The effects of κ-casein genetic variants, lactation number, pH and casein content on the renneting properties of individual milks were studied. Rennet coagulation time (RCT) and curd firmness were determined with the aid of a lactodynamograph and the results statistically analysed. Renneting properties were strongly related to κ-casein variants and lactation number, with κ-casein B and higher lactation numbers giving shorter RCT and increased curd firmness, the effects on curd firmness being more pronounced. The effect of κ-casein variant on RCT could be eliminated by reducing pH and adding CaCl2 to the milk samples. The effect on curd firmness was not altered by this treatment, however. The results show that the renneting properties of individual milks are to a considerable degree genetically controlled.

1. An analysis has been made of production records and body measurements of dairy cattle from farms and special progeny testing stations in Denmark.

2. Heritability estimates of various measurements of body size and also of gross efficiency of food conversion were all in the neighbourhood of 0·50. Body weight was more closely correlated with girth than with height.

3. Milk yield had a very high correlation, both phenotypic and genetic, with gross efficiency. This was shown on theoretical grounds to be a necessary result of any standard system of feeding.

4. Correlations between production characteristics and body measurements were slight. There was some suggestion that selection for yield would increase skeletal size but decrease fleshing.

5. The best index on which to select for efficiency of food conversion under these conditions would be the yield divided by the height.

The proportions of milk stored in the gland cistern (cisternal) and within secretory alveolar tissue (alveolar) were determined for a group of ten cows in mid lactation, yielding ∼ 20 kg milk/d. As a proportion of total milk, cisternal milk, measured at 8 h after milking, ranged from 0·09 to 0·53. Milking frequency was then reduced from twice daily to once daily on two occasions, first for 1 week and then, 2 weeks later, for 2 weeks. Milk yield was reduced during once daily milking in all cows, by an average of 22·8% during the first week (P < 0·001). The amount of decrease varied among cows in a way that was unrelated to pretreatment yield, but the immediate decrease was significantly and inversely correlated with cisternal milk proportion (r = 0·81, P < 0·01). When milking reverted to twice daily, yield recovered to a value not significantly different from pretreatment, and the degree of recovery was inversely related to cisternal milk proportion (r = 0·74, P < 0·01). With the exception of one cow, yield decreases correlated well between the two periods of once daily milking. When treatment weeks were disregarded, lactation persistency (the rate of decline in yield with time) was unaffected by once daily milking. The results show that the ability of individual cows to tolerate once daily milking is related to their cisternal storage characteristics; yield is reduced less in cows that store a greater proportion of their total milk production within the cistern. Once daily milking for up to 2 weeks had no lasting effect on milk yield or lactation persistency, suggesting that there had been no detrimental effect on udder development.

Rheological properties of whey protein concentrate (WPG) solutions were studied in steady shear, using a Bohlin VOR Rheometer, as a function of concentration, temperature, shear rate, shearing time, pH, salt type, salt concentration and solution age. At 22 °C and pH 7, the WPC solutions exhibited Newtonian behaviour up to a concentration of 10% total solids, pseudoplastic behaviour between 10 and 30% and time-dependent shear thinning at 35% and above. The apparent viscosity of solutions at 22 °C and pH 7 was linearly related to concentration up to 8%. The effect of temperature on apparent viscosity in the range 5–60 °C was closely described by the Arrhenius equation. The viscosities of WPC solutions were independent of solution age in the pH range 4–8 at all concentrations up to and including 20%, the precise pH range narrowing as concentration increased. At pH values above or below this range apparent viscosity became dependent on both pH and solution age, the age effect becoming more marked at higher WPC concentrations. Apparent viscosity at pH 7 increased markedly with both CaCl2 concentration and solution age at concentrations above 0·6 M-CaCl2, the age effect in this case increasing with CaCl2 concentration. In contrast, NaCl concentrations of up to 0·8 M-NaCl had little effect on apparent viscosity. The rheological behaviour of WPC solutions changed from time-independent to time-dependent shear thinning at high concentration, at extreme pH values, at high CaCl2 concentration (after ageing) and on heating to above ∼ 60 °C. This change is considered to be caused by the formation of structure in solutions; a 40% solution (at 22 °C and pH 6·75) exhibited classic thixotropic behaviour in a step–shear rate experiment.

