The effects of increasing concentrations of retinol, retinal and retinoic acid on proliferation of bovine mammary epithelial cells were investigated in collagen gel cultures. All retinoids significantly inhibited proliferation of mammary epithelial cells. The relative inhibitory potency of the retinoids was: retinoic acid > retinal > retinol. Maximal inhibition at 10 μg/ml corresponded to a 75–95% inhibition of proliferation obtained in basal medium. Retinol, retinal and retinoic acid also inhibited proliferation of cells growth-stimulated with insulin-like growth factor-I (IGF-I). Retinoids in highest concentrations (10 μg/ml) inhibited 68–85% of proliferation of cells obtained in culture medium containing 25 ng IGF-I/ml. Retinol and retinoic acid also inhibited proliferation of cells growth-stimulated by insulin and other growth factors from the IGF growth factor family (des(1-3)IGF-I and IGF-II), as well as growth factors from the epidermal growth factor family (EGF and TGF-α), with retinoic acid being more effective than retinol. At a concentration of 100 ng/ml, retinol and retinoic acid inhibited respectively 24–38 and 44–52% of mammary cell proliferation stimulated by growth factors of the IGF family, and at 10000 ng/ml, 61–71% of cell proliferation was inhibited. The growth-stimulating effect of insulin, EGF and TGF-α was inhibited 42–64% by retinol and retinoic acid at 100 ng/ml, and 64–84% at 10000 ng/ml. The present results show that retinol, retinal and retinoic acid are potent inhibitors of bovine mammary epithelial cell proliferation. It is suggested that retinoids may have concentration-dependent roles in regulation of pubertal mammary growth and development, indicating that the milk yield potential of heifers may be affected by vitamin A status.

Milk producers in Malaysia make extensive use of crossbred Sahiwal Friesian dairy cattle. These animals have, however, been found susceptible to lactation failure. A survey of cows in an experimental herd of F1 Sahiwal Friesian animals indicated that, in 30% of animals, milk yield decreased to negligible levels within the first 8 weeks post partum. Lactation failure was associated with a progressive increase in the amount of residual milk left in the udder after normal milking. By week 3 of lactation, residual milk volume was significantly greater than that in animals that, based on previous lactation history, were not susceptible to lactation failure, and accounted for up to 30% of milk available at the morning milking. The cellular consequences of residual milk accumulation were evident in the activities of acetyl-CoA carboxylase, fatty acid synthetase and galactosyltransferase, key enzyme markers of cellular differentiation, which decreased in glands undergoing lactation failure and were lower than values measured in tissue of control cows. Mammary cell number, estimated by tissue DNA content, was also reduced in animals undergoing lactation failure. These indices of mammary development indicate that lactation failure is the result of premature involution in susceptible animals. Premature involution is a predictable consequence of progressive milk stasis in failing lactation, and attributable to an increase in autocrine feedback by inhibitory milk constituents. The progressive increase in residual milk is, on the other hand, unlikely to be attributable to impaired mammary development. Measurements of milk storage during milk accumulation showed no differences between control and lactation failure cows in the distribution of milk between alveolar and cisternal storage compartments. We conclude that lactation failure in Sahiwal Friesian cows is due to a failure of milk removal, and probably the result of an impaired milk ejection reflex rather than to the glands' milk storage characteristics.

The aim of the present study was to examine how different types of early interaction between dairy cows and their calves influence milking/suckling-related hormone release in the cows and sucking/bucket-drinking related hormone release in calves. Eighteen cows of the Swedish Red and White breed were studied during the first week after parturition. The cows were machine milked twice daily, and allotted to one of three treatments: [1] cow and calf were kept together and the cow was allowed to suckle the calf for 30 min about 1 h before each milking; [2] cow and calf were kept together and the calf was bucket fed twice daily; and [3] cow and calf were separated immediately after parturition, and the calf was kept in a single box and was bucket fed twice daily. Blood samples were collected around day 7 from both cows and calves. The plasma levels of oxytocin, prolactin, cortisol, gastrin, cholecystokinin (CCK), somatostatin and insulin were analysed. In the cows the levels of oxytocin, prolactin and cortisol were influenced by all three treatments, except for the level of cortisol which did not respond to suckling. The main finding was that the release of oxytocin was significantly greater during suckling compared with machine milking. In the calves, the hormone levels were also influenced by the different milk feeding routines. The plasma concentrations of oxytocin, gastrin, CCK and insulin increased in response to milk ingestion in all treatments. However, during sucking, the increase of oxytocin was significantly greater than during bucket drinking. In addition, a strong correlation between oxytocin and insulin was found in response to sucking. Further, significant increases in prolactin and somatostatin, and a decrease in cortisol were found during sucking. The level of somatostatin also increased in response to bucket feeding when calves were kept separately. During bucket feeding, no significant correlation was found with oxytocin, but strong correlations between the gastrointestinal hormones gastrin, CCK, somatostatin and insulin were seen. Together these data suggest that different hormonal patterns were triggered in the cows by suckling and milking, and in the calves by sucking and bucket drinking. This is further supported by different correlation patterns observed in the calves in response to sucking and bucket feeding. The present findings imply that management routines for cows and calves during the first week after parturition have consequences for the physiology of the animals.

