We have investigated the responses of cow teats to machine milking in a study of relatively newly installed commercial milking parlours fitted with one of two types of milking cluster. The first was a common type with a large claw volume (> 200 ml), 15–16 mm i.d. long milk tube, 10 mm short pulse tube, cluster weight < 3·2 kg and used alternate pulsation. The second was a more traditional type with a 150 ml claw bowl volume, 13·5 mm i.d. long milk tube, 8 mm short pulse tube, cluster weight ∼ 3·5 kg and used simultaneous pulsation. We scored ∼ 50 cows in each of 20 herds, all within 60 s of cluster removal, for changes from the premilking teat condition: teat colour (creation of reddening or blueness), firmness, thickening at the base of the teat associated with the position of the liner mouthpiece, and whether the teat duct orifice was open. There were statistically significant differences in the proportion of cows displaying these four alterations in teat condition between herds using the two types of cluster. The more common type of cluster was always associated with better teat condition. The cause and effect of poorer teat condition have not been fully established and are likely to be multifactorial. The principal risk factors may be cluster weight, overmilking, vacuum applied during the overmilking phase and the design of the liner mouthpiece.

The effect of tension of teatcup liners on teat end condition and quantity of keratin in the teat canal was investigated. Liner tension was increased by using longer teatcup shells. The first experiment used six Holstein cows in early lactation. Left quarters were milked with liners under medium or normal tension by using Conewango liners in 142 mm shells. Right quarters were milked with liners under high tension by mounting the liners in teatcup shells 149 mm in length. By day 16, teat end condition and sensitivity to manipulation were worsened by thrice daily milking when liners were under a higher tension. Two subsequent experiments each used 12 different Holstein cows. These cows were in mid lactation and were milked twice daily for 10 or 30 d. Left quarters were milked with liners under high tension. Right quarters were milked with liners under low tension by using teatcup shells 126 mm in length. The quantity of keratin removed during milking was not influenced by liner tension; however, the quantity of keratin at the end of the experiments was increased 10–20% in teats that were milked using liners under a higher tension. Histological analysis and keratin content were consistent with epithelial hyperplasia induced by milking with liners under increased tension.

Heat (85 °C for 20 min) and pressure (600 MPa for 15 min) treatments were applied to skim milk fortified by addition of whey protein concentrate. Both treatments caused > 90% denaturation of β-lactoglobulin. During heat treatment this denaturation took place in the presence of intact casein micelles; during pressure treatment it occurred while the micelles were in a highly dissociated state. As a result micelle structure and the distribution of β-lactoglobulin were different in the two milks. Electron microscopy and immunolabelling techniques were used to examine the milks after processing and during their transition to yogurt gels. The disruption of micelles by high pressure caused a significant change in the appearance of the milk which was quantified by measurement of the colour values L*, a* and b*. Heat treatment also affected these characteristics. Casein micelles are dynamic structures, influenced by changes to their environment. This was clearly demonstrated by the transition from the clusters of small irregularly shaped micelle fragments present in cold pressure-treated milk to round, separate and compact micelles formed on warming the milk to 43 °C. The effect of this transition was observed as significant changes in the colour indicators. During yogurt gel formation, further changes in micelle structure, occurring in both pressure and heat-treated samples, resulted in a convergence of colour values. However, the microstructure of the gels and their rheological properties were very different. Pressure-treated milk yogurt had a much higher storage modulus but yielded more readily to large deformation than the heated milk yogurt. These changes in micelle structure during processing and yogurt preparation are discussed in terms of a recently published micelle model.

Ovine casein macropeptide (CMP) was characterized by anion-exchange FPLC and reversed-phase (RP) HPLC. To study heterogeneity (the degree of glycosylation and phosphorylation), CMP was desialylated with neuraminidase and dephosphorylated with acid phosphatase. Following RP-HPLC, the main CMP components were identified using either on-line or off-line mass spectrometry. The most abundant ovine CMP component was a diphosphorylated carbohydrate-free form, followed by one or two monophosphorylated and a non-phosphorylated asialo-aglyco species. Aglyco non-phosphorylated, monophosphorylated and diphosphorylated forms were in the ratio 3[ratio ]20[ratio ]77. Only ∼ 30% of ovine CMP was glycosylated. Assuming that the monosaccharide fraction of ovine CMP is composed of N-acetylgalactosamine, galactose and N-glycolylneuraminic acid, molecular masses consistent with the presence of CMP containing tetra-, tri-, di- and monosaccharide were identified.

