Twelve multiparous British Friesian cows in early (40±23 d in milk; n=6) or late (216±17 d in milk; n=6) lactation were used to study the effects of milking frequency on yield, udder volume and milk storage within the udder. After a 2 week control period of twice daily milking, diagonally opposed udder halves within a cow were milked once or twice daily for 3 weeks. Milk yield was 28–38% lower from the halves that were milked once daily than from halves that were milked twice daily. The loss of milk yield, expressed as a decrease in the relative milk yield quotient (an index that accounts for pretreatment differences), was greater for cows in early than in late lactation (0·59 v. 0·68). Empty udder-half volume was not decreased by once daily milking, suggesting that no cell loss occurred. Instead, once daily milking reduced the secretion efficiency (units of milk per unit of empty udder-half volume) by 46 and 27% respectively in early and late lactation; thus, at least part of the loss was due to reduced metabolic activity of the mammary epithelium. There were positive correlations between the relative milk yield quotient and the proportion (r=0·804) or volume (r=0·644) of cisternal milk in the glands that were milked once daily. These results confirm that, during extended milking intervals, milk loss was smallest for cows that stored a larger proportion of milk in the gland cistern.

The effects of addition of EDTA on the dissociation of caseins and foaming properties of milks (100 g solids/l) reconstituted from skim milk powders given a low-heat (72°C for 30 s) or high-heat (85°C for 30 min) treatment during powder manufacture were determined. The EDTA-induced dissociation of caseins was independent of heat treatment but in high-heat milk was accompanied by release of denatured whey proteins. EDTA changed the proportions of individual caseins in the supernatant. EDTA addition improved both foam overrun and foam stability of low- and high-heat milks. The increase in serum protein on addition of EDTA contributed to the improvement in foaming properties of milks by increasing the availability of the proteins for formation of the air–water interface.

In an approach to develop a commercial-scale process for the production of casein phosphopeptides containing the cluster sequence −SerP−SerP−SerP−Glu−Glu–, we have studied the relationship between casein hydrolysis and phosphopeptide release. The degrees of hydrolysis (DH) of casein using Novo trypsin PTN 3.0 S and pancreatin 4NF independently, at enzyme to substrate (E[ratio ]S) ratios of 1[ratio ]50–1[ratio ]1600 (by weight), were determined using the pH-stat method. Casein phosphopeptides (CPP) were selectively precipitated using Ca2+ and ethanol from the acid-clarified hydrolysates. The precipitates were analysed by high performance capillary electrophoresis to calculate individual phosphopeptide yields based on extinction coefficients of the purified peptides. Individual peptides were purified by reversed-phase HPLC and anion-exchange FPLC4 and characterized by MALDI-TOF mass spectrometry and amino acid sequence analysis. For both enzymes, lowering the E[ratio ]S ratio resulted in reductions in the DH and the release of the CPP, and an increase in peptide chain length. The longer chain length offset the reduction in release such that the gravimetric yields of CPP preparations remained relatively constant. For Novo trypsin the highest yields of the major cluster peptides (β-casein(CN)f(1–25), αs1-CNf(59–79), αs2-CNf(1–21), αs2-CNf(46–70) and related peptides) in the selective precipitates were obtained at a casein DH of 17%. At lower DH values (9–15%), there was a decrease in yield of the peptides derived from αs1-CN and αs2-CN while the yield of the β-CN-derived cluster peptides remained relatively constant. The CPP produced using pancreatin were found to be truncated at all E[ratio ]S ratios, relative to the tryptic CPP, owing to higher levels of chymotryptic and carboxypeptidase activities in pancreatin. The highest yields of the truncated forms of the major cluster peptides using pancreatin were obtained at a casein DH of 19–23%.

Polymorphism of caprine milk proteins was studied by capillary electrophoresis. Identification of casein (CN) fractions was effected by using isolated fractions from cation-exchange fast protein liquid chromatography. Genetic polymorphisms in caprine αs2-CN, αs1-CN, β-CN and κ-CN have been determined. κ-CN A and B, β-CN A and null, αs2-CN A, B and C, αs1-CN A, B, C and null, and other forms with intermediate and low αs1-CN content have been identified. The capillary electrophoresis method made it possible to analyse whole caprine milk using simple sample preparation and was rapid, automated and suitable for phenotyping studies. This method may also permit the quantitative study of different protein fractions.

