A total of 169 Lactobacillus strains from 12 species (Lb. acidophilus, Lb. brevis, Lb. buchneri, Lb. casei, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. delbrueckii, Lb. delbrueckii subsp. lactis, Lb. fermentum, Lb. helveticus, Lb. paracasei subsp. paracasei, Lb. plantarum and Lb. rhamnosus), isolated from raw milk and various milk products, and 9 Lactococcus lactis strains were evaluated for peptidase activities with five chromogenic substrates and a tryptic digest of casein. Within each species, the peptidase activity of the cell-free extracts of the strains varied. Furthermore, differences were observed between the Lactobacilhis species and Lc. lactis. Lb. helveticus had by far the highest hydrolysing activities towards all substrates, indicating the presence of powerful aminopeptidases, X-prolyl-dipeptidyl aminopeptidases and proline iminopeptidases. Lb. delbrueckii subsp. bulgaricus possessed high hydrolysing activities towards substrates containing proline, alanylprolyl–p–nitroanilide and prolyl–p–nitroanilide. On the other hand, Lb. fermentum and Lb. brevis could be considered as weakly proteolytic species. A more detailed study with highly proteolytic Lactobacillus strains indicated that at least three different proteinases or endopeptidases were present. Compared with Lc. lactis, the Lactobacillus strains had a much lower hydrolytic action on glutamyl-glutamic acid, suggesting that glutamyl aminopeptidase was absent in lactobacilli.

Cell viability, autolysis and lipolysis were studied in Cheddar cheese made using Lactococcus lactis subsp. cremoris AM2 or Lactococcus lactis subsp. cremoris HP. Cheddar cheese was made in triplicate over a 3 month period and ripened for 238 days at 8 °C. Cell viability in cheese was lower for AM2 (a non-bitter strain) than for strain HP (a bitter strain). Autolysis, monitored by the level of the intracellular marker enzyme, lactate dehydrogenase (EC 1.1.1.27) in cheese ‘juice’ extracted by hydraulic pressure, was much greater in the cheese made using AM2 than that made with HP. Lipolysis was determined by the increase during ripening of individual free fatty acids (FFA) from butyric (C4[ratio ]0) to linolenic acid (C18[ratio ]3) measured using a high performance liquid chromatographic technique. Levels of individual FFA from butyric (C4[ratio ]0) to linolenic (C18[ratio ]3) acids increased significantly (P<0·05) during ripening in cheeses made with either starter culture. Palmitic (C16[ratio ]0) and oleic (C18[ratio ]1) acids were the most abundant FFA throughout ripening in all cheeses. Levels of caprylic (C8[ratio ]0), myristic (C14[ratio ]0), palmitic (C16[ratio ]0) and stearic (C18[ratio ]0) acids were significantly higher (P<0·05) in cheeses manufactured with Lc. lactis subsp. cremoris AM2 than in cheeses manufactured with Lc. lactis subsp. cremoris HP. Differences in levels of lipolysis between strains was not due to differences in the specific lipolytic or esterolytic activities in cell free extracts of the strains as measured by activity on triolein (lipase) and p-nitrophenylbutyrate (esterase) substrates. Therefore, evidence is provided for a relationship between the extent of starter cell autolysis and the level of lipolysis during Cheddar cheese ripening.

The proportions of milk stored within the gland cistern (cisternal milk) and within secretory alveolar tissue (alveolar milk) were determined at various intervals after milking in peak lactation cows and late lactation cows. The rate of milk secretion remained constant up to 12 h in both groups. Cisternal milk remained low (600 g or less) until after 4 h in both groups, then increased gradually to values at 12 h of 5·08 (peak lactation) and 2·60 kg (late lactation). Alveolar milk exceeded cisternal milk in both groups and at all time points up to 12 h, and was greatest in the peak lactation cows. A further measurement was made at 20 h in the late lactation group. Milk secretion rate remained constant between 12 and 20 h and most of this additional milk accumulated in the cistern. Alveolar fraction, defined as alveolar milk as a proportion of total milk, ranged from 0·43 to 0·94 at 8 h. There was a highly significant correlation between 8 h and 20 h alveolar milk fraction results, but not between 1 h and 8 h values, nor between alveolar milk at 0 h (effectively residual milk) and alveolar fraction at any time point. The milk volume results were used to construct a mathematical model describing milk flow between cisternal and alveolar compartments, and hence the pattern of milk accumulation. The results are discussed in relation to current hypotheses regarding local autocrine control of milk secretion by a feedback inhibitor in milk.

