A new bioassay for the detection of penicillin in milk which can be completed in 1½ h has been developed. The test depends on measurement of pH changes in milk produced by an antibiotic sensitive organism, Streptococcus thermophilus BC. To measure the changes potentiometrically would require a very sensitive pH meter. Change in the enzymic coagulation time, which is extremely dependent on pH, was used in the present study to measure small changes in pH. The test is highly reliable and should be suitable as a milk selection test.

The 90Sr/Ca and 137Cs/K ‘observed ratios’ (OR) in the food intake of nursing mothers and in their milk have been determined. Between 4 and 7 days after parturition, groups of mothers were put on a controlled diet for 4 days. Pooled samples of milk were then taken from each group. 90Sr, Ca, 137Cs and K were determined in the pooled samples and in all foods taken during the experimental period. 90Sr/Ca ratios were determined in 4 experiments and 137Cs/K ratios in 3. The OR for the 4th day's intake did not differ significantly from average ratios for the whole 4 days, so that a state of metabolic equilibrium appears to have been reached. The 90Sr/Ca OR was fairly constant and considerably less than unity, but the 137Cs/K OR was greater than unity and varied considerably between experiments.

Since potassium, which is metabolically analogous to caesium, showed a fairly constant ratio between intake and excretion in the milk, the physiological behaviour of 137Cs must be quite different from that of 90Sr. The maternal organism therefore provides the baby with efficient protection against 90Sr but not against 137Cs.

The temperature-dependent dissociation of β-casein from the casein micelles of milk and from the soluble casein complexes of colloidal phosphate-free (CPF) milk was investigated by high-speed centrifugation and gel-filtration. The percentage of the total casein in supernatants prepared by high-speed centrifugation of mid-lactation milks increased from approximately 6 to 15% on cooling the milks from 30 to 5 °C; β-casein accounted for about 46% of this increase, while αs-and κ-casein constituted 30 and 23%, respectively. On gel-filtration both of skim-milk and CPF milk on Sepharose 2B at 0, 2, 5, 10 and 25 °C, maximum amounts of free β-casein (c. 60% of total) were obtained at 5 °C. The remainder of the β-casein appeared to be more strongly bound to the αs- and κ-casein and may be involved in the internal cohesion of casein micelles. The free β-casein of both milk preparations appeared to be in equilibrium with the bound β-casein. On Sephadex G-200 columns at 5 °C, approximately 5 and 60% of the β-casein of skim-milk and CPF milk, respectively, was eluted in the free form in the expected position for a globular protein of molecular weight about 200000. At low temperatures, particularly at 5 °C, colloidal phosphate appeared to play an integrating role in the association of over half the total β-casein with the other casein components of native micelles. However, when the equilibrium between micellar and free β-casein was disturbed by gel-filtration on Sepharose 2B, the presence of colloidal phosphate did not prevent the release of most of the β-casein from casein micelles. Some problems encountered in the use of densitometry for the estimation of individual caseins on electropherograms are described.

The susceptibility of the casein in milk to proteolysis was shown to be greatly influenced by its state of aggregation. In normal milk, where the casein is largely in micellar form, the αs1- and β-caseins are almost inaccessible to proteolysis. On removal of the colloidal phosphate, the casein micelles disintegrate, rendering the components, especially the αs1-casein, accessible to proteolysis. The role of colloidal calcium phosphate in the casein micelle is believed to be that of a non-specific aggregating agent which can be effectively replaced by calcium. Dissolved colloidal phosphate can be effectively reformed by elevation of the pH of colloidal phosphate-free (CPF) milk before equilibrium dialysis. Addition of κ-casein to CPF milk also causes aggregation of the component caseins but the micelles formed are smaller than those of normal milk.

The behaviour of micellar β-casein differs considerably from that of micellar αs1-casein. The evidence suggests that part of the β-casein freely dissociates either outside or within the micelle when the temperature is reduced. The temperature dependence of the susceptibility of β-casein to proteolysis was similar in skim-milk and in solutions of sodium caseinate, and increased as the temperature was reduced. αs1-Casein was quite resistant to proteolysis in normal milk but became susceptible when the micelle structure was disrupted on removal of colloidal phosphate.

It is concluded that limited proteolysis may prove a valuable technique in the study of casein micelle structure.

