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.

Comparisons were made of the flavour, free fatty acids and bacterial flora of commercial cheese made at different factories and experimental cheese made under aseptic conditions: (i) with δ-gluconic acid lactone instead of starter, (ii) with starter only, (iii) with starter and added floras derived from the curd of the commercial cheeses (reference flora cheeses).

Comparison of the bacterial flora of commercial and reference flora cheeses showed that replication of organisms was better with some reference floras than with others. In all the cheeses the lactobacilli increased in numbers during maturation, whilst other groups of organisms died out.

The amount of acetic acid present was influenced by the starter and by the lactobacilli. Single-strain starters produced some acetic acid, most of which was lost in the whey; commercial starters produced considerably more, due to the presence in them of Streptococcus diacetilactis. Later in maturation lactobacilli increased the acetic acid content, a greater increase being observed with homo-than with heterofermentative strains.

The initial levels of butyric and higher fatty acids in the milk varied with source of the milk and with the season, summer milk having higher levels than winter milk. During cheese-making a slight increase of these acids occurred in every cheese made with starter and a further small increase occurred during ripening. However, there was no increase in the content of these acids in the cheese made with δ-gluconic acid lactone, indicating that lactic acid bacteria were weakly hydrolysing the milk fat.

Flavour trials showed that Cheddar flavour was present not only in the reference flora and commercial cheese, but also in the cheese made with starter only. Different starters produced different intensities of flavour; one strain produced an intense fruity off-flavour. Cheeses made with δ-gluconic acid lactone were devoid of cheese flavour.

A method for the quantitative analysis of casein mixtures is described. The method uses ion-exchange chromatography at 4 °C on DEAE cellulose, Whatman DE52 in the presence of tris-chloride-urea buffer (pH 8·6), and a NaCl gradient to fractionate the alkylated casein mixtures and a micro-biuret technique to determine protein concentrations. Values for fractions representative of (1) γ-caseins, (2) κ-casein plus some unknown proteins, (3) β-casein, (4) minor αs-caseins and (5) αs1- plus αs0-caseins were obtained, and whole casein from herd bulk milk was found to consist on average of about 2, 13, 36, 11 and 38% respectively of these fractions. Agreement between duplicate fractionations was satisfactory and the recovery of material varied between 94 and 98%.

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.

Milk fat globule membranes (MFGM) were isolated from milk from cows injected intraperitoneally with dl-α-tocopherol acetate. The fatty acid composition and content, and the contents of tocopherols, retinol and β-carotene were determined and compared with the composition and content in the original cream sample. Intraperitoneal injection of 10 g dl-α-tocopherol acetate elevated the α-tocopherol content in the milk fat from 13–30 to 50–70 εg α-tocopherol/g milk fat 2–3 d after injection. The increase depended on the α-tocopherol status of the cow prior to injection. The concentrations of retinol and β-carotene in the milk fat were unchanged after the α-tocopherol injections. MFGM fatty acids made up 18–27 g/kg total fatty acids in the milk fat. However, the proportions of monounsaturated and polyunsaturated fatty acids were higher in MFGM than in total milk fat, while the proportion of saturated fatty acids was lower in MFGM (P < 0·001). Thus, the longchain polyunsaturated fatty acids in MFGM constituted ∼ 40–70 g/kg total milk fat. α-Tocopherol was the only fat-soluble vitamin detected in MFGM; γ-tocopherol, retinol and β-carotene were detected only in the cream. A significant relationship between α-tocopherol contents in cream and MFGM was found: (α-tocopherol in MFGM fatty acids) =–1652 × (1/ln(α-tocopherol in cream fatty acids)3)+ 97 (r = 0·857, P < 0·001) when the contents of α-tocopherol were expressed as εg/g fatty acid. According to this equation the upper limit for incorporation of α-tocopherol into MFGM is 97±5 εg/g MFGM fatty acids. Thus, under normal farming conditions the α-tocopherol content will be highest in MFGM. However, in cream with an α-tocopherol concentration < 15 εg/g cream fatty acids the concentration in MFGM will be lower and furthermore will decrease rapidly. The possible importance of this relationship in relation to the oxidation of milk fat is discussed.

