The interaction of sheep, horse, pig, human and guinea-pig whey proteins with fatty acids has been studied. Using gel filtration and autoradiography, it was found that sheep β-lactoglobulin and serum albumin from all species had the ability to bind fatty acids in vitro. Sheep β-lactoglobulin, isolated from milk, had ˜ 0·5 mol fatty acids bound per mol monomer protein, and albumin from sheep, horse and pig contained ˜ 4·5, 2·9 and 4·7 mol fatty acids/mol protein respectively. However, β-lactoglobulin from horse and pig milk had neither fatty acids physiologically bound nor the ability to bind them in vitro. Albumin was the only whey protein detected with bound fatty acids in these species as well as in human and guinea pig. This suggests that the ability of ruminant β-lactoglobulin to bind fatty acids was not shared by the same protein of non-ruminants.

Three major proteins from the proteose peptone of bovine milk were purified by Sephadex G-75 gel chromatography, Q-Sepharose ion-exchange and additional Sephadex G-75 gel chromatography in the presence of urea. From their mobility in a gradient SDS-PAGE, the proteins were found to have molecular masses of 17, 28 and 60 kDa. The N-terminal amino acid sequence of the 17 kDa protein was found to be homologous with a camel whey protein. This protein has not previously been described in bovine milk. From the SDS-PAGE results, the 28 kDa protein was judged to be the major protein of proteose peptone, contributing ~ 25% of the total. The N-terminal amino acid sequence showed no homology to any known protein sequence, but the amino acid composition indicated that the 28 kDa protein is identical with the PP3 component from the proteose peptone fraction of bovine milk, or part of it. The 60 kDa protein was found to be bovine osteopontin, a very highly phosphorylated protein with an Arg-Gly-Asp sequence which mediates cell attachment.

Casein micelle solvation, a micelle characteristic that is sensitive to many factors, has been measured by a centrifugation technique at 30 °C for a series of uncooled fresh skim milks at pH 6·3, 6·6, 6·9 and 7·1. The relative αs-(αs1- plus αs2-), β– and κ-casein contents of all centrifuge pellets and supernatants were determined by a standardized electrophoretic method. The calcium and phosphate contents of a number of the pellets and milk samples were also determined. Solvation of micelles from milks with various genetic variants of β-lactoglobulin (A and B), αs1-casein (A and B) and κ-casein (A and B) was often found to be lower for milks containing either the B variant of αs1-casein or the A variant of κ-casein. It was also found that these two variant caseins were associated with a lower κ-casein content of the milks and the micelles, which is consistent with the lower solvation as κ-casein is associated with smaller micelle size and greater solvation. The solvations also seemed to increase during the lactation period. It is possible that some of the other features of milk and its products that have been ascribed to the differences in functional character between the A and B variants of αs1-casein may be partly caused by the increased level of κ-casein. The reason for the association of the A variant of αs1-casein with higher concentrations of κ-casein (and micelle solvation) is not obvious but possibly the haplotype αs1-casein A, β-casein A1, κ-casein A contains a controlling sequence in the chromosomal DNA that enhances expression of the κ-casein gene.

Proteolysis during storage of UHT skim and whole milks processed by either direct or indirect systems has been studied. All the proteolysis indices determined (measurement of free amino groups, PAGE of caseins and PAGE and reversed-phase HPLC of the fraction soluble at pH 4·6) revealed greater proteolytic degradation during storage of skim milks compared with that of whole milks subjected to the same UHT treatments. Increased activities of both native milk proteinase and proteinases of bacterial origin were observed in skim UHT milks. The different behaviour of UHT skim and whole milks on storage would have to be taken into account in establishing the process conditions.

25Mg, 43Ca and 31P NMR have been used to study the binding of Mg2+ and Ca2+ ions to β-casein A1. The concentration dependence of the line width of the 25Mg NMR signal shows that β-casein contains at least two different types of binding sites for Mg2+ ions, one with strongly bound, slowly exchanging ions and one with more weakly bound ions which undergo fast exchange. The strong Mg2+ binding site has an unexpectedly high binding constant, Kbstrong 104 M–1, which has not been reported earlier. Mg2+ and Ca2+ compete for the Ca2+ binding sites of β-casein, while Na+ does not compete for these binding sites under physiological conditions. The dependence of the 43Ca NMR chemical shifts on total concentration of Mg2+ and Ca2+, in the presence of β-casein, could be equally well fitted with a model assuming up to five identical and independent sites as with a model assuming five or more sites with negative cooperativity. The proton dissociation constant, pka, for the strongest Ca2+ binding site was found to be 7·1.

