Lactococcus lactis strain AMP2I expresses OppA(D471R), a mutant oligopeptide binding OppA protein in which the aspartyl residue at position 471 was replaced by arginine. As a consequence of a different peptide transport in this strain, experimental Hispánico cheese made with Lc. lactis AMP2I had a higher content of total free amino acids than control cheese made with Lc. lactis AMP1I, an isogenic strain expressing wild-type OppA (Picon et al. 2005, 2007). In this work higher levels of diketones, hydroxy-ketones and, to a lesser extent, branched chain aldehydes were recorded for experimental cheese compared with control cheese. These differences levelled off as ripening proceeded. Strong correlations support the hypothesis that the increased levels of these volatile compounds in cheese made with Lc. lactis AMP2I are linked to higher concentrations of free amino acids threonine, valine and leucine.

Mastitis is one of the most prevalent diseases of cattle. Various studies have reported breed-dependent differences in the risk for developing this disease. Among two major breeds, Jersey cows have been identified as having a lower prevalence of mastitis than Holstein cows. It is well established that the nature of the initial innate immune response to infection influences the ability of the host to clear harmful bacterial pathogens. Whether differences in the innate immune response to intramammary infections explain, in part, the differential prevalence of mastitis in Holstein and Jersey cows remains unknown. The objective of the current study was to evaluate several parameters of the innate immune response of Holstein and Jersey cows to intramammary infection with Staphylococcus aureus, a common mastitis-inducing pathogen. To control for non-breed related factors that could influence these parameters, all cows were of the same parity, in similar stages of milk production, housed and managed under identical conditions, and experimentally infected and sampled in parallel. The following parameters of the innate immune response were evaluated: acute phase protein synthesis of serum amyloid A and lipopolysaccharide-binding protein; total and differential circulating white blood cell counts; milk somatic cell counts; mammary vascular permeability; milk N-acetyl-beta-d-glucosaminidase (NAGase) activity; and production of the cytokines, interferon (IFN)-γ, interleukin (IL)-12, tumour growth factor(TGF)-α, and TGF-β1. The temporal response of all of these parameters following infection was similar between Holstein and Jersey cows. Further, with the exception of changes in circulating neutrophils and NAGase activity, the overall magnitude of these parameters were also comparable. Together, these data demonstrate that the innate immune response of Holstein and Jersey cows to Staph. aureus intramammary infection remains highly conserved despite previously reported differences in mastitis prevalence, as well as genotypic and phenotypic traits, that exist between the two breeds.

Agents that increase natural protective mechanisms have been proposed for prevention and treatment of intramammary infections. The objective of this study was to describe the effects of a single intramammary infusion of a lipopolysaccharide (LPS)-based biological response modifier (BRM) on cellular death mechanism in uninfected and Staphylococcus aureus-infected bovine mammary glands during involution. Three groups of 12 cows, each one including 6 Staph. aureus-infected and 6 uninfected, were infused in two mammary quarters with BRM or placebo and slaughtered at 7, 14 and 21 d of involution. In infected quarters, BRM treatment produced a significant increase in percent of stained epithelial cells for the apoptosis-promoting protein Bax at every observation period. In addition, BRM produced a significant increase of immunostained stromal cells for Bax compared with placebo-treated quarters. BRM treatment produced an increase in percentages of epithelial cells staining with active caspase-3 at 7 d and 14 d of involution compared with placebo-treated quarters and a significant decrease in percentages of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive epithelial cells at 7 d and 21 d of involution. In addition, BRM treatment caused an increase in percentage of stromal cells immunostaining for active caspase-3 and TUNEL. An increase of active caspase-3 and TUNEL epithelial and stromal cell immunostaining was observed in Staph. aureus-infected compared with uninfected quarters. Cellular proliferation, determined by Ki-67 immunostaining, was increased in epithelial and stromal cells from Staph. aureus-infected compared with uninfected quarters at every observation period. These results provide new insights into the mechanism of mammary cell death in uninfected and Staph. aureus-infected bovine mammary gland during involution and illustrate the effects of LPS-based BRM on apoptosis and cell proliferation during mammary involution.

