This study was conducted to examine the effects of mastitis and stage of lactation on plasminogen activator (PA) activity in milk somatic cells. An assay System, which measures the plasmin-mediated hydrolysis of the chromogenic substrate D-valyl-leucyl-lysine p−nitroanilide, was used to assess PA activity present within milk somatic cells. Milk cell associated PA activity was increased (P < 0·05) by 50% in the presence of fibrin fragments. This suggests that milk somatic cells contain tissue PA which, unlike urokinase PA, is preferentially activated in the presence of fibrin fragments. An increase of the milk somatic cell count from < 5 × 104 to > 106 cells/ml resulted in an 8-fold increase in PA activity per cell. Elevated levels of PA activity were associated with milk somatic cells isolated from mastitic quarters obtained from cows in early (<4 months in lactation) or late lactation (>8 months in lactation). We conclude that PA activity is increased during severe mastitic inflammation. Although the physiological function of this enzyme is as yet unclear, we propose that it may be involved in the conversion of plasminogen to plasmin, contributing to the higher levels of plasmin occurring in milk isolated from mastitic quarters.

On the basis of complete permeability by high molecular weight reagents of casein micelles in milk and a uniform distribution of the 3 different casein subunits, a model of the micelle structure is proposed. It is composed of an average repeating unit of 1 κ-, 2 αs1;- and β-casein subunits assembled in a 3-dimensional network or branched polymer made of 130–130000 monomers, in which the trimers of κ-casein occupy the nodes and the copolymers of αs1;- and β-caseins make up the branches. All the associations between subunits are through non-covalent bonds. The chemical composition varies with the number of αs1;- and β;-casein subunits in the branches. This proposed structure is strongly supported by evidence from electron microscopy and a scale model has been made. It leads to an understanding of the role of κ-casein in micelle formation and opens new perspectives in explaining some properties of the caseins. It offers an interesting example of a new type of quaternary structure of protein subunits.

Reducing the colloidal calcium phosphate (CCP) content of milk by 40% or increasing it by 20% did not significantly affect the heat-induced pH-dependent dissociation of micellar κ-casein. However, changes in soluble Ca and phosphate affected the dissociation of κ-casein markedly; decreasing the phosphate concentration or increasing the Ca concentration reduced the formation of non-sedimentable N (NSN) and non-sedimentable 12% TCA-insoluble N-acetyl-neuraminic acid (NANA). Dialysis of milk against water for short periods (∼ 5 h) reduced the formation of both NSN and non-sedimentable 12% TCA-insoluble NANA, as did NaCl at concentrations above 0·05 M. Modification of protein amino groups by succinylation promoted the release of κ-casein while amidation of carboxyl groups had the opposite effect. It appears that the pH-dependent dissociation of κ-casein produced on heating milk above 90°C is controlled by electrostatic interactions. The effects of soluble ions such as Ca2+ or Na+ appear to be due to shielding of such negatively-charged groups as seryl phosphate and carboxyl on the protein, thus reducing the release of κ-casein.

Selective dry cow therapy (SDCT) has received increasing attention in recent years owing to global concerns over agricultural use of antimicrobial drugs and development of antimicrobial resistance. The objective of this study was to evaluate the effect of SDCT on milk yield and somatic cell count (SCC) in dairy herds in the USA. Cows in four Ohio dairy herds were categorized into two groups (low-SCC and high-SCC) at dry-off based on their SCC and clinical mastitis (CM) history during the lactation preceding the dry-off. Low-SCC cows were randomly assigned to receive or not to receive intramammary antibiotics at dry-off. Milk yield and SCC of these cows during the following lactation were compared using linear mixed effects models, adjusting for parity, calving season, stage of lactation, previous lactation milk yield and herd. Milk yield of untreated and treated low-SCC cows at dry-off did not differ significantly during the following lactation. Overall, treated low-SCC cows had 16% lower SCC (approximately 35 000 cells/ml, P=0·0267) than the untreated cows during the following lactation; however, the effect was variable in different herds. Moreover the impact of treatment, or the lack thereof, on milk yield varied considerably between herds. The results suggested that in some herds treating all cows at dry-off may be beneficial while in other herds leaving healthy cows without antibiotic dry cow treatment has no negative impact on milk yield or milk quality (SCC), and in fact, may be beneficial. Further studies are needed to identify characteristics of herds where treating all cows routinely at dry-off may be needed for maintaining good udder health and where switching to selective treatment of cows at dry-off would be the optimal approach to achieve best results.

