Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells.

This Research Communication reports interferences related to the administration of ivermectin in lactating dairy goats on the response of microbial tests for screening antibiotics in milk. Twenty-eight Murciano-Granadina goats, naturally infested with Sarcoptes scabiei var. caprae, were treated with a subcutaneous injection of ivermectin (200 µg/kg b.w.). To prevent re-infestation, a second dose was applied 7 d later. Individual milk samples were collected, daily, up to 15 d post-treatment. Milk samples were analysed by microbial inhibitor tests (BRT MRL, Delvotest SP-NT MCS and Eclipse 100) and ivermectin residues were quantified by HPLC. A large number of positive results were obtained for all microbial tests, especially on the first day after treatment (BRT MRL = 46·4%; Delvotest SP-NT MCS = 14·3%; and Eclipse 100 = 17·8%). However, the highest concentration of drug residues in milk (24·3 ng/ml) was detected on the tenth day after treatment, when positive outcomes were relatively lower (BRT MRL = 17·8%; Delvotest SP-NT MCS = 10·7%; and Eclipse 100 = 7·4%). Results herein suggest that factors related to the ivermectin treatment other than drug residues in milk, or alterations produced by the parasitic disease itself affecting the immune response of animals, could be the cause of false-positive results in microbial tests. It can be concluded that the application of ivermectin in dairy goats infested with sarcoptes mange during lactation produces persistent drug residues in milk, and could also cause false-positive results in microbial inhibitor tests for screening antibiotics.

Vitamin D has become one of the most discussed nutrients in human nutrition, which has led to an increased interest in milk as a vitamin D source. Problems related to fortifying milk with synthetic vitamin D can be avoided by securing a high content of natural vitamin D in the milk by supplying dairy cows with sufficient vitamin D. However, choosing the most efficient route and form of supplementation requires insight into how different vitamin D metabolites are transported in the body of cattle. There are two forms of vitamin D: vitamin D2 (D2) and vitamin D3 (D3). Vitamin D2 originates from fungi on roughage. Vitamin D3 originates either from endogenous synthesis in the skin or from feed supplements. Vitamin D2 is chemically different from, and less physiologically active than, D3. Endogenous and dietary D3 is chemically similar but dietary D3 is toxic, whereas endogenous D3 appears well regulated in the body.

Water buffalo Mozzarella cheese is more appreciated in the summer, but milk production is higher during wintertime, as water buffalo are seasonally polyoestrous animals. The aim of the study reported in this Research Communication was to evaluate the effect of the reversing of the calving period on Mozzarella cheese composition and quality traits. Ten batches of Mozzarella cheeses produced during the summer and winter periods were analysed for pH, colour, chemical composition. Seasonal differences were observed for cheese yield (26·66% in winter and 25·61% in summer), moisture content (66·54% in winter and 61·18% in summer) colour and consumer evaluation.

The objective of this study was to compare the prediction efficiency of IgG concentration in bovine colostrum by NIRS, using liquid and dried (Dry-Extract Spectroscopy for Infrared Reflectance, DESIR) samples by transflectance and reflectance modes, respectively. Colostrum samples (157), obtained from 2 commercial Holstein dairy farms, were collected within the first hour after calving and kept at −20 °C until analysis. After thawing and homogenisation, a subsample of 500 mg of liquid colostrum was placed in an aluminium mirror transflectance cell (0·1 mm path length), in duplicate, to collect the spectrum. A glass fiber filter disc was infused with another subsample of 500 mg of colostrum, in duplicate, and dried in a forced-air oven at 60 °C for 20 min. The samples were placed in cells for dry samples to collect the spectra. The spectra in the VIS-NIR region (400–2500 nm) were obtained with a NIRSystems 6500 monochromator. Mathematical treatments, scatter correction treatments and number of cross-validation groups were tested to obtain prediction equations for both techniques. Reference analysis for IgG content was performed by radial immunodiffusion. The DESIR technique showed a higher variation in the spectral regions associated with water absorption bands, compared with liquid samples. The best equation for transflectance method (liquid samples) obtained a higher coefficient of determination for calibration (0·95 vs. 0·94, respectively) and cross validation (0·94 vs. 0·91, respectively), and a lower error of cross validation (9·03 vs. 11·5, respectively) than the best equation for reflectance method (DESIR samples). In final, both methods showed excellent capacity for quantitative analysis, with residual predictive deviations above 3. It is concluded that, regarding accuracy of prediction and time for obtaining results of IgG from bovine colostrum, NIRS analysis of liquid samples (transflectance) is recommended over dried samples (DESIR technique by reflectance).

