Colostrum and milk samples from 20 goats were analysed for concentrations of immunoglobulin G (IgG), β-lactoglobulin (β-lg), α-lactalbumin (α-la) and serum albumin (CSA) throughout the first 14 milkings post partum (7 d of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means±SD) at first milking were IgG 47·9±25·5, β-lg 30·7±10·4, α-la 2·77±0·82 and CSA 2·97±2·46 mg/ml. Large variations were recorded for IgG concentrations (19·9–94·5 mg/ml) and β-lg (9·3–49·8 mg/ml). Concentrations of IgG, β-lg and CSA dropped abruptly in the subsequent milkings and α-la concentration decreased slowly. Mean IgG concentration was <2 mg/ml after 7 milkings and <1 mg/ml after 11 milkings. However, IgG concentration does not differ significantly, at the 1% level, from milkings 7–14. The contribution of β-lg to the increase in whey proteins in early milks was greater than that of IgG from milkings 5 to 14. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to milk supply.

The effects of heat treatment and homogenization of whole milk on chemical changes in the milk fat globule membrane (MFGM) were investigated. Heating at 80 °C for 3–18 min caused an incorporation of whey proteins, especially β-lactoglobulin (β-lg), into MFGM, thus increasing the protein content of the membrane and decreasing the lipid. SDS-PAGE showed that membrane glycoproteins, such as PAS-6 and PAS-7, had disappeared or were weakly stained in the gel due to heating of the milk. Heating also decreased free sulphydryl (SH) groups in the MFGM and increased disulphide (SS) groups, suggesting that incorporation of β-lg might be due to association with membrane proteins via disulphide bonds. In contrast, homogenization caused an adsorption of caseins to the MFGM but no binding of whey proteins to the MFGM without heating. Binding of caseins and whey proteins and loss of membrane proteins were not significantly different between milk samples that were homogenized before and after heating. Viscosity of whole milk was increased when milk was treated with both homogenization and heating.

The aim of this study was to test if the opioid antagonist naloxone has a beneficial effect on normalization of oxytocin (OT) release during repeated milking of cows in unfamiliar surroundings. One control milking without naloxone treatment in all cows was performed in the familiar parlour. For four successive evening milkings, cows were transported to, and milked in, the operating theatre of the research station without (control group) or with naloxone administration (1 mg/kg BW) (naloxone group) before milking. After cessation of spontaneous milk flow, but not before 3 min of milking, vaginal stimulation was applied for 2 min. After milk flow ceased again, 10 IU of OT was injected intravenously to remove the remaining milk including residual milk. Milk flow was recorded continuously and blood samples were collected via a jugular vein cannula at 1-min intervals from 1 min before the start of milking until i.v. injection of OT. The inhibition of milk ejection and its normalization during repeated milking in unfamiliar surroundings was not influenced by naloxone treatment. Concentrations of cortisol and β-endorphin during control milking and all relocations were similar in the naloxone and control groups, although their concentrations were higher after relocations than in the control. Therefore, a role of endogenous opioids in the inhibition of milk ejection in unfamiliar surroundings could not be demonstrated. In addition, the effect of exogenous ACTH1–24 (8 IU, i.v.) on the release of cortisol related to the response of cows milked in unfamiliar surroundings was studied. Cows with totally inhibited milk ejection in response to vaginal stimulation during milking after first relocation had numerically, but not significantly lower cortisol levels (8·8±3·4 ng/ml; AUC/min) in response to ACTH than did cows with at least partial milk ejection (38·7±12·9 ng/ml). Thus animals with a higher adrenal response to ACTH seemed to have less severe inhibition of milk ejection.