Changes in the contents of retinol, α-tocopherol and β-carotene in plasma, milk and milk fat from 38 Holstein–Friesian cows were followed during their first lactation, and the quantitative and kinetic relationships for secretion of α-tocopherol and β-carotene from blood into milk were determined. The cows were assigned to three groups such that all cows in the same group had the same sire. Milk yield and milk fat content differed with stage of lactation, but not according to sire. The plasma concentrations of retinol, α-tocopherol and β-carotene differed with stage of lactation; in addition, retinol and β-carotene concentration also differed according to sire. The concentrations of all three vitamins in milk and milk fat differed according to sire and stage of lactation. Furthermore, the total secretion of retinol, α-tocopherol and β-carotene into milk (expressed as mg/d) differed with sire and stage of lactation. The quantitative secretion of α-tocopherol and β-carotene from blood into milk followed Michaelis–Menten kinetics for active transport across membranes. Values of maximum secretory capacity Vmax and the half-rate constant Km for both α-tocopherol and β-carotene varied according to sire. Overall means for Vmax for α-tocopherol and β-carotene were 32·4 and 27·5 mg/d. Thus, the daily secretion of α-tocopherol and β-carotene is limited in quantity, and is independent of the yields of milk and milk fat. It follows that continuing breeding and management systems that focus solely on increasing milk and milk fat yield will result in a steady dilution in the milk fat of these vitamins and antioxidants important for the immune defence of the cows and oxidative stability of milk products. The genetic variation found offers the possibility of utilizing these variations in breeding systems.

Sixteen Holstein cows in mid-lactation were used to determine whether alterations of mammary fatty acid metabolism are responsible for the milk fat depression associated with consumption of fish oil. Cows were given a total mixed ration with no added fish oil (control), unprotected fish oil (3.7% of dry matter), or glutaraldehyde-protected microcapsules of fish oil (1.5% or 3.0% of dry matter) for 4 weeks. Milk samples were taken once a week and a mammary biopsy was taken from a rear quarter at the end of the treatment period. Milk fat content was lower in cows given unprotected fish oil (26.0 g/kg), 1.5% protected fish oil (24.6 g/kg) and 3% protected fish oil (20.4 g/kg) than in cows fed the control diet (36.0 g/kg). This was mainly due to a decrease in the synthesis of short-chain fatty acids. Consumption of protected fish oil decreased the abundance of lipogenic enzymes mRNA in the mammary gland. Acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase mRNAs for cows given 3% protected fish oil averaged only 30%, 25% and 25% of control values, respectively. Dietary addition of unprotected fish oil slightly decreased mRNA abundance of these enzymes but markedly reduced the amount of lipoprotein lipase mRNA. Milk fat content was significantly correlated with gene expression of acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase but not lipoprotein lipase. These results suggest that fish oil reduces milk fat percentage by inhibiting gene expression of mammary lipogenic enzymes.

The lactoperoxidase system is a naturally occurring antimicrobial system found in milk, with lactoperoxidase, thiocyanate and hydrogen peroxide as its components. The keeping quality of milk pasteurized at 72°C for 15 s was found to be better than that of milk heated at 80°C for 15 s. This agrees with previous findings and is usually attributed to heat shocking of spores. However, complete deactivation of lactoperoxidase occurred at 80°C–15 s, whereas at 72°C–15 s residual lactoperoxidase activity was ∼70%, which may provide an alternative explanation. Higher levels of hypothiocyanite (the major antimicrobial agent produced by the lactoperoxidase system) were also detected in milk processed at 72 than at 80°C, which supports the theory that the lactoperoxidase system has a role in the keeping quality of pasteurized milk. Of all the methods evaluated, titratable acidity and alcohol stability gave the most consistent estimates of keeping quality, while dissolved oxygen was a good indication of the onset of spoilage. Lactoperoxidase activity decreased with temperature more rapidly between 70 and 80°C than is usual for an enzyme over a 10 deg C range.

