The primary objective of this study is to validate a new fast method for determination of uric acid in milk. The method is based on an enzymatic-fluorometric technique that requires minimal pre-treatment of milk samples. The present determination of uric acid is based on the enzymatic oxidation of uric acid to 5-hydroxyisourate via uricase where the liberated hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine via peroxidase and the fluorescent product, resorufin, is measured fluorometrically. Fresh composite milk samples (n=1,072) were collected from both Jersey (n=38) and Danish Holstein (n=106) cows from one local herd. The average inter- and intra-assay variations were 7·1% and 3·0%, respectively. Percent recovery averaged 103·4, 107·0 and 107·5% for samples spiked with 20, 40 or 60 μm of standard, respectively, with a correlation (r=0·98; P<0·001) observed between the observed and expected uric acid concentrations. A positive correlation (r=0·96; P<0·001) was observed between uric acid concentrations using the present method and a reference assay. Storage at 4°C for 24 h resulted in lower (P<0·01) uric acid concentrations in milk when compared with no storage or samples stored at −18°C for 24 h. Addition of either allopurinol (a xanthine oxidase inhibitor) or dimethylsulfoxide (a solvent for allopurinol) did not affect milk uric acid concentrations (P=0·96) and may indicate that heat treatment before storage and analysis was sufficient to degrade xanthine oxidase activity in milk. No relationship was observed between milk uric acid and milk yield and milk components. Authors recommend a single heat treatment (82°C for 10 min) followed by either an immediate analysis of fresh milk samples or storage at −18°C until further analysis.

Volatile components of four batches of Manchego cheese were studied throughout their ripening process. Samples were submitted to a micro simultaneous distillation-extraction procedure, and then analysed by capillary gas chromatography (GC) and combined GC and mass spectrometry. Among other components, four homologous series of compounds were found: free fatty acids, methyl ketones, ethyl and methyl esters of even fatty acids. Ripening stage can be approximately estimated from the concentrations of some components.

The acetolactate synthase of Leuconostoc lactis NCW1 was studied. The Mn2+ content of cell free extracts was 3·2 µ/mg protein. The enzyme did not require Mn2+ for activity, had an optimum pH between 5 and 6 and was labile. Incubation at 21 °C or addition of thiamine pyrophosphate followed by storage at 4 °C stabilized the enzyme. It was allosteric with at least two binding sites for pyruvate and was inhibited by several products of glucose metabolism (6-phosphogluconate, 3-phosphoglycerate, 2-phosphoglycerate, PEP and ATP) at pH 5·4. Except for ATP, which became more inhibitory, the inhibition disappeared completely (6-phosphogluconate, 2-phosphoglycerate, and PEP) or partly (3-phosphoglycerate) at pH 4·7. The role of these compounds in the regulation of acetoin production from citrate by leuconostocs is discussed.

The ability of 42 microorganisms isolated from cheese, including strains of yeasts, Geotrichum candidum, Arthrobacter, Moraxella spp., Brevibacterium linens and Staphylococcus saprophyticus spp. to produce phenethyl alcohol (PEA) from L-phenylalanine was studied. All the yeast strains produced labelled PEA from L-[U-14C]phenylalanine. Phenylpyruvic acid was detected as an intermediate of PEA production and CO2 was produced by decarboxylation of this acid. For five strains tested, the level of PEA which had accumulated in the culture at the end of exponential growth phase represented 39–52% of the L-Phe added. None of G. candidum strains nor bacterial isolates produced PEA from L-Phe. Strains of Moraxella spp. and four strains of the S. saprophyticus group produced phenylacetaldehyde. For three strains of Arthrobacter spp., five of B. linens and all the Moraxella, 14CO2 produced from uniformly labelled L-Phe represented more than one carbon atom of the L-Phe molecule, suggesting that Phe was catabolized beyond the stage of phenylacetic acid. Production and disappearance of PEA during Camembert cheese ripening is probably related to the metabolic activity of microorganisms present at the cheese surface.

