This work describes the effect of the hydrolysis time and pressure (0·1–400 MPa) on the proteolysis of β-lactoglobulin A (β-lg A) with trypsin, either conducting hydrolysis of β-lg under pressure or hydrolysing β-lg that was previously pressure treated. Pressurisation, before or during enzyme treatments, enhanced tryptic hydrolysis of β-lg. Trypsin degraded pressure-modified β-lg and pressure-induced β-lg aggregates, favouring proteolysis to the intermediate degradation products: (Val15-Arg40), (Val41-Lys69)S-S(Leu149-Ile162) and (Val41-Lys70)S-S(Leu149-Ile162). These were further cleaved at the later stages of proteolysis to yield: (Val15-Tyr20), (Ser21-Arg40), (Val41-Tyr60), (Trp61-Lys69)S-S(Leu149-Ile162) and (Trp61-Lys70)S-S(Leu149-Ile162). Particularly, in the tryptic hydrolysates of pre-pressurized β-lg, two other fragments linked by disulphide bonds: (Lys101-Arg124)S-S(Leu149-Ile162) and (Tyr102-Arg124)S-S(Leu149-Ile162), were found. These corresponded to rearrangement products induced by SH/SS exchange between the free thiol group of Cys121 and Cys160, that normally forms the disulphide bond Cys66-Cys160. In the light of these results, structural modifications of β-lg under high pressure are discussed.

Experiments were designed to determine the cisternal milk during machine milking in unfamiliar surroundings, which has previously been shown to inhibit milking-related oxytocin (OT) release and milk ejection. The first experiment was performed with 22 cows in early, mid and late lactation. After cisternal milk was removed, 10 i.u. OT were twice injected intravenously to remove the remaining milk. Total milk yield, cisternal milk yield, cisternal milk fraction (17 and 12% in early and late lactation) and the milk yield obtained in response to the first OT injection significantly decreased from early to late lactation. Cisternal milk yield was similar in front and rear udder halves. However, owing to higher total yield, the cisternal milk fraction was lower in rear than in front halves. Cisternal milk yield and cisternal milk fraction were smaller in primiparous than in older cows. In a further experiment milk distribution in the bovine udder was evaluated after preceding teat stimulation and milk ejection. Teats of 12 cows were manually stimulated for 1 min at 15, 60 or 120 min before milking. Total milk yield was not significantly different with or without teat stimulation. However, cisternal milk yield and fraction were significantly higher in teat stimulated cows than in unstimulated controls but were similar whether cows were stimulated 15, 60 or 120 min before milking.

A total of 116 molecular species of triglycerides were identified in milk fat, using a combination of HPLC and GLC. Triglyceride composition was predicted from the random composition, which was calculated on the basis of the mole fractions of the main fatty acids making up the total triglyceride fraction. The qualitative composition of the milk fat was similar in cows', ewes' and goats' milk. In all three milks the partition number of the main triglycerides was 46, but the proportions of the triglycerides with partition numbers of 34, 38, 42, and 48 exhibited substantial differences among the milks of the three species.

We studied the mammary immune response to different mammary pathogenic Escherichia coli (MPEC) strains in cows, hypothesising that the dynamics of response would differ. E. coli is a major aetiologic agent of acute clinical bovine mastitis of various degrees of severity with specific strains being associated with persistent infections. We compared challenge with three distinct pathogenic MPEC strains (VL2874, VL2732 and P4), isolated from different forms of mastitis (per-acute, persistent and acute, respectively). A secondary objective was to verify the lack of mammary pathogenicity of an environmental isolate (K71) that is used for comparison against MPEC in genomic and phenotypic studies. Twelve cows were challenged by intra-mammary infusion with one of the strains. Cellular and chemokine responses and bacterial culture follow-up were performed for 35 d. All cows challenged by any of the MPEC strains developed clinical mastitis. Differences were found in the intensity and duration of response, in somatic cell count, secreted cytokines (TNF-α, IL-6 and IL-17) and levels of milk leucocyte membrane Toll-like receptor 4 (TLR4). A sharp decrease of TLR4 on leucocytes was observed concomitantly to peak bacterial counts in milk. Intra-mammary infusion of strain K71 did not elicit inflammation and bacteria were not recovered from milk. Results suggest some differences in the mammary immune response to distinct MPEC strains that could be correlated to their previously observed pathogenic traits. This is also the first report of an E. coli strain that is non-pathogenic to the bovine mammary gland.

