Quantification of β-lactoglobulin A and B in the milk of heterozygous animals has revealed a differential content of these two variants. Nucleotide sequence analysis of the first 733 bp of the bovine β-lactoglobulin promoter has indicated the presence of ten polymorphic sites, nine of them being allele A and B specific mutations. To study the differential expression of these alleles, constructs containing 753 bp long fragments of the bovine β-lactoglobulin A and B associated promoters were used in transient transfection experiments in HC11 cells. A differential transcription activity directed by the A and B specific promoters was consistently observed. The relative expression levels were 57% for β-lactoglobulin A and 43% for β-lactoglobulin B promoters. An allele-specific mutation has been reported to have a differential binding affinity to the activator protein-2 between the β-lactoglobulin A and B promoters. However, experiments in HC11 cells where the activator protein-2 binding sites were mutated in both β-lactoglobulin A and B promoters showed no major differences in activity between the mutated and native promoters.

The peptide Val–Arg–Arg–Pro–Asn–Leu–His–Pro–Ser–Phe–Ile–Ala–Ile–Pro–Pro–Lys–Lys–Ile, which corresponds to residues 84–101 of human κ-casein, has been synthesized and its conformation preferences determined by 1H-nuclear magnetic resonance spectroscopy in dimethyl sulphoxide. The peptide adopted a largely extended chain conformation in solution and there was evidence for the presence of a β-turn involving residues Pro87–His90 of human κ-casein. The presence of a turn in this position would make the physiologically significant Arg85 residue of human κ-casein (which is equivalent to Arg97 in bovine κ-casein) unavailable for interaction with Asp249of bovine chymosin, and may partly explain why human κ-casein is hydrolysed more slowly than its bovine counterpart by bovine chymosin.

The concentrations of cations (calcium and magnesium) and anions (inorganic phosphate, citrate and chloride) have been determined during the acidification of casein micelle suspensions in the pH range 6·7–1·5. The effects of casein concentration (27, 55, 83 and 144 g/kg) and ionic strength (0, 10 and 20 g/kg NaCl added) were investigated. Acidification resulted in solubilization of calcium, magnesium, inorganic phosphate and citrate ions. However, the solubilization curves were different and depended on the casein concentration. Increasing ionic strength by adding NaCl had no effect on acid-induced mineral solubilization. These results were compared with those obtained during milk acidification and discussed in relation to the mineral solubilization that occurs during curd acidification in cheese manufacture.

A methanol extract of green tea was fractionated on Sephadex LH-20. The compounds eluted were identified by thin layer chromatography as catechin–epicatechin, gallocatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate. When added to milk at 2·0 g/l, these polyphenols, apart from the catechin–epicatechin mixture, increased the heat stability of skim milk, particularly in the region of the minimum (pH 6·8–7·1). When added at 0·4 g/l, green tea polyphenols also increased the heat stability of concentrated milk. The effects of other phenolic compounds on the heat stability of milk were also examined. Chlorogenic acid, guaiacol, thymol, vanillin, butylene hydroxyanisole, propyl gallate and butylene hydroxytoluene did not affect the heat stability of milk or concentrated milk. Quinic acid markedly reduced the heat stability of skim milk. Pyrogallol, catechol, tannic acid, ellagic acid, phloroglucinol and gallate converted a type A heat coagulation time–pH profile to a type B profile. Ferulic acid and vanillic acid increased heat stability in the region of the maximum, with little effect on the minimum, and stability did not recover at pH values on the alkaline side of the minimum. Caffeic acid increased the heat stability of milk while the related non-phenolic compounds 2,5-dimethoxycinnamic acid and 3,4-dimethoxycinnamic acid had no effect.

