Cholesterol and phospholipid concentrations in serum lipoproteins, plasma activities of lecithin:cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) and lipoprotein lipase (LPL) activity in adipose tissue biopsies were measured ante and post partum in dairy cows given either free or restricted access to feed during the dry period. After parturition, all cows were fed ad libitum. The purpose of this study was to try to understand the earlier observed marked drop post partum in plasma triacylglycerol (TAG) in terms of lipoprotein metabolism in cows developing fatty liver post partum. As would be expected, free access to feed during the dry period induced a rise of hepatic TAG concentrations post partum associated with a decrease in plasma TAG levels. Total and free cholesterol concentrations in the VLDL, IDL, LDL and HDL2 fractions fell immediately after parturition. VLDL and IDL cholesterol concentrations remained at a constant, low level during the entire sampling period post partum, whereas the drop in LDL and HDL2 cholesterol post partum was followed by a rebound rise. Plasma LCAT and PLTP activities decreased by on average 19% and 33%, respectively, after parturition and then rose to values seen before parturition, but there was no effect of feeding regimen during the dry period. Activities of LCAT and PLTP were significantly correlated with cholesterol and phospholipid concentrations in LDL and HDL2. Plasma LCAT activity, as measured with exogenous substrate, and PLTP activity were both positively correlated with HDL3 phospholipid levels. LPL activity in adipose tissue dropped after parturition, the drop being smaller after feeding ad libitum during the dry period. It is concluded that the drop in adipose tissue LPL activity post partum is at variance with the simultaneous fall in plasma TAG. Possibly, the decrease in adipose tissue LPL activity helps to channel fatty acids away from adipose tissue into the udder. The post-partum changes in lipid transfer proteins in the blood are in line with the changes observed in the levels of the lipoproteins.

Whey protein hydrolysates were generated at different total solids (TS) levels (50–300 g/l) using the commercially available proteolytic preparation Debitrase™ HYW20, while enzyme to substrate ratio, pH and temperature were maintained constant. Hydrolysis proceeded at a faster rate at lower TS reaching a degree of hydrolysis (DH) of 16·6% at 300 g TS/l, compared with a DH of 22·7% at 50 g TS/l after 6 h hydrolysis. The slower breakdown of intact whey proteins at high TS was quantified by gel-permeation HPLC. Reversed-phase (RP) HPLC of hydrolysate samples of equivalent DH (~15%) generated at different TS levels indicated that certain hydrophobic peptide peaks were present at higher levels in hydrolysates generated at low TS. Sensory evaluation showed that hydrolysates with equivalent DH values were significantly (P<0·0005) less bitter when generated at 300 g TS/l (mean bitterness score=25·4%) than hydrolysates generated at 50 g TS/l (mean bitterness score=39·9%). A specific hydrophobic peptide peak present at higher concentrations in hydrolysates generated at low TS was isolated and identified as β-lactoglobulin f(43–57), a fragment having the physical and chemical characteristics of a bitter peptide.

Seven variables – electrical conductivity (EC), somatic cell count (SCC), N-acetyl-β-D-glucosaminidase (NAGase), lactose, protein, fat and pH – were compared in four quarter milk fractions (MF1: strict foremilk; MF2: first 12–15 ml foremilk; MF3: subsequent 40–45 ml milk; MF4: strippings) and in one cow composite milk sample (CC) per cow. The study used 142 quarters from 37 lactating cows of the German Black Pied breed. To rule out any possible effect due to management, animal physiology and analytical procedures, the collection and processing of milk samples from each cow was repeated for three consecutive days, and the means of 3-d values were used. All variables were affected significantly by milk fraction and udder health. Compared with foremilk, EC, lactose and protein levels in strippings decreased, while SCC, NAGase and fat increased. The pH of foremilk and strippings did not differ significantly in healthy or in mastitic quarters. The difference between MF1 and MF2 was significant for EC in mastitic quarters, and for SCC in healthy quarters only. In general, mastitis resulted in a significant increase in EC, SCC, NAGase and protein but in a decrease in lactose and fat contents of milk in one or more of the milk fractions studied. Comparison of cow composite milk samples from healthy and mastitic cows revealed the significance (P<0·01) of udder health for EC, SCC and lactose. Of the different parameters that can distinguish between healthy and mastitic quarters or cows, EC could be used to classify 76% of quarters and 73% of cows correctly, while the lactose content permitted correct identification of 81% of quarters and 76% of cows. NAGase and pH could be used to determine the status of 73% and 61% of quarters, respectively. In general, the correlation observed in strippings was higher than in foremilk for almost all the variables studied. Surprisingly, EC, SCC, NAGase and lactose in milk from healthy quarters of mastitic cows (with at least one mastitic quarter) differed significantly (P<0·05) from those from healthy quarters of cows with all four healthy quarters, indicating an inconsistent effect of mastitic quarters on neighbouring healthy quarters (quarter interdependence).

