Subclinical mastitis (SM) is one of the most important diseases affecting dairy ewes worldwide, with negative impact on the animal health, farm income and public health. Animals with SM often remain untreated because the disease may not be revealed. Increase in somatic cell count (SCC) and positive bacteriology for mastitis pathogens in milk samples are indicative of SM but the evidence of only one of these alterations must suggest an uncertain SM (UM). UM is defined when positive bacteriological examination (Latent-SM) or SCC>500 000 cells/ml (non-specific-SM) are detected in milk. Nevertheless, SCC and bacteriological examination are expensive, time consuming and are not yet in use at the farm level in dairy ewes. Recently, a sensitive acute phase protein, amyloid A, displaying multiple isoforms in plasma and different body fluids including mammary secretion (milk amyloid A-MAA), has been investigated as a marker of mastitis in cows and, in a few studies, in sheep. The aim of this trial was to compare the concentration of MAA of single udder-halves in ewes with healthy udder-halves (HU-control group) and naturally occurring subclinical mastitis, both confirmed (SM group) and uncertain (UM groups: Latent-SM and non-specific-SM), for monitoring udder health. The reliability of a specific ELISA kit for the measurement of MAA was also tested. During a 3-month trial period, 153 udder halves were assigned to the experimental groups based on their health status: 25 with SM, 40 with UM (11 with latent-SM and 29 with non-specific-SM) and 88 HU. SCC and bacteriological analysis were performed to establish the control and subclinical mastitis groups. MAA concentrations in milk samples were measured using a specific commercially milk ELISA kit. The data were submitted to statistical analysis. Significant (P<0·05) differences among the groups SM, non-specific-SM and HU were detected with the SM having the highest level and HU the lowest. MAA concentration is affected by the udder health status and is a useful indicator of subclinical mastitis and increased SCC in sheep.

We have investigated the capacities of Geotrichum candidum strains to produce sulphides from methionine. This attribute is very important in cheese technology because of the flavouring potential of sulphur compounds. A spectrophotometric procedure using 5,5′-dithiobis(2-nitrobenzoic acid) to determine sulphides was tested on a collection of G. candidum strains, and confirmed by gas chromatography–mass spectrometry. The strains were distinguished on the basis of their ability to produce methanethiol. Gas chromatography–mass spectrometry also made it possible to identify other sulphides, such as dimethyl disulphide, dimethyl trisulphide and dimethyl sulphide. Using sonicated cells, the specific production of these four sulphides was studied in presence of L-[S-methyl-2H]methionine. Both methanethiol and dimethyl sulphide were produced from methionine, but two different pathways were used by G. candidum.

The amido black and orange G methods for protein estimation in milk were investigated. The quantity of dye required for complete precipitation of a given quantity of milk protein was determined.

Dye samples of various degrees of purity all gave a linear relation between protein and dye precipitation, as determined by optical density, but with the less pure samples slightly less dye was bound per unit weight of protein.

Amido black, though reacting with protein in the same molar ratio as orange G, gave a much more sensitive optical indication of protein content.

In milks of individual cows there was a relatively constant relationship between dye values and Kjeldahl protein from 3 to 4 days after calving until 3 to 4 weeks before the end of lactation. Late lactation milks gave about 5% higher dye values than did normal milks of the same protein content.

The relation of dye precipitation to Kjeldahl nitrogen was the same for casein as for total nitrogen in normal milks.

Three strains of micrococci and two of pediococci were individually tested for effect on Cheddar cheese flavour by their addition to cheese milk. Only one of the micrococcal strains had a significant effect on flavour. In twenty-eight of the thirty-three trials with this strain the flavour of the experimental cheeses was appreciably better than that of the corresponding control cheeses; in the other five trials the experimental and control cheeses were similar in flavour.

This micrococcus did not grow appreciably during manufacture or ripening of the cheese, the peak number being present in the curd at the time of pressing. The count fell to less than 30% of the peak number after 1 week, and to less than 0·5% after 5 weeks.

The ways in which the micrococcus may have influenced the flavour of the cheese are discussed and attention is drawn to the possibility that bacterial action in the milk and the young cheese may play an important part in flavour development.

Casein and other milk proteins in maternal colostrum and milk, the earliest food of the newborn, should not only be considered as a nutritional supply but also as a source of biologically active peptides. Some of them isolated from casein and lactotransferrin were active on platelet function. They inhibited both aggregation of ADP-treated platelets and binding of [125I] fibrinogen to ADP-treated platelets. Their behaviour was compared to that of fibrinogen peptides possessing similar effects: once more similarities between the milk and blood-clotting phenomena could be observed.