Fractions enriched with α-lactalbumin (α-la) and β-lactoglobulin (β-lg) were produced by a process comprising the following successive steps: clarification–defatting of whey protein concentrate, precipitation of α-lactalbumin, separation of soluble β-lactoglobulin, washing the precipitate, solubilization of the precipitate, concentration and purification of α-la. The present study evaluated the performance of the process, firstly on a laboratory scale with acid whey and then on a pilot scale with Gouda cheese whey. In both cases soluble β-lg was separated from the precipitate using diafiltration or microfiltration and the purities of α-la and β-lg were in the range 52–83 and 85–94% respectively. The purity of the β-lg fraction was higher using acid whey, which does not contain caseinomacropeptide, than using sweet whey. With the pilot scale plant, the recoveries (6% for α-la; 51% for β-lg) were disappointing, but ways of improving each step in the process are discussed.

The effects of sheep αs1-casein CC, CD and DD genotypes on milk composition and cheese yield were studied. Processed bulk milk was collected from three groups of 15 ewes, carrying αs1- casein CC, CD and DD genotypes. CC milk was higher in casein content than CD or DD milk (+3·5 and +8·6% respectively), and had a higher protein[ratio ]fat ratio and a smaller casein micelle diameter. In addition, DD milk had a significantly lower αs1-casein content. The main differences were in curd formation: CC milk had better renneting properties. Cheesemaking trials, carried out in a pilot plant, showed that CC milk had better cheesemaking characteristics than DD milk, while CD milk was intermediate. Both 1 d old and fully ripened cheeses had different fat[ratio ]dry matter ratios and αs1-I-casein electrophoretic mobilities: these were lower for DD cheese. As a consequence, these genotypes could be considered as markers of milk and/or cheese quality.

β-Lactoglobulin (β-lg) in aqueous solution under pressure showed a marked depolarization of intrinsic fluorescence assigned to a gradually increased rotational diffusion of tryptophyl moieties in pressure-unfolded states. The corresponding change in anisotropy provided a new and more accurate method for determining denaturation volume which, for β-lg in neutral aqueous solution with ionic strength 0·16 (NaCl) at 25 °C, was ΔV°=−73 (SE 3) ml mol−1, corresponding to half denaturation at 123 MPa. The pressure unfolding led to exposure of hydrophobic regions to the protein–water interface that could be probed by fluorescence intensity of a β-lg–1-anilinonaphthalene-8-sulphonic acid (ANS) complex with 1:1 stoichiometry, as determined by Job's method of continuous variation. The unfolding of β-lg impaired the binding capacity of the inner calyx, with a reduction in binding capacity of 50% at 50 MPa, as shown by decreasing cis- parinaric acid fluorescence, decreasing anisotropy and decreasing radiationless energy transfer from tryptophans to this probe with increasing pressure. The pressure-induced reversible exposure of hydrophobic groups to the protein–water interface may, at least partly, explain the initial aggregation reactions, evident from increased Rayleigh scattering from ∼50 MPa, prior to irreversible pressure-induced gel formation of β-lg. Using results from this and previous studies, we propose a three step pressure denaturation model for β-lg for neutral solution at ambient temperature, including an initial pressure-melted state (up to 50 MPa) with partial collapse of the inner calyx and solvent exposure of the free thiol group, followed by a reversible denaturation with exposure of hydrophobic regions (half denaturation at 123 MPa) and with irreversible denaturation with thiol–disulphide exchange becoming increasingly important at higher pressures. Effects of pressure on β-lg, as measured by fluorescence depolarization, were found for the reversible denaturation steps to be similar to the effects of chemical denaturants but different with respect to shift in ANS emission maxima.

Changes in the distribution of minerals in skim goats' milk by high pressure (400 MPa) and/or heat (85°C for 30 min) treatment have been studied. Heat treatment caused reduced solubility of the calcium, magnesium and phosphorus, and this increased with the severity of heating. In contrast, high pressure released different levels of micellar elements into the soluble phase without causing appreciable changes in pH or ionic calcium concentration. The levels of soluble salts returned to their original values when the heated samples were subjected to high pressure. However, heating pressurized milk resulted in concentrations of soluble minerals that were lower than in control milks, and close to values found in heated milks. The salt balance in goats' milk was less affected by high pressure treatment at 75°C than was that of cows' milk. These results are discussed in relation to the effects of high pressure and heat treatment on mineral equilibrium and micellar structure.