Polymorphism of ovine αs1-caseins was studied by capillary electrophoresis at pH 3·0±0·1. Individual caseins (CN) were selected according to their genetic variants, as determined by PAGE and isoelectric focusing. The ovine caseins, containing different genetic variants of αs1-CN and αs2-CN, were fractionated by cation-exchange FPLC®. The αs1-CN variants A, B and C, and the fast moving αs2-CN variant were identified by a capillary electrophoresis method. The fast moving αs2-CN variant, so-called for its behaviour in PAGE, also had a faster electrophoretic mobility than the common αs2-CN when analysed by the present technique. The capillary electrophoresis method gave excellent, rapid, automated separation of αs1-CN and αs2-CN variants and was suitable for screening studies.

We propose an alternative method for casein genotyping, generally carried out using polymerase chain reaction followed by restriction fragment length polymorphism analysis. Application of the single-strand conformation polymorphism technique detects nucleotide changes in the fragment amplified by means of polymerase chain reaction and thus avoids the use of restriction enzymes. A 453 bp fragment from exon IV of κ-casein has been amplified. The two variants (A and B), found with the highest frequencies in most bovine breeds and included in some dairy cattle selection schemes, can be discriminated using single-strand conformation polymorphism analysis of heat denatured fragments in acrylamide–bis-acrylamide (100[ratio ]1) gels followed by silver staining. κ-Casein genotyping is therefore simplified, although variants A and E on the one hand, and B and C on the other, are not distinguishable with this technique.

Simultaneous measurement of viscosity and degree of proteolysis of κ-casein, α, in skim milk at three different concentrations of rennet and three different concentrations of casein showed that viscosity was a function of α until the former reached its minimum value. Then the relation between viscosity and α depended on the enzyme concentration. This is considered to be caused by casein micelles starting to aggregate at this point. The minimum in viscosity was found when α was between 0·6 and 0·7, indicating that casein micelles started aggregating at a lower degree of proteolysis than was believed earlier. The rate of gel firming was found to depend on the concentration of added rennet even after κ-casein was completely proteolysed. The results demonstrated that both proteolysis of κ-casein and aggregation of casein micelles must be taken into account when modelling the renneting reaction.

The growth of four Bacillus cereus strains producing diarrhoeal toxin at 32°C (F4433/73 and 29.155, isolated on the occasion of foodborne outbreaks, and F4581/76L and F4581/76R, two variants of a clinical strain), a weakly toxigenic strain isolated in routine analysis of food (3505M) and an emetic isolate (F3502/73) was investigated at low temperature. Biomass was determined by protein assay. Generation times were: for strain F3502/73, which grew at [ges ]12°C, 8·71 h (at 12°C); for other strains, which grew at [ges ]10°C, 10·2 to ∼18·9 h (at 10°C). Toxin production during growth was evaluated by a commercial kit (Oxoid) and by a toxicity test on Chinese hamster ovary cells. Strains F4433/73 and F4581/76, secreting high levels of diarrhoeal toxin during the exponential phase at 32°C, produced high levels of toxicity at 10°C until the stationary phase. Strain 29.155 had decreased toxin production at 10°C. Toxicities for cellular extracts remained low when compared with culture filtrates. A correlation was found between the toxicity values given by the two detection methods tested, and the suitability of both methods for the detection of potential poisoning isolates is discussed.

Whole cells of Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 (Lb. bulgaricus CNRZ 397) are able to hydrolyse α- and β-caseins. We have isolated a mutant of Lb. bulgaricus altered for growth in milk and unable to hydrolyse α- or β-casein. Normal growth was restored by adding amino acids or tryptone to milk. No significant difference between the peptidase activities of parent and mutant strains was observed. The cell surface caseinolytic activities of three lactobacilli species and Lactococcus lactis subsp. lactis (Lc. lactis) were compared. As expected, the characteristics of the cell surface proteinase activity of Lb. casei were similar to those of Lc. lactis. We showed that the cleavage specificities of the cell surface proteinase activities from lactobacilli were species-dependent and at least three types of activity were distinguished. The regulation of the biosynthesis of cell surface proteinase activities was medium-dependent and different within the Lactobacillus genus and even within the Lb. delbrueckii species. In contrast to Lb. bulgaricus, the cell surface proteinase activity of Lb. lactis was totally inhibited in a medium rich in peptides or amino acids. In contrast, the cell surface of Lb. helveticus probably displayed two proteinases with different cleavage specificities and with a biosynthesis regulation sensitive to different medium components.

Our aim was to develop an Emmental juice-like medium (EJLM) for the growth of propionibacteria. Cheese juices were extracted by pressing three different Emmental cheeses before entering the warm room (i.e. before propionic fermentation) and their composition was determined. They contained (g/kg) lactate 34·6–36·9, protein 23·8–29·6, NaCl 15–23, Ca 5·9–7·3 and no residual sugar, and had a pH of 5·4. In order to simulate this composition, EJLM was constituted from the microfiltrate of a milk, enriched in native casein and fermented by a Lactobacillus helveticus strain until lactose exhaustion. PO43−, K+, Mg2+, Ca2+ and Cl− were added to reach a mineral balance and an ionic strength close to those of juice. The main difference was the nitrogen content: that of juice was ∼3·5 times that of EJLM. Four Propionibacterium freudenreichii strains grew with similar growth rates in EJLM and in juice at 24°C in anaerobiosis and reached >3×109 cfu/ml. Using EJLM, Prop. freudenreichii TL 173 grew at pH 5·4 or 6·0, at 24 or 30°C, in semi-anaerobiosis (static culture) or anaerobiosis (CO2 atmosphere), showing a synergic inhibition of growth at low pH and temperature and in semi-anaerobiosis. EJLM provided a way to study propionibacteria in an environment similar to that in Emmental cheese.

Mineral acid casein, lactic casein and rennet casein curds were prepared either in the laboratory or on pilot-scale equipment. The water-holding capacities of mineral acid casein curd particles prepared in the laboratory were relatively constant for particle sizes >4 mm both after sieving and after centrifugation; however, they increased dramatically at smaller particle sizes to a maximum of 7·2 g water/g dry curd after sieving and 4·3 g water/g dry curd after centrifugation at 4060 g for the size fraction that passed a 0·85 mm screen. The mechanical strength of the centrifugally compressed curd as assessed by a penetrometer probe also decreased dramatically with decrease in particle size. This variation of mineral acid casein curd properties with particle size was attributed partly to variation in particle pH at the instant of curd formation because of imperfect mixing of acid and skim milk, i.e. localized pH gradients. Lactic casein and rennet casein curds and curd formed by acidification of skim milk with glucono-δ-lactone also produced similar but smaller variations in curd properties with particle size. These curd particles are all formed by break up of a quiescent gel and the variation in curd properties with particle size was attributed to variations in mechanical strength in the original milk gel, i.e. weak areas of the gel break up more easily, resulting in small, weak curd particles.

The effect of a range of milk heat treatments on the rheological properties, at small and large deformations, of acid skim milk gels was investigated. Gels were made from reconstituted skim milk heated at 75, 80, 85 and 90°C for 15 or 30 min by acidification with glucono-δ-lactone at 30°C. Gels were also made from skim milk powder (SMP) samples that had been given a range of preheat treatments during powder manufacture. Heating milks at temperatures [ges ]80°C for 15 min increased the storage moduli (G′) compared with unheated milk and produced gels with G′ in the range 300–450 Pa. Acid gels made from high-heat or medium-heat SMP had higher G′ than gels made from low-heat or ultra-low-heat SMP. Cooling gels to low temperatures resulted in an increase in G′. The yield stress of gels slightly decreased with mild heat treatments of milk, and then increased again to a maximum, finally decreasing slightly with very high heat treatments of milk. The strain at yielding decreased markedly with increasing heat treatment of milk, making these gels brittle and easier to fracture. We propose that denatured whey proteins aggregated with casein particles during the acidification of heated milk and were responsible for most of the effects observed in this study.

