Late blowing, caused by the outgrowth of clostridial spores present in raw milk and originating from silage, can create considerable product loss, especially in the production of hard and semi-hard cheeses. The conventional method for the isolation of Clostridium spp. from cheeses with late-blowing symptoms is very complicated and the identification of isolates is problematic. The aim of this work was the development of a multiplex PCR method for the detection of the main dairy-related clostridia such as: Cl. beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum. Samples derived from silage, raw milk and hard cheese were analysed by the most probable number (MPN) enumeration. Forty-four bacterial strains isolated from gas positive tubes were used to check the reliability of the multiplex PCR assay. The specificity of the primers was tested by individually analysing each primer pair and the primer pair combined in the multiplex PCR. It was interesting to note that the samples not identified by the multiplex PCR assay were amplified by V2–V3 16S rRNA primer pair and the sequencing revealed the aligned 16S rRNA sequences to be Paenibacillus and Bacillus spp. This new molecular assay provides a simple promising alternative to traditional microbiological methods for a rapid, sensitive detection of clostridia in dairy products.

Two types of artificial neural networks, multilayer perceptron (MLP) and self-organizing feature map (SOM) were used to detect mastitis by automatic milking systems (AMS) using a new mastitis indicator that combined two previously reported indicators based on higher electrical conductivity (EC) and lower quarter yield (QY). Four MLPs with four combinations of inputs were developed to detect infected quarters. One input combination involved principal components (PC) adopted for addressing multi-collinearity in the data. The PC-based MLP model was superior to other non-PC-based models in terms of less complexity and higher predictive accuracy. The overall correct classification rate (CCR), sensitivity and specificity of this model were 90·74%, 86·90% and 91·36%, respectively. The SOM detected the stage of progression of mastitis in a quarter within the mastitis spectrum and revealed that quarters form three clusters: healthy, moderately ill and severely ill. The clusters were validated using k-means clustering, ANOVA and least significant difference. Clusters reflected the characteristics of healthy and subclinical and clinical mastitis, respectively. We conclude that the PC based model based on EC and QY can be used in AMS to detect mastitis with high accuracy and that the SOM model can be used to monitor the health status of the herd for early intervention and possible treatment.

Colostrum and milk samples from 20 goats were analysed for concentrations of immunoglobulin G (IgG), β-lactoglobulin (β-lg), α-lactalbumin (α-la) and serum albumin (CSA) throughout the first 14 milkings post partum (7 d of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means±SD) at first milking were IgG 47·9±25·5, β-lg 30·7±10·4, α-la 2·77±0·82 and CSA 2·97±2·46 mg/ml. Large variations were recorded for IgG concentrations (19·9–94·5 mg/ml) and β-lg (9·3–49·8 mg/ml). Concentrations of IgG, β-lg and CSA dropped abruptly in the subsequent milkings and α-la concentration decreased slowly. Mean IgG concentration was <2 mg/ml after 7 milkings and <1 mg/ml after 11 milkings. However, IgG concentration does not differ significantly, at the 1% level, from milkings 7–14. The contribution of β-lg to the increase in whey proteins in early milks was greater than that of IgG from milkings 5 to 14. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to milk supply.

Staphylococcus aureus isolates produce several pathogenic factors. The combination of these products influences the pathogenic role of different isolates, but their specific effects are well known in the pathogenesis of udder infections. This study focused on the association of polymorphism of the coagulase gene, protein A gene, collagen-binding protein gene, and of fibrinogen-binding protein gene on somatic cell count (SCC) and on Staph. aureus growth rate. Fifty Staph. aureus isolates from 13 dairy cow herds, located in seven different provinces, were considered. The results showed a low frequency of cna gene, similar to the one observed in human isolates. Meanwhile, the high frequency of efb gene indirectly confirmed the role of this factor in bacterial pathogenesis, being associated with adhesion to epithelia. The association of these two single genes with SCC and growth rate showed to be not significant. The polymorphism of spa gene was confirmed to be significantly associated with inflammatory response and growth rate, albeit with a pattern different from the one suggested for human isolates. Sorting of isolates based on the clusters obtained by combining polymorphisms of spa and coa genes and the presence of cna and efb genes, showed that a single cluster (cluster V) was prevalent in the different herds and provinces, while the other six clusters identified were widely spread among the remaining 60% of the isolates. Results showed that clusters VI and VII had significantly higher growth rates at 3, 4, and 6 h in comparison with the other clusters. Meanwhile, quarters infected with these strains showed significantly lower SCC levels. The frequency of isolates from cluster V, suggested that they should possess pathogenic factors increasing their invasiveness, even if in the presence of a stronger inflammatory response. These results indirectly confirm previous findings on the different interactions between isolates and the udder immune system. They also suggest that isolates with higher growth rates and inducing a lower inflammatory response have better chances to spread among the herd. The relatively simple genomic method proposed in this study could be applied by an increasing number of diagnostic laboratories and could be useful in studying the epidemiology of Staph. aureus intramammary infections in dairy herds when collecting data from the field.

