Milk contains the alkaline proteinase plasmin and lysosomal proteinases; the significance of the latter is ill-defined. The objective of this study was to investigate composition and activities of several different proteolytic enzymes in milk samples of varying somatic cell count (SCC). Increasing milk SCC was correlated with increased plasmin, cathepsin D and cysteine protease activities, with concomitant increases in proteolysis in milk. Addition of plasmin inhibitors confirmed the heterogeneity of proteinase activities in milk, as urea-PAGE analysis of milk samples showed casein hydrolysis in milk after 7 d storage even in samples with inhibitors added; extent and heterogeneity of proteolysis was correlated with milk SCC. Rennet coagulation properties were not significantly correlated with SCC, or activities of measured enzymes. Milk of increasing SCC also exhibited decreased physical stability during incubation of milk at 37 °C. Pasteurized milk was more stable than raw milk, suggesting that the enzyme(s) or mechanisms leading to such instability are impaired by pasteurization. Overall, milk has a very heterogeneous proteolytic enzyme population, with a higher significance of non-plasmin enzymes, such as cathepsin D and cysteine proteinases, than perhaps previously recognised.

An attempt at classifying the influence of different characteristics of milk and cheesemaking on the rheological properties of Abondance cheese is presented. Abondance is a traditional farmhouse French hard cheese with protected denomination of origin (PDO). Thirty-nine cheeses made from unpasteurized cows' milk were sampled. Spline partial least squares regression was used to relate milk properties and cheesemaking practices to rheological properties of the six-month-old cheeses. These properties were the deformability modulus and the strain and stress at fracture measured by compression. Milk properties and technological practices had overall the same degree of relationship with the rheological properties investigated: plasminogen-derived activity in milk and mineral-protein equilibrium, on the one hand, and brining and resting, on the other hand. However, acidification kinetics and 1-d pH, which result from both milk properties and technological practices, showed the strongest relationships with rheological characteristics. Factors that were most appropriate for modelling Abondance rheological properties are discussed.

Psychrotrophs, particularly Pseudomonas spp. are known to be the main determinants of the shelf-life of pasteurized milk and refrigerated raw milk. It is presumed that they mainly cause spoilage through the elaboration of proteinase and lipase enzymes. At the time of this research, under the relevant European Directive, one of the means of determining the quality of pasteurized milk was the pre-incubated count, which involves incubating the milk sample for 5 d at 6°C followed by a plate count. Examination of numerous pre-incubated counts revealed a bimodal rather than a normal distribution indicating that the types of contaminants in pasteurized milk may be as important as their initial concentration. Pseudomonads that gave particularly high (>5×106 cfu/ml) and low (<103 cfu/ml) pre-incubated counts were isolated (high and low count isolates respectively). After the organisms had been subjected to a cold shock no consistent trend between the groups of isolates was detected with respect to lag phase duration. However, the high count isolates consistently had a faster exponential growth rate. Unexpectedly, with the exception of one isolate, the low count isolates produced detectable proteinase and lipase earlier. In addition, with one exception, maximal proteinase and lipase production was observed with the low count isolates. These findings indicate that there is no causal relationship between selective growth advantage and ability to produce proteinase and lipase. It also indicates that the spoilage of pasteurized milk is a complex phenomenon and is worthy of further research.

It has been hypothesized that certain consumers tolerate untreated cow's milk, but react to processed (i.e. homogenized and pasteurized) cow's milk although they do not suffer from IgE-mediated cow's milk allergy or lactose intolerance. The aim of the study was to compare the tolerance of unhomogenized and homogenized cow's milk in lactose tolerant adults who had repeatedly experienced better tolerance of unhomogenized than homogenized milk. Forty-four subjects were challenged with homogenized and unhomogenized cow's milk for five days in a randomized, double-blind, cross-over study. No differences in the symptoms during the challenges were found. Roughly half the subjects tolerated the homogenized milk better and the other half tolerated the unhomogenized milk better. The results of this study show no difference in the tolerance of homogenized and unhomogenized milk in adults with self-reported symptoms suggestive of hypersensitivity to homogenized milk.

Although widely adopted by the chemical and pharmaceutical industries in recent years, little published data is available regarding possible applications of high pressure homogenisation for dairy products. The objective of this work was to compare the effects of conventional (18 MPa, two-stage) and single or two-stage high pressure homogenisation (HPH) at 50–200 MPa on some properties of raw whole bovine milk (∼4% fat). Fat globule size decreased as HPH pressure increased and, under certain conditions of temperature and pressure, HPH yielded significantly smaller fat globules than conventional homogenisation. Fat globule size was also affected by milk inlet temperature. The pH of all homogenised milk samples decreased during 24 h refrigerated storage. Total bacterial counts of milk were decreased significantly (P<0·05) for milk samples HPH-treated at 150 or 200 MPa. Whiteness and rennet coagulation properties of milk were unaffected or enhanced, respectively, as homogenisation pressure was increased. Average casein micelle size decreased slightly when skim milk was homogenised at 200 MPa. Thus, HPH treatment has several, potentially significant, effects on milk properties.