The reason for the reported difference in spoilage behaviour of skim and whole pasteurised milks was investigated. The rates of growth of psychrotrophic bacteria were not significantly different in the two milks and the bacterial types, all pseudomonads, present at spoilage were also similar. However, when representative spoilage organisms were cultured into freshly pasteurised skim and whole milks, skim milks exhibited predominantly bitter flavours while whole milk showed mostly sour flavours. The different spoilage behaviours can be largely explained by greater proteolysis in skim milk than in whole milk, caused by higher production of protease and greater susceptibility of the protein to protease attack. In addition, lipolysis in whole milk, caused by the substantial quantities of lipase produced by spoilage pseudomonads in this milk, also contributes to the different flavours produced during cold storage of these milk types.

The effect of different parameters (pH, ultrasonic amplitude and pressure) on the resistance to heat and manothermosonication (MTS) treatments of heat resistant lipase and protease produced by Pseudomonas fluorescens B52 and NCDO 2085, respectively, were studied. Lipase B52 thermoresistance decreases with an increase of pH. However, inactivation by MTS seems to be pH independent. There were only slight increases in the MTS efficiency when increasing pressure at UHT temperatures and the effect of amplitude was different depending on treatment temperature. Protease NCDO 2085, which was very resistant to MTS at 30 °C, was very sensitive to MTS at 76 °C. Increases in applied pressure had no effect on MTS efficiency at 140 °C and its inactivation by MTS was almost temperature independent between 76–109 °C. Data obtained are compared with previous published data and inactivation mechanisms are discussed.

The composition of the volatile fraction of milk from cows was investigated in a survey of milk samples using a headspace sampling technique and gas chromatography coupled to mass spectrometry analysis (GC-MS). Milk samples were collected from 12 farms, selected for similar management, breed and level of production. Farms were also grouped according to the type of forage in the ration: (1) hay; (2) hay and maize silage; (3) hay, maize silage and grass silages. Forty-one compounds in milk were isolated and identified from GC-MS headspace analysis. Quantitatively, the most representative chemical class was ketones (eight compounds, 170 μg/kg), followed by aldehydes (nine compounds, 63 μg/kg), alcohols (eight compounds, 36 μg/kg), and lower amounts of hydrocarbons (six compounds), sulphur compounds (three compounds), esters (four compounds) and terpenes (three compounds). The novel headspace sampling technique, and the consequent reduction of sample pre-treatment, allowed the identification of low-molecular weight volatile compounds, and reduced the risk of producing artefacts during analysis.

Discriminant analysis was used to identify a classification criterion for milk samples, using type of forage in the ration as a grouping variable. Posterior probability error rate indicated that aldehydes provided one of the best discriminant criteria for grouping milks according to ration composition. When all 41 identified volatile compounds were included, discriminant analysis selected nine compounds (acetone, 2,3-butanedione, 2-butanone, ethanol, acetaldehyde, ethylacetate, ethylisovalerate, dimethylsulphone) that did not fail the tolerance test and which correctly classified 100% of the original cases.

The International Standards Organization (ISO) produces international standards for milking equipment. These describe the minimum specifications of design, installation, maintenance and testing of milking machines (BS ISO, 1996). Often these standards are adopted and exceeded by national standards. Standards are rarely mandatory and in some countries variations in interpretation and even disagreement can lead to differences in operating conditions of the milking machine.

A survey of plant test results in the UK (Berry & Scrivens, 1997) showed that, on annual or biannual testing, more than 75% of plants failed to meet the then existing standard (BS5545, 1988). In the UK, most types of plant installed since 1994 meet the newest ISO standard (BS ISO, 1996). However, one type of plant has many features different from all other types. This is a type of plant whose major variations include a smaller claw bowl volume (150 ml), a narrower long milk tube diameter (13·5 mm), 8 mm milk pulse tube, greater cluster weight (approximately 3·5 kg) and simultaneous pulsation used on a plant with milk lift and milking at an installed system vacuum of 47–48 kPa. The majority of parlours installed with this equipment at the time of this study had no form of indexing (see below).

