Bovine plasmin (PLM) and its precursor plasminogen (PLG) are present in cheese, in which the proteinase is thought to play a significant role in proteolysis during ripening. PLM in cheese is usually assayed by enzymic techniques and concentrations are generally expressed as activities. In order to determine absolute concentrations of PLM and PLG in cheese, we have applied an ELISA previously developed for the differential determination of PLM and PLG in milk. The assay used two monoclonal antibodies, one specific for PLG and the other cross reacting with PLM and PLG. PLM concentration was obtained by subtracting the PLG concentration from the value for PLM plus PLG. The assay was evaluated in cheese by determining the total amount of PLG and PLM in milk used in the manufacture of eight semi-hard cheeses on a pilot plant scale, and comparing it with the total amount of PLG and PLM present in the eight corresponding wheys and unripened cheeses. Concentrations of PLG and PLM in 19 commercial cheeses were also determined. It was thus demonstrated that the ELISA made it possible to quantify PLM and PLG in milk and cheese and that PLM and PLG concentrations in cheese were highly dependent on the technology used for cheesemaking.

Lactoferrin (Lf) is a milk iron-binding glycoprotein that plays a role in iron transport and acts as both a bacteriostatic and a growth modulating agent. The aim of this study was to investigate the nature of immune responses induced by repeated oral administration of bovine milk Lf in mice. Groups of ten female BALB/c mice were fed daily for 4 weeks with two doses of protein antigen: a low (0·05 mg/g body weight per d) or high (1 mg/g body weight per d) dose of Lf, or water as a control. A fourth group was immunized intramuscularly with 0·01 mg Lf in complete Freund's adjuvant. Anti-Lf IgA and IgG were detected in the intestinal fluid and serum of mice given Lf. Total immunoglobulins were higher in the intestinal fluid in Lf groups than in the control group. No difference could be detected in the serum. IgA and IgG secretion was enhanced in Peyer's patches and spleen from Lf-fed mice, in comparison with controls. [3H] thymidine uptake into Peyer's patch and spleen cells from both control and Lf-fed mice was enhanced by 75 μg Lf/ml in vitro, but Lf groups had a greater proliferation rate than the control group. These findings suggested that Lf could act as an immunostimulating factor on the mucosal immune system and that activation of the mucosal immune system is dependent on the ability of Lf to bind to the intestinal mucosa.

Growth and proteolytic activity of Lactococcus lactis strains were investigated in milks subjected to different heat treatments. Only 13·7% of the strains were of proteolytic phenotype. Proteinase-positive (Prt+) strains exhibited a proteolytic activity that increased when the level of heat treatment was reduced and grew to a higher level than proteinase-negative (Prt−) strains in low-heat-treated milk. When grown in autoclaved reconstituted skim milk, both Prt− and Prt+ strains had the same specific growth rate. Total DNA from four Prt+ and eleven Prt− strains was extracted and used for polymerase chain reaction amplification of fragments belonging to genes encoding for cell wall proteinase. Specific amplification products were displayed by all the Prt+ strains, and by the Prt− strain Lc. lactis subsp. lactis NWC 125. Reverse transcriptase-polymerase chain reaction was performed to detect the transcript of the proteinase genes. In addition to the Prt+ strains, the experiment also detected the specific transcript in Lc. lactis subsp. lactis NWC 125, suggesting that other structural or functional deficiencies occurred in this strain.

A fraction derived from the membrane of milk fat globules (MFGM) was isolated from fresh raw cream. The adsorption of MFGM at the interface of oil-in-water emulsions was studied as well as the stability of the emulsions under different conditions (pH, temperature treatment) and the surface potential. The emulsions prepared with MFGM isolates were stable at neutral pH and were destabilized at low pH. By analysis of the protein adsorbed at the interface by polyacrylamide gel electrophoresis, we also determined that the MFGM that covered the surface of the oil-in-water emulsion could not be displaced by surfactants such as Tweens and Triton X-100 and was not affected by the presence of other proteins such as caseins and β-lactoglobulin added to the emulsion. A strong molecular interaction between the adsorbed MFGM, composed of phospholipid and protein, may exist at the interface, rendering the newly formed membrane very little affected by the presence of other surfactants. This MFGM-stabilized emulsion was characterized by a behaviour very different from that of emulsions stabilized by other milk proteins.