Isolates of various species of coagulase-negative staphylococci (CNS) from extramammary swab samples were compared with isolates of bovine mastitis CNS species. Swab samples were taken from perineum skin and udder skin, teat apices and teat canals of lactating dairy cows of the research dairy herd of the University of Helsinki in 1999 and 2002. In addition, hands of herd staff and liners of teat cups were sampled for CNS. CNS isolates from milk samples of subclinical or clinical mastitis in the same herd were collected during 1998–2002. Species identification was performed using phenotyping (API Staph ID 32 test) and by constructing a 16 and 23S rRNA RFLP library (ribotyping). Based on phenotype, 84% of mastitis isolates and 57% of extramammary isolates were identified at species level with >90% probability. Ribotype patterns formed 24 clusters, and 15 of them included a CNS type strain. If the ribotype clusters contained isolates of both extramammary and mastitis origin, they were further typed using pulsed-field gel electrophoresis (PFGE). The predominant CNS species in mastitis, based both on phenotyping and genotyping, were Staph. chromogenes and Staph. simulans. Phenotyping failed to identify half of the extramammary isolates. Based on phenotyping, Staph. equorum and Staph. sciuri, and based on ribotyping, Staph. succinus and Staph. xylosus, were the predominant CNS species in extramammary samples. The most common species in milk samples, Staph. chromogenes, was also isolated from several extramammary samples, and five out of ten pulsotypes were shared between mastitis and extramammary isolates, indicating that strains from udder skin are highly similar. The second commonest mastitis species, Staph. simulans, was isolated only from three extramammary samples, indicating that Staph. simulans may be more specifically associated with mastitis. Consequently, the origin of CNS mastitis may vary depending on the causing CNS species.

In order to have a deeper insight into the retinol isomerization phenomenon, in this work different milk samples have been analysed for their content of trans retinol and its cis-isomers, by means of reliable HPLC techniques. Levels of the different isomers and the degree of retinol isomerization (13-cis/all-trans ratio, %) have been monitored during milk storage at different temperatures and after addition of specific microorganisms. In raw milk stored at 4°C for 96 h the degree of retinol isomerization shifted from 1·1 to 2·3%, while in raw milk stored at 22°C for 24 h it increased from 1·1 to 12·7%. Among microorganisms tested in pasteurized milk, the most active in causing an increment in the 13-cis/all-trans ratio (%), from 3·4 to 33·4% in 8 h, was Streptococcus thermophilus. The results obtained demonstrated a relationship between microbial evolution and retinol isomerization. Therefore, the determination of retinol isomers is of importance not only for a more precise evaluation of vitamin A activity but also for the evaluation of the microbiological quality of milk.

The objective was to evaluate the effect of β-lactoglobulin (β-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine β-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of β-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and β-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein:true protein ratio were found.

We targeted quantitative trait loci (QTL) for milk protein percentage (P%) in two Italian Holstein granddaughter design families using selective genotyping in combination with high throughput amplified fragment length polymorphism (AFLP) markers. A total of 64 extreme high and low sires in respect to estimated breeding value (EBV) for P% (EBVP%) were genotyped with 25 AFLP primer combinations that revealed 305 and 291 polymorphisms in the two families. Association between markers and EBVP% was investigated by a linear model only on bands having paternal origin (105 and 96 AFLP bands in family D and S, respectively). Although no marker was significantly associated with the target trait after correction for multiple comparisons, 17 AFLP markers, significant without correction for multiple tests, were considered suggestive of the presence of a QTL. Eleven of these were successfully located on six Bos taurus (BTA) chromosomes by radiation hybrid or in-silico mapping. Ten of these mapped in the immediate neighbourhood (less than 10 cM) of already described QTL for P%. Suggestive association was verified in four regions by microsatellites analysis: one on BTA 10; one on BTA 28; and two on BTA 18. Microsatellites identified significant effects by single marker and interval mapping analyses on BTA 10 and BTA 28, while they were only suggestive of the presence of QTL on BTA 18. In summary, our results firstly indicate that AFLP markers may be used to seek QTL exploiting a selective genotyping approach in GDD, a wide used experimental design in cattle; secondly, propose two approaches for AFLP mapping, namely in-silico mapping exploiting most updated release from the bovine whole genome sequencing project, and physical mapping exploiting a panel of Bovine/Hamster Radiation Hybrids; and thirdly, provide new information on QTLs for an economic important trait in a never investigated Holstein cattle population. AFLP in combination with selective genotyping can be a useful strategy for QTL searching in minor livestock species, sometimes having large economic impact in marginal areas, where more informative markers are still poorly developed.

