Two lactating mammary glands excised from 2 goats were perfused for several hours in the presence of [U-14C; 2,3-3H]-L-valine and received adequate quantities of glucose, acetate and amino acids. In the synthesized milk 96 and 89% respectively of the casein valine was derived from free plasma valine. Valine was extensively catabolized by mammary tissue, resulting in a considerable 14CO2 production and in the incorporation of 14C into milk citric acid and to a lesser extent into casein aspartic acid and glutamic acid. About 30% of the valine molecules which were taken up by the mammary gland were oxidized to CO2 and 70% were incorporated in casein as valine residues. About 10% of the plasma valine molecules were reversibly transaminated during one passage through the udder. An important amount of radioactivity of plasma was present in unknown metabolites. Only 7% of this activity was localized in isobutyrate. The radioactivity of total milk fat was very low. Mainly iso-14:0, iso-16:0 and 15:0 were labelled.

No relationship was found between the trisaturated glyceride content and the softening point (SP) of milk fat. Inter-esterification of milk fat increased its SP, but did not change the trisaturated glyceride content or the fatty acid composition of the trisaturated glyceride fraction. Inter-esterification increased the amount of both low and high and decreased the amount of medium molecular weight triglyceride species in the trisaturated glyceride fraction. The increase in the amount of high molecular weight triglyceride species was up to 3 times greater than the increase in the amount of low molecular weight triglycerides.

The triglyceride species composition of the trisaturated glyceride fraction of milk fat was found to vary seasonally. Excellent correlation was found between SP and groups of low molecular weight triglycerides, groups of high molecular weight triglycerides and some individual triglyceride species. Correlation coefficients were higher than those previously reported between groups of fatty acids and SP.

k-Casein is the main glycoprotein of cow's milk. Its polysaccharide part is O-glycosidically linked to threonine residue 133. It contains only 3 different sugars (Gal, GalNAc, NeuNAc), but a microheterogeneity has been detected at the sugar level. Two main polysaccharides have so far been characterized. The structure of the trisaccharide is NeuNAc α → 3 Gal β1 →3 GalNAc; the tetrasaccharide contains one additional sialic acid. The polysaccharide part of ovine k-casein resembles that of bovine k-casein, but contains also N-glycolyl neuraminic acid. Human k-casein contains 3 times more carbohydrate than bovine k-casein with 2 additional sugars, GlcNAc and Fuc. The various polysaccharide parts isolated from bovine colostrum k-caseinoglycopeptide are much more complex than those obtained from the normal glycopeptide, indicating an evolution of the sugar part as a function of time after parturition. Some aspects of the secondary structure of k-casein and the role of the sugar part are discussed. The carbohydrate moiety of another milk protein, human lactotransferrin, is also discussed briefly. It is comprised of 2 identical glycan groups, N-glycosidically linked to the protein, and quite different from the k-casein carbohydrate moiety.

Individual fatty acids were added to liquid skim-milk and to reconstituted dried skim-milk in concentrations similar to those produced by natural lipolysis, and their effects on deposit formation from the milk on a heated surface were determined. The use of reconstituted dried skim-milk considerably improved the reproducibility of the results. Butyric, caproic and caprylic acids did not influence the amount of deposit formation. Capric acid had a marked effect in reducing deposit at low concentrations: the effect of ageing of milk in reducing deposit formation is thought to be due to capric acid development. Laurie, myristic and palmitic acids had a slight effect, increasing deposit formation, while stearic acid caused a considerable increase. Capric acid was found to adsorb to the casein micelles in milk, and it may therefore act by interfering with casein aggregation during the build-up of deposit.

Particles as large as several µm diam. have been observed occasionally in normal milk and commonly in prepartum and postpartum colostrum. These particles can be dissociated by EDTA and their appearance closely resembles that of normal casein micelles. However, they are often too large to have been completely formed within the Golgi vesicles of mammary epithelium and hence some degree of post-secretory aggregation of caseins is thought to occur. Two possible mechanisms of post-secretory aggregation of caseins are: (1) a continuation of the normal processes of micelle assembly in the alveolus and (2) aggregation as a result of limited proteolysis of the caseins during the time the milk is in the mammary gland. Incubation of milk with fibrinolysin, however, failed to produce aggregation of normal micelles.

