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 effect of partial substitution of NaCl with KCl on texture profile, soluble Ca, K, Na, and P, and microstructure of low-moisture mozzarella cheese (LMMC) was investigated. LMMC batches were prepared using four combinations of NaCl and KCl salt viz., NaCl only, NaCl:KCl, 3:1, 1:1 and 1:3 (w/w); all used at of 46 g/kg curd and plasticised in 4% brine containing the above salt mixtures. Texture profile, microstructure, and percentages of soluble Ca, K, Na, and P were determined. There were no significant differences in hardness, cohesiveness, adhesiveness, and gumminess among the experimental LMMC batches. Environmental scanning electron microscopy images showed compact and homogeneous structure of LMMC at day 27 of storage; however, no significant difference was observed among the experimental LMMC batches. Hardness increased significantly in all experimental LMMC during storage. LMMC salted with NaCl/KCl mixtures had almost similar sensory properties compared with the control. There was no significant difference in creaminess, bitterness, saltiness, sour-acid, and vinegary taste among the experimental LMMC at the same storage period.

The present study examines the resistance of the α-lactalbumin to α-chymotrypsin (EC 3.4.21.1) digestion under various experimental conditions. Whey protein isolate (WPI) was hydrolysed using randomised hydrolysis conditions (5 and 10% of WPI; pH 7·0, 7·8 and 8·5; temperature 25, 37 and 50 °C; enzyme-to-substrate ratio, E/S, of 0·1%, 0·5 and 1%). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to analyse residual proteins. Heat, pH adjustment and two inhibitors (Bowman–Birk inhibitor and trypsin inhibitor from chicken egg white) were used to stop the enzyme reaction. While operating outside of the enzyme optimum it was observed that at pH 8·5 selective hydrolysis of β-lactoglobulin was improved because of a dimer-to-monomer transition while α-la remained relatively resistant. The best conditions for the recovery of native and pure α-la were at 25 °C, pH 8·5, 1% E/S ratio, 5% WPI (w/v) while the enzyme was inhibited using Bowman–Birk inhibitor with around 81% of original α-la in WPI was recovered with no more β-lg. Operating conditions for hydrolysis away from the chymotrypsin optimum conditions offers a great potential for selective WPI hydrolysis, and removal, of β-lg with production of whey protein concentrates containing low or no β-lg and pure native α-la. This method also offers the possibility for production of β-lg-depleted milk products for sensitive populations.

Conventional medical therapies for ulcerative colitis (UC) are still limited due to the adverse side effects like dose-dependent diarrhoea and insufficient potency to keep in remission for long-term periods. So, new alternatives that provide more effective and safe therapies for ulcerative colitis are constantly being sought. In the present study, probiotic LaBb Dahi was selected for investigation of its therapeutic effect on DSS-induced colitis model in mice. LaBb Dahi was prepared by co-culturing Dahi culture of Lactococci along with selected strain of Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3 in buffalo milk. Four groups of mice (12 each) were fed for 17 d with buffalo milk (normal control), buffalo milk plus DSS (Colitis control), Dahi plus DSS, and LaBb Dahi plus DSS, respectively, with basal diet. The disease activity scores, weight loss, organ weight, colon length, myeloperoxidase (MPO) and β-glucoronidase activity was assessed, and the histopathological picture of the colon of mice was studied. All colitis control mice evidenced significant increase in MPO, β-glucoronidase activity and showed high disease activity scores along with histological damage to colonic tissue. Feeding with LaBb Dahi offered significant reduction in MPO activity, β-glucoronidase activity and improved disease activity scores. We found significant decline in length of colon, organ weight and body weight in colitis induced controls which were improved significantly by feeding LaBb Dahi. The present study suggests that LaBb Dahi can be used as a potential nutraceutical intervention to combat UC related changes and may offer effective adjunctive treatment for management of UC.

The effect of intramammary inoculation of Lactobacillus perolens CRL 1724 on bovine udders at drying off was evaluated through histological examination of the canal and cistern tissues. The persistence of the strain in the udder 7 d post inoculation was also determined. Lb. perolens CRL 1724 was recovered from all mammary quarters and no clinical signs or teat damage were observed after inoculation of 106 cfu/ml. The udders showed a normal structural aspect and there were no modifications of the milk appearance. Lb. perolens CRL 1724 cells were evidenced on the surface of the epithelial cells of the cistern without causing any morphological modifications or cell alterations. Lb. perolens CRL 1724 produces a mild inflammatory reaction, characterized by recruitment of neutrophils to the epithelial zone and a slight hyperaemia into blood vessels. This preliminary study provides important information for further studies directed towards the inclusion of Lb. perolens CRL 1724 in the design of probiotic products for preventing bovine mastitis in non-lactating dairy cows.