The objective of this work was to describe the diversity of lactic acid bacteria in traditional raw milk Salers cheeses at the species and strain levels. The characterization of 381 strains isolated during ripening and various strain collections was investigated using physiological analysis and molecular techniques: Rep-PCR, species and genus specific amplifications and the sequence analysis of 16S rDNA for strain typing and taxonomic identification. The strains belonged to Lactobacillus plantarum, Lactobacillus paracasei, Lactococcus lactis, Lactococcus garviae, Enterococcus faecalis, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Streptococcus salivarius, Streptococcus millieri, Streptococcus macedonicus and Pediococcus pentosaceus. A wide phenotypic and genomic heterogeneity was observed within the different species (Lactobacillus plantarum, Lactobacillus paracasei and Leuconostoc mesenteroides) according to the origin and the time of ripening. The natural microflora was different from strain collection and each method must be combined to identify and characterize natural microflora. This study revealed the low selectivity of selective media used for the isolation of different groups of lactic acid bacteria except the Facultatively Heterofermentative lactobacilli medium selecting mesophile lactobacilli and SB medium selective for Enterococcus. The study reveals, for the first time, the microbial lactic acid bacteria community of Salers cheese and its diversity. A better knowledge of microbial flora will be useful to improve understanding of sensory quality of cheeses.

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.

Details are given of two simple tests for efficiency of pasteurisation. The first, test A, may be carried out completely within half an hour, and will detect any gross errors of pasteurisation technique. The second, test B, is more searching, and will reveal minor errors of technique as follows:

(a) 1½° F. below the minimum temperature for pasteurisation, or (b) heating (at 145° F.) for 20 instead of 30 min., or (c) admixture of 0·25 per cent, of raw milk with properly pasteurised milk. Taken together, the two tests form an efficient method for the laboratory diagnosis of faulty pasteurisation.

Mixtures of κ-casein and β-lactoglobulin (β-lg) were heated at 70 °C in 20 mM-imidazole buffer, pH 6·8 containing 20 mM-EGTA. Aggregation of the κ-casein/β-lg mixture occurred within 90 s and susceptibility to hydrolysis by chymosin decreased significantly within 180 s even though covalent interaction was not detected until after 4000 s. UV-absorbance indicated initial structural destabilization of the heated κ-casein/β-lg mixture followed by a return ( > 350 s) to a spectrum comparable to the native state, indicating molecular rearrangement. Apparent hydrophobicity of κ-casein decreased 80% within 250 s compared to a 38% decrease for β-lg. Under similar conditions, the κ-casein/β-lg mixture (1:1) showed a faster (2·5 times) decrease in apparent hydrophobicity than κ-casein alone with concomitant exposure of acidic (hydrophilic) groups. The results suggested that the driving force for the rearrangement was mainly hydrophobic, i.e. entropic in origin. The tendency of heated and subsequently cooled κ-casein/β-lg to aggregate reached a maximum after heating at 70 °C for 720 s.

The aim of the present work was to investigate the role of whey protein denaturation on the acid induced gelation of casein. This was studied by determining the effect of whey protein denaturation both in the presence and absence of casein micelles. The study showed that milk gelation kinetics and gel properties are greatly influenced by the heat treatment sequence. When the whey proteins are denatured separately and subsequently added to casein micelles, acid-induced gelation occurs more rapidly and leads to gels with a more particulated microstructure than gels made from co-heated systems. The gels resulting from heat-treatment of a mixture of pre-denatured whey protein with casein micelles are heterogeneous in nature due to particulates formed from casein micelles which are complexed with denatured whey proteins and also from separate whey protein aggregates. Whey proteins thus offer an opportunity not only to control casein gelation but also to control the level of syneresis, which can occur.