We have investigated the effects of adding a range of mineral salts and calcium-chelating agents on the distribution of casein and minerals between the non-pelleted and pelleted phases of milk obtained upon centrifugation at 78000 g for 90 min. Adding CaCl2 or mixtures of NaH2PO% and Na2HPO% to reconstituted skim milk (90 g milk solids/kg) at pH 6·65 increased both pelleted casein and pelleted calcium phosphate. Opposite effects were obtained by adding citrate or EDTA. The change in pelleted calcium phosphate was not simply related to casein release from the micelle. Upon adding 5 mmol EDTA/kg milk, 20% of the pelleted Ca, 22% of the pelleted phosphate and 5% of the micellar casein were removed. Increasing the concentration of EDTA to 10 mmol/kg milk decreased the pelleted Ca by 44% and the pelleted phosphate by 46%, and caused 30%of the micellar casein to be released. The effects of adding phosphate, citrate or EDTA at pH 6·65, followed by the addition of CaCl2, demonstrated the reversibility of the dissolution and formation of the micellar calcium phosphate. There were limits to this reversibility that were related to the amount of colloidal calcium phosphate removed from the casein micelles. Adding CaCl2 to milk containing [ges ] 20 mmol EDTA or [ges ] 30 mmol citrate/kg milk did not result in complete reformation of casein micelles. Light-scattering experiments confirmed that the dissolution of moderate amounts of colloidal calcium phosphate had little effect on micellar size and were reversible, while the dissolution of larger amounts of colloidal calcium phosphate resulted in large reductions in micellar size and was irreversible.

We have investigated the capacities of Geotrichum candidum strains to produce sulphides from methionine. This attribute is very important in cheese technology because of the flavouring potential of sulphur compounds. A spectrophotometric procedure using 5,5′-dithiobis(2-nitrobenzoic acid) to determine sulphides was tested on a collection of G. candidum strains, and confirmed by gas chromatography–mass spectrometry. The strains were distinguished on the basis of their ability to produce methanethiol. Gas chromatography–mass spectrometry also made it possible to identify other sulphides, such as dimethyl disulphide, dimethyl trisulphide and dimethyl sulphide. Using sonicated cells, the specific production of these four sulphides was studied in presence of L-[S-methyl-2H]methionine. Both methanethiol and dimethyl sulphide were produced from methionine, but two different pathways were used by G. candidum.

DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk, industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.

Lamb rennet pastes were prepared by the procedure most commonly used by Idiazabal cheese manufacturers. We studied the effects on their coagulating and lipolytic activities of the state of the stomach at the time of death (full of milk or empty), the amount of NaCl added, the origin of the lambs and paste storage time. Coagulating activities were generally between 155 and 363 units/g tissue. Pastes prepared from stomachs of lambs from slaughterhouse flocks had significantly higher coagulating activities than those of lambs from separate flocks. No significant decrease in coagulating activity was observed after 1 year storage at 4 °C. Chymosin represented 75–80% of the total coagulating activity with the remainder being pepsin. Rennet paste extracts with pH < 4·7 did not have increased coagulating activities when their pH was lowered to 2·0, while those with pH > 5·2 had activities 1·5-fold those before treatment. Lipase activity was higher in extracts of rennet pastes prepared using the stomachs of lambs that arrived at the slaughterhouse in the morning just prior to slaughter than in those prepared with the stomachs of lambs that had arrived on the previous evening. However, the reverse was the case for esterase activity. Activating the coagulating activity by pH cycling completely destroyed both lipolytic activities. Storage at 4 °C for > 1 year did not affect esterase activity but lipase activity decreased substantially after 4–5 months. Lipase, but not esterase, activity was responsible for the liberation of short-chain free fatty acids from ovine milk fat.

Whey protein isolate (WPI), either untreated or pretreated at 80 °C for 30 min, was incubated with a proteinase from Bacillus licheniformis until a gel was formed. Standardized reaction times, directly linked to the degree of hydrolysis, were obtained from plots of the relative amount of peptides released v. reaction time obtained under different conditions (enzyme concentration, temperature, pH, NaCl addition). This provided a connection between the gelation profile and the degree of hydrolysis. In the case of untreated WPI, gelation occurred at lower degrees of proteolysis when the enzyme concentration was decreased, demonstrating that a rate-limiting aggregation process occurred at the same time as the proteolysis in a manner similar to the renneting of milk. This was not the case for preheated WPI, when gelation was found to take place at a constant degree of proteolysis, independent of the enzyme concentration. In this case, the mechanism could be described by assuming the thermally induced aggregates present in this substrate had progressively more stabilizing peptide segments shaved off, resulting in increased attraction between individual aggregates that ultimately led to gelation. Results obtained at 40–60 °C supported this, as we found no effect of temperature on the degree of proteolysis at gelation for the untreated WPI, whereas the degree of proteolysis decreased with increasing temperature when heated WPI was hydrolysed. The effect of pH and NaCl addition on the process was to reduce repulsion between the aggregating species so that gelation was induced at a decreased degree of proteolysis.

