Confocal scanning laser microscopy (CSLM) methods were developed to identify fat and protein in cheeses, milk chocolate and milk powders. Various fluorescent probes were assessed for their ability to label fat or protein in selected food products in situ. Dual labelling of fat and protein was made possible by using mixtures of probes. Selected probes and probe mixtures were then used to study (a) structure development of Mozzarella cheese during manufacture and ripening, and (b) the distribution of fat and protein in milk chocolate made with milk powders containing varying levels of free fat. Microstructural changes in the protein and fat phases of Mozzarella cheese were observed at each major step in processing. Aggregation of renneted micelles occurred during curd formation; this was followed by amalgamation of the para-casein into linear fibres during plasticization. Following storage, the protein phase of the Mozzarella became more continuous; entrapping and isolating fat globules. Chocolate made with a high free-fat spray-dried powder blend showed a homogeneous fat distribution, similar to that of chocolate made with roller-dried milk. Chocolate made with whole milk powder containing 10 g free fat/100 g fat showed a non-homogeneous fat distribution with some fat occluded within milk protein particles. These differences in fat distribution were related to Casson yield value and Casson viscosity of the chocolates.

Severe losses are caused in some types of cheese by the growth of spore-forming anaerobes. The species most commonly involved is Clostridium butyricum and its variants, but other clostridia such as Cl. sporogenes (Bacillus putrificus of Dorner (1)) may also be concerned. Existing methods for the study of the properties of these organisms are inadequate, a fact on which Rosenberger(2) has commented. This work has been particularly concerned with devising a medium suitable for all clostridia, which is clear, simple to prepare, and which requires no special anaerobic precautions in its use.

The effect of long-term intake of two fermented milk products on the development of hypertension was compared in young spontaneously hypertensive rats (SHR). The products contained tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP), which have been shown to possess angiotensin converting enzyme (ACE) inhibitory activity. Six-week-old SHR were divided into four groups to receive orally ad libitum water, skim milk or two fermented milk products (fermented milk A or fermented milk B; the latter is commercially available in Japan with trade name Calpis®) for 14 weeks. The calculated intake of IPP was 0·4 mg/d and 0·2 mg/d in the groups receiving fermented milk A and B, respectively, whereas the corresponding amounts for VPP were 0·6 mg/d and 0·3 mg/d. Systolic blood pressure (SBP) was monitored weekly by tail-cuff method. The development of hypertension was significantly attenuated in both groups receiving fermented milk products, whereas skim milk did not affect blood pressure. The effect was detectable after 6 weeks of treatment. At the end of the experiment, the lowest blood pressure level was found in the group receiving fermented milk A: the SBP was 21 mm Hg lower than in the group receiving water and 10 mm Hg lower than in the group receiving fermented milk B. This difference could be explained by larger intake of ACE inhibitory tripeptides in the group receiving fermented milk A as compared with fermented milk B.

In this study, the effect of dahi containing probiotic Lactobacillus acidophilus NCDC14 and Lactobacillus casei NCDC19 (∼73×108 cfu/g) on progression of streptozotocin (STZ)-induced diabetes in rats (15 g/day/rat) for 28 days was investigated. Feeding of probiotic dahi significantly suppressed the incremental peaks and area under the curve and delayed reduction of insulin secretion during oral glucose tolerance test more than skim milk or control dahi. The feeding of milk products reduced the total cholesterol, triglycerides, LDL and VLDL-cholesterol and increased HDL-cholesterol levels (P<0·05). Moreover, probiotic dahi significantly suppressed STZ-induced oxidative damage in pancreatic tissues by inhibiting the lipid peroxidation and formation of nitric oxide, and preserving antioxidant pool such as glutathione content and activities of superoxide dismutase, catalase and glutathione peroxidase. The results suggest that the supplementation of probiotic Lb. acidophilus and Lb. casei with dahi cultures increased the efficacy of dahi to suppress STZ-induced diabetes in rats by inhibiting depletion of insulin as well as preserving diabetic dyslipidemia, and inhibiting lipid peroxidation and nitrite formation. This may empower antioxidant system of β-cells and may slow down the reduction of insulin and elevation of blood glucose levels.