The effect of the stage of lactation, the number of foals and age of the mare on changes in the fat content and fatty acid composition of colostrum and milk of primitive Konik horses was investigated. Colostrum and milk samples from 12 lactating mares were collected at the beginning of lactation, on the days 1 and 2 after foaling and then, starting from the first month of lactation, at 4-week intervals up to the sixth month of lactation. Significant differences were observed in fat content as well as the composition of some analysed fatty acids between colostrum and milk of mares of the Konik breed. The number of foalings and the age of mares did not have a statistically significant effect on the fat content in milk and had only a slight effect on the fatty acid composition. Milk produced by mares of the Konik breed is characterized by a considerable content of polyene fatty acids with 18 carbon atoms, a low ratio of n-6 fatty acids to n-3 fatty acids as well as low, highly advantageous values of atherogenic and thrombogenic indices.

In a previous study, ultrafiltration (UF) at 1·2×reduced residual chymosin activity and bitterness in exopolysaccharide (EPS)-positive reduced fat Cheddar cheese. The objective of this research was to study the effect of this level of concentration on the textural and functional characteristics of the reduced fat cheese. Ultrafiltration (1·2×) did not affect the hardness, cohesiveness, adhesiveness, chewiness, and gumminess of EPS-positive cheese. The 6-month old UF cheeses were springier than non-UF cheeses. However, the springiness of the EPS-positive cheese made from UF milk was much lower than that of the EPS-negative cheeses. Texture of the EPS-negative cheese was more affected by UF than that of the EPS-positive cheese. Differences were seen in the extent of flow between UF and non-UF cheeses at 1 and 3-months but not after 6 months ripening. Ultrafiltration increased the elastic modulus in the 6-month old EPS-positive cheeses. Higher body and texture scores were given to EPS-positive cheeses than the EPS-negative ones. Sensory panelists found the body of the UF and non-UF cheeses to be similar.

The objective was to evaluate the efficacy of a single dose of danofloxacin (6 mg/kg bodyweight) given by the intravenous route for the treatment of acute bovine mastitis induced by intra-cisternal infusion of an Escherichia coli strain (26 cfu into one rear quarter of each cow). Twenty-three Prim'Holstein lactating cows were inoculated. To be challenged, the mammary glands had to be productive, free of pathogenic bacteria, and with somatic cell counts (SCC) of <200 000 cells/ml. The cows were treated on an individual basis when predetermined criteria involving both systemic and local clinical signs were satisfied. Allocation to treatment, danofloxacin or negative saline control, was performed according to a randomized treatment allocation plan. Monitoring during a 21-d period after inoculation included individual clinical examination, bacteriological examination and determination of SCC. Esch. coli was isolated from the milk of all inoculated quarters at the first milking post-inoculation and, together with reference to the clinical scores; the challenge was considered to be successful in 20 of the 23 cows. On study day 7 bacteriological cure rates with danofloxacin and saline control were 89% (8/9) and 44% (4/9) respectively. On days 14 and 21 all milk samples that could be collected were negative for Esch. coli in both groups of animals. Beneficial statistically significant differences were found at the end of the observation period (days 19–21 post treatment) between cows treated with danofloxacin and saline for SCC (P=0·0091) and earlier in the study for milk production (P=0·0003) and udder inflammation (P=0·004). Obvious beneficial trends were recorded in the danofloxacin group for rectal temperature, milk quality, general behaviour and appetite. Danofloxacin-treated cows showed statistically significant lower local clinical scores and a more rapid return to pre-inoculation values. It was concluded that systemically administered danofloxacin is effective in terms of bacteriological results, milk production and both systemic and local signs when used in the treatment of induced acute Esch. coli mastitis. Danofloxacin hastens recovery and return to productivity compared with potential self cure.