Milk flow in nine primiparous cows with disturbed milk ejection (D) and in six corresponding control animals (C) with normal milk removal was recorded during machine milking and blood samples were taken before and during milking to determine plasma oxytocin, vasopressin. prolactin, cortisol, oestradiol-17β, luteinizing hormone, progesterone and β-endorphin concentrations. Manual teat stimulation before milking lasted for 1 min. After milk flow had stopped, air was blown into the vagina for 2 min. When milk flow had stopped again, 1 i.u. oxytocin and finally 10 i.u. oxytocin were injected to remove residual milk. During and after teat stimulation, oxytocin remained basal in D, but increased in C, whereas prolactin increased in both groups. While 94% of total milk was obtained in C during this period, only 9% could be removed from D, indicating lack of alveolar milk ejection. During vaginal stimulation, oxytocin increased transiently in D and more than by teat stimulation in C. This allowed the removal of 75% of milk in D, whereas almost no more milk was available in C. After oxytocin injections, 3 and 16% of residual milk were obtained in C and D respectively. Basal oestradiol-17β concentration was higher in D than in C (11·6 and 2·0 ng/1 respectively), whereas β-endorphin level was lower (24·1 and 86·6 μg/1 respectively). Basal concentration of luteinizing hormone and progesterone, and concentration of cortisol and vasopressin before and during milking were comparable in C and D. We conclude that in cows with disturbed milk ejection afferent nervous pathways to the hypothalamus were intact, because prolactin was released by teat stimulation. However, oxytocin was only released by vaginal stimulation, i.e. milk ejection was centrally inhibited during teat stimulation.

The internal arrangement of the mammary gland cavity system, cisternal and alveolar milk fractions and the characteristics of milk ejection were investigated in buffaloes. Twenty-four Murrah buffaloes in three different stages of lactation and of two age groups were used. Continuous ultrasound cross-sections during milk ejection induced by exogenous oxytocin were performed to record the latency period of milk ejection. Buffaloes had small cisterns and the cavity area in the teat and gland regions were not significantly different (P>0·05). The animals had long teat canals (3·1±0·1 cm), longer in the hind than fore quarters. Cisternal milk yield was low (0·17±0·01 kg) and cisternal fraction was only 4·9±0·1% of the total milk. The cisternal area (cm2) was 69·6±4·6, 51·61±4·8 and 26·01±4·8 while the cisternal yield (kg) was 0·32±0·05, 0·18±0·05 and 0·05±0·05 in early, mid and late lactation, respectively. A close correlation (r=0·87, P<0·05) existed between the ultrasound cisternal area and cisternal milk yield. The latency period of induced milk ejection was similar to that reported for cows (25±1 s) and was negatively correlated with milk yield (r=−0·75, P<0·05). Milk ejection occurred shortly after elevated oxytocin concentrations were present. Delayed milk ejection reported earlier in this species must therefore be due to the absence of cisternal milk and delayed oxytocin release. An increase in teat length and circumference at milk ejection was also evident in the ultrasound cross sections.

A tributyrin esterase was purified from Lactococcus lactis subsp. cremoris E8 using FPLC chromatography. This was the major esterase activity observed in strain E8 and was associated with a single protein with a subunit molecular mass of 29 kDa and a holoenzyme of molecular mass 109 kDa. The enzyme was active against tributyrin and p-nitrophenyl butyrate. The N-terminal sequence of the enzyme was determined. The enzyme had a pH optimum in the neutral range, was stable on freezing at −20 °C, and had a half life of 1 h at 50 °C.

The physicochemical characteristics and the renneting properties of milks from Lacaune ewes are reported. The ewes belonged to divergent genetic lines termed ‘low’ or ‘high’ and were selected on a combination of fat and protein yield. The 179 second to fifth parity ewes were sampled three times during lactation at about 50, 135 and 175 d in milk. Physicochemical and renneting variables were principally influenced by days in milk and somatic cell count, whereas β-lactoglobulin genotype, parity and divergent lines had relatively few effects. Fat and protein content increased throughout lactation, whereas rennet gel firming rate decreased. Salt distribution and micellar characteristics were also significantly affected by days in milk. Milks with high somatic cell count were characterized by high values of pH and soluble nitrogen content and rennet coagulation properties were significantly affected at >3×105−5×105 cells/ml milk. Divergent lines had a strong effect on milk yield, but no effect on milk composition consistent with the selection criteria. Gel firming rate was slightly higher in milks from high yield ewes. Protein content, Ca concentration, pH and micelle characteristics were the main factors related to variations in the renneting properties of the milks.