Studies of the potential of high pressure homogenisation (HPH) for the combined pasteurisation/homogenisation of raw bovine milk were undertaken. Raw milk was preheated to 45 °C and HPH-treated at 150, 200 or 250 MPa; milk outlet temperature at these pressures were 67, 76·8 and 83·6 °C, respectively, with a holding time of ~20 s. Raw and commercially pasteurized and homogenized (CPH) milk samples were analysed as controls. Fat globules in HPH samples were approximately half the size of those in CPH samples, although differences were not significant (P>0·05). β-Lactoglobulin was denatured at pressures [ges ]150 MPa, although little denaturation of α-lactalbumin was observed. Numbers of psychrotrophic bacteria in raw milk were reduced by 2·73 log cycles by HPH at 150 MPa and were uncountable following HPH at 200 or 250 MPa. Mesophilic bacterial counts were reduced by 1·30, 1·83 and 3·06 log cycles by HPH at 150, 200 or 250 MPa, respectively. No viable Staphylococcus aureus nor coliform cells remained in any HPH milk samples. HPH did not affect the colour of milk and HPH samples did not cream during refrigerated storage. The activities of plasmin, alkaline phosphatase and lactoperoxidase in milk were all greatly reduced by HPH. Pseudomonas fluorescens, inoculated into milk (~106 cfu/ml), was reduced to undetectable levels by HPH at 200 MPa (milk inlet temperature, ~10 °C); however, Ps. fluorescens proteinase was quite resistant to HPH under such conditions. Overall, owing to the significant increase in temperature and the possibility of varying the holding time, there may be potential applications for HPH as a novel liquid milk processing technique, combining many advantages of conventional homogenization and pasteurization of milk in a single process.

Autolysis of and proteolysis by various Lactococcus lactis subsp. cremoris strains were monitored in cheese ‘juice’ extracted by hydraulic pressure up to 63 d ripening. Viability was lowest for strain AM2 (non-bitter), intermediate for strain HP (bitter) and highest for the defined mixed strains G11/C25 (non-bitter). Autolysis monitored by the levels of the intracellular marker enzymes lactate dehydrogenase (EC 1.1.1.27), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and post-proline dipeptidyl aminopeptidase proceeded in the order AM2 > G11/C25 > HP. Differences in autolysis between strains did not appear to be due to differences in stabilities of the marker enzymes, populations of non-starter lactic acid bacteria or levels of the marker enzymes in the strains. Proteolysis, as measured by gel permeation FPLC and free amino acid analysis of the cheese juice was highest for AM2, intermediate for G11/C25 and lowest for HP. The results of this study provided some evidence that different Lactococcus strains used for cheesemaking had different autolytic patterns during ripening, the effects of which on ripening and flavour development have not yet been clearly demonstrated.

Lysozyme (LZ), lactoferrin (LF), lactoperoxidase (LP), immunoglobulin G and secretory immunoglobulin A were extracted from camel milk. The activity of these protective proteins was assayed against Lactococcus lactis subsp. cremoris, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and rotavirus. Comparative activities of egg white LZ, bovine LZ and bovine LF are also presented. The antibacterial activity spectrum of camel milk LZ was similar to that of egg white LZ, and differed from bovine milk LZ. Bovine and camel milk LF antibacterial activity spectra were similar. The camel milk LP was bacteriostatic against the Gram-positive strains and was bactericidal against Gram-negative cultures. The immunoglobulins had little effect against the bacteria but high titres of antibodies against rotavirus were found in camel milk. The LP system was ineffective against rotavirus.

The structure and characteristics of a model suitable for estimating digestion within the rumen and rates and patterns of nutrient entry in lactating cows are presented. The model consists of 12 state variables comprising a large particle pool, small particle pools representing insoluble dietary nutrients, soluble pools representing soluble dietary nutrients, and fermentation intermediates and end products. The model was constructed assuming continuous feeding, using Michaelis–Menten or mass action kinetics. The computer program was written in ACSL to run on a VAX computer. A fourth-order Runge–Kutta procedure was used for numerical integration.

Sensitivity and behavioural analysis demonstrated that overall stability and sensitivity of the model to individual parameters was generally satisfactory, but the need to improve the description and parameterization of aspects such as particle size in relation to availability, rate and affinity constants for amino acid degradation and rate constants for particle outflow from the rumen was established. Adjustments of the model to examine discontinuous feeding regimes were undertaken and initial results with respect to changes in fermentation rates, rumen acetate levels and microbial metabolism were considered realistic.

Comparisons with experimental data were considered satisfactory on forage-based and medium concentrate-containing diets, but with diets comprising 90% cereal, some inconsistencies, especially with respect to predictions of volatile fatty acid production rates, were observed. Reasons for this are put forward and suggestions for improvements in the model are discussed.

