Bulk milk with an apparently high electronic cell count (ECC) was found to have a normal cell count when the latter was determined directly with a microscope or when cell nuclei were counted with a Fossomatic apparatus. Particles in the form of collapsed spheres formed by aggregated casein micelles were found in bulk milk which accounted for the falsely high ECC values. The ECC value agreed with the values obtained by the other methods if the milk was heated to 55 °C for 15 min before fixation. The shape of the particles and the fact that they were only present after the milk had passed through the milking machine suggested that they may be produced as vacuum vacuoles arising from cavitation in the pump.

The concentrations of diffusible Ca and Mg in milk diffusates from 3 goats were determined before, during and after a 2-d period of starvation. Free Ca-concentrations were determined by a murexide method and compared with values calculated from a detailed model of the ion equilibria. It was shown that diffusible Ca increased as citrate increased during fasting and both decreased after refeeding, but there was a difference of about half a day in the response curves, with citrate following Ca. The difference was accounted for mainly by a change in free Ca2+ concentration which in turn was related mainly to changes in milk pH. Diffusible Mg underwent changes similar to those of diffusible Ca.

The manufacture of skim-milk powder with heat stable characteristics was investigated commercially during the course of 8 trials carried out over a 12-month period. Skim-milk was preheated to temperatures ranging from 110 to 130 °C with a 2-min holding time prior to evaporation and drying. The effect of added urea was also examined during each trial run. Heat coagulation times at 120 °C were determined upon reconstitution of the powders to 20% total solids. From February to April the heat stability of the skim-milk powders increased, with the more heat stable samples being obtained at the higher preheating temperatures. Addition of urea increased the heat stability, particularly so in those milks which had been preheated to 130 °C. The presence of added urea during preheating was not found to be necessary as an equal effect on heat stability was observed when urea was introduced at the concentrate stage before drying. Later in the season optimum heat coagulation times were obtained by maintaining preheating temperatures at 118–120 °C.

Urea stabilizes milk to heat only in the presence of a carbonyl compound, such as a reducing sugar. Low molecular weight carbonyl compounds can increase stability in the absence of urea whereas larger molecules, e.g. hexoses and reducing disaccharides, cannot. However, the efficacy of all carbonyl compounds is increased considerably in the presence of urea. Aldehydes and ketones of similar molecular weights appear to be equally effective stabilizers. The optimum concentration of lactose in milk for stability was approximately 1% irrespective of urea concentration within the range 5–20 mM. However, modifying the lactose content of concentrated milks (approximately 8.5% protein) had little effect on heat stability.

Optimum conditions have been determined for the use of Hide Powder Azure (HPA) to estimate extracellular proteinases produced by psychrotrophic Gram-negative bacteria. This assay is quicker, more precise and 3·2 times more sensitive than assays of soluble N released from casein. It provides a simple alternative method for monitoring proteinase purification schemes. With the modification described it is now possible to determine proteinase activities directly in milk. In its simplest form the HPA assay is sufficiently sensitive to detect proteinases produced by as few as 1·5 x 106 c.f.u. ml-1 psychrobtrophs without the necessity for any sample processing.

The 6 most widely used milk-clotting enzymes, i.e. chymosin, bovine and porcine pepsins and proteinases from Mucor miehei, M. pusillus and Endothia parasitica, have been purified and used to prepare rabbit antisera against each of them. The antisera were adsorbed with appropriate cross-linked antigens to remove non-specific antibodies. Monospecific antisera thus obtained were used to identify enzymes eventually added to calf or bovine rennets, using the double radial immunodiffusion technique. The threshold of sensitivity was c. 1%, expressed as clotting activity. This qualitative method complements the quantitative procedure recently proposed for determining chymosin and bovine pepsin A in commercial extracts of bovine veils.

The effects of temperature, dilution, dialysis and the presence of heparin on the stability of lipoprotein lipase (LPL) in milk, skim-milk, milk serum and casein micelles were investigated. At 4 and 20 °C milk serum was the source of the least stable LPL and casein was that of the most stable. There was little difference between LPL stability in milk and skim-milk at these temperatures, or between serum and casein LPL at 50 °C. Heparin (5 µg/ml) increased stability although the effect was less for casein LPL than for serum LPL. A 40-fold dilution of serum LPL with either simulated milk ultrafiltrate (SMUF) or 0·01 M-Tris-Cl pH 8·3 increased the loss of serum LPL, but not of casein LPL. Dialysis of skim-milk against deionized water or SMUF increased stability at 4 or 20 °C but not at 37 °C. LPL activity was more stable in diluted samples of dialysed skim-milk than in diluted samples of the same milk which had not been dialysed. Dialysis against deionized water increased lipolysis but against SMUF it did not increase. Solutions prepared by dialysing water against some milks were found to inhibit lipolysis and this effect was overcome by heparin. The possibility that milk serum contains a factor which influences LPL stability is discussed.