Two groups of eight multiparous cows with different calving periods (November or February) were managed in the same way during lactation. During four 4 week experimental periods distributed over 12 months, the cows were fed on a diet composed of hay and concentrate (70[ratio ]30) in a restricted and controlled amount which varied according to their lactation stage, so as to cover the animals' requirements correctly. The animals' average lactation stage varied according to period from 26 to 298 d. Milk from each group was processed on two occasions during each period to make Saint-Nectaire-type cheese; the cheesemaking conditions were the same throughout. Lactation stage had an important effect on milk fat, protein and calcium contents but not on the casein[ratio ]protein ratio or phosphorus content. The milk pH and the urea content were higher in late lactation. The calcium concentration of milk was higher in late lactation but the soluble fraction was higher in early lactation. Despite higher protein contents, the maximal firmness of the coagulum of late-lactation milk was not different from that of early or mid-lactation milks. pH was higher in cheeses from late-lactation milks compared with those from early and mid-lactation milks. Cheeses from early lactation milks were more yellow than the others and had a lower dry matter fat content. In sensory analysis the odour of cheeses from early and late-lactation milks was less pleasant than that of those from mid-lactation milks. Cheeses from late-lactation milks were more melting and less firm than those from early or mid-lactation milks. Their taste was more intense and more persistent. At tasting, they were less appreciated than the others. These differences were linked to increased proteolysis in the cheeses made with late-lactation milks.

The effects of varying the rennet concentration (by a factor of 216) and the coagulation method on the texture of 32 Feta cheeses produced from ultrafiltered milk were examined by rheological, sensory and chemical methods. Cheeses were obtained that varied greatly in texture but had the same gross chemical composition. All rheological measurements (uniaxial compression, strain sweep and relaxation measurement), sensory firmness and chemical measures of proteolysis depended significantly on the rennet concentration used. With increasing rennet addition, the cheeses became firmer and grittier, and less adhesive and sticky. With two of the three coagulation methods, the physicochemical state of the casein micelles at the point of coagulation varied with the rennet level: when less rennet was used, coagulation occurred at a lower pH and after longer contact between milk and NaCl. However, the effects of increased rennet concentration were also significant with the third method, where there was no effect on the pH or the period with NaCl at coagulation. Probably the primary effect observed was that an increased aggregation rate, with increased rennet level, affected the initial mode of aggregation of casein particles, leading to a denser network structure and a firmer and grittier texture. The level of micellar disintegration at coagulation (controlled by the coagulation method used) had a supplementary effect.

In Swiss-type cheese such as Emmental, proteolysis is one of the major phenomena occurring during ripening. Among the proteolytic agents involved in cheese ripening, the free enzymes originally present in milk and those arising from bacterial autolysis can act directly on the casein network. In order to understand the contribution of the bacterial enzymes and especially those arising from the thermophilic starters, the juice of an Emmental cheese entering the warm room was extracted by pressure, then sterilized by filtration and incubated at 24°C for 20 d under anaerobiosis. At different times, the peptides and free amino acids were determined in the sterile cheese juice. In parallel, in order to gather information about the nature of the enzymes present, the sterile juice was also incubated with β-naphthylamide derivatives as substrates. We have demonstrated a continuous increase in free NH2 groups and in free amino acids throughout the 20 d incubation time. The main peptidase activity was due to aminopeptidase(s) and X-prolyldipeptidyl aminopeptidase(s) whose activities were recovered after non-denaturing polyacrylamide gel electrophoresis. Most of the enzymes found in the juice would have their origin in thermophilic starters. As they are generally intracellularly located, their release could be explained by the autolysis of these starters. Finally, the main free amino acids released in the juice (Pro, Glu, Ala, Val, Leu and Lys) corresponded to those previously found in Emmental cheese, suggesting that the enzymes detected in this study participate significantly in peptide degradation during ripening.

