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.

Summer and winter milk-fat samples from 14 dairies in Sweden were fractionated by crystallization from acetone solution (1:8) at 15 °C. The composition of the major fatty acids of the parent milk fat and of the acetone insoluble fraction were examined by GLC, and the gross triglyceride pattern by TLC on plates of silicic acid treated with silver nitrate.

The fatty acid composition of the milk fat was similar to that of milk fat from other countries and varied according to season and also, to a smaller extent, from region to region. Four fractions, representing 33–45, 41–34, 18–14 and 7–6 % of the fat and which contained progressively smaller proportions of saturated acids, were obtained by TLC.

The acetone insoluble glyceride (AIG) fraction was characterized by a smaller content of short-chain fatty acids and unsaturated fatty acids, and a larger content of saturated long-chain fatty acids, than the parent milk fat. AIGs from summer milk fat contained a larger proportion of C18 acids and a smaller proportion of C6–C16 acids than AIGs from winter milk fat.

Four fractions representing 62–70, 15–8, 16–15 and 7 % of the AIG fraction were obtained by TLC. The distribution of the triglycerides in the AIG fraction differed from that in the parent milk fat, mostly in the relative amounts of glycerides in the 2 most saturated TLC fractions. The seasonal variation was largely confined to these 2 fractions.

Two fistulated adult bulls of the Zebu breed and 2 fistulated adult buffalo steers were given a diet consisting of concentrate mixture (rice bran and cotton-seed cake in equal portions +2% calcium hydroxide + 1% common salt) and straw. This was later changed to an all-roughage diet of first cut berseem (Trifolium alexandrinum) Samples of the rumen contents were taken for determination of protozoal differential counts, concentration and relative proportions of volatile fatty acids (VFA), rate of cellulose digestion, fermentation rate, and net growth of rumen micro-organisms. Further data were obtained for the 2 buffalo steers fed sweet Sudan grass. When the feed consisted of concentrate and straw there were significant differences in the numbers of protozoa and in volatile fatty acid concentration in the 2 species of animals. Higher protozoal counts were observed in the rumen of bulls (7·2−8·1 × 105 organisms/ml) than in the rumen of buffaloes (2·6−3·6 × 105 organisms/ml). Greater concentration of VFA was found in the rumen of buffaloes (5·3−11·2 mequiv/100 ml rumen liquor) than in bulls (4·8−10·4 m-equiv/100 ml rumen liquor). With berseem, the concentration of VFA in the rumen was generally higher (6·73−11·0 m-equiv/100 ml rumen liquor) and the protozoal counts were lower (2·8−5·2 × 105 organisms/ml) but there were no significant differences between bulls and buffaloes.

The rates of cellulose digestion and the maximum fermentation rates were similar in the 2 species indicating a microbial population of the same order. Higher rates of cellulose digestion and of maximum fermentation when the ration consisted of berseem than when it consisted of concentrate + straw indicated a higher concentration of micro-organisms, mostly bacteria.

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.

The hydrolysis of κ-casein by a number of rennets and other proteolytic enzymes has been followed by measuring the increase in opacity due to the formation of insoluble aggregates of para-κ-caseins. The stability of these precipitates varied markedly, some being solubilized rapidly by the further action of the enzyme. The turbidity obtained with certain enzymes was dependent upon the calcium ion concentration, indicating that the para-κ-caseins produced were not identical for all enzymes.

For high concentrations of calf rennet, the rate of aggregation was linear with respect to time. With low concentrations of enzyme, increase in turbidity was preceded by a lag period which was lengthened by decreasing the enzyme concentration or increasing the κ-casein concentration. This increase in lag is favoured by a high κ-casein/para-κ-casein ratio, suggesting that the aggregation of newly formed para-κ-casein is prevented by the unchanged κ-casein. In addition, small amounts of αs1- or β-caseins present in the κ-casein also markedly affected the aggregation of para-κ-casein, indicating that all 3 major casein components can inhibit the aggregation of para-κ-casein in the absence of calcium ions. In the light of these observations the possible role of protein-protein interactions in casein coagulation by calf rennet is discussed.

A Coulter counter was used to measure the diameters and numbers of fat globules in serial samples taken during a single milking of each of 8 cows. Milk fat percentages of samples were estimated by the Babcock method. The average globule diameter, globule number and fat percentage all increased during milking. Regression analyses indicated that fat percentage was a stronger function of fat globule diameter than of globule number. The results are discussed with relation to the rise in fat percentage that occurs during milking.

The release, by the action of rennet, of peptides which are soluble in trichloroacetic acid (TCA) has been studied using milk samples from individual cows. This release showed an initial marked increase with time of rennet action. After the milk clotted there was no further increase for at least 30 min. There was considerable variation in the total amount of N and of sialic acid released from milk samples taken from individual animals at different times during the lactation.

For a given milk sample treated with rennet, the amount of sialic acid in the filtrate obtained after precipitation with 2% TCA was similar to that obtained with 10% TCA, whereas the amount of nitrogen was much greater with the 2 % TCA. It appeared that the peptides containing sialic acid were released at a slower rate.

On average, about half the total sialic acid in the casein was recovered in the TCA filtrate after rennet action, which suggests that either a proportion of the κ-casein was not accessible to enzyme action or the technique used did not permit full recovery of the sialic acid-containing peptides.

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%.

