The effect of varying periods of energy undernutrition on milk yield and composition was determined in 30 British Friesian cows receiving a typical winterstall diet. An absence of steaming-up, and underfeeding from the beginning of lactation, depressed both milk yield and solids-not-fat (SNF) content, especially in early lactation. Initially the reduction in milk SNF content was accounted for mainly by a loss in lactose content, but as lactation progressed this effect lessened and an effect on milk protein content was established. Protein content was similarly depressed by underfeeding in mid-lactation. The restoration of normal feeding in mid-lactation after varying periods of underfeeding was associated with only small responses in SNF and protein contents.

The effect of the type of basal diet and supplementary concentrates on the extent of the increase in SNF and protein contents in response to improved feeding was examined in 2 further experiments. Responses were in all instances small and it was concluded that with the diets commonly in use in Great Britain the provision during the winter period of supplementary concentrates, whatever their composition, is unlikely to give a marked improvement in milk SNF or protein contents.

The effects on rumen fermentation and milk-fat secretion of a dietary supplement of protected tallow given to 4 Friesian cows established on a low-roughage ration and with depressed milk fat is reported. The ratios of acetate to propionate in the rumen were unaffected by the supplement and remained typical of those associated with low-roughage diets in that the proportion of propionate was increased. The supplement produced almost complete recoveries in yield and content of milk fat without any increase in intramammary fatty-acid synthesis. The recoveries were due to transfer of about 20% of the total fatty acids of the tallow supplement. These results are discussed in relation to the effects of low-roughage diets on milk-fat secretion and it is concluded that in the ‘low-fat syndrome’ the capacity of the mammary gland to absorb preformed fatty acids is not impaired.

The relative importance of β-hydroxybutyric acid (BHBA) and acetate as precursors for milk-fat synthesis was studied in lactating goats by infusing separately tracer quantities of [3−14C]DL-BHBA and [1−14C]acetate into the jugular vein, and [1−14C]butyrate into the portal vein. The concentrations and specific radioactivities of blood plasma constituents, the yields and specific radioactivities of individual milk fatty acids and the relative radioactivities of individual carbon atoms of milk fatty acids were determined.

The infusion of [1−14C]butyrate resulted in the appearance of labelled BHBA in the blood plasma which behaved almost identically with infused [14C]BHBA as a precursor for milk fatty acids.

The relative radioactivity of carbon atoms of the fatty acids of milk fat following the infusions provided direct evidence that BHBA had provided an intact 4-carbon unit at the methyl end of each fatty-acid chain. Acetate provided 2-carbon units both for the elongation of the 4-carbon units and for complete de novo synthesis. BHBA also provided 2-carbon units which behaved in a similar fashion to those from acetate.

Acetate and BHBA together accounted for all of the C4–C12 acids of milk fat, about 75% of the C14, 45% of the C16 and 10% of the C18.

The total contributions of the various precursors to the fatty acids of milk fat were: acetate 42%, BHBA 9·4% and other plasma precursors (by difference) 48·6%.

A series of experiments is described, outlining a study of 5 factors likely to influence the efficiency and reproducibility of a standardized method used for the electronic counting of somatic cells in milk. Samples containing cells covering 5 ranges from 250000 cells/ml to 1500000 cells/ml were used and the results analysed statistically.

The residual error was given special consideration, and was found not to contribute to any significant degree to the accuracy of the method. Sample volumes of 0·5 ml and 0·1 ml used for counting were compared, and there was a decrease in accuracy using the smaller volume. The influence of individual technician skill was studied with 4 workers having differing degrees of experience, and no significant addition to the overall error was observed. The effect of agitating bulk tank milk for between 0 and 2 min before sampling was examined. Variations were considerable for agitation times of less than 1 min, but the recommended mixing time of 2 min was found to be adequate with milk quantities between 450 l and 2250 l (100 and 500 gal), and at the 5 cell-count levels chosen.

The distributions of the cell counts at the 5 levels were found to be normal with the marginal exception of samples tested at 250000 cells/ml.

All aspects of the technique, sample preparation, use and calibration of the Coulter Counter, etc, should be closely followed to ensure that no inaccuracies will result from inadequate maintenance of the machine or other equipment employed in the test.

The heat produced per colony-forming unit (CFU) of growing bacteria was found to depend on the species and was greater the younger the culture. With the exception of 2 determinations out of 13, with 6 species the results show that it should be possible to detect < 5×105 CFU/ml by the heat produced. A seventh species, a micrococcus, could be detected at a lower level of CFU presumably because the unit was a clump of several cells. Gross variations which occurred with fullcream milk await investigation.

A 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.

The acid phosphatase of bovine milk was further purified to yield enzyme with an activity of about 2 units/mg. This was almost 105 times the activity present in milk and enabled a detailed study of heat inactivation to be made, together with further measurements on binding to casein substrates.

The effectiveness of caseins as inhibitors of the hydrolysis of p-nitrophenyl phosphate by acid phosphatase paralleled the phosphate content of the casein molecules, so that αs1-casein A was a more potent inhibitor with a K1 of 1·7 mM than β-casein A1A2 (K1 = 4·3 mM), which in turn was more inhibitory than κ-casein A (K1 = 5·9 mM).

