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.

The main components of bovine whole casein were characterized by electrofocusing; pi values of αs1-, β-, κ-, γ1-, γ2- and γ3-caseins were determined. A further identification of casein components was achieved by a 2-dimensional electrophoresis study. 2-Dimensional patterns of γ-caseins obtained from a hydrolysate of β-cascin by bovine plasmin are in good agreement with those of γ-caseins naturally present in whole casein.

The water-soluble extract of Cheddar cheese was fractionated by diafiltration using 10 kDa cut-off membranes. Peptides were isolated from the diafiltrate retentate by chromatography on DEAE-cellulose with a linear NaCl gradient in 50 mM-Tris-HCl, pH 8·6, and reversed-phase HPLC or electroblotting from urea-PAGE gels. Peptides were identified by determining N-terminal amino acid sequences and mass spectrometry. Most (45) of the total 51 peptides identified in the diafiltrate retentate originated from β-casein, especially from a short region in the N-terminal half of the molecule. Only six peptides originated from αs1-casein; β-lactoglobulin was also identified in the retentate. The origin of most of these peptides could be explained on the basis of known specificities of lactococcal cell envelope proteinases.

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.

To investigate matrix-specifity of probiotic effects and particularly of the reduction of antibiotics-associated diarrhea, a controlled, randomized, double-blind study was performed, in which 88 Helicobacter pylori-infected but otherwise healthy subjects were given for eight weeks either a) a probiotic fruit yoghurt “mild” containing Lactobacillus acidophilus LA-5 plus Bifidobacterium lactis BB-12, n=30), b) the same product but pasteurized after fermentation (n=29) or c) milk acidified with lactic acid (control, n=29). During week five, a Helicobacter eradication therapy was performed. Helicobacter activity was measured via 13C-2-urea breath tests and antibiotic-associated diarrhoea and other gastrointestinal complaints were recorded by validated questionnaires. In intervention group a, b and c the mean number of days with diarrhoea was 4, 10 and 10 (P<0·05), the frequency of episodes 17%, 7% and 27% (n.s.), and the change in total symptoms score before antibiotics treatment was −1·4±1·1, −1·2±1·1, 2·6±1·1 points/four weeks (P<0·05). All milk products decreased Helicobacter activity by 18 to 45% without significant differences between groups. The observed decrease in Hel. pylori activity seems to be not or not only due to probiotic bacteria but (rather) to components of acidified milk (most probably lactic acid). Fruit-yogurt-like fermented milk products with living probiotic bacteria significantly shorten the duration of antibiotics-associated diarrhoea and improve gastrointestinal complaints. Fruit yogurt-like fermented milk is a matrix suitable for probiotic bacteria.

The lactational variation in milk protein composition and renneting properties and their relationship to the cow's body condition at calving were investigated in 39 Danish Holstein first lactation cows fed on a well balanced standard diet. All milk characteristics measured were significantly affected by stage of lactation (P<0·01). Casein as a proportion of total milk nitrogen reached a maximum in mid lactation. The proportion of αs- and κ-casein in total casein decreased and the proportion of β-casein increased systematically during lactation while the proportion of γ-casein was lowest in mid lactation. The α-lactalbumin content of milk and its proportion of total whey proteins decreased during lactation. Renneting time was highest and curd firmness lowest in mid lactation. These results appeared to reflect a low degree of proteolysis in late-lactation milks compared with several other investigations, probably because of the good nutritional state of the cows. The body condition at calving affected proteolysis and the renneting properties of milk. A good body condition increased the content of whey protein in total milk nitrogen and of γ-casein in total caseins (P<0·05); in addition, curd firmness was improved (P<0·01) and aggregation time was reduced (P<0·05). We suggest that these effects were related to the fat metabolism and energy status of the cows during lactation. The interrelationships between the milk characteristics were evaluated by factor analysis to support the interpretation.

Four lactating Holstein cows (average milk yield: 20±3 l/d) were used to develop and validate a method for estimating the size of udder cisterns (Sinus lactiferi) using ultrasonography. A sectorial transducer probe of 5 MHz, placed in contact with the teat in a parallel cranial position, was used to obtain vertical scans of the udder in two perpendicular planes with the teat canal axis as reference. Udder scans for each udder quarter were taken randomly at intervals of 4, 8, 12, 16, 20 and 24 h after milking. Glandular parenchyma (echogenic) and lumen of the cisterns full of milk (anechogenic) were evident in the scans, the calculated area of the anechogenic portion being defined as cistern area. Cistern areas measured in perpendicular scans were highly correlated. Immediately after each measurement, cisternal milk was removed from each quarter using a teat cannula after i.v. injection of an oxytocin-receptor blocking agent. Alveolar milk from each quarter was then obtained by machine milking after i.m. injection of oxytocin. Cistern area and cisternal milk volume increased with length of milking interval showing a curvilinear pattern with a plateau after 16 h. Correlations between cistern area and cisternal milk volume were positive and significant (P<0·001) at all intervals but showed the highest values with the smallest residual standard deviations at 8 h (r=0·88) and 12 h (r=0·84). Since 8 h has previously been identified as a suitable time at which to determine cisternal milk volume for the purposes of defining suitability for different milking strategies, we conclude that ultrasonography provides a satisfactory, non-invasive method for determination of milk storage characteristics in dairy cows.

Optimum conditions have been determined for the use of Hide Powder Azure (HPA) to estimate extracellular proteinases produced by psychrotrophic Gram-negative bacteria. This assay is quicker, more precise and 3·2 times more sensitive than assays of soluble N released from casein. It provides a simple alternative method for monitoring proteinase purification schemes. With the modification described it is now possible to determine proteinase activities directly in milk. In its simplest form the HPA assay is sufficiently sensitive to detect proteinases produced by as few as 1·5 x 106 c.f.u. ml-1 psychrobtrophs without the necessity for any sample processing.

The milk-clotting enzyme found in the flowers of Cardo (Cynara cardunculus) was investigated as to its suitability as a substitute for traditional animal rennet used in cheese-making. The influence of milk pH, temperature and quantity on the clotting activity of this enzyme was studied. Rheological behaviour of cow's-milk and sheep-milk curds, from renneting to cutting, was determined with the Plint cheese curd torsiometer for various Ca contents and pH values. Edam, Serra and Roquefort cheeses were made and the protein breakdown which occurred in the cheese during the ripening period was determined. Animal rennet was used as a control in all the experiments. The enzyme from Cardo was a satisfactory substitute for animal rennet with cow's milk and was even more suitable for sheep's milk. It was found to be a very good clotting enzyme for soft-bodied cheese like Serra, but because of its high proteolytic activity it presented some problems in Edam cheese-making. In Roquefort cheese it gave satisfactory results, but with some loss in yield.

Bovine κ-caseinoglycopeptides (i.e. residues 106–169, CGP) were prepared from κ-casein digested with rennin and a commercial whey protein concentrate. CGP from whey protein concentrate was further divided into seven CGP fractions having different carbohydrate compositions using FPLC. Unfractionated CGP inhibited lipopolysaccharide (LPS)- and phytohaemagglutinin (PHA)-induced proliferative responses of mouse spleen cells and rabbit Peyer's patch cells. The unfractionated CGP also inhibited antibody responses to sheep red blood cells in mouse spleen cell cultures. However, seven CGP fractions having zero to five N-acetylneuraminic acid (NANA) residues had different inhibitory effects on both LPS- and PHA-induced proliferative responses of mouse spleen cells. The inhibitory effect on PHA-induced proliferative responses increased with increasing numbers of NANA residues, whereas that on LPS-induced proliferation was highest with the CGP fraction having two NANA residues. Both inhibitory effects decreased significantly after neuraminidase or chymotrypsin digestion. These findings indicate that both the carbohydrate (particularly the NANA residues) and the polypeptide portions are essential for inhibitory effects on LPS- and PHA-induced proliferative responses of mouse spleen cells.

The aim of this study was to assess the effect of chemical composition, coagulation properties, pH, and titratable acidity (TA, SH°/50 ml) of vat milk on Grana Padano cheese yield (CY) under field conditions. Twelve cheese-making sessions were carried out from February to December 2009 in a dairy cooperative of Grana Padano Consortium (Italy), for a total of 96 vats of milk processed. For each vat, samples of raw milk were collected and analysed for quality traits (fat, protein, and casein contents), pH, TA, and milk coagulation properties (MCP), measured as rennet coagulation time (RCT, min), curd-firming time (k20, min), and curd firmness (a30, mm). Cheese yield was expressed as kilograms of cheese per 100 kg milk transformed, and was measured after 2 d of drainage. Fat, protein, and casein contents were positively and strongly correlated with CY (coefficients of correlation, r = 0·72, 0·88, and 0·84, respectively; P < 0·001). Coagulation properties were moderately and significantly (P < 0·001) related to CY: milk that coagulated earlier and had stronger a30 was associated to greater CY. Cheese yield was analysed with a model that accounted for fixed effects of cheese-making day, fat and protein content, TA, and a30. Significance was found for all the effects (P < 0·05). Milk characterised by high values of a30 resulted in higher CY than milk with low values of a30, indicating that MCP could be used as indicators of cheese-making efficiency. Future research should investigate the relationships between MCP and quality of cheese, and explore the feasibility of including MCP in multiple component milk pricing system for Grana Padano cheese production.