The influence of mastitis and early lactation, and the effect of treating milk with heparin, blood serum and trypsin, on the proportion of lipoprotein lipase (LPL) activity in milk serum was investigated. The relative importance of milk serum LPL and LPL bound to micellar casein in promoting lipolysis was also examined. Colostrum contained LPL activity, 45% of which was found in the serum phase in samples obtained from the first milking post partum, but this value fell to 34% in samples taken 24 h later. The proportion of serum LPL was also increased in milks from quarters infected with Staphylococcus aureus, but not after overnight treatment of normal milk at 4 °C with 5% (w/v) blood serum or 2 µg/ml trypsin. The addition of 5 µg/ml heparin resulted in a consistent increase in serum LPL which varied between 14 and 50% of total milk LPL. Heparin did not release all the enzyme bound to casein micelles even after a second heparin treatment of resuspended micelles. Serum LPL was more effective in promoting lipolysis and was more responsive to blood serum activation than LPL bound to casein micelles. Lipolysis increased after heparin treatment but the increase was not related to serum LPL activity.

A total of 24 lactating Holstein cows averaging 620 (se=29) kg of body weight were allotted at week 17 of lactation to eight groups of three cows blocked for similar days in milk to determine the effects of feeding two sources of the plant lignan precursor secoisolariciresinol diglucoside, whole flaxseed and flaxseed meal, on concentrations of the mammalian lignans (enterodiol and enterolactone) in milk. Feed intake, digestion, milk production and milk composition were also determined to compare the use of whole flaxseed and flaxseed meal for milk production. Cows within each block were assigned to one of the three isonitrogenous and isoenergetic total mixed diets: no flaxseed product; 10% flaxseed meal; or 10% whole flaxseed in the dry matter. The experiment was carried out from week 17 to week 21 of lactation and diets were fed at ad-libitum intake. The mammalian lignan, enterodiol, was not detected in the milk of cows. Cows fed whole flaxseed and flaxseed meal had greater concentrations of enterolactone in milk than those fed the control diet. Feed intake, milk production and milk composition were also similar for all diets, indicating that both flaxseed meal and whole flaxseed are suitable feed ingredients for milk production of cows in mid lactation. The results provide new information on the conversion of plant secoisolariciresinol diglucoside from two flaxseed products into mammalian lignans in dairy cows.

A simple and rapid method for determination of retinol in whole milk is described. Milk (400 mg) was diluted with a water–methanol–ethanol mixture (55:9:36 by vol.) and saponified with potassium hydroxide in a culture tube. Retinol was extracted into 10ml heptane-di-isopropyl ether (3:1) of which 100 μl was injected into the HPLC. Fluorescence detection ensured the specificity and sensitivity. The critical points in the analysis were the extraction of retinol from fatty samples and the instability of retinol towards photo-oxidation and air oxidation in alkaline solvents. There was an average recovery of 98·5 ± 2·4% and a CV of 3·6%.

A total of 555 milk samples was collected from 53 Holstein cows for 1 year. Samples were analysed for N-acetyl neuraminic acid (NANA) content, κ-casein content and somatic cell count. The extent of glycosylation of κ-casein was estimated through the NANA content of κ-casein in each milk sample. The overall mean value for the NANA content was 63·6 ± 20 μg/mg of κ-casein with a range of 23–166 μg/mg. Least squares analysis showed significant (P < 0·01) effects of parity, test day, stage of lactation, phenotype for κ-casein, somatic cell count, and cow within phenotype. The NANA content of κ-casein decreased with increasing parity number and increased significantly when somatic cell count was above 200000 cells/ml. After reaching a minimum value between 2 and 3 months after calving, the NANA content of κ-casein increased during the later part of the lactation. NANA content was higher in the milk of cows phenotyped AB for κ-casein than for cows phenotyped for κ-casein AA, thus suggesting that B variant is more efficiently glycosylated than A variant.

Psychrotrophs, particularly strains of Pseudomonas fluorescens, dominate the microflora of refrigerated raw milk and secrete heat-stable extracellular enzymes (proteases and lipases) which survive pasteurisation and even UHT heat treatments and degrade the casein and fat components of raw milk causing a reduction in cheese yield, gelation of UHT milk and off flavours in many dairy products. These enzymes are usually produced in the late log/early stationary growth phases when the cell density is high. This fact indicated that induction of these enzymes may be a candidate for quorum sensing, a phenomenon by which bacteria can sense and respond to cell population size by means of chemical signals based on the homoserine lactone molecule. It is assumed that the production of these enzymes contributes to the selective advantage of psychrotrophs and hence have a significant effect on their growth kinetics. In the work reported here nine proprietary homoserine lactone compounds were screened, using water as a control, for their effect on the lag phase duration (LPD) and exponential growth rate (EGR) of three strains of Pseudomonas fluorescens isolated from refrigerated raw milk after 1, 3 and 5 days storage. Two compounds viz. N-benzoyloxycarbonyl-L-homoserine lactone and N-3-oxyhexanoyl-DL-homoserine lactone were found to significantly (P<0·001) reduce the LPD and increase the EGR of the three strains. Further work with these compounds is warranted, monitoring growth in parallel with enzyme production, to determine the extent to which extracellular enzyme production is mediated by the quorum sensing phenomenon in this species.

