Vers le développement de bactéries lactiques probiotiques pour améliorer la digestibilité du soja

Le développement des produits à base de soja est freiné par leur forte teneur en a-galactosides (raffinose, stachyose). Du fait de l'absence d'a-galactosidase (a-Gal) dans l'intestin grêle, ils s'accumulent dans le côlon où ils sont métabolisés par des bactéries gazogènes, entraînant des problèmes de flatulences. L'objectif de cette étude est de dégrader les a-galactosides grâce à des bactéries lactiques a-Gal+ en fermentation de lait de soja et in vivo dans la partie supérieure du tractus digestif. Des fermentations de lait de soja ont été réalisées avec L. plantarum ATCC8014, L. fermentum CRL722, B. longum R0070 et B. breve R0175. L. plantarum ATCC8014 dégrade seulement le stachyose alors que les autres souches permettent de dégrader plus de 90% des deux a-galactosides majoritaires du soja. L. fermentum CRL722, testée dans le tractus digestif du rat monoxénique placé en chambre respiratoire, permet de réduire significativement l'émission de gaz intestinaux, indiquant sa capacité à exercer l'activité a-Gal in vivo. Un modèle d'étude a été mis au point chez L. lactis afin de définir les facteurs influençant l'activité a-Gal dans le tractus digestif. Dans ce cadre, les gènes melA et melAF, codant respectivement pour l'a-Gal de L. plantarum ATCC8014 et de L. fermentum CRL722, ont été caractérisés. Nous avons choisi de faire varier la localisation cellulaire de l'enzyme chez la bactérie (cytoplasme, surnageant, ou ancrée à la paroi). Dans les conditions expérimentales appliquées, les vecteurs construits n'ont pas permis de diminuer les flatulences chez le rat nourri avec un régime riche en a-galactosides. Les améliorations à apporter à ce système sont discutées.The development of soy products has been limited because of their high a-galactosides contents (raffinose, stachyose). Since humans do not posses a-galactosidase (a-Gal) in the small intestine, they accumulate in the large intestine where they are metabolized by gasogenic microorganisms which cause flatulence problems. The objective of this study was to degrade the a-galactosides in soy using a-Gal producing lactic acid bacteria in soymilk fermentation and in vivo in the upper gastrointestinal tract. Soymilk fermentations were performed using L. plantarum ATCC8014, L. fermentum CRL722, B. longum R0070 and B. breve R0175. L. plantarum ATCC8014 only degrades stachyose whereas the other strains degraded more than 90% of the two major a-galactosides found in soymilk. The activity of L. fermentum CRL722 was evaluated in vivo in the digestive tract of monoxenic rats placed in respiratory chambers. This strain reduced significantly the intestinal gas production, suggesting that it is able to exert a-Gal activity in vivo. Also, a study model was developed in L. lactis for evaluation of factors that affect a-Gal activity in the digestive tract. For this purpose, the melA and melAF genes, which code for the a-Gal of L. plantarum ATCC8014 and L. fermentum CRL722 respectively, have been characterized. In this study, we chose to vary the cellular localization of the enzyme in the bacterium. In the applied experimental conditions, these vectors did not reduce the flatulence of rats fed an a-galactosides rich diet. Improvements to this system are discussed.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Characterization of the melA Locus for α-Galactosidase in Lactobacillus plantarum

Alpha-galactosides are abundant sugars in legumes such as soy. Because of the lack of α-galactosidase (α-Gal) in the digestive tract, humans are unable to digest these sugars, which consequently induce flatulence. To develop the consumption of the otherwise highly nutritional soy products, the use of exogenous α-Gal is promising. In this framework, we characterized the melA gene for α-Gal in Lactobacillus plantarum. The melA gene encodes a cytoplasmic 84-kDa protein whose enzymatically active form occurs as oligomers. The melA gene was cloned and expressed in Escherichia coli, yielding an active α-Gal. We show that melA is transcribed from its own promoter, yielding a monocistronic mRNA, and that it is regulated at the transcriptional level, i.e., it is induced by melibiose but is not totally repressed by glucose. Posttranscriptional regulation by the carbon source could also occur. Upstream of melA, a putative galactoside transporter, designated RafP, was identified that shows high homology to LacS, the unique transporter for both α- and β-galactosides in Streptococcus thermophilus. rafP is also expressed as a monocistronic mRNA. Downstream of melA, the lacL and lacM genes were identified that encode a heterodimeric β-galactosidase. A putative galM gene identified in the same cluster suggests the presence of a galactose operon. These results indicate that the genes involved in galactoside catabolism are clustered in L. plantarum ATCC 8014. This first genetic characterization of melA and of its putative associated transporter, rafP, in a lactobacillus opens doors to various applications both in the manufacture of soy-derived products and in probiotic and nutraceutical issues

Reduction of α-galactooligosaccharides in soyamilk by Lactobacillus fermentum CRL 722: In vitro and in vivo evaluation of fermented soyamilk

Aims: Consumption of soya-derived products has been hampered by the presence of α-galactooligosaccharides (α-GOS) because mammals lack pancreatic α-galactosidase (α-Gal) which is necessary for their hydrolysis. These sugars thus reach the large intestine causing gastrointestinal disorders in sensitive individuals. The use of lactic acid bacteria (LAB) expressing α-Gal is a promising solution for the degradation of α-GOS in soyamilk. Methods and Results: The capacity of the LAB Lactobacillus fermentum CRL 722 to properly degrade α-GOS was studied in vitro using controlled fermentation conditions and in vivo using a rat model. Lactobacillus fermentum CRL 722 was able to grow on commercial soyamilk and completely eliminated stachyose and raffinose during fermentation because of its high α-Gal activity. Rats fed soyamilk fermented by this LAB had smaller caecums compared with rats fed unfermented soyamilk. Conclusions: Soyamilk fermentation by Lact. fermentum CRL 722 results in the reduction of α-GOS concentrations in soyamilk, thus eliminating possible undesirable physiological effects normally associated with its consumption. Significance and Impact of the Study: Fermentation with Lact. fermentum CRL 722 could prevent gastrointestinal disorders in sensitive individuals normally associated with the consumption of soya-based products. This LAB could thus be used in the elaboration of novel fermented vegetable products which better suit the digestive capacities of consumers.Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Garro, Marisa Selva. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Silvestroni, Aurelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Connes, Cristelle. Institut National de la Recherche Agronomique; FranciaFil: Piard, Jean Christophe. Institut National de la Recherche Agronomique; FranciaFil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Savoy, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentin