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.

The objective was to promote a reduction in the prevalence of Staphylococcus aureus and Streptococcus dysgalactiae after 2 years of selective dry cow therapies and teat dipping/external teat sealant implementation. Three different dry cow treatments, one long-acting and two short-acting penicillin-based products were tested at herd level together with a negative control teat dipping group, an iodine teat dipping group and DryFlex™, an external teat sealant. The regimes were independently randomly allocated to 178 dairy herds. Yearly bacteriological quarter milk samples were collected from all cows at the beginning of the trial, after 1 year and 2 years. At herd level, a total of 15% of the herds showed no Staphylococcus aureus isolates after 2 years, compared with 5% at the beginning. The distribution of Streptococcus dysgalactiae infected herds remained the same after 2 years. At cow level, there were no significant differences in the reduction of Staph. aureus between the different dry cow therapies and teat dipping regimes. But there was a significant reduction of Str. dysgalactiae in the iodine teat dipping group compared with Dryflex™ and the negative control group. The proportional rate of Staph. aureus positive quarters was reduced from 65·9% to 54·9% after 2 years. As for Str. dysgalactiae, an increase was observed from 14·2% to 15·2%.

Ewes' milk samples with low (<500000 ml−1), medium (1000000–1500000 ml−1) and high (>2500000 ml−1) somatic cell counts (SCC) were used to manufacture hard ewes' cheese using the Zamorano cheese manufacturing protocol. Cheeses that had been ripened for 1, 2 and 3 months were used to obtain isoelectric ovine casein that was analysed by capillary electrophoresis. The texture of the cheeses during ripening was determined instrumentally using the Warner-Bratzler maximum shear force and assessed for sensory qualities by consumers using hedonic tests. The study revealed that the pH value and the lactose content of the milk were affected by high SCC and that the coagulation properties were dependent on the somatic cell content. The protein and moisture contents of the cheeses were unaffected by SCC but a significant increase of pH with ripening time were observed in high-SCC cheeses. The results also pointed to a significant increase in proteolysis related to SCC levels, showing that intact casein, both αs1 and β-casein, decreased as the SCC of milk increased, and that the proteolytic fragments, mainly I-αs1, increased with SCC levels. Significant differences in texture were found among the samples, the cheeses made with high levels of SCC being significantly less compact at each ripening time. The differences in texture were detected by the consumers, who reported defects in cheeses made with high levels of SCC. Indeed, high SCC cheeses were significantly less well accepted.

Thiocyanate content and lactoperoxidase activity of individual cow's milk of different breeds were determined, and the effects of different lactoperoxidase system (LP-s) activation strategies were compared. Lactoperoxidase activity varied significantly between Friesian and both Ayrshire and Tanzania Short Horn Zebu (TSHZ), but differences between Ayrshire and TSHZ were not significant. There was no significant variation in SCN− content between breeds. The LP-s was activated using three strategies based on SCN−: namely; equal concentrations of SCN− and H2O2 (7:7, 10:10, 15:15 mg/l), excess SCN− concentrations (15:10, 20:10, 25:10 mg SCN−:H2O2/l), and excess H2O2 concentrations (10:15, 10:20, 10:25 mg SCN−:H2O2/l), plus a fourth strategy based on I− (15:15 mg I−:H2O2/l). The keeping quality (KQ) was assessed using pH, titratable acidity, clot on boiling and alcohol stability tests. All activation strategies enhanced the shelf life of milk (typically increasing KQ from around 10 to around 20 h), but it was clear that the effectiveness of the LP-s depends on the type and concentrations of the activators of the system. The LP-s activated using I− as an electron donor was more effective than the LP-s activated using SCN− as an electron donor, increasing the KQ by a further 6–8 h compared with SCN−.

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.

A detailed investigation was undertaken to determine the effects of four single starter strains, Lactococcus lactis subsp. lactis 303, Lc. lactis subsp. cremoris HP, Lc. lactis subsp. cremoris AM2, and Lactobacillus helveticus DPC4571 on the proteolytic, lipolytic and sensory characteristics of Cheddar cheese. Cheeses produced using the highly autolytic starters 4571 and AM2 positively impacted on flavour development, whereas cheeses produced from the poorly autolytic starters 303 and HP developed off-flavours. Starter selection impacted significantly on the proteolytic and sensory characteristics of the resulting Cheddar cheeses. It appeared that the autolytic and/or lipolytic properties of starter strains also influenced lipolysis, however lipolysis appeared to be limited due to a possible lack of availability or access to suitable milk fat substrates over ripening. The impact of lipolysis on the sensory characteristics of Cheddar cheese was unclear, possibly due to minimal differences in the extent of lipolysis between the cheeses at the end of ripening. As anticipated seasonal milk supply influenced both proteolysis and lipolysis in Cheddar cheese. The contribution of non-starter lactic acid bacteria towards proteolysis and lipolysis over the first 8 months of Cheddar cheese ripening was negligible.

