Systematic factors affecting the activities of L-lactate dehydrogenase (LDH) and N-acetyl-β-D-glucosaminidase (NAGase) and somatic cell count (SCC), the association between the activities of LDH and NAGase and SCC with respect to udder health status, and the ability of LDH and NAGase to classify cows in udder health categories for early detection of mastitis were studied. A dataset of records from 74 Danish Holstein, 76 Danish Red and 47 Jersey cows on one research farm was used. Cows were grouped into healthy and clinically mastitic. A healthy cow was defined as having no veterinary treatment and SCC<100000 cells/ml. A clinically infected cow was one receiving veterinary treatment after showing clinical signs of mastitis and SCC >800000 cells/ml. Breed, month of production, and days in milk significantly influenced (P<0·001) LDH activity, NAGase activity and SCC in both healthy and clinically mastitic cows. In healthy cows, LDH activity, NAGase activity and SCC started at a high level immediately after calving and decreased to low levels approximately 30–40 d post partum. All the three parameters increased due to clinical mastitis. NAGase activity had numerically higher variation in healthy cows than in clinically mastitic cows (CV=56·2% v. CV=53·5%). The relationship between LDH activity and SCC was stronger in milk from clinically mastitic than from healthy cows (r=0·76 v. r=0·48 and r=0·67 v. r=0·44 for correlation of observed values and residuals, respectively). LDH activity had higher sensitivity than NAGase activity (73–95% v. 35–77%) while specificities were in a similar range (92–99%). Further, sensitivities for LDH activity were more robust to changes in the threshold value than those for NAGase activity. Opportunities for automated, in-line real-time mastitis detection are discussed.

High pressure (HP) treatment affects many constituents of milk (for reviews see Huppertz et al. 2002; Needs, 2002); particular in the properties of casein micelles in HP-treated milk differ considerably from their counterparts in untreated milk. In milk treated at 100–200 MPa, average casein micelle size differs little from that of untreated milk (Needs et al. 2000a; Huppertz et al. 2004a; Regnault et al. 2004; Anema et al. 2005), but micelle size in milk treated at 250 MPa for [ges ]15 min is considerably higher than in untreated milk, probably due to HP-induced aggregation of casein micelles (Huppertz et al. 2004a,b; Regnault et al. 2004); after treatment at 300–800 MPa, micelle size is ~50% lower than that in untreated milk (Needs et al. 2000a; Huppertz et al. 2004a,b; Anema et al. 2005). HP-induced changes in average casein micelle size are irreversible on subsequent storage, except for the increase in micelle size after treatment at 250 MPa (Huppertz et al. 2004a).

Phosphopeptides derived from digests of milk casein possess bioactive properties with gastrointestinal, immunological, vasoregulatory and nutritional activities (Clare & Swaisgood, 2000; Kitts & Weiler, 2003). Products of tryptic digestion of casein, yielding caseinphosphopeptides (CPP), bind to divalent minerals such as iron and calcium by ionic interactions that involve phosphoseryl residues (Kitts & Yuan, 1992; Aìt-Oukhartar, 2000). Distribution of phosphoserine moieties varies with the individual native caseinates, and the extent of phosphorylation directly influences CPP mineral binding affinity (e.g. αs2>, αs1>β-caseins). The anionic pentapeptide (SerP-SerP-SerP-Glu-Glu) is the distinctive feature for the major fractions of casein phosphopeptides (CPP) characterized both in vitro and in vivo. Common CPP derived from tryptic digests of whole bovine casein in vitro include, β-casein-4P (1–25), αs1-casein-5P (59–79), αs2-casein-4P (1–21) and αs2-casein-4P (46–70) (Kitts & Kwong, 2004).

