A total of 78 lactations (25 primiparous and 53 multiparous) in a herd of Murciano–Granadina dairy goats were studied over 3 years. Animals were allocated to two experimental groups: suckling (S) goats were milked once daily until weaning (week 7) and thereafter twice daily; milking (M) goats were milked twice daily from 2 d after parturition. Milk partitioning during milking (machine and machine stripping milk fractions) was recorded every week and the residual milk every 2 weeks. Milk flow rate was studied in 63 lactations on three consecutive days during week 12 or 13. Average machine and machine stripping milk fractions over 210 d were 1·09 and 0·23 1/d for the S and 1·23 and 0·28 1/d for the M group respectively. Machine milk volume and percentage were smaller in the S group during the first 7 weeks of lactation, while the machine stripping fraction was unaffected by group, indicating that this fraction was constant. The average residual milk was 11·1 and 9·2% of total milk in the S and M groups. Goats in their third lactation had the least residual milk (8·9%). Milk flow and total machine milk volume (but not milking time) were affected by parity, second and third lactation goats having higher values. Positive correlations were found between daily milk yield and milk flow characteristics. Residual milk was positively correlated with the machine stripping but not with the machine milk fraction. The results indicated that Murciano–Granadina goats can readily be machine milked, since > 80% of the milk can be obtained without massage or stripping.

Thirty-nine multiparous Holstein cows were used to measure the effect of propylene glycol treatment around parturition on milk yield, reproductive efficiency and some hormone and metabolite concentrations. Cows were assigned randomly to control (n = 19) or propylene glycol treated (n = 20) groups. Propylene glycol (300g) was administered directly mixed with the diet from day 10 prior to the expected calving date until parturition (day 0) and orally after dilution in 1 l water on days 3, 6, 9 and 12. Blood samples were collected on days −20, −5, 0, 3, 10, 25 and 50 while milk samples were taken weekly until 13 weeks post partum. Body condition scores, recorded on days −20, 15 and 50, were not affected by propylene glycol administration. Propylene glycol did not significantly affect milk yield or composition but linear somatic cell score measured from the first 13 weeks post partum was reduced by propylene glycol administration (P < 0·01). Moreover, propylene glycol reduced milk urea (−25 mg/l, P < 0·05), especially during the first 9 weeks post partum. Plasma insulin concentrations were similar in both groups during the experiment while insulin-like growth factor I (P < 0·05) and insulin-like growth factor-binding protein 3 (P < 0·001) levels were higher on days 10, 25 and 50 post partum in the propylene glycol group. Propylene glycol administration decreased plasma non-esterified fatty acid concentrations (P < 0·05 to P < 0·01) but increased total cholesterol levels (P < 0·01) after parturition while 3-hydroxy-butyrate levels were unaffected by the treatment. Changes in the hormone and metabolic concentrations after propylene glycol administration in the last few days of gestation and the first week of lactation seem to indicate that energy balance in the treated group was probably more positive than in the control group. There was also evidence that propylene glycol administration prevented fatty liver syndrome and hastened the resumption of oestrous cycles (P < 0·001).

The transport of lysine by perfused lactating rat mammary tissue has been examined using a rapid, paired-tracer dilution technique. This experimental approach allowed the characteristics of lysine transport across the blood-facing aspect of the mammary epithelium to be studied. The clearance of lysine from the perfusate was influenced by the extracellular lysine concentration in a fashion consistent with the presence of carrier-mediated transport. Replacing extracellular Na+ with N-methyl-D-glucamine had no significant effect on lysine transport. Lysine uptake was inhibited by extracellular leucine and glutamine but not by α-(methylamino)isobutyric acid. Leucine interacted with lysine transport under Na+-free conditions. It appears that the system for cationic acid transport which is situated in the blood-facing aspect of the lactating rat mammary epithelium may also accept neutral amino acids as substrates.

