Multivariate analysis of organic acids in fermented food from reversed-phase high-performance liquid chromatography data.

Multivariate calibration coupled to RP-HPLC with diode array detection (HPLC-DAD) was applied to the identification and the quantitative evaluation of the short chain organic acids (malic, oxalic, formic, lactic, acetic, citric, pyruvic, succinic, tartaric, propionic and α-cetoglutaric) in fermented food. The goal of the present study was to get the successful resolution of a system in the combined occurrence of strongly coeluting peaks, of distortions in the time sensors among chromatograms, and of the presence of unexpected compounds not included in the calibration step. Second-order HPLC-DAD data matrices were obtained in a short time (10min) on a C18 column with a chromatographic system operating in isocratic mode (mobile phase was 20mmolL-1 phosphate buffer at pH 2.20) and a flow-rate of 1.0mLmin-1 at room temperature. Parallel factor analysis (PARAFAC) and unfolded partial least-squares combined with residual bilinearization (U-PLS/RBL) were the second-order calibration algorithms select for data processing. The performance of the analytical parameters was good with an outstanding limit of detection (LODs) for acids ranging from 0.15 to 10.0mmolL-1 in the validation samples. The improved method was applied to the analysis of many dairy products (yoghurt, cultured milk and cheese) and wine. The method was shown as an effective means for determining and following acid contents in fermented food and was characterized by reducibility with simple, high resolution and rapid procedure without derivatization of analytes.

Taxonomic Identity Resolution of Highly Phylogenetically Related Strains and Selection of Phylogenetic Markers by Using Genome-Scale Methods: The Bacillus pumilus Group Case.

Bacillus pumilus group strains have been studied due their agronomic, biotechnological or pharmaceutical potential. Classifying strains of this taxonomic group at species level is a challenging procedure since it is composed of seven species that share among them over 99.5% of 16S rRNA gene identity. In this study, first, a whole-genome in silico approach was used to accurately demarcate B. pumilus group strains, as a case of highly phylogenetically related taxa, at the species level. In order to achieve that and consequently to validate or correct taxonomic identities of genomes in public databases, an average nucleotide identity correlation, a core-based phylogenomic and a gene function repertory analyses were performed. Eventually, more than 50% such genomes were found to be misclassified. Hierarchical clustering of gene functional repertoires was also used to infer ecotypes among B. pumilus group species. Furthermore, for the first time the machine-learning algorithm Random Forest was used to rank genes in order of their importance for species classification. We found that ybbP, a gene involved in the synthesis of cyclic di-AMP, was the most important gene for accurately predicting species identity among B. pumilus group strains. Finally, principal component analysis was used to classify strains based on the distances between their ybbP genes. The methodologies described could be utilized more broadly to identify other highly phylogenetically related species in metagenomic or epidemiological assessments.

α-Acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions.

Lactic Acid Bacteria (LAB) are recognized as safe microorganisms with the capacity to improve the quality of dairy products. When the LAB Lactococcus lactis is employed as starter for the production of fermented foods, high quantities of important aroma compounds such as diacetyl are generated by means of the diacetyl/acetoin pathway. Our previous results obtained with L. lactis strains report that this pathway is activated under acidic conditions. In this study, we describe the metabolism of pyruvate, a diacetyl/acetoin precursor, and its contribution to pH homeostasis in this microorganism. L lactis strain IL1403 is able to cometabolize pyruvate and glucose at low pH, producing lactate, acetate as well as diacetyl/acetoin compounds. In contrast, the als defective strain, which is incapable of producing C4 compounds, appeared sensitive to pyruvate under acidic conditions rendering it unable to grow. Accordingly, the als-mutant strain showed a simultaneous inability to alkalinize internal and external media. These results demonstrate that the decarboxylation reactions associated to the diacetyl/acetoin pathway represent a competitive advantage in a condition of intracellular pyruvate accumulation during growth at low pH. Interestingly, a genomic comparative analysis shows that this pathway has been conserved in L. lactis during the domestication of different strains. Also, our analysis shows that the recent acquisition of the cit cluster required for citrate metabolism, which contributes to diacetyl/acetoin production as well, is the specific feature of the biovar. diacetylactis. In this regard, we present for first time genetic evidence supporting the proposal made by Passerini et al. (2013) who postulated that the expression "biovar. citrate" should be more appropriate to define this specific industrial strain.