Lamb rennet pastes were prepared by the procedure most commonly used by Idiazabal cheese manufacturers. We studied the effects on their coagulating and lipolytic activities of the state of the stomach at the time of death (full of milk or empty), the amount of NaCl added, the origin of the lambs and paste storage time. Coagulating activities were generally between 155 and 363 units/g tissue. Pastes prepared from stomachs of lambs from slaughterhouse flocks had significantly higher coagulating activities than those of lambs from separate flocks. No significant decrease in coagulating activity was observed after 1 year storage at 4 °C. Chymosin represented 75–80% of the total coagulating activity with the remainder being pepsin. Rennet paste extracts with pH < 4·7 did not have increased coagulating activities when their pH was lowered to 2·0, while those with pH > 5·2 had activities 1·5-fold those before treatment. Lipase activity was higher in extracts of rennet pastes prepared using the stomachs of lambs that arrived at the slaughterhouse in the morning just prior to slaughter than in those prepared with the stomachs of lambs that had arrived on the previous evening. However, the reverse was the case for esterase activity. Activating the coagulating activity by pH cycling completely destroyed both lipolytic activities. Storage at 4 °C for > 1 year did not affect esterase activity but lipase activity decreased substantially after 4–5 months. Lipase, but not esterase, activity was responsible for the liberation of short-chain free fatty acids from ovine milk fat.

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.

We investigated the mucus-binding properties of aggregating and non-aggregating potentially probiotic strains of kefir-isolated Lactobacillus kefiri, using different substrates. All the strains were able to adhere to commercial gastric mucin (MUCIN) and extracted mucus from small intestine (SIM) and colon (CM). The extraction of surface proteins from bacteria using LiCl or NaOH significantly reduced the adhesion of three selected strains (CIDCA 8348, CIDCA 83115 and JCM 5818); although a significant proportion (up to 50%) of S-layer proteins were not completely eliminated after treatments. The surface (S-layer) protein extracts from all the strains of Lb. kefiri were capable of binding to MUCIN, SIM or CM, and no differences were observed among them. The addition of their own surface protein extract increased adhesion of CIDCA 8348 and 83115 to MUCIN and SIM, meanwhile no changes in adhesion were observed for JCM 5818. None of the seven sugars tested had the ability to inhibit the adhesion of whole bacteria to the three mucus extracts. Noteworthy, the degree of bacterial adhesion reached in the presence of their own surface protein (S-layer) extract decreased to basal levels in the presence of some sugars, suggesting an interaction between the added sugar and the surface proteins. In conclusion, the ability of these food-isolated bacteria to adhere to gastrointestinal mucus becomes an essential issue regarding the biotechnological potentiality of Lb. kefiri for the food industry.

The goal of the study was to evaluate the composition of leucocyte populations in different milk fractions as a basis on which to judge their possible role in the immune response in different compartments of the udder. The milk of one healthy quarter of nine dairy cows (SCC/quarter [les ]125000/ml; bacteriologically negative) was removed separately and during the course of milking divided into: cisternal milk (C), alveolar milk 0–25%, 25–50%, 50–75%, 75–100% (A25; A50; A75; A100, respectively) and residual milk (R). Each fraction was analysed for the main constituents, SCC and distribution of leucocyte populations and their viability. The content of fat increased steadily during milking and reached highest values in R. Protein and lactose increased from C to A25, decreased from A25 to A100 and reached their minimum in R. Na and Cl ion levels diminished from C to A25 and thereafter increased from A50 to R. Electrical Conductivity (EC) also decreased from C to A25 but remained similar within the alveolar samples and reached a minimum in R. SCC decreased from C to a minimum in A25 and increased subsequently to a significant maximum in R. Somatic cell viability increased throughout consecutive fractions with a maximum value in R. The ratio of cell populations in the various milk fractions showed a reverse trend of macrophages (M) and polymorphonuclear neutrophils (PMN). M values were highest in C while PMN levels increased to their maximum in the R fraction. The lymphocyte (L) fraction remained similar in C, A25, A50, A75 and R but was higher in A100. Proportions of L, PMN and M were, respectively, 9·3%, 38·2%, 52·3% in C, 10·9%, 64%, 25·1% in A25–A100 and 10·2%, 64·9%, 24·8% in R. Numbers of L, PMN and M in milk showed a similar pattern for all three cell types: high levels in C decreased to a minimum at A25 and increased steadily thereafter to their maxima in R. It is concluded that, for healthy quarters, M, the predominant cell type in C, are located near the teat canal, the main entrance of pathogens. Obviously they are the first immunological barriers for invading pathogens. In contrast, PMN are the most important population in the alveolar compartment. However, each leucocyte fraction had a higher concentration in C than in early alveolar fractions, thus indicating the crucial role of immune defence in the cisternal compartment.