The effects of exposure of slices of Havarti cheeses to monochromatic light of wavelengths 366 nm, 405 nm, and 436 nm, respectively, were studied by tristimulus colorimetry, solid-phase microextraction gas chromatographic analysis of volatiles, and open-end fluorescence spectroscopy. Having determined the photon fluxes of the three wavelengths by ferrioxalate actinometry, it was possible to quantify the effects of light exposure in an absolute manner. For all analyses, the most severe effects were caused by visible light, leading to colour bleaching, change in hue, riboflavin degradation, and formation of the secondary oxidation products hexanal, 1-pentanol, and 1-hexanol. Apparent quantum yields for formation of hexanal and 1-pentanol were found to be insignificantly different for 405 nm and 436 nm exposures, having values of (3–5).10−5 mol.einstein−1 and (9–13).10−5 mol.einstein−1, respectively. These compounds were not formed when exposed to 366 nm light. In contrast, 1-hexanol was formed when exposing cheese to all three wavelengths, resulting in apparent quantum yields of (2–6).10−5 mol.einstein−1. The results obtained are discussed in relation to the interplay between inherent product colorants, light sources, and transmission characteristics of the packaging materials.

The effects of dietary inclusion of freeze-dried goat and cow milk on the utilization of zinc and selenium, and on the metabolic fate of zinc, were studied in transected (control) and resected rats (resection of 50% of the distal small intestine). Intestinal resection reduced the apparent digestibility coefficient and zinc retention in the cow milk diet, whereas these biological indices were similar in transected (control) and resected rats with standard (without milk) and goat milk diets. The apparent digestibility coefficient and retention of selenium were not affected by intestinal resection in the animals fed with the three diets studied. In transected (control) and resected rats, the apparent digestibility coefficient and retention of zinc and selenium were higher for the goat milk diet than for the other two diets. Zinc deposits in the organs, expressed as μg/g dry weight were in order of decreasing concentrations: femur, testes, sternum, liver, kidney, heart, spleen, longissimus dorsi muscle and brain. Deposits were greatest with the goat milk diet, followed by the standard diet and were lowest for the rats given the cow milk diet, both for transected (control) and resected animals.

We conclude that consumption of the goat milk diet produces a greater bioavailability of zinc and selenium and a greater deposit of zinc in key organs, for both the transected (control) and the resected rats, with respect to the standard diet and the cow milk diet.

Bovine β-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6·8 for 10 min at 30, 37 and 44 °C. The extent of β-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated β-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact β-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of β-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.

The objective of this study was to determine the effect of high pressure homogenisation (HPH) on alkaline phosphatase activity and plasmin and plasminogen-derived activities in raw whole bovine milk. Milk (approximately 4% fat) was treated by two-stage conventional homogenisation (18 MPa) or single or two-stage HPH at 50, 100, 150 or 200 MPa. Inactivation of plasmin and plasminogen-derived activities was evident in conventionally homogenised samples, and increased as HPH pressure increased. Two-stage HPH reduced both activities to a greater extent than single-stage HPH. Milk inlet temperature had a significant effect on residual plasmin and plasminogen activities of HPH-treated milk samples, especially those treated at 50 MPa. Inactivation of plasmin and plasminogen on HPH-treatment (150 MPa) of milk samples of varying fat contents (0–10%) was also investigated; there was a curvilinear relationship between residual plasmin and plasminogen-derived activities and fat content in the ranges 0–2% and 0–4%, respectively, with little additional inactivation at higher fat contents. Thus, indigenous proteolytic activity of milk is clearly affected by HPH. However, all homogenised milk samples retained active alkaline phosphatase, indicating that thermal conditions during HPH did not equate to that of conventional high temperature short time pasteurisation, and that the wide range of forces experienced by milk during HPH treatment does not inactivate the latter enzyme.

The interaction of κ-casein and β-lactoglobulin is fundamental to all heat-induced modifications of milk product functionality, such as the heat stability of concentrated milks. Purified native κ-casein B and β-lg A solutions were heated at 80 °C at pH 6·7 separately and in a mixture. The circular dichroism spectra in the near UV indicated irreversible changes in the disulphide bonding patterns involving both proteins. Alkaline- and SDS-PAGE of heated samples showed that, in the presence of κ-casein, less β-lg was converted into β-lg polymers and the rate of loss of native β-lg was greater. When κ-casein was added to previously heated β-lg and the mixture was heated, the κ-casein reacted with the heat-induced β-lg polymers more readily than with the β-lg native monomers. The formation of β-lg dimers, trimers etc. was diminished. It was concluded that, when β-lg and κ-casein were heated together, β-lg formed thiol-exposed monomers, which reacted with each other or with the native κ-casein depending on the relative concentrations of β-lg and κ-casein. The products of these reactions included some disulphide-bonded 1[ratio ]1 β-lg[ratio ]κ-casein complexes, some monomer κ-casein and a range of large aggregates held together by either or both disulphide bonds and hydrophobic association.

