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.

Mari Luz Mohedano,Rosa Aznar,M Carmen Martínez-Cuesta,Pasquale Russo,Jean Guy Leblanc,Alba Yépez,Giuseppe Spano,Sara Hernández-Recio,Carmen Peláez,Teresa Requena,Paloma López

doi:10.3389/fmicb.2019.01748

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.

Highlights

Nowadays, vitamin requirement is usually adequately supplied by a balanced diet; the insufficient or reduced intake of some vitamins is an important cause of nutritional deficiencies which affects susceptible groups of the population (LeBlanc et al, 2011; Mensink et al, 2013)
We have developed the pRCR12 plasmid (Russo et al, 2015), which allows lactic acid bacteria (LAB) fluorescent-labeling with mCherry, and it has been validated for its use in L. lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, L. plantarum, Lactobacillus sakei, and Pediococcus parvulus (Mohedano et al, 2015; Russo et al, 2015; Nácher-Vázquez et al, 2017a; Perez-Ramos et al, 2017, 2018)
Bacteria were grown in defined chemically defined medium (CDM)-Rib medium lacking riboflavin and fluorescent components that could interfere with the vitamin B2 quantification

Summary

Introduction

Vitamin requirement is usually adequately supplied by a balanced diet; the insufficient or reduced intake of some vitamins is an important cause of nutritional deficiencies which affects susceptible groups of the population (LeBlanc et al, 2011; Mensink et al, 2013). During the last decade resistance to roseoflavin has been a widely employed method to obtain B2 vitamin-overproducing LAB strains (Capozzi et al, 2011; Juarez del Valle et al, 2014) These riboflavin-resistant strains are spontaneous, non-genetically modified organisms, and should be acceptable for the production of vitamin B2-enriched foods (Burgess et al, 2006). In this context, five Lactobacillus plantarum strains isolated from Argentinian “chicha” (a fermented drink) (Elizaquível et al, 2015; Jiménez et al, 2018) were used to select B2-overproducing mutants carrying punctual mutations in the RFN region, upon their treatment with roseoflavin (Yépez et al, 2019). Prior to human interventions, animal models are needed and these should be designed to mimic the conditions of how the probiotics will be administered including the dosage and mode of administration (de Moreno de LeBlanc et al, 2016)

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Results

Conclusion