A three-week algal meal supplementation of the cows' basal diet resulted in an increase in the firmness of milk fat crystallized isothermally at 5 °C for 24 h – the apparent elastic constant increased from 100 to 224 N/mm. This was accompanied by a decrease in solid fat content, from 47·7% to 44·4%. The crystallization behaviour of milk fat was also modified significantly. The rate constant of crystallization (Avrami constant) of the enriched milk fat at 19 °C was ∼20 times higher than that of control milk fat. A shorter induction time of nucleation was also observed in the temperature range [20, 27 °C]. These effects were attributed to a higher degree of supersaturation of the enriched milk fat. Enriched milk fat nucleated in a more stable β' polymorphic form at 5 °C, while control milk fat nucleated in the metastable α form, as determined by powder X-ray diffraction and differential scanning calorimetry. Changes in the microstructure of the material were observed by polarized light microscopy at 5 °C. The enriched milk fat displayed a greater amount of crystal clustering than the control. This effect was reflected in a decrease in the box-counting mass fractal dimension (Db) of the fat crystal network from 1·853 to 1·809. The decrease in Db closely predicted the observed 2·2-fold increase in the elastic constant of the fat. These changes in mechanical properties, crystallization behaviour and microstructure were driven by an increase in the 18[ratio ]1trans and a decrease in the 18[ratio ]1cis fatty acid content of the enriched milk fat.

Heat-induced gelation (80 °C, 30 min or 85 °C, 60 min) of whey protein concentrate (WPC) solutions was studied using transmission electron microscopy (TEM), dynamic rheology and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (150 g/kg, pH 6·9) were prepared by dispersing WPC powder in water (control), 10 g/kg sodium dodecyl sulphate (SDS) solution or 10 mM-dithiothreitol (DTT) solution. The WPC gels containing SDS were more translucent than the control gels, which were slightly more translucent than the gels containing DTT. TEM analyses showed that the SDS-gels had finer aggregate structure (≅10 nm) than the control gels (≅100 nm), whereas the DTT-gels had a more particulate structure (≅200 to 300 nm). Dynamic rheology measurements showed that the control WPC gels had storage modulus (G′) values (≅13500 Pa) that were ≅25 times higher than those of the SDS-gels (≅550 Pa) and less than half those of the DTT-gels after cooling. Compression tests showed that the DTT-gels were more rigid and more brittle than the control gels, whereas the SDS-gels were softer and more rubbery than either the control gels or the DTT-gels. PAGE analyses of WPC gel samples revealed that the control WPC solutions heated at 85 °C for 10 min contained both disulphide bonds and non-covalent linkages. In both the SDS-solutions and the DTT-solutions, the denatured whey protein molecules were in the form of monomers or small aggregates. It is likely that, on more extended heating, more disulphide linkages were formed in the SDS-gels whereas more hydrophobic aggregates were formed in the DTT-gels. These results demonstrate that the properties of heat-induced WPC gels are strongly influenced by non-covalent bonding. Intermolecular disulphide bonds appeared to give the rubbery nature of heat-induced WPC gels whereas non-covalent bonds their rigidity and brittle texture.

The changes in milk fat globules and fat globule surface proteins during concentration of whole milk using a pilot-scale multiple-effect evaporator were examined. The effects of heat treatment of milk at 95 °C for 20 s, prior to evaporation, on fat globule size and the milk fat globule membrane (MFGM) proteins were also determined. In both non-preheated and preheated whole milk, the size of milk fat globules decreased while the amount of total surface proteins at the fat globules increased as the milk passed through each effect of the evaporator. In non-preheated samples, the amount of caseins at the surface of fat globules increased markedly during evaporation with a relatively small increase in whey proteins. In preheated samples, both caseins and whey proteins were observed at the surface of fat globules and the amounts of these proteins increased during subsequent steps of evaporation. The major original MFGM proteins, xanthine oxidase, butyrophilin, PAS 6 and PAS 7, did not change during evaporation, however, PAS 6 and PAS 7 decreased during preheating. These results indicate that the proteins from the skim milk were adsorbed onto the fat globule surface when the milk fat globules were disrupted during evaporation.

