Microbial composition and viability of natural whey starters used in PDO Comté cheese-making.

Natural whey starters (NWS) are cultures with undefined multiple-strains species commonly used to speed up the fermentation process of cheeses. The aim of this study was to explore the diversity and the viability of Comté cheese NWS microbiota. Culture-dependent methods, i.e. plate counting and genotypic characterization, and culture-independent methods, i.e. qPCR, viability-qPCR, fluorescence microscopy and DNA metabarcoding, were combined to analyze thirty-six NWS collected in six Comté cheese factories at two seasons. Our results highlighted that NWS were dominated by Streptococcus thermophilus (ST) and thermophilic lactobacilli. These species showed a diversity of strains based on Rep-PCR. The dominance of Lactobacillus helveticus (LH) over Lactobacillus delbrueckii (LD) varied depending on the factory and the season. This highlighted two types of NWS: the type-ST/LD (LD > LH) and the type-ST/LH (LD < LH). The microbial composition varied depending on cheese factory. One factory was distinguished by its level of culturable microbial groups (ST, enterococci and yeast) and its fungi diversity. The approaches used to estimate the viability showed that most NWS cells were viable. Further investigations are needed to understand the microbial diversity of these NWS.

Whole-Genome Sequencing and Annotation of Clostridium tyrobutyricum Strain Cirm BIA 2237, Isolated from Silage.

Clostridium tyrobutyricum is the main bacterial species leading to the late blowing defect, a major cause of spoilage in semihard and hard cheeses. This study reports the complete genome sequencing, assembly, and annotation of C. tyrobutyricum strain Cirm BIA 2237, formerly called CNRZ 608, isolated from silage.

Evaluation of qPCR and plate counting for quantifying thermophilic starters in cheese.

The respective inputs of plate counting and qPCR for the quantification of starters in cheese were evaluated using hard-cooked cheeses made with various starter combinations. Five starter strains were quantified at their different growth phases, from 0.5 h to day 214 of manufacture: one strain of Streptococcus thermophilus (ST) and two strains each of Lactobacillus delbrueckii (LD) and Lactobacillus helveticus (LH). Numbers of colony-forming units (CFU) were obtained by plate counting (PC) and qPCR (GNCFU). The qPCR standard curves require a special attention since GNCFU depends on the degree of culturability of the standard culture. Discrepancies were evidenced from the vat milk to the end of ripening. During cheese making, GNCFU were lower than PC at the inoculation for all ST and LD samples and 83% of the LH samples, and during both the exponential and stationary phases for many of the ST and LD samples. During ripening which corresponds to the decline phase, GNCFU were higher than PC for 90% of the ST, 78% of the LD and 69% of the LH samples. Hypotheses are discussed to explain those discrepancies. The data provided by GNCFU and PC complement each other, providing a better description of starter growth in cheese.

Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples.

The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R(2) > 0.990), down to 22 gene copies/µL per well for Lc. lactis and 73 gene copies/µL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei.

Structure and composition of model cheeses influence sodium NMR mobility, kinetics of sodium release and sodium partition coefficients.

The mobility and release of sodium ions were assessed in model cheeses with three different lipid/protein ratios, with or without added NaCl. The rheological properties of the cheeses were analysed using uniaxial compression tests. Microstructure was characterised by confocal laser scanning microscopy. (23)Na nuclear magnetic resonance (NMR) spectroscopy was used to study the molecular mobility of sodium ions in model cheeses through measurements of the relaxation and creation times. Greater mobility was observed in cheeses containing a lower protein content and with added NaCl. The kinetics of sodium release from the cheese to an aqueous phase was correlated with the mobility of sodium ions. The highest rates of sodium release were observed with a lower protein content and with added NaCl. The water/cheese partition coefficients of sodium increased when NaCl was added or the protein content was higher. The study highlighted the effect of model cheese characteristics on molecular and macroscopic behaviours of sodium.

In vivo sodium release and saltiness perception in solid lipoprotein matrices. 2. Impact of oral parameters.

This study aimed to investigate the relationships between sodium release, saltiness, and oral parameters during the eating of lipoprotein matrices (LPM). Sodium release and saltiness relative to 10 LPM were recorded during normal mastication by five subjects with differing oral parameters (chewing efficiency and salivary flow rate). The LPM samples varied in composition (dry matter, fat, salt, and pH levels) and represented a broad range of hardness. Mastication was recorded using electromyography simultaneously with sensory assessment. Differences in chewing behavior could explain most of the variability in sodium release and saltiness among subjects. Subjects with a higher chewing force and lower salivary flow rate experienced higher levels of sodium release and saltiness. In terms of the LPM, sodium release and saltiness were affected by either chewing behavior or food composition.

In vivo sodium release and saltiness perception in solid lipoprotein matrices. 1. Effect of composition and texture.

Reducing the sodium content in foods is complex because of their multidimensional sensory characteristics and the multifunctionality of sodium chloride. The aim of this study was to elucidate how food composition may influence in-mouth sodium release and saltiness perception. Lipoprotein matrices (LPM) were produced using milk constituents and characterized by means of rheological measurements, texture, and taste sensory profiles. Texture and taste perceptions were affected differently by variations in the salt level, dry matter, and fat contents. Composition and textural changes also modified temporal sodium release and saltiness perception recorded in five subjects, but the effects varied as a function of the salt content. The water content mainly appeared to influence the amount of sodium released, whereas saltiness perception was mainly related to fat content. Elasticity, coating, and granularity were found to be correlated with temporal sodium release and/or saltiness parameters.

Investigation of surface plasmon resonance biosensor for skin sensitizers studies.

Non-animal testing methods are a current challenge in terms of the assessment of skin sensitization potential for new chemicals. Our objective was to investigate a surface plasmon resonance (SPR) biosensor to screen allergens against nucleophilic amino acids (cysteine, lysine and histidine) in a direct binding assay. Amino acids were immobilized on the sensor surface and exposed to different skin allergens (chemicals and fragrances) with varying sensitizing potential. Cysteine was found to be more reactive than lysine while histidine showed the lowest reactivity. The interactions observed were different depending on the allergen/amino acids involved. It appeared that weak allergens could quickly dissociate from the ligand, whereas strong and extreme allergens remained bound to the amino acids. The SPR report points allowed a good discrimination of the tested allergens. With this technology, we can observe low energy bindings and get information on the stability of the hapten/amino acid complex which seem relevant for the determination of skin sensitization potential. This prospective experiment showed the potential of real-time SPR to generate specific report points to refine the skin sensitization allergen assessment.