The anti-colon cancer effect of dietary fibre results in part from its fermentation into the short-chain fatty acid butyric acid (BA) by intestinal microflora. BA has potent anti-colon cancer properties owing to its ability to induce apoptosis in colon cancer cells. The colon is not the only location where BA may reach high concentrations, because dietary BA is rapidly absorbed and transported to the liver. We have investigated whether BA could induce apoptosis in transformed human liver (Hep G2) cells. Hep G2 cells treated with BA displayed acetylated histones, increased DNA fragmentation and morphological features consistent with apoptosis. These biochemical features of BA-treated liver cells are identical to those of BA-treated colon cells. In addition, we investigated whether BA present in tributyrin, a triacylglycerol more compatible for inclusion into colloidal lipid structures than BA, could also induce apoptosis in Hep G2 cells. Tributyrin induced DNA fragmentation and morphological features characteristic of apoptotic cells in Hep G2 cells. These results are a significant advance towards delivering BA via colloidal lipid particles to cancerous sites in vivo. This study showed that BA and tributyrin are potent apoptotic agents, and we suggest that sources of dietary BA, such as milk fat, may provide anti-liver cancer properties.

The development of the Maillard reaction in pasteurized, UHT and in-bottle sterilized dietetic milks was studied. In these products damage caused by heat treatments could increase as a result either of the addition of various ingredients or of manufacturing processes that alter their content of reducing carbohydrates. Protein damage was evaluated by measuring furosine by reversed-phase ion-paired HPLC. The levels of furosine detected made it possible to assess the amounts of biologically unavailable lysine. In all milks analysed blocked lysine values were <340–350 mg/g total lysine, the level at which lysine becomes the limiting amino acid in milk. Pasteurized dietetic milks had levels of blocked lysine similar to that in ordinary pasteurized cows' milk. In some UHT and in-bottle sterilized dietetic milks their different composition resulted in an increase in the blocked lysine content. In some in-bottle sterilized milks, protein damage greatly reduces the beneficial effects of milk as a dietary supplement. Lactose-free milks, which are more susceptible to protein deterioration because of their higher content of reducing carbohydrates, were also analysed after storage at 20°C and at [les ]4°C. At the end of their recommended storage times, they contained limited amounts of blocked lysine only if they had been stored at [les ]4°C.

The lactoperoxidase system is a naturally occurring antimicrobial system found in milk, with lactoperoxidase, thiocyanate and hydrogen peroxide as its components. The keeping quality of milk pasteurized at 72°C for 15 s was found to be better than that of milk heated at 80°C for 15 s. This agrees with previous findings and is usually attributed to heat shocking of spores. However, complete deactivation of lactoperoxidase occurred at 80°C–15 s, whereas at 72°C–15 s residual lactoperoxidase activity was ∼70%, which may provide an alternative explanation. Higher levels of hypothiocyanite (the major antimicrobial agent produced by the lactoperoxidase system) were also detected in milk processed at 72 than at 80°C, which supports the theory that the lactoperoxidase system has a role in the keeping quality of pasteurized milk. Of all the methods evaluated, titratable acidity and alcohol stability gave the most consistent estimates of keeping quality, while dissolved oxygen was a good indication of the onset of spoilage. Lactoperoxidase activity decreased with temperature more rapidly between 70 and 80°C than is usual for an enzyme over a 10 deg C range.

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.

