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.

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.

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.

The protein fraction of milk contains several components with physiological significance for the development of the newborn. Among them, immunomodulatory peptides and lactoferrin exemplify the complexity of biologically active substances of milk. Immunomodulatory peptides have latent activity within the native protein and are generated after proteolysis during gastrointestinal transit. Once they are generated, they modulate mucosal immunity, possibly by guiding the local immune system until it develops its full functionality. Lactoferrin is another milk bioactive compound with nutritional and health promoting properties; it modulates the microbial intestinal environment, displays anti-microbial activity against various pathogens and stimulates the establishment of beneficial microflora. The following overview focuses on the importance of immunomodulatory peptides and lactoferrin for the maturation of intestine and immune system that are functionally immature in the newborn.

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.

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 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.

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.

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 method is outlined for accelerating ripening in Swedish semihard cheese by increasing the number of lactic starter bacteria present in the cheese without impairing characteristic texture and flavour. In addition to the normal starter inoculum, suitable lactic starter bacteria whose lactic-acid-producing activity had been greatly reduced by previous sublethal heat treatment, were added to the cheesemilk. When suspensions of streptococci and lactobacilli cultivated at a constant pH were heated at 59 and 69°C respectively acid production was retarded by 5–10 h, which was found to be sufficient for the cheese-making. Proteolysis was lowered only 10–30% by these heating temperatures. Bacterial cell suspensions, prepared by the methods outlined and added to the cheesemilk, were incorporated in cheese curd to extents depending on the amount added and the type of starter. The number in the final cheese could be increased to a maximum of 4–5 times that of control cheese. No adverse effect of the extra starter bacteria on pH, fat content and water content of cheeses 24-h old was observed. Proteolysis, measured as the increase in trichloroacetic acid and phosphotungstic acid (PTA)-soluble N, increased with increasing number of cells in the cheese. Organoleptic judgements showed a positive correlation (r = 0·81) between taste and PTA-soluble N, which in turn was influenced by the number of cells in the final cheeses.

Eight strains of Geotrichum candidum isolated from cheese were compared for aroma production. Twenty-four components, including alcohols, methyl ketones, fatty acids and esters, were identified by gas–liquid chromatography and gas chromatography–mass spectrometry of the volatile compounds produced by G. candidum cultures. Strains could be differentiated by their ability to produce dimethyldisulphide, phenol, phenylethanol and methyl ketones.

Mechanisms controlling exchange and partition of minerals and β-casein between micellar and soluble phases during cooling have been studied using suspensions of milk proteins of varying concentrations in the presence of aqueous phases having varying mineral compositions. In these studies the final equilibrium between Ca and phosphate was found to be regulated by formation of Ca phosphates of varying solubilities. The effect of the total casein level on mineral and protein equilibria was also studied. In milk, part of the β-casein content is bound to micelles by hydrophobic bonds; the proportion present in this form was increased by spontaneous micellar demineralization during cooling as well as by addition of a complexant to milk. At low temperatures, an equilibrium between the micellar and the monomeric states was reached which depends upon the total casein level and on the hydrophobic-bonded β-casein content. Not all of these hydrophobic bonds were broken when milk was cooled.

A study of casein micelles was made with the electron microscope, using very thin sections cut from micelles embedded in Araldite. The micelles appear to be built up of units that are approximately spherical, about 100 Å in diameter and of about 300000 molecular weight.