We found previously that the current recommendations for Na+, K+, and Cl− contents in the diet do not meet the needs of lactating cows. The responses of cows receiving a ration with increased amounts of Na+, K+, and Cl− (E cows) were compared with those of cows consuming the same ration with a fixed concentration of these ions (C cows) between weeks 2 and 8 post partum (PP). Milk, protein, fat and lactose yields, and dry matter intake between weeks 2 and 4 PP were higher in E than in C cows. These differences did not occur between weeks 4 and 8 PP, mainly because of a higher incidence of PP complications in E cows. A greater increase in plasma insulin-like growth factor-1 concentration in E than in C animals during weeks 2 and 3 PP was consistent with the milk responses. A reduction in aldosterone concentration in E cows in weeks 2 and 3 PP was a consequence of their Na+ requirements being satisfied as a result of their enhanced Na+ intake. A subsequent elevation in aldosterone concentration in E animals was probably related to a moderate excess in K+ intake. This increase in aldosterone explains the urinary potassium loss that was detected at week 6 PP. The absence of differences between E and C cows in plasma renin activity was consistent with an absence of differences in urine volume and with the apparent utilization of the enhanced ion intake for body functions.

In order to describe the kinetics of rennet coagulation, measurements of turbidity as a function of wavelength were used to determine the weight-average degree of polymerization, x¯w, during renneting of milk at three different concentrations of enzyme and three concentrations of casein, including the normal casein concentration of milk. The change of x¯w as a function of time was described using Von Smoluchowski's equation, testing a number of expressions for the aggregation rate constant, kij. The best description was achieved when kij was taken as a function of an energy barrier against aggregation that was diminished by the proteolysis of κ-casein. The initial value of the energy barrier partly depended on the casein concentration, and had a value >25 kBT at normal casein concentration, where kB is Boltzmann's constant and T the absolute temperature. When the proteolysis of κ-casein was complete, the energy barrier was reduced to 11 kBT and was independent of casein concentration.

The effect of interactions of denatured whey proteins with casein micelles on the rheological properties of acid milk gels was investigated. Gels were made by acidification of skim milk with glucono-δ-lactone at 30°C using reconstituted skim milk powders (SMP; both low- and ultra-low-heat) and fresh skim milk (FSM). The final pH of the gels was ∼4·6. Milks containing associated or ‘bound’ denatured whey proteins (BDWP) with casein micelles were made by resuspending the ultracentrifugal pellet of heated milk in ultrafiltration permeate. Milks containing ‘soluble’ denatured whey protein (SDWP) aggregates were formed by heat treatment of an ultracentrifugal supernatant which was then resuspended with the pellet. Acid gels made from unheated milks had low storage moduli, G′, of <20 Pa. Heating milks at 80°C for 30 min resulted in acid gels with G′ in the range 390–430 Pa. The loss tangent (tan δ) of gels made from heated milk increased after gelation to attain a maximum at pH ∼5·1, but no maximum was observed in gels made from unheated milk. Acid gels made from milks containing BDWP that were made from low-heat SMP, ultra-low-heat SMP and FSM had G′ of about 250, 270 and 310 Pa respectively. Acid gels made from milks containing SDWP that were made from ultra-low-heat SMP or FSM had G′ values in the range 17–30 Pa, but gels made from low-heat SMP had G′ of ∼140 Pa. It was concluded that BDWP were important for the increased G′ of acid gels made from heated milk. Addition of N-ethylmaleimide (NEM) to low-heat reconstituted milk, to block the —SH groups, resulted in a reduction of the G′ of gels formed from heated milk but did not reduce G′ to the value of unheated milk. Addition of 20 mm-NEM to FSM, prior to heat treatment, resulted in gels with a lower G′ value than gels made from reconstituted low-heat SMP. It was suggested that small amounts of denatured whey proteins associated with casein micelles during low-heat SMP manufacture were probably responsible for the higher G′ of gels made from milk containing SDWP and from milk heated in the presence of 20 mm-NEM, compared with gels made from FSM.

