Spanish Churra sheep were studied in a daughter design for the presence on chromosome 6 of quantitative trait loci (QTL) influencing milk production traits. Eight half-sib families were genotyped for 11 microsatellites and marker-QTL effects analysed using yield deviations (YD) as quantitative measurements for the following traits: milk yield, protein yield, and protein percentage. QTL analysis was performed by interval mapping based on multimarker regression principles. Significance thresholds were estimated through a permutation test followed by a correction for multiple testing. The results suggest a region on ovine chromosome 6, close to the casein cluster, with an influence on milk traits and particularly on protein percentage. These results, the first ones reported for QTL affecting milk traits in sheep, are discussed in relation to data available for cattle, a closely related species.

In this work the purification and the complete primary structure of κ-casein from equine milk are reported for the first time. Mares' milk casein was separated by RP-HPLC into four fractions. Complete primary sequence was obtained by sequence analysis of the protein in the fastest eluting peak isolated by chromatography. This sequence was 95% identical to that reported for the C-terminal portion of the zebras' κ-casein and showed high similarity with κ-caseins from sources other than Equidae, confirming that this protein was indeed κ-casein in equine milk. The presence of post-translational modifications in equine κ-casein was investigated by mass spectroscopy, after enzymic dephosphorylation. Two main components were found, the smaller component being more abundant. Equine κ-casein was recognized by a lectin specific for one of the glucosidic bonds in the saccharide moiety of bovine κ-casein. Sequence comparison with prevision studies showed that the distribution of charged and hydrophobic regions in equine κ-casein was similar, but not identical, to that found in the bovine protein; these regions are associated with the role of κ-casein in the formation and stabilization of the micellar structure of casein in milk.

β-Lactoglobulin (β-lg) is the main protein of ruminant milk whey. Although β-lg can bind in vitro to a variety of hydrophobic substrates, mainly retinol and long-chain fatty acids, its physiological function is still unknown. In Exp. 1, we investigated the effect of β-lg on the plasma retinol concentration in preruminant calves. Holstein male calves (n = 20) were fed Holstein whole milk at 40 g/kg body weight (BW) plus vitamin A acetate (500,000 i.u.) with or without β-lg (0·4 g/kg BW). The plasma retinol concentration of 10-d-old calves was greater (P<0·05) in the β-lg-fed group than in the control group during the period from 8 to 12 h and at 24 h after the feeding. The postprandial change of plasma retinol in 40-d-old calves fed milk with β-lg was higher (P<0·05) than that in the control calves only at 12 h after the feeding. In Exp. 2, Holstein male calves (n = 18) were used to investigate the effect of β-lg on plasma triglyceride concentration and fatty acid composition. Calves were fed Holstein whole milk at 40 g/kg BW plus milk fat prepared from whole milk at 2 g/kg BW with or without β-lg (0·4 g/kg BW). Plasma triglyceride concentration at age 10 d was higher (P<0·05) in the β-lg-fed group than in the controls during the periods from 1 to 2 h and from 7 to 11 h after the feeding. At age 40 d, plasma triglyceride in the β-lg-fed group was higher (P<0·05) than in the control group only at 9 h. Ratios of palmitic, stearic, and oleic acids to total plasma lipids were higher (P<0·05) in the calves fed β-lg milk than in the control calves at age 10 d. These results suggest that β-lg enhances the intestinal uptake of retinol, triglyceride, and long-chain fatty acids in preruminant calves.

Polymorphism in the goat kappa casein gene was studied using the base excision sequence scanning (BESS) method and sequencing. Seven polymorphic sites, corresponding to single nucleotide transversions were detected. Three of these were silent mutations while the other four produced amino acid substitutions. The association between these polymorphic sites was investigated, which resulted in the identification of three goat kappa casein alleles, designated A, B, and C. Protocols for rapid genotyping of the C variant were developed by polymerase chain reaction–restriction fragment length polymorphism using Alw44I and BseNI restriction endonucleases. The occurrence of this allele was found to be very low in Spanish breeds but more frequent in the French Saanen goat. Further studies among different goat populations are necessary to establish the distribution of these alleles and their effects on the quality and functional properties of milk.

