The aim of the study was to examine the effect of morphine and naloxone pretreatment on cortisol and ACTH concentrations in response to machine milking in dairy cows. In the first part of the experiment, the effects of i.v. morphine doses (0, 21, 70 and 210 mg, one dose each day) 10 min before morning milking were studied in six Brown Swiss dairy cows. In the second part, four cows were treated 1 d after the control milking with 210 mg morphine at 10 min before milking and the next day with 210 mg naloxone at 15 min before milking followed by 210 mg morphine at 10 min before milking. In addition, four other cows were treated 1 d after the control milking with 210 mg naloxone at 10 min before milking. Pretreatment with morphine significantly suppressed the machine milking-induced increase of cortisol in blood plasma as compared with controls. Naloxone pretreatment overcame the inhibitory effect of morphine and elevated milking-induced cortisol concentrations. Naloxone administration alone significantly increased cortisol concentration resulting from milking as compared with controls. However, ACTH concentrations did not change in either control or treated animals, suggesting an ACTH-independent release mechanism for cortisol during milking. We conclude that the release of cortisol in response to machine milking seems to be modulated by endogenous opioids at the adrenal level and does not appear to be under the control of ACTH.

The mechanism of glycine transport in lactating mouse mammary gland was investigated. Three Na+-dependent systems of glycine transport, distinguished on the basis of their ionic requirement and sensitivity to 2-(methylamino)isobutyric acid (MeAIB), were A (Na+-dependent, MeAIB-sensitive); (Na++Cl−)-dependent, MeAIB-insensitive; and Na+-dependent, Cl−-independent, MeAIB-insensitive. These systems were further distinguished on the basis of inhibition analysis and sensitivity to pH of the extracellular medium and preloading mammary tissue with amino acids. The uptake of glycine via the A system (Km 0·53 mM) was inhibited by preloading mammary tissue with alanine, while glycine uptake mediated by the (Na++Cl−)-dependent, MeAIB-insensitive system (Km 0·47 mM) was downregulated by preloading mammary tissue with all amino acids (alanine, sarcosine and histidine) tested. Treatment of mammary tissue with N-ethylmaleimide inhibited the uptake of glycine via both these systems. Decreasing the pH of the extracellular medium inhibited the uptake of glycine via the A system but not the (Na++Cl−)-dependent, MeAIB-insensitive system. On the basis of ionic requirement, system A appears to comprise two components, one dependent on Na+ plus Cl− and the other on Na+ alone. Insulin upregulated the A system-mediated uptake of glycine in pregnant mouse mammary tissue cultured in vitro, while the (Na++Cl−)-dependent, MeAIB-insensitive system remained unaffected.

We have studied the effect of treatment with enrofloxacin on local and general clinical signs and chemiluminescence of circulating polymorphonuclear leucocytes during experimentally induced Escherichia coli mastitis in cows immediately after parturition. Twelve cows were infected with 104 cfu Esch. coli P4[ratio ]O32 into both left quarters. Six cows received an intravenous injection of 5 mg enrofloxacin/kg at 10 h after infection and a second enrofloxacin treatment administered subcutaneously at 30 h post infection. The other six cows were controls that received no treatment. General clinical signs (fever, tachycardia, loss of appetite, reduced rumen motility and depression) were similar in both groups. Local clinical signs, such as swelling, pain and firmness of the inflamed mammary quarters, were less severe in the treated cows. We saw no difference in the appearance of the milk[ratio ]flecks and watery or purulent milk were observed in both groups. The beneficial effects of treatment with enrofloxacin were mainly on milk production and composition. The decline in milk production and the changes in milk concentrations of lactose, Na+ and bovine serum albumin were less pronounced in the treated cows. Treatment with enrofloxacin accelerated the clearance of bacteria from the infected quarters, but had no effect on the chemiluminescence response of isolated polymorphonuclear leucocytes. The changes in the number of circulating leucocytes and the appearance of immature neutrophils in the circulation of the treated cows indicated possible beneficial effects on migration of neutrophils into the inflamed glands. Higher milk somatic cell counts in the treated cows supported this hypothesis. The results of this study indicated that treating cows that have been experimentally infected with Esch. coli mastitis after parturition with enrofloxacin reduced the severity of the disease, especially the decline in milk production and the changes in milk composition.