A study has been undertaken to assess 20 new installations for compliance with the ISO requirements (BS ISO, 1996), performance in a static test, milking performance, cow behaviour and the effects on teat condition. Compliance to standards and static test results have been reported elsewhere (Ohnstad, 1997), with no plant proving entirely satisfactory. Some plants had major problems with vacuum level, pulsation characteristics and large air leaks. The physical operation of the milking plant has important implications for cow behaviour and welfare and also ergonomics of the milking operation. The effects on teat condition have already been reported (Hillerton et al. 2000). Here the effects on cow behaviour and milking performance are assessed. Comparisons have been made in line with the International Dairy Federation recommendations (IDF, 1997) to examine the performance and interactions of the cow, the machine and the operator in evaluating the milking process.

Lactoperoxidase activity and thiocyanate content were monitored in the milks of seven individual cows and six goats during lactation. Lactoperoxidase activity exhibited a cyclic pattern with alternating peaks and troughs throughout lactation. Extremely large variations were observed between and within animals. For example, lactoperoxidase activity ranged from 0·05–5·60 U/ml for one cow on different sampling days. Variations between cows were also large, ranging from 0·98–5·1 U/ml on a specific sampling day. Mean lactoperoxidase activity in cows ranged from 1·5–2·7 U/ml with an overall mean of 2·3±1·0 U/ml while the thiocyanate concentration ranged from 6·0–10·2 μg/ml with an overall average of 8·5±5·1 μg/ml. Lactoperoxidase activity means for goats ranged from 0·04–0·16 U/ml with a grand mean of 0·1±0·06 U/ml while the thiocyanate content means ranged from 6·6–8·2 μg/ml with a grand mean of 7·0±2·59 μg/ml. There was no correlation between lactoperoxidase activity and thiocyanate content in either cows' (R2 = 0·011) or goats' (R2 = 0·015) milk. These experiments have revealed that lactoperoxidase activity is affected by many factors including the individual animals, species, feed and stage of lactation. Therefore the exogenous supply of thiocyanate and hydrogen peroxide needed to activate the lactoperoxidase system for raw milk preservation will vary in quantity depending on these factors.

Manchego cheese can be manufactured from raw or pasteurized ewes' milk. An automatic purge and trap apparatus, coupled to a GC-MS was used to isolate, identify and compare the relative amounts of the volatile components of raw and pasteurized Manchego cheese during ripening. The majority of volatile compounds were more abundant in raw milk (RM) cheeses than in pasteurized milk (PM) cheeses. Alcohols and esters predominated in the profile of RM Manchego cheese, while methyl-ketones and 2,3-butanedione were quantitatively important in PM cheeses. Branched chain alcohols were much more abundant in RM cheeses. The discriminant analysis separated 100% samples into RM or PM cheeses by using only 16 volatile compounds. Aroma intensity was correlated with esters, branched chain aldehydes and branched chain alcohols in RM cheeses, and with esters, branched chain aldehydes, 2-methyl ketones and 2-alkanols in PM cheeses. Diacetyl was positively correlated with the aroma attribute ‘toasted’ and negatively correlated with aroma quality in PM cheeses.

A homo-tetrameric ∼160-kDa cystathionine γ-lyase was purified to homogeneity from Lactobacillus reuteri DSM 20016 by four chromatographic steps. The activity was pyridoxal-5′-phosphate dependent and the enzyme catalyzed the α,γ-elimination reaction of L-cystathionine, producing L-cysteine, ammonia and α-ketobutyrate. The enzyme was active towards a range of amino acids and amino acid derivatives, including methionine. The pH and temperature optima were found to be 8.0 and 35 °C, respectively. Isoelectric pH (pI) was ∼5.0 as determined by two-dimensional electrophoresis. Sensitivity to chemical inhibitors was typical of lactococcal cystathionine γ- and β-lyases, except it was inhibited by sulphydryl reagents. The N-terminal sequence was MKFNTQLIHGGNSED, which had 100% homology with cystathionine β-lyase of Lb. reuteri 104R (Accession Number CAC05298). Lb. reuteri DSM 20016, together with 10 other strains of non-starter lactic acid bacteria, was used as adjunct starter in the production of miniature Canestrato Pugliese-like cheeses. After 40 d ripening, the water-soluble extract of the cheeses with added Lactobacillus fermentum DT41 and Lb. reuteri DSM 20016 contained the highest enzyme activities on cystathionine and methionine substrates. Determinations of methanethiol, dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide in the miniature cheeses confirmed the findings of enzyme activities.

