Mixed cultures of different EPS-producing Streptococcus thermophilus strains in combination with a Lactobacillus delbrueckii subsp. bulgaricus strain with negligible EPS-production were used for yoghurt production. The yoghurt texture was characterised with respect to sensory, rheological and microstructural properties and the EPS-concentrations were determined. The cultures resulted in yoghurts with highly different texture properties, and positive interactions between certain Streptococcus thermophilus strains were observed. The underlying properties of yoghurt texture are multidimensional, but a number of microstructural characteristics were apparent in the yoghurts with the highest mouth thickness, creaminess and viscosity. A strong protein network, not too dense and with medium size pores containing EPS, seems associated with these properties. The presence of capsular polysaccharides (CPS) also appeared to be beneficial as did a combination of EPS types, which were distributed differently in the protein network (in serum pores, respectively in association with protein). Obviously, a certain concentration of EPS must be present to provide for these effects on yoghurt texture, but other factors than concentration per se seem more important.

The five exons and the 5′ and 3′-untranslated regions (5′-UTR and 3′-UTR) of the oGH gene were screened for mutations using PCR-single strand conformation polymorphism (PCR-SSCP) procedures in 523 Serra da Estrela ewes and were found to be highly polymorphic. The region extending across and between the GH2-N and GH2-Z copies was sequenced allowing the design of primers for the specific PCR amplification of each copy. These were cloned and sequenced in 20 animals representative of all SSCP patterns. The corresponding genotypes were established for each copy following nucleotide sequencing of SSCP alleles. Twenty-four polymorphic sites were found at the GH2-N (or GH1) and fourteen at the GH2-Z copies. Eight amino acid substitutions were predicted at the GH2-N and six at the GH2-Z copies. Milk yield adjusted to 150 lactation days was analysed for the genotype of each oGH gene copy taken separately or together (associated genotypes) by restricted maximum likelihood (REML) through a univariate best linear unbiased prediction (BLUP) animal model with repeated measures. Significant associations between genotypes and milk yield were observed. Within GH2-N genotypes there was a milk yield differential of 21·4±0·2 l/150 d between the most (N7) and the least (N5) productive ones. Within GH2-Z genotypes there was a differential of 21·6±0·2 l/150 d between the most (Z8) and the least (Z1) productive ones. The effect of associated GH2-N and GH2-Z genotypes revealed a differential of 39·6±0·3 l/150 d between the most (N1+Z7) and the least (N3+Z2) productive associated genotypes. The results show that GH2-N and GH2-Z genotypes significantly affect milk yield in Serra da Estrela ewes. Moreover, the apparent joint effect of GH2-N and GH2-Z genotype could improve milk yield in 25% as compared with the mean milk production of the analysed population.

Milk fat globule membranes (MFGM) were isolated from the milk of mid-lactation Holstein cows. The purified MFGM were fractionated using 1-dimensional SDS gels. Tryptic peptides from gel slices were further fractionated on a micro-capillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Analysis of the data resulted in 120 proteins being identified by two or more unique peptide sequences. Of these 120 proteins, 71% are membrane associated proteins with the remainder being cytoplasmic or secreted proteins. Only 15 of the proteins identified in the cow MFGM were the same as proteins identified in previous mouse or human MFGM proteomic studies. Thus, the bulk of the proteins identified are new for bovine MFGM proteomics. The proteins identified were associated with membrane/protein trafficking (23%), cell signalling (23%), unknown functions (21%), fat transport/metabolism (11%), transport (9%), protein synthesis/folding (7%), immune proteins (4%) and milk proteins (2%). The proteins associated with cell signalling or membrane/protein trafficking may provide insights into MFGM secretion mechanisms. The finding of CD14, toll like receptor (TLR2), and TLR4 on MFGM suggests a direct role for the mammary gland in detecting an infection.

