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.

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.

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.

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.

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 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.

Casein, whole αs-casein and β-casein were incubated for 3 and 14 h with crystalline rennin, at pH 6·60 and 36 °C, both in phosphate buffer and in milk dialysate. Products obtained from both systems, comprising 30–83% calciumsensitive (Cas) components, gave similar patterns on starch gel electrophoresis. Whole casein and whole αs-casein were not so soluble in milk dialysate as in phosphate buffer. No significant differences in composition were observed between the Cas and the calcium-insensitive (Ca1) products from the same source.

The αs1-component of the Cas product from rennin-treated whole αs-casein had faster gel mobility in comparison to the αs1-component in the Cas product from untreated whole αs-casein. Also, αs1-casein yielded one faster-moving degradation product, while αs2,3,4 appeared unaltered after 14h. The Cas product of rennintreated β-casein also had faster mobility than untreated β-casein and yielded one faster degradation product and several minor ones of slower mobility. Arginine was the only N-terminal amino acid found in the Cas product of both rennin-treated and untreated αs - and β-caseins. The arginine content increased from 3·48 and 4·98 moles/105g to 5·12 and 6·38 moles/105g in the Cas products from rennin-treated β-and αs-caseins, respectively.

The metabolism, of pyruvate by resting whole-cell suspensions of Group N streptococci was studied over the pH range 4·0–7·0, in the presence and absence of oleic acid. In the absence of oleic acid pyruvate was utilized maximally at pH 4·5 with the formation of acetate (volatile acid), acetoin + diacetyl and carbon dioxide. The formation of acetate took precedence over the formation of acetoin + diacetyl. In the presenoe of oleic acid the utilization of pyruvate was maximal at pH 6·5 and completely inhibited at pH 4·5. The only products detected at pH 6·5 were acetoin + diacetyl and carbon dioxide. This effect of oleic acid on the metabolism of pyruvate was also obtained after treating the cells with acetone. The mechanism of action of oleic acid on cells of Group N streptococci and its possible influence on the formation of flavour compounds in cultured dairy products is discussed.

The inactivation of lactoperoxidase (LP) by cultures of Group N streptococci was originally reported by Kiermeier & Kayser (1960). However, Pickering, Oram & Reiter (1962) found that incubation of Streptococcus cremoris 803 with LP did not completely inactivate the enzyme, but only modified its activity with certain substrates. This observation was subsequently confirmed by Kiermeier & Lechner (1966, 1967). In the present investigation, several strains of Group N streptococci were found to modify LP in such a way as to cause total or partial loss of enzymic activity with guaiacol. This loss of LP activity was found to be a consequence of the production of hydrogen peroxide by these organisms.

The nutritional deficiencies of co-precipitate as a growth medium for a strain of Lactobacillus acidophilus have been studied. Acid production by this organism was insignificant in a medium composed of 4% low-calcium co-precipitate and 5% lactose.

Additions of the amino acids phenylalanine, tyrosine, valine, cysteine and threonine as well as of nicotinic acid, manganese, magnesium and citric acid all increased acid production.

When only half the milk solids of skim-milk powder was replaced by the coprecipitate, fortification with manganese only was required.

Four methods of estimating the cell content of bulk milk were studied, correlated statistically and their degree of reproducibility estimated. The relative merits of the 4 methods are discussed, and the need for defining the object of testing before selecting a test is stressed. If designed for use in a programme for mastitis control, or the control of milk quality based on cell content, each country must choose a screening test or a precise cell-counting technique, depending on the availability of laboratory resources, extension advisory services and other economic factors.

Screening tests studied were the California Mastitis Test (CMT) and the Brabant Mastitis Test (BMR), the former showing a correlation coefficient of 0·817 against the Electronic Cell Count (ECC) method in 320 samples. The more objective BMR correlated well with the ECC method, showing a correlation coefficient of 0·987 for 406 samples. Direct microscopic counts (DMC) were correlated with a centrifugal method and with a chemical method of preparing samples for electronic cell counting. For 430 samples in each case the correlation coefficients obtained were 0·930 and 0·966 respectively. The 2 ECC techniques were compared using 430 samples, and a very close relationship was found (r=0·988).

For regular examination of large numbers of milk samples using the electronic cell counter we consider the chemical method of sample preparation more suitable than the centrifugal method. Differences in the reproducibility and accuracy of both tests are negligible, but the former procedure is quicker, more economical in terms of cost, and more adaptable to a national testing programme.

Eight lactating cows were used in a Latin square experiment, to study the associative effects of level of energy and protein intake on milk yield and composition. Four diets were used, supplying 80 and 120% of estimated energy requirements and 80 and 120% of estimated protein requirements. The level of energy intake significantly affected milk yield, milk energy output, percentage butterfat, ash and non-protein nitrogen. The level of protein intake only significantly affected milk energy output and the non-protein nitrogen content of the milk. Although only the interaction of the effects of energy and protein intake on the milk content of solidsnot-fat (SNF) and ash was significant, it was evident that the effect of each of these factors on milk yield or composition was related to the level of the other in the diet.

Input-output relationships within each protein level were used to compute the response in milk energy output and bodyweight change to a change in energy intake. These showed a greater partitioning of additional energy toward milk energy output with the high than with the low protein level. Multiple regression analysis within each level of protein intake was used to partition energy intake between that used for maintenance, milk energy output and liveweight change. The results showed efficiencies of utilization of metabolizable energy for milk output of 63 and 50% on the high- and low-protein diets, respectively.

Nitrogen balance data are presented.