The effects of dietary inclusion of freeze-dried goat and cow milk on the utilization of zinc and selenium, and on the metabolic fate of zinc, were studied in transected (control) and resected rats (resection of 50% of the distal small intestine). Intestinal resection reduced the apparent digestibility coefficient and zinc retention in the cow milk diet, whereas these biological indices were similar in transected (control) and resected rats with standard (without milk) and goat milk diets. The apparent digestibility coefficient and retention of selenium were not affected by intestinal resection in the animals fed with the three diets studied. In transected (control) and resected rats, the apparent digestibility coefficient and retention of zinc and selenium were higher for the goat milk diet than for the other two diets. Zinc deposits in the organs, expressed as μg/g dry weight were in order of decreasing concentrations: femur, testes, sternum, liver, kidney, heart, spleen, longissimus dorsi muscle and brain. Deposits were greatest with the goat milk diet, followed by the standard diet and were lowest for the rats given the cow milk diet, both for transected (control) and resected animals.

We conclude that consumption of the goat milk diet produces a greater bioavailability of zinc and selenium and a greater deposit of zinc in key organs, for both the transected (control) and the resected rats, with respect to the standard diet and the cow milk diet.

A study of the hydrolysis of β-casein-B by crystalline rennin or rennet extract at pH 6·5, using a disk electrophoresis technique, showed that 3 bonds in β-casein are appreciably more sensitive than the others to rennin proteolysis, and that these bonds are probably located near the C-terminus of the protein. The most susceptible bond is hydrolysed, at 10°C, about 200 times faster than any other bond, whilst at 37°C it is hydrolysed 60 times faster. A study of the hydrolysis of this bond showed that its rate of hydrolysis at 37°C and pH 6·5 is decreased by either increased ionic strength or increased calcium ion concentration at constant ionic strength. Conformational changes in the substrate are probably responsible for these effects.

That bacteriophage is the cause of much loss to the cheese-making industry of New Zealand has been shown by Whitehead(1). In New Zealand in recent years, cheese has been made almost entirely from flash-pasteurized milk with the aid of starters prepared from single-strain cultures of lactic acid streptococci (Str. cremoris). The use of such starters has been accompanied by the occasional failure of the bulk starter to clot, or, if clotted, by failure to produce the required amount of acid in the cheese vat—the latter condition being described by Whitehead as ‘pack up’. Whitehead has traced the cause of sudden failure to air-borne infection of the bulk starter by specific bacteriophage (2), the most probable source of infection being the spray from whey separators. When adequate protection from phage contamination was provided slowness in cheese making disappeared.

Subclinical mastitis (SM) is a major concern in the dairy industry because it causes economic losses and can lead to clinical mastitis. The mechanisms of the onset and progression of SM are not fully understood, and a new procedure for the detection and appropriate prediction of SM leading to clinical mastitis is necessary for dairy cow management. Inflammatory cytokines such as interleukin (IL)-6 are known to be present in inflamed mammary glands at the onset of mastitis, and IL-6 concentrations correlate with the levels of inflammation. In this study, the detection of IL-6 was examined for the evaluation for the future prediction of SM in 77 quarter milk samples from 20 cows. IL-6 concentrations in quarter milk were measured by sandwich ELISA, and the data were compared with milk somatic cell count (SCC) levels to diagnose SM. Average IL-6 concentration was significantly higher in SM quarter milk (207·0±441·6 pg/ml) than in healthy control quarter milk (12·6±33·4 pg/ml, P<0·01). Results of the cross-tabulation table show that SM prediction accuracy based on IL-6 concentration was almost equal or superior to SM prediction accuracy based on SCC. The detection of IL-6 in milk indicated SM earlier than did the detection of elevated SCC. Thus, the detection of IL-6 in milk could be a future prediction marker for SM.