There is considerable interest in the possibility that diet-derived isoflavonoids may help in protection against a number of chronic diseases common in Western society. Based on animal studies, however, concerns have been raised that consumption of isoflavonoids by infants and young children may be undesirable. Clover contains isoflavonoids and therefore may represent, via milk, a source of isoflavonoids in the human diet. In this study the concentrations of daidzein (7, 4′-dihydroxyisoflavone), genistein (5, 7, 4′-trihydroxyisoflavone) and equol (7-hydroxy-(4′-hydroxyphenyl)chroman) were measured using HPLC in cows' milk samples obtained from 76 farms in three Australian states. In addition, concentrations were measured in samples collected from one South Australian factory both before and after pasteurization. Concentrations in all samples were found to be extremely low. The mean daidzein concentration was <5 ng/ml. Mean genistein concentrations ranged from just detectable (∼2 ng/ml) in Victorian samples collected during summer to 20-30 ng/ml in samples from all states collected during spring when isoflavonoid-containing clover is most dominant in pasture. Mean equol concentrations ranged from 45±10 ng/ml in Victorian farm samples collected during summer to 293±52 ng/ml in Western Australian samples collected in spring. The mean concentrations of genistein and equol in post-pasteurization samples collected in spring were approximately double those for samples collected in autumn. Pasteurization had no effect on isoflavonoid concentrations. We conclude that the concentrations of isoflavonoids in Australian cows' milk are low and are therefore unlikely to have any pronounced biological effects in human consumers.

Low-moisture part-skim Mozzarella cheeses were made, at pilot scale (450 kg), on five occasions at weekly intervals from milks containing κ-casein AA, AB or BB genetic variants. Compared with κ-casein A variant milks, the κ-casein B variant milks were associated with higher concentrations of casein (P<0·001), whey protein (P<0·02) and total protein (P<0·001), superior curd-forming properties (P<0·05) and increased cheese yields (P<0·05). The moisture-adjusted (to 465 g/kg) cheese yields for the κ-casein AA, AB and BB cheeses were 91·5, 100·6, and 102·5 kg/1000 kg milk respectively. κ-Casein variant had no significant effect on the proteolysis and ripening of uncooked cheese or on the functionality (melt time, flowability and stretchability) of the cooked cheese during the course of a 90 d ripening period.

To estimate the suitability of cultured milk for the subsequent growth of dairy lactic acid bacteria in cheese manufacturing, control milk was first cultured until the end of the exponential phase with one of four proteolytic (Prt+) strains of Lactococcus lactis differing in their proteolytic enzymes, and pasteurized after readjusting the pH to its initial value. Nineteen non-proteolytic (Prt−) strains of Lc. lactis and nine Leuconostoc mesenteroides strains of dairy origin were then grown in the precultured milk until the stationary phase and their growth was compared with that in control milk. Despite the accumulation of non-protein N (NPN) during preculture, the growth of most Prt− strains of Lc. lactis in precultured milk was either reduced or unchanged whereas that of Ln. mesenteroides strains was unchanged or slightly stimulated. This reduction in growth was reversed by adding an NPN source to precultured milk, indicating that it was due to the exhaustion of assimilable NPN in precultured milk. Thus, preculturing milk with Prt+ strains of Lc. lactis could not be recommended for promoting the subsequent growth of starter cultures in cheese manufacturing.

The relationship between proteolysis and texture in Feta-type cheese made from ultrafiltered cows' milk was studied using capillary electrophoresis, chemical analysis and uniaxial compression. For this purpose cheeses were made with almost identical gross chemical compositions, but wide variations in texture, obtained by varying the amount of rennet and the coagulation conditions. αs1-Casein (CN) 8P, αs1-CN 9P and αs1-CN 8P-I were degraded during ripening, while β-CN A1, β-CN A2 and para-κ-CN were slightly degraded at the highest rennet additions and longest storage time tested (39 weeks). αs1-CN 8P, αs1-CN 9P and αs1-CN 8P-I were degraded even in cheeses made without rennet, and this was ascribed to cathepsin D activity. αs1-CN 9P disappeared faster than αs1-CN 8P, which suggests that milk acid phosphatase was active during storage. During ripening, stress at fracture (σf), strain at fracture (εf), the deformability modulus (E) and work to fracture (Wf) all decreased. Since the gross chemical composition was essentially constant during storage, these changes could be ascribed purely to proteolysis. Although εf could be predicted fairly well from capillary electrophoresis results (correlation coefficient 0·85), there were no unique relationships between degradation of casein components and σf when both storage time and the amount of rennet used were varied. In the young cheese a high level of proteolysis could coexist with high values of σf. This suggests that high levels of rennet caused σf to increase, via an effect unrelated to general proteolysis, whereas general proteolysis caused the reverse effect.