In this study, the properties of casein particles reformed from alkaline disrupted casein micelles were studied. For this purpose, micelles were disrupted completely by increasing milk pH to 10·0, and subsequently reformed by decreasing milk pH to 6·6. Reformed casein particles were smaller than native micelles and had a slightly lower zeta-potential. Levels of ionic and serum calcium, as well as rennet coagulation time did not differ between milk containing native micelles or reformed casein particles. Ethanol stability and heat stability, >pH 7·0, were lower for reformed casein particles than native micelles. Differences in heat stability, ethanol stability and zeta-potential can be explained in terms of the influence of increased concentrations of sodium and chloride ions in milk containing reformed casein particles. Hence, these results indicate that, if performed in a controlled manner, casein particles with properties closely similar to those of native micelles can be reformed from alkaline disrupted casein micelles.

A washing treatment was applied to caprine cream before churning in order to improve phospholipids and MFGM protein purification from buttermilk and butter serum. Cream obtained from a first separation was diluted with water and separated a second time using pilot plant equipment. Regular and washed creams were churned to produce buttermilk and butter, from which butter serum was extracted. The washing treatment allowed a significant decrease of the casein content. As a result, the phospholipids-to-protein ratios in washed buttermilk and butter serum were markedly increased by 2·1 and 1·7-folds respectively, which represents an advantage for the production of phospholipids concentrates. However, when compared with bovine cream, lower phospholipids-to-protein ratios were observed when the washing treatment was applied to caprine cream. A higher concentration of MFGM protein and a lower retention of phospholipids during washing treatment are responsible for the lower phospholipids-to-protein ratios in buttermilk and butter serum obtained from caprine cream. The phospholipids distribution in the butter making process was similar to the one obtained from bovine regular and washed cream. Phospholipids were preferentially concentrated in the butter serum rather than the buttermilk fraction. This simple approach permitted the production of caprine and bovine butter sera extracts containing up to 180 and 240 g phospholipids/kg sera, respectively, on a dry basis.

We evaluated the effect of grazing time of day on goat milk chemical composition, renneting properties and milk fatty acid profile in a Mediterranean grazing system. Sixteen lactating Girgentana goats were divided into two experimental groups and housed in individual pens, where they received 500 g/d of barley grain. For 5 weeks the two groups were left to graze in two fenced plots on a ryegrass sward as follows: morning group (AM), from 9·00 to 13·00; afternoon group (PM), from 12·00 to 16·00. In selected herbage, water-soluble carbohydrates (WSC) increased in the afternoon (204 v. 174 g/kg dry matter, DM; P=0·01), whereas crude protein (CP) and linolenic acid decreased (respectively, 16·7 v. 19·8% DM; P<0·01 and 26·8 v. 30·4 g/kg DM; P<0·01). Pasture dry matter intake (DMI) was significantly higher in the afternoon (0·82 v. 0·75 kg/d; P=0·026). Fat corrected milk production (FCM), milk fat and lactose content were not affected by treatment, whereas protein and titrable acidity (°SH) increased in the PM group (respectively 3·56 v. 3·42%; P=0·01; 3·55 v. 3·22°SH/50 ml; P=0·01). In contrast, milk urea content was significantly higher in the AM group (381 v. 358 mg/l; P=0·037). The results seem to indicate that an improvement in ruminal efficiency might be obtained by shifting grazing time from morning to afternoon, as a consequence of a more balanced ratio between nitrogenous compounds and sugars. Indeed, the higher linolenic acid and the lower conjugated linoleic acid (CLA) (respectively 1·02 v. 0·90, P=0·037; 0·71 v. 0·81% of total fatty acids, P=0·022) in the milk of goats grazing in the afternoon seem to indicate a reduced biohydrogenation activity in the PM group.