The objective of this study was to determine whether the occurrence of a previous case of pathogen-specific clinical mastitis (CM) protects Holstein dairy cows against a recurrent case. Pathogens studied were Escherichia coli, Staphylococcus aureus, Staphylococcus spp., Streptococcus spp., Klebsiella spp., and Trueperella pyogenes. A total of 40 864 lactations (17 265 primiparous and 23 599 multiparous) from 19 835 cows from 5 large, high milk producing New York State dairy herds were analysed. We estimated the effects of parity, calving diseases, milk yield, current season and number of CM cases in the previous lactation on the risk of a first CM case using generalised linear mixed models with a log link and Poisson error distribution. The aforementioned risk factors and the occurrence of previous cases of pathogen-specific CM within the current lactation were evaluated as risks for second and third cases of pathogen-specific CM. Cows with more CM cases in the previous lactation were at greater risk of pathogen-specific CM in the current lactation. Multiparous cows were at greater risk of a second CM case if they had suffered from a first CM case that was caused by the same pathogen as the second case. In contrast, a second CM case generally put cows at greater risk of a third case, irrespective of whether the third case was caused by the same or a different pathogen. Our results showed that a previous case of pathogen specific CM does not generally protect against a recurrent case.

A high variation in immunoglobulin G1 (IgG1) concentration in first milked quarter colostrum has been reported, but BSA quarter colostrum variation is not known. The occurrence of serum albumin in milk has been attributed to increased blood-milk barrier penetration. Reports of serum albumin binding to the Fc Receptor of the neonate, the receptor thought to be responsible for IgG1 transcytosis, suggested that a correlation with the appearance of IgG1 in colostrum of dairy cows was likely. The objective of the study was to establish the quarter colostrum concentration and mass of immunoglobulins and serum albumin. First colostrum was quarter collected within 4 h of parturition from healthy udders of 31 multiparous dairy cows. Individual quarter colostrum weight was determined and a sample of each was frozen for subsequent analysis. Concentrations of immunoglobulin G1, G2, and BSA were measured by ELISA and total mass of components was calculated. In addition, colostrum was also analysed for L-lactate dehydrogenase activity. Analysis of concentration and mass of BSA, immunoglobulin G1, G2 established that the quarter variations were different by cow, quarter and quarter within cow. Partial correlations corrected for colostrum weight indicated that BSA and IgG2 concentration and mass are closely correlated while that of BSA and IgG1 concentration and mass exhibited no correlation suggesting that BSA and IgG1 may have different transport mechanisms. Interestingly, immunoglobulin G1 and G2 concentration and mass exhibited strong correlations suggesting that also some unknown mechanism of immunoglobulin G2 appearance in colostrum is occurring. Finally, no measured protein exhibited any correlation with the activity of lactate dehydrogenase in colostrum.

In cheese, a negative oxidation-reduction (redox) potential is required for the stability of aroma, especially that associated with volatile sulphur compounds. To control the redox potential during ripening, redox agents were added to the salted curd of Cheddar cheese before pressing. The control cheese contained only salt, while different oxidising or reducing agents were added with the NaCl to the experimental cheeses. KIO3 (at 0·05, 0·1 and 1%, w/w) was used as the oxidising agent while cysteine (at 2%, w/w) and Na2S2O4 (at 0·05 and 0·1%, w/w) were used as reducing agents. During ripening the redox potential of the cheeses made with the reducing agents did not differ significantly from the control cheese (E h ≈ −120 mV) while the cheeses made with 0·1 and 0·05% KIO3 had a significantly higher and positive redox potential in the first month of ripening. Cheese made with 1% KIO3 had positive values of redox potential throughout ripening but no starter lactic acid bacteria survived in this cheese; however, numbers of starter organisms in all other cheeses were similar. Principal component analysis (PCA) of the volatile compounds clearly separated the cheeses made with the reducing agents from cheeses made with the oxidising agents at 2 month of ripening. Cheeses with reducing agents were characterized by the presence of sulphur compounds whereas cheeses made with KIO3 were characterized mainly by aldehydes. At 6 month of ripening, separation by PCA was less evident. These findings support the hypothesis that redox potential could be controlled during ripening and that this parameter has an influence on the development of cheese flavour.

The periparturient period is one of the most critical periods in the productive life of a dairy cow, and is the period when dairy cows are most susceptible to developing new intramammary infections (IMI) leading to mastitis. Acute phase proteins (APP) such as haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) have been detected in milk during mastitis but their presence in colostrum and milk in the immediate postpartum period has had limited investigation. The hypothesis was tested that APP are a constituent of colostrum and milk during this period. Enzyme linked immunosorbent assays (ELISAs) were used to determine each APP's concentration in colostrum and milk collected daily from the first to tenth day following calving in 22 Holstein-Friesian dairy cows. Haptoglobin was assessed in individual quarters and composite milk samples while M-SAA3 and CRP concentration were determined in composite milk samples. Change in Hp in relation to the high abundance proteins during the transition from colostrum to milk were evaluated by 1 and 2 dimension electrophoresis and western blot. In 80% of the cows all APPs were detected in colostrum on the first day following parturition at moderately high levels but gradually decreased to minimal values in the milk by the 6th day after calving. The remaining cows (20%) showed different patterns in the daily milk APP concentrations and when an elevated level is detected could reflect the presence of IMI. Demonstration that APP are present in colostrum and milk following parturition but fall to low levels within 4 days means that elevated APP after this time could be biomarkers of post parturient mastitis allowing early intervention to reduce disease on dairy farms.

The aim of this study was to identify the differences between the main macro and micronutrients including proteins, fat, minerals and vitamins in cow and goat dehydrated fermented milks. Fermented goat milk had higher protein and lower ash content. All amino acids (except for Ala), were higher in fermented goat milk than in fermented cow milk. Except for the values of C11:0, C13:0, C16:0, C18:0, C20:5, C22:5 and the total quantity of saturated and monounsaturated fatty acids, all the other fatty acid studied were significantly different in both fermented milks. Ca, Mg, Zn, Fe, Cu and Se were higher in fermented goat milk. Fermented goat milk had lower amounts of folic acid, vitamin E and C, and higher values of vitamin A, D3, B6 and B12. The current study demonstrates the better nutritional characteristics of fermented goat milk, suggesting a potential role of this dairy product as a high nutritional value food.

Activity of lysosomal N-acetyl-β-d-glucosaminidase (NAGase) in milk has been used as an indicator of bovine mastitis. We studied NAGase activity of 808 milk samples from healthy quarters and quarters of cows with spontaneous subclinical and clinical mastitis. Associations between milk NAGase activity and milk somatic cell count (SCC), mastitis causing pathogen, quarter, parity, days in milk (DIM) and season were studied. In addition, the performance of NAGase activity in detecting clinical and subclinical mastitis and distinguishing infections caused by minor and major bacteria was investigated. Our results indicate that NAGase activity can be used to detect both subclinical and clinical mastitis with a high level of accuracy (0·85 and 0·99). Incomplete correlation between NAGase activity and SCC suggests that a substantial proportion of NAGase activity comes from damaged epithelial cells of the udder in addition to somatic cells. We therefore recommend determination of NAGase activity from quarter foremilk after at least six hours from the last milking using the method described. Samples should be frozen before analysis. NAGase activity should be interpreted according to DIM, at least during the first month of lactation. Based on the results of the present study, a reference value for normal milk NAGase activity of 0·1–1·04 pmoles 4-MU/min/μl for cows with ≥30 DIM (196 samples) could be proposed. We consider milk NAGase activity to be an accurate indicator of subclinical and clinical mastitis.