The authors report the results of a study aimed at the comparison of the basic chemical composition, the main protein fractions distribution, rennet coagulation properties and Parmigiano-Reggiano cheese yield of vat milk from Italian Brown and Italian Friesian herds. Parmigiano-Reggiano cheese factories which manufacture milk separately from Italian Brown herds and Italian Friesian herds were used in the study. Thirteen cheesemaking trials were performed at 10 different commercial cheese factories. The study was carried out from March to October 2003. For each cheesemaking trial in each factory, approximately 1100 kg milk from Italian Brown cows and from Italian Friesian cows were processed in parallel. The animals involved in the study came from farms with comparable management practices, size, location, number of lactation and days in milking. Each vat contained milk obtained by combining milk collected during the evening milking (partially skimmed milk by natural creaming) and the following morning milking (full-cream milk), from at least 2 dairy herds. Milk from Italian Brown cows is characterised by a higher casein content (27·1 v. 23·7 g/kg; P[les ]0·0001) than Italian Friesian cows' milk. Curd firming time (k20) of Italian Brown cows' milk was markedly lower than that of Italian Friesian cows' milk (6·6 v. 10·0 min; P[les ]0·001). This implies a higher rate of aggregation of para-casein micelles for Italian Brown cows' milk. The coagulum of Italian Brown cows' milk had better rheological properties and lower losses of fat in the cheese whey. Parmigiano-Reggiano cheese yield at 24 h was also higher for Italian Brown cows' milk, +0·99 kg cheese for every 100 kg vat milk.

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3–6 zones at pH 4·6–5·1, while that of kid pepsin was at pH [les ]3·0. Kid pepsin contained 0·37 molecules phosphorous per molecule and was totally inhibited by 5 μM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5·6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin <kid chymosin <kid pepsin.

Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1·1 mg/l. Within-assay and between-day variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples containing bovine Hp. Coefficients of variation varied between 3·6 and 8·6% at concentrations between 4·0 and 12 mg/l, respectively. Agreement between the results obtained by the biosensor assay and the ELISA was satisfactory; however, the results obtained by the biosensor were generally lower than the results obtained by the ELISA. Possible explanations for this observation are discussed.

The growth of five bacteria isolated from red-smear cheeses, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variabile, Microbacterium gubbeenense and Staphylococcus saprophyticus in mixed cultures with Debaryomyces hansenii on aseptic model cheese curd at 10 and 14 °C was investigated. At both temperatures, C. casei and Micro. gubbeenense had a longer lag phase than C. variabile, Brevi. aurantiacum and Staph. saprophyticus. In all cultures, lactose was utilised first and was consumed more rapidly at 14 °C than at 10 °C, i.e., 6 d at 14 °C and 10 d at 10 °C. This utilisation coincided with the exponential growth of Deb. hansenii on the cheese surface. Lactate was also used as a carbon source and was totally consumed after 21 d at 14 °C and ~90% was consumed after 21 d at 10 °C regardless of the ripening culture. Small differences (<0·5 pH unit) in the surface-pH during ripening were noticeable between ripening cultures. Differences in the colour development of the mixed cultures with the yeast control were only noticeable after 15 d for Brevi. aurantiacum and after 21 d for the other bacteria. Regardless of the organisms tested, colour development and colour intensity were also greater at 14 °C than at 10 °C. This study has provided useful information on the growth and contribution to colour development of these bacteria on cheese.

In development of fermented dairy products and protein hydrolysates with high inhibitory activity towards angiotensin-converting enzyme (ACE), it is crucial to have a reliable assay for measuring the ACE activity. In the present study, the performance of two commonly used assays based on synthetic N-derivates of tripeptides as substrates were tested with respect to reliability in determination of ACE activity per se and to the inhibitory effect of a tryptic whey protein digest and captopril. In one test, the ACE activity was calculated from the amount of hippuric acid liberated from hippuryl-His-Leu (HHL) during 30 min of incubation with ACE, as quantified after HPLC separation of reaction products. In the other assay, the ACE activity was measured directly as the rate of decrease in the absorbance at 340 nm during the first 30 min of ACE catalysed hydrolysis of furanacroloyl-Phe-Glu-Glu (FA-PGG). Both assays, in the absence of inhibitor, showed a good performance with relative standard deviation between replicate samples around 7%. In the presence of inhibitor solutions, relative standard deviations for both assays varied between 1 and 18% for the variously diluted inhibitors. Both assays gave values for the concentration of inhibitor needed to inhibit ACE by 50% similar to those previously reported for whey protein digests and captopril. Different results from the two assays, however, emphasize the importance of controlling the actual ACE-activity for comparison between assays. The limitations of each assay are discussed. Considering the fewer steps in the assay using FA-PGG as substrate, and thus less time and chemicals consumed per sample, and the simpler equipment needed, this assay is recommended for the screening of clear peptide samples.

The capability of goats to maintain milk production during water deprivation is remarkable and not yet fully understood. The aim of the present study was to investigate whether intravenous infusions of hypertonic NaCl cause release of both vasopressin and oxytocin and whether the peptides, in combination with the hyperosmolality, affect milk flow and milk composition. Six Swedish domestic landrace goats in their first to third lactation were milked every 30 min during experiments. Hypertonic NaCl (HNaCl) or isotonic NaCl (IsoNaCl) were infused for 90 min. Goats were not allowed to drink during infusions. Plasma vasopressin concentration increased during HNaCl infusions, and did not change in response to IsoNaCl infusions. Plasma oxytocin concentration did not change during either infusion. Milk flow was maintained during the infusions. Milk fat concentration decreased in the three samples taken before onset of the infusions, but then increased gradually during HNaCl infusions, while it continued to fall during the IsoNaCl infusions. Milk osmolality followed the rise in plasma osmolality during the HNaCl infusions and did not change in IsoNaCl experiments. Milk lactose concentration increased throughout both series of experiments, the concentration being higher during HNaCl infusions. Milk protein concentration did not change during HNaCl infusions, but fell in the IsoNaCl experiments. It is concluded that the hyperosmolality in combination with elevated plasma vasopressin levels did not disturb the secretory activity of the mammary cells, but rather facilitated emptying of the alveolar milk. Such a mechanism may help to explain the sustained milk production in water deprived goats.

This study investigated the application of near infrared (NIR) reflectance spectroscopy to the measurement of texture (sensory and instrumental) in experimental processed cheese samples. Spectra (750 to 2498 nm) of cheeses were recorded after 2 and 4 weeks storage at 4 °C. Trained assessors evaluated 9 sensory properties, a texture profile analyser (TPA) was used to record 5 instrumental parameters and cheese ‘meltability’ was measured by computer vision. Predictive models for sensory and instrumental texture parameters were developed using partial least squares regression on raw or pre-treated spectral data. Sensory attributes and instrumental texture measurements were modelled with sufficient accuracy to recommend the use of NIR reflectance spectroscopy for routine quality assessment of processed cheese.

Cow throughput in an automatic milking system (AMS) is limited by system parameters such as the time required for pre-milking udder preparation and cup attachment, physiological responses of the cow (such as milk let-down and milking-out rate), milking machine features and cow behaviour. A single-factor cross-over design was used to investigate the effect of pre-milking teat brushing on milk processing time in an AMS operating in an extensive grazing farming system. Teat brushing consisted of two roller brushes tracking up each teat three times (total brushing time of up to 45 s/cow). Cows were allocated to one of two treatment groups with either no brushing (NB) or brushing (B) for a 4-week period before being changed to the other treatment. Teat brushing resulted in shorter average cups-on-time (B=506·1 s, NB=541·0 s, P=0·0001), longer average milk processing time (B=10·30 min, NB=9·76 min, P=0·001) and no difference in daily milk yield (B=14·67, NB=14·71 kg/cow, P=0·826). There was no difference between the two treatments in the success of cup attachment (B=3·76%, NB=5·10% unsuccessful milking attempts, P=0·285). The estimated time cost of pre-milking teat brushing was 53 min for every 100 milkings, equivalent to an additional 5–6 milkings for every 100 milkings by an AMS. The importance of these potential time savings is discussed in relation to automatic milking in farming systems that aim for a lower per cow milking frequency and high ratio of cows to AMS.

Osteopontin (OPN) is an acidic phosphorylated glycoprotein found in many tissues and physiological fluids. OPN is known to possess multiple biological functions including bone remodeling, cell adhesion, cellular transformation and immunological mediation; these functions have been extensively reviewed (Butler et al. 1996; Sodek et al. 2000).

