Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.

The teats of 18 cows were externally exposed to infection with mastitis organisms by dipping them daily for 1 or 2 weeks in a mixed suspension of Streptococcus dysgalactiae and Str. uberis at the start, the middle or the end of the dry period. The teat sinuses of quarters which remained uninfected after external exposure at the beginning or the middle of the dry period were then infused with the same strains of streptococci. Of the 9 quarters infected following external exposure 8 occurred in animals exposed at the start and one in the middle of the dry period. Thirty-six new infections occurred after infusion of bacteria into the teat sinuses of 38 quarters. In a further trial with 10 cows, Staphylococcus aureus and Str. zooepidemicus were inoculated into the distal 3 mm of the streak canals of 5 cows immediately after drying-off and into those of 5 cows which had been dry for 28 weeks. Animals were slaughtered 48 h later and infection determined by teat puncture. Five infections occurred in cows which were at the start of the dry period and only one in the cows dry for 28 weeks. It is suggested that these differences in the rates of new infection between the early dry period and a very extended dry period are due to differences in the ease with which bacteria can penetrate the teat canal. At the later stage bacterial growth through the teat canal appeared to be inhibited.

Three microbial rennets, Noury rennet (Mucor pusillus, Lindt), Rennilase (M. miehei) and Suparen (Endothia parasitica), were assessed as calf rennet substitutes for Feta cheesemaking, in comparison with calf rennet. Cheeses prepared were all of good quality with similar organoleptic properties. Yield (referred to 55% moisture), moisture content, NaCl and protein content did not differ significantly. Different rates of proteolysis were observed in the cheeses, the highest of which was exhibited by calf rennet followed by Rennilase, Noury rennet and Suparen preparations. These calf rennet substitutes are well suited for Feta cheese manufacture.

Twenty-two Murciano-Granadina dairy goats were used to investigate the effects of organic Zn supplementation of a diet containing a high level of inorganic Zn. Goats were kept in pens, machine milked once a day throughout lactation and fed a diet based on a dehydrated mixture of whole-plant maize and alfalfa ad libitum, alfalfa pellets, barley grain and a concentrate mixture. Treatments were: (1) control, and (2) supplemented with 1 g/d Zn-Methionine (Zn-Met) included in the concentrate mixture. After parturition, goats were blocked in week 3 and dietary treatments were applied until week 23. From weeks 3–20, feed intake, milk yield, milk composition, milk somatic cell count (SCC), and udder health were measured. In week 21, all goats were injected intraperitoneally with 1 g/d DL-methionine for 5 d to establish the effects of methionine under the conditions of udder stress induced by hand milking on the second day. During weeks 22 and 23, diet digestibility, and N and Zn balance were determined. Dry matter intake, milk yield, and milk contents of total solids, fat, total and true protein, and casein did not differ between treatments, but whey protein and non-protein nitrogen contents were significantly lower for the Zn-Met group. Milk SCC tended to decrease as a result of Zn-Met supplementation but differences between treatments were not significant when halves with persistent infection were excluded. Hand milking increased SCC in both groups, but udders of supplemented goats showed a lower reaction. Apparent absorption of N significantly increased and Zn retention tended to increase in Zn-Met supplemented goats. We conclude that Zn-Met supplementation can enhance resistance to udder stress in dairy goats. Effects were attributed to the organic Zn and not to the methionine component. Zn retention and protein utilization were also improved by the Zn-Met supplement.

The effect of subclinical mastitis on levels of plasminogen and plasmin in milk from cows in a high-yielding herd was investigated. Comparisons were made with levels of milk Na, antitrypsin and N-acetyl-β-D-glucosaminidase (NAGase). In samples from mastitic quarters plasminogen activity, as measured after activation to plasmin, increased by only 21% and plasmin by 82%, while NAGase increased by 307 %. Plasminogen was the only component that was normally distributed, all other components showed more or less skewed distributions. Plasmin and plasminogen were significantly related to the other components. However, plasminogen plateaued when the other components continued to increase. There was thus no further increase in plasminogen with the severity of inflammation as with the other components. Plasmin showed a similar although less pronounced tendency. Results of treatment of mastitic whey samples with acid suggested that the non-linear increase in plasmin activity was due to interaction with acid-labile proteinase inhibitors. Mastitis led to dissociation of plasminogen and plasmin from the casein micelles. The degree of activation of plasminogen was higher with casein-associated than with soluble plasminogen in both healthy and mastitic milks. Plasmin was very closely related to milk Na, which is a sensitive indicator of epithelial integrity. It is suggested that plasmin contributes to Na leakage into milk by degrading membrane proteins of the epithelial lining. Plasminogen and antitrypsin, which are both plasma proteins, were not identically affected by stage of lactation, indicating nonidentical modes of transport from plasma to milk.

