This work evaluated the effect of adjunct autochthonous cultures on the chemical, microbiological and sensory characteristics of Fiore Sardo cheese during ripening. A total of twelve batches of cheeses were manufactured according to the technical Disciplinary of Fiore Sardo cheese, with and without different combinations of autochthonous strains isolated from the native microflora of artisanal Fiore Sardo. There were no significant differences in the cheese compositional parameters between experimental and control cheeses, but the addition of cultures led to a statistically significant decrease in pH values in experimental cheeses. The evolution of total mesophilic bacteria, total coliforms and lactic acid bacteria were significantly influenced by the addition of autochthonous cultures in most of the experimental cheeses. As for sensory characteristics, all the experimental cheeses reported significantly higher scores especially for shape, texture, interior openings, taste and aftertaste. This study demonstrated the beneficial effect of the addition of selected autochthonous cultures in accelerating the disappearance of undesirable flora and improving the typical sensory characteristics of the cheese, and confirmed the importance of ewes' milk as a source of technologically interesting strains that could be used to ensure a higher quality of artisanal cheese productions.

In order to establish cut-off values for Lactococcus lactis to six antibiotics to distinguish susceptible and intrinsically resistant strains from those having acquired resistances, the minimum inhibitory concentration (MIC) of tetracycline, erythromycin, clindamycin, streptomycin, chloramphenicol and vancomycin was determined in 93 different Lc. lactis strains using the Etest. These bacterial strains were originally isolated from dairy and animal sources in widely separated geographical locations. Cut-offs were defined on the basis of the distribution of the MICs frequency of the studied antibiotics, which in the absence of acquired determinants should approach to a normal statistical distribution. In general, the new cut-off values proposed in this study are higher than previously defined (European Commission, 2005. The EFSA Journal 223, 1–12). Based on these new values, all the strains tested were susceptible to erythromycin, chloramphenicol and vancomycin, and 79 susceptible to all six antibiotics. However, 11 strains (around 12%) were considered resistant to tetracycline (six of which had been identified after screening of a large collection of lactococci strains for tetracycline resistance) and five (5·4%) resistant to streptomycin. Of these, two fish isolates proved to be resistance to both tetracycline and streptomycin. From the tetracycline resistant strains, tet(M) and mosaic tet(L/S) genes were amplified by PCR, demonstrating they harboured acquired antibiotic resistance determinants.

Bovine milk antibodies directed against human pathogenic organisms have potential as prophylactic or therapeutic treatments of disorders affecting mucosal surfaces. The cow, however, does not naturally secrete high levels of IgA in milk, the predominant immunoglobulin of the mucosal immune system. We have patented an immunisation protocol that results in increased production of IgA in ruminant milk and in this study established that our protocol can be used on a scale sufficient to produce semi-industrial quantities of milk for processing. Cows were immunised with a common pathogenic yeast, Candida albicans and responded with high levels of antigen-specific IgA antibodies in their milk. The spray-dried milk-protein concentrate (85% protein) powder was shown to reduce adherence of Cand. albicans cells in in vitro adherence assays, demonstrating an ability to retain efficacy through the processing. These results suggest that this milk product may be of therapeutic value if the reduction in Cand. albicans adhesion observed in vitro translates to reduced colonisation in vivo.

The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.

The formation of pressure-induced casein structures (600 MPa for 30 min at 30°C) was investigated for different pressure release rates (20 to 600 MPa min−1) and casein contents (1 to 15 g/100 ml). Structures from liquid (sol) to solid (gel) were observed. The higher the protein content and the pressure release rate, the higher was the dynamic viscosity. A firm gel was built up at a casein content of 7 g/100 ml for a pressure release rate of 600 MPa min−1, while lower release rates resulted in less firm gels (200 MPa min−1) or liquid structures (20 MPa min−1). In a 5 g/100 ml casein solution and at a pressure release rate of 600 MPa min−1, casein aggregates were generated which were built from smaller casein particles with a larger hydrodynamic diameter and higher voluminosity than in the untreated solution. After a slow release rate casein micelles had a smaller hydrodynamic diameter and a lower voluminosity, but were similar in shape and diameter as compared with the micelles in solution before high pressure treatment.

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0·02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

A monoclonal antibody based capture immunoassay has been recently developed for the specific quantitation of bovine milk alkaline phosphatase (ALP) without interference by contaminating microbial or fungal ALPs (Geneix et al. 2007). This immunoassay was used to study the kinetics of ALP heat denaturation in bovine milk over a range 50–60°C for 5 to 60 min using a colorimetric quantification of the enzyme activity as a reference test. A denaturation midpoint was obtained at 56°C for a 30 min heating. Thermal inactivation was found to follow first order kinetics and is characterized by z value of 6·7 deg C (D60°C=24·6 min) and 6·8 (D60°C=23·0 min) for respectively immunoassay and colorimetric assay. The high values of enthalpy of activation and the positive values of the entropy of activation and free energy of activation indicate that during denaturation ALP underwent a large change in conformation. The results of the immunoassay were highly correlated (r=0·994) with those obtained by the colorimetric assay. A similar high correlation (r=0·998) was obtained when industrially thermized milks (62–67°C for 20–90 s) were analysed by both techniques. These results indicated that 1) thermally induced epitopic structural changes recognized by the capture monoclonal antibody are concomitant with or occur after the loss of enzymatic activity and 2) quantification of ALP by the specific immunoassay is appropriate for determining mild time/temperature treatment of milk and for the control of milk pasteurization.

A subjective cow cleanliness scoring system was validated and used to assess the cleanliness score of dairy cows at different times in the year. A longitudinal study followed a number of farms from summer to winter, and a larger, cross-sectional study assessed a greater number of farms during the housed winter period. The scoring system was demonstrated to be both a repeatable and practical technique to use on-farm and showed that cows become dirtier in the transition from summer grazing to winter housing. Although farming system (organic or conventional) had no effect on cow cleanliness when cows were at grass, when housed in the winter, organic cows were significantly more likely to be cleaner. There was a link between cow cleanliness scores and milk quality, with herds having lower bulk tank somatic cell counts (BTSCC) tending to have a lower (cleaner) median cow cleanliness score; with this relationship strongest for the organic herds. There was no significant link between cleanliness score and Bactoscan (BS) count or clinical mastitis incidence. No major mastitis pathogens were cultured from bulk tank milk samples from the quartile of herds with the cleanest cows in contrast to the quartile of herds with the dirtiest cows, where significant mastitis pathogens were cultured. Based on this study, all farms, especially organic systems, should attempt to keep cows clean as part of subclinical mastitis control.

We evaluated the impact of the dietary cation-anion difference (DCAD) on the influence of anionic salts (AS) on the metabolism of dairy cows using a study-design that included control of feed intake. Ten mature, non-lactating, non-pregnant, Holstein-Friesian-crossbreed cows received 2000 mEq of either one of the seven anionic salts tested, two combinations of the anionic salts or water as control via a rumen cannula. Salts and controls were assigned in a 10×10 Latin square design. Whole blood, serum and urine samples were taken during treatment (TP) and washout period. Samples of whole blood were tested for pH, base-excess and bicarbonate concentrations. In urine, pH and net acid-base excretion (NABE) were analysed. Calcium was measured in serum and urine. According to the different batches of hay, five groups of DCAD were created regarding cluster analysis. Changes in urine and blood parameters were statistically analysed for each DCAD group separately. The different DCAD had an impact on the amount of change in acid-base balance (ABB) and calcium metabolism and for how long these changes lasted. In the DCAD group receiving the highest amount of AS (239 mEq/kg dry matter with AS), changes of ABB were only noticeable in urine and these changes only differed from day zero in the first week of TP (P<0·05). In the other four groups changes of ABB were also visible in blood parameters, but only on a few days of TP did the deviations differ significantly (P<0·05) from day zero. Changes of ABB parameters in urine samples were more pronounced than those in blood and differed clearly from day zero (P<0·05). Parallel to the changes of ABB, calcium concentrations in these samples were significantly increased (P<0·001) in all DCAD groups. Except for the highest DCAD group, ionized calcium concentrations changed over time (P<0·020). However, the differences were very small and only differed from day zero on a few TP days. We conclude that the DCAD of a dairy cow's diet has an important impact on the effect of AS on ABB and calcium metabolism with respect to the duration and amount of change. The target regions of DCAD should be clearly below 100 mEq/kg dry matter to ensure the desired effect on ABB and calcium metabolism. Extremely negative DCAD should be avoided to minimize the risk of clinical acidosis induced by AS.