In dairy cows the period of transition from late gestation to early lactation is recognized as inducing considerable metabolic adaptation. The aim of this study was to analyse modifications in serum protein values occurring during the dry and the transition period and during lactation in a group of five Holstein cows of high average milk production. For all subjects, selected on the basis of their pregnancy status, blood samples were collected at different physiological phases: dry period (−60, −30 d to calving), transition period (almost 7 d to calving, 7 d after calving), and lactation (weeks 2, 5 and 15 after calving), for a total of eight blood samples for each cow. On each blood sample total proteins and electrophoresis analysis were performed. On the data obtained, normally distributed (P<0·05, Kolmogorov-Smirnov's test), one-way Repeated Measure Analysis of Variance (ANOVA), was applied to evaluate the influence of different stages of gestation and lactation on the considered parameters. Results showed a significant effect on total proteins, α1-globulins, β-globulins, γ-globulins and albumin/globulin ratio. Most of the detected modifications were related to the transition from gestation to lactation, indicating that it is a period of great metabolic stress for cows. On the basis of the obtained results we can affirm that the pattern of serum protein fraction rn could give information about dehydration, plasma volume expansion and hepatic function occurring during the peripartum period in dairy cows.

Considerable inhibitory antibacterial actions were exerted by the soybean 11S subunit comparable with nisin on the proliferation of total viable count, Pseudomonas count and Enterobacteriaceae count in bovine milk stored at 4 or 25 °C for 30 d and 48 h, while 7S and lysozyme were much less effective. The maximum magnitudes of bacterial reduction by 11S and nisin were in the range 2–4 log CFU/ml. The proliferation of 3 pathogenic bacteria (Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli O157:H7) artificially inoculated into raw milk stored at 4 or 25 °C were particularly and significantly (P<0·05) reduced by 11S subunit and nisin (0·5% w/v), but only slightly by 7S and moderately by lysozyme. Lactose consumption, acidity development and casein degradation during storage of bovine raw milk were attenuated during storage at 4 or 25 °C and sensorial traits were better maintained by supplementation with 11S (0·5% w/v). 11S subunit may be used a safely food preservative, if permitted.

1. The sensitivity to calcium of the caseinate-phosphate complex of milk heated at 120°C., increases to an early maximum and thereafter steadily declines, apparently in consequence of parallel changes in the sensitivity of its caseinate constituent.

2. A simultaneously developed capacity of the heated complex to bind additional colloidal phosphate appears to be unrelated to these changes in casein sensitivity.

3. Colloidal phosphate content is confirmed as a factor in the heat coagulation of milk.

4. Heat-developed acidity contributes to the heat coagulation of milk primarily by increasing hydrogen-ion concentration and only slightly, if at all, through release of calcium ions from insoluble combinations.

5. Neither serum proteins nor protein-lactose combinations play any significant part in the heat coagulation of milk.

6. The bearing of these results on the heat coagulation of milk in general and on the operation of certain high-temperature stabilization treatments is discussed.

Inhibition of milk ejection in cows by oxytocin receptor blockade (Atosiban) and α-adrenergic receptor stimulation (phenylephrine) prior to prestimulation was compared with inhibition of milk ejection in unfamiliar surroundings. In addition, Atosiban and phenylephrine were administered after a 1 min prestimulation or 1 min after the start of milking. Oxytocin concentrations increased during milking in all treatments. The spontaneously removed milk fraction (before oxytocin was injected) was similar for Atosiban and phenylephrine treatments and in unfamiliar surroundings, but lower than in controls. Peak flow rates were similar in all treatments, but reduced as compared with controls when phenylephrine and Atosiban were administered before prestimulation. Peripheral (Atosiban, phenylephrine) and central (unfamiliar surroundings) inhibition of milk ejection reduced the amount of available milk similarly. Drug treatments resulted in similar peak flow rates; however, teats were contracted after phenylephrine administration but not after Atosiban. The inhibition induced by Atosiban could be abolished by oxytocin injection, but not that induced by phenylephrine, which was antagonized by α-adrenergic receptor blockade. These results indicate that inhibition of milk ejection through activation of α-adrenergic receptors is based on blockade of milk flow into the cistern, but not through the teats.

The cheesemaking properties of recombinant chymosin from Escherichia coli were compared with those of standard rennet in parallel trials with three single strain starters. For each pair of cheeses the cheesemaking characteristics, recoveries of milk solids in the curds and compositions were similar. The rates and types of proteolysis and texture development during ripening between cheeses made with the two coagulants were not significantly different.

The main compounds responsible for the aroma of bovine, ovine, caprine and water buffalo freshly secreted milk have been identified by means of a gas chromatography–olfactometry technique. Of the fourteen odour-active volatile compounds detected, eight were present in all milks studied. Ethylbutanoate and ethylhexanoate (fruit-like aroma), among the neutral odorants, were the major contributors to the odour of bovine, ovine and caprine milk. The aroma of water buffalo milk was less dependent on ethylhexanoate and was also due to l-octen-3-ol (aroma of raw mushrooms), nonanal (freshly cut grass), indole (stable, animals) and an unidentified constituent, characterized by a retention index of 828, with a typical odour of warm milk and/or smoked cheese. Other aroma components were specific for some types of milk, contributing to the complexity and richness of flavour.