Rheological properties of whey protein concentrate (WPG) solutions were studied in steady shear, using a Bohlin VOR Rheometer, as a function of concentration, temperature, shear rate, shearing time, pH, salt type, salt concentration and solution age. At 22 °C and pH 7, the WPC solutions exhibited Newtonian behaviour up to a concentration of 10% total solids, pseudoplastic behaviour between 10 and 30% and time-dependent shear thinning at 35% and above. The apparent viscosity of solutions at 22 °C and pH 7 was linearly related to concentration up to 8%. The effect of temperature on apparent viscosity in the range 5–60 °C was closely described by the Arrhenius equation. The viscosities of WPC solutions were independent of solution age in the pH range 4–8 at all concentrations up to and including 20%, the precise pH range narrowing as concentration increased. At pH values above or below this range apparent viscosity became dependent on both pH and solution age, the age effect becoming more marked at higher WPC concentrations. Apparent viscosity at pH 7 increased markedly with both CaCl2 concentration and solution age at concentrations above 0·6 M-CaCl2, the age effect in this case increasing with CaCl2 concentration. In contrast, NaCl concentrations of up to 0·8 M-NaCl had little effect on apparent viscosity. The rheological behaviour of WPC solutions changed from time-independent to time-dependent shear thinning at high concentration, at extreme pH values, at high CaCl2 concentration (after ageing) and on heating to above ∼ 60 °C. This change is considered to be caused by the formation of structure in solutions; a 40% solution (at 22 °C and pH 6·75) exhibited classic thixotropic behaviour in a step–shear rate experiment.

A comparative study was carried out on the flavour constituents of bovine, ovine, caprine and water buffalo fresh raw milks. The volatiles were isolated from milks by means of vacuum distillation and liquid–liquid extraction. Eighty neutral volatile compounds were identified using high resolution gas chromatography (HRGC) and HRGC-mass spectrometry. About 30 of these components have not been detected previously in milk. The volatiles in milk were similar for the four species, although several quantitative differences might explain the different odours. The volatiles consisted mainly of ethyl esters, especially those derived from butyric and hexanoic acids. Dimethylsulphone alone comprised ~ 25% of the volatile components in bovine, caprine and ovine milk, but only 4% in buffalo milk. Among the aldehydes, pentanal and nonanal were the most representative. Other aldehydes were present selectively: 3-methylbutanal was found only in buffalo milk; phenylacetaldehyde and benzaldehyde were present in large quantities in caprine milk, and in trace amounts in the other milks. The content of ketones, mainly 2-methylketones, was higher in buffalo milk than in the other three types. Alcohols varied between species, from 1·5% (bovine) to 5% (buffalo). Among these, 1-octen-3-ol and phenylethanol could be particularly important for the aroma of milk. The level of l-octen-3-ol in the milks was: buffalo > ewe > goat ≫ cow. Phenylethanol was not found in ewes' and goats' milk and its concentration in buffalo milk was 100 times higher than in bovine milk. Finally, indole and 4-methylphenol, interesting because of their odorant properties, were present in higher quantities in buffalo, ewes' and goats' milks than in cows' milk.

Several mathematical models are presented in an attempt to describe the kinetics of the enzyme-induced coagulation of casein micelles. In each model the primary phase of the clotting reaction is assumed to follow first order kinetics. The only differences amongst the various models centre on the definition of the flocculation rate constant, which is defined in seven different ways. The rate constants are defined and discussed in terms of activation energy and functionality theory. The first model is such that the number of functional sites is two. The second is such that the number is much larger. The third and fourth are such that there is an exponential energy barrier, one which has a magnitude proportional to the extent of proteolysis caused by the clotting enzyme. These two definitions differ only in the pre-exponent. In one case the pre-exponent is a constant, whereas in the other it is dependent on the size of clotting particles. The fifth and sixth definitions are also energy barrier rate constants, but the energy barrier changes in an arbitrary fashion with respect to time during proteolysis. The seventh definition assumes a large number of functional sites, but such that the number increases with extent of proteolysis. In the Payens nomenclature (Payens, 1989), all models could be considered to be ‘source’ models, and all are derived using the Drake moment equation (Drake, 1972). Only the first model has a truly constant flocculation rate parameter, and only this model has a relatively simple analytical solution. All other models yield analytical solutions only by way of infinite series expansions. Thus, all models are presented in terms of power series expansions, and only through the first five time-dependent coefficients. This confines all models to the early stages of coagulation. In all cases the first three coefficients are virtually the same. The first two coefficients involve only proteolysis, and the third includes initial flocculation information. Time-dependent changes in the flocculation rate constant begin to take effect in the fourth coefficient. When the fourth coefficients of the third and seventh models are compared, a simple relationship is suggested between free energy barrier removal and functional site generation, but only assuming that the number of functionalities is large.