The authors report the results of a study aimed at the comparison of the basic chemical composition, the main protein fractions distribution, rennet coagulation properties and Parmigiano-Reggiano cheese yield of vat milk from Italian Brown and Italian Friesian herds. Parmigiano-Reggiano cheese factories which manufacture milk separately from Italian Brown herds and Italian Friesian herds were used in the study. Thirteen cheesemaking trials were performed at 10 different commercial cheese factories. The study was carried out from March to October 2003. For each cheesemaking trial in each factory, approximately 1100 kg milk from Italian Brown cows and from Italian Friesian cows were processed in parallel. The animals involved in the study came from farms with comparable management practices, size, location, number of lactation and days in milking. Each vat contained milk obtained by combining milk collected during the evening milking (partially skimmed milk by natural creaming) and the following morning milking (full-cream milk), from at least 2 dairy herds. Milk from Italian Brown cows is characterised by a higher casein content (27·1 v. 23·7 g/kg; P[les ]0·0001) than Italian Friesian cows' milk. Curd firming time (k20) of Italian Brown cows' milk was markedly lower than that of Italian Friesian cows' milk (6·6 v. 10·0 min; P[les ]0·001). This implies a higher rate of aggregation of para-casein micelles for Italian Brown cows' milk. The coagulum of Italian Brown cows' milk had better rheological properties and lower losses of fat in the cheese whey. Parmigiano-Reggiano cheese yield at 24 h was also higher for Italian Brown cows' milk, +0·99 kg cheese for every 100 kg vat milk.

The effect of heating on plasmin activity in various media, including phosphate buffer pH 7·0, skim milk, blood plasma, solutions of casein and solutions of whey proteins were investigated. Plots of log residual activity υ. heating time were linear at all temperatures from 63 to 143 °C. In buffer solutions the presence of casein led to substantial substrate protection, the Arrhenius plots being linear both in the presence and absence of casein. The activation energy, Ea, for the inactivation reaction, was 62·4 kJ/mol in buffer alone and 58·4 kJ/mol with casein present at 25 mg/ml. In skim milk, despite the presence of casein at a similar concentration, plasmin was no more stable to heat than in buffer alone, and a curved Arrhenius plot was obtained indicating a more complex inactivation mechanism. Heating in the presence of proteins having free -SH groups accelerated the inactivation of plasmin. The role of -SH groups was confirmed by experiments with added α-lactalbumin, in which no free -SH groups occur, and reduced carboxymethylated β-lactoglobulin, both of which were without effect. In blood plasma, plasmin was less stable to heat than in buffer (pH 7·0) or in skim milk. Plasminogen behaved very similarly to plasmin either when activated to plasmin with urokinase before heating or when activated afterwards. A hypothesis is presented to describe the heat inactivation and denaturation of plasmin. Technologically important findings are that in skim milk plasmin was largely unaffected by pasteurization conditions and 30–40% of its activity remained even after ultra high temperature processing conditions.

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.

In the accompanying article, we demonstrated that a live culture of Lactococcus lactis compares favourably with antibiotics for treatment of bovine mastitis in two initial field trials. In an effort to explain the mechanism involved, this study investigated the effect of culture administration on the local immune response. In this respect we initially observed that infusion of the live culture Lactococcus lactis stimulated substantial recruitment of polymorphonucleocytes (PMN) and lymphocytes to the udder. For instance, in one assay, quarters infused with the probiotic experienced a dramatic increase (~20 000-fold) in neutrophils over the first 48-h period from an average value of 83·6 cells/ml pre-treatment to 1·78×106 cells/ml 48 h post-infusion. Levels of the acute phase proteins haptaglobin and milk amyloid A were also elevated significantly in comparison with controls following infusion of the culture. The results of flow cytometric assays also demonstrated that while infusion of a live lactococcal culture led to an enhanced recruitment of PMN to the udder (from 1·85×104 cells/ml pre-infusion to 1·45×106 cells/ml 24 h post-infusion) cell-free supernatant from the same culture was not able to do so, indicating that live Lc. lactis can specifically trigger the mammary immune response to elicit PMN accumulation. These results suggest that the mechanism responsible for this probiotic treatment of mastitis is associated with stimulation of the host intramammary immune system.