PCR primers were developed for the specific detection of Clavispora lusitaniae, Debaryomyces hansenii var hansenii, Geotrichum candidum, Kluyveromyces lactis and K. marxianus and Yarrowia lipolytica, yeast species commonly found on the surface of smear cheese. Forty eight representative strains frequently found in smear cheeses or taxonomically related to the target yeasts were used as templates, to validate the designed primers. The specific and selective detection of these yeasts was effective in situ, in Livarot smear, without yeast isolation and culture and was comparable with data obtained with a conventional method. The primers described here have thus potential for PCR studies applied to cheese. It should also be possible to use some of these primers with other substrates.

This study aimed to compare Petrifilm™ Aerobic Count (AC) plates and the conventional pour plate methodology using de Mann-Rogosa-Sharpe (MRS), Kang-Fung (KF) and Kang-Fung-Sol (KFS) culture media for screening and enumeration of lactic acid bacteria (LAB) in milk. Suspensions of 10 LAB species in reconstituted powder skim milk and 30 raw milk samples, without experimental inoculation, were tested. For selective enumeration, all samples were previously diluted in MRS, KF and KFS broths and then plated in Petrifilm™ AC and conventional pour plate methodology, using the same culture media with added agar. All plates were incubated at 37°C for 48 h in anaerobic conditions. Differences in the counts were observed only for raw milk samples using KFS in conventional methodology, when compared with the counts obtained from MRS and KF (P⩽0·05). The results showed excellent correlation indexes between both methodologies using the three culture media for LAB suspensions (r=0·97 for MRS, KF and KFS). For raw milk samples, the correlation indexes were excellent (r=0·97, for MRS) and good (r=0·84 for KF, and r=0·82 for KFS), showing some interference in Petrifilm™ AC when supplements were added, especially lactic acid. These results indicate the possibility of using Petrifilm™ AC plates for enumeration of LAB in milk, even with the use of selective supplements.

Sixty dairy heifers from seven Austrian herds, with high prevalence of Staphylococcus aureus mastitis, were used in this pilot study. Heifers were randomly allocated to two groups. The treatment group received at parturition intramuscularly 10 million i.u. of penethamate hydriodide and then 24 h later, 5 million i.u.; the control group received no treatment. Bacteriological examination was conducted on 7, 14, 21, 35 and 49 d post partum (pp) and milk yield data, fat and protein contents and SCC data were collected every 5th week for the first 200 d of lactation. Occurrence of retained placenta and endometritis were recorded, and the days open of both groups were compared. No effect was observed on the postparturient genital tract health and reproduction indicators. On day 7 pp, four intramammary infections (IMI; two severe clinical; one mild clinical; and one subclinical mastitis) were detected in the untreated control group, whereas there were no IMI in the antibiotic-treated group. At subsequent samplings, there were fewer IMI in the antibiotic-treated group, which were later in lactation, less severe and less persistent. Although SCC was numerically lower in the treatment group, significant differences in SCC between groups could not be detected. Antibiotic-treated heifers produced significantly more milk during the first 15 weeks of lactation than untreated heifers. Over the whole observation period (200 d), peripartum antibiotic-treated heifers produced 323 kg more milk than heifers in the untreated control. Periparturient antibiotic treatment of heifers with penethamate hydriodide prevented IMI during the first week after parturition and achieved a significant increase in milk yield, which was found to be economically beneficial.

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0·38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (αs2-CN; f89–95), LPQNIPPL (β-CN; f70–77), SKVLPVPQK (β-CN; f168–176), LPYPYY (κ-CN; f56–61) and FLPYPYY (κ-CN; f55–61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.