The heat inactivation of acid phosphatase followed first-order kinetics at pH 4·9, 5·2 and 6·7 and values of E, the activation energy, were between 2·4×105 and 3·0×105 J mole −1 in all cases, consistent with simple protein denaturation. The presence of 1% αs1-casein A, 1% β-casein A1A2, 1% κ-casein A, 1% isoelectrically precipitated ‘whole’ casein and 1% fresh raw milk provided no substrate protection at pH 5·2 or 6·7. Acid phosphatase was somewhat less heat stable at pH 6·7 than at pH 4·9, but may be expected to survive typical milk pasteurization conditions almost completely. However, conventional milk sterilization or ultra-high-temperature (UHT) processes would be expected to give total inactivation.

The casein micelles of sheep (Ovis aries), goat (Capra hircus) and cow (Bos taurus) milks have been examined by electron microscopy. The ovine micelles were smaller than bovine micelles, most being below 80 nm diam. The caprine micelles were either large (c. 200 nm diam.), easily sedimented and electron-dense, or small (below 80 nm) and similar to the ovine micelles. Examination of the micelles using the freeze-etch technique showed, however, that they were all composed of subunits of similar dimensions (10–15 nm diam.).

Electrophoresis of the caseins showed that there were differences between them, with the caprine casein containing a smaller proportion of its protein in the more mobile components. The mineral contents of the milks were also different, but these were apparently related to the different pH and casein contents of the milks. It is likely that the diiferences in micelle size distribution are determined by the physical and chemical properties of the casein components.

The bacteriological formation of 2-butanone and 2-butanol was investigated. The data suggest that 2-butanol is formed in cheese via 2-butanone in 3 steps, each step being carried out specifically by a different microbial species. Suitable species have been isolated from a single cheese. The first step could be carried out (in the present investigation) either by certain starter organisms or by a strain of Pediococcus cerevisiae. This strain formed substantial amounts of 2,3-butylene glycol in milk, presumably from citrate, but could not metabolize this compound further. A second micro-organism, a strain of Lactobacillus plantarum, was isolated which, in Cheddar cheese, could convert 2,3-butylene glycol into 2-butanone. The final reduction of 2-butanone to 2-butanol could be readily carried out in MRS medium by a strain of L. brevis. Results from cheese made aseptically indicate that 2-butanone is formed in Cheddar cheese only when non-starter organisms are present.

Methods are described for the solubilization of the nitrogenous portion of cheese in a dissociating solvent system and separation of the solution into fractions on a Sephadex G-100 column under conditions where the proteins and peptides would be expected to be split into monomeric units. The E280 measurement of the fractions, expressed as a percentage of the total material eluted, followed a reproducible pattern, both with duplicate runs on the same sample and comparative runs on non-identical samples of the same type. With cheese curd, a small peak of undissociated material was obtained at the void volume of the column; this was followed by a main peak and a minor third peak, with a small quantity of material being eluted at larger volumes. A qualitatively similar pattern was obtained with 43-day-old cheese as the sample, but the main peak was smaller, the third peak was larger, and a relatively larger amount of material was eluted at greater volumes. There was only partial separation of the proteins on the column; the main peak contained β- and αs-caseins, their larger breakdown products and some para-κ-casein, and the third peak contained smaller breakdown products of the main casein fractions and para-κ-casein. Although the resolution was considerably inferior to that obtained by gel electrophoresis, the method has the advantage over this and other methods in that it gives a quantitative analysis of the sample based on molecular size. Thus, it is suggested that it may find use in analysing protein breakdown in cheese during ripening.

The rates of proteolysis during ripening were followed in cheeses made with either calf rennet or swine pepsin and either starter or δ-gluconic acid lactone (GAL) as a replacement for the starter. A gel-filtration column technique and starch-gel electrophoresis were used for analysis, and bacterial counts were made on all samples. Proteolysis was faster in cheeses made using GAL than in those made using starter and also slightly faster in GAL cheeses made with swine pepsin than in those made with rennet. Further, it was considerably slower in starter-containing cheeses made with swine pepsin than in those made with rennet. It is suggested that these differences were due to the much greater rate of development of acidity in cheeses made with GAL than in those made with starter, which resulted in more of the coagulant being incorporated into the curd in an active state. The rate of proteolysis in starter-containing cheeses appeared to follow a characteristic course, being initially slow, then markedly increasing with a later slow decline. It is suggested that the increase in the rate of proteolysis was due to an increase in the total activity of bacterial proteinases released by lysis of the bacteria. Indications were obtained that the coagulants and bacterial proteinases catalysed broadly similar patterns of protein breakdown in cheese, and that medium-sized peptides (mol. wt 9000–14000) were formed as definite intermediates in the process. The results also showed that rennet and swine pepsin remained active for at least 7 months in GAL cheeses, that rennet contributed significantly to proteolysis in starter-containing cheeses, and that swine pepsin was at least extensively inactivated and possibly completely inactivated during cheese-making with starter.

Treatment of unfractionated bovine casein with papain produced a digest which was bitter in taste. The bitterness was shown to be a property of a peptide originating only in β-casein and having an amino-acid composition consistent with its derivation from residues 53–79 inclusive of β-casein. The relationship existing between flavour and primary structure in this peptide is discussed briefly.