The objectives of this research were to confirm the effect of compounds derived from milk fermented by Lactobacillus helveticus (LH-2) on the nonspecific host defence system, and isolate and characterize the active peptides that mediate the immune response. The cell-free supernatant obtained from the fermented milk and its fractions were tested in vitro for immuno-modulating activity using murine macrophages (RAW 264·7 cell line). Cytokine production (Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), and Interleukin-1β (IL1-β)), nitric oxide (NO) production and phagocytosis were used as biomarkers. Macrophages stimulated with cell-free supernatant of fermented milk showed higher production of cytokines and NO compared with macrophages stimulated with LPS (Lipopolysaccharide) and a commercial immunomodulator derived from β-casein (f54-59). Phagocytosis was observed by macrophages stimulated with the supernatant. Two of nine fractions collected from the supernatant using size exclusion chromatography produced the highest response when used to stimulate macrophages. The results of the dose-response study of the effect of the fraction with the highest stimulation effect on the production of TNF-α showed a direct correlation between protein concentration and TNF-α release. The fraction contained four novel peptides, three derived from the hydrolysis of β-casein and one from the hydrolysis of α-lactalbumin. These results confirm that fermentation of milk by Lactobacillus helveticus (LH-2) results in the production of specific peptides capable of modulating macrophage activity.

Time spent lying by lactating Holstein-Friesian cows of varying body condition scores (BCS) and milk yield was measured using an animal activity monitor. A 3-week average BCS was calculated for each cow; and in total, 84 cows were selected with 28 cows each among three BCS categories (Thin: BCS<2·75; Moderate: 2·75⩾BCS<3·25; Heavy: BCS⩾3·25) and two stage of lactation categories (<150 days in milk or >150 days in milk). Cows were kept in two management systems: parlour/freestall (n=60) or automated milking system/freestall (n=24). Behaviour was recorded for 5·3±0·1 d for each cow. Production levels were considered using a 28-d rolling average of daily milk production. Cows that exhibited clinical lameness before or during the observation period were excluded from analyses. For cows exhibiting oestrus, the day prior to, day of, and day following breeding were removed. The final analysis included 77 cows (408 d of observation). A mixed model was fitted to describe average daily hours spent lying. Results demonstrated that lying time increased as days in milk (DIM) increased (P=0·05). Variables that were tested but not significant (P>0·05) were BCS category, parity category (1 or ⩾2) and 28-d rolling average daily milk production. Although a numerical trend for increasing hours spent lying with increasing BCS was observed, after accounting for other factors in the mixed model, BCS did not significantly impact lying time. Continued investigation of these management factors that impact lying time and bouts, using new technologies, more cows, and more herds will help dairy owners better manage facilities and cow movements to optimize this essential behaviour.

The pH and calcium activity of reconstituted skim milk solutions (9–21% w/w milk solids non-fat) on heating and after cooling were studied as a function of milk pH prior to heating (pH 6·2–7·2 at 25°C) and added calcium complexing agents (phosphate or EDTA). The pH decreased as the temperature was raised from 25 to 90°C and the magnitude of the pH decrease was greater with increase in initial pH at 25°C before heating or milk concentration. The pH decrease on heating from 25 to 90°C in skim milk solutions with added calcium complexing agents was lower than that of milk without the addition of these salts. The calcium activity decreased on heating from 25 to 60°C. The magnitude of the change decreased with increase in initial pH at 25°C before heating and milk concentration. The decrease in calcium activity on heating from 25 to 60°C for skim milk solutions with added calcium complexing agents was lower than that of milk solutions without the addition of calcium complexing agents. The changes in pH and calcium activity on heating milk were largely reversible after cooling the milk. The results suggested that the pH and calcium activity at high temperatures are a function of the milk composition. Knowledge of the initial pH prior to heating alone is not sufficient for predicting the changes that occur during heating.