The objective of the study was to assess the effects of intravenous infusion of triacylglycerol (TAG) emulsions derived from different lipid sources on responses to mitogens of peripheral blood mononuclear cells (PBMC) isolated from fasted dairy cows. Six multiparous, non-pregnant, non-lactating Holstein cows were used in a replicated 3×3 Latin Square design. For 4 d, cows were fasted and infused intravenously with a 20% TAG emulsions derived from tallow (TA), linseed oil (LO) or fish oil (FO). Fasting was employed to induce energy deficit and lipid mobilization. Emulsions were administered for 20 to 30 min every 4 h throughout the 4 d fast at a rate of 0·54 g TAG/kg BW/d. Blood samples were taken before the first infusion, and then every 24 h during the fast. Blood was utilized to assess DNA synthesis, IgM and interferon-gamma (IFN-γ) secretion by PBMC stimulated with mitogens. In TA infused cows there was a decline of PBMC ability to respond to mitogens, which was significant 48 h after initiation of the infusion period for DNA synthesis and IFN-γ secretion. In LO or FO infused cows, PBMC responses to mitogens were not altered during the infusion period, and in some cases PBMC responses to mitogen was improved at 72 and 96 h after initiation of treatments. Effects of TAG infusion on PBMC responses to mitogens depended on the lipid source suggesting that LO or FO can attenuate the negative effects of fasting on immune functions.

The use of artisan-produced lamb rennet pastes, but not any of the other commercial animal rennets, imparts a characteristic flavour to the cheese, so most Mediterranean ewes' milk cheeses are coagulated with this kind of rennet paste. In contrast to the advantages of using lamb or kid rennet pastes from the sensory point of view, questions are still raised as to their hygienic quality. The goal was to examine the microbiological and enzymic quality of lamb rennet pastes prepared by cheese manufacturers for their own use, and evaluate the hygienic quality of raw sheeps' milk cheeses made with them, using Idiazabal cheese as a model. Lamb rennet pastes prepared by artisan cheese makers from the Basque region of Spain (27), and Italy (8) were evaluated. For cheese making experiments 5 different lamb rennet pastes were selected among the 27 samples from the Basque Country region of Spain. Microbiological analyses were carried out on samples from rennet pastes, rennet extracts, milks and cheeses during ripening. Enzymic activities studied in rennet paste were: total coagulating strength and lipase. Analysis of variance and Student's t-tests was performed. The results show that the artisan-produced rennet pastes contain high levels of a variety of microorganisms. After 60 ripening days, which is the minimum ripening period required for Idiazabal cheese prior to its commercialization, no Eschericia coli, Clostridium, Salmonella spp. or Listeria monocytogenes were detected, and levels for the rest of the microorganisms were below the limits of the European legislative standards for cheese manufactured with raw milk. We can conclude that the use of artisan-produced lamb rennet pastes of questionable hygienic quality for the manufacture of raw milk hard cheeses yields products of good hygienic quality.

In this study, the antihypertensive activity in spontaneously hypertensive rats of two peptides isolated from β-lactoglobulin hydrolysates with thermolysin was evaluated. These peptides, with sequences LLF [β-lg f(103–105)] and LQKW [β-lg f(58–61)], showed potent in vitro ACE-inhibitory activity. Two hours after administration, both sequences caused a clear and significant decrease in the blood pressure of these rats. The impact of a simulated gastrointestinal digestion on ACE-inhibitory and antihypertensive activities of these peptides was also studied. The results showed that both fragments were susceptible to proteolytic degradation after incubation with pepsin and Corolase PP®. In addition, their in vitro ACE-inhibitory activity decreased after the simulated digestion. It is likely that fragment LQK was the active end product of the gastrointestinal digestion of peptide LQKW. The fragment LL, observed after digestion of peptide LLF, probably exert its antihypertensive effect through a mechanism of action different than ACE-inhibition.