The non-stimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated luminol-augmented cellular chemiluminescence (CL) response and viability of milk and blood polymorphonuclear leukocytes (PMN) were determined in lactating dairy cows during different stages of lactation. In the first study, ten healthy cows each in early, mid and late lactation were compared. In a second study, the same measurements as in the first study were evaluated longitudinally in 12 cows during 1 month following parturition. The CL activity and myeloperoxidase (MPO) content of milk PMN and macrophages (M) were also compared. Milk M did not possess MPO activity and were devoid of any luminol-enhanced CL. The CL activity of milk and blood PMN was significantly lower in early lactation than in mid and late lactation (P < 0·001). Whereas little changes were observed in viability of blood PMN, the viability of milk PMN was lower in early lactation than in mid and late lactation (P < 0·001). The percentage of PMN in isolated milk cells was also lower during early lactation than during mid and late lactation (P < 0·001). The CL activity in response to PMA during early, mid and late lactation increased 13, 59 and 42-fold in blood PMN and 1·7, 2·6 and 2·4-fold in milk PMN, respectively, in comparison with non-stimulated PMN. The CL activity, both in milk and blood PMN, the milk PMN viability and the percentage of milk PMN were lowest between 3 d and 11 d post partum. These observed changes immediately after calving could contribute to a higher susceptibility to mastitis in that period.

Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) αs1-casein content. Parenteral sensitization to GM1 and GM2 proteins was also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine β-lactoglobulin (β-lg), α-lactalbumin (α-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (ΔIsc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-β-lg IgG1, with an important cross reactivity between goat and cow β-lg. However, in guinea pigs fed on GM2, anti-goat β-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3·5±4·5 μA/cm2) compared with that observed in GM1 guinea pigs (8·3±7·6 μA/cm2). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats' milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.

The aim of this research was to study the effects of the CSN1AG allele on the main rennet coagulation properties of milk. The study was carried out on individual milk samples with low αs1-casein obtained from 19 Italian Brown cows heterozygous for the CSN1AG allele (seventeen CSN1A BG and two CSN1A CG) from four herds in the province of Parma (Italy). Control cows (sixteen CSN1A BB and three CSN1A BC) giving milk with normal αs1-casein levels were chosen from within the same herds in order to establish pairs of cows with identical environment and management conditions, and comparable lactation stages and numbers. Individual milk samples from single pairs of cows with somatic cell counts and lactose and chloride levels within the normal ranges were collected and analysed in parallel. Rennet coagulation properties of milk were analysed using Formagraph and Gel Tester. Milk from low αs1-casein cows was characterized by lower casein content, lower titratable acidity and a higher proportion of κ-casein in total casein. The clotting time of this milk was ∼ 23% lower than that obtained with milk from normal αs1-casein cows. Rennet curd from low αs1-casein milk was obtained more rapidly and had a higher final firmness: curd-firming time was ∼ 35% lower and curd firmness measured 30 min after rennet addition was ∼ 27 % higher compared with that for normal αs1-casein milk. In addition, curd from low αs1-casein milk had a higher resistance to compression. These results suggest that, although a role for the CSN2 locus cannot be definitely excluded, the CSN1AG allele can considerably affect the main rennet coagulation properties of milk.

Oral tolerance to foreign enteral antigens is not fully developed in early neonatal life. Epidemiological evidence supports a role for maternal milk in the development of immune responses, including oral tolerance. Formula fed infants have an increased susceptibility to food allergy and the later development of autoimmune disease. This may relate to the lack in infant formula of growth factors found in maternal milk. Bovine milk contains proteins, growth factors and cytokines. Various studies have outlined the immune modulating potential of bovine milk-derived products. Fractionated whey extracts have therapeutic potential in disease states where there is an excessive inflammatory reaction, and disease preventive potential for infants who are not breast-fed. We have shown that daily oral administration of a growth factor-enriched fraction from milk whey to naturally suckling rat pups between days 4–9 postnatal can down-regulate immune activation to a specific orally administered food antigen, ovalbumin, assessed by lymphocyte proliferation. In addition, non-specific down-regulation in the intestine was observed as assessed by the expression of MHC I. Treatment of rat pups with whey extract at the time of oral sensitisation to ovalbumin also resulted in an increased secretion of TGF-β into the culture supernatant of spleen cells incubated with specific antigen. TGF-β is an immuno-down-regulatory cytokine involved in tolerance induction. Immune modulation by extracts derived from milk whey could be of potential benefit for formula-fed and pre-term infants in reducing susceptibility to inappropriate activation to food antigens.

A new polymerase chain reaction (PCR)-based method was developed to detect cows' milk in goat cheese. This method is based on mitochondrial DNA (mtDNA) control region sequence variations. DNA extractions from 150 mg of cheese were carried out using a spin column-based method. Subsequent PCR amplifications of DNA were performed with cow specific primers, demonstrating the ability to detect cows' milk in a variety of cheeses. This simple approach provides high quality DNA, and is shown to be very sensitive, with a detection limit of less than 0·1% of cows' milk. Analysis of an agarose gel digital image allows a rough estimation of the percentage of cows' milk used in adulteration.