The effects of high rates of infusion of essential amino acids on amino acid uptake by the isolated perfused guinea-pig mammary gland were studied. Infusion of methionine, tyrosine, phenylalanine, histidine and tryptophan (designated group 1) resulted in significant increases in the uptakes of tyrosine, phenylalanine and histidine. Methionine, tryptophan and other essential amino acids were not significantly affected. Infusion of threonine, valine, isoleucine, leucine, lysine and arginine (designated group 2) resulted in significant increases in uptake of all these amino acids. Group 1 amino acid uptake was not significantly affected. Infusion of all the essential amino acids (i.e. groups 1 and 2 together) resulted in significant increases in all their uptakes. Using as index ‘the predicted rate of protein synthesis’, infusion of group 1 and 2 together led to an apparent 27% increase in protein synthesis. The above results are discussed in relation to the control of milk protein synthesis by limiting essential amino acids.

In dilute buffers ⋟ pH 5·8, chymosin hydrolysed bovine αs1-casein to αs1-I, αs1-II and αs1-III/αs1-IV in a sequential manner while at pH 4·6 αs1-casein was hydrolysed to αs1-I which was then hydrolysed to αs1-V. In the presence of 5 % (w/v) NaCl at pH 5·2, αs1-casein was hydrolysed to αs1-I which was then hydrolysed to αs1-VII and αs1-VIII. αs1-I, αs1-II and αs1-III/αs1-IV were isolated by chromatography on cellulose phosphate followed by preparative slab-gel electrophoresis; αs1-V was isolated by repeated preparative slab-gel electrophoresis and αs1-VII by gel filtration on Sephadex G-150 followed by preparative slab-gel electrophoresis. The mol. wts of the peptides, estimated by gel filtration on Sephadex G-100, were 21000, 17600, 15600, 19900 and 14600 for αs1-I, αs1-II, αs1-III/αs1-IV and αs1-V and αs1-VII respectively. Characterization of the peptides by amino acid, phosphorus and terminal residue analysis showed that they probably consisted of segments of the αs1-casein chain as follows: αs1-I: residues 24/25–199; αs1-II: residues 24/25–169; αs1-III/αs1-IV: residues 24/25–149–150; αs1-V: residues 29/33–199; αs1-VII: residues 56–179. Peptide bonds close to phosphate residues on the αs1-casein chain were not hydrolysed by chymosin at high pH values (⋟ 5·8) when the phosphate groups were charged, but became available for hydrolysis when the reaction pH was reduced. Proteolytic specificity was also modified by NaCl.

The effects of solubilization, treatment with organic solvents and storage under alkaline conditions on membrane-associated peptidases of intact cells of Streptococcus cremoris HP were studied. Differences in the response of the peptidase activities towards these membrane perturbing treatments were observed. Pyrrolidonecarboxylylpeptidase (PCP) and an endopeptidase (P50) showed 50% irreversible inhibition at the same concentration of each solvent tested. An amino- and proline iminopeptidase activity and the endopeptidase P37 were in this respect much more sensitive to the action of the solvents. Within a homologous series of n-alkanols irreversible inhibition of PCP showed a dependence on the hydrophobicity of the solvent molecules. Only P37 activity was increased considerably upon solubilization of the enzyme. Similar levels of activation were found upon treatment of cells with 3% (v/v) n-butanol at 25 °C or storage at 30 °C at an alkaline pH. Optimal activity of P50 during n-butanol treatment was at 25 °C using a concentration of 5% (v/v), but no activation was observed upon solubilization. The results are discussed in terms of enzyme–lipid interaction and accessibility of the enzymes in situ. It is concluded that the enzymes apparently occupy different positions within the membrane although they may together constitute a functional peptide-hydrolysing unit.

When individual essential amino acids were omitted for periods of 40–100 min from the infusate substrate solution in isolated perfused guinea-pig mammary gland experiments, uptake of methionine, tyrosine, phenylalanine, histidine and tryptophan (group 1) was significantly depressed by a mean of 49·8%, whereas the remaining essential amino acids (group 2) showed no significant decrease in uptake. During depletion periods oxidation of [14C\amino acids was increased. The possible significance of the differences in absorption between the 2 groups of amino acids is discussed.

