Classical methods and methods based on the use of disodium ethylene-diaminetetra-acetate (EDTA) for the semi-micro determination of calcium and magnesium in milk and milk diffusate are described. With both types of method satisfactory accuracy and precision are attainable. The use of the EDTA methods leads to greater speed and convenience but practice in recognizing the correct titration end-points is necessary.

The lipoprotein lipase and tributyrate hydrolysing activities were found to be similarly distributed in the fractions obtained when whole milk was separated into skim-milk and cream, and when the cream was washed and freed from lipid. These enzyme activities in skim-milks and in extracts of lipid-free cream could not be separated by affinity chromatography on heparin-Sepharose. The enzymes were inactivated to the same degree when incubated at 37°C in the presence of 1·5 M-NaCl, pH 8·5, and both showed the same marked decrease in stability at 4°C in 1·5 M-NaCl, when the pH was changed from 8·3 to 9·0. Irradiation of skim-milk with UV-light caused the same decrease in both lipoprotein lipase and tributyrate hydrolysing activities. An antiserum against a highly purified skim-milk lipoprotein lipase caused total inhibition of the lipoprotein lipase and tributyrate hydrolysing activities in skim-milk and in extracts of lipid-free cream. It is suggested that in bovine milk there is only one major lipase and that it is identical to lipoprotein lipase.

The solubility of milk protein concentrate (MPC) powders was influenced by the method used for preparing the concentrate, drying conditions, and the type of dryer used. Increasing total solids of the ultrafiltered concentrates (23% total solids, TS) by diafiltration to 25% TS or evaporation to 31% TS decreased the solubility of MPC powders (80–83% protein, w/w dry basis), with ultrafiltration followed by evaporation to higher total solids having the greater detrimental effect on solubility. High shear treatment (homogenisation at 350/100 bar, microfluidisation at 800 bar or ultrasonication at 24 kHz, 600 watts) of ultrafiltered and diafiltered milk protein concentrates prior to spray drying increased the nitrogen solubility of MPC powders (82% protein, w/w dry basis). Of the treatments applied, microfluidisation was the most effective for increasing nitrogen solubility of MPC powders after manufacture and during storage. Manufacture of MPC powders (91% protein, w/w dry basis) prepared on two different pilot-scale dryers (single stage or two stage) from milk protein concentrates (20% TS) resulted in powders with different nitrogen solubility and an altered response to the effects of microfluidisation. Microfluidisation (400, 800 and 1200 bar) of the concentrate prior to drying resulted in increased long term solubility of MPC powders that were prepared on a single stage dryer but not those produced on a two stage spray dryer. This work demonstrates that microfluidisation can be used as a physical intervention for improving MPC powder solubility. Interactions between the method of preparation and treatment of concentrate prior to drying, the drying conditions and dryer type all influence MPC solubility characteristics.

A study of 799 cultures of staphylococci and micrococci, isolated mainly from milk, showed that according to their general character the strains could be arranged in a virtually continuous series. At one extreme is the pathogenic staphylococcus and at the other a thermoduric saprophyte. The series consists of three main groups:

(1) The staphylococcus group in which the organisms are sugar fermenters and relatively sensitive to heat. They are, mainly at least, parasites of the animal body. Four subgroups are distinguishable. In three ammonia is formed from arginine, and in two acetoin is formed from glucose. A test for acetoin production defines those mannitol fermenters which do not produce coagulase.

(2) An intermediate group in which the organisms are obligate aerobes and do not produce acid from sugars.

(3) The dairy micrococci, a group of thermoduric sugar fermenters which occur frequently on dairy equipment and in pasteurized milk. The group comprises two species conforming to Micrococcus luteus Cohn emend. Lehmann & Neumann, and M. varians (Dyar) Migula.