Twelve cows were experimentally infected in two quarters with 1 × 104 cfu Escherichia coli per quarter and six cows were infused with 500 μg endotoxin into two quarters. Six cows infected intramammarily with Esch. coli were treated intravenously with a bactericidal antibiotic 10 h after infection and subcutaneously 20 h later. Blood and milk samples were collected from all cows at regular time intervals. Milk production decreased more rapidly, but was less pronounced, after endotoxin infusion than during Esch. coli mastitis. The milk production losses in the non-inflamed quarters were negligible in endotoxin mastitis, but were substantial during Esch. coli mastitis, probably due to more pronounced systemic effects. Reticulo-rumen motility was inhibited only during Esch. coli mastitis. Changes in plasma haptoglobin were more pronounced during Esch. coli mastitis, although they occurred sooner during endotoxin mastitis. No changes in plasma activities of enzymes such as lactate dehydrogenase, glutamic–oxaloacetic transaminase and γ-glutamyl transpeptidase were observed. Concentrations of tumour necrosis factor-α increased in both types of mastitis. Absorption of these cytokines into the circulation was highest during Esch. coli mastitis, especially in the untreated control group. We found only minor differences between the treated and untreated Esch. coli groups, but there were larger differences between the Esch. coli groups and the endotoxin group. These differences were probably due to differences in kinetics, composition and amounts of different cytokines released in the mammary gland and subsequently absorption into the circulation. Endotoxin is probably not directly responsible for the systemic changes during coliform mastitis.

An adaptive stochastic dynamic programming model was used to solve the optimum replacement decision problem for the dairy cow under a range of alternative mastitis control procedures. The model predicted that reducing milk yield losses and somatic cell count penalties by using milking machine test, post-milking teat disinfection and dry cow therapy added approximately £4, £10 and £13 respectively to an original annuity equivalent net present value for the replacement heifer of £286. Assuming that these procedures also reduced involuntary culling due to mastitis by 50% added £8·90 to the annuity. This latter figure indicated that an important part of the benefit of mastitis control procedures might come from a reduction in the culling risk of persistent clinical cases. We concluded that the strength of the dynamic programming model in this context was that it provided an integrated evaluation of the various impacts of each alternative mastitis procedure in the long term, which is essential for correct economic evaluation of mastitis.

31P-NMR has been tested as a technique for determining the phosphorus content of milk samples in both the inorganic form (Pi) and bonded to caseins as phosphoserine (SerP). 31P-NMR made possible the simultaneous determination of Pi and SerP in ∼ 1·7 h. The determination is based on measuring the area under the resonance in question and interpolating in a calibration curve prepared using external standards of known phosphorus contents and prepared in the same manner. The concentration–area plots obtained were linear in the ranges 162–1993 mg P/l for Pi and 38–402 mg P/l for casein SerP. The repeatability of the method was good. When the same milk samples were analysed by both 31P-NMR and the classic colorimetric method the correlation was good. Polyphosphates added to commercial milk were also detected, as diphosphates. The NMR response for diphosphate was linear between 58 and 576 mg P/l and preliminary results indicated that quantitative analysis could be achieved in commercial milk.

Transgenic mice were produced carrying the coding region of the rabbit κ-casein gene linked to the upstream region of the rabbit whey acidic protein gene. Mice from the highest-expressing line produced 2·5 mg rabbit κ-casein/ml in their milk. The foreign protein was associated with the casein micelles and altered micelle size, though in the high-expressing line rabbit κ-casein also segregated into the whey fraction obtained after centrifuging the milk samples. Milk from transgenic mice had the same overall protein content as that from non-transgenic mice, except for the transgene product. However, litters fed with this transgenic mouse milk grew less well than litters given milk from non-transgenic mice. This reduction in growth was not related to changes in mammary gland structure or mammary cell morphology. Preliminary results indicated that milk from the transgenic mice had a higher viscosity.

Goat whey was hydrolysed by pepsin in an ultrafiltration membrane enzymic reactor coupled with a 30 kDa mineral membrane. Peptides collected in the permeate were resolved using reversed-phase HPLC. Their sequences were determined by amino acid analysis, second order derivative spectra analysis and mass spectrometry. Owing to the resistance of β-lactoglobulin (β-lg) towards pepsin, the majority of peptides identified were derived from α-lactalbumin (α-la). Pepsin showed a broad specificity of hydrolysis sites and generated a wide range of products from dipeptides to very large peptides containing disulphide bridges. The molecular masses of peptides resulting from α-la degradation were between 150 and 6900 Da: 36% were < 600 Da, 24% were 600–2000 Da and 40% were > 2000 Da.