Differential cell count of milk is a traditional parameter for the evaluation of udder health. The literature shows great variation in differential cell counts of the milk of healthy mammary glands: macrophages range from 0% to 80%, lymphocytes from 1·5% to 79·5%, polymorphonuclear neutrophils from 3% to 95%, and epithelial cells from 1% to 19%. We conducted three studies to seek explanations for such variation. In the first, we evaluated the impact of polyethylene and glass sampling bottles. The aim of the second study was to compare the results of differential cell counts performed by three different technicians. The third study evaluated two methods of smear preparation. When polyethylene plastic bottles were used, the macrophage population was minimized but lymphocytes remained unaffected. This was shown by an exemplary flow cytometric analysis using four monoclonal antibodies against three lymphocyte surface structures. There were significant differences in the differential cell counts of 40 smears made by three technicians despite identical operating procedures. For the sediment smear, milk was centrifuged once and the sediment spread by eye on a glass slide. For the “coffee grinder” smear method, the sample was subjected to four centrifugations and then placed on a cover glass in order to spread the sediment using centrifugal force. The coffee grinder procedure led to a reduction of lymphocytes and an enrichment of polymorphonuclear neutrophils without affecting the macrophage population. Both methods made it possible to distinguish different udder health classes. It can be concluded that differential cell counts are a useful tool for comparing and monitoring udder health only if: samples are taken in a glass bottle; smears are prepared with the identical technique; and the differential cell counts are performed by a single person.

A preparation of the bacteriocin lacticin 3147 (prepared from a demineralized whey protein fermentation liquor) was combined as a powder with a bismuth-based intramammary teat seal and evaluated for its potential as an antimicrobial in non-lactating cows. The lacticin/teat seal formulation enabled significant bacteriocin release from the seal without the requirement for a surfactant. Studies in vivo in lactating cows demonstrated that this formulation was effective in reducing bacterial recoveries (~20-fold) from teats deliberately inoculated with Staphylococcus aureus after infusion. Moreover, this formulation also significantly reduced the numbers of Staph. aureus recovered from teats that were exposed to the challenge bacterium before the infusion of the teat seal preparation. The powdered preparation of lacticin 3147 did, however, cause some teat irritation as evidenced by associated rises in somatic cell count (SCC). However, this effect was short-lived and when the mean SCC readings pre-infusion and the final two readings post-infusion were compared, there was no significant difference in the immunological acceptance between treatments.

The effect of an added adjunct culture consisting of facultatively heterofermentative lactobacilli (FHL) on the volatile compounds and sensory characteristics of a Spanish ewes'-milk cheese was examined. Three cheese batches were prepared using a commercial starter, one from raw milk, another from pasteurized milk, and a third from pasteurized milk with an added culture of wild Lactobacillus. paracasei+Lb. plantarum. Analysis of the volatile compounds was carried out by the purge and trap method and gas chromatography with a mass spectrometer and disclosed a total of 86 compounds belonging to the chemical families hydrocarbons, fatty acids, esters, ketones, aldehydes, and alcohols. After ageing for 120 and 240 days, the cheese samples underwent sensory analysis by a panel of expert assessors. The attributes evaluated were characteristic odour and odour intensity and characteristic aroma and aroma intensity. Pasteurization of the milk had an effect on the formation of certain volatile compounds, adversely affecting the characteristic flavour of the cheese. Use of the adjunct culture in addition to the commercial starter improved the flavour of the cheese made from the pasteurized milk, which earned sensory scores similar to those awarded to the cheese made from the raw milk. Use of adjunct cultures consisting of indigenous FHL strains could help to conserve the traditional characteristics of Roncal cheese made from pasteurized milk, although some technical adjustments to the Regulations would be needed.