In in vivo study on 24 weaned piglets (8 per group), the survival rates of human isolates Lactobacillus gasseri K7 and LF221 were quantified by selective enumeration on MRS agar with rifampicin, and the presence of both strains in intestinal mucosa was examined. Faeces from individual animals were analysed for the number (cfu/g) of coliforms, lactobacilli, clostridia and both of the two probiotic strains during 2-weeks probiotic application period (5×1010 cfu of individual strain/day) and 1 week after the probiotic treatment was ceased. Samples of duodenum, jejunum and ileum of sacrificed animals (5th or 20th day) were also examined microbiologically. A great variability in the microflora of faeces and mucosa was observed even between equally treated animals. The survival of both Lb. gasseri strains was established by their detection in the faeces (2·5×105 to 3·3×105 cfu of K7 strain/g faeces; 4·5×105 to 5×105 cfu of LF221 strain/g). In two animals, the LF221 or K7 viable cells were found in the faeces 6 d after ceasing probiotic application. In both animals from the group fed with Lb. gasseri K7 that were sacrificed 5 d after weaning, the presence of K7 strain was found either in the mucosa of duodenum (140 cfu/10 cm2) and jejunum (170 cfu/10 cm2) or in the ileum (1600 cfu/10 cm2). LF221 cells were detected in the ileal mucosa of one piglet (820 cfu/10 cm2). The results demonstrated the capability of both tested strains of in vivo adhesion to intestinal mucosa and of temporary colonisation of the piglets' intestine.

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 °C for 2 min and 95 °C for 8 min. The milk was acidified at 40 °C to a final pH of 4·4 using 20 g glucono-delta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

Systematic factors affecting the activities of L-lactate dehydrogenase (LDH) and N-acetyl-β-D-glucosaminidase (NAGase) and somatic cell count (SCC), the association between the activities of LDH and NAGase and SCC with respect to udder health status, and the ability of LDH and NAGase to classify cows in udder health categories for early detection of mastitis were studied. A dataset of records from 74 Danish Holstein, 76 Danish Red and 47 Jersey cows on one research farm was used. Cows were grouped into healthy and clinically mastitic. A healthy cow was defined as having no veterinary treatment and SCC<100000 cells/ml. A clinically infected cow was one receiving veterinary treatment after showing clinical signs of mastitis and SCC >800000 cells/ml. Breed, month of production, and days in milk significantly influenced (P<0·001) LDH activity, NAGase activity and SCC in both healthy and clinically mastitic cows. In healthy cows, LDH activity, NAGase activity and SCC started at a high level immediately after calving and decreased to low levels approximately 30–40 d post partum. All the three parameters increased due to clinical mastitis. NAGase activity had numerically higher variation in healthy cows than in clinically mastitic cows (CV=56·2% v. CV=53·5%). The relationship between LDH activity and SCC was stronger in milk from clinically mastitic than from healthy cows (r=0·76 v. r=0·48 and r=0·67 v. r=0·44 for correlation of observed values and residuals, respectively). LDH activity had higher sensitivity than NAGase activity (73–95% v. 35–77%) while specificities were in a similar range (92–99%). Further, sensitivities for LDH activity were more robust to changes in the threshold value than those for NAGase activity. Opportunities for automated, in-line real-time mastitis detection are discussed.

The growth of five bacteria isolated from red-smear cheeses, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variabile, Microbacterium gubbeenense and Staphylococcus saprophyticus in mixed cultures with Debaryomyces hansenii on aseptic model cheese curd at 10 and 14 °C was investigated. At both temperatures, C. casei and Micro. gubbeenense had a longer lag phase than C. variabile, Brevi. aurantiacum and Staph. saprophyticus. In all cultures, lactose was utilised first and was consumed more rapidly at 14 °C than at 10 °C, i.e., 6 d at 14 °C and 10 d at 10 °C. This utilisation coincided with the exponential growth of Deb. hansenii on the cheese surface. Lactate was also used as a carbon source and was totally consumed after 21 d at 14 °C and ~90% was consumed after 21 d at 10 °C regardless of the ripening culture. Small differences (<0·5 pH unit) in the surface-pH during ripening were noticeable between ripening cultures. Differences in the colour development of the mixed cultures with the yeast control were only noticeable after 15 d for Brevi. aurantiacum and after 21 d for the other bacteria. Regardless of the organisms tested, colour development and colour intensity were also greater at 14 °C than at 10 °C. This study has provided useful information on the growth and contribution to colour development of these bacteria on cheese.