Twenty-five strains of lactobacilli isolated from dairy products, including Lactobacillus casei, Lactobacillus plantarum and Lactobacillus brevis species, were grown in semi-defined media and examined for their ability to ferment citrate and produce formate. Of 7 strains of L. casei, all utilized citrate to varying extents, as did 9 of 10 strains of L. plantarum and 3 of 8 strains of L. brevis. L. casei produced 19–35% of the theoretical yield of formate from the citrate utilized, L. plantarum 0–11% and L. brevis 0–28%. Of 2 strains of L. casei tested for their abilities to ferment citrate in the presence of 2% lactose, strain C 5 was unaffected by lactose whereas strain C 2 showed a 45% decrease in the citrate utilized. However, lactose used at 2% concentration greatly reduced formate production by L. casei C 5, which produced 62% of the theoretical yield of formate from citrate after 35 days in the absence of lactose and only 8% in its presence. The yield of diacetyl produced by L. casei C 5 from citrate plus lactose (both at 2% concentration) was 145 times that produced from citrate alone, and 800 times that from lactose alone.

Lactating mammary glands of sheep and goats were perfused for several hours in the presence of [1-14C]palmitate or [1-14C]oleate. Adequate quantities of acetate, glucose, amino acids and chylomicrons were added to the perfusate.

The fall in specific activity of [1-14C]palmitic acid or [1-14C]oleic acid across the gland and the labelling of milk triglyceride fatty acids indicates an extensive transfer of radioactivity from plasma free fatty acids (FFA). The plasma triglycerides showed large arterio-venous differences in concentration. The small [14C] incorporation in plasma triglycerides decreased across the gland. In a control experiment triglycerides were also slightly labelled.

There were no significant arterio-venous differences in cholesterol esters and their fatty acid composition showed only slight changes during passage through the gland. Their specific activity showed a small rise across the gland.

In milk components, the [14C] was mainly localized in the triglycerides. An appreciable proportion of the palmitoleate is derived from palmitate by dehydrogenation within the gland, while there is no evidence for the hydrogenation of oleic acid to stearic acid. Elongation of palmitic acid to C18-acids does not occur to any important extent. FFA are catabolized to a variable extent by the gland.

The role of FFA in labelling of milk and blood plasma fatty acid fraction is discussed.

A study was made of the utilization of citrate in milk by some lactic acid bacteria. When Streptococcus diacetilactis 1007 was grown alone or with either Streptococcus cremoris 924 or Lactobacillus casei B 142/C or with both these latter organisms, > 99% of the milk citrate was utilized within 5 days. L. casei B 142/C and L. casei/Str. cremoris utilized 57 and 14% of the citrate, respectively. When L. casei C 2 and L. casei C 5 were grown in milk in which Str. cremoris 924 had been previously grown, 94 and 64%, respectively, of the citrate was utilized after 7 days at 30°C.

Cheeses were made using a citrate-fermenting and a non-citrate-fermenting starter and citrate concentrations of the milks, wheys and curds were determined during cheese-making. With Str. cremoris 924, citrate was preferentially retained in the curd at pressing, the concentration in the curd moisture being 2·9 times that in the whey. With the mixed starter Str. cremoris 924/Str. diacetilactis 1007, the curd at pressing and from the press contained only 27 and 5%, respectively, of the citrate present in the Str. cremoris curd at these times.

Cheeses were made using Str. cremoris 924, combinations of Str. cremoris/Str. diacetilactis 1007, or with δ-gluconic acid lactone instead of starter, with and without the addition of L. casei C 5, in order to examine the ability of the latter organism to produce ‘blowing’ in the sense of distension of the Cryovac wrapping of film-wrapped cheeses. L. casei C 5 neither accelerated the decrease in cheese citrate nor produced blowing of the film-wrapping. Possible reasons for this behaviour are discussed.

The action of rennin in milk subjected to the ultra-high-temperature heat sterilization process has been studied. The primary phase of rennin action, as determined by the release of peptides soluble in trichloroacetic acid, was partially inhibited as a result of the heat treatment. This was largely due to a reduction in the release of non-carbohydrate-containing peptides from κ-casein. It is suggested that when whole milk is heated the formation of a complex between β-lactoglobulin and κ-casein proceeds more readily with the species of κ-casein which lacks carbohydrate. Evidence is presented which shows that there may be more than one type of carbohydrate moiety attached to κ-casein.

