Four Ayrshire cows in their second lactation were used in a 4×4 Latin square experiment to determine the effects of frequency of feeding on milk secretion. The animals were given a diet consisting of hay, sugar-beet pulp and dairy concentrates (34·9:19·7:45·5 on a dry-matter basis) either in 2 equal meals/d at 07.00 and 17.00 h or with the hay in 4 meals at 07.00, 12.00, 17.00 and 22.00 h and the sugar-beet pulp and concentrates in 24-h meals. Each feeding frequency was tested at 2 levels of feeding providing either 100 or 80% of the standard requirements for energy.

There were no significant effects of frequency of feeding on milk yield or on the milk content of fat, protein, lactose, solids-not-fat or total solids, but underfeeding depressed the yield of milk and of all milk constituents. Neither the frequency nor level of feeding had appreciable effects on the fatty-acid composition of the milk fat or on the blood glucose concentration, but in animals fed frequently plasma urea levels were lower than in animals fed twice daily and there was no postprandial peak in urea concentration.

Perphenazine (0·8–1mg) caused udder growth and artificial induction of lactation, when stereotaxically implanted into the median eminence of virgin female goats. Sham implantations into the median eminence or implantation of perphenazine into sites of the hypothalamus not in the immediate proximity of the median eminence were ineffective. Fourteen–twenty-two d after implantation the goats were milked for the first time. Maximal milk yields of 250–1050 ml/d were achieved 3–5 months later. Udder development and induction of lactation during the premilking period were exclusively due to the action of perphenazine. It is likely that after implantation the milking stimulus played a synergic role with perphenazine in the progressive increase and the maintenance of lactation. Apparently no side effects were caused by the drug. Implantation of small doses of perphenazine in the hypothalamus might be a suitable technique for studying the physiological effects of increased levels of endogenous prolactin in goats.

The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10·1 for p-nitrophenylphosphate, 8·6 for phosphoserine, 8·0 for phosvitin and 6·8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of αs- and β-caseins or of αs-, β- and κ-caseins and micellar casein. Mixtures of 2 components involving κ-casein were more readily dephosphorylated than αs- and β-casein mixtures. At pH 6·8, lactose, whey proteins and phosphate ions had an inhibitory effect. β-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.

The electrokinetic potential of bovine casein micelles was determined under a variety of conditions using moving boundary electrophoresis. The technique used allowed sharp stable boundaries to be formed without addition of density gradient-forming materials. Casein micelles are negatively charged and in any one milk sample all micelles had similar electrophoretic mobilities. The electrokinetic potential of micelles was markedly reduced by the action of rennet and to a lesser extent by a decrease in temperature from 30 to 6°C. Addition of sucrose to milk caused a small increase in the electrokinetic potential. Addition of anionic detergent to milk increased both the electrokinetic potential and the rennet coagulation time. Addition of cationic detergent caused a reduction in both these quantities, promoted syneresis of the rennet coagulum and caused coagulation in the absence of rennet if present in concentrations exceeding about 10 mM. Alteration of cation activities by addition of salts to milk caused complex changes, either increasing or decreasing the electrokinetic potential. The data obtained are most readily explained by a model for the casein micelle in which some, but not necessarily all, of the κ-casein is located at the micelle surface.

Two distinct types of chymosin-sensitive polypeptide were found in the soluble fraction obtained when a solution of crude κ-casein in 0·4 m-NaCl was adjusted to pH 3. One corresponded to a segment of κ-casein comprising residues 94–169. The other was very similar to the section Gln77-Val169 in the protein, but contained a phosphate group plus isoleucine and glycine in the region between the N-terminus and the chymosin-sensitive bond. If these polypeptides form as a result of a specific cleavage of κ-casein, the data indicate that there is a variant of this protein with a number of amino acid replacements on the N-terminal side of the chymosin-sensitive bond. The shorter polypeptide and κ-casein are both rapidly cleaved by chymosin at pH 6·7 which suggests that the contribution of residues 1–93 to the sensitivity of the Phe-Met bond in κ-casein is a minor one.

Slow, irreversible aggregation of whey proteins in acidified milk or whey at pH 3·4–4·6 held for up to 10 d at 35–45°C was revealed by the reduction of discrete bands in disc electrophoresis. The aggregation was confirmed by precipitation of protein observed in stored, acid whey. The rate of aggregation of all protein fractions increased with the acidity and the storage temperature. It was enhanced by the presence of casein, but was unaffected by the presence of milk fat or by pasteurization of the fresh, unacidified milk at 70°C. The effect may contribute to the physical properties of certain fresh cheeses and other cultured dairy products.

Using a standard method, the electronic cell count of formalin-treated milk usually increased during storage. This increase varied with the milk and was found to be due to a corresponding decrease in the effectiveness of the fat-dispersal procedure before counting. A more severe fat-dispersal procedure incorporated into the standard method resulted in a stable cell count being obtained. It was found that formalin must be added to milk within 24 h of milking to prevent lysis of the cells.