Dairy herds are expanding and, with increasing numbers of animals in each herd, there is a need for automatic recording of indicators in milk in order to detect mastitis, inflammation of the udder. A number of biomarkers for mastitis have been suggested over the years. Mastitis usually occurs in one of the four udder quarters and since it is now possible to milk each udder quarter separately in automated milking systems, it is important to evaluate the normal variation in the biomarkers at udder quarter level. This study evaluated the normal variations between milkings for some biomarkers in clinically healthy cows, determined by repeated somatic cell count and bacteriological analysis. The biomarkers studied were serum amyloid A (SAA), haptoglobin (Hp), lactate dehydrogenase (LDH), N-acetyl-β-d-glucosaminidase (NAGase) and alkaline phosphatase (AP), parameters that have been suggested as markers for mastitis. Ten cows were monitored on 42 consecutive milking occasions through collection of udder quarter milk samples and representative cow composite milk samples, giving a total of 2100 individual milk samples. Each cow had its individual profile for the concentrations and variations in the parameters analysed. Although there was relatively large variation between cows for the biomarkers analysed, the variation between milkings in clinically healthy quarters within cows was often below 10%. The biomarker with the lowest variation in this study was LDH. The results suggest that comparing quarters within an individual cow can identify deviations from the natural variations between milkings. This could be a valuable tool instead of, or in combination with, a cut-off value for each parameter in order to detect changes in the milk indicating mastitis.

The ability of total milk proteose peptone and individual proteose peptone components to inhibit lipolysis in milk was examined. Proteose peptone (1·5 mg/ml) when added to milk inhibited lipolysis, and component 3 was a more effective inhibitor than component 5 or component 8-fast. Inhibition by proteose peptone also occurred when the milk was treated with between 2 and 20% v/v bovine blood serum or with 5 μg/ml heparin. In vitro activity of milk lipoprotein lipase and its stability were not affected by proteose peptone. It was concluded that proteose peptone does not interact with lipase or its activator and that the mechanism of inhibition involves the substrate.

The amounts of total lipid and fatty acids consumed, leaving the stomach and excreted in the faeces were examined in 4 sheep fitted with rumen and re-entrant duodenal cannulas. Diets of high (HM1) and low (CM1) roughage content were given at 0·9 times maintenance and the low-roughage diet was also given at 1·7 and 2·3 times maintenance. With all the rations more fatty acid left the abomasum than was consumed in the food, the difference being greater on ration CM1 than on ration HM1 and increasing irregularly with the amount of the low-roughage ration fed. Of the fatty acid entering the duodenum in the chyme, 72–89% was digested in the intestine. About 80% of the increase in fatty acids in the stomach was stearic acid and most of the remainder was palmitic acid. Almost all the polyunsaturated C18 acids ingested in the food were hydrogenated in the stomach, and the amounts of oleic acid were also greatly reduced, although more oleic acid entered the duodenum in the chyme with ration CM1 than with ration HM1. The possible origins of the increase in fatty acids in the stomach are discussed.

Use of a model of the renneting reaction has led to the definition of the amounts of soluble casein present in milks of different concentrations, at the time of clotting. It is shown that, although the clotting time (RCT) is relatively little affected by the concentration of milk by ultrafiltration, the proportion of casein which is soluble at the RCT is highly dependent upon the concentrations of both milk and rennet. This may affect the structure of the curd.

The effects of the isocaloric replacement of starch in a low-fat concentrate mixture by either 5 or 10% ‘stearic acid’ (85% pure) or 10% ‘palmitic acid’ (85% pure) on the yield and composition of milk fat were investigated in a feeding experiment with 12 cows in mid-lactation. The concentrate mixtures were given with a highroughage diet that supplied daily 4·4kg of hay and 2·7kg of sugar-beet pulp.