An agar diffusion slide assay using a thin layer of calcium caseinate has been developed as a micro-method for the quantitative study of calf rennet and other proteolytic enzymes. The number and width of the precipitation zones formed as a result of proteolytic activity depended upon the enzyme used, the concentration of calcium ion, pH and temperature. The assay was highly reproducible and particularly suitable for the measurement of large numbers of samples. It was considerably more sensitive than the milk clotting technique and, in general, more sensitive than other standard methods for measuring proteolytic activity.

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.

A re-investigation has been made of the nature of the tainting substances present in butterfat possessing the weed taint which arises through the ingestion of Lepidium spp. by dairy cattle. Some 0.5 ppm of skatole were isolated from Lepidiumtainted butterfat, along with 0.3 ppm indole. Flavour evaluation tests demonstrated that the skatole was principally responsible for the flavour defect, somewhat modifying the conclusions arrived at by earlier investigators.

In order to obtain more information on the importance of the milk ejection reflex in the lactation of normal ewes, experiments were conducted to determine whether the neuroendocrine reflex is able to modify the parameters which characterize the flow rate of the milk under normal milking conditions.

Ewes were allowed to suckle their lambs freely during the 3 days following parturition. They were then milked with a machine and the individual milk production was measured volumetrically at each milking. Different fractions obtained during milking (‘machine milk’, ‘machine strippings’ and ‘hand strippings’), and the milk flow, were measured using a sensitive recording system. In further experiments, machine milking was performed after intravenous administration of oxytocin, during general anaesthesia, and after unilateral or bilateral denervation of the mammary gland.

The ewes fell into 2 categories: those giving most of their milk in 1 rapid emission, and those giving it in 2 quite separate emissions. The results indicated that the second emission found in some of the ewes was the milk emptied from the acini by the neuroendocrine ejection reflex. Disappearance of the second emission resulted from the administration of oxytocin before milking—which induces passage of the milk from the acini into the mammary cistern—and also from general anaesthesia or denervation of the mammary gland.

The flavour stability of casein and co-precipitated milk proteins has been examined organoleptically. Differences in flavour stability were correlated with processing conditions and the early stages of the non-enzymic browning reaction were implicated in the off-flavour development. The ‘gluey’ flavour present in stored casein could be removed and the flavour stability improved by treatment with activated carbon or Sephadex. However, a better solution to the problem was to avoid the development of off-flavour by careful choice of conditions of preparation, drying and storage to minimize heat damage and browning. Conditions were established under which milk protein could be expected to have flavour stability during at least 6 months storage at ambient conditions.

Unheated Coronopus-tainted milk yielded butterfat from which benzyl methyl sulphide, benzyl isothiocyanate, benzyl cyanide, indole and skatole were isolated by high-vacuum distillation. The first 3 of these compounds were also isolated from the corresponding skim-milk, buttermilk and butter serum by vacuum steam distillation: identification was by gas chromatography and mass spectrometry.

The flavour threshold of benzyl methyl sulphide in milk and butter oil was found to be respectively, 1 part in 108 and 1 part in 107, whereas the amount isolated from butterfat was approximately 1 part in 106.

This evidence, together with the similarity in flavour of the Coronopus taint in milk and butter oil with that of untainted milk and butter oil to which had been added respectively, 1 part in 107 and 1 part in 106 of benzyl methyl sulphide, demonstrates that benzyl methyl sulphide is a principal contributor to the flavour defect. A hypothesis is proposed to explain the origin of the sulphide and related components in the milk. The possibility remains that in commercial weed-tainted butter, which is made from heated cream, other compounds may be present.

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.

A study was made of the effects of the hormones, prolactin and cortisol acetate, on the activities in the mammary gland of the pseudopregnant rabbit of the enzymes concerned with lactose biosynthesis and glucose 6-phosphate metabolism, namely: UDP-glucose pyrophosphorylase, UDP-glucose-4'-epimerase, phosphofructokinase, phosphoglucomutase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Significant increases in enzyme activities were observed in rabbits examined 3 or 4 days after receiving a mammary intraductal injection of prolactin. The activities were not significantly increased at examination 2 days after injection of prolactin. Rabbits receiving cortisol acetate each day for 3 days also showed no significant increases in mammary enzyme activity. However, when prolactin and cortisol acetate were given simultaneously over a period of 3 days, increased enzyme activities were found suggesting synergistic action of the hormones.

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.

Compounds associated with the ‘gluey’ off-flavour in stored casein have been isolated and identified by a combination of gas chromatography and mass spectrometry. Most of the flavour was found in the non-acid steam-volatile fraction which contained many compounds including n-alkanals, n-alkan-2-ones, furfural and other furan derivatives, benzaldehyde, dimethyl disulphide and benzthiazole. No single compound had the typical gluey flavour, which must be a complex sensation in which the quantities of minor components may determine the specific gluey character. o-Aminoacetophenone was present in the casein in subthreshold amounts. This compound has some gluey flavour and may have a synergistic effect in enhancing the total flavour.

Experiments with continuously made curd leading to the use of a 4-min scalding period at 48°C for Cheddar curd-making are described. The short time at 48°C had only a slightly adverse effect on the starter, and the moisture content of the drained curd was within the range usually found in conventionally made curd at the same stage. The starter was allowed to grow in the drained curd during cheddaring. After 3–6 h the pH and moisture content were the same as those of normal cheddared curd.

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.