The type and relative amounts of plasminogen activator (PA) in different fractions of bovine milk obtained from 15 Holstein cows were examined. Raw milk was centrifuged to separate skim milk and a somatic cell pellet. PA was mainly localized within the casein fraction, being 42 times that in the serum, and in association with somatic cells. The predominant form of PA in milk casein was isolated from SDS-PAGE gel extracts and had a molecular mass of ∽75 kDa. Its activity was increased 41-fold (P < 0·01) in the presence of fibrin but was unaffected by the presence of amiloride, indicating that it was due to tissue-PA. The predominant forms of PA associated with milk somatic cells were isolated from SDS-PAGE gel extracts and had molecular masses of ∽ 30 and ∽ 50 kDa. The activity of both proteins was unaffected by the presence of fibrin but was dramatically reduced by the presence of amiloride, indicating that they represented urokinase-PA.

Acidic and basic fibroblast growth factors (FGF) were identified in bovine cheese whey after partial purification using a two step procedure. Cation-exchange chromatography produced a mitogen-rich extract which was loaded on to a heparin-sepharose column and eluted stepwise with 0·8, 1·2 and 2·0 M-NH4HC03. Mitogenic activity was found in all three fractions by cell growth assays using Balb/c-3T3 fibroblasts. Immunoblotting identified acidic FGF in the 1·2 M-eluate and basic FGF in the 2·0 M-eluate, but neither acidic nor basic FGF was detected in the 0·8 M-fraction. Quantitative radioreceptor assays indicated 5·8 ng of acidic FGF-like activity and 19·8 ng of basic FGF-like activity per 1 whey in the appropriate eluates. This study represents the first direct demonstration of FGF in milk.

The minimal hormonal requirements for inducing the ovine β-lactoglobulin gene have been investigated using mammary gland explants from ewes in the first half of pregnancy. Quantification of β-lactoglobulin mRNA showed that a combination of insulin, Cortisol and prolactin was required to stimulate the expression of the gene and that this response could not be enhanced by the addition of oestrogen and thyroid hormone to the culture medium. Explants cultured in the presence of insulin, Cortisol and prolactin also demonstrated the capacity to synthesize the protein. Progesterone did not inhibit the induction of the gene, which is consistent with the increase in β-lactoglobulin mRNA observed in vivo in the mammary gland during the final 2 months of pregnancy when the circulating level of progesterone is elevated.

A batch fermenter modified to simulate the physical conditions of an inflamed v. uninflamed mammary gland was utilized to evaluate the effect of oxygen tension on killing of Escherichia coli P4 by bovine polymorphonuclear neutrophil leucocytes. Leucocytosis was simulated in vitro 4 h after inoculation with Escherichia coli by adding bovine neutrophils isolated from peripheral blood to the culture medium. Experiments were conducted at 39°C in UHT treated milk. At micro-aerophilic oxygen tension (oxygen partial pressure, 3·11kPa), bacterial numbers declined during the first hour following addition of the neutrophils. Oxygen tension declined rapidly following PMN addition. Once oxygen was depleted, neutrophil activity was presumably diminished and Esch. coli numbers began to increase. Under anaerobic conditions (oxygen partial pressure, 0·17kPa), no reduction in population was observed. Photomicrographs taken at the time of neutrophil addition and at subsequent time intervals demonstrated a specific association between neutrophils and the pathogen. Subsequent lysis of neutrophils associated with Esch. coli growth was seen coincident with oxygen depletion.

Adherence of bacteria to various milk contact sites was examined by scanning electron microscopy and transmission electron microscopy. New gaskets, endcaps, vacuum breaker plugs and pipeline inserts were installed in different areas in lines carrying either raw or pasteurized milk, and a routine schedule of cleaning-in-place and sanitizing was followed. Removed cleaned and sanitized gaskets were processed for scanning or transmission electron microscopy. Adherent bacteria were observed on the sides of gaskets removed from both pasteurized and raw milk lines. Some areas of Buna-n gaskets were colonized with a confluent layer of bacterial cells surrounded by an extensive amorphous matrix, while other areas of Buna-n gaskets showed a diffuse adherence over large areas of the surface. Most of the bacteria attached to polytetrafluoroethylene (PTFE or Teflon™) gaskets were found in crevices created by insertion of the gasket into the pipeline. Examination of stainless steel endcaps, pipeline inserts, and PTFE vacuum breaker plugs did not reveal the presence of adherent bacteria. The results of this study indicate that biofilms developed on the sides of gaskets in spite of cleaning-in-place procedures. These biofilms may be a source of post-pasteurization contamination.

We have investigated the effect of culture temperature on the growth of Lactococcus lactis subsp. lactis biovar diacetilactis, on its acidifying power, on diacetyl and acetoin production, and also on the activities of the principal enzymes involved in the synthesis of these two compounds. The rates of growth and lactic acid production decreased by a factor of ∼ 2 when the temperature decreased from 30 to 18°C. At 18°C, the maximal concentration of diacetyl (0·3mM) was 1·7 times that at 30°C, while that of acetoin was unchanged (5·2 mM). These results are explained by the behaviour of the principal enzymes involved in pyruvate metabolism. The activities of lactic dehydrogenase and acetolactate synthase varied little for culture temperatures between 18 and 30·C. However, the activity of NADH oxidase for a culture temperature of 18°C was 3·7 times that for 30°C, while that of diacetyl reductase for 30°C was 2·7 times that for 18°C. The net effect of temperature on these two activities was an increase in diacetyl production at lower temperature.