The effect of milks fermented by Lactobacillus helveticus and its non-proteolytic variant on mucosal and tumoral immunity was studied. Milks fermented by Lb. helveticus wild type or its non-proteolytic variant were administered orally to mice for different periods (3, 5 and 7 d). The immune response was assessed by analysing the activity of the peritoneal macrophages, the number of cells secreting IgA associated with the gut-associated lymphoid tissue and with the bronchial-associated lymphoid tissue. The number of cells was determined by direct immunofluorescence. The antitumour activity was monitored by studying the regression of the subcutaneously implanted fibrosarcomas. After 3 d feeding of milk fermented by Lb. helveticus wild type, the number of sIgA increased significantly at both the intestinal and bronchial levels, indicating that a cellular migration had occurred. This effect was not noticeable when milk fermented by Lb. helveticus Protease (−) was orally administered. Both fermented milks (wild type or its variant) exhibited an effect on the activity of the peritoneal macrophages, which might be indirectly correlated to the regression of the fibrosarcoma. Although the mechanism by which the lactic acid bacteria enhance the immune system is not clear, this study clearly suggests that the bioactive compounds released during milk fermentation might contribute to the immunoenhancing properties of these products. By releasing biopeptide, lactic acid bacteria have important implications in modulation of the host's immune response, more specifically its cellular immune response.

Removal of colloidal phosphate leads to changes in the propertics of milk of which the increased viscosity and greatly increased sensitivity to calcium salts, and the apparently diminished degree of mutual integration of the various casein fractions are the most striking. Re-introduction of colloidal phosphate restores incompletely and only in certain instances the original properties of the milk.

These observations are interpreted as favouring the conception of chemical links between colloidal phosphate and casein in milk.

Using polymer producing (ropy) strains of lactic acid bacteria it was possible to reduce considerably the syneresis of yogurt, even with 12% total milk solids. The viscosities obtained with these strains were also similar to those obtained using normal strains and milk with 17% total solids content. The concentration of milk and the polymer produced by ropy starters had a synergic effect in increasing viscosity. Polymer production was not affected in most cases by milk concentration. One type of ropy culture (Wiesby) seemed to produce a different kind of polymer as it could not be determined by alcohol precipitation, in spite of being able to reduce syneresis and increase viscosity in yogurt. A limited number of yogurts were evaluated organoleptically, one prepared with a ropy starter strain (NCFB at 12, 14·5 and 17% total solids) and one prepared with a non-ropy strain (LL-I at 17% total solids). The results suggest that the ropy strain yogurts had different mouthfeel from the non-ropy strain yogurts; the most acceptable product overall was the ropy strain made with 12% total solids.

Binding isotherms for the calcium ion–αsl-casein system have been measured, as functions of ionic strength, temperature, and pH, and the isotherms have been analysed in terms of binding constants modified by substitution effects. The results demonstrate that the strength of binding is increased with increasing temperature and decreased by increasing ionic strength or decreasing pH, all of which may be explained semi-quantitatively. Parallel studies on the precipitability of the αsl-casein–Ca2+ complexes showed that there is considerable variation in the extent of calcium binding required to initiate precipitation of the protein, and in the calcium concentration necessary to achieve the required extent of ligand binding.

Experiments involving the depletion of milk calcium phosphate (MCP) with EDTA have shown that it has a Ca to phosphate ratio of 1·61±0·04, close to that of dental enamel. The calcium phosphate also contains small amounts of citrate (citrate/Pi = 0·097±0·011) and Mg (Mg/Pi = 0·044 ± 0·011). These experiments and model calculations indicate that the Ca binding capacity of casein is not reduced by mineralization with calcium phosphate and hence that ion binding sites and nucleation sites are distinct, or nearly so. Calculations of the ionic equilibria in the aqueous phase of milk show that there is an invariant ion activity product for a molecular formula close to that of dicalcium phosphate. It is suggested that MCP is a mixture of salts, with dicalcium phosphate a precursor of more basic salts such as octacalcium phosphate and hydroxyapatite.