An outstanding parameter in cheese making is the type of coagulant, which greatly influences the characteristics of the final products. Proteolysis is the most important set of biochemical changes during ripening of most cheeses, and is carried out, in different magnitude, by proteolytic agents originated in milk, rennet (or rennet substitute), and starter and non-starter micro-organisms (Silva & Malcata, 2000). The demand for alternative sources of milk coagulants, to replace the expensive and limited natural rennet supplies, has increased (Esteves et al. 2001). All commercial enzymes employed as milk coagulant are aspartic proteinases, which are most active at acidic pH and preferentially cleave peptide bonds between residues with hydrophobic side-chains (Silva & Malcata, 1999).

Trans fatty acids (TFA) formed during biohydrogenation by ruminant animals were quantified in cows' milk fat by means of Fourier transform i.r. (FTIR) spectroscopy. When samples were analysed as the derived methyl esters, a spectral subtraction technique resulted in TFA values that were not biased by the unspecific absorption of intact triacylglycerols or influenced by the shape of the trans band. Austrian milk fat samples contained 20–50 g TFA/kg. Results obtained by analysis of TFA as triacylglycerols compared favourably with those obtained by using methyl esters. The findings of the FTIR method were further substantiated by applying argentation chromatography in combination with capillary gas chromatography to the same set of samples. Besides trans monoenoic acids, non-methylene interrupted cis, trans octadecadienoic acids contributed significantly to the total trans content. Two novel TFA, trans-11, cis-15 octadecadienoic and cis-9, trans-13 octadecadienoic acids, were identified in milk fat by means of their retention behaviour on argentation chromatography and gas chromatography as well as by an oxidative degradation procedure.

1. Thirty-five strains of acetoin producing lactic streptococci isolated from raw milk, from starter cultures and from dairy products were investigated biochemically and serologically.

2. The strains are shown to form a homogeneous unit, very similar to Strep. lactis O.J., which species they resemble in cultural behaviour, in growth-temperature range, in tolerance of sodium chloride and heat and in carbohydrate and other fermentations. They differ from Strep. lactis essentially in their ability to utilize citrate in producing CO2 volatile acids and C4-compounds (diacetyl, etc.). With few exceptions they bear no resemblance to the enterococci.

3. Serologically the strains are shown to belong to group N and thus the relationship to Strep. lactis is clearly established.

4. The strains investigated are shown to be identical with Strep. citrophilus van Beynum & Pette and almost with certainty, though no direct comparison has yet been possible, with Strep. diacetilactis Matuszewski et al. Probably they are identical, too, with Strep. diacetyl aromaticus Karnad and Strep. lactis aromaticus Joshi & Ram Ayyar.

5. The taxonomic position of the relevant unit is discussed, and it is suggested that it be given species rank under the name Strep. diacetilactis.

Several strains of Lactococcus lactis subsp. lactis, Lactobacillus casei and Lactobacillus plantarum isolated from traditional goats' cheese have been studied for titratable acidity, proteolysis in milk and enzymic activities. Aminopeptidasc activities were measured with whole cells and cells permeabilized with Triton X-100. Caseinolytic activity was investigated using electrophoresis in polyacrylamide gel with sodium dodecyl sulphate. Lc. lactis subsp. lactis had a level of proteolytic activity in skim milk greater than that of Lb. casei, while this activity in Lb. plantarum was very low. Alanine aminopeptidase activity was almost non-existent for all strains tested, while lysine aminopeptidase activity appeared to be of fundamentally intracellular origin. Leucine aminopeptidase activity was also greater in cells that had been permeabilized than in whole cells for Lb. casei and Lb. plantarum. Lc. lactis subsp. lactis leucine aminopeptidase activity was greater in whole cells. No significant hydrolysis of casein was found with Lb. casei I FPL 725 and Lb. plantarum IFPL 722 permeabilized with Triton X-100 after 24 h incubation with whole bovine casein.

The present study identifies proline-specific peptidases found in Streptococcus cremoris AM2. These activities were present prcdominantly in the cytoplasmic fraction of the cells with minor amounts in cell membrane fraction. No evidence vvas found for such activities in either the extracellular fluid used to grow the cells, in the wash fraction or in the cell wall fraction. The involvement of these enzymes in release of free proline from proline-containing peptides is considered.

Tryptic phosphopeptides were obtained from whole bovine casein by chromatography on the anion exchange resin QAE-Sephadex A 25. Salt gradient elution of the column allowed separation of non-phosphorylated peptides from phosphorylated species. The preparations obtained contained at least seven distinct phosphopeptides of which the following casein fragments were identified: αs1(43–58):2P, αs1(59–79): 5P, αs2(46–70): 4P, β(1–28): 4P, β(2–28): 4P, and β(33–48): 1P. Fast protein liquid chromatography (FPLC) on Mono Q HR 5/5 resin showed that the phosphopeptides were eluted in the same order as from the QAE-Sephadex resin. However, on the analytical column HR 5/5 the fragments αs1(59–79): 5P and β(2–28): 4P, having the same net charge under the conditions of chromatography, co-eluted, whereas they were at least partly separated on the preparative column HR 16/10. Following enzymic dephosphorylation, the peptides eluted at lower salt strength in the gradient. FPLC on Mono Q resin thus permitted dephosphorylation to be monitored and intermediates between the parent species and the fully dephosphorylated peptide to be identified.

Simultaneous measurement of viscosity and degree of proteolysis of κ-casein, α, in skim milk at three different concentrations of rennet and three different concentrations of casein showed that viscosity was a function of α until the former reached its minimum value. Then the relation between viscosity and α depended on the enzyme concentration. This is considered to be caused by casein micelles starting to aggregate at this point. The minimum in viscosity was found when α was between 0·6 and 0·7, indicating that casein micelles started aggregating at a lower degree of proteolysis than was believed earlier. The rate of gel firming was found to depend on the concentration of added rennet even after κ-casein was completely proteolysed. The results demonstrated that both proteolysis of κ-casein and aggregation of casein micelles must be taken into account when modelling the renneting reaction.

The fatty-acid composition of 17 samples of New Zealand milk fat obtained throughout one dairy season is reported.

The weight percentage of butyric (C4:0) acid was significantly correlated with that of caproic (C6:0) acid and that of caprylic (C8:0) acid. Percentages of C6:0 and C8:0 showed a highly significant correlation with each other and with weight percentages of capric (C10:0) and lauric (C12:0) acids.

There was a highly significant negative correlation between palmitic (C16:0) and stearic (C18:0) acids and between C4:0 and C16:0 acids. Oleic (C18:1) acid showed significant negative correlations with C8:0, C10:0, C12:0, myristic (C14:0) and C16:0 acids.

The reason for the reported difference in spoilage behaviour of skim and whole pasteurised milks was investigated. The rates of growth of psychrotrophic bacteria were not significantly different in the two milks and the bacterial types, all pseudomonads, present at spoilage were also similar. However, when representative spoilage organisms were cultured into freshly pasteurised skim and whole milks, skim milks exhibited predominantly bitter flavours while whole milk showed mostly sour flavours. The different spoilage behaviours can be largely explained by greater proteolysis in skim milk than in whole milk, caused by higher production of protease and greater susceptibility of the protein to protease attack. In addition, lipolysis in whole milk, caused by the substantial quantities of lipase produced by spoilage pseudomonads in this milk, also contributes to the different flavours produced during cold storage of these milk types.

Fifty-six Holstein dairy cows from a commercial dairy herd in the Northern part of Greece were used to determine the effect of vitamin E supplementation on immune parameters, milk composition and milk quality. Cows were assigned to one of two experimental groups: control (no vitamin E supplementation) and vitamin E supplementation. Supplementation of vitamin E started 4 weeks prior to and continued up to 12 weeks after parturition. Supplementation included daily oral administration of vitamin E at 3000 i.u./cow prepartum and was reduced to 1000 i.u./cow post partum. Blood samples were collected weekly for 8 weeks starting 4 weeks before parturition, neutrophils were isolated and the following parameters were determined in neutrophils activated by phorbol myristate acetate: total cell-associated and membrane-bound urokinase plasminogen activator (u-PA) activity and superoxide production. Milk samples were collected weekly and fat, protein, lactose, somatic cell count (SCC), plasmin and plasminogen-derived activity were determined. Activated neutrophils isolated from cows that received supplemental vitamin E had higher (P<0·01) total and membrane-bound u-PA activities during the first 3 weeks after parturition and higher (P<0·01) superoxide production during week 1 prepartum and week 1 post partum compared with the corresponding values of activated neutrophils isolated from control cows. Vitamin E supplementation had no effect (P=0·28) on plasminogen-derived activity in milk. Milk obtained from cows that received supplemental vitamin E had SCC lower by 25% (P<0·05) and plasmin lower by 30% (P<0·01) than corresponding values in milk obtained from control cows. The reduction in plasmin as a result of vitamin E supplementation is very beneficial to the dairy industry because plasmin reduces the cheese-yielding capacity of milk, affects the coagulating properties of milk and its overall ability to withstand processing during cheesemaking. In conclusion, vitamin E supplementation had positive effects on the function of bovine neutrophils and milk quality in a commercial dairy herd.

Selective dry cow therapy (SDCT) has received increasing attention in recent years owing to global concerns over agricultural use of antimicrobial drugs and development of antimicrobial resistance. The objective of this study was to evaluate the effect of SDCT on milk yield and somatic cell count (SCC) in dairy herds in the USA. Cows in four Ohio dairy herds were categorized into two groups (low-SCC and high-SCC) at dry-off based on their SCC and clinical mastitis (CM) history during the lactation preceding the dry-off. Low-SCC cows were randomly assigned to receive or not to receive intramammary antibiotics at dry-off. Milk yield and SCC of these cows during the following lactation were compared using linear mixed effects models, adjusting for parity, calving season, stage of lactation, previous lactation milk yield and herd. Milk yield of untreated and treated low-SCC cows at dry-off did not differ significantly during the following lactation. Overall, treated low-SCC cows had 16% lower SCC (approximately 35 000 cells/ml, P=0·0267) than the untreated cows during the following lactation; however, the effect was variable in different herds. Moreover the impact of treatment, or the lack thereof, on milk yield varied considerably between herds. The results suggested that in some herds treating all cows at dry-off may be beneficial while in other herds leaving healthy cows without antibiotic dry cow treatment has no negative impact on milk yield or milk quality (SCC), and in fact, may be beneficial. Further studies are needed to identify characteristics of herds where treating all cows routinely at dry-off may be needed for maintaining good udder health and where switching to selective treatment of cows at dry-off would be the optimal approach to achieve best results.

Lactobacillus helveticus and Propionibacterium freudenreichii are essential starters in Swiss cheesemaking and the release of their intracellular enzymes through autolysis could significantly influence ripening. To provide evidence of this lysis, cheese made from microfiltered thermized milk inoculated with Lb. helveticus ITGLH77, Prop. freudenreichii ITGP23 and a commercial Streptococcus thermophilus was assayed. Starter viability was determined and lysis was monitored during ripening by protein analysis with SDS-PAGE of aqueous cheese extracts and by immunoblot detection of intracellular proteins: dipeptidase (PepD) for Lb. helveticus and methylmalonyl coenzyme A mutase for Prop. freudenreichii. We verified that the species specificity of these lysis markers was towards the cytoplasms of all the species currently used in Swiss cheese. Lb. helveticus exhibited an almost complete loss of viability (99·9%) from the beginning of ripening in the cold room; concomitantly PepD appeared in the cheese extracts and was detected until the end of ripening. Damaged Lb. helveticus cells were also visualized by scanning electron microscopy. In addition, free PepD was also successfully detected in commercial Swiss-related cheeses. All these results clearly demonstrated the autolysis of Lb. helveticus in Swiss cheese. Prop. freudenreichii ITGP23 grew during warm room ripening and no loss of viability was detected after maximal growth (109 cfu/g cheese). Free methylmalonyl-coenzyme A mutase was detected at the end of ripening during cold storage, when the cheese extracts were concentrated 20-fold, demonstrating that the autolysis of Prop. freudenreichii was tardy and limited.