The effects of heat treatment of milk, and a range of rennet and glucono-δ-lactone (GDL) concentrations on the rheological properties, at small and large deformation, of milk gels were investigated. Gels were made from reconstituted skim milk at 30 °C, with two levels each of rennet and GDL. Together with controls this gave a total of sixteen gelation conditions, eight for unheated and eight for heated milk. Acid gels made from unheated milks had low storage moduli (G′) of < 20 Pa. Heating milks at 80 °C for 30 min resulted in a large increase in the G′ value of acid gels. Rennet-induced gels made from unheated milk had G′ values in the range ∼ 80–190 Pa. However, heat treatment severely impaired rennet coagulation: no gel was formed at low rennet levels and only a very weak gel was formed at high levels. In gels made with a combination of rennet and GDL unusual rheological behaviour was observed. After gelation, G′ initially increased rapidly but then remained steady or even decreased, and at long ageing times G′ values increased moderately or remained low. The loss tangent (tan δ) of acid gels made from heated milk increased after gelation to attain a maximum at pH ∼ 5·1 but no maximum was observed in gels made from unheated milk. Gels made by a combination of rennet and GDL also exhibited a maximum in tan δ, indicating increased relaxation behaviour of the protein–protein bonds. We suggest that this maximum in tan δ was caused by a loosening of the intermolecular forces in casein particles caused by solubilization of colloidal calcium phosphate. We also suggest that in combination gels made from unheated milk a low value for the fracture stress and a high tan δ during gelation indicated an increased susceptibility of the network to excessive large scale rearrangements. In contrast, combination gels made from heated milk formed firmer gels crosslinked by denatured whey proteins and underwent fewer large scale rearrangements.

Mastitis can be caused by a number of bacteria (Philpot & Pankey, 1975), among which Staphylococcus aureus is one of the most important, responsible for considerable economic loss to the dairy industry (Jasper et al. 1982). Carrier rates have been studied extensively in dairy herds suffering from mastitis (Davidson, 1961, 1963).

Subtyping bacteria is an important epidemiological tool; for example, antimicrobial susceptibility patterns (antibiograms) have been used for typing Staph. aureus in human medicine (Gillespie et al. 1990). Staph. aureus of bovine origin can be divided into several categories by biotyping (Devriese, 1984). Phage typing has proved useful in differentiating mastitis strains (Mackie et al. 1987) and plasmid profiling has been valuable in epidemiological studies of bovine Staph. aureus (Baumgartner et al. 1984).

During the last 5 years genomic fingerprinting of Staph. aureus became a powerful tool for epidemiological typing. Numerous techniques for comparison of staphylococcal isolates have been developed and are becoming important in investigations of strain origin, clonal relatedness and epidemiology.

The aim of this study was to determine the epidemiology of Staph. aureus isolates from mammary glands of cows from a single herd using modern molecular typing techniques, and to assess whether the same strain constantly colonizes the udder or cows are repeatedly infected by different strains. We investigated 26 Staph. aureus isolates obtained from quarter milk samples from 16 cows. These isolates were characterized biochemically, by their antibiotic resistance pattern, by two polymerase chain reaction (PCR) methods on the basis of coagulase (Coa) gene and protein A (Spa) gene (X region) polymorphism and by macrorestriction analysis of chromosomal DNA using pulsed-field gel electrophoresis (PFGE).

The sterol fraction of milk is of nutritional interest because high levels of cholesterol in plasma (modulated by the cholesterol ingested) are associated with an increasing risk of cardiovascular disease. In addition, some sterols (ergosterol and 7-dehydrocholesterol) are provitamins (D2 and D3 respectively). At the same time, through the study of the sterol fraction, vegetable fats can be detected in milk and dairy products. Sterols are a minor fraction of total milk fat, the main sterol being cholesterol (3 mg/g fat, equivalent to 100 mg/l cows' milk). Small quantities of other sterols (7-dehydrocholesterol, 22-dehydrocholesterol, ergosterol, fucosterol, lanosterol, lathosterol, 24-methylenecholesterol) and several phytosterols have been reported in cows' milk (Walstra & Jennes, 1984). International Dairy Federation (1992) states that in the sterol profile of genuine milk fat there may appear, in addition to the peak of 7-dehydrocholesterol which ranges from 0·7 to 4% of total sterols, < 1% of minor sterols with retention times corresponding to phytosterols.