Unsatisfactory milking conditions, including high milking vacuum, ineffective pulsation, heavy clusters, unsuitable liners, poor teat preparation and over-milking, are known to create poor, but undefined, teat conditions and pose a risk to an increased probability of intramammary infection (IDF, 1994). It has been shown that poor teat condition, seen as discolouration, abnormal firmness or thickened rings of tissue distally, after cluster removal may be common in a variety of commercial milking operations (Hillerton et al. 2000). That field study was observational and teat conditions were influenced by a number of variables. A small experimental study has been undertaken to develop an experimental model, under controlled milking conditions, to allow investigation of potential causes of teat trauma. Over-milking was suggested as highly important in the field observations and so has been examined in this study.

The relative proportion of caseins to total protein is a parameter that can be used to control the protein quality in standardised milk, an increasing tendency in dairy industries. 31P-NMR was used to analyse the casein content of milk, by the quantitation of the area under the resonances belonging to SerP, and using methylenediphosphonic acid as internal standard. This procedure yielded good results, as similar values of caseins were obtained from N Kjeldahl and NMR analysis for slightly heated milk samples. Heating at 95 °C for 15 min did not alter the casein content results. Casein content of raw, pasteurised and UHT milks (25·6±1·4, 26·4±1·8, 25·5±1·6 g casein/l milk, respectively), obtained by NMR, were not significantly different, giving an average of 25·8±1·6 g casein/l for bulk liquid milk. This work concluded that 31P-NMR could be used as an alternative method to determine casein in raw, pasteurised, dry and UHT milks.

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100±21 min and a z-value of 5.9±0.3 °C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39·3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56·5% homology with bovine milk lysozyme and 30·4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.

An automatic purge and trap apparatus, coupled to a GC-MS was used to study the seasonal variability of the volatile fraction of raw milk Manchego cheese. Both season and dairy significantly affected abundance of most volatile compounds. Most aldehydes, methyl ketones, n-alcohols, and secondary alcohols reached significantly higher concentrations in spring cheeses. Branched chain alcohols showed significantly higher concentrations in autumn and winter cheeses, while significantly higher amounts of diketones were found in summer cheeses. Most ethyl esters reached higher concentrations in spring and winter cheeses and lower in autumn cheeses. Lower concentrations of α-pinene were found in spring cheeses, and higher amounts of limonene were observed in winter cheeses. Heptane and octane were significantly more abundant in summer cheeses. No significant seasonal differences were found either for quality or intensity scores.

Samples of bovine caseinomacropeptide (CMP) were isolated from κ-casein A and κ-casein B and fractionated to give aglycosylated CMP A and CMP B and monoglycosylated CMP A. The secondary structures of these three peptides were compared under neutral and acidic (pH 4·2) conditions, using two-dimensional (2D) 1H nuclear magnetic resonance (NMR) spectroscopy. The differences between the spectra at pH 4·2 and 7·0 and the spectra of the aglycosylated and glycosylated CMP A were subtle, indicating little change in backbone conformation with these changes. These results suggest that differences in the coagulation properties of milks containing either κ-casein A or κ-casein B are more likely to be related to factors, such as micelle size or charge, than to structural differences arising from altered backbone conformation of the macropeptide segments of the κ-caseins.