The effects of heat treatment and subsequent acidification of milk on the distribution of proteins, Ca and Pi, between the serum and micellar phases were examined using ultracentrifugation. After heating milk at 85 °C for 10 min, and storing for 22 h at 4, 20 or 30 °C, there was a marked increase in the concentration of κ-casein in the serum. At 4 and 20 °C there was also slightly more β-casein in the serum from heat-treated milk than in that from the corresponding raw milk. The whey proteins were extensively denatured, and were almost equally distributed between the supernatants and micellar pellets. After storage for 22 h the distribution of Ca and Pi between soluble and colloidal phases in heat-treated milk was similar to that in raw milk. After acidifying heat-treated milk by the addition of glucono-δ-lactone and storing for 22 h at 4, 20 or 30 °C there was progressive solubilization of colloidal calcium phosphate with decreasing pH, and at pH 5·0 almost all of the Ca and Pi was present in the serum. At 20 °C, and even more so at 4 °C, serum concentrations of the individual caseins increased considerably with decreasing pH, reaching maximum levels of about 25 and 40% of the total casein at pH 5·7 and 5·5 respectively, and then decreasing rapidly at lower pH. Compared with raw milk, maximum dissociation in heat-treated milks stored at 4 and 20 °C occurred at higher pH, and the overall levels of dissociation of individual caseins from the micelles were lower. At 30 °C, the concentrations of individual caseins in the serum of heat-treated milk decreased steadily as the pH was reduced, and did not show the slight increase found previously for raw milk. The role of the denatured whey proteins in interacting with κ-casein and in promoting aggregation of the micelles on acidification is discussed.

Five ovine αsl-casein variants (A–E) were identified in an Italian population sample using gel electrophoresis at alkaline pH, gel isoelectric focusing, two dimensional gel electrophoresis, and immunoblotting with polyclonal antibodies against αs1-casein. Each casein sample produced two peaks by fast reversed-phase HPLC. Gel isoelectric focusing and electrospray mass spectrometry were used to demonstrate that the first HPLC peak contained the 191 residue αsl-casein molecular species and the second the 199 residue species, in proportions of ∼20:80. Only in the case of the sample containing αsl-casein CE was the method for the separation of the single short and long forms of each variant unsuccessful. Both two dimensional electrophoresis followed by staining with polyclonal antibodies against αsl-casein and electrospray mass spectrometry showed a heterogeneity consistent with that expected from a protein chain with three levels of phosphorylation and two different lengths. However, especially for αsl-caseins D and E, a further uncharacterized heterogeneity was detected.

κ-Casein was purified from a single batch of whole acid casein (κ-A variant) using different methods in order to compare their merits in producing a purified material with a carbohydrate and phosphate heterogeneity representative of the whole κ-casein complement in milk. Ion-exchange methods of purification gave products of higher purity than precipitation techniques involving final purification by ethanol fractionation, but all methods resulted in κ-caseins of apparently similar heterogeneity and chemical composition. The purified κ-caseins were hydrolysed with chymosin and the derived macropeptides isolated. These were all virtually identical as determined by reversed-phase chromatography and gel electrophoresis. Some observations on chymosin hydrolysis of κ-casein were made. In addition to formation of the major para-κ-casein (Glu1–Phe105) and macropeptide (Met106–Val169), chymosin hydrolysis at pH 6·6 also resulted in two minor para-κ-caseins with N-termini corresponding to Phe18 and Ser33 of κ-casein. At pH 5·5 and 4·5 para-κ-casein was rapidly hydrolysed into at least six fragments, one of which had an N-terminus corresponding to Trp76 of κ-casein. At pH 6·6, 5·5 and 4·5 the κ-casein macropeptide was stable to chymosin, but at pH 2·3 it was hydrolysed by chymosin into fragments with N-termini corresponding to Met106, He125, Ala138, Val139, Thr145 and Glu147 of κ-casein.

A microparticle-enhanced nephelometric immunoassay has been developed for the determination of caseinomacropeptide (CMP) in bovine milk. It is based on the nephelometric quantification of the competitive immunoagglutination of a microparticle–CMP conjugate with an anti-κ-casein (κ-CN) antiserum. This one step immunoassay was sensitive (detection limit in reaction mixture, 16μg/l), accurate (linear recovery of CMP in dilution overloading) and reproducible (CV 7–14% for within and between run precision). Because of the specificity of the polyclonal antiserum used, it was necessary to separate CMP from κ-CN by ultrafiltration before the quantification of bovine milk CMP. Under the conditions of milk ultrafiltration used, κ-CN was entirely retained (> 99·5%) but the concentration of CMP measured in milk ultrafiltrates was underestimated (by ∼25%) compared with its concentration in whole milk. Microparticle-enhanced nephelometric immunoassay of CMP, with a calibration range from 0·32 to 20 mg/1 for 20- fold diluted milk ultrafiltrate, allowed contamination of bovine milk by rennet whey as low as 5 ml/1 to be detected. Applied to ultrafiltrates from milk stored at 4 °C, this immunoassay also detected proteolysis of κ-CN not revealed by measurement of κ-CN concentration in milk. A statistical lower limit of 3·21 mg/1 was determined as the increase in CMP concentration in milk ultrafiltrates that indicated probable κ-CN proteolysis in the milk sample. Previously demonstrated to be an easy to perform method for assaying the main proteins of bovine milk, microparticle-enhanced nephelometric immunoassay thus also appeared to be appropriate to quantify CMP so as to detect slight contamination of milk by whey and to indicate the proteolysis of κ-CN during milk storage at low temperature.