The objective of this study was to examine the quarter health status of quarter-individually and conventionally milked cows. The MultiLactor®, a quarter-individual milking system (MULTI), has single guided tubes which provide milking on the quarter level with a low system vacuum level (37 kPa), sequential pulsation and periodic air inlet. The conventional milking system (CON) was equipped with a milking cluster where the system vacuum level was adjusted to 40 kPa. A total of 84 German Holstein cows, randomly divided into two groups, were included in the study. Over a period of 32 trial weeks, quarter foremilk samples were taken every week to determine somatic cell count (SCC). Bacteriological examinations and udder palpation were conducted at three different times. During the trial period, median SCC of quarter foremilk samples in both groups did not exceed the threshold value of 100 000 cells/ml. The results of the F test showed that the milking system (P = 0·0587) and days in milk (DIM) (P = 0·8066) had no significant effects on the quarter health status. On the other hand, lactation (P = 0·0396), quarter health status in the previous week (P < 0·0001) and trial week (P = 0·0061) affected quarter health status significantly. The estimated probabilities of the occurrence of a suspicious quarter (SCC > 100 000 cells/ml) were 19·97% (CON) and 31·72% (MULTI). However, the test of differences in the Least Square Means (LSM) showed no significant differences (P = 0·0585) between CON and MULTI. The estimated probability of quarters becoming suspicious during the first lactation was 12·51% for both groups. With an increasing number of lactation, the probability of a quarter becoming suspicious clearly increased (2nd lactation: 32·73% and 3rd lactation: 36·19%). The results also showed that the percentage of quarters with bacteriological findings revealed a stronger increase over time for MULTI than for CON.

The objective was to identify predisposing factors for increased risk of involuntary culling in adult Holstein–Friesian dairy cows. Data were sourced from Scotland's Rural College (SRUC) Dairy Research Centre. Between September 2003 and August 2010 175 cows were culled, a herd culling rate of 33·7%. The major reasons for involuntary culling were fertility (27·4%) and udder problems (26·9%). In the analysis, the culled cows were matched with their cohorts that survived to a later lactation. To identify predisposing factors, a binary logistic model was applied. Cows with higher than average body condition score (BCS) at last service were five times [Odds Ratio (OR) = 4·8] more likely to be culled due to infertility. Cows with low protein yield on day 60 ± 5 in lactation were ten times less likely (OR = 0·1) to be culled. In first lactation heifers, only BCS at last service increased the risk of involuntary culling due to infertility (OR = 13·0). A high milk yield acceleration was a significant (P = 0·04) factor in increasing the risk, five times (OR = 5·2) more, of culling cows due to udder problems. In conclusion, a high BCS at last service, high milk protein yield at around day 60 in lactation and acceleration of milk yield after calving exposed cows to a risk of being culled involuntarily. In practice, monitoring of traits that indicate metabolic imbalance could assist identifying cows at high risk of being culled and contribute to reducing the associated risk through a more effective timely decision.

Hispánico cheese, manufactured from a mixture of cow and ewe milk, is representative of cheese varieties made using milk from more than one animal species in Mediterranean countries. The shortage of ewe milk production in autumn hinders the uniformity of Hispánico cheese composition throughout the year. To surmount this inconvenience of ewe milk seasonality, curds made in spring from raw and pasteurized ewe milk were stored frozen and used four months later for the manufacture of Hispánico cheese. Experimental cheeses were made by mixing fresh curd from pasteurized cow milk with thawed curd from raw or pasteurized ewe milk, and control cheese from a mixture of pasteurized cow and ewe milk in the same proportion. Characteristics of experimental and control cheeses throughout a 60-d ripening period were investigated. On the one hand, the experimental cheese containing frozen curd from raw ewe milk showed the highest counts of staphylococci, Gram-negative bacteria and coliforms, the highest levels of aminopeptidase and esterase activity, and the highest concentrations of free amino acids, free fatty acids, alcohols and esters. On the other, the experimental cheese containing frozen curd from pasteurized ewe milk had concentrations of free amino acids, free fatty acids and volatile compounds similar to those of control cheese, with the only exception being a higher level of ketones. Flavour intensity reached the highest scores in the experimental cheese containing frozen curd from raw ewe milk, followed by the experimental cheese containing frozen curd from pasteurized ewe milk. Flavour quality scores of both experimental cheeses were similar, and lower than those of control cheese.