The current status of knowlege of the major proteins of milks of various species is evaluated. Most of the non-bovine milk proteins are homologous with the recognized families of those of Bos taurus, αs1-caseins, αs2-caseins, β-caseins, K-caseins, β-lactoglobulins, and α-lactalbumins, each family representing a separate genetic locus specific to the mammary gland. No prominent milk protein not homologous to one of these families has yet been discovered in milk of any species. Genetic polymorphism resulting from substitutions in the polypeptide chains and various degrees of post-translational phosphorylation, glycosylation, and proteolysis have been identified in milk proteins of several species. Total protein production ranges among species from about 0.5 to 10 g/d per kg0.75 maternal weight. Proportions of the several proteins vary greatly among species, but few accurate analytical data are available except for total casein and total whey protein contents.

Microbial thermograms have been used to investigate the growth of Streptococcus thermophilus and Lactobacillus bulgaricus in single and mixed culture in milk medium. Growth has been observed with different ratios of the strains in the inoculum and, at one ratio, the effect of the amount of inoculum. The effect of serial sub-culture both with and without pH control has been observed, and stimulation of Str. thermophilus by cell-free fitrates of L. bulgaricus has been examined. The development of the technique is discussed in terms of the application of microbial thermograms in selecting cultures for incorporation into mixtures and evaluating their performance.

The relationship between extent of κ-casein proteolysis and aggregatability of milk casein micelles has been studied using chymosin either bound to porous glass supports or free in solution. Both enzyme preparations demonstrated that, overall, 86–90 % of the κ-casein had to be destroyed before any aggregation could occur. Based on these results, a mathematical model of chymosin action on casein is described involving (i) the attack on κ-casein by chymosin, via a Michaelis–Menten mechanism (ii) the probability that sufficient κ-casein on any micelle is destroyed to allow aggregation, and (iii) the aggregation of para-casein micelles by a von Smoluchowski mechanism.

The effect of proteolytic, psychrotrophic strains of Pseudomonas fluorescens, Ps. putida and Acinetobacter spp. on cheese-making with stored milk has been investigated. Ps. fluorescens and Ps. putida growing for 72 h in raw milk at 7·5 °C to levels of approx. 107 colony-forming units/ml caused a low degree of β- and к-casein breakdown detectable by gel electrophoresis, but this was insufficient to affect N losses in whey or cheese yields. Variations in cheese-making times with pasteurized milks were not attributable to the counts of psychrotrophs in the corresponding raw milks. The water-soluble and TCA-soluble N fractions of maturing cheeses were unaffected by psychrotroph counts in raw milks, but small differences in levels of casein fractions of cheeses made from milks stored for 72 h were detected by quantitative polyacrylamide gel electrophoresis. The incidence of casein breakdown in raw milk and subsequently in cheese were not necessarily related. None of the cheeses developed off flavour related to excessive protein breakdown but many became lipolytically rancid, despite the selection of strains with low lipolytic activity on a diagnostic medium. It is concluded that the numbers of psychrotrophic bacteria likely to occur in stored raw milk under commercial conditions are unlikely to cause significant changes in the yields or quality of Cheddar cheese through their proteolytic activity.

Powders were prepared from whole and skim-milk using reverse osmosis (RO) as a first stage followed by either evaporation and spray drying or spray drying alone. They were compared with powders made by the conventional method of evaporation and spray drying. Measurements were made of powder solubility, free fat content, whey protein denaturation, vitamin content, powder morphology and bacterial count. The free fat contents of powders prepared from whole milk concentrated by RO were high; otherwise, there were no significant differences in the properties of powders made from milks concentrated initially by RO and those prepared by conventional evaporation. The results indicate that it is feasible to use RO for the manufacture of skim-milk powder and for dried whole milk production where higher free fat levels are not detrimental.

Pancreatic lipase deacylation was used to study the variation in distribution of the fatty acids at position sn-2 of milk fat triglycerides of a series of butters and of the milk obtained from a cow on a restricted feeding regimen. Selected samples were analysed for stereospecific distribution of their triglyceride fatty acids which were found to be esterified in a non-random manner. The percentage of a fatty acid at position sn-3 generally decreased with increase in the chain length of the acid; the reverse occurred at position sn-1. There was no major variation in the stereospecific distribution of fatty acids throughout the year; the variation that did occur was exhibited mainly by the medium chain length acids. Variation was greater at positions sn-1 and sn-3 than at position sn-2 and when the proportion of an acid varied at position sn-1 it usually varied at position sn-3 in the reverse manner.