On the basis of complete permeability by high molecular weight reagents of casein micelles in milk and a uniform distribution of the 3 different casein subunits, a model of the micelle structure is proposed. It is composed of an average repeating unit of 1 κ-, 2 αs1;- and β-casein subunits assembled in a 3-dimensional network or branched polymer made of 130–130000 monomers, in which the trimers of κ-casein occupy the nodes and the copolymers of αs1;- and β-caseins make up the branches. All the associations between subunits are through non-covalent bonds. The chemical composition varies with the number of αs1;- and β;-casein subunits in the branches. This proposed structure is strongly supported by evidence from electron microscopy and a scale model has been made. It leads to an understanding of the role of κ-casein in micelle formation and opens new perspectives in explaining some properties of the caseins. It offers an interesting example of a new type of quaternary structure of protein subunits.

A method is described for canning and sterilizing concentrated whole milk containing 36·5% total solids. The product had acceptable organoleptic properties and did not form sediment or undergo age-thickening during storage. The method involved initially increasing the pH of the raw milk and forewarming. The product was not homogenized nor were polyphosphates added.

Electron microscopy was used to study the effect of addition of NaOH, forewarming, homogenization, concentration, and heat sterilization on the structure of whole milk during the production of 4 heat-sterilized concentrates that showed different degrees of physical stability. The samples were prepared for electron microscopy by adding fixative either to liquid or freeze-dried material, and then embedding for sectioning.

Noticeable structural changes occurred during forewarming of samples containing added NaOH, during homogenization, and during heat sterilization. Addition of NaOH and forewarming decreased the closeness of packing of the subunits in the casein micelles. Homogenization reduced the average size of the fat globules, and protein became attached to their surfaces. Heat sterilization caused coalescence of protein. In non-homogenized sterilized concentrate with added NaOH, protein bodies of about 100 times the volume of the original casein micelles were formed; these were free-floating and the concentrate was stable. In non-homogenized sterilized concentrate with no added NaOH the protein bodies were about 15 times the size of the casein micelles and bridged to each other, thereby forming a sediment consisting of large irregular particles. Very large protein bodies containing fat globules formed during heat sterilization of the homogenized samples, both in the presence and in the absence of NaOH, and were responsible for the formation of sediment in these 2 products.

Needle crystals observed in most samples were identifed as CaCO3. H2O by selected area diffraction; KC1 crystals were also detected.

A study has been made of the use of 2,6-dichlorophenolindophenol (DCP) and also of 2,4-dinitrophenylhydrazine (DNPH) with and without chromatography of the DNPH derivatives, for the determination of ascorbic acid (AA) and dehydroascorbic acid (DHA) in evaporated milks, and of AA in AA-fortified sterilized milks.

In the DNPH method, when interfering compounds were removed by the chromatographic separation of the DNPH derivatives, absorption curves typical of the pure DHA derivative were obtained.

The DCP method gave erroneously high values for the AA and DHA content, and the lower values obtained by the DNPH method with chromatography of the DNPH derivatives were judged correct. Thus for an evaporated milk, the values for vitamin C content (AA + DHA) were, in mg/100ml reconstituted milk, 0·88 (DCP method), 0·66 (DNPH method without chromatography), and 0·48 (DNPH method combined with chromatography). The AA contents (mg/100 ml) of the 2 fortified sterilized milks were 3·63 and 5·78 (DCP method); 3·01 and 4·28 (DNPH method combined with chromatography).

The AA contents of the 5 different evaporated milks, determined by chromatography of the DNPH derivatives, ranged from 0·07 to 0·63 mg/100 ml reconstituted milk. The DHA content was negligible.

As judged by the shape of the absorption curves after chromatography of the DNPH derivatives, it is concluded that this method is the most reliable of those studied.

A comparison was made of the effects of direct and of indirect ultra-high-temperature (UHT) processing of milk, under standardized operating conditions giving equal sporicidal effects, on some of the more labile water-soluble vitamins and on vitamin A and carotene. The effects of processing per se were negligibly small, and the method of processing was important only in so far as the presence of residual oxygen in the sterilized milk has been found to cause losses of folic acid and ascorbic acid during storage subsequent to sterilization. The incorporation of a de-aerator vessel, to reduce the oxygen level in the indirectly heated milk and so eliminate the adverse effects of oxygen during storage, had no effect on the vitamin loss occurring during heat treatment. It is concluded that milk produced by indirect heating plant incorporating a de-aerator should be similar in vitamin content to milk produced on a direct heating plant, both immediately after processing and after storage.

The milking characteristics of 4 transparent and 8 conventional rubber teatcup liners in new condition were compared for peak flow rate, machine time and strip yield, using 30 cows in two 15×15 Latin squares. The milking characteristics of transparent liners were similar to those of normal liners at the same cluster weight. The 4 transparent liners were then compared with 4 of the conventional liners using 16 cows in two 8×8 Latin squares, to observe the movement of teats within the liners throughout milking. The various analyses indicated significant differences between liners but the ranked liner mean values showed the transparent liners widely distributed in the range. It was concluded that findings from studies of teatcup action using transparent liners could be expected to apply also to conventional liners.

The conventional liners were selected to form groups differing mainly in one physical characteristic only (stiffness of the mouthpiece, wall thickness, bore and rubber hardness). Within groups, there were no significant differences in milking properties of practical importance, but between groups there were small differences in machine time and strip yield. Two properties of the liners which appeared important in controlling movement of the teat into the liner were bore of the barrel and friction, bore having the greater influence at the beginning and friction being the dominant influence at the end of milking. With the transparent liners it could be seen that the end of the teat was frequently bathed in milk. Both with transparent and with conventional liners it was surprising how often the teat penetrated so deeply that complete collapse of the liner in each pulsation cycle was prevented.