Caseinomacropeptide (CMP), a 7-kDa phosphoglycopolypeptide fragment released from κ-casein during milk renneting, is heterogeneous with respect to post-translational glycosylation. Several studies have reported that CMP has growth-promoting activity on lactic acid bacteria belonging to the genera Bifidobacterium. The aim of this study was to evaluate the effect of glycosylation and sequence variations between bovine and caprine CMP on the growth of two probiotics: Lactobacillus rhamnosus RW-9595-M and Bifidobacterium thermophilum RBL67. The growth-promoting activities of CMP (mixture of glycosylated (gCMP) and non-glycosylated (aCMP) fractions), aCMP and gCMP were measured in a basal minimal culture medium using turbidimetric microplate assay at 37 °C. Supplementation of the culture media at 2 mg/ml significantly improved maximum growth by 1·5 to 1·8 times depending on the strain, the additive (CMP, aCMP, gCMP), and the bovine or caprine origin (P < 0·05). CMP preparations also decreased the time needed to reach the inflexion point of the growth curve and increase the cell density at that time (P < 0·05). The effects of CMP preparations were dose dependent and significantly superior to the effect of bovine β-lactoglobulin added to the culture media. As gCMP and aCMP were as efficient as bovine and caprine CMP (P > 0·1), it was concluded that the presence of oligosaccharides linked to CMP was not essential for growth-promoting activity of CMP.

Kefir is a fermented-milk beverage originating and widely consumed in the Caucasus as well as in Eastern Europe and is a source of bacteria with potential probiotic properties. Enterohaemorrhagic Escherichia coli producing Shiga toxin is commonly associated with food-transmitted diseases; the most prevalent serotype causing epidemics is Esch. coli O157:H7. The aim of this study was to evaluate the antagonism of Lactobacillus plantarum isolated from kefir against the action on Vero cells of supernatants of the Esch. coli O157:H7 strain 69160 expressing the type-II Shiga toxin (Stx2) and to study the role of the Lactobacillus cell wall in that inhibition. Spent culture supernatants of Esch. coli O157:H7 strain 69160 led to cytotoxic effects on cultured eukaryotic cells as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide–cleavage assay or by lactate-dehyrogenase release. Lb. plantarum CIDCA 83114 reduced the cytotoxic activity of Stx present in strain-69160 supernatants, and this protection was markedly higher than those of Lactobacillus kefir CIDCA 83113 and 8348 and Lb. delbrueckii subsp. bulgaricus CIDCA 333. This antagonism of cytotoxicity was mimicked by Lb. plantarum cell walls but was reduced after heating or protease treatments, thus indicating a protein or peptide as being involved in the protection mechanism. The cell surface of the lactobacilli bound the subunit B of Stx thereby decreasing the cytotoxicity. These interactions could constitute the first step in preventing the damage induced by Esch. coli O157:H7 supernatants, thus representing a valuable means of potentially mitigating the noxious effects of this food pathogen.

The shortcomings of Staphylococcus aureus vaccines to control bovine mastitis have been attributed to insufficient capacity of the vaccines to induce opsonizing antibodies and to stimulate cellular immune responses. Types of antigen, administration route and adjuvant used in a vaccine formulation have been identified as critical factors for the development of opsonic antibodies. Current commercially available vaccines for Staph. aureus bovine mastitis control are formulated with Al(OH)3 and oil-based adjuvants. The aim of this study was to evaluate the immune response of heifers immunized with a Staph. aureus CP5 whole cell vaccine formulated either with Al(OH)3 or ISCOMATRIX™. Twenty primigravid Holstein dairy heifers in the last trimester of gestation were immunized either with a vaccine formulated with ISCOMATRIX™ (n = 6), Al(OH)3 (n = 7), or saline solution (placebo) (n = 7). Immunization was carried out 38 and 10 d before calving. Heifers vaccinated with Staph. aureus adjuvanted with ISCOMATRIX™ responded with significantly higher levels of anti-bacterin and anti-CP5 IgG and IgG2 in sera than animals in the Al(OH)3 or control groups. Animals in the ISCOMATRIX™ group responded with significantly higher anti-bacterin specific IgG in whey than animals in the Al(OH)3 and control groups, detected from the first week post calving until 60 d of lactation. Sera from animals inoculated with Staph. aureus in ISCOMATRIX™, obtained 7 d post partum, significantly increased both the number of neutrophils ingesting bacteria and the number of bacteria being ingested by the neutrophils, compared with sera obtained from heifers vaccinated with Al(OH)3 or non-vaccinated controls. These features coupled to safety of the ISCOMATRIX™ formulation, warrant additional studies.

