Genomic diversity in Fructobacillus spp. isolated from fructose-rich niches.

The Fructobacillus genus is a group of obligately fructophilic lactic acid bacteria (FLAB) that requires the use of fructose or another electron acceptor for their growth. In this work, we performed a comparative genomic analysis within the genus Fructobacillus by using 24 available genomes to evaluate genomic and metabolic differences among these organisms. In the genome of these strains, which varies between 1.15- and 1.75-Mbp, nineteen intact prophage regions, and seven complete CRISPR-Cas type II systems were found. Phylogenetic analyses located the studied genomes in two different clades. A pangenome analysis and a functional classification of their genes revealed that genomes of the first clade presented fewer genes involved in the synthesis of amino acids and other nitrogen compounds. Moreover, the presence of genes strictly related to the use of fructose and electron acceptors was variable within the genus, although these variations were not always related to the phylogeny.

Exploring the Genome of Fructobacillus tropaeoli CRL 2034, a Fig-Origin Strain that Produces High Levels of Mannitol from Fructose.

We report the draft genome sequence of Fructobacillus tropaeoli CRL 2034, a strain isolated from ripe fig in Tucumán province, Argentina. The interest in studying the genome of this fructophilic lactic acid bacterium strain was motivated by its ability to produce high levels of mannitol from fructose. This polyol has multiple industrial applications; however, it is mainly used as low calorie sugar in the food industry. The assembled genome of this strain consists of a 1.66-Mbp circular chromosome with 1465 coding sequences and a G+C content of 44.6%. The analysis of this genome supports the one step reaction of fructose reduction to mannitol by the mannitol 2-dehydrogenase enzyme, which together with a fructose permease, were identified as involved in mannitol synthesis. In addition, a phylogenetic analysis was performed including other Leuconostocaceae members to which the Fructobacillus genus belongs to; according to the 16S rRNA gene sequences, the strain CRL 2034 was located in the Fructobacillus clade. The present genome sequence could be useful to further elucidate regulatory processes of mannitol and other bioactive metabolites and to highlight the biotechnological potential of this fruit-origin Fructobacillus strain.

Major role of lactate dehydrogenase D-LDH1 for the synthesis of lactic acid in Fructobacillus tropaeoli CRL 2034.

The enzymes D- and L-lactate dehydrogenase are involved in the reduction of pyruvate to D(+)- and L(-)-lactate, respectively. The fig-origin strain Fructobacillus tropaeoli CRL 2034 produces D- and L-lactic acids in a 9:1 ratio. In this work, two D-ldh (ldh1 and ldh2) and one L-ldh (ldh3) genes were found in the CRL 2034 genome. ldh1 and ldh2 are homologous (79% identity) and organized as contiguous operons, each gene containing 996 base pair (bp) and encoding for a 331-amino acid (aa) protein (74% identity). In contrast, ldh3 is a 927-bp gene coding for a 308-aa protein. The identity between ldh1/ldh2 and ldh3 was lower than 48%. To elucidate the role of these genes in the synthesis of lactic acid by the Fructobacillus strain, plasmid insertion mutants in each gene were generated and characterized. The growth kinetic parameters were affected only in CRL2034 ldh1::pRV300 cells, this mutant showing the lowest total lactic acid production (4.50 ± 0.15 versus 6.36 ± 0.67 g/L of wild-type strain), with a D/L ratio of 7.1:2.9. These results showed that the ldh1 gene is primarily responsible for lactic acid production by the studied strain. A comparative analysis among strains of the five Fructobacillus species revealed that the identity of D-LDH proteins was higher than 70%, while the identity of L-LDH was over 60%. Finally, phylogenetic analysis of D- and L-LDHs revealed that only D-LDH phylogeny was consistent to the phylogenetic evolution among Fructobacillus and evolutionarily related genera. Key Points •F. tropaeoli CRL 2034 harbors three ldh genes in its genome. •ldh1 and ldh2 encode D-lactate dehydrogenase; ldh3 encodes L-lactate dehydrogenase. •Gene ldh1 plays the major role in lactic acid production by strain CRL 2034. •Fructobacillus D-LDH phylogeny was consistent to phylogenetic evolution.

Survival of selenium-enriched lactic acid bacteria in a fermented drink under storage and simulated gastro-intestinal digestion.

