Using an ultrafiltration membrane (molecular cut-off, 3000), low molecular weight compounds in bovine milk were collected (YM-3 filtrate). A hydrogen peroxide (H2O2)-like substance was generated in the YM-3 filtrate. This substance was undetected at 0 h, but increased in a time-dependent manner, peaking after 2 h of incubation at 38°C. After incubating the YM-3 filtrate with catalase and lactoperoxidase, the signal showing the presence of this substance disappeared. The substance was quantified using one chemiluminescence and three colorimetric H2O2 detection systems. In all systems, their estimates were within the same range. The amount of substance, as estimated by the chemiluminescence H2O2 detection system, was correlated with that estimated by the other three colorimetric systems (r=0·98, 0·95 and 0·87). The substance was eluted at the same position as H2O2 by gel filtration on Superdex 30. Thus, the substance had the same characteristics as H2O2. An H2O2-generating substance in either the YM-3 filtrate or whey had a molecular mass of about 600. In this study, we clarify that bovine milk is capable of generating H2O2 by utilizing a low molecular weight compound. Thus, we present a new type of H2O2-supplying system in bovine milk.

The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55°C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20°C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16–18%) and PA (38–62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40°C and at the pressures 450 and 650 MPa at 55°C. Synergistic negative effects on the amount of native β-lactoglobulin were observed at 450 and 650 MPa and on the amount of native α-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native β-lactoglobulin and α-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage.

Olive tree leaves (OTL) and grape marc (GM) are by-products with high linolenic (LNA) and linoleic (LA) acid content, respectively, which can be used as dietary ingredients to increase the cis-9 trans-11 conjugated linoleic acid (CLA) content of milk fat in sheep and goats. An experiment was conducted with 16 Friesian ewes and 16 Alpine goats to study the effect of OTL and GM inclusion in sheep and goat diets on their milk fatty acid profile, with emphasis on cis-9, trans-11 CLA and vaccenic acid (VA). Ewes and the goats were fed the control (C) diets from parturition to the 90 days in milk (DIM) and then both groups were divided into two sub-groups (treatments). The control groups of both species continued to be fed the C diets, whereas the treated groups were gradually switched over a 2-week period (DIM=91–105) from the C diets to that of treatment 1, which contained air-dried OTL. These OTL diets were fed ad libitum for 1 month (DIM=106–135). After that period, the same treated groups, after 2 weeks of gradual adaptation (DIM=136–150), were switched to treatment 2, which contained air-dried GM. The GM diets were fed ad libitum for 1 month (151–180 DIM). Concentrations of polyunsaturated fatty acids (PUFA) and mono-unsaturated fatty acids increased significantly in milk fat of sheep fed OTL v. C. For goats, only the PUFA in milk fat was increased by feeding OTL compared with C. Relative to C, GM increased significantly the concentration of PUFA only in milk fat of sheep. OTL and GM diets increased the cis-9, trans-11 CLA and VA content in milk fat, compared with C, only in sheep. GM caused a sharp increase in 18:0 only in sheep milk fat, while the OTL diet increased significantly the 18:0 in milk fat of goats. GM and OTL diets also had opposite effects on the 18:1/18:0 ratio of sheep milk fat. In conclusion, OTL and GM, when included in sheep diets altered the milk fatty acid profile with a pronounced increase in cis-9, trans-11 CLA and VA contents. The results show that the response of sheep and goats to OTL and GM diets was different, suggesting a species difference that needs further investigation.

Somatic cell count (SCC) in milk is considered to be a valuable indicator of cow mastitis. For assessment of SCC in milk, the bioluminescent assay based on determination of ATP from somatic cells ([ATPsom]) in milk was proposed earlier. However, this assay is still not widely used in practice owing to lower reliability compared with conventional methods such as direct microscopy and flow cytometry. We revised the bioluminescent SCC assay and developed a simple protocol based on determination of the total non-bacterial ATP concentration in milk. It was shown that the novel ATP-releasing agent Neonol-10 (oxy-ethylated iso-nonyl phenol) has superior performance providing 100% lysis of somatic cells while not disrupting bacterial cells of milk at a concentration of 1·5% w/w. There was high correlation (R2=0·99) between measured bioluminescence and SCC as measured by direct microscopy. The observed detection limit of the bioluminescent milk SCC assay was as low as 900 cell/ml, time of analysis was 2–3 min per sample. The proposed method has high potential for on-site mastitis diagnostics.

Two angiotensin converting enzyme (ACE)-inhibitory peptides were isolated from enzyme modified cheese (EMC) and their amino acid sequences were identified as Leu-Gln-Pro and Met-Ala-Pro. The EMC was prepared by a combination of Protease N, Umamizyme, and Flavourzyme 500L. Both peptides were derived from β-casein, f 88-90 and f 102-104, respectively. Met-Ala-Pro showed strong ACE inhibitory activity (IC50=0·8 μm) and antihypertensive activity in spontaneously hypertensive rats (SHR) after single oral administration. The IC50 value of Met-Ala-Pro was not affected by pre-incubation with ACE, suggesting that this peptide was a true ACE-inhibitory peptide. We report here, for the first time antihypertensive peptides from EMC.