Lactobacillus fermentum Lf2 is a strain which is able to produce high levels (approximately 1 g/l) of crude exopolysaccharide (EPS) when it is grown in optimised conditions. The aim of this work was to characterize the functional aspects of this EPS extract, focusing on its application as a dairy food additive. Our findings are consistent with an EPS extract that acts as moderate immunomodulator, modifying s-IgA and IL-6 levels in the small intestine when added to yogurt and milk, respectively. Furthermore, this EPS extract, in a dose feasible to use as a food additive, provides protection against Salmonella infection in a murine model, thus representing a mode of action to elicit positive health benefits. Besides, it contributes to the rheological characteristics of yogurt, and could function as a food additive with both technological and functional roles, making possible the production of a new functional yogurt with improved texture.

In the current study the microbiological, sensory and chemical properties of 24 kefirs (12 producers) from Estonian, Latvian and Lithuanian retail market were determined using gas chromatography (GC), high performance liquid chromatography (HPLC-MS/MS-Q-TOF and LC-ion trap MS/MS), spectrophotometry and other methods. Antihypertensive, angiotensin-converting enzyme (ACE) inhibiting, antioxidant and antibacterial peptides were found in the kefir samples. According to the results of principal component analysis of 200 most abundant compounds obtained with HPLC-MS/MS-Q-TOF analysis, Estonian kefirs differed from the rest. Kefirs of Latvian and Lithuanian origin showed similarities in several characteristics, probably related to the starter cultures and technological processes. The fatty acids composition of all Baltic kefirs was uniform. The antioxidant capacity of the kefirs varied slightly, whereas intermediate positive correlation (r = 0·32, P < 0·05) was found between antioxidativity and total bacterial count. The lipid oxidation level, estimated as the content of linoleic and oleic acid primary oxidation products, oxylipins, was very low in all studied kefirs. Only one third of analysed kefirs met the requirements of the minimum sum of viable microorganisms, indicated in the Codex Standard for Fermented Milks.

The urokinase-plasminogen activator (u-PA), its receptor (u-PAR) and the inhibitors of u-PA (PAI-1 and PAI-2) provide a multi-molecular system in leucocytes that exerts pleiotropic functions influencing the development of inflammatory and immune responses. The objective of the present study was to examine the ability of water soluble extracts (WSE) obtained from traditional Greek yoghurt made from bovine or ovine milk to modulate the expression of u-PA, u-PAR, PAI-1 and PAI-2 in ovine monocytes and neutrophils. WSE were obtained from 8 commercial traditional type Greek yoghurts made from ovine or bovine milk. WSE upregulated the expression of all 4 u-PA related genes in monocytes but the upregulation was much higher in the PAI-1 (10-fold) than in u-PA and u-PAR (3–4 fold) thus, shifting the system towards inhibition. In line with this observation, WSE reduced total and membrane-bound u-PA activity in monocytes. In neutrophils, WSE caused small (50–60%) but significant (P < 0·05) reductions in expression of u-PAR and PAI-2 but had no effect on expression of u-PA, PAI-1 and on total cell-associated and membrane-bound u-PA activity. WSE from yoghurts made from bovine or ovine milk were essentially equally effective in affecting the u-PA system except for the u-PAR gene in ovine neutrophils that was affected (reduced) by the ovine and not the bovine WSE. In conclusion, peptides present in WSE modulated the expression of u-PA related genes but the effect was much more prominent in monocytes than in neutrophils.

The aim of this Research Communication was to investigate the changes in physicochemical, colour, apparent viscosity and texture properties in low-fat goat milk yogurts prepared with cupuassu pulp by the addition of inulin (SI), maltodextrin (SM), whey protein (SW) and skim milk powder (SP). Three batches of each cupuassu goat milk yogurt were prepared and analysed on the 1st day of storage by pH, proximate composition, colour, apparent viscosity, and texture. In comparison to yogurts from whole (W) or skimmed milk (S), all of the fat replacers improved the physicochemical properties (P < 0·05). The addition of the carbohydrates (inulin and maltodextrin) and proteins (whey protein and skim milk powder) also influenced the colour of the low-fat cupuassu goat milk yogurt (P < 0·05). All fat replacer treatments (SI, SM, SW and SP) presented a higher (P < 0·05) apparent viscosity than W and S yogurts. However, only the addition of skim milk powder increased the texture parameters (firmness and consistency) (P < 0·05). These results suggest that skim milk powder can be used to improve the texture properties of low-fat cupuassu goat milk. Furthermore, inulin, maltodextrin, and whey protein can potentially be applied in the goat dairy industry to increase the viscosity of yogurts.

Staphylococcus aureus is arguably the most important pathogen involved in bovine mastitis. The aim of this study was to determine the virulence gene profiles of 124 Staph. aureus isolates from subclinical mastitis in cows in eastern Poland. The presence of 30 virulence genes encoding adhesins, proteases and superantigenic toxins was investigated by PCR. The 17 different combinations of adhesin genes were identified. Occurrence of eno (91·1%) and fib (82·3%) genes was found to be common. The frequency of other adhesion genes fnbA, fnbB, ebps were 14·5, 50, 25%, respectively, and for cna and bbp were 1·6%. The etA and etD genes, encoding exfoliative toxins, were present in genomes of 5·6 and 8·9% isolates, respectively. The splA and sspA, encoding serine protease, were detected in above 90% isolates. The most frequent enterotoxin genes were sei (21%), sem (19·4%), sen (19·4%), seg (18·5%) and seo (13·7%). The tst gene was harboured by 2·4% isolates. The 19 combinations of the superantigenic toxin genes were obtained and found in 35·5% of isolates. Three of them (seg, sei, sem, sen, seo; sec, seg, sei, sem, sen, seo and seg, sei, sem, sen) were the most frequent and found in 16·1% of the isolates. The most common virulotype, present in 17·7% of the isolates, was fib, eno, fnbB, splA, splE, sspA. The results indicate the variation in the presence of virulence genes in Staph. aureus isolates and considerable diversity of isolates that are able to cause mastitis in cows.

Antibiotics are commonly used in poultry feed as growth promoters. This practice is questioned given the arising importance of antibiotic resistance. Antimicrobial peptides can be used as food additives for a potent alternative to synthetic or semi-synthetic antibiotics. The objective of this study was to develop a peptide production method based on membrane adsorption chromatography in order to produce extracts with antimicrobial activity against avian pathogens (Salmonella enterica var. Enteritidis, Salmonella enterica var. Typhimurium, and two Escherichia coli strains, O78:H80 and TK3 O1:K1) as well as Staphylococcus aureus. To achieve this, buttermilk powder and purified lactoferrin were digested with pepsin. The peptide extracts (<10 kDa) were fractionated depending on their charges through high-capacity cation-exchange and anion-exchange adsorptive membranes. The yields of cationic peptide extracts were 6·3 and 15·4% from buttermilk and lactoferrin total peptide extracts, respectively. Antimicrobial activity was assessed using the microdilution technique on microplates. Our results indicate that the buttermilk cationic peptide extracts were bactericidal at less than 5 mg/ml against the selected avian strains, with losses of 1·7 log CFU/ml (Salm. Typhimurium) to 3 log CFU/ml (E. coli O78:H80); viability decreased by 1·5 log CFU/ml for Staph. aureus, a Gram-positive bacterium. Anionic and non-adsorbed peptide extracts were inactive at 5 mg/ml. These results demonstrate that membrane adsorption chromatography is an effective way to prepare a cationic peptide extract from buttermilk that is active against avian pathogens.

Colostrum and milk feeding are key factors for the newborn ruminant survival, affecting the future performance of the animal. Nowadays, there is an increasing interest in the potential of feeding newborn ruminants (mainly goat kids and lambs) with colostrum and milk from other more productive ruminant species (mainly cows). Although some studies regarding differences between colostrum and milk from these three species have been performed, herein we conduct for the first time a comparison using a proteomics 2-Dimensional Electrophoresis gel-based approach between these three ruminant species. In this study colostrum and milk samples from six Holstein cows, six Canarian sheep and six Majorera goats were used to determine the chemical composition, immunoglobulin G (IgG) and M (IgM) concentrations and proteomics profiles. Results showed that in general sheep colostrum and milk contained higher fat, protein and lactose percentages compared to bovine and goat samples. Additionally, no differences in the IgG or IgM concentrations were found among any of the three studied species, with the exception of sheep colostrum that showed the highest IgM concentration. With reference to the proteomics-based approach, some high abundant proteins such as serum albumin precursor, beta-caseins or different immunoglobulins components were found in colostrum, milk or even both. Nevertheless, differences in other proteins with immune function such as serotransferrin or lactoperoxidase were detected. This study shows that despite the similar immunoglobulin concentrations in colostrum and milk from the three studied species, differences in several immune components can be detected when these samples are studied using a proteomics approach. Finally, this study also provides a base for future investigation in colostrum and milk proteomics and metabolomics.

Sweet cheese whey has been used to obtain whey cheese without the addition of milk. Pre-treated whey was concentrated by nanofiltration (NF) at different concentration ratios (2, 2·5 and 2·8) or by reverse osmosis (RO) (2–3 times). After the concentration, whey was acidified with lactic acid until a final pH of 4·6–4·8, and heated to temperatures between 85 and 90 °C. The coagulated fraction (supernatant) was collected and freely drained over 4 h. The cheese-whey yield and protein, fat, lactose and ash recoveries in the final product were calculated. The membrane pre-concentration step caused an increase in the whey-cheese yield. The final composition of products was compared with traditional cheese-whey manufacture products (without membrane concentration). Final cheese yields found were to be between 5 and 19·6%, which are higher than those achieved using the traditional ‘Requesón’ process.

Burrata is an Italian fresh ‘pasta filata’ cheese made from cow's milk and cream that is rapidly spreading in Europe. It has very high caloric content, and a technological protocol was developed for producing a reduced-fat type and fortifying it with polyunsaturated fatty acids (PUFA) of vegetable origin. A satisfactory reduced-fat prototype was obtained by using a 14% fat cream, which was specifically developed by diluting double cream with a suspension of carob seed flour. The composition of the new cheese changed with respect to the control, but the sensory characteristics were not impaired. Moisture increased from 62·6 to 68·4%, fat on dry matter decreased from 59·1 to 34·7%, and the caloric content decreased from 1060·8 to 718 J/100 g. Proteolysis and lipolysis were not affected by the technological modifications: after 7 d storage, the electrophoretic pattern of caseins and the free fatty acids profile of experimental and control cheeses were not significantly different. Fortification of reduced-fat Burrata with PUFA was obtained by using two commercial formulates available at a compatible price with the current economic values of the cheese. The two formulates derived from flaxseeds and Carthamus tinctorius oil and allowed enrichment in C18 :3 : n3 (α-linolenic acid, ALA), and 9cis,11trans- and 10trans,12cis- conjugated linoleic acid (CLA), respectively. Fortification was easy to perform under a technical point of view, but the negative sensory impact limited fortification at a maximum of 7·0 mg g-1 fat ALA and 6·8 g-1 fat CLA.