The interaction of lactoferrin from human and bovine milk with the human promonocytic cell line U937 has been studied. Both human and bovine Fe-lactoferrins bound to the cells. Binding of bovine lactoferrin was inhibited by excess bovine lactoferrin but not by human lactoferrin, suggesting that the binding mechanisms for the two proteins are different. Binding of human but not bovine lactoferrin was inhibited by bovine lactoperoxidase, while a 20-fold excess of human IgA inhibited binding of human but not bovine lactoferrin. Human and bovine α-lactalbumins, bovine β-lactoglobulin, and human lysozyme had no effect on binding of lactoferrin from either species. Samples of bovine Fe- and apolactoferrin in capillary tubes were exposed to temperatures of 72 °C for 20 s, 85 °C for 20 min or 137 °C for 8 s. All the heated samples inhibited binding of native Fe- and apolactoferrin, though to a lesser extent than the native proteins. Both heated and native lactoferrins enhanced [3H]thymidine incorporation by U937 cells, except for Fe-lactoferrin heated at 85 °C for 20 min, which was inhibitory. These results suggest that heat treatment of lactoferrin under conditions used for industrial processing does not greatly affect its ability to interact with and stimulate monocytic cells, and that other milk proteins in general do not interfere with lactoferrin–monocyte interactions. It may thus be feasible to incorporate biologically active lactoferrin into infant formulas.

The main compounds responsible for the aroma of bovine, ovine, caprine and water buffalo freshly secreted milk have been identified by means of a gas chromatography–olfactometry technique. Of the fourteen odour-active volatile compounds detected, eight were present in all milks studied. Ethylbutanoate and ethylhexanoate (fruit-like aroma), among the neutral odorants, were the major contributors to the odour of bovine, ovine and caprine milk. The aroma of water buffalo milk was less dependent on ethylhexanoate and was also due to l-octen-3-ol (aroma of raw mushrooms), nonanal (freshly cut grass), indole (stable, animals) and an unidentified constituent, characterized by a retention index of 828, with a typical odour of warm milk and/or smoked cheese. Other aroma components were specific for some types of milk, contributing to the complexity and richness of flavour.

Defatted whey was obtained by aggregating residual fat to calcium phosphate precipitates and separating the precipitate by membrane microfiltration (pore diameter 0·2 μm). When ultrafiltering this defatted whey the performance of an inorganic membrane (molecular mass cut-off, 10 kDa) was limited by the large concentration of Ca and phosphates. Consequently, the influence of the aggregation pH (either decreasing or constant) on membrane fouling has been studied for ultrafiltration (UF) of defatted sweet whey and defatted whey UF retentates (protein content up to 30g l–1). In all experiments protein rejection was 100%. When pH was kept constant during the pretreatment, membrane fouling was significantly lowered. Hydraulic resistances ascribed to irreversible fouling were in good agreement with fouled membrane analyses performed by i.r. and X-ray photoelectron spectroscopies. They showed that provided a low Ca and phosphate content was maintained in the microfiltrate, which was achieved at constant pH, no apatite was detected within the membrane, and proteins were less fouling. On the other hand, the amount of fouling material depended on the transmembrane pressure gradient along the hydraulic path. On the membrane surface, the higher the pressure, the higher the fouling. In the membrane bulk, the fouling heterogeneity depended on the ability of the defatted whey to precipitate apatite. If it did, the higher the pressure, the higher the calcium phosphate and the protein fouling. With other phosphate structures, the bulk fouling depended on the barrier formed by surface fouling layers and the protein concentration polarization layer, which were more resistant to solute and solvent transfer under higher pressure, where they were thicker.

Component PP3 is a phosphorylated glycoprotein with an apparent molecular mass of 28 kDa isolated from the proteose peptone fraction of bovine milk. The function of the protein is not known. The primary structure has been determined and shown to contain 135 amino acid residues (EMBL accession no. P80195). It was phosphorylated at Ser29, Ser34, Ser38, Ser40 and Ser46. Two O-linked carbohydrate groups were found at Thr16 and Thr86, while one N-linked carbohydrate group was present at Asn77. Thr16 was only ∼ 50% glycosylated. The amino sugar detected by the amino acid analyser at Thr86 was mainly galactosamine but a small amount of glucosamine was also present. The amino sugars found in the carbohydrate group linked to Asn77 were both glucosamine and galactosamine. A fragment of PP3 has been isolated from milk and shown to correspond to residues 54–135. This fragment was probably generated by plasmin hydrolysing the Arg53–Ser54 bond.