The aim of this study was to assess the effect of milk source and of cheese production protocol on proteolytic and lipolytic pattern of cheese during ripening. The study involved six dairy factories located in Monti Dauni Meridionali area of Southern Italy; three dairy factories processed the milk produced by their own cow herds, while the other three dairy factories processed the milk collected in other dairy farms located in the neighbouring area. Cow milk processed to cheese had different nutritional parameters and hygienic quality. Caciocavallo cheese showed differences in the evolution of proteolysis during ripening and in the intensity of the lipolytic process detected at the end of ripening. The main factors influencing Caciocavallo cheese features were the quality of the starting milk, differences in technological steps such as milk heating, type of starter cultures and coagulant used.

For its characteristics, donkey milk has been proposed as an alternative to goat or artificial milk to feed allergic infants. Therefore, it is important to increase our knowledge on health and immunological characteristics of donkey milk. Ten donkeys, bred as companion animals, were enrolled in this study and sampled once a month, for eight months. Milk (10 ml) was collected from each half udder for somatic cell count (SCC), bacteriological analysis and total bacteria count (TBC). The major pathogens were tested for antimicrobial susceptibility, and Staphylococcus aureus isolates were further genotyped by nanoarray analysis. Whey lysozyme and NAGase (NAG) activities were also assessed. Overall, 101 half-udder milk samples were taken. They showed very low values of TBC (<250 cfu/ml) and SCC (<50 000 cells/ml) and a minor prevalence of pathogens: Staph. aureus was isolated only from 5 milk samples (3 animals), Streptococcus equi from 2 samples and Str. equisimilis from a single sample. All the isolates were sensitive to all antibiotic classes used in veterinary medicine. None of the Staph. aureus isolates were shown to harbour genes coding for any enterotoxin, toxic-shock syndrome toxin or antibiotic resistance. Lysozyme levels were always very high (4000–5000 U/ml), while NAG values were mostly low (<50 U/ml), out of the last part of lactation. The results of this study confirmed the low prevalence of intramammary infections in donkey and the absence of food-borne pathogens, suggesting that donkey milk could be a safe food, if the mammary gland is healthy and the animals are milked in proper hygienic conditions.

Two greatly related Lactobacillus plantarum bacteriophages (named FAGK1 and FAGK2) were isolated from Kefir grains of different origins. Both phages belonged to the Siphoviridae family (morphotype B1) and showed similar dimensions for head and tail sizes. The host range of the two phages, using 36 strains as potential host strains, differed only in the phage reactivity against one of them. The phages showed latent periods of 30 min, burst periods of 80±10 min and burst size values of 11·0±1·0 PFU per infected cell as mean value. Identical DNA restriction patterns were obtained for both phages with PvuI, SalI, HindIII and MluI. The viral DNA apparently did not present extremes cos and the structural protein patterns presented four major bands (32·9, 35·7, 43·0 and 66·2 kDa). This study reports the first isolation of bacteriophages of Lb. plantarum from Kefir grains and adds further knowledge regarding the complex microbial community of this fermented milk.

Three models for the estimation of milk, fat and protein daily yield (DY) based on a.m. (AM) or p.m. (PM) milkings were compared. A total of 518 766 test-day records from 5078 dairy cattle farms obtained between March 2004 and April 2008 were analysed. The DY model was a linear model with DY as a dependent variable. In the PYR model and the DYR model, partial yield ratios (AM:DY and PM:DY) and daily yield ratios (DY:AM and DY:PM), respectively, were used as a dependent variable in the first step. In the second step, DY was estimated as a partial yield divided (PYR model) or multiplied (DYR model) by the estimated yield ratio from the first step. Models included the effect of partial yield (only in the DY model), milking interval, stage (month) of lactation and parity. Analysis of variance indicated that partial yield was the most important source of variation for the DY model whereas milking interval had the biggest effect in the PYR model and the DYR model. Differences in accuracy (correlation between the true and the estimated DY) between the models were negligible. On the other hand, models differed in the amount of bias (average error). The DYR model on average overestimated DY by 0·13 kg, 0·01 kg and 0·01 kg for milk, fat and protein, respectively. For the other two models the overall bias was almost zero. However, the DY model overestimated low and underestimated high DY owing to the well known regression property. The DYR model progressively overestimated high DY. These problems were not observed with the PYR model which seemed to be the best model. In this paper a relatively old topic was analysed and discussed from a new point of view, where the estimation of DY is based on modelling biologically more stable partial yield ratios rather than yield values from a.m. or p.m. milking.