The effect of yogurt on the inhibition of colon tumours induced by 1,2-dimethylhydrazine in BALB/c mice has been studied, and the hypothesis examined that yogurt induces a great reduction in the inflammatory immune response and inhibits tumour growth. Mice were assigned to five experimental groups: a control group fed with a conventional balanced diet, and four other test groups that received yogurt supplements for 2, 5, 7 or 10 consecutive days. At the end of each feeding period, mice were given subcutaneous injections of 1,2-dimethylhydrazine (20 mg/kg) once a week for 8 weeks. After tumour induction, yogurt was given again for 2, 5, 7 or 10 consecutive days each 10 d for 20 weeks. By week 20, 70% of the animals in the control group had developed colorectal tumours. From week 8, there was a considerable infiltration of mononuclear cells into the lamina propria of the large intestine. There was an increase in the number of IgG-producing cells and a slight increase in the IgA-secreting cells, and of CD8+ but not CD4+ T lymphocytes, a high level of β-glucuronidase activity in the intestinal fluid and leucocytosis with neutrophilia in the blood. However, in the test groups given yogurt tumour growth was inhibited, the effect being more evident with 7 or 10 d treatment. The inflammatory immune response as measured by the characteristics we assessed was also reduced, with an increase in the IgA-secreting cells and in CD4+ T lymphocytes. The blood count was similar to that of normal animals and no colorectal tumours were observed in week 20. We suggest that one of the mechanisms by which yogurt exerts antitumour activity is through its immunomodulator activity, by reducing the inflammatory immune response, which was markedly increased when the carcinogen was administered.

A microbial sensor based on a carbon dioxide electrode coupled with immobilized Saccharomyces cerevisiae (baker's yeast) has been used for the determination of sucrose in dairy products. The sensor was used as the detector in a flow injection analysis system. Calibration curves for sucrose were established from 1 to 100 g/l. Determinations for several dairy products containing added sucrose gave good agreement with the concentrations given by manufacturers. Typically, the se of the method was shown to be <5% of the calculated mean.

Citrate metabolism by resting cells of Propionibacterium freudenreichii subsp. shermanii was investigated. In vivo13C nuclear magnetic resonance spectroscopy was used to study the pathway of citrate breakdown and to probe its utilization, non-invasively, in living cell suspensions. [2,4-13C]citrate was metabolized by resting cells to glutamate labelled in positions 2 and 4. In the presence of lactate or pyruvate, its rate of consumption was faster, but it was still converted to glutamate. No catabolic pathway other than the first third of a turn of the tricarboxylic acid cycle was used by Prop. freudenreichii subsp. shermanii to degrade citrate.

The volatile concentrate obtained from Grana Padano cheese by vacuum distillation was fractionated by continuous liquid–liquid extraction into neutral and acid fractions. Both were analysed by high resolution gas chromatography (HRGC), HRGC–mass spectrometry, and HRGC–olfactometry. A total of 67 components were identified in the neutral extract (22 esters, 13 alcohols, 12 ketones, 6 aldehydes, 5 nitrogen-containing compounds, 3 lactones and 6 miscellaneous compounds) and 16 in the acid extract. Esters were the predominant constituents of the neutral fraction, whose major components were ethyl butanoate and ethyl hexanoate. HRGC–olfactometry of the neutral compounds demonstrated that 23 were odour-active: ethyl butanoate, 2-heptanol, 3-methylthiopropanal, 1-octen-3-ol, ethyl hexanoate and nonanal being the most potent odorants. n-Butanoic and n-hexanoic acids were the main volatile free fatty acids identified in the acid extract as having an important odour with a high olfactometric index. The backbone of Grana Padano cheese aroma seemed to consist of these acids and 14 potent neutral odorants imparting fruity, green, nutty and coconut notes. The concentration of volatile components responsible for the fruity and green notes was inversely proportional to the length of ripening, whereas the concentration of volatile agents with spicy, nutty and earthy notes tended to increase during maturation. In a comparison of the olfactometric profile, the Grana Padano cheese aroma was found to be more complex than an imitation Grana Padano cheese produced with similar technology but outside the area of the genuine cheese. Some of the main metabolic pathways for the biosynthesis of cheese aroma are reviewed briefly to indicate the possible origin of the compounds identified.