A rapid method of performing the Limulus amoebocyte lysate (LAL) test in milk is proposed using the Toxinometer ET-201. This instrument measured the increase in turbidity due to the interaction between the endotoxins of the Gram-negative bacteria and the LAL reagent, monitored the ratio Rt of the sequential to the initial transmission at 12 s intervais and quantified endotoxins by determination of the reaction time Tr required to obtain a 5% decrease in Rt. There was a good correlation between the toxinometrically determined endotoxin concentrations and the number of Gram-negative bacteria (SD, 0·18 log(plate count units)), and the repeatability (CV, 6·10%) was high. The assay may be useful for screening raw materials for UHT milk production, as the endotoxin content of the raw material is related to the rest proteinase activity in the UHT milk.

A total of 204 psychrotrophic isolates from raw ewes' milk (hand and machine milked) were identified by conventional methods. In addition, a numerical taxonomic study was conducted on 180 of these isolates and 19 reference strains. Three of the isolates were yeasts. Using identification schemes, 54 isolates were assigned to genera of Gram-negative aerobic rods (Pseudomonas, Acinetobacter, Flavobacterium, Moraxella and Psychrobacter), 48 were Enterobacteriaceae (Entero-bacter, Hafnia, Klebsiella, Citrobacter and Serratia) and one was identified as Aeromonas hydrophila. The 98 Gram-positive isolates were identified as Enterococcus, Streptococcus, Leuconostoc, Lactococcus, Bacillus, Staphylococcus, Micrococcus, Aureobacterium, Kurthia and Microbacterium. At the 82% similarity level (SSM), 18 clusters were formed. Cluster I included 34 strains of Lactococcus, Streptococcus and Leuconostoc. Most of the 35 strains in cluster II were Enterococcus. Clusters III and IV were identified as Kurthia and Microbacterium respectively. Cluster V was identified as Aureobacterium and cluster VI consisted of coagulase-negative staphylococci. Gram-negative isolates formed 12 clusters: Aeromonas (one cluster), Enterobacteriaceae (two clusters), Flavobacterium (two clusters), Pseudomonas and Psychrobacter immobilis (three clusters) and Acinetobacter (four clusters). Non-motile variants of Ps. fragi were found. Enterococcus and Enterobacteriaceae did not have significant spoilage properties. As expected, Gram-negative aerobic rods were proteolytic and/or lipolytic even at low temperature. Contamination with certain types of psychrotrophs (Gram-negative aerobic rods and enterococci) seemed to be associated with the milking method. The isolate of Aes. hydrophila had properties associated with virulence.

The thermal gelation of a commercially available whey protein concentrate was studied by oscillatory rheometry using a Bohlin rheometer. Gelation time increased with decreasing protein concentration with a critical protein concentration (at infinite gelation time) of 6·6%. The effect of temperature in the range 65–90 °C on gelation time was described by an Arrhenius equation with an activation energy of 154 kJ/mol. Gelation time was a minimum at pH 4–6, the isoelectric region of the whey proteins. Small additions of NaCl or CaCl2 dramatically decreased gelation time. Higher protein concentrations always produced higher storage modulus (G′) values after any heating time. Loss modulus (G″) v. time curves exhibited maxima at relatively short times for protein concentrations of 30 and 35%. G′ values for 10% protein concentration increased with temperature for heating times up to 59·5 min. G′ values at 59·5 min for 25% protein concentration were higher at 78 °C than at either 85 or 90 °C. The results are discussed in terms of current theories for biopolymer gelation.

Whey protein solutions of different chemical compositions were filtered through a large pore size ultrafiltration membrane (molecular mass cut-off 100 kDa). The influences of pH, ionic environment and Ca chelation on the performance characteristics of the membrane (filtration rate, total and individual whey protein permeability) were examined. Increase in pH in the range 5·0-10·0 resulted in a gradual increase in filtration rate and individual whey protein permeability. The presence of Ca in the whey protein solution had a negative effect on the performance characteristics of the membrane. Addition of EDTA to protein solution improved the filtration rate but had no effect on the protein permeation. Fractionation of the low molecular mass whey proteins (β-lactoglobulin and α-lactalbumin) from high molecular mass proteins (bovine serum albumin, lactoferrin and immunoglobulins) was best achieved at pH 8·0.