The inclusion of 5 or 10% ‘stearic acid’ in the concentrate mixture decreased the concentrations and yields of 10:0, 12:0, 14:0, 14:1, 16:0 and 16:1 in the milk fat but increased the concentrations and yields of 18:0 and 18:1. When the concentrate mixture contained 10% ‘stearic acid’ the decreases in the yields of the shorter chain acids and the increases in the yields of 18:0 and 18:1 in the milk fat were more pronounced than when the concentrate mixture contained 5% ‘stearic acid’. The inclusion of 10% ‘stearic acid’ in the concentrate mixture also decreased the concentrations of 6:0 and 18:2 in the milk fat. The inclusion of 10% ‘palmitic acid’ in the concentrate mixture decreased the concentrations of 6:0, 8:0, 10:0, 12:0, 14:0, 14:1, 18:0 and 18:2 in the milk fat but increased the concentrations of 16:0 and 16:1. This dietary treatment also reduced the yields of 6:0, 10:0, 12:0, 14:0 and 18:2 but increased the yields of 4:0, 16:0 and 16:1 in the milk fat. The inclusion of 5% ‘stearic acid’ or 10% ‘palmitic acid’ in the concentrate mixture increased the yield of total milk fat.

The inclusion of 10% ‘palmitic acid’ or 10% ‘stearic acid’ in the concentrate mixture increased the relative proportions of propionic and iso-butyric acids in the rumen liquor but decreased the proportions of η-butyric and valeric acids. The inclusion of ‘stearic acid’ at the level of 5% in the concentrate mixture increased the relative proportions of iso-butyric and iso-valeric acids but decreased the proportion of butyric acid in the rumen liquor.

Milk from cows fed a low-fat diet high in cereals designed to stimulate fat synthesis de novo was lower in unsaturated fatty acids (21·3%) than milk from cows fed a diet high in fat, mainly from roasted soy beans (41·3% unsaturated fatty acids). Buttermilk from the more unsaturated milk was less oxidatively stable during storage (at 4 °C, followed for 11 d) than buttermilk from the more saturated milk, as monitored both by primary lipid oxidation products (lipid hydroperoxides) and by the secondary lipid oxidation product, hexanal. Fat-soluble antioxidants, β-carotene and α-tocopherol, analysed by HPLC, were not consumed during storage for either of the two types of buttermilk. In contrast, the antioxidative capacity of the serum phase decreased during storage as evaluated in a radical scavenging assay based on the semi-stable water-soluble radical nitrosodisulphonate (Fremy's salt). The time course for the decrease in water-soluble antioxidants was very similar for the two types of buttermilk suggesting that oxidation is initiated in the serum phase independently of fatty acid composition.

Effects of six different milking intervals on the distribution of milk between cistern and alveoli were studied in a randomized, incomplete Latin Square experiment with four lactating Holstein cows. Cisternal and alveolar milk was measured by udder quarter at 4, 8, 12, 16, 20 and 24-h intervals with a 3-d interperiod of regular milking. Cisternal milk was evacuated using a cannula after injection of an oxytocin-receptor blocking agent, followed by an injection of oxytocin to remove the alveolar fraction. Milk samples from each fraction and quarter were collected for analysis. Cisternal and alveolar milk increased with milking interval and represented on average 30 and 70% of the milk stored in the udder, respectively. Fat content in alveolar milk remained constant during the first 16 h, increasing rapidly thereafter, reaching its maximum at 24 h (6·95%). Fat content in cisternal milk decreased with milking interval and reached its minimum at 24 h (0·96%). Total fat yield tended to increase for cisternal milk with longer milking intervals, but it increased markedly for alveolar milk, showing that fat globules did not pass freely from alveoli to cistern between milkings. Milk protein content was greater in rear quarters than in front quarters for both milk fractions. Milk protein content increased in the cisternal milk fraction and tended to increase in the alveolar milk fraction with longer milking intervals, but values did not differ between cisternal and alveolar fractions or between front and rear quarters. Total protein yield increased with milking interval in both fractions, indicating that casein micelles passed more freely than fat globules from the alveolar to the cisternal compartment. In conclusion, the short-term effects of milking intervals in milk composition were explained by the changes observed in alveolar and cisternal milk ratio.

