A methodology for the selection and characterization of riboflavin-overproducing Weissella cibaria strains after treatment with roseoflavin

Abstract

Fermentative processes by lactic acid bacteria can produce metabolites of interest to the health and food industries. Two examples are the production of B-group vitamins, and of prebiotic and immunomodulatory dextran-type exopolysaccharides. In this study, three riboflavin- and dextran-producing Weissella cibaria strains (BAL3C-5, BAL3C-7 and BAL3C-22) were used to develop a new method for selection and isolation of spontaneous riboflavin-overproducing W. cibaria mutants. This method was based on the selection of strains resistant to roseoflavin. The DNA sequencing of the FMN riboswitch of bacterial cell populations treated with various roseoflavin concentrations, revealed the existence of at least 10 spontaneous and random point mutations at this location. Folding and analysis of the mutated FMN riboswitches with the RNA fold program predicted that these mutations could result in a deregulation of the rib operon expression. When the roseoflavin-treated cultures were plated on medium supporting dextran synthesis, the most promising mutants were identified by the yellow color of their mucous colonies, exhibiting a ropy phenotype. After their isolation and recovery in liquid medium, the evaluation of their riboflavin production revealed that the mutant strains synthesized a wide range of riboflavin levels (from 0.80 to 6.50 mg/L) above the wild-type level (0.15 mg/L). Thus, this was a reliable method to select spontaneous riboflavin-overproducing and dextran-producing strains of W. cibaria. This species has not yet been used as a starter or adjunct culture, but this study reinforces the potential that it has for the food and health industry for the production of functional foods or as a probiotic. Furthermore, analysis of the influence of FMN present in the growth medium, on rib mRNA and riboflavin levels, revealed which mutant strains produce riboflavin without flavin regulation. Moreover, the BAL3C-5 C120T mutant was identified as the highest riboflavin-overproducer. Determination of its chromosomal DNA sequence and that of BAL3C-5, revealed a total identity between the 2 strains except for the C120T mutation at the FMN riboswitch. To our knowledge, this work is the first demonstration that only a single alteration in the genome of a lactic acid bacteria is required for a riboflavin-overproducing phenotype.

Leuconostoc mesenteroides and Liquorilactobacillus mali strains, isolated from Algerian food products, are producers of the postbiotic compounds dextran, oligosaccharides and mannitol

“…In this study, the result presented revealed that among the six LAB tested only B12 and 09 produced significant levels of riboflavin. Moreover, the increase of vitamin B 2 concentration detected in cultures grown in RAMSR (results not shown) supports that there was not a detrimental feedback repression of the synthesis of this molecule by these LAB when the flavin is present in the environment, as previously observed for L. plantarum M5MA1-B2 (Mohedano et al 2019 ), Lactobacillus fermentum PBCC11.5 (Russo et al 2014 ), 8 W. cibaria strains (Diez-Ozaeta et al 2023 ; Hernández-Alcántara et al 2022 ) and Leuconostoc citreum BAL3C-4 and Leuconostoc falkenbergence (VSL11h-8 and VSL14h-1 strains) (Llamas-Arriba et al 2021 ). In the case of the Algerian LAB tested here, for B12 and O9, the absence of riboflavin in the medium did not hinder their growth.…”

Biotechnological Potential and Safety Evaluation of Dextran- and Riboflavin-Producing Weisella cibaria Strains for Gluten-Free Baking

“…W. cibaria BAL3C-5 C120 is a riboflavin-overproducing strain selected by roseoflavin treatment of the dextran-producing BAL3C-5 strain isolated from a rye sourdough [ 37 ]. The selected BAL3C-5 C120T constitutive mutant is able to produce high concentrations of dextran and riboflavin in growth medium [ 28 ]. Therefore, it could be a good candidate for the development of functional GF bread.…”

“…Weissella cibaria BAL3C-5 previously isolated from rye sourdough [ 37 ] and its roseoflavin-resistant riboflavin-overproducing W. cibaria BAL3C-5 C120T [ 28 ] were used in this work. LAB strains were grown at 30 °C in De Man–Rogosa–Sharpe (MRS) broth (Oxoid, Basingstoke, UK).…”

“…Furthermore, dextrans synthesized by W. cibaria have been reported to be bioactive compounds with immuno-stimulant and anti-inflammatory properties [ 25 ], suggesting their possible ability to improve the functionality of bakery products. In recent studies, W. cibaria strains isolated from a rye sourdough made in Spain and the corresponding roseoflavin-resistant riboflavin-overproducing derivatives have been employed to biofortify in situ with riboflavin and dextran wheat laboratory bread [ 26 , 27 , 28 ]. Riboflavin (vitamin B 2 ) is an essential compound mainly involved in the respiratory chain, which exerts antioxidant effects and is synthesized by food-grade micro-organisms [ 29 ].…”

“…In this study, the dextran-producing W. cibaria BAL3C-5 and its derivative, the strain BAL3C-5 C120T (to our knowledge, this is the described W. cibaria strain producing the highest riboflavin levels) [ 28 ], have been used as coadjuvant together with a commercial bakery yeast to ferment pilot scale GF dough obtained by mixing different combinations of corn or rice GF flour with ones from pseudocereals or legumes. The riboflavin and dextran production has been determined in laboratory-scale bread.…”