Volatile concentrates were obtained by vacuum distillation from both natural and creamy Gorgonzola cheese and isolated by continuous liquid–liquid extraction. Both were analysed by high resolution gas chromatography (HRGC), HRGC–mass spectrometry and HRGC–olfactometry. A total of 63 components were identified in the neutral extract of the natural type (21 esters, 13 ketones, 14 alcohols, 5 aldehydes, 1 sulphur compound, 7 aromatic compounds and 2 terpenes) and 52 in the creamy type (17 esters, 12 ketones, 10 alcohols, 5 aldehydes, 1 sulphur compound, 5 aromatic compounds and 2 terpenes). Ketones, whose major components were 2-nonanone and 2-heptanone, were the predominant constituents of the neutral fraction. By olfactometric analysis of the neutral extracts, 23 odour-impact compounds were found in the natural and 21 in the creamy Gorgonzola cheese. 1-Octen-3-ol, ethyl hexanoate, 2-nonanone, 2-heptanone, 2-heptanol, ethyl butanoate, 2-nonanol and 4-methylanisole were the key odorants of the natural cheese, whereas 2-heptanone, 2-heptanol, ethyl butanoate, 3-methyl thiopropanal and an unidentified constituent with a fruity odour were characteristic of the creamy Gorgonzola cheese. On the basis of high odour unity values, 2-nonanone, 1-octen-3-ol, 2-heptanol, ethyl hexanoate, methylanisole and 2-heptanone were the most important odorants of natural and creamy Gorgonzola cheese aroma.

TWO models of lactating dairy cows were constructed by combining mechanistic models of digestion and metabolism reported previously and by adding elements to allow simulation of changes in tissue metabolic capacities over time. One model (day version) had an integration interval of 0·005 d and was suitable for simulation of within-day dynamics of nutrient supply and partition. The other (lactation version) had an integration interval of 1 d and was suitable for simulation of full lactation cycles. A number of simulation analyses were conducted to characterize and evaluate the models, to examine quantitative and dynamic properties of mechanisms which influence partition of nutrients, to identify aspects requiring further study, and to illustrate the potential usefulness of mechanistic, as compared to empirical, models in analyses of energy balance in lactating dairy cows.

Occurrence of milk ejection and course of milk removal were investigated in 18 dairy cows at milking intervals of 4, 8 and 12 h in early, mid or late lactation. Milk ejection occurred fastest in early lactation at a milking interval of 12 h and was delayed at short milking intervals and in late lactation. Storage capacity of the udder was estimated and the actual milk yields of experimental milkings were calculated as a percentage of storage capacity, i.e. degree of udder filling. It was shown that the occurrence of milk ejection after the start of teat stimulation is a function of udder filling. The relationship between the degree of udder filling and the delay from the start of milking until commencement of milk ejection followed a linear regression curve. Changes in occurrence and course of milk ejection have to be considered in practical milking, mainly in late stages of lactation and after short milking intervals. In automatic milking systems where variable and sometimes extremely short milking intervals occur, the duration of pre-milking udder preparation should be adapted to the expected milk yield at each individual milking procedure.

The colloidal properties of micellar casein are reviewed. It is shown that the behaviour of intact micelles is much at variance with the predictions from the Schulze–Hardy rule, and that therefore their stability cannot be explained by the principles of the DLVO theory. Towards electrolyte, micelles behave as a protein rather than a lyophobic colloid.

Casein is a strong protective colloid. In the micelle, however, it does not completely cover the inorganic constituent which remains sensitive to changes in the ionic environment.

The rate theory of the enzyme-induced clotting of casein micelles is summarized. It is shown that the lag phase in the clotting is due to the second order of the coagulation reaction. Flocculation rate constants of micelles have been deduced from clotting times. Their relatively low values can be attributed to an orientational constraint. Practical consequences of the theory with respect to clot structure, gelation of sterilized products and cheese manufacture are discussed.