A milk-related model system, 2 mM-trilinolein in 0·1 M-piperazine-N, N′-bis-2-ethanesulphonic acid buffer, pH 6·8, stabilized by lysophosphatidylcholine was used to assess the effect of milk proteins on lipid oxidation in emulsion, catalysed by Cu2+ ions. In some experiments the trilinolein was replaced by sunflower oil (2 g 1-1). Oxidation at 25°C was assessed by measurement of the rate of O2-utilization and by the thiobarbituric acid assay. The features, relevance and reliability of the model are described in terms of the observed effects in the absence and presence of added protein. Casein was antioxidative at concentrations relevant to those in bovine milk, human milk, infant milk formulations and ‘high linoleic acid’ milk. Whole whey protein was a less effective inhibitor of lipid oxidation at similar concentrations. Purified α-lactalbumin was also marginally antioxidative, and its effect was independent of the molar Cu2+: protein ratio between 1:1 and 3:1.

The milk-related model system described by Allen & Wrieden (1982) was used to assess the effects of a number of purified milk metalloproteins, at concentrations relevant to those in milk and dairy products, on triglyceride oxidation in emulsions. Oxidations were monitored by the rate of O2 utilization (ROU) and by the thiobarbituricacid (TBA) assay. The proteins lactoferrin, lactoperoxidase, superoxide dismutase and xanthine oxidase were used either on their own or in conjunction with 10 μM-Cu2+ or Fe2+. Lactoferrin alone had little effect on oxidation, although both the iron-free and iron-saturated protein were able to protect the lipid to some extent from Fe2+-catalysed oxidation. Cu2+-catalysed oxidation was slightly promoted. Lactoperoxidase was pro-oxidative in the absence and presence of added Cu2+ or Fe2+. Heat treatment at 72°C/20 s had little effect, but oxidation was greatly reduced by treatment at 80°C/20 s. Superoxide dismutase strongly reduced the rate of lipid oxidation in the absence of added metals, but had no effect in the presence of 10 μM-Cu2+. xanthine oxidase had little effect on lipid oxidation in the absence of added metals, but was strongly pro-oxidative in the presence of 10 μ-Cu2+. Similar effects were observed down to 1 μM-Cu2+. The enzymic activity of xanthine oxidase was rapidly eliminated by 10 μM-Cu2+, but not by 10 μM-Fe2+. A moderate pro-oxidative effect was observed in the presence of 10 μM-Fe2+. Heat treatment of xanthine oxidase at 80°C/20 s reduced its lipid pro-oxidative effect in the presence of Cu2+ more effectively than treatment at 72°C/20 s.

With either Na caseinate or skim-milk powder, 1% (w/v) additions of pepsin, chymotrypsin or Alcalase and an incubation time of 24 h at 37°C gave the best hydrolysates for subsequent plastein production. In spite of the fact that peptide species which were produced by treatment with the 3 enzymes were entirely different, they were all capable of producing qualitatively similar amounts of plastein when concentrated and further incubated with proteinase, suggesting that the identity of peptide components was relatively unimportant. αs1-, β- and κ- caseins, Na-caseinate and skim-milk powder all led to plastein products with broadly similar properties, confirming the comparative unimportance of peptide composition. This also indicated that for most practical applications fractionation of initial protein mixtures (as in many food products) would not be justified. Optimum peptide concentration for plastein formation with pepsin and caseinate hydrolysates was 20–35% (w/v) with an optimum pH range of 4–5; the best peptide molecular weight varied between 400 and 800 and the optimum temperature was either 37°C for 24 h or 50°C for 4–6 h. Higher temperatures (70°C) gave more rapid plastein formation but poorer yields. Lower temperatures (e.g. 20°C) gave similar yields, but incubation times required to be extended to at least 48 h. The same proteinase should be used for plastein synthesis as that used in the initial hydrolysis stage, or else further hydrolysis (over hydrolysis) could occur during the plastein formation stage causing lower yields due to differences in the specificity of the 2 enzymes. Even under ideal conditions plastein yields never exceeded 27% of the weight of peptide taken and at the present time it seems unlikely that the plastein reaction will have anything more than curiosity value. Economics must preclude its application in low-cost, high volume situations such as generally exist within the food industry.

Chemical and physical properties of plasteins derived from caseins and skim-milk powder have been investigated. Plasteins were very similar in properties regardless of starting material or of the proteinase used in their production. Plastein formed from Na caseinate hydrolysate at pH 5 was stable over the pH range 1·5–9·0 and had the properties of a thixotropic gel. More extreme pH values caused dissolution as did various chaotropic reagents. 8 M-urea disrupted plastein structure almost entirely, as did various organic acids at concentrations of 50% (v/v) and strongly ionic detergents. Non-ionic or weakly cationic detergents and 6 M-guanidine hydrochloride were less effective. Treatment with strong salt solutions, Ca2+ and EDTA had no effect. Gel filtration experiments demonstrated that plasteins were completely disrupted under dissociating conditions and peptide mapping confirmed that all the peptides originally present in the hydrolysate were also present in plastein. Amino group contents were unchanged on plastein formation. These experiments together with gel electrophoresis studies clearly showed that only hydrophobic and ionic bonding mechanisms were involved in plastein formation and no detectable peptide bond formation or transpeptidation occurred. In accordance with this, amino acid analysis suggested that hydrophobic peptides were preferentially incorporated. Implications for use of plasteins in food products are discussed.