Various species of coagulase-negative staphylococci (C-NS) are reported to be common in milk and on the teat skin of domestic ruminants. The commonest C-NS species in mastitic milk of cows varies between reports, with Staphylococcus simulans (Jarp, 1991) in one and Staph. hyicus in another (Watts & Washburn, 1991). The teat skin of heifers may be colonized by Staph. xylosus or Staph. chromogenes, while Staph. chromogenes and Staph. warneri are reported as frequent isolates from teat canals and secretion (Boddie & Nickerson, 1986; White et al. 1989). Staph. haemolyticus was isolated frequently from the nares, the teat skin and the milk of goats (Valle et al. 1991), although others reported Staph. xylosus (Bedidi-Madani et al. 1992) or Staph. epidermidis and Staph. capitis (Kalogridou-Vassiliadou, 1991) as the most predominant C-NS in goats' milk. Staph. simulans has been found experimentally to be pathogenic for the mammary gland of meat ewes (Fthenakis & Jones, 1990), but little is known about the prevalence of this species in ewes' milk collected from cases of naturally occurring subclinical mastitis (SCM). The aim of the present investigation was the identification of the commonest C-NS species in ewes' milk collected from field cases of SCM or predominating in the ewes' environment.

Elevated free fatty acid (FFA) levels in milk can impair the flavour quality and shelf life of milk and milk products and thus have implications for the dairy industry. An increase in milk FFA is induced when the protective fat globule membrane is disrupted and the fat exposed to the action of lipoprotein lipase (EC 3.1.1.34). Various aspects of FFA development have been reviewed (Deeth & Fitz-Gerald, 1976; Fleming, 1979; International Dairy Federation, 1980). Mechanical factors that contribute to FFA development are directly related to poor milking systems and procedures (O'Halloran et al. 1975; Judge et al. 1977; Fleming, 1991). Mechanical abuse of milk in many modern milking systems arises from air admission to the milking system, height of milklines and pump operation. Surveys and experience (Fleming et al. 1996) showed that the quality of the components used and the overall maintenance of many milking machines was inadequate. In continental Europe low-level milking systems are generally recommended in preference to higher lines. However, low-level systems are considerably more expensive as units have to be doubled up compared with the mid-level system.

Some of the machine factors contributing to excessive FFA development in milk can be eliminated by proper design, installation, maintenance and operation; however, the benefits of design changes remain a source of debate. The purpose of this investigation was to determine the relative effects on milk FFA of various components of a modern mid-level milking system (a pipeline milking system with the milkline 1·2–1·5m above cow standing level) and then to compare the optimal mid-level system with direct-to-line and low-level milking systems.

Control of environmental mastitis remains a problem on many modern dairy farms. These infections are often transmitted between milkings, and milking hygiene will not prevent new infection. Consequently, the control of environmental mastitis has prompted dairy managers to develop new approaches that either limit bacterial contamination of teat ends between milkings or directly increase the cow's resistance to infection. Intervention strategies that may decrease the incidence of environmental mastitis include improved sanitation of housing areas, decreased water use in udder preparation, optimal dietary concentrations of vitamin E and selenium, and use of R-mutant vaccines (Smith et al. 1984, 1985; Weiss et al. 1990; Smith & Hogan, 1993; Tyler et al. 1993).