Digestion of bovine αs1-casein with bovine trypsin produced peptide(s) with an inhibitory effect on concanavalin A-induced proliferation of mouse spleen cells. One of these peptides was isolated from the αs1-casein digest following ultrafiltration, hydroxyapatite chromatography and reversed-phase HPLC, and amino acid composition and sequence analyses showed it to be sequence 59–79 from the phosphoserine-rich region of αs1-casein. The isolated peptide significantly inhibited the concanavalin A-induced proliferation of mouse spleen cells and rabbit Peyer's patch cells, whereas it enhanced the lipopolysaccharide- and phytohaemagglutinin-induced proliferation of both cells. The peptide displayed mitogenic activity in the cell cultures without the commercial mitogen, and significantly enhanced immunoglobulin production. Moreover, residues 1–25 from the phosphoserine-rich region of bovine β-casein had a similar effect on the proliferation of mouse spleen cells and rabbit Peyer's patch cells stimulated or not stimulated by the commercial mitogen. These results indicate that caseinophosphopeptides may act as a humoral immunostimulator in cell cultures.

Homocitrulline arises from the reaction between cyanate and the ε-amino group of lysine residues; in milk, cyanate derives from heat-induced urea breakdown. Since homocitrulline levels were unknown in heated milk, its formation was studied in the temperature range 100–150°C. Firstly, an analysis method based on ion-exchange chromatography using an amino acid analyser was developed. Homocitrulline, liberated from milk protein by enzymic hydrolysis, was eluted well separated from other amino acids and could be readily distinguished using this technique. Secondly, homocitrulline levels were determined for various time–temperature combinations in samples of milk, milk to which 10 mm-urea had been added, and milk that was made urea-free. No homocitrulline was formed in urea-free milk, while homocitrulline formation was stimulated by urea addition. Upon prolonged heating, we found extensive subsequent breakdown of homocitrulline. The kinetics of homocitrulline formation was quite complicated, but an approximation was possible as the initial formation reaction could be modelled using zero order reaction kinetics. The apparent activation energy for homocitrulline formation was estimated at ∼90 kJ/mol. In general, the levels of homocitrulline to be expected in heated milk appeared to be quite low: ∼0·3 mm in in-bottle sterilized milk, and <0·01 mm in UHT sterilized milk.

7-Ketocholesterol and 7-ketositosterol were chosen as reliable markers of the oxidation of cholesterol and phytosterols in infant milk formulas and infant milk cereals. A reversed-phase HPLC method was developed to measure them simultaneously in infant formulas. This method was then tested on a wide range of infant milk formulas and milk cereals on sale in Italy whose lipid composition is representative of the most common commercial formulas. The analytical results revealed no significant differences in the extent of oxidation of cholesterol and sitosterol. As the level of 7-ketocholesterol often followed the cholesterol level, a cholesterol content similar to that of human milk produced amounts of cholesterol oxides with possible negative effects on infant health. In contrast, the low cholesterol content of milk cereals never produced amounts of cholesterol oxides high enough to cause concern. The contents of phytosterols and hence their oxides were always low.

The aim of this study was to examine the effect of bovine colostral whey proteins from cows immunized with Streptococcus mutans/Strep. sobrinus on the adherence and aggregation of caries-inducing bacteria, i.e. mutans streptococci. Both adherence and aggregation are important phenomena in the bacterial colonization of the human oral cavity. In all adherence experiments there was a significant difference between treatments by immune product (IP; from immunized cows) and a control product (CP; a similar product from non-immunized cows). The adherence of 35S-labelled Strep. mutans cells (serotype c) to parotid saliva-coated hydroxyapatite (SHA) was dose-dependently inhibited by both IP and CP if SHA was coated with either product before exposure to bacteria, but markedly lower concentrations of IP than CP were effective. When instead of SHA the bacterial cells were pretreated with IP or CP, only IP strongly and dose-dependently inhibited streptococcal adherence. When bacteria, IP or CP, and SHA were incubated simultaneously, a significant difference between IP and CP treatments was again found. Further, IP effectively aggregated both Strep. mutans and Strep. sobrinus cells, whereas hardly any effect was seen with CP. Both IP and CP aggregated the control bacterium Strep. sanguis, which affected the adherence of the pretreated bacteria.