The objective of the present work was to identify the compositional parameters of raw milk that affected ethanol stability at natural pH when natural milk conditions were not modified. Heat stability, measured as coagulation time (CT), was included in the analysis to verify relation to alcohol test. Statistical models were proposed for alcohol and heat (CT) stabilities. Milk samples of good hygienic quality from dairy farms were classified in two groups according to their alcohol stability. Unstable samples to ethanol (72%, v/v) presented lower values of pH, somatic cells count, casein and non-fat-solids relative to ethanol stable samples (ethanol at 78%, v/v or more); whereas freezing point, chloride, sodium and potassium concentrations were higher in the unstable group. Logistic regression and multiple regression were applied to modelling alcohol and heat stability behaviour respectively. Chloride, potassium, ionic calcium and somatic cell count were included in the alcohol regression model, whereas calcium, phosphorous, urea, pH and ionic calcium were part of CT model. Ionic calcium was the only measured variable that contributed to both models; however coagulation time was noted to be more sensitive to ionic calcium than alcohol. The relation between ionic strength and casein was found to contribute to the alcohol model but not to the CT model. However, the interaction calcium plus magnesium plus phosphorous and casein contributed only to CT model.

Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were used to monitor changes in the secondary structure and thermal stability of β-lactoglobulin A and B in the presence of sodium dodecyl sulphate (SDS), N-ethylmaleimide (NEM), urea and cysteine. An increase in the thermal stabilities of both proteins was noted in the presence of 10 mM-SDS. In the presence of 50 mM-SDS, there was extensive denaturation of both variants. In general, the β-strand/β-sheet regions in the secondary structure of both variants were very susceptible to denaturation by SDS and cysteine, suggesting that these regions may be held by hydrophobic and disulphide bonds. At ambient temperature and physiological pH, a notable difference was observed in the 1636 and 1627 cm−1 regions of the FTIR spectra of the two β-lg variants. The results suggest possible differences in the nature of the β-sheet/β-strand distribution/content of the two proteins. Urea and NEM at a concentration of 50 mM, had little effect on the secondary structure and denaturation of both variants. New findings are presented which further indicate that although the β-lg B variant showed greater thermal stability than the A variant in all the cases studied, its denaturation temperature and secondary structure were affected to a greater extent by the protein perturbants than β-lg A.

Pressure processing of foodstuff has been applied to produce or modify proteinaceous gel structures. In real pressure processing the treatment is non-isothermal, due to the adiabatic nature of the process and the heat loss from the product to the vessel. In order to estimate the effect of pressurization on milk constituents pressure and temperature dependent kinetics were determined separately from each other. In a detailed kinetic study whey protein isolate was treated under isobaric (200 to 800 MPa) and isothermal conditions (−2 to 70 °C), and the resulting degree of denaturation of β-lactoglobulin A and B and α-lactalbumin was analysed. Kinetic parameters of denaturation were estimated using a one step non-linear regression method which allowed a global fit of the whole data set. The isobaric isothermal denaturation of β-lactoglobulin and α-lactalbumin was found to follow third and second order kinetics, respectively. Isothermal pressure denaturation of both β-lactoglobulin fractions do not differ significantly and were characterized by an activation volume decreasing with increasing temperature from −10 to about −30 ml mol−1, which demonstrates that the denaturation rate is accelerated with increasing temperature. The activation energy of about 70 to 100 kJ mol−1 obtained for β-lactoglobulin A and B is not dependent to a great extent on the pressure which indicates that above 200 MPa denaturation rate is limited by the aggregation rate while pressure forces unfolding of the molecule.

