RESUMO
Among the isoflavones and isoflavone-derived metabolites, equol, which in the human gut is synthesised from daidzein by minority bacterial populations, shows the strongest estrogenic and antioxidant activity. The beneficial effects on human health of isoflavone consumption might be partially or indeed totally attributable to this equol. Although some of the bacterial strains involved in its formation have been identified, the interplay between the composition and functionality of the gut microbiota and equol producer phenotype has hardly been studied. In this study, after shotgun metagenomic sequencing, different pipelines for the taxonomic and functional annotation of sequencing data were used in the search for similarities and differences in the faecal metagenome of equol-producing (n=3) and non-producing (n=2) women, with special focus on equol-producing taxa and their equol-associated genes. The taxonomic profiles of the samples differed significantly depending on the analytical method followed, although the microbial diversity detected by each tool was very similar at the phylum, genus and species levels. Equol-producing taxa were detected in both equol producers and non-producers, but no correlation between the abundance of equol-producing taxa and the equol producing/non-producing phenotype was found. Indeed, functional metagenomic analysis was unable to identify the genes involved in equol production, even in samples from equol producers. By aligning equol operons with the collected metagenomics data, a small number of reads mapping to equol-associated sequences were recognised in samples from both equol producers and equol non-producers, but only two reads mapping onto equol reductase-encoding genes in a sample from an equol producer. In conclusion, the taxonomic analysis of metagenomic data might not be suitable for detecting and quantifying equol-producing microbes in human faeces. Functional analysis of the data might provide an alternative. However, to detect the genetic makeup of the minority gut populations, more extensive sequencing than that achieved in the present study might be required.
RESUMO
γ-Aminobutyric acid (GABA), an amino acid not used in protein synthesis, intervenes in several physiological functions and has both diuretic and calming effects in humans. Lactic acid bacteria (LAB) strains that produce GABA could be exploited for the manufacture of health-promoting GABA-enriched dairy products. In this study, 262 LAB strains isolated from traditional dairy products made from raw milk without starter cultures were screened for GABA production in culture media supplemented with 1% monosodium glutamate (MSG) using an enzymatic (GABase) method. About half of the strains (123) were found to be GABA producers. Of these, 24, among which were 16 Lactococcus lactis subsp. lactis and three Streptococcus thermophilus strains, produced >1 mM of GABA (range 1.01-2.81 mM) and were selected for further characterisation. GABA production was confirmed in most strains by culturing in 5 mM MSG followed by HPLC quantification. A majority of the strains were confirmed to be GABA producers by this method, although lower production levels were recorded. Using species-specific primers, the gene encoding glutamate decarboxylase (GAD) was PCR-amplified in all but one of the GABA producers analysed. Amplicons sequences were compared to one another and to those held in databases. Except for one Lactobacillus brevis strain, none of the 24 GABA producers investigated produced toxic biogenic amines, such as tyramine, histamine or cadaverine. They were therefore considered safe. Either alone, in mixtures, or in combination with industrial starter or adjunct cultures, these strains might be useful in the development of health-oriented dairy products.