The increase in levels of free fatty acids (FFA) in morning and afternoon milk after 48 h cold storage at 5 °C was determined at two stages of lactation in milk from 96 cows. Lipolysis was positively correlated to day of lactation (r = 0·6, P < 0·001), while the correlations between lipolysis and milk yield and fat content respectively were due to these factors being related to day of lactation. In afternoon milk the level of FFA increased almost three times above that in morning milk. Lipolysis was closely related to the lipoprotein lipase (LPL) activity associated with the cream fraction (r = 0·8, P < 0·001). Afternoon milk had higher LPL activity in the cream fraction than morning milk. Lipase activity in the cream increased with increasing stage of lactation. Cooling of milk increased LPL activity associated with the fat phase. This effect was greater in afternoon milk from late lactation than in morning milk from early lactation. When heparin was added to milk the LPL activity associated with the fat increased substantially; again, this effect was greater in afternoon milk from late lactation than in morning milk from early lactation. Lipolysis was higher in heparinized milk than in normal milk, and there was a close correlation between lipolysis and LPL activity associated with the fat (r = 0·82, P < 0·001). Characteristics of the milk fat globule influencing the attraction of LPL were found to be fundamentally important to lipolysis.

The ‘mushroom compound’ previously reported as a gas chromatographic fraction of oxidized dairy products has been identified as oct-l-en-3-ol. The alcohol was found to have a flavour threshold value of 1 part in 109 in water, 1 part in 108 in skim-milk, and 1 part in 107 in butterfat. A mechanism for the formation of oct-1- en-3-ol is discussed.

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.

The effects of increased somatic cell count, whether caused by infection or by experimental infusion of bacterial endotoxin, on the distribution in milk of caseins between the micellar and soluble forms were investigated. The relationship of somatic cell count to some cheese-making parameters was also studied. With quite modestly elevated cell counts (2–3 × 106/ml) increases of up to 37% in total casein in the soluble phase were observed, most of which was contributed by β-casein, while κ- and αs1-caseins increased only slightly. With storage at 4°C, the concentrations of all the caseins, Ca and phosphate in the soluble phase increased substantially during the first 48 h, but this was followed by a slight decline on further storage. Rennet clotting time, losses of fat in whey, curd moisture, and losses in curd yield and rigidity were all greater the higher the somatic count. Clear differences were detectable in these parameters between milks of very low cell count (e.g. 5 × 104 cells/ml) and milks with counts more typical of those found in bulk supplies (e.g. about 5 × 105 cells/ml). If these findings can be reproduced in commercial practice even a modest reduction in bulk milk somatic cell counts might be expected to bring definite benefits.

Cows were fed either 75 or 100% of the recommended intake levels for protein and 100% of recommended energy levels (Agricultural Research Council, 1965) from 8 weeks pre-calving until 14 weeks post calving. From 14 weeks post calving and to the end of lactation all the cows received 100% of the recommended protein and energy intakes.

The mean of the 305-d milk yields of the 2 groups was not significantly different and although cows on the lower protein intake produced less lactose during the first 14 weeks of lactation there was no significant difference in total lactose, fat, protein or total solids production between the groups. In both groups blood packed-cell volume, red cell count and haemoglobin decreased during the first 10 weeks of lactation and then began to increase in the high-protein group. The cows receiving the low-protein diet showed a similar increase only when they received the high-protein ration from 14 weeks post calving. The mean interval from calving to conception was 27·5 weeks in the high-protein group and 20 weeks in the low-protein group.

It is concluded that feeding 75% of protein requirements to dairy cows during the first 14 weeks of lactation does not reduce milk yield or quality significantly and probably has no adverse effect on fertility.

The aim of the research described here was to compare different methods of body temperature (BT) measurements in dairy cows. It was hypothesised that reticular temperature (RET) values reflect the physiological status of the animals in an equivalent way to rectal (RT) and vaginal (VT) measurements. RT, VT and RET temperatures of twelve lactating Holstein–Friesian cows were measured over five consecutive days in June and October 2013. While RT and VT were manually measured three times a day, RET was automatically recorded at 10 min intervals using a bolus in the reticulum. For comparison with RT and VT, different RET values were used: single values at the respective recording times (RET-SIN), and mean (RET-MEAN) and median (RET-MED) values of 2 h prior to RT and VT measurements. Overall, body temperatures averaged 38·1 ± 0·6, 38·2 ± 0·4, 38·7 ± 0·9, 38·5 ± 0·7 and 38·7 ± 0·5 °C for RT, VT, RET-SIN, RET-MEAN and RET-MED, respectively. RT and VT were lower than all RET measurements, while RET-SIN and RET-MED were higher than RET-MEAN (P < 0·001). RET-MEAN and RET-MED values were higher in the morning, whereas RT and VT were greatest in the evening (P < 0·001). Overall, records of RT and VT were strongly correlated (r = 0·75; P < 0·001). In contrast to RET-SIN and RET-MEAN, RET-MED was higher correlated to RT and VT. In June, coefficients were higher between all methods than in October. Relation of barn T to RT and VT was stronger when compared to RET measurements. RET-SIN was higher correlated to barn T than RET-MEAN or RET-MED. Correlation between VT and barn T was strongest (r = 0·48; P < 0·001). In summary, RET-MED showed highest correlation with VT and RT. However, single RET measurements (influenced by water or feed intake) can lead to extreme variations and differences to single VT and RT values.