The somatic cell count (SCC) in milk is associated with increasing proteolytic degradation of caseins and it has been suggested that enzymes derived from somatic cells contribute to a lower yield and poorer quality of cheese. It is essential to increase the knowledge on naturally occurring milk proteinase activities to better understand how to improve the technological quality of milk. The aim of this work was to identify peptides actually present in milk as a result of proteolysis at different levels of SCC and to assign these peptides to potential responsible proteases where possible. Peptide fractions were prepared from acid whey by ultrafiltration at a molecular cut-off value of 10 000 Da. The peptides were separated using capillary reversed phase high performance liquid chromatography (RP-HPLC) and identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (MALDI-TOF MS/MS). Peptides identified ranged in mass from 1023 to 2000 Da, and originated from αS1-, αS2- or β-casein. Possible responsible proteases that could be suggested when examining the peptide cleavage sites included plasmin, cathepsin B, D and leukocyte elastase. The results indicated that plasmin was primarily responsible for the observed proteolysis in milk at low cell count, whereas the cathepsins and elastase became implicated at elevated cell count. Specificity and activity of cathepsins and elastase has earlier mainly been studied in model systems, whereas less is known about their activities in milk itself. This is also the first indication of involvement of elastase in milk proteolysis through the unequivocal determination of cleavage sites.

The dynamic viscosity coefficients η of creams containing up to 50% fat have been determined at temperatures of approximately 40, 50, 60, 70 and 80°C. All the creams behaved as Newtonian liquids, the shear stress being proportional to rate of shear up to the maximum rate used of 100 sec−1. At a given temperature a linear dependence of log η on (φ+φ5/3) was obtained for φ < 0·4, where φ is the concentration (w/w) of fat. Interpolation formulae have been derived to enable η to be calculated at any temperature between 40 and 80°C and for any fat content up to 40%. Formulae for the density ρ of cream have also been deduced to permit kinematic viscosities η/ρ to be computed. Nomograms have been constructed to enable η and ρ to be readily determined without the use of the interpolation formulae and when slight loss in accuracy is unimportant.

The yield and composition of milk from Friesian cows grazing either perennial ryegrass (Lolium perenne) (G, ten cows) or white clover (Trifolium repens) (C, nine cows) were evaluated between d 21 and 129 post partum. The two forages, of similar digestible energy content, were the sole source of nutrients and were offered ad lib. (exp. 1). Digestion and flow at the duodenum were measured on 13 occasions in early lactation with comparable cows fitted with rumen and duodenal cannulae and grazing similar forages (exp. 2). The gross milk yield (22·2, G; 25·0, C, kg/d P < 0·05) and the yield of protein (0·66, G; 0·77, C, kg/d, P < 0·01) were higher, but the protein content was similar and the fat content lower for cows fed C compared with G. Cows fed G were heavier at the beginning of the experiment and lost weight more rapidly than cows fed C. Milk energy output, adjusted for tissue energy gain or loss, was 83·9 for cows fed C compared with 71·8 MJ/d for cows fed G (P < 0·001), during the period of tissue energy repletion (weeks 11–18). From week 18 to the end of lactation all cows from exp. 1 were fed silage ad lib. and 502 kg dry matter of concentrates. The total (305 d) difference in lactation response to grazing C compared with G was 931 1 (5657 1, C; 4726 1, G); this was a direct response during the experiment of 301 1 and a residual response of 630 1. In exp. 2, more organic matter (6·47, G; 8·01, C, kg/ d, P < 0·001), and more non-ammonia N (433, G; 575, C, g/d, P < 0·001) entered the duodenum of cows grazing C compared with G.

We assessed polymorphisms in exon IV of the κ-casein gene (CSN3) in ten different breeds of domestic goat (Capra hircus) from three continents and in three related wild caprine taxa (Capra <ibex> ibex, Capra <ibex> sibirica and Capra aegagrus). Thirty-five DNA samples were sequenced within a 558 bp fragment of exon IV. Nine polymorphic sites were identified in domestic goat, including four new polymorphisms. In addition to four previously described polymorphic positions, a total of 13 polymorphisms allowed the identification of 13 DNA variants, corresponding to 10 protein variants. Because of conflicting nomenclature of these variants, we propose a standardized allele designation. CSN3*A, CSN3*B, and CSN3*D were found as widely distributed alleles in European goat breeds. Within Capra ibex we identified three variants and showed that the sequence of Capra aegagrus is identical to the most common Capra hircus variant, consistent with Capra aegagrus being the wild progenitor of domestic goats. A dendrogram was drawn to represent the molecular network between the caprine CSN3 variants.