A method is described for the quantitative estimation of a mixture of whey proteins by spectrophotometric scanning of the stained protein bands following polyacrylamide-gel electrophoresis. The incorporation of β-lactoglobulin A as an internal standard improves the accuracy of the technique. The method has been used to measure residual native whey proteins in milk after heating. Results are presented for whey protein denaturation in skim-milk after heat treatment at 130 and 140 °C for various periods of time.

Milk permeate was separated at various temperatures by means of a hollow fibre ultrafiltration unit coupled to a stainless steel heat exchanger. Milk samples conditioned at 4°C were heated to 20, 40, 60, 80, 85 or 90°C prior to ultrafiltration. Ca, P, Mg, Na, K and citrate concentrations were measured in the permeate samples. Ca and P contents of the permeate decreased as the temperature increased. The pH was measured after cooling the permeate to room temperature. Smaller losses of Mg and citrate were also observed with increase in temperature. Na and K levels were not affected. A two-step time-concentration relationship was apparent for the species under study. An initial sharp decrease in concentration occurred in the first minute of holding time and was followed by a slower reaction. The possible occurrence of a two-step mechanism in the heat-induced salt balance changes is discussed. Dicalcium phosphate precipitation is believed to be coupled with tricalcium citrate precipitation upon heating.

Eight cows were machine milked either in an operating theatre or in their familiar barn. During the experiments, milk flow curves were recorded and blood samples were taken for determination of concentrations of oxytocin, prolactin, cortisol and β–endorphin. The milking cluster was attached without udder preparation. After cessation of milk flow, air was blown into the vagina for 2 min. When milk flow had stopped again, 1 i.u. oxytocin and finally 10 i.u. oxytocin were injected to remove the remaining milk. After the start of milking, oxytocin remained basal in unfamiliar, but increased in familiar surroundings. Therefore, during normal milking only 9% of total milk was removed in unfamiliar, whereas 79% was available in familiar surroundings. In response to subsequent vaginal stimulation in the operating theatre, oxytocin increased transiently in five cows and 15–71% of the milk was removed in these animals. In the other three cows in the operating theatre, oxytocin remained basal during vaginal stimulation, and no more milk was available. After injection of 1 i.u. oxytocin, 56 and 11%, and after injection of 10 i.u. oxytocin, 13 and 8% of milk was removed in unfamiliar and familiar surroundings respectively. Concentrations of prolactin increased during the course of milking in both treatments. Premilking concentrations of cortisol and β–endorphin were elevated in unfamiliar as compared with familiar surroundings. During the course of milking, cortisol increased slightly and β–endorphin decreased in unfamiliar, whereas both hormones increased markedly during milking in familiar surroundings. We conclude that disturbed milk removal in unfamiliar surroundings is due to central inhibition of oxytocin release during normal milking and partly also to a response to vaginal stimulation. This blockade is possibly associated with elevated concentrations of β–endorphin.

As a result of multiple QTL-mapping projects in recent years, a quantitative trait locus for milk fat percentage and milk yield has been described on BTA14. Recent reports name the acyl-CoA[ratio ]diacylglycerol acyltransferase (DGAT1) gene on BTA14 as a potential candidate gene, with a nonconservative substitution of lysine by alanine (K232A) producing a major effect on milk composition and yield. DGAT1K appears to be the ancestral allele and the K232A substitution probably occurred after the divergence of the Bos indicus and Bos taurus lineages. These findings prompted us to genotype 1748 DNA samples of 38 different Bos taurus and Bos indicus cattle breeds from 13 countries on five continents (Europe, Africa, Asia, North America and South America), to examine the occurrence of the DGAT1 polymorphism and characterize the K232A substitution in cattle breeds of different origins and selected for different purposes (e.g., beef, dairy and dual purpose). Calculating pairwise FST values for pooled subpopulations showed least divergence for Bos indicus breeds with high milk fat percentage. Fixation of DGAT1A was found in some Bos taurus breeds and fixation of DGAT1K in one Bos indicus breed. Breeds of no known organized breeding background from the Near East domestication centre of Bos taurus and taurine African N'Dama cattle were found to possess intermediate frequencies of DGAT1K. While beef breeds tended to harbour higher DGAT1A levels, dairy cattle showed everything from very low levels of DGAT1K to unexpectedly high frequencies of this allele.

