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Bioengineering of probiotics allows the improvement of their beneficial characteristics. In this work, we develop a molecular tool that would allow the activation of desirable traits in probiotics once they reach the intestine. The activity of upstream regions of bile-inducible genes of Lactobacillus casei BL23 and Lactobacillus plantarum WCFS1 was analyzed using plasmids encoding an anaerobic fluorescent protein as reporter. The promoter P16090 from Lb. casei BL23 was selected and its bile induction confirmed in Lb. casei BL23, Lb. plantarum WCFS1, and in Lactobacillus rhamnosus and Lactobacillus reuteri strains. However, the induction did not occur in Lactococcus lactis MG1363 or Bifidobacterium strains. Studies with different bile compounds revealed the importance of cholic acid in the bile induction process. Induction of fluorescence was also confirmed for transformed Lb. casei BL23 under simulated colonic conditions and in the presence of intestinal microbiota. The developed vector, pNZ:16090-aFP, constitutes a promising tool suitable for the expression of genes of interest under intestinal conditions in probiotic strains of the species Lb. casei, Lb. plantarum, Lb. rhamnosus, and Lb. reuteri.

Phytoestrogens (isoflavones, ellagitannins, and lignans) are polyphenols with an estrogenic/antiestrogenic function present in fruits, vegetables, and foods derived from them. Some microorganisms are capable of transforming isoflavones, ellagitannins, and lignans into equol, urolithins, and enterolignans, respectively. These compounds are more bioavailable, they have more estrogenic/antiestrogenic and antioxidant activity than their precursors, and they are associated with beneficial effects for human health. Extracts of soy- bean and lignans and ellagic acid were incubated with feces, or bacteria isolated from feces, and later the phytoestrogens were extracted twice with diethyl ether and twice with ethyl acetate, and the solvents were evaporated at room temperature under a N2. Subsequently, phytoestrogens were detected by HPLC-PAD and chromatographic peaks were identified by HPLC-ESI/M, and they were confirmed by comparison of retention times and characteristics of UV spectra with those of standards. Analysis of phytoestrogens by means of HPLC- PAD allowed the simultaneous detection of isoflavones, ellagitannins, and lignans metabolized, or not, by intestinal microbiota with high sensitivity (LOD: equol 0.11 mg/L; urolithin A 0.17 mg/L, enterodiol 0.15 mg/L). Assays were linear up to at least 20 mg/L in all standards with a high correlation (equol >0.999; urolithin A >0.998, enterodiol >0.999). Therefore, the described method constitutes an affordable and easy alternative method for the detection of phytoestrogen metabolism. Moreover, this method could be applied to the detection of isoflavones, lignans, and ellagitannins in food., This work was supported by RM2012-00004-00-00. J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO, Spain).

While many compounds have been reported to change in laboratory based drought-stress experiments, little is known about how such compounds change, and are significant, under field conditions. The Pisum sativum L. (pea) leaf metabolome has been profiled, using 1D and 2D NMR spectroscopy, to monitor the changes induced by drought-stress, under both glasshouse and simulated field conditions. Significant changes in resonances were attributed to a range of compounds, identified as both primary and secondary metabolites, highlighting metabolic pathways that are stress-responsive. Importantly, these effects were largely consistent among different experiments with highly diverse conditions. The metabolites that were present at significantly higher concentrations in drought-stressed plants under all growth conditions included proline, valine, threonine, homoserine, myoinositol, γ-aminobutyrate (GABA) and trigonelline (nicotinic acid betaine). Metabolites that were altered in relative amounts in different experiments, but not specifically associated with drought-stress, were also identified. These included glutamate, asparagine and malate, with the last being present at up to 5-fold higher concentrations in plants grown in field experiments. Such changes may be expected to impact both on plant performance and crop end-use.

Inhibitory activity of reuterin (β-hydroxypropionaldehyde) combined with the antimicrobial peptide nisin or the lactoperoxidase system (LPOS) against food-borne Gram-negative pathogens in milk refrigerated at 4 and 8 °C was investigated. At 4 °C, reuterin (8 AU/ml) and LPOS were bactericidal against Salmonella enterica, Campylobacter jejuni, Aeromonas hydrophila and Yersinia enterocolitica, whereas the only effect recorded for nisin was a slight inhibition of Escherichia coli O157H7. At 8 °C, reuterin was bactericidal against all the Gram-negative pathogens studied. Same results were attained with LPOS, except for its effect on Y. enterocolitica which was only bacteriostatic. The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin. A strong synergistic bactericidal activity of reuterin in combination with LPOS on E. coli O157H7 and S. enterica was observed in milk at 4 °C, and against all the Gram-negative bacteria assayed in milk refrigerated at 8 °C. The application of both antimicrobials would be a useful means to inhibit pathogenic microorganisms, which may be present in milk due to postpasteurization contamination. © 2007 Springer-Verlag.