Twenty-three strains of lactic acid streptococci were shown to be types of Streptococcus cremoris or Str. lactis by cultural, biochemical and serological means. The antibiotics produced by the cremoris strains could be differentiated readily from the lactis antibiotics, of which at least five different varieties are described. A cross-test and a resistant-strain test were used to differentiate the antibiotics.

Continuous measurements of curd-firming using a commercial vibratingreed viscometer and a pressure transmission system (PTS) developed at this Institute were compared both in the laboratory and during Cheddar cheesemaking. The coefficient of variation about the mean of the curd-firmness measured in reconstituted skim-milk 40 min after renneting was 3·5% with the viscometer and 2·6% with the PTS. The effects of changes in conditions on the curd-firming rate of raw whole milk were determined. As the rennet concentration was raised the rates increased linearly as measured by the viscometer, but non-linearly as measured by the PTS. Addition of CaCl2 increased the rates measured by both instruments rapidly at first then more slowly at higher levels. Raising the temperature increased the rate exponentially in the case of the viscometer but more slowly with the PTS. With both instruments the curd-firming rates of milk at pH 5·8 were 6·5 times those at pH 6·7 but the rates fell slightly at pH 5·6. In experimental cheesemaking, the mean readings of both instruments at the subjectively determined cutting time of the curd had the same coefficient of variation.

The commercial viscometer is probably more suitable for laboratory investigations of the earlier stages of the coagulation of milk by rennet whilst the PTS may prove the more useful in indicating the optimum cutting time of the curd during cheesemaking.

The effect of brining time and smoking on the free fatty acid content of Idiazabal cheese during ripening was examined. The main free fatty acids considered underwent at least some increase during the first stage of ripening before day 90 and tended to level off around a constant value towards the end of the ripening period. There were significant differences in free fatty acid levels during ripening among cheeses with different brining times and between smoked and unsmoked cheeses. Brining time and smoking exerted marked effects on lipolytic activity during cheese ripening, depending upon the free fatty acid involved and ripening time. In general, brining and smoking led to increases in free fatty acid levels at the end of the ripening period; the different behaviour of butyric acid may be due to a specific lipolytic activity.

Of 161 cultures of aerobic sporeformers isolated from raw and pasteurized milk 96 were found to be B. cereus, 27 B. mycoides, 17 B. subtilis, 11 B. sphaericus, 3 B. megatherium, 2 B. pumilus, 1 B. firmus and 4 were unidentified strains.

Typical broken cream followed by rapid sweet curdling of sterile milk was produced by 36 of 39 strains of B. cereus and 5 strains of B. mycoides examined. Three strains of B. cereus failed to produce broken cream. These 3 strains were atypical in that they failed to produce lecithinase, unlike the remaining 93 strains isolated during this study. Other species of aerobic sporing bacilli tested, including 17 strains of B. subtilis, 10 of B. sphaericus, 2 of B. megatherium, 2 of B. pumilus and 1 of B. firmus, all failed to produce broken cream and in fact produced no visual change in milk within 48 hr. at 22° C.

Under the conditions of our experiments, 48 out of 60 samples of fresh farm milk and 12 out of 14 samples of freshly bottled pasteurized milk developed broken cream within 18 hr. at 22° C. followed by sweet curdling within a further 24 hr. Strains of B. cereus or B. mycoides were isolated from all samples showing broken cream, but only 3 atypical strains of B. cereus were isolated from the 14 samples which did not develop the fault.

It seems that the production of broken cream is in some way associated with the production of lecithinase by B. cereus and B. mycoides. This view is supported by the fact that three non-lecithinase producing strains of B. cereus were also unable to produce broken cream. However, attempts to reproduce the fault by adding a concentrated culture filtrate to sterile milk or cream were unsuccessful.

Because of the widespread distribution of spores of B. cereus and B. mycoides in both raw and pasteurized milk, the absolute prevention of broken cream does not seem possible, but preliminary observations suggest that maintenance of milk at 5° C. or lower from the time of pasteurization until delivery to the consumer may sufficiently delay the onset of the fault to obviate the difficulty in practice except in very hot weather in households where insufficient care is taken to keep milk cool.