Fractions enriched with α-lactalbumin (α-la) and β-lactoglobulin (β-lg) were produced by a process comprising the following successive steps: clarification–defatting of whey protein concentrate, precipitation of α-lactalbumin, separation of soluble β-lactoglobulin, washing the precipitate, solubilization of the precipitate, concentration and purification of α-la. The present study evaluated the performance of the process, firstly on a laboratory scale with acid whey and then on a pilot scale with Gouda cheese whey. In both cases soluble β-lg was separated from the precipitate using diafiltration or microfiltration and the purities of α-la and β-lg were in the range 52–83 and 85–94% respectively. The purity of the β-lg fraction was higher using acid whey, which does not contain caseinomacropeptide, than using sweet whey. With the pilot scale plant, the recoveries (6% for α-la; 51% for β-lg) were disappointing, but ways of improving each step in the process are discussed.

Sixty female dairy calves (body weight, BW, 43·2±0·58 kg and age 9·8±0·61 d) were arranged in two groups to compare the short-term and long-term effects of an enhanced-growth feeding programme (EF) with those of a conventional-growth feeding programme (CF). After 1 week of adaptation to a milk replacer (MR), CF calves were fed 4 l/d of MR (25% crude protein, CP; 19·2% ether extract) at 12% dry matter (DM) from days 1 to 27 and 2 l/d at 12% DM from days 28 to 34, and the EF calves were offered the same MR at 18% DM: 4 l/d from days 1 to 6, 6 l/d from days 7 to 13, 7 l/d from days 14 to 20, 6 l/d from days 21 to 27 and 3 l/d from day 28 to weaning at day 34 of the study (50 d of age). Individual calf starter (20·7% CP) intake was recorded daily from the beginning until day 41 of study (57 d of age). Then, calves were placed in groups of six and they received a total mixed ration (TMR) containing 18·5% CP until day 56 d of study (72 d of age). Then, heifers were moved to larger pens and were fed the same TMR in both treatments at each subsequent stage of growth throughout the study. Calves were weighed weekly until day 56 of study and before every pen change (days 94, 149, 200, 387 of study). When heifers were 400 d old and weighed >380 kg, they were moved to a breeding pen where oestruses were checked three times a day. Heifers were inseminated 12 h after the detection of oestrus. One month before calving, heifers were returned to their original farm and milk yield at 305 days in milk was recorded from 28 cows. Starter intake was greater (P<0·001) in CF than in EF calves (0·79 v. 0·29±0·043 kg/d, respectively) during the preweaning period, but TMR consumption was similar in both treatments from days 42 to 56 of study. BW of EF calves was greater (P<0·01) than that of CF calves at weaning (76·4 v. 71·6±1·10 kg, respectively), but BW was not different at day 387 of study (405 d of age) (406·3 v. 401·3±4·05 kg, respectively). There were numerical differences in age at first breeding, fertility at first artificial insemination, age at pregnancy, and milk yield but some of these differences might have reached statistical significance with more replication.

Four enzymes of citrate metabolism (viz. citrate lyase, acetolactate synthase, diacetyl reductase and acetoin reductase) were constitutively present in cells of several strains of Streptococcus lactis subsp. diacetylactis. In strain DRC1, which was studied in detail, diacetyl reductase and acetoin reductase were partly repressed and acetolactate synthase partly induced by growth on citrate. The stage of growth also affected the formation of each enzyme. The buffer species affected the activity of acetolactate synthase, diacetyl reductase and acetoin reductase.