Gelation of α-lactalbumin (α-la) incubated with a protease from Bacillus licheniformis (BLP) at 50 °C for 4 h was monitored using small oscillatory shear and the large deformation properties of final gels were characterized by uniaxial compression. Transmission electron microscopy was used to visualize the microstructure. Gels made from α-la (10 g/l) using BLP were almost transparent, although somewhat whitish, and they were more than 20 times stiffer (measured as complex modulus) than equivalent gels made from β-lactoglobulin (β-lg) at the same concentration. The microstructure of the gels consisted of non-branching, apparently hollow strands with a uniform diameter close to 20 nm, similar in overall structure to microtubules. Adding Ca2+ in amounts of 50 or 100 mM changed the spatial distribution of the strands and resulted in a reduction in the failure stress recorded in uniaxial compression. Apart from affecting the microstructure, Ca2+ was shown to be essential for the formation of the gels. It is proposed, that the mechanism behind the self-assembly of the partially hydrolysed α-la into long tubes is a spatially restricted creation of ionic bonds between Ca2+ and carboxyl acid groups on peptide fragments resulting from the action of BLP on α-la. Proteolysis of α-la with BLP in the presence of Ca2+ thus results in formation of a strong gel with a microstructure not previously observed in food protein systems.

The bioavailability of dietary ionised calcium is affected by intestinal basic environment. Calcium-binding peptides can form complexes with calcium to improve its absorption and bioavailability. The aim of this study was focused on isolation and characterisation of a calcium-binding peptide from whey protein hydrolysates. Whey protein was hydrolysed using Flavourzyme and Protamex with substrate to enzyme ratio of 25 : 1 (w/w) at 49 °C for 7 h. The calcium-binding peptide was isolated by DEAE anion-exchange chromatography, Sephadex G-25 gel filtration and reversed phase high-performance liquid chromatography (RP-HPLC). A purified peptide of molecular mass 204 Da with strong calcium binding ability was identified on chromatography/electrospray ionisation (LC/ESI) tandem mass spectrum to be Glu-Gly (EG) after analysis and alignment in database. The calcium binding capacity of EG reached 67·81 μg/mg, and the amount increased by 95% compared with whey protein hydrolysate complex. The UV and infrared spectrometer analysis demonstrated that the principal sites of calcium-binding corresponded to the carboxyl groups and carbonyl groups of glutamic acid. In addition, the amino group and peptide amino are also the related groups in the interaction between EG and calcium ion. Meanwhile, the sequestered calcium percentage experiment has proved that EG-Ca is significantly more stable than CaCl2 in human gastrointestinal tract in vitro. The findings suggest that the purified dipeptide has the potential to be used as ion-binding ingredient in dietary supplements.

A new method for measuring the activity of mesophilic and thermophilic lactic acid bacteria is proposed based on measuring the pH of cultures at extremely short intervals (30–90 s) during growth and calculating several kinetic parameters, i.e. the maximum acidification rate (Vm), the time and pH at which Vm occurred, the time or pH range during which the observed rates were greater than Vm/2. These were easily calculated by coupling the pH meter to a microcomputer.

A new method for characterization of acid production by dairy starter cultures is presented. Microplates with integrated optical pH sensors are developed. Two fluorophores, a pH-sensitive and a pH-insensitive one are immobilised at the bottom of a polystyrene 96-well microtitre plate. The pH-insensitive fluorophore serves as an internal reference and makes calibration unnecessary. The sensor measures pH accurately in optically well-defined media. Particles and fluorophores contained in the bulk medium disturbed the measurements. Despite these disturbances it was possible to clearly sense differences in inoculum type and in inoculum sizes of cultures of Lactococcus lactis and of Streptococcus thermophilus at 30 and 37°C. Besides a pH-related signal there is information about other changes during milk fermentation. The cultivation results were compared with those from the established CINAC-method. From this comparison it can be concluded that the new method can be used reliably to characterize particularly a large number of strains for screening purposes but also for quality control.