Acid-precipitated rabbit ‘whole casein’ was digested by trypsin, chymotrypsin, pepsin, and clostripain to screen for possible peptides with antibacterial properties. The peptide fragments were separated by reversed-phase chromatography. The collected fractions were pooled and their antibacterial properties tested against Escherichia coli, Bacillus subtilis and Staphylococcus lentus. Three antibacterial peptide fragments derived from tryptic digestion of rabbit casein were isolated and identified. Their sequences were found as follows: HVEQLLR (residues 50–56 of β-casein), ILPFIQSLFPFAER (residues 64–77 of β-casein), and FHLGHLK (residues 19–25 of αs1-casein). The three peptides were synthesized and found to exert antibacterial effect against Gram positive bacteria only. Proteolytic digestion of rabbit casein by chymotrypsin, pepsin and clostripain yielded several peptide fragments with antibacterial activity. Since antibiotic peptides can be released from casein during the digestion of milk proteins, our results suggest a possible antibacterial function of rabbit caseins. It is conceivable that antibacterial peptides can be generated by endopeptidases of the mammalian gastrointestinal tract possibly providing protection for new-born rabbits against aggression of micro-organisms.

The enhancement of the strength of set acid gels by heating milk was related to rheological parameters (water retention capacity, storage modulus) of corresponding stirred gels. To obtain accurate rheological data from stirred gel it was necessary to maintain a constant granulometry of gel particles and to recognize time after stirring as a contributing factor. Two hours after stirring, the gel exhibited a higher storage modulus when milk was heated above 80 °C. A measurement of viscosity of just-stirred yoghurt was sufficient to predict correctly the quality of a stirred gel analysed by viscoelastic measurements. Increased resstance to syneresis of just-stirred gels was related to higher viscosity. The quantity of β-lactoglobulin (β-lg) bound to casein micelles explains the improvement of these gel qualities. We have considered that the structure of the initial firm gel (mesostructure level) was conserved in fragments within the stirred gel. Consequently, the explanation given by various authors for the effect of heating milk on the properties of set gels can also be applied to stirred gels. The same mechanism, described in literature for structure formation of set gels from acidified milk is purposed to explain the role of heating milk on the recovery of gel structure after stirring. The β-lg association with casein micelles during heating favoured micelle connections during the acidification. It also favoured the association of gel fragments after stirring during the recovery in gel structure.

Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including αs1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro αs1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.

The production of similar or even identical bacteriocins by different lactic acid bacteria is not a rare event. To take advantage of this finding, genetic determinants of the Lactobacillus K7 bacteriocins were tested for putative homologies with previously described bacteriocins of the Lactobacillus acidophilus group through polymerase chain reaction (PCR). Among specific primer pairs of seven known bacteriocins, derived from their respective sequences, only acidocin LF221 A and B primers amplified fragments in chromosomal DNA of K7 strain that revealed strong similarity over small regions of LF221 bacteriocins. Treatment of Lactobacillus K7 with ethidium bromide and mitomycin C was ineffective in generating non-bacteriocinogenic derivatives and had no impact on plasmid loss either. Classification studies elucidated Lactobacillus K7 as a member of the Lactobacillus gasseri species.

Nutritional deficiency is commonly associated with impaired immune response and the relation between infection and malnutrition is synergic. Probiotics, especially lactic acid bacteria are immunomodulatory. The aim was to determine whether optimal doses of Lactobacillus casei and yogurt, used as adjuvants in a re-nutrition diet in a non-severe malnutrition experimental model, protect against Salmonella typhimurium and Escherichia coli. Groups of malnourished mice were used, which were re-nourished with milk for 7 or 14 d. After that, both groups of mice received the optimal doses of Lb. casei and yogurt supplements. We measured IgA+ and IgG+-B cells and phenotypic markers of T lymphocytes; CD3+, CD4+ and CD8+ cells. We also determined αβ and γδ T cell receptor (TCR). The ability to protect against Sal. typhimurium and Esch. coli infections and specific S-IgA were assessed. Probiotics complemented the effects of the re-nutrition diet, by stimulating recuperation of the activity of immune cells that improved protection against infections.