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.

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.

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.

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.

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.

Mastitis is the most frequent and prevalent production disease in dairy herds in developed countries. Based on a milk somatic cell count (SCC) of either >300 000 or <200 000 cells/ml in this study, we defined the quarter as either inflamed or uninflamed, respectively. The electrical conductivity (EC) of milk was used as an indicator of udder epithelial cell damage. We determined the amount of H2O2 produced by utilizing a small molecular weight compound in milk, and examined the characteristics of H2O2 production and EC in milk from inflamed and uninflamed quarters. In cows with milk of delivery grade (control population), H2O2 production and EC were 3·6±1·3 nmol/ml and 5·4±0·4 mS/cm (mean±sd), respectively. In 37 inflamed quarter milk samples, the production of H2O2 was 1·9±1·0 nmol/ml and was significantly smaller than that in the control population (P<0·01). Production of H2O2 was moderately but significantly correlated with EC (r<−0·71). In 20 cows with inflamed quarters, the production of H2O2 in milk from inflamed quarters was significantly smaller than that in milk from uninflamed quarters (P<0·01). In 18 out of 20 cows, milk from inflamed quarters showed the smallest H2O2 production among all tested quarters in each cow. We conclude that inflammation caused a decrease in H2O2 production in milk. In this study, we present parameters for evaluating the lactoperoxidase/H2O2/thiocyanate antibacterial defence system in bovine milk.

The objective was to describe the physicochemical changes during the early phase of subclinical mastitis and to associate them with pathological findings. A Mannheimia haemolytica strain was deposited into one teat duct of 25 ewes and the clinical, bacteriological, cytological, physicochemical (pH, milk composition), gross-pathological and histological findings were subsequently recorded. The organism was consistently isolated from samples of teat duct material (140/150) but not from mammary secretion (50/150). California Mastitis Test (CMT) scores increased (>1) and remained high (143/150 samples) after challenge; polymorphonuclear neutrophils (PMN) predominated in milk films, but the proportion of lymphocytes and macrophages progressively increased. Increased pH values (>7·0) were recorded in the mammary secretion from the challenged side. Furthermore, content of fat, total proteins and lactose therein decreased markedly. Histological changes (leucocytic infiltration, destruction of epithelial cells) were observed in the mammary parenchyma of the ewes. The present results confirm that the reduction of milk constituents is the effect of cellular damage and can occur soon after infection.

The changes in milk fat globules and fat globule surface proteins of both low-preheated and high-preheated concentrated milks, which were homogenized at low or high pressure, were examined. The average fat globule size decreased with increasing homogenization pressure. The total surface protein (mg m−2) of concentrated milk increased after homogenization, the extent of the increase being dependent on the temperature and the pressure of homogenization, as well as on the preheat treatment. The concentrates obtained from high-preheated milks had higher surface protein concentration than the concentrates obtained from low-preheated milks after homogenization. Concentrated milks heat treated at 79°C either before or after homogenization had greater amounts of fat globule surface protein than concentrated milks heat treated at 50 or 65°C. This was attributed to the association of whey protein with the native MFGM (milk fat globule membrane) proteins and the adsorbed skim milk proteins. Also, at the same homogenization temperature and pressure, the amount of whey protein on the fat globule surface of the concentrated milk that was heated after homogenization was greater than that of the concentrated milk that was heated before homogenization. The amounts of the major native MFGM proteins did not change during homogenization, indicating that the skim milk proteins did not displace the native MFGM proteins but adsorbed on to the newly formed surface.

This study investigated the evolution of trans-9 trans-11 conjugated linoleic acid (CLA) from cis-9 trans-11 CLA during methylation and its avoidance through a rapid base methylation of milk fat. The study examined three conditions shown to result in loss of cis-9 trans-11 CLA during methylation namely: temperature, methylation time, water contamination in old reagents and acidic conditions. Three techniques currently used for the conversion of milk fat into fatty acid methyl esters for analysis of CLA content by gas liquid chromatography and a fourth procedure designed to eliminate acidic conditions and to limit methylation temperature and time were used. The four methods were: (i) acidic methylation (AM); (ii) acidic and basic bimethylation with fresh reagents (FBM); (iii) acidic and basic bimethylation with pre-prepared reagents (PBM) and (iv) basic methylation (BM). Each regime was carried out on six milk samples over two periods and methylated 1 ml freeze-dried milk (n=12 per regime). Total CLA was not different across methylation regimes (0·30 mg/ml). Isomer cis-9 trans-11 was higher (P<0·01) with BM than the other regimes and lowest with AM: 21·2, 17·8, 18·8 and 14·7 mg/100 ml for BM, FBM, PBM and AM, respectively. The inverse relationship was shown for trans-9 trans-11 with higher (P<0·001) amounts with AM than the other regimes and lowest with BM: 0·57, 2·55, 2·36 and 3·69 mg/100 ml for BM, FBM, PBM and AM, respectively. The trans-10 cis-12 isomer was also shown to alter with methylation procedure being higher (P<0·001) with AM than the other regimes: 0·43, 0·47, 0·29 and 1·20 mg/100 ml for BM, FBM, PBM and AM, respectively. Validation with known CLA free fatty acid and triacylglycerol standards confirmed that AM resulted in conversion of cis-9 trans-11 to trans-9 trans-11, and also elevated trans-10 cis-12 whilst BM of triacylglycerol CLA did not isomerise cis-9 trans-11 and was comparable to FBM.