The viability of five single-strain and one three-strain probiotic cultures was assessed during Pategrás cheese ripening. Probiotics were inoculated into cheese-milk after a pre-incubation step – intended to improve their survival – or directly as a lyophilised culture; control cheeses without probiotics were also obtained. pH of probiotic and control cheeses was similar, except in probiotic cheeses containing the strain Lb. acidophilus B or the mixed culture. In these cases, the probiotic cheeses were more acid than their respective control cheeses. All the probiotics tested maintained counts above 107 cfu/g during the shelf-life settled for the product. Strains of the Lb. casei group: Lb. paracasei, Lb. casei and Lb. rhamnosus reached and kept the highest cell concentration during cheese ripening, followed by Lb. acidophilus and bifidobacteria. The direct addition of the probiotic cultures was more efficient than their inoculation after a pre-incubation step, for all the probiotics assayed. We have provided evidence that support the use of Pategrás cheese as a performing food-based vehicle for probiotic bacteria.

Ninety-nine individual milk samples from 37 cows in lactation week 10–35, selected for producing well or poorly/non-coagulating milk, were compared regarding protein composition, total calcium content, casein micelle size, pH, and coagulating properties after addition of 0·05% CaCl2. The results showed that a low κ-casein concentration in milk was a risk factor for non-coagulation. CaCl2 addition improved coagulating properties (coagulation time, curd firmness) of nearly all samples and eliminated differences between poorly/non-coagulating and well-coagulating milk, particularly regarding curd firmness. A second, independent data set with 18 non-coagulating or well-coagulating milk samples were analysed for protein composition, where indications of a similar association with κ-casein was observed.

To investigate the relationships between milk flow traits and udder health status in primiparous cows, 74 primiparous Holstein cows were randomly selected in 5 herds and monitored monthly throughout the whole lactation. A total of 2902 quarter milk samples were collected for bacteriological analyses and the determination of lysozyme, N-acetyl-β-glucosaminidase (NAGase) and somatic cell count (SCC). Milk flow curves of the whole udder of each cow were registered with continuous electronic milk flow meters. Teat conditions and teat thickness changes during milking were assessed monthly. Quarters, udders and cows were classified as healthy, latent, inflamed and subclinical depending on SCC and the results of bacteriological analyses. Lysozyme in milk, teat apex score and teat thickness change did not vary with udder health status while NAGase in milk significantly increased as udder health status worsened (P<0·001). Milk production (P<0·001) and time of plateau phase (P<0·05) were significantly lower in subclinical cows in comparison with the others. Animals with a high frequency of bimodal curves in the first 100 days in milk showed the worst udder health status during the whole lactation (P<0·01). Moreover, cows classified as subclinical in the first 3 months of lactation had higher peak milk flow than healthy cows (3·81 v. 3·48 kg/min; P<0·05) and shorter duration of plateau phase, expressed both as minutes and as percentage of time of milk flow (pTPL; P<0·001). Multivariate logistic analysis showed udder health status to be associated with duration of plateau phase, time of milk flow, bimodality and duration of overmilking phase. With short time of plateau phase (pTPL <25%), short time of milk flow (<5 min), presence of bimodality and long overmilking phase (>0·8 min) there was an increased risk of poor udder health status. These milk flow traits can be predictive indicators of udder health status; time of plateau phase, expressed as percentage of time of milk flow, can also be a useful parameter for animal selection.