Plasmin (EC 3.4.21.7) was added to cheese milk to assess its contribution to Cheddar cheese ripening; the activity of plasmin in the cheese was increased by levels ranging from 1·5 to 6 times that in the control cheeses. Even at the highest level of added plasmin, no activity was found in the whey. β-Casein was degraded faster in the experimental cheeses than in the controls, and the concentration of γ-caseins increased concomitantly. The total N in the water-soluble extract was up to ˜ 20% higher in the experimental than in the control cheeses but phosphotungstic acid-soluble N was not affected by the plasmin activity in the cheese. Several differences were apparent in the gel electropherograms of the water-soluble extracts of the experimental and control cheeses; some peptides were present at higher concentrations in the former, others in the latter, suggesting that plasmin contributes to both the formation and degradation of water-soluble peptides in cheese. The organoleptic quality of the plasmin-enriched cheeses was judged superior to that of the controls and ripening was considerably accelerated; a plasmin level 3–4 times the indigenous value appeared to be optimal. No bitterness was detected in any of the cheeses.

The aggregation of casein micelles in milk by the action of rennet, examined by electron microscopy and viscometry, was correlated with visually-observed coagulation. The spatial relationships between micelles were quantified from measurements made on micrographs. Aggregation did not start until about 60% of the rennet-clotting time, when the enzymic action was almost complete. The micelles tended to form chains which then linked into a loose network. This became more extensive with time as the average size of the aggregates increased. By the clotting time, most micelles were in contact with another and a network was beginning to form. Initially, micelles probably linked by bridges. These then appeared to contract, bringing the particles into contact and eventually causing partial fusion.

The effects of manual teat stimulation compared with stimulation by the liner of a conventional milking machine and the influence of milk ejection on the course of milk flow during the entire milking were investigated. Twelve cows were milked with or without prestimulation on three quarters and milk flow was recorded while intramammary pressure (IMP) was recorded in the left front quarter and blood samples for the determination of oxytocin (OT) were taken at 1 min intervals. OT concentrations increased very similarly in response to manual teat stimulation or liner stimulation and remained elevated during the entire milking procedure. Milk flow curves were generally bimodal without stimulation and not bimodal during milking after prestimulation. Milk yield was not significantly lower in milkings without than with stimulation, whereas machine-on time was prolonged and peak milk flow rate was reduced during milking without stimulation. Time to reach milk flow plateau, time to reach peak flow rate and IMP profiles were similar in both treatments if time was calculated from the start of stimulation. IMP increased similarly in response to manual or liner teat stimulation. In milkings with and without prestimulation IMP did not reach its maximum until the start of milking. In bimodal milk flow curves the second increase of milk flow occurred concomitantly with the start of IMP increase; ejection IMP generally occurred concomitantly with the milk flow plateau. In some cases during milking without prestimulation only small and transient OT release was observed. In response, IMP did not increase to its maximum and increased further in response to a stripping-related OT release. During the course of milking IMP decreased slightly because of the emptying of the three milked quarters. After the end of milking, and with OT concentrations decreasing to the premilking values, IMP again decreased slightly to another plateau, probably owing to myoepithelial relaxation. Thus the course of milk ejection, i.e. the IMP profile, influenced milk removal markedly during early milking.

A longitudinal study involving 73 primiparous (PP) and 47 multiparous (MP) Holstein cows was conducted over an 8-month period to assess the associations between locomotion score (LCS) and milk production, dry matter intake (DMI), feeding behaviour, and number of visits to an automatic milking system (AMS). Twice weekly, all cows were locomotion scored (scale 1–5) by the same observer. Individual eating behaviour and individual feed consumption at each cow visit to the feed troughs, individual milk production, the time of milking, and the number of milkings for each cow were recorded for the day of locomotion scoring and the day before and after. Dependent variables, such as milk yield, DMI, etc. were modelled using a mixed-effects model with parity, LCS, days in milk (DIM), the exponential of −0·05 DIM, and the interaction between parity and LCS, as fixed effects and random intercepts and random slopes for the linear and the exponential of −0·05DIM effects within cow. LCS did not affect time of attendance at feed troughs, but affected the location that cows occupied in the feed troughs. The time devoted to eating and DMI decreased with increasing LCS. Milk production decreased with LCS>3. The number of daily visits to the AMS also decreased with increasing LCS. The cows with high LCS were fetched more often than the cows with low LCS. Overall, PP cows were more sensitive to the effects of increasing LCS than were MP cows. The decrease in milk production observed with increasing LCS seemed to be affected similarly by the decrease in DMI and by the decrease in number of daily visits to the AMS. A further economic loss generated by lame cows with AMS will be associated with the additional labour needed to fetch them.