Twenty-eight Brown cows were maintained on a mountain pasture for a period of 40 days and assigned to 4 groups following a factorial design 2 stocking density (0·7 and 1·4 cows/ha)×2 supplement levels (2·4 and 4·8 kg organic matter (OM)/d). Herbage intake, animal body condition score (BCS), milk yield, milk chemical and coagulation properties, cheese composition, rheology and sensory characteristics were measured. The average herbage intake was 12·2 kg OM/d, with a significant effect related to stocking density (low, 13·1 v. high, 11·4 kg OM/d). BCS variation was always negative and changed with supplement level, although with no statistical significance (−0·43 points on average). Milk yield was lower for the group with lower availability of herbage (low supplement and high stocking density: 15·9 kg/d), whereas it was comparable among others groups (16·9 kg/d on average). The group with high supplement and low stocking density produced milk with worse cheese making properties. Cheese composition analyses showed an effect of supplement level on calcium content, and on parameters for degree of ripening, such as nitrogen fractions and lipolysis index. As for the textural parameters, hardness and gumminess were found to be higher with the low level of supplement. The different level of supplementation could differentiate the groups on the basis of the sensory perception of cheese.

A treatment containing a live food-grade organism, Lactococcus lactis DPC3147, was compared with conventional antibiotic therapy for its potential to treat bovine chronic subclinical or clinical mastitis in two separate field trials. Effects on disease symptoms and bacteriology were monitored in response to infusion with the culture in each trial. In the first trial, the live culture treatment was compared with an intramammary antibiotic (n=11 quarters for each treatment). Results from this small trial demonstrated that the live culture had potential to be as effective at eliminating chronic subclinical infections as an antibiotic treatment. By day 12, 7 of the 11 quarters treated with the live culture were pathogen-free compared with 5 of the 11 antibiotic-treated infected quarters. Somatic cell counts (SCC) remained relatively unchanged regardless of treatment: average log SCC pre- and post-treatment in the lactococci-treated group were 6·33±0·41 (day 0) and 6·27±0·43 cells/ml (day 12) and average log SCC pre- and post-treatment in the antibiotic-treated group were 6·34±0·37 and 6·22±0·46 cells/ml on day 0 and on day 12, respectively. In the second trial, the live culture was compared with an intramammary antibiotic for the treatment of naturally occurring clinical mastitis cases (n=25 quarters for each treatment). Following a 14-d experimental period, similar bacteriological responses were observed in 7 out of 25 live culture treated quarters and 9 out of 25 antibiotic-treated quarters. Additionally, 15 of 25 cases treated with the culture and 18 of 25 cases treated with the antibiotic did not exhibit clinical signs of the disease following treatment. The results of these trials suggest that live culture treatment with Lc. lactis DPC3147 may be as efficacious as common antibiotic treatments in some instances.

In the accompanying article, we demonstrated that a live culture of Lactococcus lactis compares favourably with antibiotics for treatment of bovine mastitis in two initial field trials. In an effort to explain the mechanism involved, this study investigated the effect of culture administration on the local immune response. In this respect we initially observed that infusion of the live culture Lactococcus lactis stimulated substantial recruitment of polymorphonucleocytes (PMN) and lymphocytes to the udder. For instance, in one assay, quarters infused with the probiotic experienced a dramatic increase (~20 000-fold) in neutrophils over the first 48-h period from an average value of 83·6 cells/ml pre-treatment to 1·78×106 cells/ml 48 h post-infusion. Levels of the acute phase proteins haptaglobin and milk amyloid A were also elevated significantly in comparison with controls following infusion of the culture. The results of flow cytometric assays also demonstrated that while infusion of a live lactococcal culture led to an enhanced recruitment of PMN to the udder (from 1·85×104 cells/ml pre-infusion to 1·45×106 cells/ml 24 h post-infusion) cell-free supernatant from the same culture was not able to do so, indicating that live Lc. lactis can specifically trigger the mammary immune response to elicit PMN accumulation. These results suggest that the mechanism responsible for this probiotic treatment of mastitis is associated with stimulation of the host intramammary immune system.