Accelerated ripening was successfully achieved in Feta cheese by the addition of a heat-shocked culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, or neutral or acid microbial proteinase. Optimum levels of microbial proteinases were established at which trichloroacetic acid-soluble N (TCA-N) compounds were increased by about 35% in 40 d. Treated cheeses of this age had flavour intensities and body and texture qualities typical of 80 d-old control cheeses. Any further increase in TCA-N by adding larger amounts of enzyme resulted in bitterness. Heat-shocked culture treated cheeses developed similar characteristics to those prepared with neutral or acid proteinases. In contrast to the control cheese, it was necessary in the experimental cheeses to add minute amounts of lamb lipase (0·1–0·2 g/100 kg milk) to the cheese milk to keep the flavour balance. Best organoleptic characteristics were obtained from cheese treated with heat-shocked culture, followed by those treated with neutral or acid proteinase.

A yogurt mix (2 g fat and 17 g solids/100 g) was supplemented with an algae oil emulsion to provide 500 mg ω-3 fatty acids per 272 g serving of yogurt white mass. The emulsion was added to the yogurt mix either before or after the homogenization step and prior to pasteurization. It was then flavoured with a strawberry fruit base and fermented and stored for up to three weeks. The oxidative deterioration of the products was determined by hydroperoxide measurements and by trained and consumer sensory evaluations. The hydroperoxide content of the supplemented yogurts increased over the storage treatment and was unaffected by the stage of addition. The trained panel could distinguish a stronger fishy flavour in both of the supplemented yogurts after 22 days storage, but the consumer panel rated both control and supplemented samples similarly, as ‘moderately liked’.

A treatment containing a live food-grade organism, Lactococcus lactis DPC3147, was compared with conventional antibiotic therapy for its potential to treat bovine chronic subclinical or clinical mastitis in two separate field trials. Effects on disease symptoms and bacteriology were monitored in response to infusion with the culture in each trial. In the first trial, the live culture treatment was compared with an intramammary antibiotic (n=11 quarters for each treatment). Results from this small trial demonstrated that the live culture had potential to be as effective at eliminating chronic subclinical infections as an antibiotic treatment. By day 12, 7 of the 11 quarters treated with the live culture were pathogen-free compared with 5 of the 11 antibiotic-treated infected quarters. Somatic cell counts (SCC) remained relatively unchanged regardless of treatment: average log SCC pre- and post-treatment in the lactococci-treated group were 6·33±0·41 (day 0) and 6·27±0·43 cells/ml (day 12) and average log SCC pre- and post-treatment in the antibiotic-treated group were 6·34±0·37 and 6·22±0·46 cells/ml on day 0 and on day 12, respectively. In the second trial, the live culture was compared with an intramammary antibiotic for the treatment of naturally occurring clinical mastitis cases (n=25 quarters for each treatment). Following a 14-d experimental period, similar bacteriological responses were observed in 7 out of 25 live culture treated quarters and 9 out of 25 antibiotic-treated quarters. Additionally, 15 of 25 cases treated with the culture and 18 of 25 cases treated with the antibiotic did not exhibit clinical signs of the disease following treatment. The results of these trials suggest that live culture treatment with Lc. lactis DPC3147 may be as efficacious as common antibiotic treatments in some instances.