A study was made of the changes in the iodine value of milk fat from cows following the feeding of linseed oil (450g) at intervals of 6 days. Three cows in midlactation and of differing age and breed were used. The experiment consisted of a series of 3 collection periods, each of 6 days' duration, with 12 days between periods. Three doses of oil were given (6 days apart) between periods 1 and 2, the last dose of oil being given immediately before the start of period 2. The mean iodine values for the 3 periods were 35·5, 40·5 and 34·7 respectively. There was a distinct pattern of change in iodine value with time after feeding the oil, a maximum of 45·5 being attained on the second day with a subsequent decline to 38·5 on the sixth day. Analysis of variance showed that these effects were significant and due to oil-feeding, whereas changes in yield and content of fat and production of milk could be accounted for by normal decline in lactation. It was concluded that the periodic feeding of a large dose of linseed oil produces a significant increase in the iodine value of milk fat and avoids the possible disadvantages of continuous fat supplementation.

The modification of arginine residues in casein by treatment with glyoxal at pH 8·6 resulted in an inhibition of the coagulation of rennin-treated casein. The effective residues are on the κ-casein fraction and inhibition of coagulation was complete when about 1·5 residues of arginine/mole of κ-casein had been altered. It is suggested that the arginine forms part of a positively charged region that also includes lysine and histidine residues, and that this region is important in the coagulation of the casein.

The release, as a result of heat treatment on cow's milk, of peptides and glycopeptides which are soluble in the 12% trichloroacetic acid (TCA) filtrate of the milk has been studied. The results showed that the amount of peptides released increased with temperature and duration of the heat treatment. There were differences between milks in their response to a given heat treatment. Glycopeptides were released at temperatures as low as 50 °C but not at 37 °C. A comparison was made of the glycopeptides released by heat treatment and of those released from κ-casein by the action of rennin on the milk. This showed that N-acetyl neuraminic acid, D-galactose and 2-acetamido-2-deoxy-D-galactose were invariably present in both groups of glycopeptides. D-Mannose was also present in the glycopeptides released by heat but was detected on the glycopeptides released by rennin in only one of 6 experiments. It is suggested that glycopeptides released by heat may be derived from κ-casein but that a definite conclusion cannot be reached at present because of lack of information on the carbohydrates attached to other milk proteins.

A study has been made of methods using 2,4-dinitrophenylhydrazine (DNPH) or 2,6-dichlorophenolindophenol (DCP) for the determination of vitamin C (ascorbic acid+dehydroascorbic acid) in raw, UHT processed, evaporated and sterilized milk.

Interfering substances were not detected in milk that had received a heat treatment no more severe than 145°C for 4 s (UHT process), so that either reagent could be used.

With more drastic heat treatment, interfering substances were formed and only the DNPH method with column and thin layer chromatography of the DNPH derivatives was specific for vitamin C. With in-bottle sterilized milk, the values for ascorbic acid were (in mg/100 ml) 1·16 (DCP method with H2S reduction); 0·58 (DCP method with Escherichia coli reduction); 0·64 (DNPH method); 0·33 (DNPH method combined with chromatography).

In our experience the DNPH method combined with chromatography of the derivatives is highly specific for vitamin C and should be used to check the results obtained by other and simpler methods.

The effects of intravenous infusion of insulin and of sodium succinate on milk yield and composition and on the yields of milk constituents was investigated in the goat. Depression of plasma glucose concentration to less than 40 mg/100 ml in response to infusion of insulin was associated with a decrease in milk volume and lactose yield, and increases in the contents of fat and casein and in the yield of fat. The increase in fat yield was the result of an increased output of C12–C18 fatty acids; the output of C4–C10 acids was slightly reduced. Infusion of succinate caused a reduction in the yields of milk and of lactose, protein and fat. There was no consistent change in milk composition.

The origin of the effects is discussed.

Nuclear magnetic resonance was used to follow the decrease in moisture in curd cooled to solidify the fat as whey was exuded. The results did not correlate closely with those of moisture determinations by drying at 98 °C partly because of the relaxing effect of the solid curd structure when in close proximity to the whey. This effect was diminished as the whey left the curd, so that whey out of the curd was more readily saturated with radio-frequency energy and therefore absorbed less energy than whey in the curd when high intensity fields were used. The saturation effect could also be used to follow approximately the exudation of the whey.