By its numerous properties and importance in cheese technology (production of colour, flavour, bacteriocins and resistance to salt) Brevibacterium linens is a major cheese ripening bacteria. However, the genetic approach of such biological functions has been hindered, up to now, by the lack of tools necessary to realise genetic modifications in this species. Our objective was to demonstrate that it is possible to genetically modify several strains exhibiting interesting technological properties, especially the production of sulphur compounds. We worked with a phenotypically and genetically diverse collection of 11 strains. We genetically transformed several Brevi. linens with acceptable rates with plasmids classically used to transform lactic acid bacteria and other Gram+ bacteria. These results open up new prospects to investigate the most interesting Brevi. linens metabolic pathways both at the biochemical and genetic level.

The involvement of Staphylococcus aureus exosecretions in bovine udder infection (Younis et al. 2003) suggests that four different monomer protein bands appearing between 36 and 31 kDa, are associated with the severity of the cow's infection response. Three out of these four bands have been identified by means of protein sequencing. Band B, with a MW of 35 kDa was identified as Panton-Valentaine leucocidin LukF'-PV chain- Staph. aureus; band C, with a MW of 32 kDa was identified as leucocidin chain LukM precursor- Staph. aureus; and band D was found to be similar, but not identical, to phosphatidylinositol-specific phospholipase-C-X. Bands B and C were purified by gel filtration using FPLC. The ability of these proteins to induce udder inflammation in vivo, and proliferation response in vitro and cytokine secretion were tested for both the crude exosecretions and purified bands. Three cows were inoculated intracisternally, with three quarters receiving either 0·007–0·008 mg (as total proteins) of Staph. aureus FR2449/1 bacterial exosecretion, pooled fraction 39–41 (bands B and C), or culture broth medium. The fourth quarter was left free as a control. Quarters that received fraction 39–41 of Staph. aureus FR2449/1, exhibited induced inflammation, which was indicated by increased somatic cell count and enhanced NAGase activity that was significantly higher than that of the original Staph. aureus FR2449/1 bacterial exosecretion. Proliferation tests of bovine blood lymphocytes in vitro showed that the pooled fraction 39–41 stimulated bovine proliferation of mononuclear cells much more than the original Staph. aureus FR2449/1 bacterial exosecretion. Secretion of TNF-α, IL-1β, IL-6 and IL-8 was in accordance with the contents of LukF'-PV and LukM precursor in the exosecretions. The results suggest that LukM/LukF' induce inflammation into the udder by a mechanism similar to that of LPS or by a unique mechanism(s) which requires further investigation.

Kefir is a fermented milk produced by the action of lactic acid bacteria, yeasts and acetic acid bacteria, trapped in a complex matrix of polysaccharides and proteins. Beyond its inherent high nutritional value as a source of proteins and calcium, kefir has a long tradition of being regarded as good for health in countries where it is a staple in the diet. However, published human or animal feeding trials to substantiate this view are not numerous. The aim of this work was to determine the immunomodulating capacity of kefir on the intestinal mucosal immune response in mice and to demonstrate the importance of dose and cell viability on this response. BALB/c mice were fed with commercial kefir ad libitum (diluted 1/10, 1/50, 1/100 or 1/200) or pasteurized kefir (diluted 1/6, 1/10, 1/50, 1/100) for 2, 5 or 7 consecutive days. At the end of each feeding period, the bacterial translocation assay was performed in the liver. Small intestine structure was studied by haematoxilin-eosin staining and light microscopy. The number of IgA+ and IgG+ cells was also determined. For the functional doses chosen, cytokines (IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α and IFN-γ) were determined. Kefir and pasteurized kefir were able to modulate the mucosal immune system in a dose-dependent manner. Kefir was administred 10-times more diluted than pasteurized kefir, but it induced an immunomodulation of similar magnitude, indicating the importance of cell viabilty. The results suggest that a Th1 response was controlled by Th2 cytokines induced by kefir feeding. Pasteurized kefir would induce both Th2 and Th1 responses. This is the first study in vivo regarding the mechanisms involved in the immunomodulating capacity of the oral administration of kefir containing viable or heat-inactivated bacteria at different doses.