We have studied the contents of trace elements of nutritional or toxicological interest in 90 samples of whole, low-fat, skim, condensed, evaporated and powdered milks. Slurries of the samples were prepared with Triton X-100 and analysed using electrothermal atomic absorption spectrometry. The temperature–time programme of the graphite oven was optimized for each element, and the accuracy, precision, selectivity and sensitivity of the method were verified. Concentrations of the trace elements we investigated were: Pb 0–0·211 μg/g, Cd 0–28·985 ng/g, Al 0·528–4·025 μg/g, Cu 0·041–0·370 μg/g, Cr 0–0·177 μg/g, Mn 0·024–0·145 μg/g, Se 0–237·333 ng/g, Zn 0·297–0·827 μg/g and Ni 0·058–1·750 μg/g. (A value of zero indicates that the element was undetectable by our methods.) Concentrations of the pairs of elements Cu–Cd, Mn–Cd, Mn–Cu, Zn–Mn, Ni–Cu, Ni–Mn and Ni–Zn were significantly correlated (P<0·001). Linear discriminant analysis confirmed the separation between the six types of milk analysed.

Recombinant human αs1-casein expressed in Escherichia coli was purified and digested with trypsin in an attempt to find peptides with angiotensin-I-converting enzyme (ACE) inhibitory activity. Three novel ACE inhibitory peptides, A-II, B-II and C, were isolated and their amino acid sequences identified as Tyr–Pro–Glu–Arg (residues 8–11), Tyr–Tyr–Pro–Gln–Ile–Met–Gln–Tyr (residues 136–143) and Asn–Asn–Val–Met–Leu–Gln–Trp (residues 164–170) respectively. ACE inhibitory activities were measured for the corresponding synthetic peptides, and the ACE IC50 (the amount of peptide causing 50% inhibition of ACE activity) values of A-II, B-II and C estimated to be 132·5, 24·8 and 41·0 μmol/l respectively. Peptides A-II and C were resistant to further digestion by pepsin, whereas peptide B-II was hydrolysed. All three peptides were resistant to digestion by chymotrypsin. These ACE inhibitory peptides may prove useful for oral administration in the treatment of hypertension.

Twenty-four dairy Leuconostoc strains isolated from French commercial starters and four reference strains were screened for 58 morphological and biochemical characters. Hierarchical clustering analysis was performed on the results of dextran production and carbohydrate fermentation tests. Most strains fitted into three clusters at a similarity level of 65% and were identified as belonging to the species lactis or mesenteroides. Fourteen strains were then selected and further characterized for their technological properties. They were examined for growth, acidification kinetics and proteolytic capacity when growing in milk, and peptidase activities and diacetyl production after growth in MRS broth. Principal component analysis was carried out to group strains and compare their technological properties. This allowed selection of strains of dairy interest. Special attention was given to the flavouring capacity of Leuconostoc lactis isolates that could be useful in designing commercial starters.

The two glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Lactoferrin is a single chain glycoprotein of molecular mass 80 kDa with two iron-binding sites and two structural lobes connected by a short peptide. Purified samples of lactoferrin, isolated from buffalo colostrum, were subjected to hydrolysis using trypsin, chymotrypsin, pepsin, subtilisin and proteinase K. The first three proteinases produced two major fragments of approximately 35 and 23 kDa together with small molecular mass peptides. Trypsin and chymotrypsin partly digested lactoferrin, while pepsin converted all the intact lactoferrin into fragments. Subtilisin hydrolysis produced fragments of 40 and 26 kDa together with low molecular mass peptides. However, SDS-PAGE of the proteinase K hydrolysis product gave a clear band at 40 kDa together with a band indicating a substantial quantity of low molecular mass peptides (<14·4 kDa). Upon ion-exchange chromatography this product gave two major fractions, which were further purified by gel filtration and identified as the C and N lobes from their N-terminal sequences. Thus, the 40 kDa band in SDS-PAGE of the proteinase K hydrolysis product contained two fragments of equal molecular mass. On further hydrolysis with proteinase K, the N lobe was completely hydrolysed into low molecular mass peptides, while only a small fraction of the C lobe was converted into small products. This suggested that an inhibitory fragment was present in the C lobe that was released on hydrolysis to small fragments and prevented complete digestion of the C lobe by high-affinity binding to the active site of proteinase K. This fragment was isolated from the lactoferrin–proteinase K complex and its sequence determined to be Val–Ala–Gln–Gly–Gly–Ala–Ala–Gly–Leu–Ala. Circular dichroism studies indicated a high α-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein.

