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The use of lactic acid bacteria for developing functional foods is increasing for their ability to synthesize beneficial metabolites such as vitamin B (riboflavin, RF) and postbiotic compounds. Here, the spontaneous mutant FS54 B2 was isolated by treatment of the dextran-producing Weissella confusa FS54 strain with roseoflavin. FS54 B2 overproduced RF (4.9 mg/L) in synthetic medium. The FMN riboswitch is responsible for the regulation of RF biosynthesis, and sequencing of the coding DNA revealed that FS54 B2 carries the G131U mutation. FS54 B2 retained the capacity of FS54 to synthesize high levels of dextran (3.8 g/L) in synthetic medium. The fermentation capacities of the two Weissella strains was tested in commercial oat-, soy- and rice-based beverages. The best substrate for FS54 B2 was the oat-based drink, in which, after fermentation, the following were detected: RF (2.4 mg/L), dextran (5.3 mg/L), potential prebiotics (oligosaccharides (panose (5.1 g/L), isomaltose (753 mg/L) and isomaltotriose (454 mg/L)) and the antioxidant mannitol (16.3 g/L). pH-lowering ability and cell viability after one month of storage period were confirmed. As far as we know, this is the first time that an RF-overproducing W. confusa strain has been isolated, characterized and tested for its potential use in the development of functional beverages.

Leuconostoc lactis is found in vegetables, fruits, and meat and is used by the food industry in the preparation of dairy products, wines, and sugars. We have previously demonstrated that the dextransucrase of Lc. lactis (DsrLL) AV1n produces a high-molecular-weight dextran from sucrose, indicating its potential use as a dextran-forming starter culture. We have also shown that this bacterium was able to produce 10-fold higher levels of dextran at 20°C than at 37°C, at the former temperature accompanied by an increase in dsrLL gene expression. However, the general physiological response of Lc. lactis AV1n to cold temperature in the presence of sucrose, leading to increased production of dextran, has not been yet investigated. Therefore, we have used a quantitative proteomics approach to investigate the cold temperature-induced changes in the proteomic profile of this strain in comparison to its proteomic response at 37°C. In total, 337 proteins were found to be differentially expressed at the applied significance criteria (adjusted p -value ≤ 0.05, FDR 5%, and with a fold-change ≥ 1.5 or ≤ 0.67) with 204 proteins overexpressed, among which 13% were involved in protein as well as cell wall, and envelope component biosynthesis including DsrLL. Proteins implicated in cold stress were expressed at a high level at 20°C and possibly play a role in the upregulation of DsrLL, allowing the efficient synthesis of the protein essential for its adaptation to cold. Post-transcriptional regulation of DsrLL expression also seems to take place through the interplay of exonucleases and endonucleases overexpressed at 20°C, which would influence the half-life of the dsrLL transcript. Furthermore, the mechanism of cold resistance of Lc. lactis AV1n seems to be also based on energy saving through a decrease in growth rate mediated by a decrease in carbohydrate metabolism and its orientation toward the production pathways for storage molecules. Thus, this better understanding of the responses to low temperature and mechanisms for environmental adaptation of Lc. lactis could be exploited for industrial use of strains belonging to this species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Besrour-Aouam, de Los Rios, Hernández-Alcántara, Mohedano, Najjari, López and Ouzari.)

Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter P dsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the P dsrLL -dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20°C than at 37°C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc , AV1n was transformed with plasmids carrying either the P dsrLL - mrfp fusion or the P dsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from P dsrLL upon growth at low temperature (20°C versus 30°C and 37°C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from P dsrLL . A different behavior was detected, when expression from P dsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30°C and 20°C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications.