The phenomenon of rennet hysteresis in heated milk is essentially due to reversible heat-induced changes of calcium phosphate equilibrium which affect the second or calcium ion stage of the rennet coagulation. The phenomenon is shown not only by heated milk but also by all heated caseinate systems provided they contain sufficient calcium to permit of coagulation with rennet and enough phosphate to ensure some degree of colloidal phosphate precipitation during the heating process; its occurrence does not require the presence of micellar casein or of β-lactoglobulin, or the initial presence of colloidal phosphate. Nevertheless, rennet hysteresis is greatly increased in these heated systems if β-lactoglobulin is present owing to the formation of a calcium caseinate/β-lactoglobulin complex which in its renneted condition is much less sensitive to calcium ions than is calcium para-caseinate. The resulting relative prolongation of the second stage of the rennet coagulation renders this phenomenon more apparent by increasing the proportion of the total time occupied by the hysteresis effect.

The maximum and minimum heat stability exhibited by most milks over a relatively narrow range of pH values is shown also by synthetic colloidal calcium caseinate-calcium phosphate systems and even by simple caseinate systems, provided all possess adequate contents of β-lactoglobulin, soluble calcium and phosphate. The phenomenon is not, however, dependent on the presence of the characteristic micellar structure of the casein of milk. The minimum stability observed, usually around pH 6·9, is the most characteristic feature of the phenomenon and arises from heat induced deposition of calcium phosphate on a caseinate/β-lactoglobulin complex. This reaction, which tends to occur to a marked degree at relatively high pH values and calcium ion concentrations, sensitizes the complex to precipitation by calcium ions. The precise pH values at which the maximum and minimum stabilities occur can vary depending on the salt composition of the serum, since the latter can influence the solubility of calcium phosphate.

Batch methods applicable on a large scale are described for the purification of crude κ- and βκ-Casein, dissolved in urea-containing buffer, was freed from αs- and β-caseins by treatment with calcium phosphate gel and recovered in about 60% yield. β-Casein was freed from most impurities by adsorption on to calcium phosphate gel at pH 7·8 in the presence of urea and elution with 6 M-urea–N-NH4OH at 4°C. The recovery was about 50%.

The effects of various factors on gel formation in the California Mastitis Test (CMT) were studied to obtain information on the mechanism of the reaction. Death of the leucocytes in milk, produced either by storing the milk at 5°C or by freezing and thawing, markedly reduced the viscosity of the gel formed by the milk with CMT reagent. Samples of skim-milk to which leucocytes were added gave increasingly viscous gels with CMT reagent as the pH was increased to 10·0. Soluble casein increased the viscosity of the gel formed by leucocytes with CMT reagent. The viscosity of the gel was decreased by the addition of CaCl2 and NaCl. On microscopic examination the gel formed in the CMT appeared as an irregularly arranged fibrillar network containing DNA. Deoxyribonuclease I rapidly eliminated the gel-forming property of leucocyte nuclei with CMT reagent, and EDTA inhibited this enzyme activity. Crude protease, trypsin, papain and bacterial protease decreased the gelforming property to different degrees. The presence of EDTA with these enzymes resulted in the formation of a viscous, ropy gel before the addition of CMT reagent. It was concluded that at least a part of the protein of the DNA-protein complex of leucocyte nuclei, in addition to the native polymer of DNA, was necessary for gel formation in the CMT reaction.

The effects of the isocaloric replacement of starch in the concentrate by either 5 or 10% ‘stearic acid’ (85% pure) or by 10% ‘palmitic acid’ (85% pure) on the yield and composition of milk were investigated in a feeding experiment with 12 cows in mid-lactation. The concentrate mixtures were given with a high-roughage diet that supplied 4.4 kg of hay and 2·7kg of sugar-beet pulp/day.

The inclusion of 5% ‘stearic acid’ in the concentrate mixture resulted in increases in the yields of milk, milk fat, solids-not-fat (SNF) and lactose and a reduction in the content of protein in the milk, whereas, when the level of ‘stearic acid’ in the concentrate was increased to 10%, there was an increase in only the yield of milk and a reduction in the contents but not in the yields of SNF and protein in the milk. The inclusion of 10% ‘palmitic acid’ in the concentrate mixture also reduced the contents of SNF and protein in the milk but produced increases in the yields of milk, milk fat and lactose and also in the content of fat in the milk.

None of the dietary treatments resulted in any changes in the concentration of blood glucose.

The effects of the isocaloric replacement of starch in a low-fat concentrate mixture by either 5 or 10% ‘stearic acid’ (85% pure) or 10% ‘palmitic acid’ (85% pure) on the yield and composition of milk fat were investigated in a feeding experiment with 12 cows in mid-lactation. The concentrate mixtures were given with a highroughage diet that supplied daily 4·4kg of hay and 2·7kg of sugar-beet pulp.

The inclusion of 5 or 10% ‘stearic acid’ in the concentrate mixture decreased the concentrations and yields of 10:0, 12:0, 14:0, 14:1, 16:0 and 16:1 in the milk fat but increased the concentrations and yields of 18:0 and 18:1. When the concentrate mixture contained 10% ‘stearic acid’ the decreases in the yields of the shorter chain acids and the increases in the yields of 18:0 and 18:1 in the milk fat were more pronounced than when the concentrate mixture contained 5% ‘stearic acid’. The inclusion of 10% ‘stearic acid’ in the concentrate mixture also decreased the concentrations of 6:0 and 18:2 in the milk fat. The inclusion of 10% ‘palmitic acid’ in the concentrate mixture decreased the concentrations of 6:0, 8:0, 10:0, 12:0, 14:0, 14:1, 18:0 and 18:2 in the milk fat but increased the concentrations of 16:0 and 16:1. This dietary treatment also reduced the yields of 6:0, 10:0, 12:0, 14:0 and 18:2 but increased the yields of 4:0, 16:0 and 16:1 in the milk fat. The inclusion of 5% ‘stearic acid’ or 10% ‘palmitic acid’ in the concentrate mixture increased the yield of total milk fat.

The inclusion of 10% ‘palmitic acid’ or 10% ‘stearic acid’ in the concentrate mixture increased the relative proportions of propionic and iso-butyric acids in the rumen liquor but decreased the proportions of η-butyric and valeric acids. The inclusion of ‘stearic acid’ at the level of 5% in the concentrate mixture increased the relative proportions of iso-butyric and iso-valeric acids but decreased the proportion of butyric acid in the rumen liquor.

The effects of the isocaloric replacement of starch in a low-fat concentrate mixture by either 5 or 10% ‘stearic acid’ (85% pure) or by 10% ‘palmitic acid’ (85% pure) on the composition of the plasma lipids were investigated in a feeding experiment with 12 cows in mid-lactation. The concentrate mixtures were given with a high roughage diet that supplied daily 4·4 kg of hay and 2·7 kg of sugar-beet pulp. A study was made of the relationships between the compositions of the plasma and milk lipids.

The inclusion of 10% ‘stearic acid’ or 10% ‘palmitic acid’ in the concentrate mixture increased the concentration of total plasma fatty acids. Irrespective of dietary treatment, about 40% of the total plasma fatty acids occurred in the cholesteryl ester fraction, 54% in the phospholipid fraction, 3% in the triglyceride fraction and 3% in the unesterified fatty acid fraction. There was a positive curvilinear relationship between the concentration of unesterified fatty acids in the plasma and the yield of total milk fatty acids.

In the plasma triglycerides, the concentrations of 16:0 and 16:1 were decreased and the concentration of 18:0 was increased when the concentrate mixture contained ‘stearic acid’; the concentration of 16:0 was increased and the concentrations of 18:0, 18:1 and 18:2 were decreased when the concentrate mixture contained ‘palmitic acid’. Similar changes were observed in the compositions of the plasma unesterified fatty acids when the cows were given the different diets.

In the plasma cholesteryl esters, the concentration of 16:0 was decreased and the concentrations of 18:3 and 20:4 were increased when the concentrate mixture contained stearic acid; the concentrations of 16:1, 18:3 and 20:4 were increased and the concentration of 18:2 was decreased when the diet was supplemented with palmitic acid. The addition of stearic acid to the diet increased the concentration of 18:0, 18:1 and 18:3 in the plasma phospholipids but decreased the concentrations of 16:0, 18:2, 20:3 and 20:4. When the diet contained palmitic acid the concentrations of 16:0, 16:1, 18:1 and 18:3 in the plasma phospholipids were increased but the concentrations of 18:0, 18:2 and 20:3 were decreased.

The major fatty acid circulating in the plasma of the cows was 18:2, which accounted for about 45% of the total plasma fatty acids. Only about 0·7% of the total plasma 18:2 occurred in the plasma triglycerides.

The results are discussed in relation to the changes in the composition of the milk fatty acids produced by the cows when they were given the experimental diets.