The protein and peptide fraction of human milk samples collected from mothers of pre- and full-term infants in the first week after parturition was analysed by use of liquid chromatography-mass spectrometry and tandem mass spectrometry. By characterising the peptide sequence, we defined the pathway of casein hydrolysis which leads to the formation of small peptides through intermediate oligopeptides. It was found that the action of a plasmin-like enzyme acting on specific lysine residues is the primary step in casein degradation. This is followed by endopeptidases and/or exopeptidases mediated cleavage of the oligopeptides which, in turn, produces a multiplicity of short peptides differing by one or more amino acid residues. In this process, a series of potentially bioactive peptides (opioid, phosphopeptides) and their precursors are produced.

Acidity is an environmental condition commonly encountered by lactic acid bacteria and bifidobacteria in the gastrointestinal tract and fermented foods. In the present study, 22 strains of Bifidobacterium were screened for acid tolerance in artificial gastric juice (AGJ, pH 3·0) and fermented milk. AGJ tolerance was found to be strain-specific, with a pronounced variation among the strains. Several strains with a high survival rate in AGJ that belonged to Bifid. longum, Bifid. breve and Bifid. adolescentis were selected. Among them, only strain BL1 of Bifid longum was found to possess a high survival rate in fermented milk during refrigerated storage. Strain BL1 exhibited a survival rate of more than 25% in AGJ at pH 3·0 for 2 h and maintained a viable cfu level of more than 108 per gram of product in fermented milk (pH 4·6) under refrigerated conditions for 2 weeks. The acid tolerance of strain BL1 was found to depend on the final growth pH (<4·5). Rapid loss of acid tolerance was observed when the cells were shifted from acid to neutral conditions by addition of NaOH. Strain BL1 cells were able to maintain much higher intracellular pH under acid conditions, in comparison with those of AGJ sensitive mutant (BL1-S) or cells that lost acid tolerance following pH shifting from acid to neutral conditions. These results suggested that a cytoplasmic pH homeostasis system may function in the acid tolerance response in this strain.

Details in the hydrolysis of α-lactalbumin known to result in formation of highly ordered nanotubules, was investigated by incubation of solutions with 10 g α-lactalbumin/l with a specific protease from Bacillus licheniformis (BLP). After 50 min of incubation, soluble aggregates were formed, the concentration of which increased until precipitation occurred after 200 min. The latter aggregates were dissolved in urea or at low pH, like the nanotubules characteristic of gels formed by the action of BLP on α-lactalbumin at 100 g/l. On the molecular level, α-lactalbumin was initially cleaved into two large hydrophobic fragments with masses of 11·6 and 11·3 kDa, which in turn were cleaved in a stepwise manner into the ultimate fragment of 8·8 kDa. This fragment was the predominating component in the insoluble aggregates, and was identified as the sequences 26–37 and 50–113 of α-lactalbumin linked together by a disulphide bond. Cleavage of α-lactalbumin into this fragment probably created new hydrophobic surfaces and new calcium binding sites allowing its association into ordered structures.

In this study, a two-plasmid system for enhanced and consistent biosynthesis of the model lactococcal bacteriocin lactococcin A in non-producing Lactococcus lactis hosts was developed. The system comprised a plasmid carrying the genes lcnA and lciA under the control of the nisin-inducible nisA promoter, and a second plasmid harbouring the lcnC and lcnD genes. The introduction of both plasmids into two strains containing the nisRK genes required for nisin-controlled expression, Lc. lactis FI5876 (a nisin A-producer strain) and FI7847, resulted in production of extracellular lactococcin A at a higher level than that for the parental strain, Lc. lactis WM4. In addition, transformation of the nisin-producing host with both plasmids led to a high-level production of both lactococcal bacteriocins, which may provide a means to exploit their complementary properties in cheese ripening.