Time spent lying by lactating Holstein-Friesian cows of varying body condition scores (BCS) and milk yield was measured using an animal activity monitor. A 3-week average BCS was calculated for each cow; and in total, 84 cows were selected with 28 cows each among three BCS categories (Thin: BCS<2·75; Moderate: 2·75⩾BCS<3·25; Heavy: BCS⩾3·25) and two stage of lactation categories (<150 days in milk or >150 days in milk). Cows were kept in two management systems: parlour/freestall (n=60) or automated milking system/freestall (n=24). Behaviour was recorded for 5·3±0·1 d for each cow. Production levels were considered using a 28-d rolling average of daily milk production. Cows that exhibited clinical lameness before or during the observation period were excluded from analyses. For cows exhibiting oestrus, the day prior to, day of, and day following breeding were removed. The final analysis included 77 cows (408 d of observation). A mixed model was fitted to describe average daily hours spent lying. Results demonstrated that lying time increased as days in milk (DIM) increased (P=0·05). Variables that were tested but not significant (P>0·05) were BCS category, parity category (1 or ⩾2) and 28-d rolling average daily milk production. Although a numerical trend for increasing hours spent lying with increasing BCS was observed, after accounting for other factors in the mixed model, BCS did not significantly impact lying time. Continued investigation of these management factors that impact lying time and bouts, using new technologies, more cows, and more herds will help dairy owners better manage facilities and cow movements to optimize this essential behaviour.

The effect of preheat treatment, evaporation and drying in a commercial plant on the denaturation of βlactoglobulin and α-lactalbumin, their incorporation into the casein micelle and the heat stability characteristics of the milks and powders were determined. Preheat treatments between 110 °C for 2 min and 120 °C for 3 min denatured between 80 and 91% of β-lactoglobulin and between 33 and 45% of α-lactalbumin. Evaporation increased the extent of denaturation but spray drying did not increase it further. The incorporation of α-lactalbumin and βlactoglobulin into the micelles was markedly less than the amount that denatured and was not a constant ratio to it. Heat coagulation times at 140 °C of milks, concentrates and powders diluted to the original milk concentration were measured as a function of pH. In general, the greater the collective heat treatment, the shorter the time required to achieve coagulation. Spray drying shifted the peak positions in the pH-heat coagulation time profiles. In contrast, heat coagulation times (measured at 120 °C) of concentrates and powders diluted to 20% total solids content increased with the severity of the preheat treatment. Surprisingly, spray drying markedly increased the heat coagulation times of the diluted concentrates.

The effects of high intensity pulsed electric field (HIPEF) treatments at room or moderate temperature on water-soluble (thiamine, riboflavin, ascorbic acid) and fat-soluble vitamins (cholecalciferol and tocopherol) were evaluated and compared with conventional thermal treatments. Vitamin retention was determined in two different substrates, milk and simulated skim milk ultrafiltrate (SMUF). Samples were subjected to HIPEF treatments of up to 400 μs at field strengths from 18·3 to 27·1 kV/cm and to heat treatments of up to 60 min at temperatures from 50 to 90 °C. No changes in vitamin content were observed after HIPEF or thermal treatments except for ascorbic acid. Milk retained more ascorbic acid after a 400 μs-treatment at 22·6 kV/cm (93·4%) than after low (63 °C-30 min; 49·7% retained) or high (75 °C-15 s; 86·7% retained) heat pasteurisation treatments. Retention of ascorbic acid fitted a first-order kinetic model for both HIPEF and thermal processes. First-order constant values varied from 1·8×10−4 to 1·27×10−3 μs−1 for the HIPEF treatments (18·3–27·1 kV/cm) and, for thermal processing ranged from 5×10−3 to 8×10−2 min−1 (50–90 °C). No significant differences were found between the results obtained after applying HIPEF treatments at room or moderate temperature. However, results depended on the treatment media. A beneficial effect of natural skim milk components, mainly proteins, was observed on the preservation of ascorbic acid, since skim milk retained more ascorbic acid than SMUF after HIPEF treatments.