The effect of milks fermented by Lactobacillus helveticus and its non-proteolytic variant on mucosal and tumoral immunity was studied. Milks fermented by Lb. helveticus wild type or its non-proteolytic variant were administered orally to mice for different periods (3, 5 and 7 d). The immune response was assessed by analysing the activity of the peritoneal macrophages, the number of cells secreting IgA associated with the gut-associated lymphoid tissue and with the bronchial-associated lymphoid tissue. The number of cells was determined by direct immunofluorescence. The antitumour activity was monitored by studying the regression of the subcutaneously implanted fibrosarcomas. After 3 d feeding of milk fermented by Lb. helveticus wild type, the number of sIgA increased significantly at both the intestinal and bronchial levels, indicating that a cellular migration had occurred. This effect was not noticeable when milk fermented by Lb. helveticus Protease (−) was orally administered. Both fermented milks (wild type or its variant) exhibited an effect on the activity of the peritoneal macrophages, which might be indirectly correlated to the regression of the fibrosarcoma. Although the mechanism by which the lactic acid bacteria enhance the immune system is not clear, this study clearly suggests that the bioactive compounds released during milk fermentation might contribute to the immunoenhancing properties of these products. By releasing biopeptide, lactic acid bacteria have important implications in modulation of the host's immune response, more specifically its cellular immune response.

Proteolysis of sodium caseinate solution (24·5 g/l) induced by heat treatment at 120 °C at different pH values was studied by measuring nitrogen content and relative fluorescence intensity in the 4% trichloroacetic acid filtrates. The low molar mass peptides corresponding to the soluble nitrogen were identified using liquid chromatography/tandem mass spectrometry. Increase in proteolysis, deduced from the increase in soluble nitrogen content, was observed with heating time (10, 20 and 30 min) and pH (6·0, 7·0, 8·0 and 9·0). The fluorescence measurements showed that the release of peptides containing tryptophan was minimal at pH ∼ 7·0. In parallel, eighteen low molar mass peptides were characterized, of which four came from κ-casein, nine from β-casein and five from αs1-casein. Peptides were preferentially released under alkaline conditions.

Confocal scanning laser microscopy (CSLM) methods were developed to identify fat and protein in cheeses, milk chocolate and milk powders. Various fluorescent probes were assessed for their ability to label fat or protein in selected food products in situ. Dual labelling of fat and protein was made possible by using mixtures of probes. Selected probes and probe mixtures were then used to study (a) structure development of Mozzarella cheese during manufacture and ripening, and (b) the distribution of fat and protein in milk chocolate made with milk powders containing varying levels of free fat. Microstructural changes in the protein and fat phases of Mozzarella cheese were observed at each major step in processing. Aggregation of renneted micelles occurred during curd formation; this was followed by amalgamation of the para-casein into linear fibres during plasticization. Following storage, the protein phase of the Mozzarella became more continuous; entrapping and isolating fat globules. Chocolate made with a high free-fat spray-dried powder blend showed a homogeneous fat distribution, similar to that of chocolate made with roller-dried milk. Chocolate made with whole milk powder containing 10 g free fat/100 g fat showed a non-homogeneous fat distribution with some fat occluded within milk protein particles. These differences in fat distribution were related to Casson yield value and Casson viscosity of the chocolates.

The effect of β-lactoglobulin and heat-induced precipitation of calcium phosphate on the pH dependence and mechanism of thermal coagulation of milk throughout the pH range 6·3–7·3 was studied using serum protein-free milk and sodium caseinate as models for micellar and non-micellar milk protein systems respectively. It appears that the specific effect of β-lactoglobulin at the pH of maximum stability may be related to its ability to chelate calcium. The effect of β-lactoglobulin at the pH of minimum stability does not appear to be directly related to heat-induced dissociation of κ-casein or micellar integrity but may be due to its ability to sensitize casein micelles to heat-induced precipitation of calcium phosphate, by increasing micellar hydrophobicity. The extent of heat-induced precipitation of calcium phosphate, as a function of pH, is an inverse reflection of the pH dependence of heat stability. Micellar integrity appears to play a critical role in the heat stability of milk but for reasons not previously appreciated.

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 probiotic Lactobacillus rhamnosus HN001 is known to enhance immunity in animal models, following oral delivery. In this study, we investigated the immunoenhancing effects of HN001 delivered to mice in different milk-based substrates, including: whole (full-fat) milk supplemented with HN001; fermented milk supplemented with HN001; or whole milk which had been part-fermented by HN001. Direct oral feeding of mice with HN001 in whole milk was shown to enhance the phagocytic activity of blood and peritoneal cells. Similar effects on phagocytosis were observed when HN001 was offered to mice in the form of a milk- or fermented milk-based diet. The degree of immune enhancement conferred by HN001 was similar whether the probiotic was used as an additive or as a fermentative agent. These studies confirm that Lb. rhamnosus HN001, derived originally from dairy food, enhances immune function following oral delivery in different milk bases.