A simple, rapid and sensitive spectrophotometric assay was developed and evaluated for monitoring proteolysis during cheese ripening, based on the fact that α-amino groups released by hydrolysis of cheese proteins react with trinitrobenzenesulphonic acid to form products that absorb strongly at 420 nm. A linear relationship was shown to exist between A420 and concentration of free α amino groups up to 0·5 HIM (r = 0·999, 38 df, P < 0·001). Repeatability of the method was satisfactory. The coefficient of variance was 0·53% for amino acid solutions and 1·19% for cheese extracts. Average recovery of glycine added to the cheese was 104 ± 2·9%. A comparison of the above method with that of determination of water-soluble N to total N ratio showed that there was good agreement between these two methods of assessment of proteolysis in cheese (r = 0·857, 32 df, P < 0·001). Mainly Feta and Teleme cheese were examined, but a similar correlation was obtained with hard Greek cheeses. Analytical conditions of the procedure are discussed.

Data are presented which show that the use of glucono-δ-lactone as a chemical acidulant in cheese manufacture results in a much more rapid decrease in pH than that occurring in biologically acidified cheese. One of the consequences of the excessive early acid development was a very marked increase in the level of proteolysis during cheese-manufacturing operations. Consequently, chemically acidified cheese, as manufactured by the technique of Mabbitt, Chapman & Berridge (1955) (expt 17), does not appear to be a suitable ‘reference cheese’, especially for studies in which proteolysis during ripening is under investigation. The technique of Mabbitt et al. Avas modified to simulate the pH development pattern of starter cheese more closely; the level of proteolysis in curd made by the modified method was approximately equal to that in starter curd.

An attempt was made to link the propensity to develop bitterness of cheese made with fast acid-producing cultures with excessive early proteolysis in such cheese. The results show that fast- and slow-culture strains differed significantly in their acid-producing capability only after the cooking stage and consequently such cheeses differed little with respect to early proteolysis. Further, cheeses made with 0·5, 1·0, 2·0 or 4·0% of ML8 culture which differed considerably in rate of acid development and consequently in the level of early proteolysis did not develop bitterness, even when 4·0% starter was used.

It is concluded that gross proteolysis and bitterness are not correlated.

The combination of ultrasound and heat (thermoultrasound) is an emerging food preservation technique that retains higher quantities of bioactive components compared with current thermal pasteurization practice, but has not yet been assessed for pasteurizing human milk. Artificially contaminated human milk samples were treated with ultrasound (20 kHz, 150 watts) with and without heating. The retention of four human milk proteins was quantified by biochemical assay and laser scattering particle sizing was used to determine the extent of homogenization. While ultrasonic treatment was effective at inactivating Escherichia coli (D4 °C=5·94 min), Staphylococcus epidermidis exhibited resistance (D4 °C=16·01 min). Thermoultrasonic treatment was considerably more effective (Esch. coli D45 °C=1·74 min, D50 °C=0·89 min; Staph. epidermidis D45 °C=2·08 min, D50 °C=0·94 minutes) with a predicted retention (2·8 min treatment, 50°C) of secretory IgA lysozyme, lactoferrin and bile salt stimulated lipase of 91, 80, 77, and 45%, respectively. Homogenization of the milk samples occurred after 5 min and 2 min of ultrasonic and thermoultrasonic treatment, respectively. Thermoultrasonic treatment is an effective method for pasteurizing donor human milk and retaining a greater proportion of bioactive components compared with current practices. However, further studies are required to assess the practicality of applying this technique routinely to donor human milk.

The effects of stage of lactation and of age on milk and composition yield of cows kept under controlled conditions of feeding and management were investigated. Two groups each of 3 Friesian heifers were used: one group (A) was given a daily diet of 18 lb hay and concentrates at the rate of 3·8 lb/10 lb of milk and kept on experiment throughout the first 3 lactations; a second group (B) was given the same amount of hay and a constant daily amount of concentrates of 10·7 lb and was kept on experiment throughout the 1st lactation only.

The total yields of milk and peak yields for the 1st lactation were similar in both groups. In all the cows, fat content was high early and late in lactation and varied little throughout the mid-lactation period. With cows of group A, the content of solids-not-fat declined until about the 5th week of lactation and then remained steady until a marked rise occurred late in lactation. With cows of group B, however, the content of solids-not-fat was at a minimum between the 5th and 10th weeks of lactation and then increased progressively until late lactation when a more marked rise occurred. Lactose content increased rapidly in the first few days of lactation and then remained constant for several months, eventually declining late in lactation. Characteristic changes were also observed in the contents of total protein, and of the various protein fractions, and of the major minerals of milk.

The concentrations of all major constituents in milk secreted from the 2nd–5th months of lactation tended to decrease from lactation to lactation.

The results are discussed in relation to the development of knowledge of the changes in milk composition throughout lactation.

The specificity of chymosin on αs1-casein was shown to be dependent on the reaction pH and on the state of aggregation of the substrate. In aqueous solution αs1-casein was optimally hydrolysed to αs1-I at pH 5·8; if the casein was solubilized in the isoelectric region by the use of 5 M-urea, optimum proteolysis occurred at pH 2·8. Hydrolysis of αs1-I to yield αs1-II, αs1-III and αs1-IV occurred at pH values > 5·8 in the presence or absence of urea. In the isoelectric region αs1-II, αs1-III and αs1-IV were not formed in the absence of urea where the substrate was aggregated: instead a peptide αs1-V was produced; at the same pH and using urea as a solubilizing agent αs1-II, αs1-III and αs1-IV were formed together with a further peptide αs1-VI.

Hydrophobic and hydrophilic peptides in the water-soluble fraction of 20 batches of Hispanico cheese made from pasteurized and raw milk were determined by reversed-phase HPLC, with detection at 214 and 280 nm. Cheese flavour characteristics were evaluated by a sensory panel, and regressions of bitterness scores on levels of hydrophobic and hydrophilic peptides and their ratio were calculated. The best fitting relationship for pasteurized milk cheese was the linear regression of mean panel bitterness scores on hydrophobic peptides at 280 nm (r2=0·791). The determination coefficient for the regression of hydrophobic peptides at 280 nm on panellist bitterness scores (r2=0·356) was lower, owing to individual differences in the perceived intensity of bitterness. In the case of raw milk cheese, the respective determination coefficients were 0·203 for panel scores and 0·034 for individual panellist scores.

The interaction of κ-casein and β-lactoglobulin is fundamental to all heat-induced modifications of milk product functionality, such as the heat stability of concentrated milks. Purified native κ-casein B and β-lg A solutions were heated at 80 °C at pH 6·7 separately and in a mixture. The circular dichroism spectra in the near UV indicated irreversible changes in the disulphide bonding patterns involving both proteins. Alkaline- and SDS-PAGE of heated samples showed that, in the presence of κ-casein, less β-lg was converted into β-lg polymers and the rate of loss of native β-lg was greater. When κ-casein was added to previously heated β-lg and the mixture was heated, the κ-casein reacted with the heat-induced β-lg polymers more readily than with the β-lg native monomers. The formation of β-lg dimers, trimers etc. was diminished. It was concluded that, when β-lg and κ-casein were heated together, β-lg formed thiol-exposed monomers, which reacted with each other or with the native κ-casein depending on the relative concentrations of β-lg and κ-casein. The products of these reactions included some disulphide-bonded 1[ratio ]1 β-lg[ratio ]κ-casein complexes, some monomer κ-casein and a range of large aggregates held together by either or both disulphide bonds and hydrophobic association.

Considering that several health promoting properties are associated with kefir consumption and a reliable probiotic product requires a complete identification of the bacterial species, the present work evaluates several proved markers of probiotic potential of eleven isolates of homofermentative lactobacilli isolated from kefir grains and molecular identification and genotypic diversity. Using restriction analysis of amplified ribosomal DNA (ARDRA) and analysis of the 16S–23S rRNA internal spacer region we confirmed that all homofermentative lactobacilli belong to the species Lactobacillus plantarum. RAPD-PCR analysis allowed the discrimination of lactobacilli in five clusters. All isolates exhibited high resistance to bile salt. High survival after one hour of exposure to pH 2·5 was observed in Lb. plantarum CIDCA 8313, 83210, 8327 and 8338. All isolates were hydrophilic and non autoaggregative. Isolate CIDCA 8337 showed the highest percentage of adhesion among strains. All tested lactobacilli had strong inhibitory power against Salmonella typhimurium and Escherichia coli. Seven out of eleven isolates showed inhibition against Sal. enterica and five isolates were effective against Sal. gallinarum. Only CIDCA 8323 and CIDCA 8327 were able to inhibit Sal. sonnei. We did not find any correlation between the five clusters based on RAPD-PCR and the probiotic properties, suggesting that these isolates have unique characteristics.