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6·7) and skimmed milk of 6·5 and 7·6 °C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 °C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.

Dialysing milk against phosphate-free sera showed that the transition in the ethanol (EtOH) stability/pH profile was associated with the soluble phosphate component of the milk serum. Sigmoidal behaviour similar to that of milk was reproduced when the EtOH stability of artificial mixtures of casein, Ca and phosphate was measured as a function of pH. A mechanism for the s coagulation of skim-milk is discussed.

The effect of preheating skim milk and artificial micelle milk on curd syneresis was studied. The inhibition of syneresis caused by heat was dependent on the presence of β-lactoglobulin (β-lg) and to a lesser extent α-lactalbumin. The degree of inhibition increased with increasing amounts of added β-lg and preheating temperature. This agrees with the hypothesis that the detrimental effect of preheating on syneresis is due to complex formation between β-lg and κ-casein. This complex appeared to be mediated via thiol–disulphide exchange and its formation appeared to interfere with the micelle–micelle interactions responsible for syneresis.

The complexing of whey proteins in hydrochloric acid whey by carboxymethyl cellulose (CMC) has been studied. The efficiency of precipitation of CMC (the ability to form insoluble complexes with whey proteins) increased with increasing degree of substitution (DOS) of CMC. At a DOS of greater than 1·4, efficiencies of precipitation of whey protein greater than 90% were achieved without the need for dilution of whey. CMC of high DOS would thus be useful in whey protein reclamation schemes. The amount of CMC necessary for maximum efficiency of precipitation also increased with increasing DOS. The composition of the CMC-protein complex varied little over the range pH 2·8–4·0. Under optimum conditions for binding, the complex contained 61–68% protein and more than 95% of the CMC originally in solution was precipitated in the complex.

Coagulation of para-casein micelles was monitored using a Platelet Aggregometer at 37·8 °C and pH 6·7. The macropeptide released by chymosin was determined quantitatively with fluorescamine. The aggregatability of para-casein micelles integrated over the complete hydrolysis of κ-casein was calculated from the amount of κ-casein hydrolysed during the clotting time (Tc). Low concentrations of CaCl2 enhanced the rate of κ-casein hydrolysis but increases in CaCl2 concentration above 8 mM caused marked decreases in this rate. Calcium chloride enhanced the ability of para-casein micelles to aggregate. Little aggregation of para-casein micelles occurred at 0·6 mM-CaCl2 even after all of the κ-casein was hydrolysed. As the concentration of CaCl2 was increased from 3 to 60 mM, aggregation of para-casein micelles took place at progressively lower levels of κ-casein hydrolysis and the percentage hydrolysis at the Tc decreased markedly from 71±7% to 26±8%. The combined influence of CaCl2 on the velocity of the chymosin-catalysed reaction and on para-casein micelle aggregatability accounted for its effect on the average rate of coagulation (calculated by the reciprocal of the Tc). Results are consistent with the hypothesis that electrostatic repulsions and ionic bonding are involved in the interaction between chymosin and κ-casein.

Effects of pH, added Ca, rennet concentration, temperature, concentration and dilution of milk, on rennet clotting and coagulum development have been investigated. Rennet clotting time (RCT) was decreased by reduced pH, by increased temperature and by increased concentrations of rennet or added Ca. Only extremes of concentration or dilution of milk increased RCT. Strength of milk coagulum at 1 h after RCT was increased by reduction in pH and by increased concentrations of added Ca. Increased temperatures decreased coagulum strength whereas increased rennet concentrations had little effect. A curvilinear relationship between total casein concentration and coagulum strength was found for Friesian and Jersey milks. Two phases of coagulum development after clotting were observed. Changes in pH particularly affected the first phase whereas changes in temperature particularly affected the second. Effects of rennet, casein and Ca were similar for both phases.