Folding variants of α-lactalbumin (α-la) are known to induce cell death in a number of cell types, including mammary epithelial cells (MEC). The native conformation of α-la however has not been observed to exhibit this biological activity. Here we report that native bovine α-la reduced the viability of primary bovine mammary epithelial cells (BMEC) and induced caspase activity in mammospheres, which are alveolar-like structures formed by culturing primary BMEC on extracellular matrix in the presence of lactogenic hormones. These observations suggest a possible role for bovine α-la in involution and/or maintaining the luminal space in mammary alveoli during lactation. In addition, co-incubation of bovine α-la in an in-vitro mammosphere model resulted in decreased β-casein mRNA expression and increased αs1- and κ-casein mRNA expression. This differential effect on casein expression levels is unusual and raises the possibility of manipulating expression levels of individual caseins to alter dairy processing properties. Manipulation of α-la levels could be further investigated for its potential to enhance milk protein expression and/or improve lactational persistency by influencing the balance between proliferation and apoptosis of BMEC, which has a major influence on the milk-producing capacity of the mammary gland.

In this study, the effect of dahi containing probiotic Lactobacillus acidophilus NCDC14 and Lactobacillus casei NCDC19 (∼73×108 cfu/g) on progression of streptozotocin (STZ)-induced diabetes in rats (15 g/day/rat) for 28 days was investigated. Feeding of probiotic dahi significantly suppressed the incremental peaks and area under the curve and delayed reduction of insulin secretion during oral glucose tolerance test more than skim milk or control dahi. The feeding of milk products reduced the total cholesterol, triglycerides, LDL and VLDL-cholesterol and increased HDL-cholesterol levels (P<0·05). Moreover, probiotic dahi significantly suppressed STZ-induced oxidative damage in pancreatic tissues by inhibiting the lipid peroxidation and formation of nitric oxide, and preserving antioxidant pool such as glutathione content and activities of superoxide dismutase, catalase and glutathione peroxidase. The results suggest that the supplementation of probiotic Lb. acidophilus and Lb. casei with dahi cultures increased the efficacy of dahi to suppress STZ-induced diabetes in rats by inhibiting depletion of insulin as well as preserving diabetic dyslipidemia, and inhibiting lipid peroxidation and nitrite formation. This may empower antioxidant system of β-cells and may slow down the reduction of insulin and elevation of blood glucose levels.

Coagulase gene restriction fragment length polymorphism (RFLP), six-locus variable-number tandem-repeat analysis patterns (MLVA) and detection of enterotoxin genes (se) (sea, sec, sed, seg, seh, sei, sej and sel) were used to determine the phylogenetic relationship among isolates of Staphylococcus aureus isolated from dairy products from different regions of Italy. A total of 25 Staph. aureus were subtyped into 16 coagulase genotypes by RFLP, and MLVA revealed marked genomic variability. Furthermore, 17 of the isolates harboured at least one toxin gene, with the predominance of sea, sed and sej among cow isolates and sec-sel among the goat and sheep strains. Combined RFLP, MLVA polymorphism and se genes were found to be useful techniques for discriminating several genetic variants in Staph. aureus isolates.