The in vivo formation of biologically active caseinopeptides was studied. It was proved that bioactive peptides were released in the small intestine of minipigs in the course of luminal digestion of diets containing bovine casein. An opioid peptide and a phosphopeptide were isolated from jejunal chyme and were chemically characterized. The opioid peptide has been identified as a fragment of β-casein (60–70). This peptide, named β-casomorphin-ll, displayed substantial opioid activity in an opiate receptor-binding assay. The caseinophosphopeptide has been shown to be a fragment of αs1-casein (66–74). Casein-derived phosphopeptides exhibit a potent ability to form soluble complexes with Ca and trace elements. Evidence exists that casomorphins and caseinophosphopeptides participate in the regulation of nutrient entry.

Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) αs1-casein content. Parenteral sensitization to GM1 and GM2 proteins was also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine β-lactoglobulin (β-lg), α-lactalbumin (α-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (ΔIsc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-β-lg IgG1, with an important cross reactivity between goat and cow β-lg. However, in guinea pigs fed on GM2, anti-goat β-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3·5±4·5 μA/cm2) compared with that observed in GM1 guinea pigs (8·3±7·6 μA/cm2). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats' milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.

The current status of knowlege of the major proteins of milks of various species is evaluated. Most of the non-bovine milk proteins are homologous with the recognized families of those of Bos taurus, αs1-caseins, αs2-caseins, β-caseins, K-caseins, β-lactoglobulins, and α-lactalbumins, each family representing a separate genetic locus specific to the mammary gland. No prominent milk protein not homologous to one of these families has yet been discovered in milk of any species. Genetic polymorphism resulting from substitutions in the polypeptide chains and various degrees of post-translational phosphorylation, glycosylation, and proteolysis have been identified in milk proteins of several species. Total protein production ranges among species from about 0.5 to 10 g/d per kg0.75 maternal weight. Proportions of the several proteins vary greatly among species, but few accurate analytical data are available except for total casein and total whey protein contents.

Colostrum and milk samples from 60 Holstein–Friesian cows were analysed for concentrations and yields of immunoglobulin G (IgG), β-lactoglobulin (β-lg), α-lactalbumin (α-la) and serum albumin (BSA) throughout the first 16 milkings post partum (8 d of lactation) using a single radial immunodiffusion assay. Concentrations (mg/ml, means±SD) at first milking were IgG 59·8±28·5, β-lg 14·3±4·6, α-la 2·04±0·6, BSA 1·21±0·44. Large variations were recorded for IgG concentrations (15·3–176·2 mg/ml) and yields (0·2–925 g). Cows in their first lactation produced significantly lower concentrations and yields of colostral IgG than cows in later lactations. A colostral yield of IgG below the 100 g required to prevent calf hypo-γ-globulinaemia was found in 18·3% of the cows. The concentrations of IgG, β-lg and BSA dropped abruptly in subsequent milkings and α-la concentration decreased slowly. The mean IgG concentration was <2 mg/ml after eight milkings and <1 mg/ml after fifteen milkings. However, IgG concentration did not differ significantly, at the 1% level, during milkings 11–15. 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 the milk supply.

Improved methods have been evolved for the separation of the total protein, casein, albumin, globulin, and proteose-peptone substances of milk.

I. The use of acetic acid-sodium acetate solutions for the precipitation of casein is elucidated, and it is shown that the maximum precipitation of casein is rapidly effected from milk samples of varying casein content by the addition to 10 ml. of milk of about 80 ml. of water at 40° C. and 1·0 ml. of 10% acetic acid solution, followed, after 10 min., by 1·0 ml. of N sodium acetate solution.

This maximum precipitation was found to be 1·0–1·4% greater than by Moir's method, and 2·4–3·8% greater than by the method of the Association of Oflicial Agricultural Chemists.

A procedure is suggested for the determination of casein which avoids the tedious transfer and washing of the precipitate, and gives enhanced ease, accuracy and speed of working.

II. The advantages of trichloroacetic acid for the precipitation of proteins in determinations of the total protein and the non-protein nitrogenous substances of milk are discussed.

The trichloroacetic acid methods at present in use are shown not to give complete precipitation, and for this a rapid method employing, at room temperature, a final concentration of 12% acid in the milk-acid mixture is recommended.