1. This paper is Part I of a series dealing with the relation between the chemical composition of milk and the stability of the caseinate complex to ethanol (Part II), rennet (Part III) and heat (Part IV).

2. A description is given of the milk samples, which were taken from herd bulk milk, from individual cows in different stages of lactation and also from cows with subclinical mastitis.

3. The methods used to measure the stability of the caseinate complex to the three coagulating agents are described, together with the methods used to make a detailed chemical analysis of the milk samples.

4. The chemical composition of the samples is given and also the relation between composition and stage of lactation, and the interrelations of the concentrations of certain milk constituents.

The authors thank Miss M. H. King and Miss R. E. M. Stevenson for technical assistance, Mr N. H. Strachan and Mr R. C. Voss (West of Scotland Agricultural College) for the sodium and potassium determinations and Dr P. S. Blackburn for the differential cell counts and bacteriological examination of the milks.

A subjective test for the determination of the stability of milk protein to heat is described. In the test, the time required for particles of coagulated protein to become visible throughout a 2·5-ml sample of separated milk maintained at 135°C in a glass tube rocking at 8 c/min is taken as a measure of stability. The precision of the test was such that single determinations were generally adequate.

Coagulation time decreased by about 12% as rocking speed was increased over the range 4–12 c/min and increased by a factor of about 3 for a decrease in heating temperature of 10 degC over the range 140–105 °C; with some milks the Q10 °C value increased to 5–8 a temperature decreased. As sample volume was increased over the range 1–3 ml coagulation time increased, especially With milks whose coagulation was poor (initial clots small). This volume effect appeared to be a consequence of the accompanying decrease in the proportion of headspace oxygen to volume of milk.

Bovine β-lactoglobulin (β-lg), α-lactalbumin (α-la) and bovine serum albumin (BSA), dispersed in ultrafiltration permeate, that had been prepared from whey protein concentrate solution (100 g/kg, pH 6·8), were heated at 75 °C. The consequent protein aggregation was studied by one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). When 100 g β-lg/kg permeate solution was heated at 75 °C, cooled and examined, large aggregates were observed. These aggregates were partially dissociated in SDS solution to give monomers, disulphide-bonded dimers, trimers and larger aggregates. When mixtures of β-lg and α-la or BSA were heated, homopolymers of each protein as well as heteropolymers of these proteins were observed. These polymer species were also observed in a heated mixture of the three proteins. Two-dimensional PAGE of mixtures demonstrated that these polymers species contained disulphide-bonded dimers of β-lg, α-la and BSA, and 1:1 disulphide-bonded adducts of α-la and β-lg, or BSA. These results are consistent with a mechanism in which the free thiols of heat-treated β-lg or BSA catalyse the formation of a range of monomers, dimers and higher polymers of α-la. It is likely that when whey protein concentrate is heated under the present conditions, BSA forms disulphide-bonded strands ahead of β-lg and that α-la aggregation with β-lg and with itself is catalysed by the heat-induced unfolded BSA and β-lg.

A comparison of literature results on the voluminosity of casein micelles yields large differences, largely related to the method of determination. After applying some corrections, methods based on hydrodynamic radius yield a value of roughly 3·9ml/g dry casein, other methods (microscopy, sediment volume) about 2·2. Roughly half of the discrepancy can be explained by the micelles being not perfectly spherical. To explain the remaining difference, it is assumed that the micelles are hairy, i.e. they have molecular chains protruding into the milk serum. The hairyness would increase with decreasing temperature and be largely removed by the action of rennet. It would cause some entropic repulsion between micelles at close approach.

High isostatic pressures up to 600 MPa were applied to samples of skim milk before addition of rennet and preparation of cheese curds. Electron microscopy revealed the structure of rennet gels produced from pressure-treated milks. These contained dense networks of fine strands, which were continuous over much bigger distances than in gels produced from untreated milk, where the strands were coarser with large interstitial spaces. Alterations in gel network structure gave rise to differences in rheology with much higher values for the storage moduli in the pressure-treated milk gels. The rate of gel formation and the water retention within the gel matrix were also affected by the processing of the milk. Casein micelles were disrupted by pressure and disruption appeared to be complete at treatments of 400 MPa and above. Whey proteins, particularly β-lactoglobulin, were progressively denatured as increasing pressure was applied, and the denatured β-lactoglobulin was incorporated into the rennet gels. Pressure-treated micelles were coagulated rapidly by rennet, but the presence of denatured β-lactoglobulin interfered with the secondary aggregation phase and reduced the overall rate of coagulation. Syneresis from the curds was significantly reduced following treatment of the milk at 600 MPa, probably owing to the effects of a finer gel network and increased inclusion of whey protein. Levels of syneresis were more similar to control samples when the milk was treated at 400 MPa or less.