The caseinolytic activities at pH 6·8 of polymorphonuclear (PMN) and mononuclear leucocyte homogenates (equivalent to a level of 106 cells/ml milk) were less than the levels of natural milk proteinase activity found in milk from healthy cows. Bulk milks contained ∼ 4 times more milk proteinase activity than the composite milks from individual healthy cows. Isolated blood leucocytes, when added to raw milk of good bacteriological quality and stored at 5 °C, did not readily degenerate and had no detectable effect on the milk proteins even when these cells were completely disrupted by homogenization of the milk. Pasteurization of milk which contained leucocytes caused loss of cell vitality. Extracellular proteinases of psychrotrophic bacteria growing in milk were not detected until the early stationary phase of growth. The total viable count at which this occurred varied greatly. Proteinase production by a pure culture of Pseudomonas fluorescens was not detected in milk stored at 5 °C until a viable count of ∼ 109 colony forming units (c.f.u.)/ml was obtained, whilst normal bulk milks stored at 5 °C produced detectable levels of extracellular proteinase(s) when the psychrotrophic flora reached 107–108 c.f.u./ml. Casein proteolysis by PMN and mononuclear leucocyte homogenates resulted in similar polypeptide maps, but plasmin and bacterial proteinase isolated from a strain of Serratia marcescens resulted in polypeptide maps different from each other and from that produced by the leucocyte proteinase(s). The rate of proteolysis of caseins by the different proteinase sources appeared to be in the order αsl- > β- > > κ-casein for the leucocyte extracts, β- > αsl- > > > κ-casein for bovine plasmin and β- ≈ κ- > αsl-casein for S. marcescens proteinase.

Subclinical mastitis is a costly disease and its diagnosis is difficult. Besides the somatic cell count (SCC) and bacteriology, the differential inflammatory cell count (DICC) is a meaningful tool for mastitis detection. As microscopy is very subjective because of the low number of events to be counted, flow cytometry has often been proposed for the differentiation of milk cells. The objective of this study was to determine whether it is possible to identify subclinical mastitis in cattle at an early stage by a simple and fast flow cytometric method. The aim was to identify the main leucocyte populations in flow cytometric dotplots (polymorphonuclear neutrophils (PMN), lymphocytes and macrophages) and, with these, to elaborate a method of mastitis prognostics. Milk from 15 German Holstein cows was sampled in cross-sectional studies and SCC determined. After preparation, the milk cells were incubated with different specific antibodies that bind to different cell types and also to propidium iodide (PI), which differs between viable and non-viable cells. This procedure made it possible to localize cell types in a flow cytometric dot plot and to differentiate between viable and non-viable PMN. Percentages of viable PMN can be determined by a procedure consisting of a simple centrifugation, incubation with PI, and flow cytometric measurement. So it is possible to quickly determine the stage of the inflammation even in quarters with a low SCC.

Milk obtained from cows which were either infected by clinical mastitis or had been subjected to intramammary infusion of Escherichia coli endotoxin possessed high counts of somatic cells and very high levels of proteinase activity which hydrolysed the caseins almost completely in a few hours at 37 °C. The rate of hydrolysis of β-casein was slightly greater than that of αs1-casein, but in both cases hydrolysis was enhanced by 6 cycles of freezing and thawing to disrupt somatic cell membranes. A study of the relationship between proteinase activity and cell count suggested that only some of the proteinase activity originated in the somatic cells and also that the identity of the cells making up the total cellular population was important. Maximum proteolysis occurred at 50–60 °C, but the temperature-activity curve was a broad peak. Likewise the pH versus activity plot was very broad and was almost flat over the pH range 6–9. Experiments with a number of inhibitors of proteinases failed to give a clear cut pattern of inhibition. All evidence obtained was consistent with the view that several different enzymes with different pH and temperature optima and different specificities contributed to the overall hydrolysis of caseins in these milks. From electrophoretic band patterns one of these enzymes was clearly plasmin, but in high cell count milks other proteinases also became significant.

