Dextranos de bacterias lácticas aisladas de productos cárnicos: caracterización y aplicaciones
Some lactic acid bacteria (LAB) synthesize exopolysaccharides (EPS) that improve the rheological properties of fermented foods, which is of interest to the food industries. Moreover, some EPS have demonstrated beneficial health properties, such as antitumor, immunomodulatory, hypocholesterolemic or prebiotic activities. Thus, LAB are good candidates for the development of functional foods. Among the EPS, dextrans have various industrial applications outside of the food sector. It is known that these EPS are synthesized by species of the genera Lactobacillus, Leuconostoc, Oenococcus, Streptococcus and Weisella. However, little is known about the regulation of expression of the dsr genes, which encode the dextransucrases that synthesise dextrans. The present doctoral work has characterized at the molecular, physiological, metabolic and physicochemical levels, two EPS: EPS-LS and EPS-LM produced respectively by Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10, both isolated from meat products. The biological functionality of these polymers as antiviral agents and immunostimulants, as well as the potential of the Lb. sakei MN1 as a probiotic against fish pathogens, has been investigated. The physiological and physicochemical studies revealed that in the presence of sucrose as carbon source Lb. sakei MN1 and Lc. mesenteroides RTF10 produce dextrans with α-(1-6) linkages in their main chain and 3% (EPS-LS) or 9% (EPS-LM) of branches with α- (1-3) linkages. Analysis by electron microscopy showed that the EPS are associated with the cell wall or cells surrounding. A further study showed that the genetic determinants for the production of EPS-LS and EPS-LM, are located respectively in the pMN1 (13.7 kbp) and the pRTF10 (20.6 kbp) plasmids. Furthermore, the determination of the complete nucleotide sequence of pMN1 (11,126 bp) revealed that it belongs to a family of plasmids which replicate by the theta type mechanism and whose prototype is pUCL287. The plasmid carries a replicon (the origin of replication and the repA and repB genes), the dsrLS gene and 7 open reading frames. Analysis of the dsrLS gene expression at the transcriptional level showed that the dextransucrase DsrLS is synthesized from two transcripts: one monocistronic and other polycistronic including repA, repB and dsrLS. As far as we know, this is the first instance of synchronised expression of a dextransucrase and the machinery of plasmid replication. In addition, the expression of both mRNAs does not increase when sucrose is present in the growth medium, revealing that this hexose is not an inducer of DsrLS expression. Bioinformatic analysis of the dsrLS gene product revealed that it is a peptide composed of 1,767 amino acids with a molecular mass of 190,039 Da and that contains a leader peptide at its amino terminus, indicating that DsrLS is an extracellular protein. 3D models of DsrLS structure based on amino acid homology and structural conformation indicate that the protein is a dimeric enzyme, whose substrate is sucrose and which has dextransucrase activity. Lb. sakei MN1 and Lc. mesenteroides RTF10 cultures grown in defined medium have been used for production of EPS-LS and EPS-LM. The polymers have been purified from the culture supernatants by ethanol precipitation, dialysis and chromatographic fractionation, with a recovery of 80% and with a purity of 99%. Both dextrans have shown in vitro antiviral activity against infection by two salmonid viruses: infectious hematopoietic necrosis virus (IHNV) and Infectious pancreatic necrosis virus (IPNV). Furthermore, it has been demonstrated that EPS-LS has antiviral activity in vivo, and acts as an immunostimulant of the immune response of rainbow trout (Oncorhynchus mykiss). Furthermore, it was shown that in vitro Lb. sakei MN1 has the ability to aggregate and to form biofilms when grown in the presence of glucose but not in the presence of sucrose, condition in which produces dextran. This behaviour has been validated by use of a gnotobiotic zebrafish (Danio rerio) larva model. Moreover, this model has demonstrated that Lb. sakei MN1 colonizes the larval intestine and it is able to compete in this habitat with the fish pathogen Vibrio anguillarum. For all the above mentioned reasons, both Lb. sakei MN1 for its probiotic potential as competitor with bacterial pathogens, and the EPS-LS for its immunomodulatory and antiviral activities, have potential application for the development of functional feed for salmonids.