The aim of the study was to establish in vitro a bovine mammary epithelial cell (MEC) clone, able to respond to mitogenic growth factors and to lactogenic hormones. Mammary tissue from a 200-d pregnant Holstein cow was used as a source of MEC, from which a clone was established through a process of limiting dilution. When plated on plastic, the cells assumed a monolayer, cobblestone, epithelial-like morphology, with close contact between cells. Inclusion of IGF-1 and EGF in the media significantly increased the number of cells 5 d after plating. All cells stained strongly for cytokeratin and moderately for vimentin at young and old passage stages, indicating the epithelial nature of this cell clone. When the cells were plated at a high density on a thin layer of a commercial extracellular matrix preparation (Matrigel), lobular, alveoli-like structures developed within approximately 5 d, with a clearly visible lumen. When cells were plated onto Matrigel in differentiation media (containing lactogenic hormones), detectable quantities of α-casein were present in the media and particularly on the lumen side of the structures. Omission of one of the lactogenic hormones (insulin, prolactin or hydrocortisone) reduced α-casein release to the limit of detection of the assay used. Lactoferrin was also produced when the cells were plated on Matrigel, again principally on the lumen side of the lobules, though this was independent of the lactogenic hormones. By passage 40, the cells had senesced, and it was not possible to induce α-casein or lactoferrin production. This study notes the establishment of a functional bovine mammary epithelial cell clone, which is responsive to mitogenic and lactogenic hormones and an extracellular matrix.

1. The variation in the stability of milk protein to ethanol and the relationship between milk composition and ethanol stability were examined.

2. Samples of herd bulk milk were very similar in stability to ethanol; the range of aqueous ethanol solutions required to coagulate the caseinate complex in an equal volume of milk was only 80–84% (v/v) ethanol. Samples from individual cows showed a wide variation in stability; coagulation was caused by ethanol solutions ranging in strength from 66 to 90% (v/v) ethanol.

3. Colostrum was very unstable to ethanol but stability rapidly increased during the post-colostrum period to higher levels in mid-lactation. Late lactation and subclinical mastitis milk showed no definite bias towards stability or instability.

4. The strength of ethanol required to coagulate the caseinate complex in an equal volume of milk was inversely related to the concentration of ionized calcium in the milk. The correlation coefficient was –0·76 (significant at 0·001 level) indicating that approximately 60% of the variation in stability was accounted for by the variation in the concentration of ionized calcium.

5. The relationships between the concentrations of other milk constituents and stability to ethanol could be attributed to the interrelations of the concentrations of these constituents and the concentration of ionized calcium.

Optimising the number of replacement heifers needed will have positive economic and environmental consequences on herds that rear their own young stock. The number of heifers needed to be kept is closely related with the number of culled dairy cows in the herd. This study therefore looked at the variation that exists in culling rate and herd level factors associated with it. A dataset from 1903 dairy herds available included information at animal level (dates of culling, slaughter/death) and herd level (characteristics of reproduction, performance, health) over the years 2007 to 2010. The average culling rate for slaughter/death was used and was defined for each year as percentage of the herd size that died within 30 d after they were culled. The analysis of the association between average culling rate for slaughter/death and the characteristics of the herd was performed using a mixed model. The results showed that the average culling rate for slaughter/death was 25·4% and varied between 23% (2007) and 28% (2010). More than 70% of the herds have an average culling rate for slaughter/death of less than 30%, showing that there is room for lowering the average culling rate for slaughter/death. A higher average culling rate for slaughter/death is associated with a longer average calving interval, a higher average 305-d protein production, a higher average somatic cell count (SCC), a higher percentage of new high SCC, a more than 5% decrease in herd size, and herds that bought more than 1% of animals per year. A lower average culling rate for slaughter/death is associated with a longer average age, herds that bought less than 1% of animals per year and a more than 5% increase in herd size. In conclusion, the average culling rate for slaughter/death is associated with fertility, udder health and openness of the herd.