Fresh buckwheat (Fagopyrum esculentum) and chicory (Cichorium intybus) had been shown to have the potential to improve certain milk quality traits when fed as forages to dairy cows. However, the process of ensiling might alter these properties. In the present study, two silages, prepared from mixtures of buckwheat or chicory and ryegrass, were compared with pure ryegrass silage (Lolium multiflorum) by feeding to 3 × 6 late-lactating cows. The dietary dry matter proportions realised for buckwheat and chicory were 0·46 and 0·34 accounting also for 2 kg/d of concentrate. Data and samples were collected from days 10 to 15 of treatment feeding. Buckwheat silage was richest in condensed tannins. Proportions of polyunsaturated fatty acids (PUFA) and α-linoleic acid in total fatty acids (FA) were highest in the ryegrass silage. Feed intake, milk yield and milk gross composition did not differ among the groups. Feeding buckwheat resulted in the highest milk fat concentrations (g/kg) of linoleic acid (15·7) and total PUFA (40·5; both P < 0·05 compared with ryegrass). The concentration of α-linolenic acid in milk fat was similar across treatments, but its apparent recovery in milk relative to the amounts ingested was highest with buckwheat. The same was true for the occurrence of FA biohydrogenation products in milk relative to α-linolenic acid intake. Recovery of dietary linoleic acid in milk remained unaffected. Feeding buckwheat silage shortened rennet coagulation time by 26% and tended (P < 0·1) to increase curd firmness by 29%. In conclusion, particularly buckwheat silage seems to have a certain potential to modify the transfer of FA from feed to milk and to contribute to improved cheese-making properties.

Indirect comparisons from studies in vivo have suggested that caprine mammary tissue is less sensitive than bovine mammary tissue to the anti-lipogenic effect of long-chain fatty acids (LCFA), including specific rumen biohydrogenation (RBH) intermediates of polyunsaturated fatty acids (PUFA). Our objective was to investigate the effects on lipogenesis of 18-carbon LCFA differing in the degree of unsaturation and/or double bond conformation using cultured slices of bovine and caprine mammary tissues. Mammary tissues were collected from five multiparous Holstein × Normande cows and six multiparous Alpine goats in mid lactation. The expression of genes involved in milk component synthesis was measured in tissues collected at slaughter and after slice preparation: FASN, SCD1, CD36, SREBF1 and PPARG1 mRNA levels were higher in bovine than caprine samples, whereas the opposite was observed for CSN2 mRNA levels. Bovine and caprine mammary slices were incubated for 20 h in a medium with BSA (control), cis-9-18 : 1, 18 : 2n-6, 18 : 3n-3, cis-9, trans-11-CLA, or trans-10, cis-12-CLA (the latter at 3 increasing concentrations: C1 (0·11 mm), C2 (0·16 mm), C3 (0·37 mm)). Lipogenesis was estimated by measuring the incorporation of 14C-acetate into total lipid. Significant differences of individual LCFA (P < 0·05) were observed between species: bovine tissue showed a decrease in total lipogenesis with 18 : 2n-6, 18 : 3n-3, trans-10,cis-12-CLA (C2 and C3) while caprine tissue showed an increase after treatment with 18 : 3n-3, cis-9, trans-11-CLA or trans-10, cis-12-CLA (C3). These results were not related to the mRNA abundance of our set of genes in the mammary slices after incubation. In conclusion, this study demonstrates that caprine mammary slices reacted differently from bovine mammary slices to the anti-lipogenic activity of specific LCFA and suggests that regulation of lipogenesis via other genes and/or at protein level and enzyme activity may be involved.

Kefir is a dairy product obtained by fermentation of milk with a complex microbial population and several health-promoting properties have been attributed to its consumption. In this work, we tested the ability of different kefir-isolated bacterial and yeast strains (Lactobacillus kefir, Lb. plantarum, Lactococcus lactis subps. lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) or a mixture of them (MM) to antagonise the cytopathic effect of toxins from Clostridium difficile (TcdA and TcdB). Cell detachment assays and F-actin network staining using Vero cell line were performed. Although incubation with microbial cells did not reduce the damage induced by C. difficile spent culture supernatant (SCS), Lc. lactis CIDCA 8221 and MM supernatants were able to inhibit the cytotoxicity of SCS to Vero cells. Fraction of Lc. lactis CIDCA 8221 supernatant containing components higher than 10 kDa were responsible for the inhibitory activity and heating of this fraction for 15 min at 100 °C completely abrogated this ability. By dot-blot assay with anti-TcdA or anti-TcdB antibodies, concentration of both toxins seems to be reduced in SCS treated with Lc. lactis CIDCA 8221 supernatant. However, protective effect was not affected by treatment with proteases or proteases-inhibitors tested. In conclusion, we demonstrated that kefir-isolated Lc. lactis CIDCA 8221 secreted heat-sensitive products able to protect eukaryotic cells from cytopathic effect of C. difficile toxins in vitro. Our findings provide new insights into the probiotic action of microorganisms isolated from kefir against virulence factors from intestinal pathogens.

The robustness of in-line raw milk analysis with near-infrared spectroscopy (NIRS) was tested with respect to the prediction of the raw milk contents fat, protein and lactose. Near-infrared (NIR) spectra of raw milk (n = 3119) were acquired on three different farms during the milking process of 354 milkings over a period of six months. Calibration models were calculated for: a random data set of each farm (fully random internal calibration); first two thirds of the visits per farm (internal calibration); whole datasets of two of the three farms (external calibration), and combinations of external and internal datasets. Validation was done either on the remaining data set per farm (internal validation) or on data of the remaining farms (external validation). Excellent calibration results were obtained when fully randomised internal calibration sets were used for milk analysis. In this case, RPD values of around ten, five and three for the prediction of fat, protein and lactose content, respectively, were achieved. Farm internal calibrations achieved much poorer prediction results especially for the prediction of protein and lactose with RPD values of around two and one respectively. The prediction accuracy improved when validation was done on spectra of an external farm, mainly due to the higher sample variation in external calibration sets in terms of feeding diets and individual cow effects. The results showed that further improvements were achieved when additional farm information was added to the calibration set. One of the main requirements towards a robust calibration model is the ability to predict milk constituents in unknown future milk samples. The robustness and quality of prediction increases with increasing variation of, e.g., feeding and cow individual milk composition in the calibration model.

Ovarian steroids, oestradiol and progesterone, are required for normal mammary growth at puberty and during pregnancy. They contribute to mammary parenchyma development by stimulating mammary epithelial cell (MEC) proliferation. However several studies demonstrate that oestradiol negatively affects milk production during the declining phase of lactation, but the oestradiol effect on MEC in lactating mammary gland remains unclear. The objective of this study was to investigate the differential effect of oestradiol on bovine MECs mimicking two physiological statuses: active and early apoptotic MECs. We demonstrated that oestradiol has a major effect on early apoptotic MECs and might accelerate MEC apoptosis by activation of caspases rather than by inducing apoptosis in active MECs. Early apoptotic MECs could be compared with senescent cells in the late-lactation mammary gland. These results suggest that the negative effect of oestradiol on milk production during the declining phase of lactation would be due to an enhancement of apoptotic processes in MECs.

The aim of the study was to identify the species of Enterobacteriaceae present in Montasio cheese and to assess their potential to produce biogenic amines. Plate count methods and an Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) approach, combined with 16S rDNA sequencing, were used to investigate the Enterobacteriaceae community present during the cheesemaking and ripening of 6 batches of Montasio cheese. Additionally, the potential decarboxylation abilities of selected bacterial isolates were qualitatively and quantitatively assessed against tyrosine, histidine, ornithine and lysine. The most predominant species detected during cheese manufacturing and ripening were Enterobacter cloacae, Escherichia coli and Hafnia alvei. The non-limiting physico-chemical conditions (pH, NaCl% and aw) during ripening were probably the cause of the presence of detectable levels of Enterobacteriaceae up to 120 d of ripening. The HPLC test showed that cadaverine and putrescine were the amines produced in higher amounts by almost all isolates, indicating that the presence of these amines in cheese can be linked to the presence of high counts of Enterobacteriaceae. 44 isolates produced low amounts of histamine (<300 ppm), and four isolates produced more than 1000 ppm of this amine. Only 9 isolates, belonging to the species Citrobacter freundii, Esch. coli and Raoultella ornithinolytica, appeared to produce tyramine. These data provided new information regarding the decarboxylase activity of some Enterobacteriaceae species, including Pantoea agglomerans, Esch. fergusonii and R. ornithinolytica.