The effectiveness of three natural compounds in slowing down the deterioration of microbial quality of Fior di latte cheese is addressed. In particular, the control of the growth of spoilage microorganisms and the determination of the Microbiological Stability Limit (MAL) were the main goals. A Central Composite Design (CCD) strategy was adopted to highlight possible combined effects between three essential oils: sage and two types of lemon. Results showed an increase in the MAL value for all cheese samples packaged with the antimicrobial compounds, compared with the control cheese. Moreover, the combinations of the three essential oils indicated that the substances can act in a synergistic or antagonistic way, depending on their concentrations. In particular, a synergistic effect was evident when the three compounds were used at about 3000 ppm.

The effects of dry period omission on oxidative stress and metabolic indicators around calving were studied. Seventeen Italian Friesian cows were randomly assigned to two groups, homogeneous for milk yield and parity, and managed either with a traditional 55-d dry off period (n=8) or continuously milked till parturition (n=9). Between 60 d before expected calving and 90 d after calving, body condition (BCS) was recorded and blood samples were collected to measure cortisol, urea, cholesterol, glucose, NEFA, triglycerides, insulin, malondialdehyde (MDA), total glutathione (GSH) and glutathione peroxidase (GPx) activity. BCS changes after calving were not different between the two groups. The normally dried group showed lower (P<0·05) glucose concentrations on day 7 before calving, greater (P<0·01) non-esterified fatty acid concentrations at 7 d and 15 d after calving, and greater (P<0·01) triglyceride concentrations for all the period before calving. On the other hand, plasma MDA was not different between groups. On average, plasma GSH concentrations were greater in continuously milked cows after calving (P<0·05), while plasma GPx was greater with continuous milking up to parturition (P<0·01). The results confirmed that omitting the dry period leads to an improved energy balance. The degree of oxidative stress was not detrimental for animal health, and the slight modifications of GPx observed prepartum were possibly related to continuous milk secretion. The differences in plasma GSH observed after calving may depend upon sulphur amino acid sparing in continuously milked cows.

The present study aimed to assess the effect of information about organic production on Pecorino cheese liking and consumer willingness to pay. Mean scores of perceived liking were similar for organic cheese (OC) and conventional cheese (CC). Expected liking scores were higher for OC than for CC (P<0·001). For OC the expected liking was significantly higher (P<0·001) than the perceived liking expressed in blind conditions (negative disconfirmation), whereas for CC the expected liking was significantly lower (P<0·001) than the perceived liking expressed in blind conditions (positive disconfirmation). Consumers assimilated their liking for OC in the direction of expectations, as the difference actual vs. perceived liking was significant (P<0·001). However the assimilation was not complete, as also the difference actual liking vs. expected liking was significant (P<0·001). Consumers showed a willingness to pay OC (mean±se=4·20±0·13 €/100 g) higher than the local retail price for conventional (1·90 €/100 g) and even organic cheese (3·00 €/100 g). We conclude that the information about organic farming can be a major determinant of cheese liking and consumer willingness to pay, thus providing a potential tool for product differentiation, particularly for small scale and traditional farms.

Two feeding systems trials were carried out to determine the antioxidant activity of soft goats' milk cheeses, and also to evaluate the presence of bioactive polyphenolic compounds. Two groups (A and B), each one with 20 (BW 50±5 kg) French Alpine goats were employed. All animals had between 70 to 80 milking days and were milked once a day. Group A included daily grazing on shruby rangeland vegetation, and group B was kept in full indoor confinement. Thirty kg of goats' milk from each group were collected; 15 kg from each were pasteurized and the remaining 15 kg were not pasteurized (raw), resulting in four treatments (two feeding system×two milk treatments). Soft goat's cheese antioxidant activity can be modified by the animals feeding system; grazing management represents a better option than indoor feeding to produce a healthy profile of bioactive compounds; providing an increase of total polyphenol, hydroxycinnamic acids and flavonoid concentrations. Pasteurization did have a significant effect on these metabolites, and diminished total polyphenol concentration. More research is needed to elucidate the potential of soft goat's cheese as a functional food.