The water-holding capacities of mineral acid casein (MAC) curd, rennet casein curd and whey protein concentrate (WPC) gel were determined as a function of pH and temperature by drainage of liquid on a stationary, horizontal screen. As precipitation pH was varied over the range 4·3–5·55, the water-holding capacity of MAC curd ranged from 2·4 to 5·2 g water/g dry casein with minimum water-holding capacity at precipitation pH 5·3. Samples of dried MAC curd over the same range of precipitation pH did not vary significantly in water-holding capacity, and all absorbed ∼2·0 g water/g dry casein. Both rennet casein curd and WPC (170 g total solids/kg) gels increased in water-holding capacity as the pH of the suspending liquid increased in the range 3·5–10, but the changes with pH were much smaller than for MAC curd. For both types of casein curd and for WPC gel, water-holding capacity decreased with increase in temperature in the range 5–90 °C. The results are interpreted largely in terms of changes in electrostatic interactions with pH and changes in hydrophobic interactions with temperature.

Fourier transform infrared spectroscopy and differential scanning calorimetry were used as complementary techniques to study changes in the secondary structure of β-lactoglobulin under various physicochemical conditions. The effects of pH (3–9), NaCl (0–2 M), and lactose, glucose and sucrose (100–500 g/l) in the temperature range 25–100 °C on the conformation sensitive amide I band in the i.r. spectrum of β-lactoglobulin in D2O solution were examined. The 1692 cm−1 band in the amide I band profile had not been definitively assigned in previous studies of the i.r. spectrum of β-lactoglobulin. The decrease in this band at ambient temperature with time or upon mild heating was attributed to slow H-D exchange, indicating that it was due to a structure buried deep within the protein. The disappearance of the 1692 cm−1 band on heating was accompanied by the appearance of two bands at 1684 and 1629 cm–1, assigned to β-sheets. The 1692 cm−1 band was therefore attributed to a β-type structure. β-Lactoglobulin showed maximum thermal stability at pH 3 and was easily denatured at pH 9. On denaturation, the protein unfolded into more extensive random coil structures at pH 9 than at pH 3. After 10 h at pH 9 (25 °C), β-lactoglobulin was partly denatured. Heating to 60–80 °C generally resulted in the loss of secondary structure. At all pH values studied, two new bands at 1618 and 1684 cm−1, characteristic of intermolecular β-sheet structure and associated with aggregation, were observed after the initial denaturation. Differential scanning calorimetry studies indicated that the thermal stability of β-lactoglobulin was enhanced in the presence of sugars. The Fourier transform i.r. results obtained provide evidence that sugars promoted the unfolding of β-lactoglobulin via multiple transition pathways leading to a transition state resisting aggregation.

Hydrolysis of β-lactoglobulin B (β-lg B) by pepsin, a process slow at ambient conditions, is facilitated at a moderately high hydrostatic pressure such as 300 MPa, corresponding to an apparent volume of activation ΔV# = −63 ml mol−1 at pH 2·5, 30 °C and Γ/2=0·16. Digestion of β-lg by trypsin and thermolysin is likewise enhanced by pressure, and the pressure effect has been traced to pressure denaturation of β-lg B, which by high-pressure fluorescence spectroscopy has been shown to have a large negative volume of reaction, ΔV° = −98 ml mol−1, at pH 6·7, 30 °C and Γ/2 = 0·16. Pressure denaturation is only slowly reversed following release of pressure and the enhanced digestibility is maintained at ambient pressure for several hours.