Staphylococcus aureus isolates produce several pathogenic factors. The combination of these products influences the pathogenic role of different isolates, but their specific effects are well known in the pathogenesis of udder infections. This study focused on the association of polymorphism of the coagulase gene, protein A gene, collagen-binding protein gene, and of fibrinogen-binding protein gene on somatic cell count (SCC) and on Staph. aureus growth rate. Fifty Staph. aureus isolates from 13 dairy cow herds, located in seven different provinces, were considered. The results showed a low frequency of cna gene, similar to the one observed in human isolates. Meanwhile, the high frequency of efb gene indirectly confirmed the role of this factor in bacterial pathogenesis, being associated with adhesion to epithelia. The association of these two single genes with SCC and growth rate showed to be not significant. The polymorphism of spa gene was confirmed to be significantly associated with inflammatory response and growth rate, albeit with a pattern different from the one suggested for human isolates. Sorting of isolates based on the clusters obtained by combining polymorphisms of spa and coa genes and the presence of cna and efb genes, showed that a single cluster (cluster V) was prevalent in the different herds and provinces, while the other six clusters identified were widely spread among the remaining 60% of the isolates. Results showed that clusters VI and VII had significantly higher growth rates at 3, 4, and 6 h in comparison with the other clusters. Meanwhile, quarters infected with these strains showed significantly lower SCC levels. The frequency of isolates from cluster V, suggested that they should possess pathogenic factors increasing their invasiveness, even if in the presence of a stronger inflammatory response. These results indirectly confirm previous findings on the different interactions between isolates and the udder immune system. They also suggest that isolates with higher growth rates and inducing a lower inflammatory response have better chances to spread among the herd. The relatively simple genomic method proposed in this study could be applied by an increasing number of diagnostic laboratories and could be useful in studying the epidemiology of Staph. aureus intramammary infections in dairy herds when collecting data from the field.

Indigenous L-lactate dehydrogenase (LDH) in milk originates mainly from somatic cells, leucocytes and invading microorganisms. Its activity may be used for detection of mastitis. However, existing methods to measure LDH activity in milk both need pretreatment of the samples and still suffer from methodological problems. The present paper describes a fast, reliable method for determination of LDH activity, suitable for milk samples. The method is based on fluorometric determination of enzyme kinetics when L-lactate is converted to pyruvate. The assay uses raw milk without pretreatment and the method is easily adjustable to large-scale analyses on micro assay plates. Detection is based on (straight line) linear response within 4–7 min of initiation of the reaction. A substrate concentration of 35 mM in the reaction mixture was considered to be optimal for the assay. Intra plate assay precision was approx. 6% (CV) and the inter plate precision approx. 10%. Known inhibitors of LDH activity (oxidative direction), i.e., oxalic acid, oxamate, and pyruvate, were tested in different concentrations in order to verify the specificity of the response. The detailed kinetics of samples analysed indicated that the isoenzyme composition may have differed between milk samples, and that this composition may have been altered in high activity samples.

The impact of dietary components on the immune system is gaining increased attention in the effort to develop safe food products, some even with health-promoting potential, as well as to improve the basic understanding of the immunomodulatory potential of common food components. In such studies, which are mainly based on experiments in vitro, it is important to be able to differentiate nonspecific activation of immune cells induced by dietary components from ex vivo restimulation of antigen-specific cells that might be present in cell cultures owing to prior dietary exposure to the antigens in cell donors. Focusing on the immunostimulatory potential of cows' milk proteins and peptides, we studied the impact of prior dietary exposure to cows' milk on proliferation of murine immune cells upon ex vivo stimulation with bovine milk proteins. Nonspecific proliferation induced by β-casein peptides was further assessed on cells from mice bred on a cows'-milk-free diet. Regarding the dietary effect, we found that prior oral intake of cows' milk proteins affected cell proliferation induced by culturing with cows' milk proteins in vitro, as spleen cells from mice fed a milk-containing diet showed a significantly greater proliferative response than did cells from mice bred on a cows'-milk-free diet. Studies of immune enhancing potentials of β-casein peptides showed that some peptides stimulate proliferation of immune cells nonspecifically. In conclusion, these findings stress the importance of employing immune cells from mice unexposed to cows' milk for studies of the immunomodulating capacity of cows' milk proteins and peptides, in order to rule out the interference caused by antigen-specific immune responses. By using such cells, we here show that some β-casein peptides possess the potential to induce proliferation in immune cells in a nonspecific manner.