This study investigated the protective capacity of the oral administration of fermented milk containing the probiotic strains; Lactobacillus casei, Lb. delbrueckii subsp. bulgaricus and Streptococcus thermophilus, against enteroinvasive Escherichia coli infection in a murine (BALB/c mice) model. Mice were fed for 2, 5 or 7 consecutive days with fermented milk diluted to a concentration of viable Lb. casei, Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus of 107 cfu/ml. Phagocytic activity of peritoneal macrophages and the number of IgA+ cells in small and large intestine were determined at the end of the feeding periods. For the preventive effect against Esch. coli, animals were fed for 5 days (selected dose). Mice were challenged with an infective dose of enteroinvasive Esch. coli of 108 cfu/mouse. The colonization of liver and spleen and the secretory IgA specific for the pathogen in the intestinal fluid were determined (ELISA test). Results showed that the unspecific immune response enhanced itself after 5 consecutive days of the administration of this fermented milk (increase in the percentage of phagocytosis and number of IgA+ cells in the small intestine). Treated animals showed less Esch. coli colonization of liver than control mice and a higher secretory anti-Esch. coli IgA in the intestinal fluids. These results suggest that the protection against enteroinvasive Esch. coli infection observed for the fermented milk containing probiotic bacteria may be associated with an enhance of the intestinal mucosa immunity.

A database consisting of 35291 milking records from 83 cows was built over a period of 10 months with the objectives of studying the effect of teat cup attachment failures and milking interval regularity on milk production with an automated milking system (AMS). The database collected records of lactation number, days in milk (DIM), milk production, interval between milkings (for both the entire udder and individual quarters in case of a teat cup attachment failure) and average and peak milk flows for each milking. The weekly coefficient of variation (CV) of milking intervals was used as a measure of milking regularity. DIM, milking intervals, and CV of milking intervals were divided into four categories coinciding with the four quartiles of their respective distributions. The data were analysed by analysis of variance with cow as a random effect and lactation number, DIM, the occurrence of a milking failure, and the intervals between milkings or the weekly CV of milking intervals as fixed effects. The incidence of attachment failures was 7·6% of total milkings. Milk production by quarters affected by a milking failure following the failure was numerically greater owing to the longer interval between milkings. When accounting for the effect of milking intervals, milk production by affected quarters following a milking failure was 26% lower than with regular milkings. However, the decrease in milk production by quarters affected by milking failures was more severe as DIM increased. Average and peak milk flows by quarters affected by a milking failure were lower than when milkings occurred normally. However, milk production recovered its former level within seven milkings following a milking failure. Uneven frequency (weekly CV of milking intervals >27%) decreased daily milk yield, and affected multiparous more negatively than primiparous cows.

Lactating heifers were treated for 4 weeks with recombinant bovine growth hormone (bGH, n = 9) or were untreated (n = 9). In addition, two mammary glands of each heifer were milked four times daily rather than the normal twice daily for the same 4 weeks, and for the following 2 weeks. Over the 4 weeks, milk yield was increased 12·8% by bGH, 14·0% by frequent milking and 28·5% by the combined treatment. The effect of bGH as administered here was slower in onset than that of frequency, but eventually produced a higher peak yield. ANOVA revealed significant effects of each stimulus independently and an additive, but not synergic effect of the combined treatment. The effect of the combined treatment tended to persist beyond the end of treatment; most of this response was related to the milking frequency component rather than the bGH. Mammary differentiation was assessed in biopsies of mammary tissue obtained prior to and at the end of treatment. Mammary enzyme activities (expressed on a per cell basis) indicated minimal differentiative response to either treatment, but synthesis rates for lactose and casein determined in vitro were increased by bGH treatment. Histological examination revealed a stimulatory effect of milking frequency on epithelial cell size. The results indicate that these two galactopoietic stimuli operate through independent mechanisms, and neither stimulus alone is sufficient to maximize milk yield in dairy heifers.

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29–fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35·6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 °C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7·0. The ratio of milk clotting to proteolytic activity was 3·03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

Comparable cheeses were made using single strains of Streptococcus cremoris and Str. lactis as starters. The three Str. lactis strains imparted a characteristic rather abnormal flavour to the cheese, whereas the three strains of Str. cremoris all gave cheeses of normal flavour. The abnormal flavour caused by the lactis cultures increased in intensity as ripening proceeded, and differed slightly from strain to strain.

Bacteriophage usually developed slightly in the whey and in the curd during the manufacturing process, but never to such a degree as to delay acid production unduly. The extent of phage development did not have a significant influence on cheese flavour.

All the lactis strains survived in much larger numbers in the cheese than the cremoris strains. The simplest explanation of the data is that the ‘lactis’ flavour was a direct effect, due to flavour substances produced by the Str. lactis cells present in the young cheese.