Cathepsin D is an indigenous aspartic proteinase in bovine milk. By competitive enzyme-linked immunosorbent assay the amount of immunoreactive cathepsin D and procathepsin D in bovine skim milk was estimated to be 0·4 μg/ml. Immunoreactive cathepsin D purified from whey consisted of a small fraction of mature cathepsin D, but the major form was the proenzyme procathepsin D. A preparation of bovine milk procathepsin D was, like mature cathepsin D, able to degrade purified αs1-, αs2-, β- and κ-casein and α-lactalbumin, while β-lactoglobulin was resistant to cleavage. The cleavage sites in these proteins were determined and compared with those of chymosin. Cathepsin D was capable of generating the αs1-I, β-I, β-II and β-III fragments originally described from the action of chymosin on the respective caseins, and these fragments were subjected to further proteolysis. Cathepsin D was also able to liberate the caseinomacropeptide from purified κ-casein, and to coagulate bovine skim milk. This demonstrated that milk contains an indigenous coagulation enzyme present mainly in the whey fraction.

A tributyrin esterase was purified from Lactococcus lactis subsp. cremoris E8 using FPLC chromatography. This was the major esterase activity observed in strain E8 and was associated with a single protein with a subunit molecular mass of 29 kDa and a holoenzyme of molecular mass 109 kDa. The enzyme was active against tributyrin and p-nitrophenyl butyrate. The N-terminal sequence of the enzyme was determined. The enzyme had a pH optimum in the neutral range, was stable on freezing at −20 °C, and had a half life of 1 h at 50 °C.

Polyclonal antibodies raised against partly purified aminopeptidase were specific to cell-free extracts of Lactobacillus casei subsp. pseudoplantarum UL 137, and did not cross react with any other proteins in Cheddar cheese, at least during the first week of maturation. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed in order to quantify cell-free extracts in the cheese curd, and was also used to determine the efficiency of encapsulation of cell-free extracts in liposomes. This method was very sensitive and exhibited a detection limit of ∼10 μg total protein/g cheese and ∼1 μg total protein/ml liposome suspension. The efficiency of encapsulation of cell-free extracts into liposomes was ∼55–60%. The retention of liposome-encapsulated cell-free extracts was ∼14 times that of non-encapsulated extracts.

Some media available for the isolation and enumeration of starter cultures employed for the manufacture of cheese, yogurt and bio-yogurt were examined. Reddy's medium or a modification of Elliker's medium was found to be most satisfactory for Lactococcus spp., while trypticase phytone yeast (TPY) agar with a mixture of antibiotics proved suitable for the discrete enumeration of Bifidobacterium spp. The inclusion of Prussian blue (PB) in reinforced clostridial medium or tryptone proteose peptone yeast extract (TPPY) agar gave excellent differential counts for the starter bacteria in yogurt even when the culture was imbalanced, while TPPY (PB) agar allowed the visible separation of all four of the organisms that might be found in a typical bio-yogurt, namely Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, a, Bifidobacterium sp. and Lb. acidophilus. It was noted that variation among different strains of any given species could change the expected reactions, so for quality control purposes the suggested media may need to be modified to cope with the specific cultures in use.

In a dairy spread (800 g lipid/kg, 10 g salt/kg) based on 750 g milk fat/kg and 250 g rapeseed oil/kg fat in 15 g extruded catering packaging, there was a more significant accumulation of cholesterol oxidation products than in butter (minimum 800 g lipid/kg, 12 g salt/kg) in 10 g extruded catering packaging when stored at 4 or at 20 °C. There was a lag phase of 7 weeks in cholesterol oxidation in dairy spread stored at 4 °C, while no lag phase was observed for storage at 20 °C. Total concentrations of oxysterols were, however, very similar for dairy spread stored at 4 and 20 °C after 13 weeks storage (∼ 12 μg/g milk lipid); storage at – 18 °C almost prevented cholesterol oxidation (∼ 4 μg/g milk lipid). For butter, cholesterol oxidation was less pronounced at 4 °C (< 3 μg/g milk lipid) than at – 18 °C (∼ 4 μg/g milk lipid) and 20 °C (∼ 7 μg/g milk lipid). 7-Ketocholesterol was the dominant oxidation product, with 1·3 and 5·7 μg/g milk lipid in butter and dairy spread respectively after 13 weeks storage at 4 °C.