The contamination of pasteurized milk by Bacillus cereus during the filling process was studied in two dairy plants. Samples of pasteurized milk were taken at four different sites along the production line. The samples were stored at 7 °C for 7 d, or at 10 °C for 5 d, before plate counting and random selection of B. cereus isolates. Isolates of B. cereus were typed by the polymerase chain reaction (PCR)-based method randomly amplified polymorphic DNA (RAPD). Samples taken at three different sites between the pasteurizer and the filling machine were all holding similar low concentrations of B. cereus, while an increase of the B. cereus count was seen in the consumer packages. More B. cereus of different RAPD types was growing in the consumer packages than in samples taken just before the filling machine. Several RAPD types found in the consumer packages were not detected in the samples taken just before the filling machine.

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine κ-casein due to the silent substitution Ile/ Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8–14% and 86–92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.

Polymorphism in the goat kappa casein gene was studied using the base excision sequence scanning (BESS) method and sequencing. Seven polymorphic sites, corresponding to single nucleotide transversions were detected. Three of these were silent mutations while the other four produced amino acid substitutions. The association between these polymorphic sites was investigated, which resulted in the identification of three goat kappa casein alleles, designated A, B, and C. Protocols for rapid genotyping of the C variant were developed by polymerase chain reaction–restriction fragment length polymorphism using Alw44I and BseNI restriction endonucleases. The occurrence of this allele was found to be very low in Spanish breeds but more frequent in the French Saanen goat. Further studies among different goat populations are necessary to establish the distribution of these alleles and their effects on the quality and functional properties of milk.

Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) αs1-casein content. Parenteral sensitization to GM1 and GM2 proteins was also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine β-lactoglobulin (β-lg), α-lactalbumin (α-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (ΔIsc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-β-lg IgG1, with an important cross reactivity between goat and cow β-lg. However, in guinea pigs fed on GM2, anti-goat β-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3·5±4·5 μA/cm2) compared with that observed in GM1 guinea pigs (8·3±7·6 μA/cm2). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats' milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.

A new polymerase chain reaction (PCR)-based method was developed to detect cows' milk in goat cheese. This method is based on mitochondrial DNA (mtDNA) control region sequence variations. DNA extractions from 150 mg of cheese were carried out using a spin column-based method. Subsequent PCR amplifications of DNA were performed with cow specific primers, demonstrating the ability to detect cows' milk in a variety of cheeses. This simple approach provides high quality DNA, and is shown to be very sensitive, with a detection limit of less than 0·1% of cows' milk. Analysis of an agarose gel digital image allows a rough estimation of the percentage of cows' milk used in adulteration.

Bipolar-membrane electroacidification (BMEA) technology, which uses the property of bipolar membranes to split water and the demineralization action of cation-exchange membranes (CEM), was tested for the production of acid casein. BMEA has numerous advantages in comparison with conventional isoelectric precipitation processes of proteins used in the dairy industry. BMEA uses electricity to generate the desired ionic species to acidify the treated solutions. The process can be precisely controlled, as electro-acidification rate is regulated by the effective current density in the cell. Water dissociation at the bipolar membrane interface is continuous and avoids local excess of acid. In-situ generation of dangerous chemicals (acids and bases) reduces the risks associated with the handling, transportation, use and elimination of these products. The aim of this study was to evaluate the performance of BMEA in different conditions of added ionic strength (μadded = 0, 0·25, 0·5 and 1·0 M) and added salt (CaCl2, NaCl and KCl).The combination of KCl and μadded = 0·5 M gave the best results with a 45% decrease in energy consumption. The increased energy efficiency was the result of a decrease in the anode/cathode voltage difference. This was due to an increase of conductivity, produced by addition of salt, necessary to compensate for the lack of sufficiently mobile ions in the skim milk. However, the addition of salts, irrespective of type or ionic strength, increased the required operation time. The protein profile of isolates were similar under all experimental conditions, except at 1·0 M-CaCl2.