The volatile concentrate obtained from Grana Padano cheese by vacuum distillation was fractionated by continuous liquid–liquid extraction into neutral and acid fractions. Both were analysed by high resolution gas chromatography (HRGC), HRGC–mass spectrometry, and HRGC–olfactometry. A total of 67 components were identified in the neutral extract (22 esters, 13 alcohols, 12 ketones, 6 aldehydes, 5 nitrogen-containing compounds, 3 lactones and 6 miscellaneous compounds) and 16 in the acid extract. Esters were the predominant constituents of the neutral fraction, whose major components were ethyl butanoate and ethyl hexanoate. HRGC–olfactometry of the neutral compounds demonstrated that 23 were odour-active: ethyl butanoate, 2-heptanol, 3-methylthiopropanal, 1-octen-3-ol, ethyl hexanoate and nonanal being the most potent odorants. n-Butanoic and n-hexanoic acids were the main volatile free fatty acids identified in the acid extract as having an important odour with a high olfactometric index. The backbone of Grana Padano cheese aroma seemed to consist of these acids and 14 potent neutral odorants imparting fruity, green, nutty and coconut notes. The concentration of volatile components responsible for the fruity and green notes was inversely proportional to the length of ripening, whereas the concentration of volatile agents with spicy, nutty and earthy notes tended to increase during maturation. In a comparison of the olfactometric profile, the Grana Padano cheese aroma was found to be more complex than an imitation Grana Padano cheese produced with similar technology but outside the area of the genuine cheese. Some of the main metabolic pathways for the biosynthesis of cheese aroma are reviewed briefly to indicate the possible origin of the compounds identified.

The rates of denaturation by heat of α-lactalbumin and β-lactoglobulin in skim-milk were measured by an immunodiffusion method over a wide range of temperatures. Both reactions showed an unusual temperature dependence.

The denaturation of α-lactalbumin is a first-order reaction; between 90 and 155 °C the kinetic constant k1 in s−1 is given by the equation

where T is the temperature in °K.

The denaturation of β-actoglobulin in skim-milk is second order with respect to time, and the kinetic constant k2 in 1g–1 s–1 is given by 2 equations, valid for different temperature ranges. Between 68 and 90 °C,

Between 90 and 135 °C

Results obtained by the immunodiffusion method agreed well with those found by salt fractionation of the milk proteins. The denaturation rates decreased when a specific reagent for sulphydryl groups was added, suggesting that such groups are involved in the denaturation of both proteins.

Kefir is a fermented milk produced by the action of lactic acid bacteria, yeasts and acetic acid bacteria, trapped in a complex matrix of polysaccharides and proteins. Beyond its inherent high nutritional value as a source of proteins and calcium, kefir has a long tradition of being regarded as good for health in countries where it is a staple in the diet. However, published human or animal feeding trials to substantiate this view are not numerous. The aim of this work was to determine the immunomodulating capacity of kefir on the intestinal mucosal immune response in mice and to demonstrate the importance of dose and cell viability on this response. BALB/c mice were fed with commercial kefir ad libitum (diluted 1/10, 1/50, 1/100 or 1/200) or pasteurized kefir (diluted 1/6, 1/10, 1/50, 1/100) for 2, 5 or 7 consecutive days. At the end of each feeding period, the bacterial translocation assay was performed in the liver. Small intestine structure was studied by haematoxilin-eosin staining and light microscopy. The number of IgA+ and IgG+ cells was also determined. For the functional doses chosen, cytokines (IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α and IFN-γ) were determined. Kefir and pasteurized kefir were able to modulate the mucosal immune system in a dose-dependent manner. Kefir was administred 10-times more diluted than pasteurized kefir, but it induced an immunomodulation of similar magnitude, indicating the importance of cell viabilty. The results suggest that a Th1 response was controlled by Th2 cytokines induced by kefir feeding. Pasteurized kefir would induce both Th2 and Th1 responses. This is the first study in vivo regarding the mechanisms involved in the immunomodulating capacity of the oral administration of kefir containing viable or heat-inactivated bacteria at different doses.