Lactobacillus helveticus and Propionibacterium freudenreichii are essential starters in Swiss cheesemaking and the release of their intracellular enzymes through autolysis could significantly influence ripening. To provide evidence of this lysis, cheese made from microfiltered thermized milk inoculated with Lb. helveticus ITGLH77, Prop. freudenreichii ITGP23 and a commercial Streptococcus thermophilus was assayed. Starter viability was determined and lysis was monitored during ripening by protein analysis with SDS-PAGE of aqueous cheese extracts and by immunoblot detection of intracellular proteins: dipeptidase (PepD) for Lb. helveticus and methylmalonyl coenzyme A mutase for Prop. freudenreichii. We verified that the species specificity of these lysis markers was towards the cytoplasms of all the species currently used in Swiss cheese. Lb. helveticus exhibited an almost complete loss of viability (99·9%) from the beginning of ripening in the cold room; concomitantly PepD appeared in the cheese extracts and was detected until the end of ripening. Damaged Lb. helveticus cells were also visualized by scanning electron microscopy. In addition, free PepD was also successfully detected in commercial Swiss-related cheeses. All these results clearly demonstrated the autolysis of Lb. helveticus in Swiss cheese. Prop. freudenreichii ITGP23 grew during warm room ripening and no loss of viability was detected after maximal growth (109 cfu/g cheese). Free methylmalonyl-coenzyme A mutase was detected at the end of ripening during cold storage, when the cheese extracts were concentrated 20-fold, demonstrating that the autolysis of Prop. freudenreichii was tardy and limited.

The cysteine proteinase from Micrococcus sp. INIA 528 was added at three concentrations to pasteurized cows' milk to study its effect on the ripening process of Hispanico cheese. Dry matter was higher in whey from experimental cheeses than in whey from control cheese manufactured from milk with no added proteinase. No differences in cheese proteolysis were found 24 h after manufacture. Residual αs- and β-caseins were significantly lower, and N soluble at pH 4·6 and in trichloroacetic acid significantly higher, in experimental cheeses than in control cheese from day 15. Hydrophobic and hydrophilic peptides in the water-soluble fraction of cheese measured by absorption at 280 nm were significantly higher in experimental cheeses than in control cheese from days 1 and 15 respectively. Texture was less firm in experimental than control cheese from day 30. Bitterness, detected in cheese with the highest levels of added cysteine proteinase, adversely affected cheese flavour. Flavour intensity was significantly higher from day 30 in cheeses with added proteinase than in control cheese.

We have investigated accelerating the ripening of Pecorino Umbro cheese by adding crude cytoplasmic extract from Pseudomonas fluorescens, non-starter lactic acid bacteria (NSLAB) or cheese slurry. Microbiological and biochemical analyses and sensory evaluation were carried out on control and experimental cheeses over 28 d ripening. In the cheeses containing NSLAB or slurry, counts of mesophilic lactobacilli ranged from log 7·6 at day 1 to ∼log 8·6 cfu/g after 28 d ripening, ∼2 log cycles higher than in the control cheese. All the experimental cheeses contained higher levels than the control of total free amino acids and N soluble at pH 4·6 and in 120 g trichloroacetic acid/l. Compared with the control, higher aminopeptidase and dipeptidase activities were found in the cheeses containing NSLAB and slurry, and especially in those containing the Pseudomonas enzyme. The cheeses containing NSLAB or slurry were characterized by an accumulation of short peptides (Mr<2000) detected by FPLC. Although the cheese containing enzyme had an atypical flavour, the addition of mesophilic lactobacilli reduced from 60 to 28 d the ripening period of Pecorino Umbro cheese, without the appearance of off flavour.

Bovine plasmin (PLM) and its precursor plasminogen (PLG) are present in cheese, in which the proteinase is thought to play a significant role in proteolysis during ripening. PLM in cheese is usually assayed by enzymic techniques and concentrations are generally expressed as activities. In order to determine absolute concentrations of PLM and PLG in cheese, we have applied an ELISA previously developed for the differential determination of PLM and PLG in milk. The assay used two monoclonal antibodies, one specific for PLG and the other cross reacting with PLM and PLG. PLM concentration was obtained by subtracting the PLG concentration from the value for PLM plus PLG. The assay was evaluated in cheese by determining the total amount of PLG and PLM in milk used in the manufacture of eight semi-hard cheeses on a pilot plant scale, and comparing it with the total amount of PLG and PLM present in the eight corresponding wheys and unripened cheeses. Concentrations of PLG and PLM in 19 commercial cheeses were also determined. It was thus demonstrated that the ELISA made it possible to quantify PLM and PLG in milk and cheese and that PLM and PLG concentrations in cheese were highly dependent on the technology used for cheesemaking.

