Discontinuous PAGE at alkaline and acid pH, polyacrylamide gel isoelectric focusing, two dimensional electrophoresis and immunoblotting have been used to study the heterogeneity of sheep caseins. Three main phenotypes were selected either because of mobility at alkaline and acid pH of the individual αs components or because of their relative intensity. On electrophoresis at alkaline pH, one phenotype showed two distinct bands of lower electrophoretic mobility than β,- and β2-caseins. A detailed study of these components using immunospecific detection with β-casein antiserum showed that these minor components of ovine casein may have a polypeptide chain similar to that of β1- and β2-caseins. Complete electrophoretic patterns of the casein components in some individual milks are also presented.

The degradation of bovine β-casein by plasmin was used as a model system to investigate the applicability of an enzyme recycle reactor for the continuous production and isolation of peptide fractions. A simple, ultrafiltration-type membrane reactor was used incorporating a commercially available hollow-fiber module normally employed for dialysis purposes (artificial kidney). The results were compared with those obtained in a batch reactor process, using HPLC to monitor the course of proteolysis.

Peptide fragments were isolated and were characterized using amino-acid analysis and N-terminal end-group determination. Sixteen peptide fragments were identified, which together accounted for virtually all the potential cleavage sites in β-casein. In the system studied the N-terminal half of β-casein appeared to be more sensitive to plasmic hydrolysis than the rest of the molecule.

Forty-four goats were milked and milk flow recorded without or with 1 min manual prestimulation in early, mid and late lactation. Ultrasound measurements of cross sections of the whole mammary gland were performed in a water bath. In additional experiments with 15 goats, milk flow was recorded and frequent blood samples were taken for the determination of oxytocin and prolactin concentrations. Milk yield increased from the first to the third lactation and decreased markedly during the course of lactation. Average and peak milk flow rates were closely related to the actual milk yield. The ultrasound cisternal area was 27·4±1·5% of the entire udder half cross section. Milking characteristics were scarcely different without or with prestimulation, although oxytocin was released within 30 s after the start of prestimulation, whereas oxytocin concentrations without prestimulation increased only after the start of milking. Concentrations of prolactin were higher during July and August than in April, and increased similarly with or without prestimulation during milking. In contrast to dairy cows, prestimulation and an opportune release of oxytocin during milking does not significantly influence the course of milk flow in goats, and this is probably because large amounts of cisternal milk allow milk ejection to be induced only after the start of milking without causing bimodal or otherwise reduced milk flow.

Pulsation rates of 40, 60 and 80 cycles/min were combined with pulsator ratios of 50, 60, 70 or 80% in two experiments with different liners. Machine-induced, short-term changes in teat thickness of 14 cows were compared with milk flow rate characteristics and machine strip yields to evaluate the effectiveness of pulsation in relation to liner type. Post-milking teat thickness increased progressively as the b phase of the pulsation cycle was lengthened, and as the d phase was shortened, at all pulsator rates and with either liner. Teat thickness values increased significantly (P < 0·05) when the d phase was <15%. For ratios of 50 and 60%, teat thickness decreased progressively as pulsation rate was increased. At the higher ratios, thickness values were lowest at 60 cycles/min. Pulsation settings that tended to increase teat thickness values also increased both peak milk flow rates and machine strip yields. The influence of liner type on teat thickness changes appeared to be at least as important as the influence of pulsator ratios and greater than the effect of pulsation rate. If so, then international standards for acceptable pulsation characteristics cannot be defined solely in terms of pulsator settings. A limit of ±5% for machine-induced changes in thickness of the teat apex would be an additional guideline for effectiveness of pulsation in relation to both liner type and vacuum level. This could provide a basis for a dynamic test applied to milking cows under field conditions.

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.

Citrate utilization and diacetyl, acetoin and acetaldehyde production by 2 strains each of Leuconostoc cremoris and Streptococcus diacetilactis in milk were studied. With the leuconostoc bacteria no growth and little citrate utilization occurred unless a stimulant (yeast extract) was present, when complete utilization of citrate without concomitant production of diacetyl or acetoin was obtained. The addition of Mn2+ stimulated growth and citrate utilization in the presence of yeast extract. Addition of citric acid after 65-h growth resulted in diacetyl and acetoin production. Destruction of diacetyl and acetoin occurred when the citric acid level fell to c. 1000 and 600 μg/g in the case of Leuc. cremoris FR8-1 and CAF1, respectively. Only strain FR8-1 produced acetaldehyde. In contrast, Str. diacetilactis produced diacetyl, acetoin and acetaldehyde concomitant with citrate utilization.