It is fairly easy to control the enzymic hydrolysis of proteins in alkaline conditions by measuring the base consumption required to keep the pH constant in the reactor. Unfortunately, however, base consumption is not related in any simple way to the degree of hydrolysis reached at any given moment and to establish this relationship it is essential to find out the mean pK of the α-amino groups released during the hydrolytic process. We have shown here that the correct mean pK value varies according to the pH of the working conditions and that the relationship between these values may depend upon the kind of protein and protease used. We have put forward a method for determining this relationship experimentally by using a given protein–protease system, consisting of an alkaline titration of the raw protein and when partially hydrolysed. We have tested the results predicted by our theoretical model by applying it to the hydrolysis of whey proteins with a bacterial protease from Bacillus licheniformis at 50 °C, pH 8·0. This model can easily be applied to any hydrolytic process involving the appearance of functional groups that are partially protonizable under the working conditions in question in order to follow the kinetics of the reaction via the consumption of the neutralizing agent required to keep pH constant.

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6·7) and skimmed milk of 6·5 and 7·6 °C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 °C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.

Gelation of α-lactalbumin (α-la) incubated with a protease from Bacillus licheniformis (BLP) at 50 °C for 4 h was monitored using small oscillatory shear and the large deformation properties of final gels were characterized by uniaxial compression. Transmission electron microscopy was used to visualize the microstructure. Gels made from α-la (10 g/l) using BLP were almost transparent, although somewhat whitish, and they were more than 20 times stiffer (measured as complex modulus) than equivalent gels made from β-lactoglobulin (β-lg) at the same concentration. The microstructure of the gels consisted of non-branching, apparently hollow strands with a uniform diameter close to 20 nm, similar in overall structure to microtubules. Adding Ca2+ in amounts of 50 or 100 mM changed the spatial distribution of the strands and resulted in a reduction in the failure stress recorded in uniaxial compression. Apart from affecting the microstructure, Ca2+ was shown to be essential for the formation of the gels. It is proposed, that the mechanism behind the self-assembly of the partially hydrolysed α-la into long tubes is a spatially restricted creation of ionic bonds between Ca2+ and carboxyl acid groups on peptide fragments resulting from the action of BLP on α-la. Proteolysis of α-la with BLP in the presence of Ca2+ thus results in formation of a strong gel with a microstructure not previously observed in food protein systems.

A detailed kinetic study of hydroxymethylfurfural, lactulose and furosine formation was performed upon heating milk at temperatures between 90 °C and 140 °C. In case of prolonged heating, formation kinetics could be described by a fractional conversion model. Considering only the first phase of the model, kinetics could be simplified to a pseudo-zero order model. A first assessment of kinetic parameters was made by isothermal experiments. Data were analysed using both a 2-step linear and a 1-step non-linear regression method. Only for furosine, did the global 1-step regression approach seem to give better results than the individual 2-step regression approach. Next, the estimated parameters kref and Ea were re-evaluated under non-isothermal conditions by subjecting milk to a time variable temperature profile. Given the complexity of Maillard reaction, it seemed better to estimate kinetic parameters under non-isothermal conditions when using a simplified model. Formation of hydroxymethylfurfural, lactulose and furosine was characterized by an Ea value of 90·2 kJ/mol (k110 °C = 1·2 μmol/l, min), 99·1 kJ/mol (k110 °C = 51·5 mg/l, min) and 88·7 kJ/mol (k110 °C = 16·3 mg/100 g protein, min) respectively. Additionally, 90% joint confidence regions were constructed in order to obtain an accurate representation of the statistical confidence associated with the simultaneously estimated parameters.

In the present study, 124 enterococcal strains, isolated from traditional Italian cow, goat and buffalo cheeses, were characterized using phenotypic features and randomly amplified polymorphic DNA polymerase chain reaction (RAPD–PCR). The RAPD–PCR profiles obtained with four primers and five different amplification conditions were compared by numerical analysis and allowed an inter- and intraspecific differentiation of the isolates. Whole-cell protein analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used as a reference method for species identification. The strains were identified as Enterococcus faecalis (82 strains), E. faecium (27 strains), E. durans (nine strains), E. gallinarum (four strains) and E. hirae (two strains). Species recognition by means of RAPD–PCR was in agreement with the SDS–PAGE results except for eight strains of E. faecium that clustered in separated groups. On the other hand, phenotypic identification based on carbohydrate fermentation profiles, using the rapid ID 32 STREP galleries, gave different results from SDS–PAGE in 12·1% of the cases. The majority of the strains had weak acidifying and proteolytic activities in milk. One E. faecium strain showed vanA (vancomycin resistance) genotype while four strains showed a β-haemolytic reaction on human blood. Several strains showed antagonistic activity towards indicator strains of Listeria innocua, Clostridium tyrobutyricum and Propionibacterium freudenreichii subsp. shermanii.