Osteopontin (OPN) is an acidic phosphorylated glycoprotein found in many tissues and physiological fluids. OPN is known to possess multiple biological functions including bone remodeling, cell adhesion, cellular transformation and immunological mediation; these functions have been extensively reviewed (Butler et al. 1996; Sodek et al. 2000).

Raw whole milk of high microbial quality ([les ]4×104 cfu/ml) was processed using a ~15 l/h homogeniser with a high pressure (HP) valve immediately followed by cooling heat exchangers. The effects of homogenisation between 100 and 300 MPa (HP valve) with an initial milk temperature Tin=4 °C or 24 °C was investigated on the inactivation of: (i) endogenous alkaline phosphatase (ALP); (ii) endogenous milk flora and (iii) two Gram positive (Listeria innocua and Micrococcus luteus) and one Gram negative (Pseudomonas fluorescens) strains inoculated into milk. Temperatures T1 and T2 measured before and immediately after the HP valve, and fat globule size distributions were also determined. ALP activity slightly decreased after homogenisation above 250 MPa when Tin=4 °C (corresponding T2>58 °C), but markedly decreased above 200 MPa when Tin=24 °C (T2>60 °C). In contrast to inactivation induced by continuous short-time thermal treatments, ALP inactivation induced by HP homogenisation was clearly due to mechanical forces (shear, cavitation and/or impact) in the HP valve and not to the short ([Lt ]1 s) residence time at temperature T2 in the same valve. Inactivation of the three exogenous microorganisms led to similar conclusions. Homogenisation at 250 MPa or 300 MPa (Tin=24 °C) induced a 2–3 log cycle reduction of the total endogenous milk flora and a 1·5–1·8 log cycle reduction of inoculated List. innocua. Higher reduction ratios (2–4 log cycles) were obtained for the two other microorganisms. The highest levels of ALP inactivation corresponded to the highest extents of microbial reduction. Running the milk twice or three times through the homogeniser (recycling), keeping temperature T1≈29 °C and pressure=200 MPa, increased homogenisation efficiency.

Difficulties with the analysis of opaque milk and dairy products can be resolved by rendering the sample transparent. This allows direct photometric measurements of the reaction mixtures, and all sample pre-treatment steps become unnecessary. For this, several reagents have been reported in literature. Among them, the ‘Clarifying Reagent’ is mainly described here: its composition is given because it is now released from patenting restrictions. Its main property is a low absorbance between 340 and 800 nm. So it can be used in many chemical and enzymatic colorimetric measurements for estimating the quality of milk and dairy products.

Nutritional status has a major impact on the immune system. Probiotic effects ascribed to fermented dairy products arise not only from whole microorganisms but also from metabolites (peptides, exopolysaccharides) produced during the fermentation. We recently demonstrated the immunomodulating capacity of kefir in a murine model. We now aimed at studying the immunomodulating capacity in vivo of the products derived from milk fermentation by kefir microflora (PMFKM) on the gut. BALB/c mice received the PMFKM for 2, 5 or 7 consecutive days. IgA+ and IgG+ cells were determined on histological slices of the small and large intestine. IL-4, IL-6, IL-10, IL-12, IFNγ and TNFα were determined in the gut, intestinal fluid and blood serum. IL-6 was also determined in the supernatant of a primary culture of small intestine epithelial cells challenged with PMFKM. PMFKM up-regulated IL-6 secretion, necessary for B-cell terminal differentiation to IgA secreting cells in the gut lamina propria. There was an increase in the number of IgA+ cells in the small and large intestine. The increase in the number of IgA+ cells was accompanied by an increase in the number of IL-4+, IL-10+ and IL-6+ cells in the small intestine. Effects of PMFKM in the large intestine were less widely apparent than the ones observed at the small intestine lamina propria. All cytokines that increased in the small intestine lamina propria, also did so in blood serum, reflecting here the immunostimulation achieved in the gut mucosa. We observed that the PMFKM induced a mucosal response and it was able to up and down regulate it for protective immunity, maintaining the intestinal homeostasis, enhancing the IgA production at both the small and large intestine level. The opportunity exists then to manipulate the constituents of the lumen of the intestine through dietary means, thereby enhancing the health status of the host.