Commercial bulk milk contained, in its acid-soluble nucleotide fraction, small quantities of CMP and AMP as well as a high proportion of orotate (503±16 μg/g of total solids). These components were altered by thermal treatment during processing. Standard sterilization and spray drying of milk lowered significantly the CMP, AMP, and orotate concentrations. HTST pasteurization caused a slight decrease in CMP and AMP concentrations, but the orotate content remained unchanged. The UHT sterilization seemed to be the thermal treatment in which the acid-soluble nucleotides of milk underwent least change. It is therefore concluded that the decrease in concentration of the nucleotides of milk is more directly related to the time for which the milk remains at the processing temperature than to the temperature itself.

The acid-soluble ribonucleotides of human milk were measured by enzymic and ion-exchange chromatographic procedures at different stages of lactation. Human colostrum and milk contained CMP, AMP, GMP, UMP, GDP-mannose, UDP-N-acetyl-glucosamine, UDP-N-acetyl-galactosamine, UDP-glucose, UDP-galactose, UDP and other minor nucleotides. Cytidine and adenosine derivatives were present in relatively higher amounts than in milk from ruminant species. The nucleotide concentration decreased with advancing lactation. Human milk at 3 months of lactation contained about 10 µmol/100 ml nucleotides, which represented about 75% of the amount of nucleotides present in human colostrum. Human milk did not contain orotic acid. The UDP-glucose/UDP-galactose ratio was constant during lactation and was similar to that of milks from ruminant species.

The efficiencies of small capacity homogenizing valves of the high pressure type have been studied in relation to their design. The performance of a valve was strongly affected by the magnitude of the effective inlet diameter D0 of the seating for a given exit diameter D when D0/D > 0·5. The indications are that narrow mating faces situated such that D0/D → 1 should maximize performance. Milk fat globule dispersion varied regularly with a valve's face angle; optimum angles for maximum disruptive effect were about +35° and –30°. Perforated metal insets interposed between flat faces of a valve piece and its seat noticeably improved fat dispersion; reductions in mean globule diameter of up to 25% were obtained.

Variations in the sensitivity of different milks to homogenization were observed but avoided by appropriate experimental procedures.

The performance characteristics of a valve accepting a high rate of milk flow and operating at Reynolds’ numbers (Re) of 6000–15000 have been studied. Features of valve design which improved homogenizing efficiency were identified. As with small valves operating at lower Re numbers, efficiency increased as D0/D increased beyond 0·5; for D0/D ≈ 0·7, a 15% reduction in mean globule size was obtained. Changes of face angle up to 45° had virtually no effect on milk fat globule dispersion. The use of impact rings produced only small improvements in dispersion. The incorporation of perforated insets led to substantial globule size reductions of 15–25% or the equivalent in reductions of homogenizer energy consumption of 12–35%.

The Limulus test was used to investigate the occurrence of Gram-negative bacterial lipopolysaccharide (LPS) in milk and dairy products. This test specifically detects the extremely heat-resistant LPS, and so determines the total accumulated quantity of LPS in a product whether it has been heat-treated or not. A positive correlation between number of psychrotrophic bacteria and amount of LPS in fresh farm milk was found (r = 0·63). Fresh farm milk and freshly produced market milk contained on average < 1 ng LPS/ml. The Limulus test is considered highly suitable for use in the dairy industry, being a method which will characterize quickly and specifically the hygienic level of a product by the extent of the cumulative contamination by Gram-negative bacteria.

In this method, the curd, formed in a beaker, was retained after cutting by means of a wire grid placed on top of it. This enabled the whey to be poured off easily at timed intervals for measurement of its volume. The method gave results reproducible within 1·5%. Increasing the amount of rennet added to the milk increased the rate of syneresis of the curd slightly. Adding CaCl2 to the milk, raising the temperature and decreasing the pH, all increased the rate of syneresis. Addition of fat, as cream, to skim-milk decreased the rate of syneresis proportionally. The rate of syneresis was decreased if the milk was concentrated by ultrafiltration and increased if the milk was diluted with permeate. In all instances the early stages of syneresis followed first-order kinetics, the rate depending on the amount of whey remaining within the curd. The later stages of syneresis appeared to depend on hydrophobic forces. Probably, the increasing aggregation of the casein within the curd caused the whey to be squeezed out.

Cephalotyre (Ras) cheese slurries developed within 7 d of storage at 30 °C a flavour similar to that of conventional Ras cheese ripened for 2 months. Addition of a proteinase/lipase mixture, trace elements or Na citrate to fresh cheese slurries before storage at 30 °C enhanced flavour development. Additive-treated slurries showed after storage for 5 d at 30 °C a strong flavour similar to that of 3–4 months Ras cheese with increased protein degradation, acidity development, volatile fatty acid formation and higher bacterial counts. The effectiveness of the additives in enhancing flavour development and related chemical changes was in the order: proteinaseòlipase mixture > trace elements > Na citrate.