Dairy managers are encouraged to provide cows with clean, fresh, palatable feed immediately after milking. This practice is thought to provide cows with an incentive to remain standing for an extended interval, permit teat sphincters to close, and limit teat end contamination and new infections when cows lie down. In one recent study, cows that had access to feed remained standing for a significantly longer time than did cows that were denied access to feed (48 v. 21 min; Tyler et al. 1997). The purpose of the present study was to substantiate that feed availability could be used to extend postmilking standing time in a larger population of cows maintained under different management conditions.

The enterococci have traditionally been used as indicators of faecal contamination because they are common inhabitants of the human and animal intestinal tract. In addition, some strains are well documented as opportunistic pathogens, and have been implicated in endocarditis, infant diarrhoea and other conditions. However, other strains are widespread in foods, particularly in milk and dairy products, where they are considered desirable microflora. In fact, through their proteolytic and lipolytic abilities, they play an important role in cheese ripening, contributing to the development of the organoleptic properties characteristic of certain cheeses (Villani et al. 1993).

Production of antimicrobial substances is one of the mechanisms by which microorganisms can exert a probiotic effect in a host. In this context, a significant number of bacteriocin-producing enterococci of dairy origin have been isolated in recent years (Giraffa, 1995). Production of these antimicrobial peptides or proteins is a common phenotype among lactic acid bacteria, and this is the application of enterococcal bacteriocins of special interest in dairy systems. Firstly, they show activity against a broad spectrum of spoilage and pathogenic organisms of concern in dairy industries, such as Listeria monocytogenes. Secondly, they are inactivated by human gastric enzymes but not by some enzymes that, like rennet, are frequently used in dairy plants. Finally, their marked heat stability enables them to be used in a wide variety of dairy products (Giraffa, 1995).

The genes that encode the biosynthesis of some enterocins or enterococcins, such as enterocin AS-48 (Martínez-Bueno et al. 1994), have been sequenced, allowing their rapid detection by molecular biology techniques such as the polymerase chain reaction (PCR) (Joosten et al. 1997). Enterococcal bacteriocins that have been genetically characterized have been shown to be plasmid-encoded (Clewell, 1993).

In this paper, we report a simple method for the isolation of plasmid DNA from dairy enterococci, using a combination of lysozyme and glass beads (Frère, 1994; Reinkemeier et al. 1996) to achieve cell lysis. Plasmid DNA was used in dot-blot and Southern hybridization analyses to identify enterocin AS-48-encoding dairy enterococci by using a specific PCR-generated probe. In addition, a more rapid detection method based on colony hybridization was also developed.

Polyclonal antibodies specifically directed towards native casein fractions have been recently used to solve some analytical problems such as localization of casein antigenic sites (Otani et al. 1985; Ametani et al. 1987), identification of casein variants (Moio et al. 1989a; Chianese et al. 1992), detection of bovine casein adulterating goats', ewes' and water-buffalo milk and cheese (Elbertzhagen & Wenzel, 1982; Bernhauer et al. 1983; Aranda et al. 1988; Moio et al. 1992; Rolland et al. 1993, 1995; Addeo et al. 1995b), evaluation of the efficiency of chromatographic fractionation of casein (Addeo et al. 1992) and detection of casein proteolysis in cheese (Addeo et al. 1995a). However, the use of polyclonal antibodies for immune analysis is often limited owing to nonspecific binding. The use of monoclonal antibodies may overcome this problem to some extent. Although the binding affinity of a hyperimmune serum is seldom attainable with a monoclonal antibody, an alternative approach consists of the isolation of specific antibodies by affinity chromatography (Johnstone & Thorpe, 1982). In a single step 1000–10000-fold purification has been achieved.

In this paper a fast protein liquid chromatography (FPLC) tandem immunoaffinity is used to enhance the specificity of bovine αs1-casein (CN) polyclonal antiserum. In the first column a caprine whole casein lacking αs1-CN was bound to a solid phase matrix and in the second column bovine αs1-CN was bound. After elution of macromolecular contaminants by a washing step, the two columns were detached and the purified bovine αs1-CN antibodies were eluted from the second column by a simple pH change.