When skim milk at pH 6·55 was heated (75 to 100 °C for up to 60 min), the casein micelle size, as monitored by photon correlation spectroscopy, was found to increase during the initial stages of heating and tended to plateau on prolonged heating. At any particular temperature, the casein micelle size increased with longer holding times, and, at any particular holding time, the casein micelle size increased with increasing temperature. The maximum increase in casein micelle size was about 30–35 nm. The changes in casein micelle size were poorly correlated with the level of whey protein denaturation. However, the changes in casein micelle size were highly correlated with the levels of denatured whey proteins that were associated with the casein micelles. The rate of association of the denatured whey proteins with the casein micelles was considerably slower than the rate of denaturation of the whey proteins. Removal of the whey proteins from the skim milk resulted in only small changes in casein micelle size during heating. Re-addition of β-lactoglobulin to the whey-protein-depleted milk caused the casein micelle size to increase markedly on heat treatment. The changes in casein micelle size induced by the heat treatment of skim milk may be a consequence of the whey proteins associating with the casein micelles. However, these associated whey proteins would need to occlude a large amount of serum to account for the particle size changes. Separate experiments showed that the viscosity changes of heated milk and the estimated volume fraction changes were consistent with the particle size changes observed. Further studies are needed to determine whether the changes in size are due to the specific association of whey proteins with the micelles or whether a low level of aggregation of the casein micelles accompanies this association behaviour. Preliminary studies indicated lower levels of denatured whey proteins associated with the casein micelles and smaller changes in casein micelle size occurred as the pH of the milk was increased from pH 6·5 to pH 6·7.

An objective method for determining the coagulum cutting time is needed to improve consistency and processing efficiency of goats' cheese. A fibre optic sensor was used to measure the backscatter of near-infrared radiation at 880 nm during the coagulation of skimmed goats' milk for the purpose of predicting coagulum cutting time. A randomised block design, replicated three times, was used to test the effect of three protein concentrations (3, 5 and 7% (w/w)) on diffuse reflectance parameters for cutting time prediction of milk coagulated at five different temperatures (20, 25, 30, 35 and 40°C) to assure a wide range of coagulation rates. The inclusion of a protein term in the existing algorithms was essential to reduce the standard error of prediction to under 6·2 min. An algorithm including a time-based parameter and a protein term, Tcut=β0T2 min (1+γ% Protein) was found to predict cutting time with a SEP of 2·42 min and an R2 of 0·98. γ was considered constant (γ=−0·0674, goats' milk) representing the protein effect on β0. Algorithms using response-based parameters (such as change in reflectance ratio) and the composition parameter protein required additional regression parameters such as temperature and an intercept term to predict the cutting time with the same precision as algorithms using only time-based parameters. Time-based parameters were found to decrease proportionally with increasing temperature and decreasing protein concentration. Response-based and mixed-based parameters were found to decrease with decreasing temperature. Reflectance ratio at cutting time did not significantly change with protein concentration for skimmed goats' milk. The activation energy of κ-casein hydrolysis was calculated based on changes in reflectance profile parameters and was found to be in the range 63–72 kJ mol−1.

Simplified designs of milk yield recording based on the yield of a single monthly milking, adjusted or not for interval between milkings and for production level, were simulated and evaluated for 3173 ewe-test-day records belonging to 155 lactations of Churra dairy ewes. Losses of precision associated with simplified methods were evaluated by comparing estimated lactation yields with those observed both in a reference plan, where the two daily milkings were recorded at weekly intervals, and in the official A4 milk recording (monthly records of the two daily milkings). Estimates of lactation yields were less precise when the usual monthly designs were compared with a weekly sampling of both a.m. and p.m. milkings. The losses of precision were high at 9·4–36·2% including the A4 plan. The yield from only the milking period was more predictable than milk yield from the whole lactation (suckling and milking periods) and should consequently be adopted in dairy ewes. All options with one daily milking every month were more accurate when the corresponding plan was based on, or began with, the a.m. milking (loss of precision 14·9–15·8%). There was no evidence of improvement in sampling accuracy by adjusting for the preceding interval between milkings or production level. For practical and economic reasons, the design alternating a.m. and p.m. milkings every month, without adjustment, is suggested for ovine milk recording.

In the context of the general applicability of hydroxymethylfurfural (HMF), lactulose and furosine as time-temperature integrators (TTIs) for thermal processing of milk, the influence of milk fat content was studied. Formation kinetics were analysed for milk with fat content of 4·0±<0·1%. In previous experiments, it was observed that, under isothermal and non-isothermal heating conditions, formation of the three chemical compounds could be described by pseudo-zero order kinetics. Since the kinetic model was known, the experimental design could be simplified. Data were analysed by a non-linear regression procedure and results were evaluated by construction of joint confidence regions and temperature time tolerance (TTT-) diagrams. Formation kinetics of HMF and lactulose was not affected by milk fat content. Regarding furosine, significant differences were observed between kinetic parameters in whole, semi-skimmed and skimmed milk. The observed differences however were negligible in the context of process impact evaluation.

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.