Low-fat Gouda-type cheese (200 g fat/kg dry matter) was made on a pilot plant scale from cheesemilk to which heat-treated lactobacilli and/or Neutrase encapsulated in dehydrated rehydrated vesicles had been added. In cheese containing added Neutrase, there was increased proteolysis after the first day following cheesemaking. The cheese with added heat-treated lactobacilli developed increased amounts of amino N as well as increased amounts of acetaldehyde and other, unidentified, volatile compounds. The addition of heat-treated lactobacilli also influenced the texture of the cheese which was firmer but less cohesive.

The modulating effect of bovine milk casein components and their digests on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced or not induced by mitogens has been studied with a colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. All the casein components and their digests tested had little mitogenic effect on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells. Intact κ-casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and Peyer's patch cells induced by mitogens such as lipopolysaccharide from Salmonella typhimurium, concanavalin A, phytohaemagglutinin and pokeweed mitogen. In contrast, intact αsl-casein and β-casein had little effect. κ-Casein had an inhibitory effect after digesti on by pancreatin or trypsin, but not after pepsin or chymotrypsin digestion. Both pancreatin and trypsin digests of αsl-casein and -casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced by mitogens, whereas pepsin and chymotrypsin digests of both caseins were without effect. Moreover, the trypsin digest of each casein component had an inhibitory effect on mouse spleen lymphocyte proliferation in the absence of mitogen. Since trypsin is a major proteinase in pancreatin, the substrate specificity of trypsin seems to be important for the formation of the inhibitory peptides from casein components. These observations suggest that intact κ-casein and some peptides formed from milk casein components by the action of trypsin may suppress the immune responsiveness of neonates.

Unlike that of other lactic acid bacteria, the growth of Leuconostoc spp. in milk is poor and the resulting acidification cannot be used to distinguish different strains. An acidification test based on the use of high initial numbers (109 cfu/ml) has been developed and proved to be an efficient tool for discriminating between 110 Leuconostoc strains isolated from French raw milk cheeses. The pH values after 24 h ranged from 6.55 to 4.05 and distinguished four acidification groups. All 34% of the strains that acidified milk to at least pH 5.1, coagulating it and should be considered as Lac+. The differences in rates and degrees of acidification could not be related to the proteolytic activity which was, from all 27 representative strains tested, similar to that of Lactococcus lactis Prt– variants.

The influence of diet quality and stage of lactation on Cheddar cheese moisture and yield were segregated and the effects of other potential variables (cow breed, milk protein genotype, age and level of mastitis) were minimized. An experiment was conducted using milk of cows in mid lactation or late lactation fed on high or low quality diets. Increased moisture in cheese was largely due to the influence of cow diet. Milk from cows given high quality diets produced lower moisture cheese. Cheese yield was influenced by diet and stage of lactation. Cows in late lactation fed on high quality diets always gave milks yielding the most cheese. Fat and protein recovery in cheese was not influenced by diet or stage of lactation. The results suggest that low quality diets may induce changes in the complex interactions of milk proteins and other components that affect cheesemaking.

Bovine κ-caseinoglycopeptides (i.e. residues 106–169, CGP) were prepared from κ-casein digested with rennin and a commercial whey protein concentrate. CGP from whey protein concentrate was further divided into seven CGP fractions having different carbohydrate compositions using FPLC. Unfractionated CGP inhibited lipopolysaccharide (LPS)- and phytohaemagglutinin (PHA)-induced proliferative responses of mouse spleen cells and rabbit Peyer's patch cells. The unfractionated CGP also inhibited antibody responses to sheep red blood cells in mouse spleen cell cultures. However, seven CGP fractions having zero to five N-acetylneuraminic acid (NANA) residues had different inhibitory effects on both LPS- and PHA-induced proliferative responses of mouse spleen cells. The inhibitory effect on PHA-induced proliferative responses increased with increasing numbers of NANA residues, whereas that on LPS-induced proliferation was highest with the CGP fraction having two NANA residues. Both inhibitory effects decreased significantly after neuraminidase or chymotrypsin digestion. These findings indicate that both the carbohydrate (particularly the NANA residues) and the polypeptide portions are essential for inhibitory effects on LPS- and PHA-induced proliferative responses of mouse spleen cells.

Studies on the immunomodulatory properties of dietary whey proteins in mice are reported. Ingestion of bovine milk whey proteins, either as a supplement in an adequately balanced commercial diet or as the only protein source in a balanced diet, consistently enhanced secondary humoral antibody responses following systemic immunization with ovalbumin, when compared with other protein sources such as soyabean protein isolate and ovine colostral whey proteins. After 5–8 weeks of feeding, dietary milk whey proteins enhanced cell-mediated immune responses as revealed by footpad delayed-type hypersensitivity responses, and concanavalin A-induced spleen cell proliferative responses. To monitor nutritional effects of milk whey proteins, live weight, leucocyte counts and clinical changes of diet-fed mice were examined. The present results confirm other previous results that dietary bovine milk whey proteins have immunoenhancing properties in mice and these properties are unlikely to be related solely to the nutritional effects.