The experiment used 45 Comisana ewes, divided into 3 groups of 15. The aim was to determine the effects of two different stocking densities and two different housing conditions on welfare, and on production performance of dairy ewes. The stocking densities tested were: high stocking density (1·5 m2/ewe, HD group) and low stocking density (3 m2/ewe, LD group); the two housing conditions tested were: ewes housed indoors (LD group, 3 m2/ewe) and ewes allowed to use an outdoor area (LDP group, 3 m2/ewe divided into 1·5 m2/ewe indoors and 1·5 m2/ewe outdoors). At the beginning of the experiment, and then every 2 months, the cell-mediated immune status of sheep was evaluated. One month after the beginning of the experiment, and 20 d later, the ewes were injected with chicken egg albumin (OVA) to assess their humoural immune responses. Starting from the beginning of the experiment and then monthly, behavioural activities of ewes were monitored using 15-min scans. After lamb weaning, milk yield from individual ewes was measured and milk composition analysed weekly. Housing conditions (low density reared ewes indoors v. low density reared ewes with free access to an outdoor area) affected cell-mediated response, which was higher in LDP than in LD ewes. Concentrations of anti-OVA IgG were mainly influenced by space allowance, with higher antibody titres in LD than in HD ewes throughout the experiment. Both housing conditions and space allowance affected sheep behavioural activities: a greater proportion of LDP ewes displayed standing and drinking behaviours than LD ewes, and a greater proportion of LD ewes was observed walking than HD ewes. Ewes allowed access to the outdoor area had a higher protein content and lower somatic cell count in their milk, whereas reduced space allowance led to a reduction in milk yield and an increase in somatic cell count of milk. Results indicate that both increased space allowance and availability of outdoor area can improve the welfare and production performance of the lactating ewe.

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRα, PPARγ, SREBP1 and the milk proteins lactoferrin and α-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and α-lactalbumin. ABCG1, ABCG5, LXRα, PPARγ and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.

One of the most important stages governing the accuracy of lactose determinations in milk is the preparation of the protein-free serum. Factors influencing the lactose content of the serum are: (1) the complete precipitation of protein, (2) the formation of lactose-protein complexes, and (3) the degree of hydration of the protein coagulum.

Experiments are described which show how these factors are influenced by different clearing agents operating at different pH values. On the basis of the results obtained a reagent containing zinc acetate, phosphotungstic acid and acetic acid is suggested; it reduces to a minimum any errors introduced by the above factors.

The effect of teat washing and drying on bacterial numbers in bulk milk was compared with that of no teat preparation in eight commercial herds over one year. Using in-line milk samplers, milk was collected at various points during its passage through the milking plant and the samples were used to establish the relative significance of the sources of contamination of raw milk. Teat washing and drying of cows housed during winter reduced the total counts by 40% and streptococcal and coliform counts by 50%. Bacterial counts were significantly lower in cows at pasture during the summer and there was no reduction in count due to teat washing and drying. Bacteriological counts increased at each stage as the milk passed through the milking machine. The milking equipment significantly increased the total colony count by between 2000 and 3000/ml, and the bulk tank added a further 1500 to 2000/ml. The mean rinse bacterial counts of the milking equipment were higher in summer than winter, averaging 4·4 x 107 bacteria/m2 compared with 3·5 x 107/m2 respectively. Although this level of bacterial contamination of the equipment is high by current standards, very low bulk milk bacterial counts were nevertheless achieved, particularly in the summer. This confirms that organisms from this source are not a major contaminant of the bulk milk. There was a very poor correlation between rinse counts and the bulk milk bacterial count, but a strong correlation (0·98) between total and streptococcal counts of the bulk milk. The unreliability of the use of rinse techniques to assess the contribution of milking equipment to bacterial counts of raw milk is emphasized.

Total and differential cell counts were obtained for 1710 samples of milk taken from cows in which the stage of lactation was known. A bacteriological examination of the samples was also made. The total and differential cell counts considered together showed no marked advantage over the total cell count alone in the diagnosis of mastitis, except in milk of late lactation.