In this study, effects of high pressure (HP) on some constituents and properties of buffalo milk were examined. HP treatment at 100–600 MPa for 30 min affected casein micelle size only slightly, whereas treatment at 800 MPa increased it by ~35%. Levels of non-micellar αs1- and β-caseins were increased by treatment [ges ]250 MPa, and were highest after treatment at 400–800 MPa. The level of non-micellar calcium increased with increasing pressure up to 600 MPa. The L*-value of the milk decreased gradually with increasing pressure, from ~82 for untreated milk to ~65 for milk treated at 800 MPa. Milk pH was increased by ~0·07 units after treatment at 100–800 MPa, with no significant difference between treatment pressures. Denaturation of α-lactalbumin occurred at pressures [ges ]400 MPa, and reached >90% after treatment at 800 MPa, whereas β-lactoglobulin (β-lg) was denatured >100 MPa, reaching ~100% after treatment at 400 MPa; after treatment [ges ]400 MPa, all β-lg was associated with the casein micelles. The rennet coagulation time of buffalo milk increased with increasing pressure, whereas the strength of the coagulum formed decreased after treatment at 250–800 MPa. Overall, HP treatment affected many constituents and properties of buffalo milk; some of these effects have also been observed in the milk from other species, but the extent of the effects, and the pressure at which they occurred, differed considerably.

This study demonstrated that both chymosin and salt-in-moisture (SM) were important factors for proteolysis in the manufacture of ultrafiltrated white-salted cheese, with significant effects on water-soluble nitrogen and nitrogen soluble in trichloroacetic acid. In contrast, the levels of free amino acids were not significantly affected by chymosin and salt treatments. The cheeses made using high levels of chymosin with low SM had lower levels of residual αs1- and β-casein at the end of ripening. On texture profile analysis, the hardness and fracturability of the cheeses significantly increased with SM and decreased during ripening. Increases in chymosin significantly contributed to the overall weakening of the structure throughout ripening. Bitter flavour was detected after 12 weeks in the cheese made with the higher chymosin level and lower SM, which could be the result of accumulation of γ-casein fractions. The sensory data indicated that the hedonic responses for low chymosin with low SM cheeses were good and acceptable in flavour, which may be due to the moderate levels of proteolysis products.

This study investigated the protective capacity of the oral administration of fermented milk containing the probiotic strains; Lactobacillus casei, Lb. delbrueckii subsp. bulgaricus and Streptococcus thermophilus, against enteroinvasive Escherichia coli infection in a murine (BALB/c mice) model. Mice were fed for 2, 5 or 7 consecutive days with fermented milk diluted to a concentration of viable Lb. casei, Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus of 107 cfu/ml. Phagocytic activity of peritoneal macrophages and the number of IgA+ cells in small and large intestine were determined at the end of the feeding periods. For the preventive effect against Esch. coli, animals were fed for 5 days (selected dose). Mice were challenged with an infective dose of enteroinvasive Esch. coli of 108 cfu/mouse. The colonization of liver and spleen and the secretory IgA specific for the pathogen in the intestinal fluid were determined (ELISA test). Results showed that the unspecific immune response enhanced itself after 5 consecutive days of the administration of this fermented milk (increase in the percentage of phagocytosis and number of IgA+ cells in the small intestine). Treated animals showed less Esch. coli colonization of liver than control mice and a higher secretory anti-Esch. coli IgA in the intestinal fluids. These results suggest that the protection against enteroinvasive Esch. coli infection observed for the fermented milk containing probiotic bacteria may be associated with an enhance of the intestinal mucosa immunity.

While the effect of long-chain fatty acids on adipose tissue (AT) lipogenic activities has been described in non-lactating ruminants (Vernon, 1977), little is known about their effects on the mammary gland and the AT in lactating animals. However, in cows in mid lactation, duodenal rapeseed oil infusion decreased the rate of fatty acid (FA) synthesis in AT and increased milk yield of long-chain FA (18[ratio ]1, 18[ratio ]2 and 18[ratio ]3) and decreased medium-chain FA (14[ratio ]0 and 16[ratio ]0), suggesting a depressive effect of fat feeding on mammary lipid synthesis de novo (Chilliard et al. 1991). On the other hand, in goat species, the addition of vegetable lipids to the diet led to an increase in the milk fat content and yield (Chilliard et al. 2003) suggesting that the possible negative effect of long-chain FA on FA synthesis in the lactating mammary gland could be more than compensated by increasing the supply of FA brought to the mammary gland for milk synthesis. Elsewhere, AT from various anatomical sites are characterized by different FA composition in goat (Bas et al. 1987) together with different patterns of lipogenic gene expression in sheep (Barber et al. 2000). These results suggest that each AT site is characterized by a specific metabolism. However, in lactating ruminants, few data are available on the extent of expression and regulation of genes coding for lipogenic enzymes in AT. Therefore, the current study was performed in three lipogenic tissues of lactating goats, namely the mammary gland, an internal AT site (perirenal AT) and an external AT site (subcutaneous AT).