Pooled milk samples from 115 Bergamasca meat sheep were collected aseptically five times from lambing to weaning to determine the prevalence of intramammary infection, somatic cell counts and milk quality parameters (protein, fat and lactose), and effects of infection on lamb weight gain. The global prevalence of subclinical intramammary infection was 51·2%. The Staphylococcus genus was responsible for the greatest prevalence (53·3% among infected udders). Staphylococcus aureus was isolated in 8·4% of infected milk samples. Infection status had significant effects on fat and protein percentage and on somatic cell count. Lamb growth was greatest for lambs of ewes with no infection and decreased as the number of infected samples increased. No significant differences were detected in the growth of lambs with dams infected by different bacterial species.

Milk quality is measured by a variety of parameters and one of these is the number of somatic cells in the milk. Within the bovine dairy industry there are maximum acceptable levels on bulk milk cell counts and these are being introduced into caprine dairy production. The reference method for cell counting in goats is the direct microscope (DM) method using pyronin Y-methyl green stain, which stains cell DNA. The use of the DeLaval cell counter (DCC) on goats' milk was compared with this reference method. Samples from 102 udder halves were cell counted using the direct microscope method and the DeLaval cell counter (DCC). Each sample was counted twice by two different observers by the DM method and once using DCC. DCC showed large coefficients of regression (1·03) and correlation (0·95) when compared with the DM cell count (F1,199=1080·0; P<0·001). Goat, udder half and reader type were significant factors.

We used a Mannheimia haemolytica isolate to study differences in susceptibility to experimental mastitis between two breeds of dairy sheep. The isolate was deposited into the teat duct of Karagouniko (K, n=8) or Frisarta (F, n=8) ewes. The animals were monitored by means of clinical, bacteriological, cytological and pathological methods. K ewes did not develop any systemic or mammary clinical signs, whilst F ewes became ill and developed acute clinical mastitis 12 h later (P<0·001). Bacteria were isolated from 34/48 samples from K ewes and from 46/46 samples from F ewes. Positive California mastitis test (CMT) results were 17/24 samples from K ewes and 23/23 samples from F ewes; leucocytes were seen in Giemsa-stained films. Total pathology score summed over all group K ewes was 41 (maximum possible: 128); Man. haemolytica was isolated from 12/24 tissue samples. Total pathology score summed over all group F ewes was 93; Man. haemolytica was isolated from 24/24 tissue samples. Hyperplastic lymphoid nodules consisting of lymphocytes and plasma cells with germinal activity were characteristically present at the border between teat duct–teat cistern of group K ewes; no such structures were observed in teats of group F ewes. The results identified differences in susceptibility/resistance to a mastitis pathogen among animals of the two breeds. Defence mechanisms of the teat appeared to be inadequate against the invading organisms; as lymphoid nodules have been considered important defensive mechanisms of the ovine teat, their observed lack in Frisarta ewes might have predisposed them to development of mastitis.