To isolate and identify antioxidant peptides from enzymatically hydrolysed whey protein, whey protein isolate was hydrolysed by different protease (trypsin, pepsin, alcalase 2·4L, promatex, flavourzyme, protease N). The hydrolysate generated by alcalase 2·4L had the highest antioxidant activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, superoxide radicals and in a linoleic acid peroxidation system induced by Fe2+. The IC50 values of DPPH and superoxide radical scavenging activities of the hydrolysate decreased significantly (6·89 and 38·88%, respectively) after treatment with macroporous adsorption resin. Seven different peptides showing strong antioxidant activities were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography and high-performance liquid chromatography. The molecular mass and amino acids sequences of the purified peptides were determined using a Quadrupole time-of-flight mass spectrometer (Q-TOF MS). One of the antioxidative peptides, Trp–Tyr–Ser–Leu, displayed the highest DPPH radical scavenging activity (IC50=273·63 μm) and superoxide radical scavenging activity (IC50=558·42 μm). These results suggest that hydrolysates from whey proteins are good potential source of natural antioxidants.

Forty-two multiparous dairy cows of three different breeds (Holstein, Montbéliarde and Tarentaise) were fed on the same type of forage (natural grassland) preserved in the form of either hay (H) or silage (GS), according to a changeover design (two 4 week periods). The proportion of concentrate in the diet and the energy and nitrogen contents were similar in both treatments. The milk produced by these cows was used for the manufacture of Saint-Nectaire type cheeses, under controlled and identical cheesemaking technological conditions. More cheese was produced with the H treatment milk. The cheeses made with the GS treatment milk were more yellow and tended to be more bitter. The other chemical and sensory characteristics did not differ much between the two treatments. Of the 51 volatile compounds identified, four were in significantly higher proportion in the GS than in the H cheeses. Cheeses produced from Tarentaise cows' milk were more yellow and their pH was higher than those made with the milk of Holstein or Montbéliarde cows. The cheeses from Montbéliarde and Tarentaise cows' milk were firmer, more melting and tastier than those made with the milk of Holstein cows. Although some trends were apparent, there were no significant differences in cheese volatile compounds for different breeds.

The sizes of the casein particles in milk heated at 130°C were measured as functions of heating time and of initial pH. The effect of adding 10 mM-urea to the milks was also studied. Turbidities of the milks before and after dispersing the caseinate particles in 8 m-urea/EDTA/β-mercaptoethanol were measured, the latter measurement giving an indication of the extent of covalent cross-linking. Urea was found to modify the behaviour of the milk: diameters of the caseinate particles were considerably altered, and the extent of covalent cross-linking was diminished. These results agree with the observation that more protein is solubilized from the casein micelles when urea is present, indicating that a competitive reaction scheme involving cross-linking in opposition to micellar dissociation may be responsible for the ultimate precipitation of milk by heat.

By resuspending casein micelles in whey and dialysate it is shown that the role of whey proteins in the ethanol (EtOH)-induced coagulation of skim-milk is minimal. Experiments involving the interchange of milk sera indicated that the position of the EtOH stability/pH profile along the pH axis was governed by the diffusible constituents of the milk serum phase. The identities of those serum components governing the shape and position of the EtOH stability/pH profile were investigated. The addition of Ca2+ caused a shift in the entire profile to higher pH. Reduction of the available Ca2+ by addition of EDTA (up to 5 ran) shifted the profile to lower pH. The addition of phosphate (up to 5 mM) or citrate (up to 1 mM) had no effect on the profile, though higher concentrations of citrate (up to 5 mMi) caused slight shifts to lower pH. When equimolar amounts of Ca and phosphate were added, the system showed a shift in profile approximately equivalent to that of the free Ca introduced. Increasing the ionic strength of a milk by the addition of NaCl did not shift the profile, but decreased the maximum EtOH stability of the high pH arm of the sigmoidal profile. The EtOH stability/pH profile retained the same sigmoidal shape in all cases.

Late blowing, caused by the outgrowth of clostridial spores present in raw milk and originating from silage, can create considerable product loss, especially in the production of hard and semi-hard cheeses. The conventional method for the isolation of Clostridium spp. from cheeses with late-blowing symptoms is very complicated and the identification of isolates is problematic. The aim of this work was the development of a multiplex PCR method for the detection of the main dairy-related clostridia such as: Cl. beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum. Samples derived from silage, raw milk and hard cheese were analysed by the most probable number (MPN) enumeration. Forty-four bacterial strains isolated from gas positive tubes were used to check the reliability of the multiplex PCR assay. The specificity of the primers was tested by individually analysing each primer pair and the primer pair combined in the multiplex PCR. It was interesting to note that the samples not identified by the multiplex PCR assay were amplified by V2–V3 16S rRNA primer pair and the sequencing revealed the aligned 16S rRNA sequences to be Paenibacillus and Bacillus spp. This new molecular assay provides a simple promising alternative to traditional microbiological methods for a rapid, sensitive detection of clostridia in dairy products.