Lactuloselysine (ε-N-deoxylactulosyl-L-lysine) and fructoselysine (ε-N-deoxyfructosyl-L-lysine), formed by Maillard reaction, were identified in enzymic hydrolysates of casein from stored ultra-heat-treated (UHT) milk. Furosine (ε-N-(2-furoylmethyl-L-lysine) and pyridosine (ε-(3-hydroxy-4-oxo-6-methyl-1-pyridinyl)-L-norleucine) were identified in the corresponding acid hydrolysates. Enzymic hydrolysis of the caseins, gel filtration and preparative scale amino acid analysis were used to isolate the compounds which were then identified by reference to synthesized authentic compounds. Lactuloselysine formation was extensive and involved 10–30% of lysine residues in UHT milks stored at 30–37 °C for 6 months to 3 years. Fructoselysine concentration was generally about 10% of the lactuloselysine concentration.

To study the possible presence of staphylococcal enterotoxin A in raw goats' milk lactic cheese, milk was inoculated with an enterotoxigenic Staphylococcus aureus strain to a final concentration of 4, 5 and 6 log(cfu/ml). Cheese was prepared following industrial specifications and ripened for 42 d. Detection of the enterotoxins was by the Vidas Staph enterotoxin test (BioMérieux) and by an indirect double-sandwich ELISA technique using anti-enterotoxin monoclonal antibodies. Staphylococcal counts declined markedly after draining, and by the end of ripening they had disappeared from some cheeses. In contrast, aerobic mesophilic organisms grew well. The level of staphylococcal enterotoxin A recovered varied from 1 to 2·5 ng/g cheese made with an initial population of 105 or 106 cfu/ml. Only traces of enterotoxin A (0·5 ng/g) were detected in cheeses made with the lowest Staph. aureus inoculum used in this study. Enterotoxin A was also detected in cheeses from which Staph. aureus had disappeared.

Mastitis is one of the most prevalent diseases of cattle. Various studies have reported breed-dependent differences in the risk for developing this disease. Among two major breeds, Jersey cows have been identified as having a lower prevalence of mastitis than Holstein cows. It is well established that the nature of the initial innate immune response to infection influences the ability of the host to clear harmful bacterial pathogens. Whether differences in the innate immune response to intramammary infections explain, in part, the differential prevalence of mastitis in Holstein and Jersey cows remains unknown. The objective of the current study was to evaluate several parameters of the innate immune response of Holstein and Jersey cows to intramammary infection with Staphylococcus aureus, a common mastitis-inducing pathogen. To control for non-breed related factors that could influence these parameters, all cows were of the same parity, in similar stages of milk production, housed and managed under identical conditions, and experimentally infected and sampled in parallel. The following parameters of the innate immune response were evaluated: acute phase protein synthesis of serum amyloid A and lipopolysaccharide-binding protein; total and differential circulating white blood cell counts; milk somatic cell counts; mammary vascular permeability; milk N-acetyl-beta-d-glucosaminidase (NAGase) activity; and production of the cytokines, interferon (IFN)-γ, interleukin (IL)-12, tumour growth factor(TGF)-α, and TGF-β1. The temporal response of all of these parameters following infection was similar between Holstein and Jersey cows. Further, with the exception of changes in circulating neutrophils and NAGase activity, the overall magnitude of these parameters were also comparable. Together, these data demonstrate that the innate immune response of Holstein and Jersey cows to Staph. aureus intramammary infection remains highly conserved despite previously reported differences in mastitis prevalence, as well as genotypic and phenotypic traits, that exist between the two breeds.

Pressure processing of foodstuff has been applied to produce or modify proteinaceous gel structures. In real pressure processing the treatment is non-isothermal, due to the adiabatic nature of the process and the heat loss from the product to the vessel. In order to estimate the effect of pressurization on milk constituents pressure and temperature dependent kinetics were determined separately from each other. In a detailed kinetic study whey protein isolate was treated under isobaric (200 to 800 MPa) and isothermal conditions (−2 to 70 °C), and the resulting degree of denaturation of β-lactoglobulin A and B and α-lactalbumin was analysed. Kinetic parameters of denaturation were estimated using a one step non-linear regression method which allowed a global fit of the whole data set. The isobaric isothermal denaturation of β-lactoglobulin and α-lactalbumin was found to follow third and second order kinetics, respectively. Isothermal pressure denaturation of both β-lactoglobulin fractions do not differ significantly and were characterized by an activation volume decreasing with increasing temperature from −10 to about −30 ml mol−1, which demonstrates that the denaturation rate is accelerated with increasing temperature. The activation energy of about 70 to 100 kJ mol−1 obtained for β-lactoglobulin A and B is not dependent to a great extent on the pressure which indicates that above 200 MPa denaturation rate is limited by the aggregation rate while pressure forces unfolding of the molecule.

1. Spray-dried (Kestner process), roller-dried and evaporated milks were prepared commercially from one batch of raw milk.

2. The biological value of the proteins (nitrogen), vitamin B1 and the growth-promoting properties of the milks were measured by tests on rats.

3. Vitamin A and carotene, riboflavin and vitamin C were measured by physical and chemical methods at intervals in the course of storage for a year.

4. No significant difference was found between the biological values of the proteins (nitrogen) of the processed milks. The values ranged from 80 for evaporated milk to 84 for spray-dried milk.