The behaviour of Streptococcus thermophilus in combination with Lactococcus lactis subsp. cremoris or subsp. lactis mesophilic starters in experimental Cheddar cheese is reported. In a standard manufacturing procedure employing a 38 °C cook temperature, even very low levels (0·007%) of Str. thermophilus combined with normal levels of the mesophilic starter (1·7%) resulted in increased rates of acid production, the formation of significant amounts of galactose (∼ 13 mmol/kg cheese), and populations nearly equivalent to those of the mesophilic lactic starter in the curd before salting. At a 41 °C cook temperature, the Str. thermophilus attained a higher maximum population (∼ log 8·2 colony forming units (cfu)/g) than the Lc. lactis subsp. cremoris (∼ log 6·8 cfu/g) and formed more galactose (∼ 28 mmol/kg). Lactobacillus rhamnosus, deliberately added to a cheese made using Str. thermophilus starter and which contained 24 mmol galactose/kg at day one, utilized all the galactose during the first 3 months of cheese ripening. Adventitious non-starter lactic acid bacteria had the potential to utilize this substrate too, and a close relationship was demonstrated between the increase in this flora and the disappearance of the galactose. Some possible consequences for cheese quality of using Str. thermophilus as a starter component are discussed.

During a routine inspection of Cheddar cheese manufactured at a commercial factory in New Zealand, some lots of 6-month-old cheese were found to have developed a pinkish colouration on the surface of the 20 kg blocks of cheese. Colouration did not always occur uniformly on all six faces of the rectangular cheese block, or even on a single face of the block. Furthermore, not all blocks from within the same day's manufacture were equally affected. When an affected block was removed from its bag and cut across, colouration was sometimes found to penetrate approximately 1–2 cm down into the cheese. In those blocks where a plug of cheese had been removed previously, a pinkish zone surrounded the plug-hole cavity.

The pinkish colouration was observed to fade slowly (over about 12–24 h) when the cheese surface was exposed to air.

Annatto, known to cause pink discolouration in “coloured” Cheddar cheese (Govindarajan & Morris, 1973) and in processed cheese made using coloured Cheddar, was not used in the manufacture of the present cheeses and could therefore be excluded as a cause of the colouration.

The flavour profiles of all affected cheeses were considered by experienced industry cheese graders to be easily within the normal range of flavour profile expected for a cheese of this type i.e. there was no evidence of any off-flavour development.

The present short communication describes the microbiological and chemical investigations carried out to determine the origin and nature of the pinkish colouration in Cheddar cheese.

Numerous methods based on DNA analysis have been employed in the food industry to monitor adulterations of food products of animal origin. Among them the most frequently used are: polymerase chain reaction (PCR) amplification of a marker gene fragment(s) with universal primers, or amplification of DNA with species-specific primers. PCR-products of different origin can be discriminated by size, restriction fragment length polymorphism (RFLP) or single stranded conformational polymorphism (SSCP) analysis. These methods have been used for identification, and differentiation between, the animal origins of raw or heat-treated meat and meat products (Chikuni et al. 1994; Meyer et al. 1994, 1995; Zehner et al. 1998; Behrens et al. 1999; Guoli et al. 1999; Hopwood et al. 1999; Matsunaga et al. 1999; Wolf et al. 1999). These approaches are also applicable to the analysis of dairy products. However, adulterations of goats' milk and its products are traditionally tested by immunological and/or electrophoretic methods (Amigo et al. 1992; Levieux & Venien, 1994; Mimmo & Pagani, 1998). So far, only a few DNA-based techniques designed to detect the presence of bovine DNA in goats' milk have been described (Plath et al. 1997; Branciari et al. 2000). This paper presents a one-step PCR procedure for detection of adulteration of goats' milk with cows' milk. The method, employing bovine-specific primers for amplification of a 274 bp fragment of cytochrome b DNA, seems to be simple, fast, specific and sensitive.