Analysis by gas chromatography–mass spectrometry of pasteurized milk with a fruity (pineapple-like) off-odour and a sour, rancid and soapy taste indicated the presence of concentrations at μg/ml levels of ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, octanoic acid, decanoic acid and dodecanoic acid. The off-odour and taste were attributed to the presence of these compounds in the milk. Microbiological examination confirmed that the milk was also contaminated with a series of psychrotrophic bacteria including Yersinia intermedia, Pseudomonas putida and Rahnella aquatilis. Growth of isolates of these bacteria in UHT milk at 23 °C for 7 d showed that Yer. intermedia produced significant quantities of the C4–C12 alkanoic acids; Ps. putida produced only small quantities of these acids and Rah. aquatilis produced none. In addition, Yer. intermedia and Ps. putida also produced small but significant quantities of the corresponding ethyl esters. In milk inoculated with both Yer. intermedia and Ps. putida, the quantity of ethyl esters produced was greater than that found in cultures containing only one of the isolates. These studies indicated that Yer. intermedia was the principal source of the alkanoic acids in the tainted milk and that the major producer of the corresponding ethyl esters was Ps. putida. This is the first report that Yer. intermedia and Ps. putida can cause an off-odour or taste in dairy products.

Mutants of Lactococcus lactis producing excess carbon dioxide could be isolated on LDHA-20 agar (described by El Attar et al. Journal of Dairy Research67 641–646 2000). The use of these mutants in the manufacture of Roquefort cheese has the potential to improve the formation of openings in this cheese. The aim of this work was to examine the stability of these mutants, their enzymic activities and their metabolism of lactose and citrate during growth in milk. They produced less l-lactate than the parent strain and their lactate dehydrogenase activity was lower. Nevertheless none of the mutants produced no L-lactate at all and the most active gas generators among them generally produced 30–50 mM-L-lactate. Unexpectedly, all the strains produced some D-lactate, some > 10 mM. We found that carbon dioxide production by the mutants could be determined indirectly by assaying acetoin, citrate and 2,3-butanediol by high-performance liquid chromatography. Generally, spontaneous mutants were more stable than those obtained after treating with nitrosoguanidine or u.v. irradiation.

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 °C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology180 5947–5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.

We have investigated the influence of partial hydrolysis with an immobilized proteinase from Bacillus licheniformis on the thermal gelation of isolated β-lactoglobulin B. Gelation behaviour was determined by dynamic rheological measurements (small deformation) and the gels were characterized with respect to microstructure and water-holding properties. A fine-stranded gel with a complex modulus of ∼ 2000 Pa was formed from β-lactoglobulin (50 g/l in 75 mM-Tris-HCl, pH 7·5). Limited hydrolysis prior to thermal gelation resulted in coarser gels with thicker protein strands and larger pores. Gel structure correlated with its permeability, proton mobility and water-holding capacity. Total gel stiffness increased with low degrees of hydrolysis, but decreased after prolonged hydrolysis. Maximal gel stiffness was 1·5-fold that of gels made from unhydrolysed β-lactoglobulin. This was much lower than the stiffening effect obtained after partial hydrolysis of whey protein isolate, showing that the gel strengthening effect of partial hydrolysis was dependent on the protein composition and/or the hydrolysis and gelation conditions. A mechanism to explain the observed effects of hydrolysis on gelation and gel properties is presented.

The aim of this work was to study the spin–spin (T2) relaxation components of one hard cheese and three soft cheeses to characterize fat and water states. NMR signals were measured at 6 °C with a 0·47 T NMR device. The transverse relaxation decay was fitted using the Marquardt method. The T2 relaxometric behaviour of the cheeses under consideration was characterized by four relaxation components. To understand the chemical composition of each NMR component, we studied anhydrous milk fat extracted from each cheese analysed. At 6 °C, the fat was 60% crystalline. In cheese, the solid fat was found mainly in the shorter relaxation component with a T2 of 17 μs. The intensity of the NMR relaxation with a T2 > 1 ms was explained by the amount of water, liquid fat and proteins, and the associated relaxation time varied as a function of the process used. The composition of each relaxation component was confirmed by the temperature effect and the influence of the fat content on the NMR cheese signal. NMR relaxometry was able to provide information on water behaviour (i.e. the quantity and level of interactions with proteins) and on the solid[ratio ]liquid ratio of anhydrous milk fat in the cheese.