Values for the cholesterol content of goats' milk vary considerably, from 211 mg/l (Pantulu et al. 1975) to 125 mg/l (Lu, 1993), partly owing to the use of different analysis techniques. Some of these values were obtained using non-specific colorimetric methods, which are inaccurate in the presence of cholesterol precursors or phytosterols (Clark et al. 1983; Haugh & Harzer, 1984). Some minor peaks have been assumed to be sterols but have not been identified (García-Olmedo & Barrera, 1985).

Conventional methods of sample preparation for sterol analysis prior to gas chromatography (GC), which involve saponification of fat with or without isolation of the sterol fraction by thin layer chromatography, are tedious and time-consuming. Transesterification with KOH–methanol has been successfully used as a rapid alternative for obtaining the unsaponifiable fraction.

This paper describes the identification of sterols (cholesterol and other minor sterols) in goats' milk fat using an alkali-catalysed transesterification procedure prior to GC and GC–mass spectrometry (GC–MS) analysis.

Among specific genes that may affect economically important traits in sheep, the β-lactoglobulin (LGB) locus has been extensively studied. Polymorphism has been detected in several breeds, but studies of the effect of LGB alleles on milk production traits have given conflicting results. Some found that LGB polymorphism significantly affects milk yield (Bolla et al. 1989; Herget et al. 1995; Fraghì et al. 1996), fat and protein content (Garzon & Martínez 1992; Giaccone et al. 1997; Kukovics et al. 1998), only fat content (Pirisi et al. 1998) and cheese yield and composition (Di Stasio et al. 1997; Rampilli et al. 1997). However, other studies failed to detect any effect of the gene on milk production traits (Barillet et al. 1993; Recio et al. 1997). These inconsistencies, similar to those reported for dairy cattle, can be explained by breed differences, population size, frequency distribution of the genetic variants and a failure to consider relationships among animals (Sabour et al. 1996).

Moreover, both the production data considered and the methods used for statistical analysis could be further causes of conflicting results (Ng-Kwai-Hang, 1997). Investigations of the relationships between milk protein polymorphism and milk production usually consider accumulated yields for standardized lactation lengths, assuming that environmental effects average out over a lactation. Such an assumption is not always valid, because there can be marked effects peculiar to individual test day (TD) measures that may not average out (Jamrozik & Schaeffer, 1997). The direct modelling of TD measures offers the advantage of a more accurate removal of environmental variation from phenotypic observations (Stanton et al. 1992). However, particular attention to the temporal dependence of the covariance structure among TD is required. In TD analysis performed by mixed linear models a simple covariance structure, known as compound symmetry, is usually assumed. This structure assumes an equal variance for all TD and an equal correlation between all pairs of TD within each lactation. An initial drawback of this assumption arises because of the heterogeneity of variance throughout lactation. Moreover, since TD values within a lactation are a sequence of repeated measures taken on the same experimental unit (Van der Werf & Schaeffer, 1997), measures close in time are likely to be more highly correlated than measures far apart in time. All these potential patterns of correlation and variation may combine to produce a complicated structure of covariance among TD that, when ignored, may result in inadequate analysis or incorrect conclusions (Littel et al. 1998). In particular, there can be marked differences in the estimates of the fixed factors considered in the analysis; such a bias is enhanced when the data structure is highly unbalanced, as in the case of studies on relationships between milk protein polymorphisms and milk production traits.

A possible solution can be found in the property of mixed linear models to assume different (co)variance structures in order to find the one that best fits experimental data. The aim of the present study was to test the possible influence of the statistical model used on the results when the relationships between β-lactoglobulin polymorphism and milk production traits in dairy ewes were analysed. With this aim in view, TD measures were directly modelled with mixed linear models and the effects of alternative (co)variance structures on fixed factors estimates were compared.

In the Iberian Peninsula, the proteinases present in the flowers of members of the Cynara genus, C. cardunculus, C. humilis and C. scolymus, have for many years been successfully used in the manufacture of traditional cheeses from ovine and/or caprine milk on individual farms (Vieira de Sá & Barbosa, 1972; Trujillo et al. 1994). In Portugal, C. cardunculus is the species most frequently employed. Although commercial thistle was tentatively assumed to be pure in taxonomic terms, accurate analyses have shown that the flowers of C. cardunculus are often mixed with flowers of C. humilis (Pires et al. 1994). The clotting activity of C. humilis is due to an aspartic proteinase, currently designated cardosin A and similar to another enzyme obtained from C. cardunculus. This enzyme is similar in specificity and activity to chymosin (Pires et al. 1994).