Bovine and caprine milks have a similar overall gross composition, but vary considerably in the ratios of their casein components. These differences cause significant changes in the ability of caseins to bind and stabilize calcium (Ca). It might be expected that these in vitro variations, which are thought to be due to differences in casein phosphopeptides (CPP) content, could lead to in vivo differences in the digestion and absorption of Ca. To test this hypothesis three milks with different casein ratios [bovine (B), caprine high in αs1-casein (CH) and caprine low in αs1-casein (CL)] were compared with regard to Ca absorption and deposition in growing male rats. For comparison, each milk was Ca-fortified (BCa-milk, CHCa-milk, and CLCa-milk) and CPP, prepared by enzymatic hydrolysis from the respective caseins (extrinsic CPP), were added to both native and Ca-milks. The effects of added CPP (extrinsic) could then be compared with intrinsic CPP released from the gastrointestinal digestion of caseins. Total gastric Ca was sampled at 15, 30 and 60 min after ingestion. No differences were found among the native milks with or without CPP, but the Ca from all Ca-milks (regardless of casein type) appeared to clear the stomach more rapidly and this was enhanced by the extrinsic CPP. The total intestinal Ca was not different among the native milks±CPP, however, it rose more rapidly with Ca fortification, and was higher at 30 min for all CPP-Ca-milks. At 60 min the total intestinal Ca level fell for the CPP-Ca-milks while all others continued to rise. These observations suggest that the CPP in Ca-milks enhance gastric clearance and uptake from the intestine. Ca availability from BCa-milk, CHCa-milk, and CLCa-milk with and without CPP was estimated by both plasma and femur uptake of 45Ca. Ca availability was enhanced at 5 h in the plasma in each case by added CPP. In all cases CPP stimulated Ca availability in the femur, but the CL-CPP was higher (P<0·05) than that of either CH-CPP or B-CPP (extrinsic CPP). Based on the results of this study we can conclude that the addition of CPP will have beneficial effect on the absorption of Ca in growing rats from CaCO3 added to bovine and caprine milks.

We investigated the effects of a probiotic fermented milk and inulin on gastrointestinal function and microecology. The study was double-blinded and comprised 66 healthy adults (22 male, 44 female), mean age 40 years (range, 22–60 years). After a 12-d baseline period the subjects were randomized to consume, for 3 weeks, 3×200 ml daily of either (1) a fermented milk with probiotics (Bifidobacterium longum BB536, Bifidobacterium spp. 420 and Lactobacillus acidophilus 145), (2) a fermented milk with the same probiotics plus 4 g inulin, or (3) a control fermented milk. During the last 7 d of the baseline and the intervention periods, the subjects kept a record of their defaecation frequency and gastrointestinal symptoms, and collected all their faeces. Intestinal transit time, stool weight and faecal enzyme activities were measured. Thirty-nine subjects were randomized to give faecal samples for analysis of pH and microbes, including lactobacilli, bifidobacteria, coliforms, Escherichia coli, Bacteroides and Clostridium perfringens. Consumption of fermented milk with probiotics or with probiotics and inulin increased the faecal number of lactobacilli (P=0·009, P=0·003) and bifidobacteria (P=0·046, P=0·038) compared with the baseline. Compared with the control fermented milk, both active products increased lactobacilli (P=0·005, ANCOVA). Subjects consuming fermented milk with probiotics and inulin suffered from gastrointestinal symptoms, especially flatulence, more than the others (P<0·001). In conclusion, the probiotic fermented milk product had a positive effect by increasing the number of lactobacilli and bifidobacteria in the colon. Inulin did not alter this effect but it increased gastrointestinal symptoms.

α1-Acid glycoprotein (AGP) is a lipocalin that is produced mainly by the liver and secreted into plasma in response to infections and injuries. In this study, we evaluated AGP isoforms that can be detected in bovine milk. We found that milk-AGP content is made up of at least two isoform groups, a low MW group (44 kDa) that is produced in the mammary gland (MG-AGP), and a higher MW group (55–70 kDa), that is produced by somatic cells (SC-AGP). Identical SC-AGP isoforms can be found both in milk and blood PMN cells. Analysis of the mammary tissue cDNA showed that the sequence of the MG-AGP isoform is identical to that of plasma AGP. Each group contains several proteins with different MWs and different isoelectric points, as shown by 2D-electrophoresis. The glycosylation patterns of these isoforms were analysed by means of specific lectin binding, to evaluate the degree of sialylation, fucosylation and branching. The MG-AGP glycan pattern was identical to plasma AGP produced by the liver. Several differences were detected, however, between plasma and SC-AGP isoforms, the most evident being the strong degree of fucosylation and the elevated number of di-antennary glycans in SC-AGP. Immunohistochemistry showed that AGP is found in all tissues that make up the mammary gland, but that it is most likely produced for the main part by the alveoli.