The timely ingestion and absorption of colostral immunoglobulin is a critical determinant of neonatal calf health. Calves are born without appreciable concentrations of the serum immunoglobulins needed to protect against pathogenic bacteria, viruses and protozoa (Tyler & Parish, 1995). The beneficial effect of passive transfer of colostral immunoglobulin also extends beyond the neonatal period and persists into juvenile and adult life (Robison et al. 1988; Tyler et al. 1998; DeNise et al. 1989). Calves with failure of passive transfer, defined as serum protein < 50 g/l or serum IgG < 10 g/l, have increased mortality risks that persist until 10 weeks of age (Tyler et al. 1998).

Several diseases are potentially spread by the ingestion of colostrum, including bovine leukosis and Johne's disease (Perrin & Polack, 1988; Streeter et al. 1995). In one study 22% of latently infected cows were demonstrated to shed Mycobacterium paratuberculosis in their colostrum (Streeter et al. 1995). Optimal programmes to prevent and eradicate these diseases generally include the provision that calves are given colostrum derived from cows of known negative disease status. Pasteurization or heat treatment of colostrum may provide a mechanism whereby calves are provided with protection against neonatal disease without creating undue potential for infection by chronic, economically relevant diseases.

Attempts to heat disinfect colostrum are common in goat herds (MacKenzie et al. 1987). Pasteurization has been demonstrated to be effective against the caprine arthritis–encephalomyelitis virus (Adams et al. 1983; MacKenzie et al. 1987). Although Myco. paratuberculosis appears to resist pasteurization, this form of processing has been demonstrated to decrease the likelihood of positive colostral cultures for Myco. paratuberculosis under experimental conditions (Meylan et al. 1996). Sterilizing the milk and colostrum given to calves is a logical and reasonable strategy to prevent transmission of infectious microorganisms. The potential disadvantage of heat treating colostrum is that the immunoglobulins in colostrum may become denatured (Smith & Sherman, 1994). Pasteurization causes only a slight decrease in the colostral concentration of IgG in cattle (Meylan et al. 1996); however, the biological behaviour of these pasteurized immunoglobulins has not been critically examined. Therefore, we cannot be completely confident that immunoglobulin absorption, persistence in serum and biological activity are unchanged by this processing.

The goal of this study was to determine the effect of pasteurization at 76 and 63 °C on the absorption of IgG from colostrum. Should these procedures decrease immunoglobulin absorption, the use of pasteurization in disease eradication programmes would require increased efforts to optimize the passive transfer of immunoglobulin.

Psychrotrophic bacteria, capable of growing in milk during refrigerated storage before processing, produce thermostable proteinases that strongly influence the keeping quality of long life dairy products (Cousin, 1982). This is the case with UHT milk, where the activity of residual or reactivated bacterial proteolytic enzymes is linked to flavour deterioration and gelation. Furthermore, since most of these enzymes are acid proteinases with a broad specificity and particularly active against κ-casein (Fairbairn & Law, 1986), their hydrolysis products can interfere with detecting the fraudulent addition of rennet whey (López-Fandiño et al. 1993a, b; Recio et al. 1996, 2000). This is usually based upon detecting, by reversed-phase (RP-) HPLC (Olieman & van Riel, 1989; Van Riel & Olieman, 1995a) or capillary electrophoresis (CE) (Van Riel & Olieman, 1995b), caseinmacropeptide (CMP), the 106–169 fragment of κ-casein released by chymosin during the initial stages of cheesemaking (for review, see López-Fandiño & Olano, 1999). Nevertheless, it is not yet clear whether degradation of κ-casein by psychrotrophic enzymes gives rise to hydrolysis products that are identical to those produced by chymosin or just indistinguishable by CE (Van Riel & Olieman, 1995b; Recio et al. 1996).

In the present investigation, we studied CMP-like degradation products produced by the action of extracellular psychrotrophic proteinases on κ-casein by RP-HPLC and CE. Electrospray ionization–mass spectrometry (ESI–MS) was used for peptide identification. Characterization of breakdown products specific to psychrotrophic enzymes may help the investigation of their residual activity remaining in milk after processing, as well as provide an indication of the suitability of CMP as an indicator of adulteration of UHT milk with whey solids.