The bovine β-casein (CSN2) gene has been shown to span a region of 8·5 kb, containing nine exons and eight intervening introns (Bonsing et al. 1988; Martin et al. 2002). The exons range in size from 24 to 498 bp; the introns, however, are much larger and account for 85% of the gene. Twelve genetic variants in the coding sequence of the β-casein gene have been reported (Farrell et al. 2004). The A2 allele of the β-casein gene has been associated with a higher milk production (Lin et al. 1986; Bech & Kristiansen, 1990) while the B variant has been associated with an increase in protein content and better cheesemaking properties (Marziali & Ng-Hang-Kwai, 1986). The β-casein gene codes for a protein of 209 amino acids with varying regions at codons 67, 106 and 122. The A1 variant differs from A2 at position 67, where a histidine replaces a proline (Lien et al. 1992). The β-casein A2 variant has histidine and the A3 variant has glycine at position 106 (Lien et al. 1992); the β-casein A2 variant has serine at position 122 and the β-casein B variant has arginine at this codon (Stewart et al. 1987; Damiani et al. 1992).

Raw whole milk of high microbial quality ([les ]4×104 cfu/ml) was processed using a ~15 l/h homogeniser with a high pressure (HP) valve immediately followed by cooling heat exchangers. The effects of homogenisation between 100 and 300 MPa (HP valve) with an initial milk temperature Tin=4 °C or 24 °C was investigated on the inactivation of: (i) endogenous alkaline phosphatase (ALP); (ii) endogenous milk flora and (iii) two Gram positive (Listeria innocua and Micrococcus luteus) and one Gram negative (Pseudomonas fluorescens) strains inoculated into milk. Temperatures T1 and T2 measured before and immediately after the HP valve, and fat globule size distributions were also determined. ALP activity slightly decreased after homogenisation above 250 MPa when Tin=4 °C (corresponding T2>58 °C), but markedly decreased above 200 MPa when Tin=24 °C (T2>60 °C). In contrast to inactivation induced by continuous short-time thermal treatments, ALP inactivation induced by HP homogenisation was clearly due to mechanical forces (shear, cavitation and/or impact) in the HP valve and not to the short ([Lt ]1 s) residence time at temperature T2 in the same valve. Inactivation of the three exogenous microorganisms led to similar conclusions. Homogenisation at 250 MPa or 300 MPa (Tin=24 °C) induced a 2–3 log cycle reduction of the total endogenous milk flora and a 1·5–1·8 log cycle reduction of inoculated List. innocua. Higher reduction ratios (2–4 log cycles) were obtained for the two other microorganisms. The highest levels of ALP inactivation corresponded to the highest extents of microbial reduction. Running the milk twice or three times through the homogeniser (recycling), keeping temperature T1≈29 °C and pressure=200 MPa, increased homogenisation efficiency.

The influence of ethanol on the rennet-induced coagulation of milk was studied to investigate potential synergistic effects of these two mechanisms of destabilisation on the casein micelles. Addition of 5% (v/v) ethanol reduced the rennet coagulation time (RCT) of milk, whereas higher levels of ethanol (10–20%, v/v) progressively increased RCT. The temperature at which milk was coagulable by rennet decreased with increasing ethanol content of the milk. The primary stage of rennet coagulation, i.e., the enzymatic hydrolysis of κ-casein, was progressively slowed with increasing ethanol content (5–20%, v/v), possibly due to ethanol-induced conformational changes in the enzyme molecule. The secondary stage of rennet coagulation, i.e., the aggregation of κ-casein-depleted micelles, was enhanced in the presence of 5–15% ethanol, the effect being largest at 5% ethanol. Enhanced aggregation of micelles is probably due to an ethanol-induced decrease in inter-micellar steric repulsion. These results indicate an interrelationship between the effects of ethanol and chymosin on the casein micelles in milk, which may have interesting implications for properties of dairy products.

Bovine milk contains a number of indigenous proteolytic enzymes, of which plasmin is the most important (Grufferty & Fox, 1988; Bastian & Brown, 1996; Kelly & McSweeney, 2003). Plasmin (EC 3.4.21.7) is a serine proteinase with pH and temperature optima of 7·5 and 37 °C, respectively. In milk, most of the plasmin is present as its inactive precursor, plasminogen, which is converted to active plasmin by plasminogen activators (PA) present in milk, e.g., urokinase-type (u-PA) and tissue-type PA (t-PA) (Bastian & Brown, 1996). Since plasmin, plasminogen and PA are associated with casein micelles, they are incorporated into cheese curd, while plasmin inhibitors and inhibitors of PA are lost with the whey. Plasmin incorporated in cheese curd acts on its substrate, the caseins, contributing significantly to primary proteolysis during ripening (Upadhyay et al. 2004b).

