Genomic Insights Into Five Strains of Lactobacillus plantarum With Biotechnological Potential Isolated From chicha, a Traditional Maize-Based Fermented Beverage From Northwestern Argentina.

Lactic acid bacteria (LAB) are indigenous microorganisms that have been involved in food fermentations throughout history to preserve food and also to give special characteristics to them. The traditional fermented foods that are still being elaborated in indigenous populations around the world are a potential source of LAB with important biotechnological properties and/or beneficial to health. In a previous work, LAB biodiversity associated with chicha, a traditional maize-based fermented beverage from Northwestern Argentina, was studied, both by culture dependent and independent methods. From that study, 392 isolates were recovered, mostly members of Lactobacillus and Leuconostoc. Biotechnological characterization of representative isolates led to the selection of five strains belonging to the species Lactobacillus plantarum for their ability to produce vitamin B2 (riboflavin) and vitamin B9 (folates), their antimicrobial properties and antibiotics susceptibility. In this work, we present the Whole Genome Sequences (WGS) of these five strains that have been deposited in the Spanish Type Culture Collection: M5MA1 (= CECT 8962), M9MM1 (= CECT 8963), M9MM4 (= CECT 8964), M9MG6 (= CECT 8965), and M9Y2 (= CECT 8966), and a detailed description of their characterization, through a genomic approach, analyzing the genes responsible for these biotechnological properties, making a comparative study of the five genomes and reporting the aspects related to food safety, in accordance with the recommendations of the European Food Safety Authority (EFSA FEEDAP Panel, 2018) aiming at their use in the design of functional foods. The analysis unveiled, for the five strains, the complete set of genes for folate and riboflavin biosynthesis, the absence of pathogenic factors, the presence of CRISPR and cas genes, phage sequences, insertion elements and an aminoglycosides resistance gene, aadA, whose resistance could not be proved phenotypically in any strain. Genomic comparisons showed that strain CECT 8962 was significantly different in terms of genetic content and allowed the identification of carbohydrates metabolism and membrane transport related genes as the main components of the unique and accessory genome.

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.

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.

In situ riboflavin fortification of different kefir-like cereal-based beverages using selected Andean LAB strains.

Cereal-based functional beverages represent social, economic, and environmental sustainable opportunities to cope with emerging trends in food consumption and global nutrition. Here we report, for the first time, the polyphasic characterization of three cereal-based kefir-like riboflavin-enriched beverages, obtained from oat, maize and barley flours, and their comparison with classical milk-based kefir. The four matrices were successfully fermented with commercial starters: i) milk-kefir and ii) water-kefir, proving the potential of cereal ingredients in the formulation of dairy-like fermented beverages with milk-kefir starter behavior better in these matrices. In the light of their potentiality, seven riboflavin-producing Andean Lactic Acid Bacteria (LAB) were tested for tolerance to food stresses commonly encountered during food fermentation. Moreover, the LAB strains investigated were screened for spontaneous riboflavin overproducing derivatives. Lactobacillus plantarum M5MA1-B2 with outstanding response to stress, was selected to improve riboflavin content in an in situ fortification approach. The combination of L. plantarum M5MA1-B2 riboflavin overproducing strain with milk kefir starter in oat, lead to cover, for one serving of 100 g, 11.4% of Recommended Dietary Allowance (RDA). Besides, addition of L. plantarum M5MA1-B2 improved performance of water kefir in oat and maize matrices. Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) analysis provided the on-line Volatile Organic Compounds profiles supporting the best combination of starter, LAB and cereal matrix for novel functional foods development.

Acetobacter musti sp. nov., isolated from Bobal grape must.

An acetic acid bacterium (strain Bo7T), obtained during a study of the microbial diversity of spontaneous fermentations of Bobal grape must, was subjected to a taxonomic study using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences allocated strain Bo7T to the genus Acetobacter, and revealed Acetobacter aceti and Acetobacter oeni to be nearest neighbours (99.57 % 16S rRNA gene sequence similarity between strain Bo7T and A. oeni CECT 5830T, and 98.76 % between strain Bo7T and A. aceti CECT 298T). Cells of strain Bo7T are Gram-negative, motile rods, catalase-positive and oxidase-negative. The DNA G+C content of strain Bo7T was 58.0 mol%. DNA-DNA hybridizations demonstrated that strain Bo7T belongs to a single novel genospecies that can be differentiated from its nearest phylogenetic neighbours by the following phenotypic characteristics: no production of 5-keto-d-gluconic acid from d-glucose, growth with glycerol but not with methanol or maltose as sole carbon sources, and growth on yeast extract with 30 % d-glucose. The major fatty acid was C18 : 1ω7c/C18 : 1ω6c (summed feature 8; approx. 56 %); other fatty acids in significant amounts (>5 %) were C16 : 0 2-OH (11 %), C16 : 0 (7 %), C14 : 0 2-OH (7 %) and C14 : 0 3-OH/iso-C16 : 1 I (summed feature 2; 6 %). The results obtained indicate that strain Bo7T represents a novel species of the genus Acetobacter, for which the name Acetobacter musti sp. nov. is proposed. The type strain is Bo7T ( = DSM 23824T = CECT 7722T).

Pyrosequencing vs. culture-dependent approaches to analyze lactic acid bacteria associated to chicha, a traditional maize-based fermented beverage from Northwestern Argentina.

The diversity of lactic acid bacteria (LAB) associated with chicha, a traditional maize-based fermented alcoholic beverage from Northwestern Argentina, was analyzed using culture-dependent and culture-independent approaches. Samples corresponding to 10 production steps were obtained from two local producers at Maimará (chicha M) and Tumbaya (chicha T). Whereas by culture-dependent approach a few number of species (Lactobacillus plantarum and Weissella viridescens in chicha M, and Enterococcus faecium and Leuconostoc mesenteroides in chicha T) were identified, a higher quantitative distribution of taxa was found in both beverages by pyrosequencing. The relative abundance of OTUs was higher in chicha M than in chicha T; six LAB genera were common for chicha M and T: Enterococcus, Lactococcus, Streptococcus, Weissella, Leuconostoc and Lactobacillus while Pediococcus only was detected in chicha M. Among the 46 identified LAB species, those of Lactobacillus were dominant in both chicha samples, exhibiting the highest diversity, whereas Enterococcus and Leuconostoc were recorded as the second dominant genera in chicha T and M, respectively. Identification at species level showed the predominance of Lb. plantarum, Lactobacillus rossiae, Leuconostoc lactis and W. viridescens in chicha M while Enterococcus hirae, E. faecium, Lc. mesenteroides and Weissella confusa predominated in chicha T samples. In parallel, when presumptive LAB isolates (chicha M: 146; chicha T: 246) recovered from the same samples were identified by ISR-PCR and RAPD-PCR profiles, species-specific PCR and 16S rRNA gene sequencing, most of them were assigned to the Leuconostoc genus (Lc. mesenteroides and Lc. lactis) in chicha M, Lactobacillus, Weissella and Enterococcus being also present. In contrast, chicha T exhibited the presence of Enterococcus and Leuconostoc, E. faecium being the most representative species. Massive sequencing approach was applied for the first time to study the diversity and evolution of microbial communities during chicha manufacture. Although differences in the LAB species profile between the two geographically different chicha productions were observed by culturing, a larger number for predominant LAB species as well as other minorities were revealed by pyrosequencing. The fine molecular inventory achieved by pyrosequencing provided more precise information on LAB population composition than culture-dependent analysis processes.