A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native Mr of 50 000–55 000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the purification procedures at 4°C and its stability was greatly improved when 10 ml glycerol/l and 2 mm-dithiothreitol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro–Leu–Gly–NH2, Pro–Leu–Gly–Gly and Pro–His–Pro–Phe–His–Leu–Phe–Val–Tyr. This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr–Pro–Phe–Pro–Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides.

Various methods were used to study 42 Gram-positive cocci belonging to the family of Micrococcaceae and isolated from dairy products. Only a few strains could be identified using the ATB 32 biochemical micromethod rapid gallery system (API). All strains were subjected to conventional biochemical tests. The results were then analysed by a numerical method using the Sokal–Michener similarity coefficient. The 42 strains were distributed into 10 clusters at the Euclidean distance of 3·2. Most (75%) of the isolates were identified at the species level, but Micrococcus luteus could not be differentiated from Mc. lylae within the Micrococcus genus. An identification method based on the analysis of ribotype patterns was then applied to the isolates and these were compared with the patterns of collection strains. Cellular DNA was cleaved by the restriction enzymes SacI and KpnI and probed with the peroxidase-labelled 16S+23S rRNA of Escherichia coli. Ribotype patterns were analysed using a Dice coefficient and the unweighted pair group method using arithmetic averages, making dendrogram construction possible. Of the strains investigated, >84% were characterized to the species level. The remaining strains were found to belong to the Arthrobacter group or were atypical Micrococcus species. Of the isolates, 52% belonged to Micrococcus luteus, 17% were identified as Kocuria varians and 7% were assigned to the Brachybacterium genus.

A chemiluminescent method for determining xanthine oxidase (XOD) activity was developed and applied to the assay of milk enzyme activity using a photomultiplier luminometer. Various kinds of milk and cream samples were analysed for XOD content. In pasteurized milk, XOD activity depended on the fat content and in UHT milk it disappeared owing to the heat treatment. Milk sample preparation was very simple, requiring only homogenization at 40°C followed by a 1[ratio ]10 dilution with UHT (‘XOD-free’) milk. The assay was carried out at 25°C. The response obtained from XOD standard solutions in milk was linear from 0·1 to 500 enzyme units (U) l−1, but for the actual milk samples values ranged only from 1 to 135 U l−1. The detection limit at 2 SD was 0·1 U l−1 in milk, while in buffer it was 100 times lower. The intra-assay and interassay CV for XOD activity in milk were 6–12%.

A study was undertaken to compare the chemical and sensory characteristics of Abondance cheeses made with milk from animals grazing areas within the same highland pasture, but with different predominant plants. Nine cheeses made during the last 3 d of three successive 7 d periods were evaluated. The animals grazed on the southern side of the highland pasture during the first period (15–21 June), on the northern side during the second period (22–29 June) and returned to the southern side for the third period (30 June–6 July). The gross composition of the cheeses did not vary between periods. ‘North’ cheeses contained more plasmin, γ-casein, αs1-I-casein and water-soluble N than ‘south’ cheeses. Both sensory and instrumental measurements indicated that north cheeses were less firm, stickier and more easily fractured than south cheeses. North cheeses were also more salty, bitter and persistent. Their overall aroma was more intense and they had more intense sour, burnt, toasted, fermented vegetable and sweat aromas, but less intense toffee, exotic fruit and acid milk aromas. The texture differences noted between the cheeses from milk produced on the two areas may come from differences in primary proteolysis, partly due to different amounts of plasmin and plasminogen in milk and in cheeses. The aroma differences were related to differences in volatile compounds. Some compounds had a microbial origin, while some others may have come from the pasture.