We report here a comparative analysis of the growth, acidification capacity, and chemical and microbiologic composition between kefir grains after 20 subcultures in whey at 20, 30, and 37°C and the original kefir grains coming from milk along with a determination of the microbiological composition of the fermented whey as compared with that of traditional fermented milk.

When fermentation was carried out repeatedly at 30 or 37°C, kefir grains changed their kefir-like appearance, exhibited reduced growth rates, had a lower diversity of yeasts and water content, and a higher protein-to-polysaccharide ratio compared with the original kefir grains. In contrast, at 20°C kefir grains could remain in whey for prolonged periods without altering their acidification capacity, growth rate, macroscopic appearance or chemical and microbiologic composition—with the only difference being a reduction in certain yeast populations after 20 subcultures in whey. At this incubation temperature, the presence of Lactobacillus kefiranofaciens, Lb. kefir, Lb. parakefir, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces unisporus, and Sac. cerevisiae was detected in kefir grains and in fermented whey by denaturing-gradient-gel electrophoresis (DGGE). In whey fermented at 20°C the number of lactic-acid bacteria (LAB) was significantly lower (P<0·05) and the number of yeast significantly higher (P<0·05) than in fermented milk. Since the DGGE profiles were similar for both products, at this temperature the microbiologic composition of fermented whey is similar to that of fermented milk. We therefore suggest a temperature of 20°C to preserve kefir grains as whey-fermentation starters.

Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.

Two constituents of the proteose-peptone fraction of bovine milk have been isolated and characterized. Component 5 (PP5) has been shown to represent residues 1–105 and 1–107 of the β-casein amino acid sequence, while component 8-fast (PP8F) corresponds to residues 1–28 of β-casein. Thus, these proteose-peptones represent the N-terminal portions of the β-casein molecule, produced by proteolytic cleavages which form the γ1-, γ2- and γ3-caseins from the C-terminal part. The continuing formation of the total proteose-peptone fraction, PP5, PP8F and the γ-caseins during storage of raw milk at 18 or 37 °C has also been demonstrated

High pressure homogenization (HPH) is one of the most promising alternatives to traditional thermal treatment for food preservation and diversification. In order to evaluate its potential for the production of fermented milks carrying probiotic bacteria, four types of fermented milks were manufactured from HPH treated and heat treated (HT) milk with and without added probiotics. Microbiological, physicochemical and organoleptic analyses were carried out during the refrigerated period (35 d at 4°C). HPH application to milk did not modify the viability of the probiotic cultures but did increase the cell loads of the starter cultures (ca. 1 log order) compared with traditional products. The coagula from HPH-milk was significantly more compacted (P<0·05) (higher firmness) than that obtained with HT-milk, and it had the highest values of consistency, cohesiveness and viscosity indexes compared with fermented milks produced without HPH treatment. All the samples received high sensory analysis scores for each descriptor considered. HPH treatment of milk can potentially diversify the market for probiotic fermented milks, especially in terms of texture parameters.

The bacteriological formation of 2-butanone and 2-butanol was investigated. The data suggest that 2-butanol is formed in cheese via 2-butanone in 3 steps, each step being carried out specifically by a different microbial species. Suitable species have been isolated from a single cheese. The first step could be carried out (in the present investigation) either by certain starter organisms or by a strain of Pediococcus cerevisiae. This strain formed substantial amounts of 2,3-butylene glycol in milk, presumably from citrate, but could not metabolize this compound further. A second micro-organism, a strain of Lactobacillus plantarum, was isolated which, in Cheddar cheese, could convert 2,3-butylene glycol into 2-butanone. The final reduction of 2-butanone to 2-butanol could be readily carried out in MRS medium by a strain of L. brevis. Results from cheese made aseptically indicate that 2-butanone is formed in Cheddar cheese only when non-starter organisms are present.