Cows were given either a low-fat concentrate mixture or a concentrate mixture that contained 5 or 10% ‘stearic acid’ (85% pure) or 10% ‘palmitic acid’ (85% pure). The concentrate mixtures were given with a high roughage diet that supplied 4·4 kg of hay and 2·7 kg of sugar-beet pulp/day. Blood samples were taken from the cows on each dietary treatment and the plasma phospholipids were analysed.

Phosphatidyl choline accounted for 70% of the plasma phospholipids when the cows were given the low-fat diet and about 86% of the plasma phospholipids when the cows were given the diets supplemented with the fatty acids. The inclusion of the fatty acids in the concentrate mixtures decreased the relative proportions of the plasma phosphatidyl ethanolamine, sphingomyelin and lysophosphatidyl choline but the relative proportion of the plasma phosphatidyl serine remained unchanged.

When the diet was supplemented with stearic acid the concentrations of 18:0 and 16:0 in the phosphatidyl choline were unaltered but the concentration of 18:1 was increased and the concentration of 18:2 was decreased. When the diet was supplemented with palmitic acid the concentrations of 16:0 and 18:1 in the phosphatidyl choline were increased and the concentrations of 18:0 and 18:2 were decreased. In contrast, the inclusion of stearic acid in the diet increased the concentration of 18:0 in the phosphatidyl serine and decreased the concentration of 16:0; the concentrations of 18:1 and 18:2 were unchanged. The fatty acid composition of the plasma phosphatidyl ethanolamine was unaffected by dietary treatment. The effects of diet on the fatty acid compositions of the phosphatidyl choline and lysophosphatidyl choline were similar. Supplementation of the diet with stearic acid increased the concentrations of 18:0 and 18:1 in the plasma sphingomyelin and decreased the concentrations of 16:0 and 18:2. The addition of palmitic acid to the diet increased the concentration of 16:0 in the sphingomyelin but it decreased the concentrations of the other constituent fatty acids.

(1) A survey of the composition of the milk of individual cows in 4 commercial herds in the West Riding of Yorkshire was undertaken from October 1967 to May 1968.

(2) In 3 of the 4 herds there was a decline in solids-not-fat (SNF) content throughout the winter feeding period. Milk protein content was at a minimum in January–February but in all 3 herds lactose content also declined and in 2 of the herds this was the major cause of the fall in SNF content.

(3) The decline in lactose content was the result partly of the characteristic effect of an advance in lactation, partly of an increase in the incidence of mastitis throughout the winter period. In all herds there was a significant inverse relationship between lactose content and cell count in the bulked milk of individual cows.

(4) In 3 of the herds autumn-calving (October–December) animals showed a pronounced fall in milk protein content in the early months of lactation which was not observed in summer-calving (July–September) animals. There was no response in SNF or protein content to supplementary concentrates offered towards the end of the winter-feeding period, whereas with the commencement of grazing in the spring there was an immediate increase in both protein and SNF contents.

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.

The dynamic viscosity coefficients η of creams containing up to 50% fat have been determined at temperatures of approximately 40, 50, 60, 70 and 80°C. All the creams behaved as Newtonian liquids, the shear stress being proportional to rate of shear up to the maximum rate used of 100 sec−1. At a given temperature a linear dependence of log η on (φ+φ5/3) was obtained for φ < 0·4, where φ is the concentration (w/w) of fat. Interpolation formulae have been derived to enable η to be calculated at any temperature between 40 and 80°C and for any fat content up to 40%. Formulae for the density ρ of cream have also been deduced to permit kinematic viscosities η/ρ to be computed. Nomograms have been constructed to enable η and ρ to be readily determined without the use of the interpolation formulae and when slight loss in accuracy is unimportant.

The milk-clotting and proteolytic activities of rennet, bovine pepsin and porcine pepsin were compared. The milk-clotting activity of porcine pepsin was extremely pH-dependent around pH 6·6 and coagulation did not occur above pH 6·68. The clotting activity of bovine pepsin was slightly more dependent on pH than that of rennet but no rapid drop-off in activity occurred as with porcine pepsin. Temperature influenced the clotting activity of rennet and bovine pepsin similarly but the behaviour of porcine pepsin was markedly different.

For equal milk-clotting activity, the proteolytic activities of rennet and bovine pepsin were approximately equal and substantially lower than that of porcine pepsin. Electrophoretic examination showed that the proteolysis products of rennet and bovine pepsin were similar and quite different from those produced by porcine pepsin.