Selenium (Se), which is present as SeCys in seleno-proteins, is involved in cancer prevention, thyroid functioning, and pathogen inhibition. Se is incorporated in the diet through Se-containing foods. Some lactic acid bacteria (LAB) can biotransform selenite (toxic) into Se-nanoparticles (SeNPs) and Se-amino acids. To exert their beneficial properties in the host, bacteria should survive the harsh conditions of the gastrointestinal tract and during food storage. We evaluated whether selenization of LAB influenced bacterial growth and survival during gastrointestinal digestion and after storage when present in a fermented fruit juice-milk (FJM) beverage. Lactobacillus brevis CRL 2051 and Fructobacillus tropaeoli CRL 2034 were grown in MRS with and without selenite, and used to inoculate the FJM matrix. Selenization had no effect on LAB growth (9.54-9.9 log CFU/mL) in the FJM drink. The presence of SeNPs was confirmed for both selenized strains in the FJM beverage; however, the highest Se concentration (100 µg/L) was detected for the fermented beverage with selenized L. brevis. Under storage conditions 1.1 log CFU/ml decrease in cell count of selenized cells of L. brevis was observed, while no effect on cell viability was detected for non-selenized L. brevis or both selenized and control cells of F. tropaeoli. Resistance of L. brevis during digestion of the fermented FJM beverage was not affected by selenization. Contrarily, an increase (1 log CFU/mL) in the resistance of F. tropaeoli was observed when cells were selenized. After digestion, Se was detected in the soluble fraction of the beverage fermented by both strains, being higher for L. brevis (23.6 µg/L). Although selenization did not exert a drastic effect on strains´ survival during storage and digestion, microbial selenization previous to food fermentation could be an interesting tool for Se enrichment avoiding thus the addition of toxic Se salts.

Encapsulación de Weissella paramesenteroides y su aplicación en residuos de pescado / Weissella paramesenteroides encapsulation and its application in the use of fish wastes

The goal of the present study was to evaluate the encapsulation of Weissella paramesenteroides, isolated from bee bread, as a technological tool for its use in biological fish silage. The pH decrease in fish silages using the bacteria encapsulated and in a non-encapsulated form was compared. W. paramesenteroides showed a good performance in the development of bioEnc ap su lati on; logical fish silage. The alginate encapsulation method showed an encapsulation efficacy of 85% and provides a reliable technological application.

El objetivo del presente estudio fue evaluar la encapsulación de Weissella paramesenteroides, aislada a partir del pan de polen, como herramienta tecnológica para su uso en la elaboración de ensilado biológico de pescado. Se comparó el descenso de pH para los ensilados utilizando la bacteria encapsulada y no encapsulada. W. paramesenteroides mostró un buen desempeño en el desarrollo de ensilado biológico de pescado. El método de encapsulación con alginato mostró una eficacia del 85% y puede ser utilizado para su aplicación tecnológica.

Enhanced mannitol biosynthesis by the fruit origin strain Fructobacillus tropaeoli CRL 2034.

Mannitol is a natural low-calorie sugar alcohol produced by certain (micro)organisms applicable in foods for diabetics due to its zero glycemic index. In this work, we evaluated mannitol production and yield by the fruit origin strain Fructobacillus tropaeoli CRL 2034 using response surface methodology with central composite design (CCD) as optimization strategy. The effect of the total saccharide (glucose + fructose, 1:2) content (TSC) in the medium (75, 100, 150, 200, and 225 g/l) and stirring (S; 50, 100, 200, 300 and 350 rpm) on mannitol production and yield by this strain was evaluated by using a 22 full-factorial CCD with 4 axial points (α = 1.5) and four replications of the center point, leading to 12 random experimental runs. Fermentations were carried out at 30 °C and pH 5.0 for 24 h. Minitab-15 software was used for experimental design and data analyses. The multiple response prediction analysis established 165 g/l of TSC and 200 rpm of S as optimal culture conditions to reach 85.03 g/l [95% CI (78.68, 91.39)] of mannitol and a yield of 82.02% [95% CI (71.98, 92.06)]. Finally, a validation experiment was conducted at the predicted optimum levels. The results obtained were 81.91 g/l of mannitol with a yield of 77.47% in outstanding agreement with the expected values. The mannitol 2-dehydrogenase enzyme activity was determined with 4.6-4.9 U/mg as the highest value found. To conclude, F. tropaeoli CRL 2034 produced high amounts of high-quality mannitol from fructose, being an excellent candidate for this polyol production.

Characterization of strains of Weissella fabalis sp. nov. and Fructobacillus tropaeoli from spontaneous cocoa bean fermentations.

Six facultatively anaerobic, non-motile lactic acid bacteria were isolated from spontaneous cocoa bean fermentations carried out in Brazil, Ecuador and Malaysia. Phylogenetic analysis revealed that one of these strains, designated M75(T), isolated from a Brazilian cocoa bean fermentation, had the highest 16S rRNA gene sequence similarity towards Weissella fabaria LMG 24289(T) (97.7%), W. ghanensis LMG 24286(T) (93.3%) and W. beninensis LMG 25373(T) (93.4%). The remaining lactic acid bacteria isolates, represented by strain M622, showed the highest 16S rRNA gene sequence similarity towards the type strain of Fructobacillus tropaeoli (99.9%), a recently described species isolated from a flower in South Africa. pheS gene sequence analysis indicated that the former strain represented a novel species, whereas pheS, rpoA and atpA gene sequence analysis indicated that the remaining five strains belonged to F. tropaeoli; these results were confirmed by DNA-DNA hybridization experiments towards their respective nearest phylogenetic neighbours. Additionally, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved successful for the identification of species of the genera Weissella and Fructobacillus and for the recognition of the novel species. We propose to classify strain M75(T) ( = LMG 26217(T)  = CCUG 61472(T)) as the type strain of the novel species Weissella fabalis sp. nov.