To determine the effect of feeding propionibacteria on metabolic indicators during lactation, multiparous and primiparous Holstein cows were fed one of three dietary treatments in a 2×3 factorial design from 2 weeks prepartum to 30 weeks post partum: (1) Control (primiparous n=5, multiparous n=8) fed a total mixed ration (TMR); (2) high-dose group (primiparous n=6, multiparous n=5) fed TMR plus 6×1011cfu/head daily (high-dose P169) of propionibacterium strain P169; or (3) low-dose group (primiparous n=8, multiparous n=6) fed TMR plus 6×1010 cfu/head daily (low-dose P169) of P169. Blood samples were collected weekly and analysed for plasma concentrations of glucose, insulin, insulin-like growth factor-I (IGF-I), leptin, nonesterified fatty acids (NEFA) and cholesterol. Between weeks 25 and 30, all groups received bovine somatotropin (bST) every 2 weeks. Low-dose P169 multiparous cows had lower (P<0·05) plasma insulin and glucose concentrations than high-dose P169 multiparous cows, whereas high-dose P169 primiparous cows had lower glucose but greater insulin concentartions than low-dose P169 primiparous cows (P<0·05). Plasma insulin[ratio ]glucose molar ratios were 13–18% lower (P<0·05) in low-dose P169 cows than in control or high-dose P169 cows. Plasma IGF-I, NEFA and leptin levels did not differ among diet groups between weeks 1 and 25. Low-dose P169 multiparous cows had 25% greater plasma cholesterol levels than high-dose P169 and control multiparous cows, but cholesterol levels in primiparous cows did not differ. During bST treatment, high-dose P169 multiparous cows and low-dose P169 primiparous cows had lower IGF-I levels than their respective controls and, regardless of parity, high-dose P169 cows had greater NEFA than control cows. Although supplemental feeding of P169 altered plasma hormones and metabolites, the particular effects were dependent on dose of P169 and parity of cows.

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.

Effects of ventilation rate and of dietary protein level in an intensive dairy sheep system on the features of Canestrato Pugliese cheese were studied. Cheeses were manufactured from the bulk milk obtained from ewes subjected to four different experimental treatments: (1) low dietary crude protein (CP) of 13% in dry matter (DM) at a low ventilation rate (23·5 m3/h per ewe) (LPLV); (2) low dietary CP at a moderate ventilation rate (47 m3/h per ewe) (LPMV), (3) moderate dietary CP of 16% DM at a low ventilation rate (MPLV); and (4) moderate dietary CP dietary at a moderate ventilation rate (MPMV). Bulk milk and cheeses (at 1, 15, 45 and 90 d of ripening) were analysed for chemical composition, N fractions and plasmin-plasminogen activities. The pH 4·6-soluble and insoluble N fractions were analysed by urea-PAGE. Bulk milk from ewes receiving the low CP diet displayed higher casein and lower urea contents. Ewes subjected to the low ventilation rate displayed a higher plasminogen activity in milk, whereas no differences emerged among treatments in the conversion of zymogen to plasmin. During ripening the plasmin-plasminogen system in cheese did not display significant changes across treatments. At 90 d of ripening, the cheese produced with milk from ewes receiving the low CP diet and exposed to the low ventilation rate displayed more markedly stained bands in the area of γ-caseins in pH 4·6-insoluble N fraction. As from 15 d of ripening, the pH 4·6-soluble fraction showed a greater number of bands in the LPMV and MPMV than in the LPLV and MPLV cheeses. The results showed that Canestrato Pugliese cheese manufactured with milk from ewes fed the low CP diet and exposed to the moderate ventilation rate was characterized by higher protein and casein content in the fresh cheese and by a greater proteolysis after 90 d of ripening.

Milk fermented with mixed dahi cultures NCDC167, Lactococcus lactis ssp diacetylactis NCDC60 and two probiotic strains; Lactobacillus acidophilus NCDC14 and Lb. casei NCDC19 were evaluated after fermentation (14 h) and during 8 d storage at 7 °C. The β-galactosidase activity was found to increase after fermentation leading to the hydrolysis of lactose and production of glucose, galactose and oligosaccharides; that subsequently decreased during storage. The viable counts of lactococci and lactobacilli decreased during storage yet remained >106 cfu/ml after storage. The results of present study indicate that all the selected cultures have ability to produce oligosaccharides (prebiotics) due to transgalactosidal and lactose hydrolysis activities of β-galactosidase. The cultures developed an active synbiotic formula by maintaining sufficient probiotic viable counts to exert health benefits to the consumers.