The objective was to compare the prevalence of subclinical mastitis (SM) and of udder pathogens in 60 Swiss organic (OP) and 60 conventional production systems (CP). Cows (n=970) were studied for SM prevalence and udder pathogens at median 31 d and 102 d post partum. Cows showing a [ges ]1+ positive California Mastitis Test (CMT) in at least one quarter were considered to have SM. Cow-level prevalences of SM for visits at 31 d and 102 d post partum (39% and 40% in OP and 34% and 35% in CP) were similar, but quarter-level prevalences of SM were higher (P<0·02) in OP than CP (15% and 18% in OP and 12% and 15% in CP). Median somatic cell counts in milk at 31 d post partum were higher (P<0·05) in OP than CP cows (43000 and 28000 cells/ml, respectively), but were similar at 102 d post partum in OP and CP cows (45000 and 38000 cells/ml, respectively). In milk samples from quarters showing a CMT reaction [ges ]2+ the prevalences of coagulase negative staphylococci were lower (P<0·05) at 102 d post partum, whereas prevalences of non-agalactiae streptococci were higher (P<0·05) in OP than in CP cows at 31 d and 102 d post partum. In conclusion, under Swiss conditions, subclinical mastitis is a greater problem in organic than in conventional production systems, but differences are not marked.

The aim was to select potentially probiotic lactobacilli from 88 strains isolated from unpasteurized milk and cheese products, and to incorporate these bacteria in a viable state into a soft cheese, without changing its quality. The survival of these bacteria was assessed in acidic and bile conditions, after freezing at −80 °C. Four strains from unpasteurized Camembert – two Lactobacillus plantarum strains and two Lb. paracasei/casei strains – were identified and typed by PCR and PFGE and were found to display potentially probiotic characteristics in addition to resistance to low pH and bile. These characteristics were resistance to lysozyme, adhesion to CACO-2 cells, antimicrobial effects against common foodborne pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli), innocuity following the ingestion of high doses by mice and appropriate antibiotic susceptibility profiles. The potential of Lb. plantarum strain UCMA 3037 for incorporation into a soft cheese (Pont-l'Eveque registered designation of origin (RDO)) was investigated. This strain grew well and survived in sufficient numbers (more than 107 cfu/g throughout the shelf-life of the product) in the cheese. This strain did not change the quality score of the product until the best before date (75 days after manufacture). Thus, unpasteurized Camembert is a natural source of potentially probiotic lactobacilli, which could be used as an additive in the development of potentially probiotic soft cheeses. Further work is required to demonstrate the persistence and efficacy of these strains in the human host upon ingestion.

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.

Two experiments were conducted to investigate possible changes of milking-related oxytocin release (Expt 1) and of intramammary pressure and milking characteristics (Expt 2) throughout entire lactations in German Braunvieh dairy cows. Mean oxytocin concentrations after stimulation at onset of milking increased from 18·3 ± 15·9 to 30·7 ± 24·1 pg/ml in Expt 1 and decreased from 23·9 ± 17·6 to 15·4 ± 9·1 pg/ml in Expt 2, respectively, but remained above the level necessary to elicit complete milk ejection in both trials. Premilking baseline intramammary pressure had its maximum in early lactation until about month 4 and then decreased to ∼50% of its initial level. Ejection pressure followed a similar pattern, but dropped only to ∼75% of its maximum. This was due to the constant elevation of pressure increase, reaching its highest level in late lactation. Time from commencement of stimulation until maximum pressure exceeded 1 min in almost all instances even in early lactation and increased throughout lactation. Despite the normal decrease of milk yield average milk flow fell only slightly while maximum flow rate remained almost constant. Pressure increase, milk yield and milk flow were not different after 1 min and after extended stimulation. Thus there were no indications of a decreasing sensitivity of the milk ejection reflex during lactation, and milking characteristics were positively affected by intense teat stimulation. Suggestions for practical dairying are made.

1. Certain starter cultures have been found to be inhibited in the presence of milk peroxidase.

2. A high correlation has been established between the inactivation of milk peroxidase and of the inhibitory action by use of critical values of any of the following: temperature, pH, concentration of either sodium azide or hydrogen peroxide.

3. The inhibitory action can be overcome by the addition of certain reducing substances such as cysteine and sodium hydrosulphite.

4. The inhibitory action is exhibited by the corresponding separated milk and whey.

5. It is suggested that this inhibition may be due to the formation of a specific inhibitory oxidation product having a quinonoid structure.

6. The addition of 1–2% of pasteurized milk or whey to autoclaved milk caused total inhibition, whereas such additions to milk heated to 190° F. showed no such effect. This is attributed to the presence of SH groups in the latter milk capable of reducing the oxidation product.

7. It is suggested that the inhibition due to peroxidase is identical with that ascribed to lactenin 2, and that the inhibition described in our previous paper as due to ‘agglutinin’ is identical with that ascribed to lactenin 1.