Group N streptococci formed acetaldehyde and ethanol from glucose. As the enzymes aldehyde dehydrogenase, phosphotransacetylase and acetate kinase were present this would enable these organisms to reduce acetyl-CoA to acetaldehyde and convert acetyl-CoA to acetyl phosphate and acetate. A pentose phosphate pathway which converted ribose-5-phosphate to glyceraldehyde-3-phosphate was also present. Acetaldehyde could not be formed via the hexose monophosphate shunt or by direct decarboxylation of pyruvate, as the enzymes phosphoketolase and α-carboxylase were absent. Phosphoketolase activity was induced in Streptococcus lactis subsp. diacetylactis after growth on D-xylose. Group N streptococci also contained an NAD-dependent alcohol dehydrogenase which reduced acetaldehyde to ethanol while both NAD- and NADP-dependent alcohol dehydrogenase activities were found in Leuconostoc cremoris.

Group N streptococci were found to cleave threonine to form acetaldehyde and glycine. Threonine aldolase, the enzyme catalysing this reaction, was found in all strains except Streptococcus cremoris Z8, an organism which had been shown previously to have a nutritional requirement for glycine. The enzyme was strongly inhibited by glycine and cysteine. The inhibition showed characteristics of allosteric inhibition and was pH-dependent. Inhibition by glycine, but not by cysteine, was highly specific. Analogues and derivatives of cysteine which contained a thiol group and a free amino group inhibited the activity of threonine aldolase. The presence of a carboxyl group was not necessary for inhibition. The cleavage of threonine by wholecell suspensions was stimulated by either an energy source to aid transport, or by rendering the cells permeable to substrate with oleate. Threonine did not appear to be degraded by enzymes other than threonine aldolase, as threonine dehydratase activity was low and NAD- and NADP-dependent threonine dehydrogenases were absent.

Monospecific rabbit antibodies were produced against bovine chymosin (rennin) and bovine pepsin A and used for quantitative measurements of these enzymes by rocket immunoelectrophoresis. Bovine pepsin B was identified and measured quantitatively in tandem crossed immunoelectrophoresis, using a polyspecific antibody preparation. Chromatographically purified chymosin B and bovine pepsins A and B were used as standards. The contents of chymosin and bovine pepsin A were also determined by 2 forms of DEAE chromatography, and by an assay utilizing the ratio of milk-clotting activity at pH 6·0 and 6·5. Results obtained by measurements on 3 bovine rennets (calf rennet, adult bovine rennet, and a mixture of such types) by the different methods of analysis were in good agreement. The advantages of the immunochemical methods are direct and simultaneous identification and quantitation of enzymes.

Proteolysis in aseptic, chemically acidified (GDL) cheese and in starter cheese made under controlled bacteriological conditions (i.e. free of non-starter micro-organisms) was measured by gel electrophoresis, the formation of pH 4·6- and 12% TCA-soluble N, gel filtration and the liberation of free amino acids. The results show that rennet was mainly responsible for the level of proteolysis detected by gel electrophoresis, pH 4·6-soluble N and gel filtration i.e. large, medium and small peptides. However, rennet alone was capable of producing only a limited range of free amino acids; only methionine, histidine, glycine, serine and glutamic acid were produced at quantifiable levels (> 0·2 μmoles/g) in GDL cheese; it is suggested that free amino acids in Cheddar cheese are mainly the result of microbial peptidase activity. The levels of free amino acids in the starter cheese were considerably lower than values reported for commercial Cheddar.

Three techniques were used for preparing Cheddar cheese specimens for examination by scanning electron microscopy. Resulting micrographs prepared from fresh cheese made with calf rennet revealed that while all the techniques were satisfactory, different structural features were observed depending upon the method used. A modified critical point drying technique and a freeze-drying method displayed surface features whereas trypsin hydrolysis showed internal microstructure. The surface microstructure of the cheese was found to be formed of protein aggregated and fused into structural units of 1·5 μm. The internal microstructure appeared to be formed of thin compact walls. Cross sections prepared by freeze-drying displayed arrays in the protein matrix and locations where fat globules had been embedded.

Calf rennet, bovine pepsin, and porcine pepsin were used to produce cheese curd, using the same milk and lactic culture for each. Specimens were prepared for scanning electron microscope examination by a modified critical-point drying technique.

From examination of the micrographs, the curd made with bovine and porcine pepsin were similar in structure and in orientation of the coagulated protein, whereas the curd produced with rennet was different, having a more compact and organized structure.

Differences in flavour scores and in the concentrations of free fatty acids, methyl ketones and H2S were measured in Cheddar cheeses containing various groups of non-starter bacteria or starter streptococci alone, made under controlled bacteriological conditions by the aseptic vat technique. The non-starter bacteria were made up of lactobacilli, leuconostocs, pediococci, micrococci and Gram-negative rods isolated in commercial creameries from raw milk or fresh cheese curd. These were added to the experimental cheese as single groups or as complete floras (reference floras). Several bacterial groups influenced the measured concentrations of the flavour compounds, but flavour differences were not correlated with these chemical differences. Only cheese containing a curd-derived whole reference flora or cheese made in open vats in the N.I.R.D. Experimental Dairy had significantly better flavour than starter-only cheese, but this improvement was not attributable to any particular group of bacteria.

It has been reported previously (Annan & Manson, 1969) that chromatography of the αs-casein complex of bovine milk on columns of sulphoethyl Sephadex C-50 resulted in fractionation of the complex into a main component, αs1-casein and a number of minor components one of which was designated αs0-casein. The present communication describes the preparation of αs0-casein from unfractionated casein in an amount and state of purity which render possible its further characterization.