Bipolar-membrane electroacidification (BMEA) technology, which uses the property of bipolar membranes to split water and the demineralization action of cation-exchange membranes (CEM), was tested for the production of acid casein. BMEA has numerous advantages in comparison with conventional isoelectric precipitation processes of proteins used in the dairy industry. BMEA uses electricity to generate the desired ionic species to acidify the treated solutions. The process can be precisely controlled, as electro-acidification rate is regulated by the effective current density in the cell. Water dissociation at the bipolar membrane interface is continuous and avoids local excess of acid. In-situ generation of dangerous chemicals (acids and bases) reduces the risks associated with the handling, transportation, use and elimination of these products. The aim of this study was to evaluate the performance of BMEA in different conditions of added ionic strength (μadded = 0, 0·25, 0·5 and 1·0 M) and added salt (CaCl2, NaCl and KCl).The combination of KCl and μadded = 0·5 M gave the best results with a 45% decrease in energy consumption. The increased energy efficiency was the result of a decrease in the anode/cathode voltage difference. This was due to an increase of conductivity, produced by addition of salt, necessary to compensate for the lack of sufficiently mobile ions in the skim milk. However, the addition of salts, irrespective of type or ionic strength, increased the required operation time. The protein profile of isolates were similar under all experimental conditions, except at 1·0 M-CaCl2.

It is fairly easy to control the enzymic hydrolysis of proteins in alkaline conditions by measuring the base consumption required to keep the pH constant in the reactor. Unfortunately, however, base consumption is not related in any simple way to the degree of hydrolysis reached at any given moment and to establish this relationship it is essential to find out the mean pK of the α-amino groups released during the hydrolytic process. We have shown here that the correct mean pK value varies according to the pH of the working conditions and that the relationship between these values may depend upon the kind of protein and protease used. We have put forward a method for determining this relationship experimentally by using a given protein–protease system, consisting of an alkaline titration of the raw protein and when partially hydrolysed. We have tested the results predicted by our theoretical model by applying it to the hydrolysis of whey proteins with a bacterial protease from Bacillus licheniformis at 50 °C, pH 8·0. This model can easily be applied to any hydrolytic process involving the appearance of functional groups that are partially protonizable under the working conditions in question in order to follow the kinetics of the reaction via the consumption of the neutralizing agent required to keep pH constant.

A detailed kinetic study of alkaline phosphatase, lactoperoxidase and β-lactoglobulin was carried out in the context of identifying intrinsic time–temperature indicators for controlling the heat processing of milk. The heat inactivation or denaturation of alkaline phosphatase, lactoperoxidase and β-lactoglobulin under isothermal conditions was found to follow first order kinetics. Experimental results were analysed using both a two step linear regression and a one step non-linear regression method. Results obtained using the two statistical techniques were comparable, but the 95% confidence interval for the predicted values was smaller when the one step non-linear regression method was used, indicating its superiority for estimating kinetic parameters. Thermal inactivation of alkaline phosphatase and lactoperoxidase was characterized by z values of 5·3 deg C (D60 °C = 24·6 min) and 4·3 deg C (D71 °C = 38·6 min) respectively. For the denaturation of β-lactoglobulin we found z values of 7·9 deg C (D75 °C = 49·9 min) in the temperature range 70–80 °C and 24·2 deg C (D85 °C = 3·53 min) in the range 83-95 °C. Dref and z were evaluated under dynamic temperature conditions. To estimate the statistical accuracy of the parameters, 90% joint confidence regions were constructed.

Rapid and sensitive assays for plasmin, plasminogen and plasminogen activators (PA) were developed and applied to bovine milk. The reaction medium was clarified by addition of a dissolving agent after hydrolysis of a fluorescent substrate specific for plasmin. This final step enabled the use of larger sample amount with higher substrate concentration than other methods, and avoided previous sample preparation. The use of 4 g gelatin/l in buffers preserved plasmin activity, thus avoiding risks of overestimation of the assays results. Sensitivity, detection level, repeatability and analysis run time of plasmin and plasminogen assay were improved over previous enzymatic methods with synthetic substrates. The PA assay was assessed by measuring conversion of exogenous plasminogen into plasmin. A new kinetic approach was used to enable the direct determination of global PA activities on raw milk samples without interference from indigenous plasmin.

The effects of administration of Lactobacillus gasseri SBT0270 on serum lipids and bile acids, faecal bile acids and microflora were estimated in hypercholesterolemic rats. An effective dose of strain SBT0270 to exert its hypocholesterolemic effect was 109 viable cells/d. The dose of 109 cells/d did not affect the faecal coliform counts, but the number of faecal lactobacilli in rats fed this dose was significantly higher than that in the control group observed at the end of feeding period. Hypocholesterolemic effect of Lb. gasseri SBT0270 was attributed to its ability to suppress the reabsorption of bile acids into the enterohepatic circulation and to enhance the excretion of acidic steroids in faeces of hypercholesterolemic rats.