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.

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.

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.

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 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.

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.

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.

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.

Sub-clinical mastitis (SCM) affects milk composition. In this study, we hypothesise that large-scale mining of milk composition features by pattern recognition models can identify the best predictors of SCM within the milk composition features. To this end, using data mining algorithms, we conducted a large-scale and longitudinal study to evaluate the ability of various milk production parameters as indicators of SCM. SCM is the most prevalent disease of dairy cattle, causing substantial economic loss for the dairy industry. Developing new techniques to diagnose SCM in its early stages improves herd health and is of great importance. Test-day Somatic Cell Count (SCC) is the most common indicator of SCM and the primary mastitis surveillance approach worldwide. However, test-day SCC fluctuates widely between days, causing major concerns for its reliability. Consequently, there would be great benefit to identifying additional efficient indicators from large-scale and longitudinal studies. With this intent, data was collected at every milking (twice per day) for a period of 2 months from a single farm using in-line electronic equipment (346 248 records in total). The following data were analysed: milk volume, protein concentration, lactose concentration, electrical conductivity (EC), milking time and peak flow. Three SCC cut-offs were used to estimate the prevalence of SCM: Australian ≥ 250 000 cells/ml, European ≥200 000 cells/ml and New Zealand ≥ 150 000 cells/ml. At first, 10 different Attribute Weighting Algorithms (AWM) were applied to the data. In the absence of SCC, lactose concentration featured as the most important variable, followed by EC. For the first time, using attribute weighted modelling, we showed that the concentration of lactose in milk can be used as a strong indicator of SCM. The development of machine-learning expert systems using two or more milk variables (such as lactose concentration and EC) may produce a predictive pattern for early SCM detection.

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.

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.

The variation in plasmin activity and proteose-peptone (PP) content in milks from 81 cows of four breeds was statistically analysed. Plasmin activity increased with parity and stage of lactation and differed between breeds, but the breed effect was removed when adjustment was made for differences in milk casein content. The negative correlation found between plasmin activity and casein content seemed to be expressed only under assay conditions and was thus probably an artefact. The linear regressions of proteose-peptones in fresh (PP) and cold-stored milks (ΔPP) on plasmin activity were highly significant (P < 0·001). However, the variation in plasmin activity explained only 38 and 33%, respectively, of the variation in PP and ΔPP. At apparent zero plasmin activity, PP and ΔPP were significantly different from zero (P < 0·001) and the intercepts represented 77 and 46%, respectively, of the averages. PP and ΔPP were significantly higher in milks containing the BB genotype of ²-lactoglobulin than in milks with the AA genotype.

The infra-red, absorption of milk and its main constituents has been recorded and it has been found possible to determine quantitatively the fat, protein and lactose contents from measurements of the intensities of the respective absorption peaks at 5·73, 6·46 and 9·6 μm. Solids-not-fat (SNF) content can be obtained from a single intensity measurement at 7·9 μm where the fat shows no attenuation and where the protein and lactose extinction coefficients are approximately equal. Photometric errors due to natural variations in fat globule size distribution can be eliminated by prior homogenization. Based on these observations, an automatic infra-red milk analyser (IRMA) has been constructed, which can determine the fat, protein, lactose and SNF in a pre-warmed milk sample in under 1 min. Preliminary tests carried out on 50 milk samples from individual cows, representing 3 different breeds, gave standard deviations from the chemical analyses for the percentages of fat, protein, lactose and SNF of 0·10, 0·07, 0·07 and 0·19, respectively. The standard deviations for the percentages of fat and SNF on 60 farm bulk milk samples were found to be 0·08 and 0·12.