The experiment, which lasted 53 d, was conducted during the winter (February and March) of 2004 and used 48 Comisana ewes in mid lactation. A 2×2 factorial design was used, with ewes receiving two levels of dietary crude protein (CP) (moderate, 16% CP v. low, 13% CP) in the dry matter (DM) and being exposed to two ventilation rates (moderate, 47 m3/h v. low, 23·5 m3/h per ewe) for each dietary treatment. Air concentrations of NH3 and of microorganisms were measured twice weekly. Milk yield was recorded daily. Individual milk samples were analysed weekly for composition and fortnightly for bacteriological characteristics. After the last milk sampling (day 49 of the study period), four animals from each group were placed in a metabolism box and their individual faeces and urine were collected for three consecutive days. Amounts of urine and faeces excreted, and urinary and faecal N outputs were measured. The 16% CP diet resulted in a lower milk casein content and a higher milk urea concentration than the 13% CP diet, as well as in a reduced gross efficiency of utilization of dietary N, a greater amount of N excreted and a higher total coliform concentration in milk. The moderate ventilation rate resulted in higher yields of milk, irrespective of CP content. Significant interactions of CP level×ventilation rate were found for the amounts of urine, of total water and of faecal N, and for mesophilic concentration in milk, the highest values being displayed by the ewes fed the 16% CP diet and exposed to the low ventilation rate. The moderate dietary CP level and low ventilation rate had a deleterious effect on air concentrations of microorganisms and ammonia. Results suggested that a reduction of dietary CP level from 16 to 13% of DM had no detrimental effect on ewe milk yield in mid lactation and could even improve some of its nutritional and hygienic characteristics. Our findings also indicate that the choice of a proper ventilation rate is critical for high efficiency of production in the lactating ewe, especially in intensively managed flocks receiving diets high in CP.

Difficulties with the analysis of opaque milk and dairy products can be resolved by rendering the sample transparent. This allows direct photometric measurements of the reaction mixtures, and all sample pre-treatment steps become unnecessary. For this, several reagents have been reported in literature. Among them, the ‘Clarifying Reagent’ is mainly described here: its composition is given because it is now released from patenting restrictions. Its main property is a low absorbance between 340 and 800 nm. So it can be used in many chemical and enzymatic colorimetric measurements for estimating the quality of milk and dairy products.

Biogenic amines (BA) are toxic substances that appear in foods and beverages. Tyramine is the most abundant BA in cheeses. A PCR method was developed to detect the presence of tyramine-producing bacteria during cheese manufacture and ripening. Six different batches of a farmhouse blue cheese were analysed by PCR. Tyramine concentrations were also determined by HPLC. The PCR method was able to anticipate tyramine accumulation in the cheeses; the presence of tyramine-producing microorganisms in the early stages of manufacture correlated well with a high concentration of BA in mature cheese samples.

Casein phosphopeptide amorphous calcium phosphate nanocomplexes (CPP-ACP) in chewing gum, lozenges and mouthrinses have been shown to remineralize enamel subsurface lesions in human in situ experiments. The aim of this double-blind, randomized clinical study was to investigate the capacity of CPP-ACP added to bovine milk to remineralize enamel subsurface lesions in situ. Ten subjects drank milk containing either 2·0 or 5·0 g CPP-ACP/l or a control milk whilst wearing removable appliances with enamel slabs containing subsurface demineralized lesions. Each 200 ml milk sample was consumed once a day for each weekday over three consecutive weeks. After each treatment and one weeks rest the subjects crossed over to the other treatments. At the completion of the treatments the enamel slabs were removed and remineralization determined using microradiography and microdensitometry. The results demonstrated that all three milk samples remineralized enamel subsurface lesions. However, the milk samples containing CPP-ACP produced significantly greater remineralization than the control milk. The remineralising effect of CPP-ACP in milk was dose-dependent with 2·0 and 5·0 g CPP-ACP/l producing an increase in mineral content of 70 and 148%, respectively, relative to the control milk. The differences in remineralization following exposure to the three milk samples were all statistically significant (P<0·001). In conclusion, this study shows that the addition of 2·0–5·0 g CPP-ACP/l to milk substantially increases its ability to remineralize enamel subsurface lesions.