The composition of colostrum is quite different from that of mature milk. In particular, colostrum contains relatively high concentrations of proteins responsible for the immunological defence of newborns, such as lactoferrin, lactoperoxidase, lysozyme and immunoglobulins (Farkye, 1992; Cals et al. 1994; Telemo & Hanson, 1996; Wang et al. 1997). Whereas lactoferrin and lactoperoxidase are synthesized in mammary epithelial cells (Cals et al. 1994; Molenaar et al. 1996), the immunoglobulins are derived from blood serum (Larson, 1992). Immunoglobulin A (IgA), predominantly found in milk, participates in the development of the gastrointestinal system and the immune system in newborn infants. It is transported by a system that involves formation of a complex with the polymeric IgA receptor (pIgR) exposed on the basolateral aspect of mammary gland epithelial cells, followed by internalization and release into milk as secretory IgA (Rosato et al. 1995; De Groot et al. 1999). Consequently, expression of pIgR in mammary epithelial cells also contributes to the development of the immune system. These proteins specifically expressed in the early stage of lactation are of interest. However, few studies have been carried out compared with those focusing on the major proteins in mature milk, in part owing to problems of mammary gland availability.

Imamura et al. (1996) reported the isolation of mRNA from bovine mammary epithelial cells derived from colostrum and showed that it is possible to detect αs1-casein mRNA, predominantly expressed in the bovine mammary gland, through the application of reverse transcriptase polymerase chain reaction (RT-PCR). We have applied this procedure to porcine colostrum, and successfully analysed the coding sequence of pIgR. In this paper we describe the possibility of detecting gene expression in mammary epithelial cells present in colostrum and the porcine pIgR sequence obtained is compared with those of other animal species.

Milk-clotting enzymes are used during the production of cheese to coagulate the casein, allowing the formation of a three-dimensional network that entraps the milk fat. Commercially available milk-clotting enzymes differ with respect to source, specificity, optimum pH and thermostability. All are acid proteinases that can cleave κ-casein resulting in the coagulation of milk. Chymosin (EC 3.4.23.4) is specific for the Phe–Met bond in κ-casein at the natural pH of milk (6·7). Recombinant chymosin is available commercially from a variety of sources and has a maximum activity at 40 °C. Recombinant chymosins are purified from the fermentation of recombinant strains of Aspergillus niger, Asp. oryzae or Kluyveromyces marxianus. These enzyme preparations are chemically and functionally identical to calf chymosin. Rennets are purified from the abomasum of bovines and can contain from 60 to 100% chymosin with the remainder being primarily bovine pepsin (Wigley, 1996). Microbial proteinases (EC 3.4.23.6) are generally more proteolytic than chymosin, with varying heat stability. These enzymes liberate more non-protein N from casein and can cleave α- and β-casein as well as κ-casein at the natural pH of milk. Acid proteinases from Cryphonectria parasitica are more heat labile than those from Rhizomucor miehei, which are characterized as thermostable (Ernstrom & Wong, 1974).

The objective of this research was to characterize milk-clotting enzymes with respect to thermal inactivation in skim milk. This information has applications in milk and whey processing.

One of the characteristics of Roquefort cheese is the presence of irregularly shaped openings. Although many factors affect the development of opening in blue-veined cheeses (Martley & Crow, 1996), the limiting step in the case of Roquefort cheese is the production of CO2 by lactic acid bacteria (J.-P. Reverbel, pers. comm.). Most of the opening occurs after moulding; the process is difficult to control and many manufacturing runs result in cheeses with an insufficient opening. Concentrated suspensions of Leuconostoc strains are used to increase the production of CO2 (Devoyod & Muller, 1969), but it would clearly be useful to have microorganisms that produce larger quantities of the gas. McKay & Baldwin (1974) isolated a spontaneous mutant of Lactococcus lactis that produced more acetoin and CO2 than the parent strain. This mutant was lactate dehydrogenase (LDH)-deficient, which favoured the conversion of pyruvate into end products other than lactate. Following nitrosoguanidine mutagenesis, we isolated three Lc. lactis mutants whose LDH activities were reduced to varying extents and which produced varying amounts of CO2 (Boumerdassi et al. 1997). Subsequent work showed that these mutants were unstable on successive subculture in milk or synthetic broth (El Attar et al. 2000).

The aim of our current work was to select a large number of Lc. lactis mutants producing excess CO2. This would increase the probability of selecting stable mutants and also provide a collection of strains with differing gas production activities. Currently available screening methods are, however, unsuitable for processing large numbers of mutants, which is why we have developed an improved screening method.