The electrokinetic potential of bovine casein micelles was determined under a variety of conditions using moving boundary electrophoresis. The technique used allowed sharp stable boundaries to be formed without addition of density gradient-forming materials. Casein micelles are negatively charged and in any one milk sample all micelles had similar electrophoretic mobilities. The electrokinetic potential of micelles was markedly reduced by the action of rennet and to a lesser extent by a decrease in temperature from 30 to 6°C. Addition of sucrose to milk caused a small increase in the electrokinetic potential. Addition of anionic detergent to milk increased both the electrokinetic potential and the rennet coagulation time. Addition of cationic detergent caused a reduction in both these quantities, promoted syneresis of the rennet coagulum and caused coagulation in the absence of rennet if present in concentrations exceeding about 10 mM. Alteration of cation activities by addition of salts to milk caused complex changes, either increasing or decreasing the electrokinetic potential. The data obtained are most readily explained by a model for the casein micelle in which some, but not necessarily all, of the κ-casein is located at the micelle surface.

The proteins of bovine milk are the best-characterized food-protein system. Lactoproteins, which have high biological value, contribute ∼25% of dietary protein in North West Europe, North America and Oceania. However, in protein-rich western diets, milk proteins are frequently more highly valued for their functional properties than for their nutritional qualities. The remarkably high heat stability of the caseinate system permits the manufacture of a range of sterilized, concentrated and dehydrated products while its gelation on very limited proteolysis is the basis of cheese manufacture. Skim-milk powders, caseinates and whey protein concentrates are the most flexible and widely used functional proteins in food processing.

This communication reviews recent studies on milk proteins with respect to molecular and colloidal properties; coagulation by Ca2+, heat and ethanol; and functional properties and their chemical and enzymic modification.

In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66–71 kDa as measured by SDS–PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.

Fermentation of deproteinised whey with kefir grains CIDCA AGK1 was studied focusing on polysaccharide production from lactose. Kefir grains were able to acidify whey at different rates depending on the grain/whey ratio. During fermentation, kefir grains increased their weight and a water-soluble polysaccharide was released to the media. Exopolysaccharide concentration increased with fermentation time, reaching values of 57·2 and 103·4 mg/l after 5 days of fermentation in cultures with 10 and 100 g kefir grains/l, respectively. The polysaccharide fraction quantified after fermentation corresponded to the soluble fraction, because part of the polysaccharide became a component of the grain. Weight of kefir grains varied depending on the time of fermentation. Polysaccharide production was affected by temperature. Although the highest concentration of polysaccharide in the media was observed at 43 °C at both grain/whey ratios, the weight of the grains decreased in these conditions. In conclusion, kefir grains were able to acidify deproteinised whey, reducing lactose concentration, increasing their weight and producing a soluble polysaccharide.

Skim milks were pre-acidified to pH 6·4 and concentrated by ultra-filtration to give retentates with protein levels of 210 g/1. Retentates were blended with skim milk and cream to give standardized milks with protein levels ranging from 30 to 82 g/1. These were used for the manufacture of Cheddar cheese in conventional equipment. Increasing milk protein level resulted in reduced gelation times, increased curd firming rates and a decrease in the set-to-cut time when cutting at equal firmness values (i.e. elastic modulus, G′, ∼ 16 Pa). As the curd firming rates increased with milk protein level, it became increasingly difficult to cut the curd cleanly, without tearing, before the end of the cutting cycle. Reflecting the tearing of curd, and consequent curd particle shattering, fat losses in the running wheys were greater than those predicted on the basis of volume reduction (due to ultrafiltration) for milks with protein levels > 50 g/1. Reduction of setting temperatures, in the range 31–27 °C, and the level of added rennet brought the set-to-cut times and curd firming rates of concentrated milks closer to those of the control milk. While increasing milk protein level in the range 30–70 g/1 had little effect on cheese composition, it resulted in slower proteolysis and maturation.