Nineteen calves were reared in individual pens (IP), and twenty calves were grouped in four pens of five calves each (GP) to study the effects of grouping on behaviour and performance. All calves were on an enhanced-growth feeding programme and had unlimited access to starter. Total dry matter intake (DMI) body weight (BW), serum urea, non-esterified fatty acid (NEFA), cortisol, insulin, and glucose concentrations, and immune response to vaccination were measured. Behaviour patterns categorized as: non-nutritive oral behaviour, cross-sucking, inter-sucking, and self-grooming, were monitored 20 min following the morning and afternoon milk replacer consumption, and at the same times after weaning. There were no differences in final BW and starter DMI between treatments. The decrease of average daily gain (ADG) during the week following weaning was more pronounced in GP than in IP calves. Serum NEFA concentration increased at the beginning of the study in IP calves but serum urea concentrations were greater in GP than in IP calves the week after weaning. Self-grooming increased in both treatments throughout the study but GP calves increased non-nutritive oral behaviour and decreased cross-sucking and inter-sucking behaviours throughout the study. Rearing calves in groups following an enhanced-growth feeding programme did not increase starter intake. Furthermore, cross-sucking and inter-sucking behaviours in calves reared in groups did not seem to cause increased metabolic or immunological distress.

The objective was to quantify the strength of the relationship between body condition score (BCS) and live weight (LW) in pasture-based Holstein-Friesian dairy cattle, and to determine the kg LW per unit BCS. A total of 26021 test-day records with information on both BCS (1–10 scale, where 1 is emaciated and 10 is obese) and LW across 1110 lactations from one research farm were used in the analysis. Correlation and regression analyses were used to determine the degree of association between BCS and LW in different parities, stages of the inter-calving interval and years. Correlations between BCS and LW were relatively consistent, with the mean correlation between BCS and LW across all data of 0·55 implying that differences in BCS explain approximately 30% of the variation in LW. Significantly different regressions of LW on BCS were present within stage of inter-calving interval by parity subclasses. Excluding calving, LW per unit BCS varied from 17 kg (early to mid lactation in parity 1) to 36 kg (early lactation in parity 4 and 5). However, LW per unit BCS was greatest at calving varying from 44 kg in first parity animals to 62 kg in second parity animals. On average, 1 BCS unit equated to 31 kg LW across all data.

Extrinsic factors such as maternal microbiota, bacterial load of the environment, diet and medication modulate the intestinal microbiota. Maturation and function of the immune system is influenced by established gut microbiota. In this work we describe the immunological effects of the dietary deprivation of fermented foods of healthy volunteers. Significant decreases in faecal lactobacillus and total aerobes counts and concentration of short chain fatty acids were observed following deprivation of fermented food of the normal diet. Moreover, a decrease in phagocytic activity in leukocytes was observed after two weeks of restricted diet. Therefore, the dietary deprivation of fermented foods could induce a decrease in innate immune response that might affect the capacity to respond against infections. The ingestion of a probiotic product containing the strains Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711 or a standard yogurt containing a conventional starter Lactobacillus delbrueckii sp. bulgaricus counteracted the fall in the immune response, although the probiotic product was more effective than the standard yogurt.

A study was carried out to determine genetic variants of β-lactoglobulin gene and to explore associations between these and milk composition traits in riverine buffalo. Single strand conformation polymorphism was employed to detect the genetic variants of the gene. Two fragments of this gene i.e. 119 bp of exon I and 400 bp spanning exon IV and intron IV were included in the study. For 119 bp fragment, three alleles namely, A, B and C were observed in all the buffalo breeds whereas four alleles (A, B, C and D) were detected for 400 bp fragment. The frequency distribution of alleles was different in different breeds of buffaloes for both the fragments. For exon I fragment, the milk composition traits such as total SNF, protein, solid, fat and whey protein yield were found to be significantly (P<0·05) associated with genotypes in Murrah and Bhadawari buffalo whereas in Mehsana breed genotypes were significantly (P<0·05) co-related with total SNF, solid and fat yield. Genotypes of 400 bp fragment, only total fat yield in Mehsana buffalo was found to be significantly (P<0·05) associated with genotypes.