5. The proteins (nitrogen) of evaporated milk were significantly less digestible (true digestibility 91) than those of spray-dried milk (true digestibility 94). The difference between either of those and roller-dried milk (true digestibility 93) was not significant.

About 90% of the Zn in bovine skim milk was sedimented by ultracentrifugation at 100000 g for 1 h. About half of the non-sedimentable Zn was non-dialysable, indicating that it was associated with protein, probably non-sedimented casein micelles. Casein micelles incorporated considerable amounts of Zn added to skim milk as ZnCl2, and at Zn concentrations ≥ 16 mM coagulation of casein micelles occurred. Ca was displaced from casein micelles by increasing ZnCl2 concentration and ˜ 40% of micellar Ca was displaced by 16 mM-ZnCl2. Micellar Zn, Ca and P1 were gradually rendered soluble as the pH of milk was lowered and at pH 4·6 > 95% of the Zn, Ca and P1 were non-sedimentable. These changes were largely reversible by readjustment of the pH to 6·7. About 40% of the total Zn in skim milk was non-sedimentable at 0·2 mM-EDTA and most of the remainder was gradually rendered soluble by EDTA over the concentration range 1–50 mM. This indicates that there are two distinct micellar Zn fractions. No micellar Ca or P1 was solubilized at EDTA concentrations up to 1·0 mM, indicating that both colloidal calcium phosphate (CCP) and casein micelles remained intact under conditions where the more loosely bound micellar Zn fraction dissolved. Depletion of casein micelles of colloidal Ca and P1 by acidification and equilibrium dialysis resulted in removal of Zn, and in colloidal Pi-free milk non-dialysable Zn was reduced to ·-2 mg/1 (˜ 32% of the original Zn). Thus, ˜ 32% of the Zn in skim milk is directly bound to caseins, while ˜ 63% is associated with CCP. Over 80% of the Zn in colloidal Pi-free milk was rendered soluble by 0·2 mM-EDTA, indicating that the casein-bound Zn is the loosely bound Zn fraction in casein micelles. A considerable fraction of the Zn in acid whey (pH 4·6) co-precipitated with Ca and Pi on raising the pH to 6·7 and heating for 2 h at 40 °C, indicating that insoluble Zn phosphate complexes form readily under these conditions. Studies on dialysis of milk against water, or dilution of milk or casein micelles with water, showed that CCP and its associated Zn is very stable and dissolves only very slowly at pH 6·6. The nature of Zn binding in casein micelles may help to explain the lower nutritional bioavailability of Zn in bovine milk and infant formulae compared with human milk.

The major whey proteins IgG, serum albumin and α-lactalbumin were purified from camel milk using gel permeation and ion-exchange chromatography. Specific antisera against each of them were raised and used to quantify their heat denaturation in early or mature milk over a range of 60–90 °C for 10–60 min using the single radial immunodiffusion technique. The heat denaturation midpoints for the mature milk heated 30 min were 67·2, 73·0 and 77·5 °C for IgG, albumin and α-lactalbumin respectively. The early milk was more sensitive to heat treatment with coagulation at low temperature and heat denaturation midpoints of 64·8, 71·6 and 72·6 °C respectively. This difference was related to the high IgG content of the early milk (12·6 mg/ml v. 0·5 mg/ml for the mature milk) and stresses the importance of monitoring the IgG level of milk to assess the absence of colostrum.

Male dairy calves may be transported from their farm of origin at a young age. This process may involve an extended period off feed and indirect consignment through an intermediate facility, prompting potential welfare concerns. To assess the impact of transport, 59 male Holstein-Friesian dairy calves (5–9 d old) were either (1) held in situ on farm (control); (2) transported for 6 h; (3) transported for 12 h; or (4) transported for 1 h to a holding facility where they were kept for 6 h and then transported for 5 h. All treatments included a 30-h period of feed (milk) withdrawal, and calf responses were measured over time from before their last feed until the completion of the study after the transport and feed withdrawal periods. Apart from increases in serum creatine kinase in calves transported for 12 h, transported calves generally did not differ in blood concentrations of glucose, beta-hydroxybutyrate, lactate, total protein or in packed cell volume, compared with controls (P>0·05). Calf responses to the indirect consignment treatment did not differ from those of other transported calves. Withdrawal of feed for 30 h caused calves to lose 6% of body weight; blood glucose varied from 3·96 mmol/l immediately before daily feeding to 5·46 mmol/l at 3 h post feeding, and then declined to 3·43 mmol/l at 30 h. Calves lay down for 22–32% of the time during transport, and did not show a rebound effect in lying behaviour post arrival in comparison with controls. Best practice transport of 6–12 h duration, including indirect consignment via a holding facility, did not significantly affect calf blood biochemistry and metabolism in comparison with untransported animals. However, extending the time off feed beyond the daily feeding interval resulted in reduced blood glucose concentrations, suggesting that time off feed needs to be carefully managed in young transported dairy calves.