The action of cardosin A from C. cardunculus upon ovine and caprine caseins has been reported recently (Ramalho-Santos et al. 1996; Simo4es, 1998; Sousa & Malcata, 1998), but as yet there is no information on the proteolytic activity of the proteinase from C. humilis upon caseins from milks other than bovine. Caseins from small ruminants' milks are the usual substrates of cardosin during milk coagulation and cheese ripening, and sodium caseinate represents an intermediate system between isolated caseins and the cheese matrix that is free from interference by fat. Thus ovine and caprine caseinates may be useful substrates for investigating the proteolytic activity of cardosin.

The aim of the present study was to compare the action of pure cardosin A, obtained from C. humilis, on caprine and ovine caseinates, and to assess the in vitro contribution of this enzyme to the overall proteolytic action of thistle rennet.

Bacillus cereus is a common contaminant in raw milk. The spores survive pasteurization and psychrotrophic strains of B. cereus often limit the keeping quality of pasteurized milk stored at > 6 °C (Griffiths, 1992). High numbers of B. cereus in pasteurized milk are most frequent when the cows are grazing (Slaghuis et al. 1997), mainly owing to increased levels of spores in raw milk resulting from teat contamination by soil (Christiansson et al. 1999). However, high numbers can also be found in pasteurized milk while the cows are housed indoors, and this is probably caused by additional contamination at the dairy plant (te Giffel et al. 1996; Larsen & Jørgensen, 1997; Lin et al. 1998). There is little information available about the sites of recontamination in the dairy. The use of typing techniques capable of discrimination below the species level, such as fatty acid profiles and random amplification of polymorphic DNA–polymerase chain reaction (RAPD–PCR), could be helpful in demonstrating contamination routes (Lin et al. 1998; Nilsson et al. 1998).

Spores of B. cereus are very hydrophobic and readily adhere to surfaces of steel, glass and rubber (Rönner et al. 1990), and short cleaning-in-place programmes do not always eliminate all the spores (Rönner & Husmark, 1992). Spores adhering to surfaces are more difficult to eliminate by disinfectants than spores in solution (te Giffel et al. 1995). Many B. cereus spores germinate rapidly in milk upon heat activation and, if allowed to propagate undisturbed on surfaces, may form biofilms that are extremely difficult to eliminate (Mosteller & Bishop, 1993; Wirtanen et al. 1996; Kumar & Anand, 1998).

This paper describes how we demonstrated the involvement of a pasteurizer in the contamination of pasteurized milk by B. cereus in a commercial dairy plant using a combination of classic microbiological analyses and typing of strains by RAPD–PCR.

In response to nutrition guidelines recommending a reduction in saturated fats in human diets, the dairy industry has developed new products containing unsaturated fats to satisfy the demand of the more health conscious consumer. The fatty acid composition of milk, naturally rich in saturated fatty acids (SFA), can be modified either through genetic selection of dairy cows or by changing feed composition (Palmquist et al. 1993). For example, a number of dairy products including butter (Wood et al. 1975; Badings et al. 1976), Gouda (Badings et al. 1976) and Cheddar (Wong et al. 1973; Lightfield et al. 1993) containing increased amounts of linoleic acid (18[ratio ]2n–6) have been made from the milk of cows given diets supplemented with unsaturated lipids. However, dairy farmers would prefer to produce milk as cheaply as possible, leaving it to food technologists to modify milk components at the post- production stage (Banks, 1987). Therefore, dairy products made from skim milk combined with a fat mixture could be attractive, but little information is available on this type of modified product. One major problem related to the introduction of unsaturated fats into dairy products is the possible alteration of their properties. Indeed, Badings (1970) reported that butter enriched in polyunsaturated fatty acids (PUFA) has reduced flavour quality and shelf life. It is well known that PUFA are easily oxidized and can form undesirable compounds such as peroxides and aldehydes. Moreover, PUFA are more likely to be oxidized as free fatty acids (FFA) than to be integrated into a triacylglycerol structure. Therefore, when a dairy product is made by recombining skim milk with unsaturated fats, such as the soft ripened cheese in this study, it is important to consider both lipolysis and oxidative stability of the lipid fraction. This was our objective in this study.