A holistic approach of a mould cheese ripening is presented. The objective was to establish relationships between the different microbiological and biochemical changes during cheese ripening. Model cheeses were prepared from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions. Two cheese-making trials with efficient control of environmental parameters were carried out and showed similar ripening characteristics. K. lactis grew rapidly between days 1 and 6 (generation time around 48 h). G. candidum grew exponentially between days 4 and 10 (generation time around 4·6 d). Brevi. linens also grew exponentially but after day 6 when Pen. camemberti mycelium began developing and the pH of the rind was close to 7. Its exponential growth presented 3 phases in relation to carbon and nitrogen substrate availability. Concentrations of Pen. camemberti mycelium were not followed by viable cell count but they were evaluated visually. The viable microorganism concentrations were well correlated with the carbon substrate concentrations in the core and in the rind. The lactose concentrations were negligible after 10 d ripening, and changes in lactate quantities were correlated with fungi flora. The pH of the inner part depended on NH3. Surface pH was significantly related to NH3 concentration and to fungi growth. The acid-soluble nitrogen (ASN) and non-protein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until day 6, and then increased rapidly to follow the fungi concentrations until day 45. The ASN and NPN indexes and NH3 concentrations in the core were lower than in the rind and they showed the same evolution. G. candidum and Pen. camemberti populations have a major effect on proteolysis; nevertheless, K. lactis and Brevi. linens cell lysis also had an impact on proteolysis. Viable cell counts of K. lactis, G. candidum, Pen. camemberti and Brevi. linens were correlated with the environmental conditions, with proteolytic products and with carbon substrate assimilation. NH3 diffusion from surface to the cheese core during ripening was highly suspected. Interaction phenomena between microorganisms are discussed.

In this study, high pressure (HP)-induced denaturation of α-lactalbumin (α-la) and β-lactoglobulin (β-lg) in dairy systems was examined. In both milk and whey, β-lg was less baroresistant than α-la; both proteins were considerably more resistant to HP-induced denaturation in whey than in milk. HP-induced denaturation of α-la and β-lg increased with increasing proportion of milk in mixtures of milk and whey. Addition of a sulphydryl-oxidising agent, KIO3, to milk or whey increased HP-induced denaturation of β-lg, but reduced the denaturation of α-la. Denaturation of both α-la and β-lg was prevented by adding a sulphydryl-blocking agent, N-ethylmaleimide, to milk or whey prior to HP treatment, highlighting the crucial role of sulphydryl-disulphide interchange reactions in HP-induced denaturation of α-la and β-lg. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of α-la and β-lg significantly. The higher level of HP-induced denaturation of α-la and β-lg in milk than in whey may be the result of the abscence of the casein micelles and colloidal calcium phosphate from whey, which facilitate HP-induced denaturation of α-la and β-lg in milk.

Effects of high pressure (HP) on average casein micelle size and denaturation of α-lactalbumin (α-la) and β-lactoglobulin (β-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by ∼25%, while treatment at [ges ]300 MPa irreversibly reduced it to ∼50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times [ges ]2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7·0 partially stabilised micelles against HP-induced disruption. Denaturation of α-la did not occur [les ]400 MPa, whereas β-lg was denatured at pressures >100 MPa. Denaturation of α-la and β-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured β-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.

In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66–71 kDa as measured by SDS–PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.

High pressure (HP) processing has attracted considerable interest in dairy research in recent years; its effects on cheese have been reviewed by O'Reilly et al. (2001). HP treatment of cheese may influence several of its properties, e.g., for hard and semi-hard cheeses, such as Cheddar and Gouda, the most notable effects are on the texture and rate of ripening. HP treatment increased elasticity and improved organoleptic properties of Gouda cheese (Kolakowski et al. 1998). Messens et al. (2000) reported that, immediately after treatment at 400 MPa, Gouda cheese was less rigid and solid-like and more viscoelastic than untreated cheese. HP treatment also enhances the functional properties of Mozzarella cheese (Johnston & Darcy, 2000; O'Reilly et al. 2002).

