We assessed polymorphisms in exon IV of the κ-casein gene (CSN3) in ten different breeds of domestic goat (Capra hircus) from three continents and in three related wild caprine taxa (Capra <ibex> ibex, Capra <ibex> sibirica and Capra aegagrus). Thirty-five DNA samples were sequenced within a 558 bp fragment of exon IV. Nine polymorphic sites were identified in domestic goat, including four new polymorphisms. In addition to four previously described polymorphic positions, a total of 13 polymorphisms allowed the identification of 13 DNA variants, corresponding to 10 protein variants. Because of conflicting nomenclature of these variants, we propose a standardized allele designation. CSN3*A, CSN3*B, and CSN3*D were found as widely distributed alleles in European goat breeds. Within Capra ibex we identified three variants and showed that the sequence of Capra aegagrus is identical to the most common Capra hircus variant, consistent with Capra aegagrus being the wild progenitor of domestic goats. A dendrogram was drawn to represent the molecular network between the caprine CSN3 variants.

The dynamic viscosity coefficients η of creams containing up to 50% fat have been determined at temperatures of approximately 40, 50, 60, 70 and 80°C. All the creams behaved as Newtonian liquids, the shear stress being proportional to rate of shear up to the maximum rate used of 100 sec−1. At a given temperature a linear dependence of log η on (φ+φ5/3) was obtained for φ < 0·4, where φ is the concentration (w/w) of fat. Interpolation formulae have been derived to enable η to be calculated at any temperature between 40 and 80°C and for any fat content up to 40%. Formulae for the density ρ of cream have also been deduced to permit kinematic viscosities η/ρ to be computed. Nomograms have been constructed to enable η and ρ to be readily determined without the use of the interpolation formulae and when slight loss in accuracy is unimportant.

The complexing of whey proteins in hydrochloric acid whey by carboxymethyl cellulose (CMC) has been studied. The efficiency of precipitation of CMC (the ability to form insoluble complexes with whey proteins) increased with increasing degree of substitution (DOS) of CMC. At a DOS of greater than 1·4, efficiencies of precipitation of whey protein greater than 90% were achieved without the need for dilution of whey. CMC of high DOS would thus be useful in whey protein reclamation schemes. The amount of CMC necessary for maximum efficiency of precipitation also increased with increasing DOS. The composition of the CMC-protein complex varied little over the range pH 2·8–4·0. Under optimum conditions for binding, the complex contained 61–68% protein and more than 95% of the CMC originally in solution was precipitated in the complex.

The effect of preheating skim milk and artificial micelle milk on curd syneresis was studied. The inhibition of syneresis caused by heat was dependent on the presence of β-lactoglobulin (β-lg) and to a lesser extent α-lactalbumin. The degree of inhibition increased with increasing amounts of added β-lg and preheating temperature. This agrees with the hypothesis that the detrimental effect of preheating on syneresis is due to complex formation between β-lg and κ-casein. This complex appeared to be mediated via thiol–disulphide exchange and its formation appeared to interfere with the micelle–micelle interactions responsible for syneresis.

Coagulation of para-casein micelles was monitored using a Platelet Aggregometer at 37·8 °C and pH 6·7. The macropeptide released by chymosin was determined quantitatively with fluorescamine. The aggregatability of para-casein micelles integrated over the complete hydrolysis of κ-casein was calculated from the amount of κ-casein hydrolysed during the clotting time (Tc). Low concentrations of CaCl2 enhanced the rate of κ-casein hydrolysis but increases in CaCl2 concentration above 8 mM caused marked decreases in this rate. Calcium chloride enhanced the ability of para-casein micelles to aggregate. Little aggregation of para-casein micelles occurred at 0·6 mM-CaCl2 even after all of the κ-casein was hydrolysed. As the concentration of CaCl2 was increased from 3 to 60 mM, aggregation of para-casein micelles took place at progressively lower levels of κ-casein hydrolysis and the percentage hydrolysis at the Tc decreased markedly from 71±7% to 26±8%. The combined influence of CaCl2 on the velocity of the chymosin-catalysed reaction and on para-casein micelle aggregatability accounted for its effect on the average rate of coagulation (calculated by the reciprocal of the Tc). Results are consistent with the hypothesis that electrostatic repulsions and ionic bonding are involved in the interaction between chymosin and κ-casein.