Sicilo-Sarde dairy flock test-day data were analysed (1) to check predicting ability of ten milk recording procedures, based on information from a single monthly or bimonthly milking and (2) to look for the best pair of factors for predicting daily milk yield from either morning or afternoon milkings for the alternate testing method (AT). Prior to analyses, data were adjusted for variation factors with known significant effect. Number of test-day observations was 4755 from 458 lactations of 288 ewes. Losses of precision associated with simplified methods were evaluated by comparing estimated lactation yields with those observed both in a reference plan, where the two daily milkings were recorded at 2-weekly intervals and in the official A4 milk recording (monthly recording of the two daily milkings). Ranking of simplified plans based on their accuracy was still the same under either monthly or twice-monthly frequency. Estimates of lactation yields showed more lack of precision when the usual monthly designs were compared with twice-monthly sampling of both a.m. and p.m. milkings. Losses of precision ranged from 5·1%, for the A4 plan, to 14·6%. Thus, milk production should be estimated from both mikings when their measurement is economically feasible. When only one milking is weighed, designs based on—or beginning with—the a.m. milking were more accurate. There was no evidence of improvement in sampling accuracy by adjustment for milking interval or production level. For practical and economic reasons, the design alternating a.m. and p.m. milkings, without adjustment, is suggested for ovine milk recording. In such a case, new factors for estimating daily production from the measured milking (1·7, 2·3) should be used rather than the factors currently in use (2, 2) because they improved the loss of precision by more than 13% in a monthly recording system.

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.

Conjugated linoleic acid (CLA) has a range of biological properties, including effects on lipid metabolism, milk and body composition in animals. This study investigated the effects of dietary CLA on lactating rats and development of the suckling pups. Dams were fed either a control diet or the same diet supplemented with 25 g/kg of a fat supplement containing 540 g CLA/kg (final concentration of 13·5 g CLA/kg diet) from parturition to the 15th day post-partum. The CLA mixture used in this study contained the following isomers (per 100 g): cis-9, trans-11 (24 g); cis-10, trans-12 (35 g); cis-8, trans-10 (15 g); cis-11, trans-13 (17 g) and others (9 g). On d 15 post partum, CLA supplementation reduced milk fat content by 33% and pup growth by 21%. The milk fatty acid profile, with decreased content of short and medium chain acids, suggests CLA inhibition was more pronounced for de novo lipid synthesis. Consistent with these results, activities of fatty acid synthase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were reduced by CLA treatment in the mammary gland and liver. In contrast, the activity of NADP-malate dehydrogenase was unchanged.

The proportion of different strains of Staphylococcus aureus was tested in four groups of lactating dairy cows in six herds with a high overall prevalence of Staph. aureus using random amplified polymorphic DNA PCR. Group 1 included primiparous cows in early lactation (<50 days in milk, DIM). Group 2 consisted of primiparous cows in late lactation (>250 days in milk). Groups 3 and 4 were multiparous cows in the respective stages of lactation. Eight cows from each group on each farm were tested. Overall quarter prevalence of Staph. aureus ranged from 23·4 to 32·0% in the herds. Of the 130 isolates included in the analysis 86·9% were high prevalence strains (more than three isolates per herd), while 13·1% were strains that were only identified in one or two samples. Low prevalence strains were found in all six herds. The proportion of low prevalence strains was higher in multiparous than in primiparous cows (odds ratio, OR 4·4, 1·2–16·6). It is concluded that low prevalence Staph. aureus strains are common even in herds with a high prevalence of Staph. aureus and that their frequency is lower in primiparous cows than in older cows.

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.

Reducing the dietary cation-anion difference (DCAD) has been shown to be an effective means of preventing parturient paresis in confinement systems where cows are offered a total mixed ration containing DCAD-reducing mineral compounds (anionic salts). Such a supplementation strategy is not possible in cows being group fed forages precalving, and little is known about the effect of supplementing these cows with large amounts of anionic salts twice daily.

Eight non-lactating, pregnant Holstein-Friesian cows were allocated to two levels of DCAD (−20 and +18 meq/100 g DM) for 24 d, with an intensive Ca balance undertaken in metabolism stalls following a 2-week acclimatization to diet. The basal diet was 3 kg DM of crushed barley and 7 kg DM of pasture-hay. Urine and faeces were collected separately, weighed daily for 5 d and analysed for Ca content. Urinary Ca, creatinine and hydroxyproline concentration and plasma Ca concentration were determined during the period of the balance study. The diurnal pattern in urine and rumen pH was determined over 2 d. Decreasing DCAD reduced (P<0·001) the pH of urine, and increased (P<0·05) Ca absorption. Plasma Ca concentration was not affected by DCAD, and DCAD did not affect the output of urinary hydroxyproline, a marker of bone resorption. Twice-daily supplementation of anionic salts was sufficient to reduce the pH of blood and increase gastrointestinal Ca absorption. There was no diurnal variation in the pH of urine, suggesting that time of sampling to determine efficacy of DCAD in reducing systemic pH was not important.