Flavour generation in cheese is a major aspect of ripening. In order to enhance aromatic qualities it is necessary to better understand the chemical and microbiological changes. Experimental Camembert-type cheeses were prepared in duplicate from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions. Two replicates performed under controlled conditions of temperature (12 °C), relative humidity (95±2%), and atmosphere showed similar ripening characteristics. The evolutions of metabolite concentrations were studied during ripening. The volatile components were extracted by dynamic headspace extraction, separated and quantified by gas chromatography and identified by mass spectrometry. For each cheese the volatile concentrations varied with the part considered (rind or core). Except for ethyl acetate and 2-pentanone, the volatile quantities observed were higher than their perception thresholds. The flavour component production was best correlated with the starter strains. During the first 10 days the ester formations (ethyl, butyl and isoamyl acetates) were associated with the concentrations of K. lactis and G. candidum. The rind quantity of esters was lower than that observed in core probably due to (1) a diffusion from the core to the surface and (2) evaporation from the surface to the chamber atmosphere. G. candidum and Brev. linens association produced 3 methyl butanol and methyl 3-butanal from leucine, respectively. DMDS came from the methionine catabolism due to Brev. linens. Styrene production was attributed to Pen. camemberti. 2-Pentanone evolution was associated with Pen. camemberti spores and G. candidum. 2-Heptanone changes were not directly related to flora activities while 2-octanone production was essentially due to G. candidum. This study also demonstrates the determining role of volatile component diffusion.

Osteopontin (OPN) is an acidic phosphorylated glycoprotein found in many tissues and physiological fluids. Bovine OPN is a mature protein comprising 262 amino acids with a calculated molecular weight of 29 kDa. However, SDS-PAGE analysis reveals that the protein isolated from milk migrates to a molecular mass of 60 kDa (Sørensen & Petersen, 1993; Bayless et al. 1997). Bovine milk OPN is phosphorylated at 27 serine residues and one threonine residue (Sorensen et al. 1995); three O-glycosylated threonines were also identified, but no asparagine residues were glycosylated in spite of the presence of three putative N-glycosylation sites. In contrast, eight phosphates are recognized in bovine bone OPN (Salih et al. 1996), and 12 phosphoserines and one phosphothreonine are proposed in addition to five O-linked oligosaccharides and at most one N-linked oligosaccharide in the case of rat bone OPN (Prince et al. 1987). Thus, the possibility of tissue or species-specific differences in post-translational modification has been discussed.

In a study of the evolution of conjugated linoleic acid (CLA) during cheese production, the influence of Emmental cheese processing on the CLA content and the CLA isomer composition was evaluated. The use of raw and thermised milk, changes of processing temperature and the effect of propionic acid bacteria (PAB) were investigated. The content of CLA in raw milk was 8·6±1·9 mg/g fat and in the ripened cheese at 70 d was 8·6±1·6 mg/g fat, under normal processing conditions. No changes in the CLA content and CLA isomer composition were observed during Emmental cheese manufacturing process. Changes in cooking and moulding temperatures did not influence the CLA content. CLA content of cheese made from microfiltered milk with two different Propionibacterium freudenreichii strains was very close to cheeses made without PAB. CLA levels seem to be stable in this type of dairy product under the conditions examined.

High pressure processing was investigated for controlling Cheddar cheese ripening. One-month- or 4-month-old Cheddar cheeses were subjected to pressures ranging from 200 to 800 MPa for 5 min at 25 °C. The number of viable Lactococcus lactis (starter) and Lactobacillus (nonstarter) cells decreased as pressure increased. Subsequent storage of the control and pressure-treated cheeses at 10 °C caused viable cell counts to change in some cases. Free amino acid content was monitored as an indicator of proteolysis. Cheeses treated with pressures [ges ]400 MPa evolved free amino acids at significantly lower rates than the control. No acceleration in free amino acid development was observed at lower pressures. Pressure treatment did not accelerate the rate of textural breakdown compared with the non-pressure treated control. On the contrary, pressure treatment at 800 MPa reduced the time-dependent texture changes. Results indicate that high pressure may be useful in arresting Cheddar cheese ripening.

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.