Effects of pH, added Ca, rennet concentration, temperature, concentration and dilution of milk, on rennet clotting and coagulum development have been investigated. Rennet clotting time (RCT) was decreased by reduced pH, by increased temperature and by increased concentrations of rennet or added Ca. Only extremes of concentration or dilution of milk increased RCT. Strength of milk coagulum at 1 h after RCT was increased by reduction in pH and by increased concentrations of added Ca. Increased temperatures decreased coagulum strength whereas increased rennet concentrations had little effect. A curvilinear relationship between total casein concentration and coagulum strength was found for Friesian and Jersey milks. Two phases of coagulum development after clotting were observed. Changes in pH particularly affected the first phase whereas changes in temperature particularly affected the second. Effects of rennet, casein and Ca were similar for both phases.

We report here a comparative analysis of the growth, acidification capacity, and chemical and microbiologic composition between kefir grains after 20 subcultures in whey at 20, 30, and 37°C and the original kefir grains coming from milk along with a determination of the microbiological composition of the fermented whey as compared with that of traditional fermented milk.

When fermentation was carried out repeatedly at 30 or 37°C, kefir grains changed their kefir-like appearance, exhibited reduced growth rates, had a lower diversity of yeasts and water content, and a higher protein-to-polysaccharide ratio compared with the original kefir grains. In contrast, at 20°C kefir grains could remain in whey for prolonged periods without altering their acidification capacity, growth rate, macroscopic appearance or chemical and microbiologic composition—with the only difference being a reduction in certain yeast populations after 20 subcultures in whey. At this incubation temperature, the presence of Lactobacillus kefiranofaciens, Lb. kefir, Lb. parakefir, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces unisporus, and Sac. cerevisiae was detected in kefir grains and in fermented whey by denaturing-gradient-gel electrophoresis (DGGE). In whey fermented at 20°C the number of lactic-acid bacteria (LAB) was significantly lower (P<0·05) and the number of yeast significantly higher (P<0·05) than in fermented milk. Since the DGGE profiles were similar for both products, at this temperature the microbiologic composition of fermented whey is similar to that of fermented milk. We therefore suggest a temperature of 20°C to preserve kefir grains as whey-fermentation starters.

The objective was to measure teat canal length and diameter, teat diameter and teat wall thickness by ultrasonographic scanning in order to determine the differences in bovine breeds, and to study the influence of teat canal length and diameter on the occurrence of mastitis. A total of 269 lactating dairy cows of four different breeds (Brown Swiss, Simmental, Simmental crossbred with Red Pied, and Holstein-Friesians) from seven Upper Austrian dairy farms were examined. Average teat canal length of Brown Swiss animals was shortest (15·7 mm) followed by Holstein-Friesians (17·2 mm) and Simmental (18·3 mm). These differences in teat canal length were highly significant (P[les ]0·001). There was no significant difference in teat canal length between pure-bred and crossbred Simmentals. Differences of teat canal diameter between breeds were significant (P[les ]0·05). Brown Swiss animals had the largest diameters (2·0 mm) and Holstein-Friesians the smallest (1·7 mm). Differences in teat diameter between Brown Swiss, Holstein-Friesian and Simmental were also significant. No differences were found between the pure-bred and crossbred Simmental cows. The narrowest teats were in Holstein-Friesians and the widest in Simmental. Holstein-Friesians also exhibited the thinnest teat walls while the Simmental had the thickest ones. Teat canal length and diameter were correlated with udder health. Teat canals of healthy udders tended to be longer (17·4 mm) and narrower (1·8 mm) than teat canals of infected udders (15·8 mm, 2·1 mm; P[les ]0·001). A logistic regression model showed significant effects of teat canal length, teat canal diameter and lactation number on udder health.

Zn binding by whole bovine and human casein and by purified bovine caseins and whey proteins was investigated by equilibrium dialysis. Bovine αs1 casein had the greatest Zn-binding capacity (˜ 11 atoms Zn/mol). Protein aggregation was observed as Zn concentration was increased and- the protein precipitated at a free Zn concentration of 1·7 mM. Zn binding increased with increasing pH in the range 5·4–7·0 and decreased with increasing ionic strength. Competition between Zn and Ca was observed for binding to αs1-casein indicating common binding sites for these two metals. Bovine β-casein bound up to 8 atoms Zn/ mol and precipitated at a free Zn concentration of ˜ 2·5 mM, while K-casein bound 1–2 atoms Zn/mol. Whole bovine and human casein bound 5–8 atoms Zn/mol and precipitated at a free Zn concentration of ˜ 2·0 mM. Scatchard plots for Zn binding to caseins showed upward convexity, possibly due to Zn-induced association of caseins. Apparent average association constants (K¯app) for all caseins were similar (log K¯app 3·0–3·2). Enzymic dephosphorylation of αs1- or whole bovine casein markedly reduced, but did not eliminate, Zn binding. Thus, phosphoserine residues appeared to be the primary Zn-binding sites in caseins. With the exception of bovine serum albumin. which bound over 8 atoms Zn/mol, the bovine whey proteins, β-lactoglobulin, α-lactalbumin and lactotransferrin, had little capacity for Zn binding.

Two constituents of the proteose-peptone fraction of bovine milk have been isolated and characterized. Component 5 (PP5) has been shown to represent residues 1–105 and 1–107 of the β-casein amino acid sequence, while component 8-fast (PP8F) corresponds to residues 1–28 of β-casein. Thus, these proteose-peptones represent the N-terminal portions of the β-casein molecule, produced by proteolytic cleavages which form the γ1-, γ2- and γ3-caseins from the C-terminal part. The continuing formation of the total proteose-peptone fraction, PP5, PP8F and the γ-caseins during storage of raw milk at 18 or 37 °C has also been demonstrated

Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.

High pressure homogenization (HPH) is one of the most promising alternatives to traditional thermal treatment for food preservation and diversification. In order to evaluate its potential for the production of fermented milks carrying probiotic bacteria, four types of fermented milks were manufactured from HPH treated and heat treated (HT) milk with and without added probiotics. Microbiological, physicochemical and organoleptic analyses were carried out during the refrigerated period (35 d at 4°C). HPH application to milk did not modify the viability of the probiotic cultures but did increase the cell loads of the starter cultures (ca. 1 log order) compared with traditional products. The coagula from HPH-milk was significantly more compacted (P<0·05) (higher firmness) than that obtained with HT-milk, and it had the highest values of consistency, cohesiveness and viscosity indexes compared with fermented milks produced without HPH treatment. All the samples received high sensory analysis scores for each descriptor considered. HPH treatment of milk can potentially diversify the market for probiotic fermented milks, especially in terms of texture parameters.

Cows with subclinical intramammary infections were identified by milk bacteriology. The mastitis pathogens included Staphylococcus aureus (n=9), Streptococcus uberis (n=10) and coagulase-negative staphylococci (n=10). Samples of first fore milk, main flow milk and strippings milk fractions were collected from each quarter and laboratory measurements were made of electrical conductivity, milk fat concentration and somatic cell count. Conductivity measurements were corrected for milk fat concentration and within-cow inter-quarter conductivity ratios calculated. Repeatability estimates of all measurements between days were calculated. In the case of infected quarters, all conductivity values decreased markedly (P<0·05) from first fore milk to main flow milk fractions. Conductivity differences between quarters of infected cows were substantially lower during the main milk flow phase. For quarters infected with Staph. aureus an increase in conductivity was observed (P<0·05) from main flow to strippings fractions. For uninfected quarters, conductivity declined as milk fat concentration increased with successive milk fractions. Variation, both within and between milk fractions, was greater for somatic cell count than for conductivity. Differences in conductivity between milk fractions from individual infected quarters were not accounted for by changes in fat concentration and may result from the mixing of milk from infected and uninfected regions of the gland. Localized infection may produce a decrease in conductivity between fore milk and mid-flow fractions while differential drainage from an infection site in the secretory tissue may additionally produce an increase in conductivity from mid-flow to strippings fractions. Such changes may thus provide information on the location and magnitude of an infection. The results clearly demonstrate the importance of the milk fraction when using conductivity as a diagnostic of intramammary infection, the highest diagnostic sensitivity being achieved by using first fore milk samples.

The bacteriological formation of 2-butanone and 2-butanol was investigated. The data suggest that 2-butanol is formed in cheese via 2-butanone in 3 steps, each step being carried out specifically by a different microbial species. Suitable species have been isolated from a single cheese. The first step could be carried out (in the present investigation) either by certain starter organisms or by a strain of Pediococcus cerevisiae. This strain formed substantial amounts of 2,3-butylene glycol in milk, presumably from citrate, but could not metabolize this compound further. A second micro-organism, a strain of Lactobacillus plantarum, was isolated which, in Cheddar cheese, could convert 2,3-butylene glycol into 2-butanone. The final reduction of 2-butanone to 2-butanol could be readily carried out in MRS medium by a strain of L. brevis. Results from cheese made aseptically indicate that 2-butanone is formed in Cheddar cheese only when non-starter organisms are present.

A homo-tetrameric ∼160-kDa cystathionine γ-lyase was purified to homogeneity from Lactobacillus reuteri DSM 20016 by four chromatographic steps. The activity was pyridoxal-5′-phosphate dependent and the enzyme catalyzed the α,γ-elimination reaction of L-cystathionine, producing L-cysteine, ammonia and α-ketobutyrate. The enzyme was active towards a range of amino acids and amino acid derivatives, including methionine. The pH and temperature optima were found to be 8.0 and 35 °C, respectively. Isoelectric pH (pI) was ∼5.0 as determined by two-dimensional electrophoresis. Sensitivity to chemical inhibitors was typical of lactococcal cystathionine γ- and β-lyases, except it was inhibited by sulphydryl reagents. The N-terminal sequence was MKFNTQLIHGGNSED, which had 100% homology with cystathionine β-lyase of Lb. reuteri 104R (Accession Number CAC05298). Lb. reuteri DSM 20016, together with 10 other strains of non-starter lactic acid bacteria, was used as adjunct starter in the production of miniature Canestrato Pugliese-like cheeses. After 40 d ripening, the water-soluble extract of the cheeses with added Lactobacillus fermentum DT41 and Lb. reuteri DSM 20016 contained the highest enzyme activities on cystathionine and methionine substrates. Determinations of methanethiol, dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide in the miniature cheeses confirmed the findings of enzyme activities.

A routine method giving accurate results was required for the determination of the glyceride content of eluate fractions obtained in the silicic acid chromatography of lipid mixtures. The technique of determining glyceride glycerol by saponification, oxidation of the liberated glycerol to formaldehyde, reaction of the formaldehyde with chromotropic acid and measurement of the resulting chromogen at 570 mμ has been investigated. Procedures based on this principle have appeared in the literature but none of these has been found to be entirely satisfactory. Accordingly, a modification of the method has been developed that is capable of giving results of a high degree of accuracy.

1. The acid phosphatase content of milk is reasonably constant from quarter to quarter in normal uninfected cows.

2. The acid phosphatase content of milk from infected quarters is enhanced in comparison with the uninfected quarters. The differences found were from 4·1 to over 50 enzyme units per 100 ml. milk.

3. It has been shown that a Streptococcus agalactiae suspension is capable of hydrolysing phenylphosphate at pH 4–1, and it is suggested that this organism is largely responsible for the enhanced enzyme content of the milk from the infected udder.

Interest in selective dry cow therapy (SDCT) has been increasing owing to concerns over development of antimicrobial resistance. Implementation of SDCT, however, requires a quick and cost-effective on-farm method for identifying cows for treatment and cows that can be left without treatment. The objective of the present study was to evaluate the use of clinical mastitis (CM) history and somatic cell counts (SCC) from monthly Dairy Herd Improvement (DHI) records in identification of infected and uninfected cows at dry-off. A total of 647 Holstein cows were classified as uninfected or infected at dry-off based on CM history and varying number of monthly SCC records (with three different SCC cut-offs). Cows were considered uninfected based on the following criteria: (1) SCC <100 000 cells/ml and no CM during the lactation; (2) SCC <200 000 cells/ml and no CM during the lactation; (3) as criterion two, but additionally a cow was also considered uninfected if it experienced a case of CM during the first 3 months of the lactation and the SCC was <100 000 cells/ml for the rest of the lactation; (4) SCC <300 000 cells/ml and no CM during the lactation; otherwise they were considered infected. Infected and uninfected cows at dry-off were most efficiently identified using three months' SCC records with a threshold of 200 000 cells/ml for cows without CM during the lactation and a threshold of 100 000 cells/ml during the rest of lactation for cows with CM during the first 90 days in milk. Moreover, this criterion also most efficiently identified cows infected with major pathogens only at dry-off. The success of the criteria used for identifying infected and uninfected cows will, however, depend on herd characteristics, such as prevalence of infection and type of pathogens present in the herd.

Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by β-galactosidase, named transglycosylation. Lactose is the main constituent of whey permeate (WP), normally wasted output from the cheese industry. Therefore, the main goal of this work was to optimise the synthesis of GOS in WP using β-galatosidase from Aspergillus oryzaea. WP and whey permeate enzymatically treated (WP-GOS) were used as culture media of Lactobacillus plantarum 299v. Lb. plantarum 299v attained the stationary phase in approximately 16 h, reaching 3·6 and 4·1×108 CFU/ml in WP and WP-GOS, respectively. The in situ synthesised GOS were not consumed during growth. No significant differences were observed in the growth kinetics of microorganisms in both media. After fermentation, microorganisms were dehydrated by freeze-drying and spray-drying and stored. The recovery of microorganisms after fermentation, dehydration and storage at 4 °C for at least 120 d was above 108 CFU/g. These studies demonstrated that WP is an appropriate substrate for the synthesis of GOS and the obtained product is also adequate as culture medium of Lb. plantarum 299v. The coexistence of GOS and dehydrated viable probiotic microorganisms, prepared using an effluent as raw material, represents the main achievement of this work, with potential impact in the development of functional foods.