Casein phosphopeptides (CPP) which develop in Grana Padano cheese at different ages were isolated by precipitation with Ba2+ and analysed by HPLC. Profiles were complex throughout the period between 4 and 38 months. CPP in a cheese sample 14 months old were identified by a combination of fast atom bombardment–mass spectrometry and Edman degradation. They were found to consist of a mixture of components derived from three parent peptides, β-CNf(7–28)4P, αs1-CNf(61–79)4P and αs1-CNf(7–21)4P. In total, 45 phosphopeptides were identified: 24 from β-CN, 16 from αs1-CN and 5 from αs2-CN. The presence of aminopeptidase activity during cheese ripening was deduced from the presence of a number of CPP of different lengths with the loss of one or more residues from the N-terminus. The longest had C-terminal lysine and seemed to be progressively hydrolysed by carboxypeptidases A and B to shorter peptides. CPP in cheese appeared to be shortened plasmin-mediated products. Moreover, those most resistant to further hydrolysis contained at least three closely located phosphoserine residues. The anticariogenic activity of CPP is also discussed.

Control programmes for contagious mastitis pathogens, primarily Streptococcus agalactiae and Staphylococcus aureus, consisting of milking hygiene, treatment or isolation of infected cows and post-milking teat disinfection are relatively effective (McDonald, 1970; Natzke, 1977; Fox & Gay, 1993). Similar control programmes are often ineffective in the prevention of intramammary infections caused by environmental bacteria (Eberhart, 1977; Smith et al. 1985; Erskine et al. 1991; Smith & Hogan, 1993). This disparity of success relates to the fact that the principal source of environmental mastitis pathogens is the cow's environment, rather than another cow with an intramammary infection (Eberhart, 1977; Smith et al. 1985). Infections of environmental origin are particularly noteworthy because they may remain common in well managed herds, and these infections often cause severe clinical signs and high case fatality rates (Eberhart, 1977; Smith et al. 1985; Erskine et al. 1991; Smith & Hogan, 1993).

Control programmes for environmental mastitis usually include premilking teat disinfection, decreased use of water in udder preparation, increased concentrations of dietary vitamin E and selenium, improved sanitation of the environment and modification of cow behaviour in the post-milking period (Eberhart, 1977; Smith et al. 1984, 1985; Weiss et al. 1990; Erskine et al. 1991; Smith & Hogan, 1993). Environmental infections are thought to result when bacteria lacking specific virulence factors penetrate the teat sphincter (Eberhart et al. 1979; Smith et al. 1985). The bulk of these exposures are thought to occur between milkings. Control of these infections is problematic because the cow's teat sphincter remains open after mechanical milking (McDonald, 1975a). Presumably, cows are predisposed to intramammary infections when the open teat sphincter is exposed when cows lie down. Hence, dairy managers are advised to offer fresh feed to cows immediately after milking to increase the likelihood that the teat sphincter will close at least partly before cows become recumbent.

The purpose of this study was to determine the effect of feed availability on post-milking behaviour in dairy cows. Specifically, the measured dependent variable was the length of time that cows remained standing after leaving the milking parlour.

Milk production in Ireland, New Zealand and Australia is seasonal, with the majority of cows calving in spring. This pattern of production makes the maximum use of grazed grass, and in Ireland >80% of total milk for manufacturing is produced between April and November inclusive. Such a seasonal pattern of production results in a large variation in the gross composition of milk due to stage of lactation effects (Phelan et al. 1982). Some studies have investigated the relationship between milk composition and its processing characteristics (O'Keeffe et al. 1982; Grandison et al. 1984); however, in these studies the effects of diet and lactation stage were not segregated. Kefford et al. (1995) attempted to segregate the effects of diet and stage of lactation and concluded that diet quality (type and quantity) had a larger effect on Cheddar cheese quality than the stage of lactation.