The aim of the present study was to identify the nature of bonds established between protein particles after stirring that are responsible for the texture improvement of stirred yoghurts, called rebodying. Using a constant model yoghurt at pH 4·4, the effects of changes in the physicochemical conditions at stirring were studied on the subsequent rebodying. Short term rebodying was measured as the changes in viscoelastic properties at 4°C during 20 h after stirring, while long-term rebodying was measured as the viscosity changes during 28 d storage at 4°C. Moreover, stirred gels obtained from either set gels that were allowed time or not for ionic equilibration were compared. Increasing or decreasing ionic strength did not change the properties of stirred gels. Calcium chloride addition significantly decreased G′0 h, G′20 h and tan20 h but did not induce changes in the gel microstructure as observed by confocal scanning microscopy. Yoghurt rebodying could not be explained by fulfilling ionic equilibrium. Moreover, N-ethyl maleimide addition had no effect on the stirred yoghurt. Attractive electrostatic and disulphide interactions were not involved in the gel rebodying and increasing calcium concentration in the set gel limited rebodying.

A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Φ-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages.

The roles of the pro-adipogenic ligands of the transcription factor Peroxisome Proliferator Activated Receptor gamma (PPARG) in regulating innate immune responses in bovine mammary epithelial cells (bMEC) were investigated using quantitative real-time PCR. The analyses revealed that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) enhanced the expression of Interleukin 8 (IL-8) and Chemokine (C-X-C motif) ligand 6 (CXCL6) in these cells in a dose-dependent manner. 15d-PGJ2 also induced the expression of transcripts encoding proteins involved in oxidative stress, including Ferritin heavy chain and Superoxide dismutase 1, as well as substantial microfilament reorganization. In contrast, synthetic PPARG agonists displayed a different and much smaller range of effects on the cells, causing down-regulation of Interleukin 1-beta, Interleukin 6 and IL-8 and increased expression of Chemokine (C-C motif) ligand 2 (CCL2) and Tumour necrosis factor alpha (TNFα). In an independent analysis, the cells were pre-incubated with PPARG agonists followed by lipopolysaccharide stimulation. This study revealed that troglitazone increased the responsiveness of the cells to lipopolysaccharide resulting in up-regulation of Interleukin 1-beta, TNFα, IL-8, CCL2 and CXCL6 while 15d-PGJ2 caused down-regulation of TNFα, CCL2 and CXCL6. These findings are relevant to understanding the anti-inflammatory potential of the PPARG ligands and underline different mechanisms of action of 15d-PGJ2 and troglitazone in bMEC. Furthermore, the present results demonstrate that the generation of pro-inflammatory mediators can be modulated by currently available therapeutic agents and may therefore be of value in the treatment of mastitis in ruminants.

The effect of selenium supplementation of feed on the Se content in bovine milk, whey and plasma, and on the distribution of Se, Zn and Cu in whey and plasma was investigated. In a cross-over study two groups of cows were given a basal feed with 0·16 ppm selenite (approx. 3 mg Se/d) with or without 25 mg yeast Se/d for 2 weeks. In the supplemented group the Se content increased 10-fold in milk, 10-fold in whey and 2-fold in plasma, and after the cessation of the supplementation, selenium in milk decreased with a calculated half-life of 3·5 d. In another experiment, two groups of cows were given either 100 mg yeast Se/d for 1 week or only the basal feed. The increase in Se content in both whole and defatted milk was 40–50-fold, and in whey it was approx. 20-fold. Size-exclusion chromatography of whey using inductively coupled plasma mass spectrometry for detection showed that supplementation increased the proportion of Se in the β-lactoglobulin-α-lactalbumin fraction. Distribution of Cu and Zn was essentially unaffected. In plasma, supplementation increased the Se content in all major Se fractions like selenoprotein P, albumin and low-molecular-weight compounds, but the distribution profiles of Zn and Cu underwent no major changes. The study showed for the first time the rapid kinetics of the Se increase and decrease in milk after the initiation and cessation of supplementation, respectively, and the preferential appearance of Se in the β-lactoglobulin-α-lactalbumin fraction of whey. Milk highly enriched in selenium will be a useful tool for different research purposes.