Changes in the teat apex before and after different milking treatments were measured with a spring-loaded caliper device known as a cutimeter which could detect changes in thickness of the tissues of the teat end, presumably due to congestion and/or oedema, with a high degree of accuracy (± 2%) and repeatability (r = 0·99). Teat end thickness increased with increasing vacuum level. The mean increase immediately after milking with a conventional cluster was 2% for 24 teats milked at 30 kPa, 8% at 50 kPa and 21% at 70 kPa. At these vacuum levels, the mean increases for the same teats milked with an unconventional (PKME) teatcup were 10, 18 and 25% respectively. Cyclic application of 35 kPa positive pressure to the pulsation chamber of a conventional teatcup operating at 50 kPa reduced teat end thickness by 8% compared with the mean premilking value. Although most teats returned to within ± 2% of their premilking thickness values by 1 h after milking, differences were apparent between different milking systems for up to 4 h postmilking.

The stability of camel α-lactalbumin (α-la) against heat denaturation was measured, using circular dichroism (CD) and fluorescence spectroscopy, as well as differential scanning calorimetry (DSC). The experiments were performed in the presence of saturating concentrations of calcium as well as in the presence of EDTA, yielding to the apo form of α-la. The change in heat capacity (ΔCp) suggests a greater contribution of hydrophobic interactions to the stability of holo camel α-la than in its bovine counterpart. Overall the results obtained in this study suggest a greater stability of camel α-la than the bovine protein in both holo and apo states. Also CD experiments showed similar secondary structure for camel and bovine α-la and secondary structure of camel α-la was better preserved than that of bovine α-la during heat denaturation. The differences in thermal stability between the proteins from two species can be primarily ascribed to the difference in the quantity of hydrophobic interactions involved in their folding.

Grazing cows could produce milk with a higher proportion of polyunsaturated fatty acids, which is beneficial to human health, compared with non-grazing cows, though grazing alone could compromise milk production. Under oceanic climate conditions, a study involving 15 dairy cows, fed total mixed ration (TMR) ad libitum in combination with different grazing times of 12 h (TMR12), 6 h (TMR06) and zero grazing time (TMR00) with the aim to evaluate different strategies on the fatty acids profile of milk and milk production. No differences were seen between the treatments with respect to milk yield (34·4±6·3 kg/d) or milk protein content (30·4±1·8 g/kg). The milk produced by the TMR12 cows had less total fat (36·2 vs. 38·2 g/kg) and saturated fatty acid (FA, 69·39 vs. 71·44 g/100 g FA) than that produced by the TMR00 cows. The concentration of vaccenic acid in the TMR06 and TMR12 milk was twice that of the TMR00 milk (4·22, 4·09 and 2·26 g/100 g FA respectively). Linear increases in conjugated linoleic (CLA) and linolenic acids were observed with increasing grazing time. Pasture was an important source of FA especially C18:3 for TMR06 and TMR12 cows. Under oceanic climatic conditions, the grazing of dairy cows as a complement to feeding with TMR can improve the FA profile of milk and increase its CLA content.

The principal aim of this work was to compare Caciotta cheeses obtained from cow milk previously subjected to high pressure homogenisation (HPH) at 100 MPa with those produced from raw (R) or heat-treated (P) cow milk. HPH had both direct and indirect effects on cheese characteristics and their evolution during ripening. In particular, HPH treatment of milk induced a significant increase of the cheese yield; moreover, it affected the microbial ecology of both curd and cheese. Compared with the thermal treatment, the HPH treatment resulted in a decrease of about one log cfu/g of yeast and lactobacilli cell loads of the curd. The initial milk treatment also affected the evolution over time and the levels attained at the end of ripening of all the microbial groups studied. In fact, lactobacilli, microstaphylococci and yeast cell loads remained at lower levels in the cheeses obtained from HPH milk with respect to the other cheese types over the whole ripening period. Moreover, HPH of milk induced marked and extensive lipolysis. Cheeses from HPH milk showed the presence of high amounts of free fatty acids immediately after brining. The electrophoretic patterns of the different cheese types showed that Caciotta made from HPH-treated milk was characterized by a more extensive and faster proteolysis as well as a significant modification of its volatile molecule profile. The results obtained and the sensory analysis indicated that HPH treatment of milk was able to differentiate Caciotta cheese or to modify its ripening patterns.