitivity detection of NAGase and the absence of turbidity problems, it is considered to be superior to a previously reported spectrophotometric procedure (Kitchen, 1976). The correlation coefficient between the somatic cell count and the fluorimetric procedure using 243 quarter fore-milk samples was 0·86. Distribution studies on bovine milk and mammary gland homogenates indicated that this enzyme activity was located predominantly in the soluble whey protein fraction and the post-microsomal supernatant. Mammary gland secretory cells contained high levels of NAGase and appeared to be the major source of the enzyme in milk whilst NAGase from other sources (white blood cells, blood serum) contributed only a minor proportion (5–15%) of the total activity in milk. The implications of these findings on the value of the NAGase test as a means of mastitis diagnosis are discussed.

Differential counts of electron microscope sections of cell pellets isolated from bovine udder secretions showed that no secretory epithelial cells and very few ductal epithelial cells were present at any stage. The predominant cell type was the macrophage in dry and lactating cows or the polymorphonuclear leucocyte (PMNL) in colostrum. Lymphocytes were also present but no plasma cells were found. The macrophages took up polystyrene latex particles (as did the PMNL) and adhered toglass in culture. Neither macrophage-nor PMNL-rich cell suspensions produced any increase in free fatty acid levels when incubated with fresh milk.

A description is given of various methods for the ultrafiltration and dialysis of milk and of the composition of the sera obtained. Ultrafiltrate prepared by the procedure recommended is reasonably representative of the aqueous phase of milk, but its content of lactose and citric acid, and consequently also of calcium, is determined to a slight degree by the sieving phenomenon known to occur often in ultrafiltration. The composition of diffusate obtained from milk at 20°C is not thought to be controlled to any significant extent by a Donnan effect and is regarded as identical with that of the aqueous phase of milk. The lactose content of diffusate suggests that about 2% of the water in milk is bound to protein, and allowance should be made for this when calculating the concentrations of the soluble constituents in milk from the composition of diffusate. Diffusate prepared from milk at 3°C contains slightly more total calcium, ionized calcium and phosphorus than diffusate prepared at 20°C. These differences are attributed to a change in the partition of calcium and phosphorus between the disperse and aqueous phases at the lower temperature, an explanation that is supported by the reversibility of the change. The composition of diffusate prepared by the procedure recommended indicates that about 5% of the sodium and about 6% of the potassium and citric acid in milk are in the disperse phase.

The strength of binding of the individual caseins and the nature of the bonding within bovine casein micelles were examined through dissociation of the micelles by dialysis of skim milk either against phosphate-free buffers containing 3 or 6 mm-CaCl2, or against buffers that were nearly saturated with respect to micellar calcium phosphate, but which had a free Ca2+ concentration in the range 0·4–5·9 mm. Dissociation was followed by ultracentrifuging the dialysed milks and determining the partition of the total and the individual caseins between the pellet and serum. During dialysis against the phosphate-free buffers both colloidal Ca and Pi in the milks decreased and about 30 % of the Pi could be removed without significant casein dissociation. With further loss of Pi, however, increasing dissociation occurred and the proportions of the individual caseins retained in the casein pellet were in the order αs2- > αs1- > β- ≈ κ-casein. Dialysis against the calcium phosphate buffers resulted in no loss of colloidal Pi but colloidal Ca increased with the free Ca2+ concentration of the buffer. Little change in the casein partition occurred in the presence of more than 1 mm free Ca2+, but serum casein increased markedly at lower levels, and the strength of binding of the individual caseins in the pelleted casein was in the order αs2-> αs1- > β- > κ-casein. In both types of buffer, dissociation is considered to occur through the breaking of linkages between the caseins and inorganic constituents. Analysis of the amino acids in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles suggests that these linkages involve the phosphate centres of the caseins.

Milk fatty acid (FA) profile is a dynamic pattern influenced by lactational stage, energy balance and dietary composition. In the first part of this study, effects of the energy balance during the proceeding lactation [weeks 1–21 post partum (pp)] on milk FA profile of 30 dairy cows were evaluated under a constant feeding regimen. In the second part, effects of a negative energy balance (NEB) induced by feed restriction on milk FA profile were studied in 40 multiparous dairy cows (20 feed-restricted and 20 control). Feed restriction (energy balance of −63 MJ NEL/d, restriction of 49 % of energy requirements) lasted 3 weeks starting at around 100 days in milk. Milk FA profile changed markedly from week 1 pp up to week 12 pp and remained unchanged thereafter. The proportion of saturated FA (predominantly 10:0, 12:0, 14:0 and 16:0) increased from week 1 pp up to week 12 pp, whereas monounsaturated FA, predominantly the proportion of 18:1,9c decreased as NEB in early lactation became less severe. During the induced NEB, milk FA profile showed a similarly directed pattern as during the NEB in early lactation, although changes were less marked for most FA. Milk FA composition changed rapidly within one week after initiation of feed restriction and tended to adjust to the initial composition despite maintenance of a high NEB. C18:1,9c was increased significantly during the induced NEB indicating mobilization of a considerable amount of adipose tissue. Besides 18:1,9c, changes in saturated FA, monounsaturated FA, de-novo synthesized and preformed FA (sum of FA >C16) reflected energy status in dairy cows and indicated the NEB in early lactation as well as the induced NEB by feed restriction.

Maternal milk should not only be considered as a nutrient, but also as a protecting agent against aggressions from the neonate's new environment. Breastfeeding facilitates transmission of a passive immunity by multifunctional factors which have a direct effect on the neonate's resistance to bacterial and viral infections. Among these factors are the main milk proteins, the caseins: during enzymic digestion of human and bovine caseins, immunomodulating peptides are released. Corresponding synthetic peptides stimulated in vitro phagocytic activity of murine and of human macrophages and exerted in vivo a protective effect against Klebsiella pneumoniae infection of mice. These data suggest that casein peptides may exert a stimulating function on the immune system of the newborn.

Adherence of bacteria to various milk contact sites was examined by scanning electron microscopy and transmission electron microscopy. New gaskets, endcaps, vacuum breaker plugs and pipeline inserts were installed in different areas in lines carrying either raw or pasteurized milk, and a routine schedule of cleaning-in-place and sanitizing was followed. Removed cleaned and sanitized gaskets were processed for scanning or transmission electron microscopy. Adherent bacteria were observed on the sides of gaskets removed from both pasteurized and raw milk lines. Some areas of Buna-n gaskets were colonized with a confluent layer of bacterial cells surrounded by an extensive amorphous matrix, while other areas of Buna-n gaskets showed a diffuse adherence over large areas of the surface. Most of the bacteria attached to polytetrafluoroethylene (PTFE or Teflon™) gaskets were found in crevices created by insertion of the gasket into the pipeline. Examination of stainless steel endcaps, pipeline inserts, and PTFE vacuum breaker plugs did not reveal the presence of adherent bacteria. The results of this study indicate that biofilms developed on the sides of gaskets in spite of cleaning-in-place procedures. These biofilms may be a source of post-pasteurization contamination.

The caseinate complex in bovine milk was partitioned by differential centrifugation at both 20 and 4 °C into 4 micellar fractions and a fraction representing serum casein, and the protein composition of the fractions determined. At both temperatures the relative amount of κ-casein in the micellar caseins increased markedly and that of β-casein decreased appreciably with decreasing micelle size. The relative amount of αs2-casein also tended to decrease with decreasing micelle size, but the relative amounts of αs1- and γ-caseins, and an unidentified casein fraction, showed little systematic variation. The serum casein differed appreciably in composition from the micellar caseins, being very rich in β-casein and comparatively poor in αsl- and αs2-caseins, and the amount present at 4 °C was considerably greater than at 20 °C, with the increase being due almost entirely to β-casein, but with γ-casein also making a significant contribution. The changes in the composition and distribution of micellar and serum caseins induced by cooling milk at 4 °C were completely reversible when the milk was re-equilibrated at 20 °C for 18 h.

The effects of heat treatment and homogenization of whole milk on chemical changes in the milk fat globule membrane (MFGM) were investigated. Heating at 80 °C for 3–18 min caused an incorporation of whey proteins, especially β-lactoglobulin (β-lg), into MFGM, thus increasing the protein content of the membrane and decreasing the lipid. SDS-PAGE showed that membrane glycoproteins, such as PAS-6 and PAS-7, had disappeared or were weakly stained in the gel due to heating of the milk. Heating also decreased free sulphydryl (SH) groups in the MFGM and increased disulphide (SS) groups, suggesting that incorporation of β-lg might be due to association with membrane proteins via disulphide bonds. In contrast, homogenization caused an adsorption of caseins to the MFGM but no binding of whey proteins to the MFGM without heating. Binding of caseins and whey proteins and loss of membrane proteins were not significantly different between milk samples that were homogenized before and after heating. Viscosity of whole milk was increased when milk was treated with both homogenization and heating.

The effects of minerals on casein micelle stability of individual cows' milk, throughout a complete lactation, were investigated. Calcium and calcium ions, magnesium, phosphorus, sodium, potassium and citrate contents were analysed, together with the following physical properties of milk; pH, ethanol stability, rennet clotting time and coagulum firmness. There was an inverse non-linear relationship between free calcium ion concentration and ethanol stability (ES; r=0·84). Rennet coagulation time showed a weaker relationship with free calcium ion concentration (r=0·44) but a stronger relationship with pH (r=0·66). In addition, samples containing higher amounts of free calcium ions produced a firmer gel. Citrate in natural samples acts as a stabilizing factor, as it slightly improves milk stability. Potassium, on the other hand, exhibited a negative correlation, but only with rennet clotting time (r=−0·52). Throughout lactation the average values were; free Ca2+ concentration 1·88 mM, pH 6·63, ES 83·2% and clotting time 13·6 min. The equilibrium relationship between pH and free Ca2+ concentration was investigated by adjusting milk pH from 5·9 to 7·1, using acid and alkali. There was a good inverse linear relationship between pH and log (free Ca2+) for individual milk samples, with a gradient of −0·62 and a standard deviation of 0·042.

The proteolytic specificity of chymosin (EC 3.4.23.4) on bovine αs1-casein at 30°C in phosphate buffer, pH 6·5 and at pH 5·2 in the presence of 5% (w/v) NaCl was investigated. Peptides (pH 4·6-soluble) were isolated by reversed-phase HPLC and identified from their amino acid sequence; the identity of some peptides was confirmed by mass spectrometry and/or amino acid composition. The small peptides produced at pH 6·5 were Arg1–Phe23, Phe24–Phe28, Phe24–Leu40(?), Phe150–Phe153, Phe150–Leu156, Tyr154–Tyr159, Tyr154–Trp164, Asp157–Trp164 and Tyr165–Trp199. The same peptides, except Tyr154–Trp164, were produced at pH 5·2 in the presence of NaCl and, in addition, the peptides Arg1–Leu11, Phe24–Phe32, Lys102–Leu142, Ala143-Leu149 and Tyr165-Phe179. The rates of production of individual peptides differed under the two conditions studied but Arg1-Phe23 and Tyr165–Trp199 were the first and second peptides produced under both conditions. Pathways are proposed to interpret the proteolysis of αs1-casein in solution under the conditions of this study.