The effects of varying the rennet concentration (by a factor of 216) and the coagulation method on the texture of 32 Feta cheeses produced from ultrafiltered milk were examined by rheological, sensory and chemical methods. Cheeses were obtained that varied greatly in texture but had the same gross chemical composition. All rheological measurements (uniaxial compression, strain sweep and relaxation measurement), sensory firmness and chemical measures of proteolysis depended significantly on the rennet concentration used. With increasing rennet addition, the cheeses became firmer and grittier, and less adhesive and sticky. With two of the three coagulation methods, the physicochemical state of the casein micelles at the point of coagulation varied with the rennet level: when less rennet was used, coagulation occurred at a lower pH and after longer contact between milk and NaCl. However, the effects of increased rennet concentration were also significant with the third method, where there was no effect on the pH or the period with NaCl at coagulation. Probably the primary effect observed was that an increased aggregation rate, with increased rennet level, affected the initial mode of aggregation of casein particles, leading to a denser network structure and a firmer and grittier texture. The level of micellar disintegration at coagulation (controlled by the coagulation method used) had a supplementary effect.

The relationship between proteolysis and texture in Feta-type cheese made from ultrafiltered cows' milk was studied using capillary electrophoresis, chemical analysis and uniaxial compression. For this purpose cheeses were made with almost identical gross chemical compositions, but wide variations in texture, obtained by varying the amount of rennet and the coagulation conditions. αs1-Casein (CN) 8P, αs1-CN 9P and αs1-CN 8P-I were degraded during ripening, while β-CN A1, β-CN A2 and para-κ-CN were slightly degraded at the highest rennet additions and longest storage time tested (39 weeks). αs1-CN 8P, αs1-CN 9P and αs1-CN 8P-I were degraded even in cheeses made without rennet, and this was ascribed to cathepsin D activity. αs1-CN 9P disappeared faster than αs1-CN 8P, which suggests that milk acid phosphatase was active during storage. During ripening, stress at fracture (σf), strain at fracture (εf), the deformability modulus (E) and work to fracture (Wf) all decreased. Since the gross chemical composition was essentially constant during storage, these changes could be ascribed purely to proteolysis. Although εf could be predicted fairly well from capillary electrophoresis results (correlation coefficient 0·85), there were no unique relationships between degradation of casein components and σf when both storage time and the amount of rennet used were varied. In the young cheese a high level of proteolysis could coexist with high values of σf. This suggests that high levels of rennet caused σf to increase, via an effect unrelated to general proteolysis, whereas general proteolysis caused the reverse effect.

A microbial sensor based on a carbon dioxide electrode coupled with immobilized Saccharomyces cerevisiae (baker's yeast) has been used for the determination of sucrose in dairy products. The sensor was used as the detector in a flow injection analysis system. Calibration curves for sucrose were established from 1 to 100 g/l. Determinations for several dairy products containing added sucrose gave good agreement with the concentrations given by manufacturers. Typically, the se of the method was shown to be <5% of the calculated mean.

Control of environmental mastitis remains a problem on many modern dairy farms. These infections are often transmitted between milkings, and milking hygiene will not prevent new infection. Consequently, the control of environmental mastitis has prompted dairy managers to develop new approaches that either limit bacterial contamination of teat ends between milkings or directly increase the cow's resistance to infection. Intervention strategies that may decrease the incidence of environmental mastitis include improved sanitation of housing areas, decreased water use in udder preparation, optimal dietary concentrations of vitamin E and selenium, and use of R-mutant vaccines (Smith et al. 1984, 1985; Weiss et al. 1990; Smith & Hogan, 1993; Tyler et al. 1993).

Dairy managers are encouraged to provide cows with clean, fresh, palatable feed immediately after milking. This practice is thought to provide cows with an incentive to remain standing for an extended interval, permit teat sphincters to close, and limit teat end contamination and new infections when cows lie down. In one recent study, cows that had access to feed remained standing for a significantly longer time than did cows that were denied access to feed (48 v. 21 min; Tyler et al. 1997). The purpose of the present study was to substantiate that feed availability could be used to extend postmilking standing time in a larger population of cows maintained under different management conditions.

The principles of today's machine milking techniques have developed since the early 1900s. The original intention to imitate the sucking action of the calf had to be abandoned owing to technical difficulties. Further developments were made on a largely empirical basis and milking technique became a specific complex of tasks, problems and solutions. Consequently, the sucking of the calf was rejected as a model for machine milking. The sucking behaviour and the application of vacuum and/or pressure by the sucking mammalian offspring have been adapted through evolution. Any eventual changes due to ‘recent’ breeding and development of the milking machine remain marginal compared with evolutionary changes.

Since the cineradiographic techniques used by Ardran et al. (1957, 1958) it was believed that sucking calves use mainly pressure to transfer a squirt of milk from the proximally closed teat cistern and through the teat canal. The calf creates the pressure by compressing the teat between the tongue and the hard palate from the base of the teat towards the teat end. Cowie (1977) summarized these results as follows: ‘Sucking, that is the production of vacuum within the mouth cavity, is not an essential feature in suckling, although it aids the process. … The act of suckling is thus analogous to hand milking …’. These conclusions were based on visually analysed recordings. McDonald & Witzel (1966) measured pressure in teat cisterns and vacuum at the teat end simultaneously during suckling. Average maximum pressure in the teat cistern was 36·6 kPa and vacuum at the end of the teat averaged −34·6 kPa. These authors concluded that the average differential pressure across the teat canal was 71 kPa. However, this method of calculation gives only the maximal pressure difference and not the average pressure applied by the calf. They noted that vacuum in the teat cistern occurred during the resting phase of a suckling cycle. The calf's sucking technique for single teats during a meal has been analysed (Mayntz, 1996). Further details of pressure and/or vacuum application during suckling at a specific teat remain unknown.

The objective of the present study was to extend the current knowledge about milk extraction by sucking calves through continuous and simultaneous measurements of positive and negative pressure in the teat cistern and in the mouth cavity.

Economic losses caused by enzootic bovine leukosis (EBL) have been of interest since World War II, when the neoplastic form of EBL increased dramatically in Europe. Olson (1974) and House et al. (1975) showed that animals with lymphosarcoma caused by the bovine leukosis virus (BLV) had reduced milk yields, a less efficient reproductive performance and high veterinary costs and mortality rates, while many carcasses were rejected at slaughter. However, the actual impact of BLV infection in cattle without lymphosarcoma is not clear. The purpose of the study reported here was to compare some productive and reproductive responses of cattle that were antibody-positive (BLV+) or negative (BLV−) for BLV.

Holstein dairy cows in commercial dairy farms were used in this study. Blood samples were collected and subjected to BLV serological examination by the agar gel immunodiffusion test of Miller & van der Maaten (1976). Animals were then grouped as BLV+ or BLV− according to their serological response to the BLV antigen. Productive and reproductive histories were obtained from individual animal records and the following factors were considered: milk production, calving interval and birth rate. For milk production, we had the daily milk yields of 547 animals, and for calving interval the time between two successive parturitions for 444 cows. These values were examined by ANOVA and when this was significant a Student's t test was carried out for each age group. Birth rates, the percentage of animals that calved in 1 year, were available for 557 animals and were examined with the Z-two proportion test. For all analyses, P<0·05 was considered significant.

Bovine milk contains a variety of proteins and peptides that are biologically active (Ogra & Ogra, 1978; Duncan & McArthur, 1981; Newby et al. 1982; Juto, 1985; Stoeck et al. 1989; Mincheva-Nilsson et al. 1990; Watson, 1990; Barta et al. 1991; Politis et al. 1991; Fiat et al. 1993). Our laboratory has a long-term interest in some purified milk proteins, particularly lactoferrin (LF), lactoperoxidase (LP) and β-casein (β-CN), which have been shown to be immunologically significant. Some of our recent studies on these bovine milk proteins, particularly β-CN, indicated that their in vitro immunological effects did not always parallel their in vivo activities (Wong et al. 1996a, b; 1997a, b). This study was designed to investigate and compare the capacity of these purified bovine milk proteins to modulate a range of components that are vital to in vivo immune responses in sheep, with a view to providing further information on their potential in biomedical applications. To achieve this objective, a sensitive lymphatic cannulation model was employed that allows in vivo immune components and their functions to be measured in lymph collected under physiological conditions.