Spectrophotometric assays of β–galactosidase (EC 3.2.1.23) and phospho-β–galactosidase (EC 3.2.1.85) activity were used to survey the lactose utilization pathways of lactic acid bacteria and bifidobacteria. β–Galactosidase activity was found in all six genera represented (Lactococcus, Streptococcus, Leuconostoc, Lactobacillus, Pediococcus and Bifidobacterium) while phospho-β–galactosidase was restricted to the lactococci, two Lactobacillus and two Leuconostoc species. A number of strains of Lactococcus lactis, Lactobacillus casei and Leuconostoc spp. contained both enzymes. Enzyme activities varied when cells were grown on different sugars, but in general were low or absent for cells grown on glucose compared with lactose. Two lactose-related compounds, lactulose and galactosyl lactose, believed to be specific growth factors for bifidobacteria, supported growth amongst a wide range of lactic acid bacteria in addition to bifidobacteria. Growth on galactosyl lactose was restricted to some but not all strains containing β–galactosidase, implying that the presence of β–galactosidase is insufficient by itself to ensure utilization of galactosyl lactose. DNA fragments that encoded the Lactococcus lactis subsp. cremoris phospho-β–galactosidase gene or the β–galactosidase genes of Streptococcus salivarius subsp. thermophilus or Lactobacillus delbrueckii subsp. bulgaricus were isolated and used as probes in DNA-DNA hybridizations. Little or no hybridization was detected between these probes and plasmid or genomic DNA isolated from heterologous species, despite the presence of the corresponding enzyme activity in the strains probed.

After treatment with mitomycin C, 12 of the 120 strains of the French Collection of Lactic Acid Bacteria (CNRZ) belonging to the species Streptococcus salivarius subsp. thermophilus were found to be inducible and lysogenic. The corresponding temperate phages were multiplied on indicator strains and further characterized. It is noteworthy that some of the indicators were themselves lysogenic. The temperate phages belonged to Bradley's group B, or to the Siphoviridae family of the International Committee on Taxonomy of Viruses. Seven of them were shared among the two protein profiles previously established for virulent phages of Str. salivarius subsp. thermophilus. Seven different DNA restriction profiles were found for ten native temperate phages examined just after mitomycin C induction. Genome sizes varied from 40 to 45 kb and were classified into four related homology groups by DNA cross-hybridization, but there was no clear-cut relationship with the protein clusters previously shown. DNA homology with representatives of virulent phages was also found. The DNA restriction profiles of seven native temperate phages, examined just after mitomycin C induction of the lysogenic hosts, were noticeably different from those of the corresponding phages once they had been propagated on lysogenic indicators. Moreover, the host range of the latter phages was extended to a greater number of sensitive strains. The possible role of lysogenic strains of Str. salivarius subsp. thermophilus in the occurrence of phage outbreaks affecting this dairy streptococcal species is discussed.

Bovine colostrum contains transforming growth factor (TGF)-β-like activity. High levels of this activity are found in early colostrum (within 12 h after parturition); however, it decreases rapidly after 30 h. In this study, the activity in early colostrum was purified by a sequence of decaseination, DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, precipitation at neutral pH, further gel filtration and reversed-phase FPLC. A homodimeric 23 kDa protein was identified that retained the TGF-β-like activity. Its activity was completely neutralized by a specific anti-TGF-β2 antibody. These results indicate that the TGF-β-like growth factor found in bovine colostrum is chemically and antigenically related to TGF-β2. The physiological roles of the TGF-β2-related growth factor are discussed.

The extracellular proteinase from Pseudomonas fluorescens No. 33 was purified to electrophoretic homogeneity by a procedure including precipitation with HC1 and (NH4)2SO4, and column chromatography. The enzyme was purified 170-fold giving a yield of 7 % of the original activity. The molecular mass of the purified enzyme was 48000 by SDS-PAGE. The optimum pH and temperature for the hydrolysis of casein were 8·0–9·8 and 30–35 °C respectively. The enzyme was more thermostable in synthetic milk salts solution than in 0·1 M-sodium phosphate buffer, but was heat-labile at 50 °C in both buffer Systems. The activity was inhibited by o−phenanthroline, Hg2+, Cu2+, Fe2+ and, to a lesser extent, Ni2+. Caseins were susceptible to the proteinase, but degradation patterns were dependent on the form of the casein.