The association of lipase with casein micelles and soluble casein complexes was investigated by gel-filtration on Sephadex G-200 and Sepharose 2B columns which were equilibrated with synthetic milk serum. Gel-filtration indicated that the molecular weight of casein micelles in milk is > 108 whereas the casein in colloidal phosphate-free milk is present as soluble complexes of molecular weight ca. 2×106 containing αs-, β- and κ-casein. The soluble complexes appear to be stabilized in the micelle by colloidal calcium phosphate linkages. On addition of pancreatic lipase to milk, activity was impaired due to binding of the enzyme both to micellar and to soluble casein complexes. The enzyme dissociated from the latter during gel-filtration on Sepharose 2B columns. The binding of lipase to casein was not dependent on the presence of colloidal phosphate and consequently complete micellar structure is not essential for association of lipase with casein. Binding of the lipase to phosvitin did not result in a loss of enzyme activity. Lipases in milk appear to be involved in the equilibrium between micellar and soluble casein. The activity of lipases in milk is apparently influenced by this equilibrium. Some problems encountered in the use of gel-filtration to study the interactions of lipases with caseins are described.

A laboratory technique using glass and stainless steel slides has been developed to study the build-up of milk film on hard surfaces and its removal with detergent-disinfectant mixtures. Soiling and cleansing treatments were of similar duration to those used in practice but were repeated 6 or 7 times a day for 4 days to simulate 12 or 14 days' milking and washing procedures in the farm dairy; no mechanical assistance was used with the cleaning solutions. Soiling was followed immediately by a pre-rinse, wash and final rinse, allowing a short period for drying before further re-soiling. The technique eventually developed involved treatments repeated 18 times, using solutions at 15·5 °C. The weight of milk film and its content of fat, mineral and ‘protein’ were determined.

Under the conditions of the experiments the following were observed.

(1) The deposit left by all treatments consisted mainly of fat with a smaller proportion of protein and still less of mineral matter.

(2) Glass accumulated slightly less deposit than did stainless steel with an aquablast finish.

(3) Removal of milk film was more complete when sodium hypochlorite was added to certain detergent solutions than when the materials were used separately.

(4) With the treatments studied, the use of the detergent solution from a previous treatment as a pre-rinse did not result in a reduction in the residual film.

(5) A final rinse with 0·1% phosphoric acid virtually eliminated the mineral matter from the residual film.

(6) The substitution of a warm (46°C) for a cold (15·5°C) pre-rinse resulted in a reduction of 40% in residual film and the further use of a warm final rinse effected a reduction of 64%.

(7) The addition of 0·05% wetting agent (Lissapol NDB, I.C.I. Ltd.) to the prerinse effected only marginal improvement in the treatments studied.

(8) Omission of the pre-rinse and corresponding lengthening of the detergent treatment had no consistent effect.

A method is described for estimating the growth of Group N streptococci in milk and other media by measuring the 260 nm-absorbing material extracted from the cells with 0·1 M-NaOH at 100°C. A good correlation between the absorbance at 260 nm of the hot alkali extracts and the dry weight of the cells was obtained for cultures of Streptococcus lactis during the logarithmic and early stationary phases of growth.

The effect of layer thickness and orientation, and of pressure, temperature, and disturbance on the drainage rates of continuously made curd were determined. The effects of heat treatment of the milk and adjustments of its pH were briefly examined. An increase in temperature of the curd caused the expected increase in drainage rate as did also the application of pressure. Vertical layers of curd drained more quickly than almost horizontal layers, and keeping curd in whey retarded its drainage. The effects of pH change over the narrow range of interest (6·1–6·5) were relatively small. Pasteurization of the milk had no effect. Disturbance of the curd had the most pronounced effect in increasing its rate of drainage.

Neutral lipids and phospholipids, firmly bound to rennet- or acidprecipitated casein and also to α-, β- and κ-casein, have been detected by histochemical means. On sections of the caseins they appeared as areas of variable shape and size, uniformly distributed. In cheeses they partially disappeared, probably owing to the action of the milk and bacterial lipases. This led to new patterns of distribution of the lipids in the sections.

Lecithin, cephalin, sphingomyelin, cerebrosides (2 spots), ceramides (1 spot), phosphatidylserine and phosphatidylinositol were identified in the phospholipid fraction of the casein by 2-dimensional thin-layer chromatography. In the bound phospholipid fraction of 3 cheeses the same phospholipids were found together with 1 new unidentified spot.

Lactose in the plasma of 12 lactating goats and one cow with healthy udders has been measured by a sensitive and specific enzymic assay. Mean values (±S.E.) for arterial and venous concentrations were 52±11 µM and 70±11 µM respectively. Simultaneous measurement of mammary blood flow, milk yield and milk lactose concentration showed that 2·70±0·25% (mean±S.E.) of the total lactose production passed into the plasma. This rose to 42·7% in a goat when milking was suspended for 46 h. No lactose was found in the plasma of male and non-lactating female goats, but it was detected 4 days before parturition in a nulliparous goat.