Changes in chemical composition, proteolysis, lipolysis, texture, melting and sensory properties of low-fat Kashar cheese made with three different fat replacers (Simplesse® D-100, Avicel Plus® CM 2159 or β-glucan) were investigated throughout ripening. The low-fat cheeses made with fat replacers were compared with full- and low-fat counterparts as controls. Reduction of fat caused increases in moisture and protein contents and decreases in moisture-in-non fat substance and yield values in low-fat cheeses. The use of fat replacers in the manufacture of low-fat Kashar cheese increased water binding capacity and improved overall quality of the cheeses. Use of fat replacer in low-fat cheese making has enhanced cheese proteolysis. All samples underwent lipolysis during ripening and low-fat cheeses with fat replacers had higher level of total free fatty acid than full- or low-fat control cheeses. Texture attributes and meltability significantly increased with addition of fat replacers. Sensory scores showed that the full-fat cheese was awarded best in all stages of ripening and low-fat variant of Kashar cheeses have inferior quality. However, fat replacers except β-glucan improved the appearance, texture and flavour attributes of low-fat cheeses. When the fat replacers are compared, the low-fat cheese with Avicel Plus® CM 2159 was highly acceptable and had sensory attributes closest to full-fat Kashar cheese.

The non-stimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated luminol-augmented cellular chemiluminescence (CL) response and viability of milk and blood polymorphonuclear leukocytes (PMN) were determined in lactating dairy cows during different stages of lactation. In the first study, ten healthy cows each in early, mid and late lactation were compared. In a second study, the same measurements as in the first study were evaluated longitudinally in 12 cows during 1 month following parturition. The CL activity and myeloperoxidase (MPO) content of milk PMN and macrophages (M) were also compared. Milk M did not possess MPO activity and were devoid of any luminol-enhanced CL. The CL activity of milk and blood PMN was significantly lower in early lactation than in mid and late lactation (P < 0·001). Whereas little changes were observed in viability of blood PMN, the viability of milk PMN was lower in early lactation than in mid and late lactation (P < 0·001). The percentage of PMN in isolated milk cells was also lower during early lactation than during mid and late lactation (P < 0·001). The CL activity in response to PMA during early, mid and late lactation increased 13, 59 and 42-fold in blood PMN and 1·7, 2·6 and 2·4-fold in milk PMN, respectively, in comparison with non-stimulated PMN. The CL activity, both in milk and blood PMN, the milk PMN viability and the percentage of milk PMN were lowest between 3 d and 11 d post partum. These observed changes immediately after calving could contribute to a higher susceptibility to mastitis in that period.

The aim of this study was to evaluate the antioxidant, antihypertensive and immunomodulatory characteristics of skim milk fermented with Lactobacillus delbrueckii ssp. bulgaricus LB340. Supernatants obtained from the ferments after centrifugation were subjected to ultrafiltration and yielded four peptidic fractions of 10–5 kDa, 5–3 kDa, 3–1 kDa, and <1·0 kDa. Peptides in 5–3 kDa range exhibited a good antioxidant activity. The peptides (<1·0 k) was applied to Superdex-30 G column fractionation and produced six fractions (F1–6). Fraction F2 presented the highest angiotensin I-converting enzyme inhibition activity with IC50 of 67·71±7·62 mg/ml. Moreover, fraction F6, which displayed a good immunomodulatory activity, had a positive effect on murine spleen lymphocyte proliferation with Stimulation Index of 0·729±0·123. The present data showed the potential of the milk fermented with Lactobacillus delbrueckii ssp. bulgaricus LB340 as a functional food, however, further research is needed to evaluate the biofunctional activity of this fermentation product in vivo using model animal.

The fact that rumen micro-organisms have a modifying effect on the utilization of food nitrogen by ruminants has been recognized for very many years (references to early work are given in Hungate, 1966) but until about 1947, when this subject was reviewed by McNaught & Smith (1947), many of the processes involved were not well understood. Since that time a large amount of work has been done in this field and more recent reviews (Chalmers & Synge, 1954; Blackburn, 1965; Hungate, 1966; Waldo, 1968) present a much clearer picture. Most of the work covered by these authors will be mentioned only briefly in the present review and attention will be directed mainly to developments reported in the last few years. Few references to work published before about 1964 will be given as they may readily be found in later publications.