The nutritive value of the diet of cows fed on grazed grass can change owing to changes in grass supply or quality. In the current study, changes in grass supply were achieved by altering the daily herbage allowance (DHA) to the herds. The objective of the current study was to investigate the effect of varying the DHA in the range 16–24 kg grass dry matter (DM), which is typical of the variation in pasture allowance in Ireland in mid lactation, on milk composition and its processing characteristics.

Emotional stress caused by moving to unfamiliar surroundings inhibits oxytocin (OT) release and milk ejection in dairy cows, while blood concentrations of β-endorphin and cortisol are elevated (Bruckmaier et al. 1993). β-Endorphin and cortisol concentrations decrease with repeated relocation to unfamiliar surroundings while milking-related OT release and milk ejection gradually normalize (Bruckmaier et al. 1996). Exogenous cortisol has previously been shown to exert no inhibiting effect on milk ejection in cows (Mayer & Lefcourt, 1987). Therefore, it seems likely that the inhibition of milk ejection in cows – as in other species – is mediated by endogenous opioid peptides. The opioid antagonist naloxone abolishes the inhibiting effect of endogenous opioid peptides on OT release in several species (Haldar et al. 1982; Seckl & Lightman, 1987; Gilbert et al. 1992; Aurich et al. 1996).

Milk ejection is inhibited by α-adrenergic receptor stimulation in the mammary gland without reduction of OT release (Bruckmaier et al. 1997). Opioid-induced inhibition of OT release may be mediated by noradrenergic neurons via β-adrenergic receptors (Crowley & Armstrong, 1992). If adrenergic receptors are involved in the blockade of OT release in cows during milking in unfamiliar surroundings, the inhibition may be abolished by adrenergic blocking agents.

Cortisol synthesis is diminished by administration of metyrapone (Martindale, 1989). Because cortisol has a negative feedback effect on ACTH release, the release of ACTH and concomitantly of β-endorphin is elevated by metyrapone in the rat (Pettibone & Mueller, 1984). Thus, milk ejection should be indirectly inhibited by metyrapone administration via enhanced release of β-endorphin in the familiar barn.

Experiments were designed to investigate the importance of elevated concentrations of β-endorphin in unfamiliar surroundings for disturbed milk ejection in dairy cows.

During lactogenesis in the goat, the onset of secretion of calcium into milk occurs at parturition (Thompson et al. 1995) at approximately the same time as the onset of secretion of parathyroid hormone-related protein (PTHrP) by the mammary gland (Ratcliffe et al. 1992); these events may be unrelated or PTHrP may be involved in calcium transport from blood to milk.

Parturition in goats is initiated by fetal secretion of cortisol (Flint et al. 1978) and maternal secretion of cortisol also increases (Paterson & Linzell, 1971). Injecting cortisol locally into the sinus of a mammary gland of the late-pregnant goat when the tight junctions between secretory epithelial cells appear to be ‘loose’, and injectate can reach the basolateral surfaces of secretory cells, stimulates an early tightening of these junctions (Thompson, 1996) as occurs naturally at parturition. This tightening can be produced by an increased concentration of ionized calcium in the extracellular fluid of the gland (Neville & Peaker, 1981).

The experiments reported here were undertaken to determine if cortisol injection stimulates the mammary gland to secrete both PTHrP and calcium before parturition.

Plasmin (PLM; E.C. 3.4.4.14) is the major proteolytic enzyme normally present in bovine milk. From a technological point of view, PLM activity in milk is detrimental as it increases the proteolysis of casein to proteose peptones, and this results in reduced storage time, taste defects, loss of cheese yield and quality, and changes in the physicochemical properties of milk (for review, see Fox, 1992). Therefore a reduction of PLM activity in milk would be desirable. Plasminogen (PLG), the zymogen of plasmin, and PLM content are affected by several physiological and environmental factors, and by genetic factors such as breed (Richardson, 1983; Schaar, 1985; Politis et al. 1989; Benslimane et al. 1990). We have addressed the question of PLG polymorphism in dairy cattle.