The suitability of bovine pepsin as a rennet substitute is discussed.

The folate in cow's milk was strongly and specifically bound to a minor whey protein (FP), forming a complex of primary M of about 38000, but exhibiting concentration-dependent reversible aggregation. The binding protein was present in excess, and the milk had the capacity to bind about 50 μg added folic acid/l. An enriched concentrate of FP was prepared by ammonium sulphate fractionation—FP was precipitated at between 50 and 60% saturation—and further purified by chromatography in DEAE-cellulose and filtration in Sephadex gel G150. Its identity as a distinct minor whey protein was confirmed by comparative starch gel electrophoresis at various pH values.

Some properties of the protein are described, and its physiological significance discussed.

The vitamin content of ultra-high-temperature (UHT) processed milk was compared with that of the original raw milk. Three processes were used. In the first, which caused no change in oxygen content, the milk was heated and cooled in a plate-type heat exchanger. In the second, the milk was again heated indirectly and then evaporatively cooled, leaving in the milk about one-third of the initial oxygen content. In the third process the milk was heated by direct steam injection and cooled by evaporation and contained little or no residual oxygen.

On processing and during subsequent storage for 90 days there was no loss of vitamin A, carotene, vitamin E, thiamine, riboflavine, pantothenic acid, biotin or nicotinic acid. There was little or no loss of vitamin B6 or vitamin B12 on processing, but up to 50% of each of these vitamins was lost during 90 days' storage. All the dehydroascorbic acid (DHA) and about 20% of the ascorbic acid (AA) was lost on processing. There was no further loss of AA during 90 days’ storage when no residual oxygen was present, but in milks containing more than about 1 ppm oxygen all the AA was lost within 14 days. About 20% of the folic acid was lost on processing; thereafter, as with ascorbic acid, the extent of the loss on storage depended on the residual oxygen content of the milk: in the absence of oxygen the folic acid was stable.

A study was made of the changes in the pathways of glucose catabolism in slices of lactating rat mammary gland which occurred when acetate or pyruvate was added to the medium.

When acetate was added there was an increase in the rates of oxidation of the C(2) and C(6) atoms of glucose but little change in that of the C(1) atom. The amounts of these carbon atoms which were incorporated into fatty acids, lactic acid and glyceride glycerol were reduced, but the results showed that the relative proportion of the glucose converted to these substances which passed through the pentose phosphate pathway was increased.

When pyruvate was added, the rates of oxidation of the C(1), C(2) and C(6) atoms were all reduced to about the same extent. The incorporation of the glucose carbon into fatty acids was almost abolished and its incorporation into lactic acid and glyceride glycerol was very much reduced. The results showed that most of the glucose incorporated into these compounds under these conditions passed through the pentose phosphate pathway.

Evidence is presented which suggests that the triose phosphates are not in complete isotopic equilibrium in lactating rat mammary tissue.

These results are discussed in relation to the existence of 2 pools of acetyl coenzyme A, one intra- and the other extra-mitochondrial, and to the possible inhibition of certain steps of the glycolytic pathway.

A study was made of the changes in the rates of oxidation of the C(1), C(2) and C(6) atoms of glucose and in the pathways of glucose catabolism in sheep udder tissue in vitro which occurred when acetate and pyruvate were added.

Whereas in rat mammary tissue the rate of oxidation of the C(1) atom of glucose was very much greater than that of the C(6) atom, the ratio of the rates of oxidation of these 2 atoms in sheep tissue was less than 2 when glucose was the only substrate.

The addition of acetate resulted in an unequal stimulation of the oxidation of these 2 atoms, with the result that the ratio of their rates of oxidation was about doubled. The rate of oxidation of the C(2) atom was also increased.

Acetate also increased the participation of the pentose phosphate pathway in glucose catabolism as measured by the incorporation of the C(1) and C(6) atoms of glucose into fatty acids, lactic acid and glycerol.

Pyruvate produced little effect on the rate of oxidation of the C(1) atom but somewhat depressed that of the C(6) atom of glucose. At the same time, it caused a large increase in the participation of the pentose phosphate pathway.

These results are discussed with reference to re-cycling of glucose carbon in the pentose phosphate pathway and to the relationship between that pathway and fatty acid synthesis. It is noted that the incorporation of glucose carbon into the 3 intermediates used gave values for the participation of that pathway which were in better agreement than was obtained in rat tissue. It is concluded that triose phosphates are more nearly in equilibrium in sheep than in rat mammary tissue.