For the milk samples it was found that change in triglyceride fatty acid composition was not associated with random increases or decreases of fatty acids at the 3 stereospecific positions. With changing triglyceride fatty acid composition (6:0, 8:0, 10:0, 12:0, 14:0, and 16:0 decreasing and 18:1 increasing) the percentage of 6:0, 8:0, 10:0, 12:0, 14:0 and 16:0 acids at position sn-3 decreased while the percentage of 18:0 and 18:1 acids increased. These changes were compensated for by changes in the opposite direction at positions sn-1 and sn-2.

Using gel electrophoresis a genetic polymorphism of αs2-casein (Cn) was discovered in individual milk samples from 2 bovine breeds of the eastern part of France (Vosgienne and Montbéliarde). The 3 observed phenotypes (Plate 1) are determined by 2 co-dominant alleles at an autosomal locus. The αs2-Cn A variant was the only one known up to now in European breeds (reference variant) and ±s2-Cn D is a new variant, whose bands overlap the β-casein A band at pH 8.6, and migrate faster than αs2-Cn A at pH 3.0.

The sequence of the polypeptide chain αs2-Cn D differs from that of αs2-Cn A by the deletion of a very acidic nonapeptide, which includes a cluster of 3 phosphoseryl residues. Due to the characteristics of the reference sequence, this deletion cannot be exactly located but it involves residues 50–58, or 51–59, or 52–60.

A genetic analysis shows that locus αs2-Cn is closely linked to the cluster αsl-Cn–β-Cn-K-Cn. The 4 casein species are thus synthesized by 4 closely linked loci.

An insoluble benzoquinone derivative, obtained by reacting benzoquinone with Sepharose 4B and subsequent deactivation with ethanolamine, selectively absorbed chymosin from a mixture of milk-clotting enzymes and other proteins such as in rennets. This selective absorption has been used for developing a purification method and for quantitatively estimating the chymosin content of commercial rennets. Thus, from a powdered calf rennet, it was possible to obtain in one step an 8-fold purified chymosin free of bovine pepsins. The proposed analytical test involves 2 milk-clotting activity measurements and a chromatographic elution of the sample on a quinonated Sepharose column. It can be applied directly to commercial rennets, because it does not require pre-dialysis of the sample against the elution buffer. Furthermore, a combination of quinonated Sepharose and DEAE-cellulose chromatography gave a method for isolation of bovine pepsin I from rennets.

The heat denaturation of Fe-saturated lactoferrin (If) and Fe-free lactoferrin (apo-lf) was studied using the methods of micro-complement fixation and fluorescence. It was established that the change in conformation of apo-lf, induced by iron binding, conferred a higher heat stability to the molecule: the changes were observed at temperatures above 40 °C for apo-lf and above 60 °C for If. The Fe-binding ability of the protein was partially independent of the degree of denaturation. Fluorescence analyses indicated that tryptophan residues were probably not directly involved in the metal binding. There was no evidence of antibodies interfering with the binding sites.

Lipid and aqueous extracts of bovine milk and 5 fractions prepared from bovine milk, including a membrane-rich fraction (MRF) isolated during butter oil production, were analysed for neutral lipid, phospholipid, protein and glycoprotein. The MRF showed an 8·5-fold enrichment in the phospholipid: protein ratio compared to whole milk. The percentage distribution of the 3 major phospholipid classes, phosphatidyl ethanolamine, phosphatidyl choline and sphingomyelin was 30, 36, 31 % respectively, the values of which differ slightly from those of whole milk. Fatty acid analysis of the major neutral and phospholipid classes indicated a predominance of palmitate, stearate and oleate. SDS polyacrylamide-gel electrophoresis in polyacrylamide gradients indicated that the percentage protein having a mol. wt in excess of 75000 increased by a factor of 2 in the MRF when compared to milk. Glycoprotein profiles of the MRF and its precursor cream differ significantly from the patterns evident in whole milk and skim-milk.

Two constituents of the proteose-peptone fraction of bovine milk have been isolated and characterized. Component 5 (PP5) has been shown to represent residues 1–105 and 1–107 of the β-casein amino acid sequence, while component 8-fast (PP8F) corresponds to residues 1–28 of β-casein. Thus, these proteose-peptones represent the N-terminal portions of the β-casein molecule, produced by proteolytic cleavages which form the γ1-, γ2- and γ3-caseins from the C-terminal part. The continuing formation of the total proteose-peptone fraction, PP5, PP8F and the γ-caseins during storage of raw milk at 18 or 37 °C has also been demonstrated