Glucansucrase approaches in novel homopolysaccharide producer lactic acid bacteria isolated from Argentinean fruits

Abstract

Certain lactic acid bacteria (LAB) are capable of producing an extracelular α–D–glucan homopolysaccharide (HoPS) formed by glucose units when growing in sucrose-enriched media. Their structural diversity is linked to their vast commercial value and applications in the food, cosmetic, medical, and biotechnology fields, reaching much attention in recent years. In this sense, this HoPS type is synthesized in large amounts by extracellular free or cell-anchored enzymes called glucansucrases (GSs: dextransucrase, mutansucrase, alternansucrase, and reuteransucrase) belonging to the GH70 family. Usually, these GSs use the glycosidic bond energy from sucrose to extend the polymerization of glucosyl units. Previously, we reported the bioprospection of thirty-one HoPS producer LAB strains, isolated from wild fruits of northern Argentina and belonging to the Weisella (W.) and Leuconostoc (Ln.) genera in a sucrose- enriched MRS medium (MRS-S). In addition, the HoPS from the selected producer strains (W. cibaria FMy 2-21-1, W. confusa CRL951, Ln. pseudomesenteroides F-G2-22, Ln. citreum F-Cq1-496 and Ln. dextranicum CRL983) were purified and subsequently characterized, turning out to be all dextrans (α–glucan type). Thereby, we aimed to identify the GS encoding genes in the aforementioned five LAB strains and evaluate the GS activity in both extracellular contexts (supernatant and cell pellet) of MRS-S cultures. Finally, we selected one strain with the highest GS activity for further studies: morphology (TEM), polymer production (PAS reaction), bacterial growth (CFU.mL-1 ), sugar consumption (HPLC), and acidifying activity: pH and organic acids production (HPLC). Total DNA was extracted following the protocol described by Pospiech and Neumann for Gram positive bacteria and PCR reactions were performed using oligonucleotides designed to amplify regions within specific GS genes. The resulting amplicons were sequenced and used as query the BLAST tool in the NCBI database (https://www.ncbi.nlm.nih.gov/). The screening for putative GS genes revealed a positive signal for all HoPS+ strains that possess high sequence identity with GS genes from LAB strains of the same species. For example, the sequence analysis for FMy 2-21-1 and CRL983 strains showed 92.77% and 95.77% similarity to the gene coding for a GS of W. cibaria LBAE-K39 (GU237484.3) and Ln. dextranicum strain DSM 20484 (CP012009.1), respectively. Extracellular GS activity, either in the culture supernatant or cell pellets, were determined in an enzymatic reaction mixture with 5% sucrose through the Somogyi - Nelson assay. The extracellular activities showed large variation among the strains, from 5.24 U/Log(CFU.mL-1 ) to 10.66 U/Log(CFU.mL-1 ) corresponding to CRL983 and F-Cq1- 496 strains, respectively. By contrast, only Ln. citreum F-Cq1-496 exhibited activity in cell pellet, which was higher than in supernatant [32.55 U/Log(CFU.mL-1 ) and 10.66 U/Log(CFU.mL-1 ), respectively]. Therefore, we selected the F-Cq1-496 strain to study their metabolic activity in MRSS culture at 30ºC (0, 3, 6, 10 and 16 h). At the end of fermentation, the LAB strain grew more than three logarithmic units of CFU.mL-1 (3.33 Log(CFU.mL-1 ) and reduced the pH of the culture medium by two pH units (2.16). The increase in acidity was associated with the production of lactic acid (3.33 g.L-1 ) and acetic acid (2.17 g.L-1 ). Moreover, the polysaccharide production was 609 mg.L-1 in concomitance with a drastic decrease of sucrose. Finally, mannitol production (>1.5 g.L-1 at 24 h) was observed associated to decreasing levels of fructose released from sucrose. Therefore, fructose can either be used as a carbon source to obtain ATP or as an electron acceptor to synthesize mannitol and regenerate NAD+ via the mannitol dehydrogenase reaction. TEM micrograph showed a strong interaction between the LAB strain and the polymer matrix generated. In conclusion, GS genes were detected in all studied LAB strains and though the extracellular enzyme activities were varied, only Ln. citreum F-Cq1-496 exhibited GS activity in their cellular pellet. Therefore, Ln. citreum strain was selected to evaluate different fermentation parameters, exhibiting the highest polymer production with effective performance in the studied sucrose-enriched medium.