Real-Time Detection of Riboflavin Production by Lactobacillus plantarum Strains and Tracking of Their Gastrointestinal Survival and Functionality in vitro and in vivo Using mCherry Labeling

Abstract

Some strains of lactic acid bacteria (LAB) produce riboflavin, a water-soluble vitamin of the B complex, essential for human beings. Here, we have evaluated riboflavin (B2 vitamin) production by five Lactobacillus plantarum strains isolated from chicha , a traditional maize-based fermented alcoholic beverage from north-western Argentina and their isogenic riboflavin-overproducing derivatives previously selected using roseoflavin. A direct fluorescence spectroscopic detection method to quantify riboflavin production in bacterial culture supernatants has been tested. Comparison of the efficiency for riboflavin fluorescence quantification with and without prior HPLC fractionation showed that the developed method is a rapid and easy test for selection of B2 vitamin-producing strains. In addition, it can be used for quantitative detection of the vitamin production in real time during bacterial growth. On the basis of this and previous analyses, the L. plantarum M5MA1-B2 riboflavin overproducer was selected for in vitro and in vivo studies after being fluorescently labeled by transfer of the pRCR12 plasmid, which encodes the mCherry protein. The labeling did not affect negatively the growth, the riboflavin production nor the adhesion of the strain to Caco-2 cells. Thus, L. plantarum M5MA1-B2[pRCR12] was evaluated for its survival under digestive tract stresses in the presence of microbiota in the dynamic multistage BFBL gut model and in a murine model. After exposure to both models, M5MA1-B2[pRCR12] could be recovered and detected by the pink color of the colonies. The results indicated a satisfactory resistance of the strain to gastric and intestinal stress conditions but a low colonization capability observed both in vitro and in vivo . Overall, L. plantarum M5MA1-B2 could be proposed as a probiotic strain for the development of functional foods.

Screening and Spontaneous Mutation of Pickle-Derived Lactobacillus plantarum with Overproduction of Riboflavin, Related Mechanism, and Food Application

“…This bacterial suspension was further used for the fermentation experiments. Subsequently, 5 mL of the heated soymilk was inoculated with 4% (v/v) of the bacterial suspension and then fermented at different temperatures (23,30,37,44, and 50 • C) and for different times (4,8,12,16,20, and 24 h). For each fermentation experiment, 5 mL of heated aseptic soymilk without bacterial inoculation was used as a control.…”

“…The wild-type RYG-YYG-9049 and its M10 strains were applied to ferment soymilk to increase its riboflavin content. The effects of main fermentation parameters, including temperature (23,30,37,44, and 50 • C) and time (4,8,12,16,20, and 24 h) were investigated on riboflavin production. A decrease in the pH of the fermented soymilk was observed during the fermentation process ( Figure 5A), but this decrease was very slight at a fermentation temperature of 50 • C because of no accumulation of lactic acid, indicating that the strain could not grow or even died at 50 • C. Therefore, riboflavin in soymilk fermented at 50 • C was detected.…”

“…Therefore, the use of the RYG-YYG-9049-M10 strain can also be considered as a biotechnological strategy to obtain riboflavin-enriched soymilk. However, for a strain destined for industrial food production, its survival and functionality under in vitro and in vivo conditions should be tested in the future in order to evaluate whether it can be considered as a probiotic strain for food production [37]. In addition, the adaptability of the strain during food production and storage also needs to be considered, because food stresses, such as pH, freezing, temperature, and osmotic pressure, may have a significant influence on its performance [36].…”

The Ability of Riboflavin-Overproducing Lactiplantibacillus plantarum Strains to Survive Under Gastrointestinal Conditions

“…To follow the growth and fluorescence of L. plantarum strains in real time, the method described by Mohedano et al (2019) was used. Simultaneous spectrophotometric monitoring of growth at 37 • C and of the fluorescence of the bacterial cultures was performed using the Varioskan Flask System (Thermo Fisher Scientific, USA) and sterile 96-well optical white w/lid cell culture polystyrene plates (Thermo Fisher Scientific, USA).…”

“…These vitamin B2-overproducing LAB have also been used to standardize fluorescent detection of riboflavin production in real time during growth (Mohedano et al, 2019) and also showed the potential of strains L. plantarum M5MA1-B2 and M9MG6-B2, as producing high levels of riboflavin and possessing good technological properties. Both strains carry a different punctual mutation in the same position (G19A and G19C in the M5MA1-B2 and M9MG6-B2 strains, respectively) located in the regulatory region of the rib operon (Mohedano et al, 2019). Furthermore, the comparative analysis of the genomes of five of the parental riboflavin producing strains, as well as assessing some criteria to determine their food safety, revealed a great similarity between the strains studied.…”