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SUMMARYThe influence of mastitis and early lactation, and the effect of treating milk with heparin, blood serum and trypsin, on the proportion of lipoprotein lipase (LPL) activity in milk serum was investigated. The relative importance of milk serum LPL and LPL bound to micellar casein in promoting lipolysis was also examined. Colostrum contained LPL activity, 45% of which was found in the serum phase in samples obtained from the first milking post partum, but this value fell to 34% in samples taken 24 h later. The proportion of serum LPL was also increased in milks from quarters infected with Staphylococcus aureus, but not after overnight treatment of normal milk at 4 °C with 5% (w/v) blood serum or 2 µg/ml trypsin. The addition of 5 µg/ml heparin resulted in a consistent increase in serum LPL which varied between 14 and 50% of total milk LPL. Heparin did not release all the enzyme bound to casein micelles even after a second heparin treatment of resuspended micelles. Serum LPL was more effective in promoting lipolysis and was more responsive to blood serum activation than LPL bound to casein micelles. Lipolysis increased after heparin treatment but the increase was not related to serum LPL activity.

SUMMARYSkim-milk was fractionated by permeation chromatography on CPG-10 (300 nm) at 20 °C with synthetic milk serum. The elution profile, which was highly reproducible, showed 3 partly resolved peaks. On treatment with 0.1% glutaralde-hyde before chromatography, a similar profile was obtained indicating no appreciable micellar dissociation during chromatography of the milk. The casein composition of the eluent fractions, determined by quantitative chromatography on hydroxyapatite, showed that 12.1% of total casein was associated with the low molecular weight peak. The micelle content of ²- and κ-casein increased with increasing elution volume, and the ±s/κ ratio showed a progressive diminution from 4·3 to 2·3. Large differences were found in the colloidal phosphate contents and the weight average diameters of the micelle fractions which clearly show that CPG-10 fractionates on the basis of molecular size.

SummaryAeration increased the growth and lipase production in milk byPseudomonas fluorescensstrain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented ∼ 71 % of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8·0 and 35 °C; it had aKmon tributyrin of 3·65 mM. and was inhibited by concentrations of substrate > ∼ 17 mM. The enzyme was very stable over the pH range 6–9; it was relatively heat-labile in phosphate buffer in the temperature range 60–80 °C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100–150 °C: theDvalues at 150 °C were ∼ 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the correspondingZvalues in the temperature range 100–150 °C were ∼ 40 and ∼ 42 °C and theEafor inactivation were 7·65 × 104J mol-1and 6·97 × 104J mol-1respectively.

SUMMARYA comparison of literature results on the voluminosity of casein micelles yields large differences, largely related to the method of determination. After applying some corrections, methods based on hydrodynamic radius yield a value of roughly 3·9ml/g dry casein, other methods (microscopy, sediment volume) about 2·2. Roughly half of the discrepancy can be explained by the micelles being not perfectly spherical. To explain the remaining difference, it is assumed that the micelles are hairy, i.e. they have molecular chains protruding into the milk serum. The hairyness would increase with decreasing temperature and be largely removed by the action of rennet. It would cause some entropic repulsion between micelles at close approach.

SummaryBovine milk systems were analysed for adenosine 5′-triphosphate (ATP) using the luciferase-ATP reaction in a liquid scintillation counter. Approximately 0·2 µmol ATP/1 milk serum were evident both in whole milks and the corresponding skim-milks. ATP was not detectable in skim-milk ultrafiltrates. These findings indicated that ATP was present in a non-dialysable portion of skim-milk. Centri-fugation of whole milks from individual cows at 5500 g for 15 min at 10°C yielded skim-milks essentially devoid of somatic cells and bacteria. However, the ATP in the skim-milks decreased by less than 20% compared with the whole milks indicating that the calcium phosphate-citrate (CPC)–caseinate micelles were the source of the ATP. ATP was not detectable in colloidal phosphate-free milk, from which CPC had been removed, confirming that the ATP was sequestered in the constituent CPC. Likewise, the occurrence of significant amounts of Mg, another potent stabilizer of amorphous calcium phosphate (ACP) in other biological systems, was confirmed in the colloidal phosphate of milk. From 0·13 to 0·31 µmole ATP/1 (mean, 0·23) was found in the 9 milk samples studied. The discovery of small but appreciable levels of ATP in the CPC of milk provides further evidence for the analogy previously shown to exist between the CPC complex of milk and the ACP which accumulates in mitochondria. The latter has been postulated to provide an essential precursor for crystalline bone salts to form in ordered calcification processes. The implications of these findings in the biosynthesis of milk are briefly discussed.

SummaryA method for the quantitative partition of the albumin fraction of milk serum proteins is described. The partition, which is carried out by chromatography on a column of Sephadex G-100 (2·5×90cm) at 4°C in the presence of 0·02 m-sodium phosphate buffer (pH 6·7) containing 0·1 m-NaCl, yields 4 fractions. By gel electrophoresis it was shown that the fractions represent, in order of elution from the Sephadex, the following: fraction 1, a mixture of the largest proteins present, probably comprising γ-globulins, proteose-peptones and lactoferrin; fraction 2, bovine serum albumin and bovine serum transferrins; fraction 3, β-lactoglobulins; and fraction 4, α-lactalbumins. The protein contents of the various fractions were determined by nitrogen analysis and the results showed that good agreement was obtained between values from duplicate fractionations, and also that the recovery of material was satisfactory. The partition of the albumin fraction was not complete, but at least 90% of the material was found in fractions which represent essentially single proteins. The influences of some aspects of the pretreatment of samples, buffer composition and column preparation are described.

SummaryThe effect that changes in composition which occur in milks possessing high cell counts have on milk lipolysis has been investigated. High cell counts were produced either by intramammary infusion ofEscherichia coliendotoxin orStreptococcus agalactiaeor by addition of washed cells which were isolated from milk obtained from quarters infused with endotoxin. Free fatty acid levels in milk were measured in terms of acid degree value (ADV) either as initial ADV measured immediately after milking or ADV developed after a prescribed incubation period.There was an increase in initial ADV after the infusion either of endotoxin or ofStr. agalactiaerelative to a control quarter. This increase appeared to be associated with changes in cell count, but in absolute terms the influence of cells on ADV became less as cell count increased. Neither type of infusion had any effect on lipoprotein lipase activity. The addition of washed cells to normal milk resulted in an increase in developed ADV, but the increment was not as large as that produced by the addition of 1% blood serum. When cream and skim-milk from endotoxin-treated quarters and control quarters were mixed in different combinations with and without additional cells, developed ADV was higher in those samples containing endotoxin cream and those with added cells. Milk from a quarter treated with endotoxin was diluted with its own skim-milk to produce different cell counts and ADV was determined after various time intervals at 4 and 37 °C. Lipolysis increased with increasing cell count, but a depression in lipolytic rate was noted after incubation for 6 h at 4 °C and 20 min at 37 °C.The proportion of skim-milk lipoprotein lipase activity in milk serum was larger both in milks possessing high cell counts and in normal milk adjusted to between 5 and 20 mM-NaCl by addition of solid NaCl. These levels of NaCl inhibited lipolysis.The possible direct and indirect effects of high cell count on milk lipolysis are discussed.