In dilute buffers ⋟ pH 5·8, chymosin hydrolysed bovine αs1-casein to αs1-I, αs1-II and αs1-III/αs1-IV in a sequential manner while at pH 4·6 αs1-casein was hydrolysed to αs1-I which was then hydrolysed to αs1-V. In the presence of 5 % (w/v) NaCl at pH 5·2, αs1-casein was hydrolysed to αs1-I which was then hydrolysed to αs1-VII and αs1-VIII. αs1-I, αs1-II and αs1-III/αs1-IV were isolated by chromatography on cellulose phosphate followed by preparative slab-gel electrophoresis; αs1-V was isolated by repeated preparative slab-gel electrophoresis and αs1-VII by gel filtration on Sephadex G-150 followed by preparative slab-gel electrophoresis. The mol. wts of the peptides, estimated by gel filtration on Sephadex G-100, were 21000, 17600, 15600, 19900 and 14600 for αs1-I, αs1-II, αs1-III/αs1-IV and αs1-V and αs1-VII respectively. Characterization of the peptides by amino acid, phosphorus and terminal residue analysis showed that they probably consisted of segments of the αs1-casein chain as follows: αs1-I: residues 24/25–199; αs1-II: residues 24/25–169; αs1-III/αs1-IV: residues 24/25–149–150; αs1-V: residues 29/33–199; αs1-VII: residues 56–179. Peptide bonds close to phosphate residues on the αs1-casein chain were not hydrolysed by chymosin at high pH values (⋟ 5·8) when the phosphate groups were charged, but became available for hydrolysis when the reaction pH was reduced. Proteolytic specificity was also modified by NaCl.

Coagulation time–pH curves with a coagulation-time minimum around pH 6·8 (type A curve) could progressively become type B (no minimum) as the heating temperature was decreased from 150 to 130°C. The short coagulation time that most milks have when pH is around 6·8 was found to be the result of a ‘premature’ coagulation, probably caused by calcium phosphate deposition on the larger caseinate micelles. This is followed by a second coagulation, not visually detected, that coincides with the coagulation time that would be expected if no coagulation-time minimum existed on the coagulation time-pH curve. The coagulation time of milks giving type A and type B curves may therefore not be comparable.

Forewarming milk for 30 min at 80°C can introduce or accentuate a coagulationtime minimum when the milk is subsequently heated at a higher temperature. The effects of adding β-lactoglobulin, copper and N-ethylmaleimide on the heat stability of milk were examined and explanations proposed for these effects.

Specific PCR, amplifying a fragment of the mitochondrially encoded cytochromeb gene, are developed for discrimination of chicken, turkey, pig, cow and sheep. These PCR tests could be applied for detection and discrimination of animal food ingredients. For origin identification of milk, the bovine PCR is applied on DNA extracted with the DNeasy™ Tissue kit. A PCR of 30 cycles reached a sensitivity of 720 somatic cells in the PCR reaction corresponding to 2·9 × 104 cells per ml milk. A PCR of 40 cycles could detect 0·72 to 0·144 somatic cells in the PCR reaction corresponding to 29 to 2·9 cells per ml milk.

The in vivo action of gastric proteinases on bovine milk proteins was studied in rats fed with skim-milk, by analysing gastric contents after 30, 60 and 240 min of digestion. Gastric proteolysis was already marked after 30 min of digestion and liberated a large number of peptides with different molecular weights. αs1-Casein and β-casein were still the main components of the stomach contents together with α-lactalbumin and β-lactoglobulin, which were little degraded, even after 240 min of digestion. A statistical analysis (multivariate method), made on several parameters (such as pH, N, and NPN) showed changes in the stomach contents during the digestion. The amino acid composition of the protein fraction was close to that of the diet, whilst that of the non-protein fraction was very different, being between the amino acid composition of the endogenous proteins and that of the diet.