The structure development of a soft cheese curd model has been studied in relationship to its rheological properties and its biochemical characteristics (pH, amount and partition of minerals, casein proteolysis) at different technical steps including cutting, drawing, three turns and demoulding. Scanning electron microscopy was used to observe structural changes during the drainage of a fat-free soft cheese. The micrographs provided visual evidence of changes in the casein matrix from casein particles aggregated in clusters to uniform strands observed at the demoulding. The initial increase of loss tangent and of the exponent of the power law between G′ and G′′ and frequency (that were maximal at the second turn) was related to the solubilization of micellar calcium phosphate, while intact caseins and large casein fragments accumulated in the curd. After the second turn, the strength, Youngs' and loss moduli of the curd increased greatly. The hydrolysis of αs1-casein into αs1-I-CN f(24–199) may facilitate the rearrangement of casein particles within the curd. The pH-induced solubilization of calcium phosphate continued throughout the manufacture process but was unexpectedly incomplete at the end of the drainage. Combination of electron microscopic observations with dynamic rheological measurements and chemical and biochemical assessments provided increased knowledge about the structure of soft cheese during drainage, an important but poorly understood cheese making stage.

Transglutaminase (TGase) is an enzyme that cross-links many proteins, including milk proteins. In this study, the effects of TGase on some physico-chemical properties of milk were studied. TGase-treated milk was not coagulable by rennet, which was due to failure of the primary (enzymic) stage of rennet action rather than the non-enzymic secondary phase. Dissociation of TGase-treated casein micelles by urea or sodium citrate or removal of colloidal calcium phosphate by acidification and dialysis was reduced, presumably due to the formation of cross-links between the caseins. Casein micelles in TGase-treated milks were also resistant to high pressure treatment and to hydrolysis by plasmin. Results of the present study show that milk proteins are fundamentally modified by the action of TGase, which may have applications in the manufacture of functional proteins for use as novel food ingredients.

Experimental Camembert cheeses were made to investigate the effects on browning of the following factors: inoculation with Yarrowia lipolytica, the use of Penicillium candidum strains with different proteolytic activity, the addition of tyrosine, and the addition of Mn2+, thus leading to 16 different variants of cheese. Two physical colour parameters were used to describe browning, depending on the location in the cheeses: a whiteness index for the outside browning (mould mycelium), and a brownness index for the inside browning (surface of the cheese body). Mn2+ promoted a significant increase of browning at both locations, whereas Yar. lipolytica had the opposite effect. Outside browning was significantly more intense when using the Pen. candidum strain with higher proteolytic activity. A significant interaction was found between Yar. lipolytica and Pen. candidum. The yeast had no effect in combination with a low proteolytic strain of Pen. candidum, but significantly reduced proteolysis and browning in combination with a high proteolytic strain of Pen. candidum. We further confirmed that both strains of Pen. candidum were able to produce brown pigments from tyrosine, and thus both are presumably responsible for the browning activity in this type of cheese.

Milk and yogurt constitute a major source of dietary protein. The nutritive value of dietary proteins is linked to subsequent postprandial amino acid availability in the portal blood (Rérat, 1988). Portal absorption of nutrients cannot be studied in humans, but pigs provide a valid model for studying protein digestion in humans (Rowan et al. 1994).

Since stable isotopes are suitable to distinguish the exogenous from endogenous protein fraction in the intestinal lumen, intrinsic isotopic labelling of milk proteins has been considered a useful technique for nutritional investigations (Gaudichon et al. 1995; Gaudichon et al. 1999; Mahé et al. 1994). Recently, the use of 15N-labelled milk proteins were used to distinguish exogenous from endogenous N fractions in the human intestine after ingestion of 15N-milk or 15N-yogurt (Gaudichon et al. 1995). These authors pointed out that the jejunal flux of 15N was different for milk and yogurt. It is known that milk proteins and lactose undergo preliminary hydrolysis during lactic fermentation (Tamine & Deeth, 1980). It is also suggested that lactic fermentation enhances the nutritional value of milk proteins (Vass et al. 1984).