The aim of the study was to test the effect of rumen-inert fat supplements of different chemical forms or containing different unsaturated/saturated (U/S) fatty acid contents on milk production, milk composition and liver and blood metabolic variables of high-yielding dairy cows in the peripartal period. Thirty Holstein-Friesian dairy cows were divided into three equal groups and fed a corn silage-based diet, without fat supplementation (control) or supplemented with 11·75 MJ NEl per day of calcium soaps of palm oil fatty acids (CAS; U/S=61/39) or with 11·75 MJ NEl per day of hydrogenated palm oil triglyceride (HTG; U/S=6/94). Each diet was fed from 25±2 d prior to the expected calving to 100±5 d post partum. Compared with the control, both CAS and HTG supplementation resulted in an increase of the average milk yield. Milk fat content and fat-corrected milk yield were higher in the HTG group but lower in the CAS group than in the control group. In all groups liver triglyceride concentrations (TGL) increased from 15 d prepartum to 5 d post partum, and then decreased thereafter. At 5 d TGL was lower in the HTG group than control or CAS cows. No significant differences were detected in TGL among dietary treatments at 15 d prepartum and 25 d post partum. Higher plasma glucose and insulin and lower non-esterified fattay acids and β-hydroxybutyrate concentrations and aspartate aminotransferase activity were measured in the HTG group than in the control or CAS groups at 5 d or 25 d post partum. Our results show that HTG may provide a better energy supply for high-yielding dairy cows in negative energy balance than CAS around calving.

Non-specific (innate) immune response plays a major role in defending the udder from bacterial invasion. Moreover, recent investigations suggest that mammary gland epithelial cells (MGEC) could have a large and important role as a source of soluble components of immune defences. Despite many attempts to find other ways to control/prevent mastitis (i.e. vaccine) antimicrobial therapy is still the most used and effective means of curing clinical and subclinical mastitis. However, drug concentrations and therapy durations are far from the optimal in order to reduce costs. Therefore, efficacy of antimicrobial therapy is dependent not only on the substance activity but also on the positive interactions with the host innate immune response. Surprisingly, information on these interactions is rather scarce in the mastitis field. A simple experimental model was developed based on BME-UV cell line, Staphylococcus aureus as a challenge and a macrolide as an antimicrobial to assess the interactions among epithelial cells, Staph. aureus and the potential effects of antimicrobials on the immune system. The results of this study confirmed that tylosin has good antimicrobial activity against both intracellular and extracellular Staph. aureus in bovine MGEC without affecting cell functions. In this study, a significant down-regulation of IL-1 and IL-6 was observed, while TNF and IL-8 expression rate numerically increased, but differences were not significant. To our knowledge, this is the first paper assessing the concentration of two lysosomal enzymes, lysozyme and N-acetyl-β-d-glucosaminidase (NAGase), in Staph. aureus-stimulated MGEC. The results of this study confirmed that tylosin could have a significant effect on the release of these enzymes. Moreover, even if both enzymes have a similar substrate as a target, the results suggest different secretion mechanisms and an influence of antimicrobial treatment on these mechanisms. Successful mastitis cure is the result of achieving the optimal efficiency of both innate immune defences and therapeutical activities, by means of killing bacteria without eliciting an excessive inflammatory response. Therefore, antimicrobials for mastitis therapy should be selected not only on bacterial sensitivity, but also for their positive interactions with the innate immune response of the mammary gland. This study showed that an in-vitro model based on Staph. aureus challenge on MGEC could be helpful in assessing both the intracellular and extracellular activity of antimicrobials and their influence on epithelial cell immune and inflammatory response.

This study compares the colouring capacity of Brevibacterium aurantiacum (BA), Brevibacterium BL and Arthrobacter species AS in relation to deacidified media made from lactic curd (Epoisses), mixed curds (Munster) and rennet curds (Livarot or Reblochon). BA colouring capacity proved to be constant, leading to a dark orange colour, irrespective of the deacidified media. However, it gave too dark a colour for Reblochon. The strains BL and AS were not adapted to the colouring of Epoisses deacidified medium. On the Livarot or Munster deacidified medium, these two strains provided a light yellow orange colour range that was not suitable for these cheeses. However, these two strains (BL and AS) produced a suitable colour for Reblochon deacidified medium.

Retinol-binding protein 4 (RBP4) is a plasma protein involved in retinol transportation, and recent evidence in rodents suggests that RBP4 is also a metabolic regulator that modifies insulin sensitivity. To assess how RBP4 levels are regulated in ruminants, we determined the RBP4 concentrations in bovine plasma and milk using Western blot analysis. Plasma RBP4 levels in non-pregnant non-lactating (control) cows were around 45 μg/ml, which were sustained during 60-h fasting, but decreased significantly 4 h after lipopolysaccharide (LPS) administration. Basal plasma retinol concentration was around 30 μg/dl, but this decreased to approximately one-third and one-half of these values during fasting and 8 h after LPS challenge, respectively. Plasma RBP4 and retinol levels in cows 3–6 d before parturition were comparable to those of the controls. However, on the day of parturition both were significantly decreased and had returned to basal levels by two weeks after calving. Interestingly, RBP4 was clearly detected in colostrum (16·4±5·6 μg/ml) but was only faintly detected in milk from cows at 7 d and 15 d after calving. Retinol concentrations in colostrum were almost 10-fold higher than those in plasma, while those in milk were comparable to those in plasma. These results suggest that RBP4 and retinol levels are independently regulated under physiological and pathophysiological conditions and that RBP4, like retinol, is transferred from maternal stores to calves through colostrum.

Addition of methylated β-lactoglobulin (Met-BLG) in the medium of MDCK cell lines infected with influenza virus subtype H1N1 reduced hemagglutination activity (HA) in a concentration dependent manner. Antiviral activity of Met-BLG depended on its concentration, viral load, and duration of infection. Using 17 μg/ml of Met-BLG inhibited 50% of HA of H1N1 grown in MDCK cells at 1 MOI after 24 h incubation at 37°C and in 5% CO2. Extension of incubation time enhanced antiviral action since the same concentration of Met-BLG inhibited about 61% of viral activity after 48 h. This viral inhibition was accompanied by a protection of MDCK cells as observed by using neutral red or by direct microscope examination. Reduction of viral RNA replication upon the addition of Met-BLG (50 μg/ml) was observed by real time-PCR showing a reduction of viral log value of about 0·9. When viral stock solution was mixed with 25 μg/ml Met-BLG in absence of cell lines, the morphology and viability of virus particles were significantly affected as observed by electron microscopy, and the number of intact virus particles was reduced by roughly 65%.

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. It is characterized by a wide array of virulence factors. The differences among strains jeopardize the development of effective vaccines against Staph. aureus mastitis. We tested the immunogenicity of a peptide subunit vaccine coding for three different adhesion factors, fibrinogen-binding protein (Efb), fibronectin-binding protein A (FnbpA) and clumping factor A (ClfA). Then we evaluated the influence of some virulence factors on the ability of specific anti-adhesin antibodies to react with sixteen Staph. aureus strains isolated from bovine subclinical mastitis. Immunization with the recombinant adhesins stimulated a strong humoural (IgG and IgA) and mucosal IgA immune response in all animals tested. Hyperimmune serum recognized with diverse efficiency the sixteen Staph. aureus strains and this circumstance correlated well with the level of expression of adhesins. Among the different virulence factors considered to classify strains, spa gene polymorphisms showed the strongest influence on isolate reactions to hyperimmune serum. Our results indicate the importance of a disease- and environment-specific analysis of isolates. Thus, as opposed to other pathogens to obtain an effective vaccine we should characterize multiple strains and identify the prevalent virulence factors expressed.