III. An accurate method for the precipitation of globulin uncontaminated with either albumin or casein is described.

IV. Methods are given for the precipitation and separation of the albumin and proteose-peptone substances.

Heat-induced gelation (80 °C, 30 min or 85 °C, 60 min) of whey protein concentrate (WPC) solutions was studied using transmission electron microscopy (TEM), dynamic rheology and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (150 g/kg, pH 6·9) were prepared by dispersing WPC powder in water (control), 10 g/kg sodium dodecyl sulphate (SDS) solution or 10 mM-dithiothreitol (DTT) solution. The WPC gels containing SDS were more translucent than the control gels, which were slightly more translucent than the gels containing DTT. TEM analyses showed that the SDS-gels had finer aggregate structure (≅10 nm) than the control gels (≅100 nm), whereas the DTT-gels had a more particulate structure (≅200 to 300 nm). Dynamic rheology measurements showed that the control WPC gels had storage modulus (G′) values (≅13500 Pa) that were ≅25 times higher than those of the SDS-gels (≅550 Pa) and less than half those of the DTT-gels after cooling. Compression tests showed that the DTT-gels were more rigid and more brittle than the control gels, whereas the SDS-gels were softer and more rubbery than either the control gels or the DTT-gels. PAGE analyses of WPC gel samples revealed that the control WPC solutions heated at 85 °C for 10 min contained both disulphide bonds and non-covalent linkages. In both the SDS-solutions and the DTT-solutions, the denatured whey protein molecules were in the form of monomers or small aggregates. It is likely that, on more extended heating, more disulphide linkages were formed in the SDS-gels whereas more hydrophobic aggregates were formed in the DTT-gels. These results demonstrate that the properties of heat-induced WPC gels are strongly influenced by non-covalent bonding. Intermolecular disulphide bonds appeared to give the rubbery nature of heat-induced WPC gels whereas non-covalent bonds their rigidity and brittle texture.

The effects of heat and pH on sulphydryl (–SH) and disulphide (–SS–) groups of β-lactoglobulin (β-lg) A have been studied by heating at different temperatures and pH values in air and at pH 6·9 in the absence of air. At pH 6·9 under aerobic conditions a decrease of –SH groups and an increase of –SS– groups was observed with increasing time and temperature. Only small changes were found under anaerobic conditions. At pH from 3·0 to 9·8 the –SH level decreased while the –SS– level increased up to pH 6·9 and then dropped rapidly. In addition to –SH/–SS– interchange there were reactions to other sulphur-containing compounds as seen from the losses in the total amount of –SH plus –SS– sulphur. The results of gelchromatographic investigations suggest that –SH-initiated –SS– exchange-reactions play an important role in the formation of high molecular weight polymers of β-lg A during heat treatment, and that the formation of intermediates depends on the presence of air.

The protein and peptide fraction of human milk samples collected from mothers of pre- and full-term infants in the first week after parturition was analysed by use of liquid chromatography-mass spectrometry and tandem mass spectrometry. By characterising the peptide sequence, we defined the pathway of casein hydrolysis which leads to the formation of small peptides through intermediate oligopeptides. It was found that the action of a plasmin-like enzyme acting on specific lysine residues is the primary step in casein degradation. This is followed by endopeptidases and/or exopeptidases mediated cleavage of the oligopeptides which, in turn, produces a multiplicity of short peptides differing by one or more amino acid residues. In this process, a series of potentially bioactive peptides (opioid, phosphopeptides) and their precursors are produced.

Lactoferrin (Lf) is a milk iron-binding glycoprotein that plays a role in iron transport and acts as both a bacteriostatic and a growth modulating agent. The aim of this study was to investigate the nature of immune responses induced by repeated oral administration of bovine milk Lf in mice. Groups of ten female BALB/c mice were fed daily for 4 weeks with two doses of protein antigen: a low (0·05 mg/g body weight per d) or high (1 mg/g body weight per d) dose of Lf, or water as a control. A fourth group was immunized intramuscularly with 0·01 mg Lf in complete Freund's adjuvant. Anti-Lf IgA and IgG were detected in the intestinal fluid and serum of mice given Lf. Total immunoglobulins were higher in the intestinal fluid in Lf groups than in the control group. No difference could be detected in the serum. IgA and IgG secretion was enhanced in Peyer's patches and spleen from Lf-fed mice, in comparison with controls. [3H] thymidine uptake into Peyer's patch and spleen cells from both control and Lf-fed mice was enhanced by 75 μg Lf/ml in vitro, but Lf groups had a greater proliferation rate than the control group. These findings suggested that Lf could act as an immunostimulating factor on the mucosal immune system and that activation of the mucosal immune system is dependent on the ability of Lf to bind to the intestinal mucosa.