Investigating luxS gene expression in lactobacilli along lab-scale cocoa fermentations.

Previous metagenomic analyses have suggested that lactobacilli present potential for Quorum Sensing (QS) in cocoa fermentation, and in the present research, laboratory scale fermentations were carried out to monitor the expression of luxS, a universal marker of QS. For that, 96 h-fermentations were studied, as follows: F0 (non inoculated control), F1 (inoculated with yeasts, lactic acid bacteria, and acetic acid bacteria), F2 (inoculated with yeasts and acetic acid bacteria), F3 (inoculated with yeasts only). The parameters evaluated were: plate counting, quantification of key enzymes and analysis of volatile organic compounds associated with key sensory descriptors, using headspace gas chromatography-mass spectrometry (GC-MS). Furthermore, QS was estimated by the quantification of the expression of luxS genes by Reverse Transcriptase Real-Time PCR. The results demonstrated that microbial succession occurred in pilot scale fermentations, but no statistical differences for microbial enumeration and α-diversity index were observed among experiments and control. Moreover, it was not possible to make conclusive correlations of enzymatic profile and fermenting microbiota, likely due to the intrinsic activity of plant hydrolases. Regarding to the expression of luxS genes, in Lactiplantibacillus plantarum they were active along the fermentation, but for Limosilactobacillus fermentum, luxS was expressed only at early and middle phases. Correlation analysis of luxS expression and production of volatile metabolites evidenced a possible negative association of Lp. Plantarum with fermentation quality. In conclusion, these data corroborate former shotgun metagenomic analysis by demonstrating the expression of luxS by lactobacilli in pilot scale cocoa fermentation and evidence Lp. Plantarum is the main lactic acid bacteria related to its expression.

Comparative pangenomic analyses and biotechnological potential of cocoa-related Acetobacter senegalensis strains.

Acetobacter senegalensis belongs to the group of acetic acid bacteria (AAB) that present potential biotechnological applications, for production of D-gluconate, cellulose and acetic acid. AAB can overcome heat and acid stresses by using strategies involving the overexpression of heat-shock proteins and enzymes from the complex pyrroquinoline-ADH, besides alcohol dehydrogenases (ADH). Nonetheless, the isolation of A. senegalensis and other AAB from food may be challenging due to presence of viable but non-culturable (VBNC) cells and due to uncertainties about nutritional requirements. To contribute for a better understanding of the ecology of AAB, this paper reports on the pangenome analysis of five strains of A. senegalensis recently isolated from a Brazilian spontaneous cocoa fermentation. The results showed biosynthetic clusters exclusively found in some cocoa-related AAB, such as those related to terpene pathways, which are important for flavour development. Genes related to oxidative stress were conserved in all the genomes, with multiple clusters. Moreover, there were genes coding for ADH and putative ABC transporters distributed in core, shell and cloud genomes, while chaperonin-encoding genes were present only in the core and soft-core genomes. Regarding quorum sensing, a response regulator gene was in the shell genome, and the gene encoding for acyl-homoserine lactone efflux protein was in the soft-core genome. There were quorum quenching-related genes, mainly encoding for lactonases, but also for acylases. Moreover, A. senegalensis did not have determinants of virulence or antibiotic resistance, which are good traits for strains intended to be applied in food fermentation.

Metagenome-Assembled Genomes Contribute to Unraveling of the Microbiome of Cocoa Fermentation.

Metagenomic studies about cocoa fermentation have mainly reported on the analysis of short reads for determination of operational taxonomic units. However, it is also important to determine metagenome-assembled genomes (MAGs), which are genomes deriving from the assembly of metagenomics. For this research, all the cocoa metagenomes from public databases were downloaded, resulting in five data sets: one from Ghana and four from Brazil. In addition, in silico approaches were used to describe putative phenotypes and the metabolic potential of MAGs. A total of 17 high-quality MAGs were recovered from these microbiomes, as follows: (i) for fungi, Yamadazyma tenuis (n = 1); (ii) lactic acid bacteria, Limosilactobacillus fermentum (n = 5), Liquorilactobacillus cacaonum (n = 1), Liquorilactobacillus nagelli (n = 1), Leuconostoc pseudomesenteroides (n = 1), and Lactiplantibacillus plantarum subsp. plantarum (n = 1); (iii) acetic acid bacteria, Acetobacter senegalensis (n = 2) and Kozakia baliensis (n = 1); and (iv) Bacillus subtilis (n = 1), Brevundimonas sp. (n = 2), and Pseudomonas sp. (n = 1). Medium-quality MAGs were also recovered from cocoa microbiomes, including some that, to our knowledge, were not previously detected in this environment (Liquorilactobacillus vini, Komagataeibacter saccharivorans, and Komagataeibacter maltaceti) and others previously described (Fructobacillus pseudoficulneus and Acetobacter pasteurianus). Taken together, the MAGs were useful for providing an additional description of the microbiome of cocoa fermentation, revealing previously overlooked microorganisms, with prediction of key phenotypes and biochemical pathways. IMPORTANCE The production of chocolate starts with the harvesting of cocoa fruits and the spontaneous fermentation of the seeds in a microbial succession that depends on yeasts, lactic acid bacteria, and acetic acid bacteria in order to eliminate bitter and astringent compounds present in the raw material, which will be further roasted and grinded to originate the cocoa powder that will enter the food processing industry. The microbiota of cocoa fermentation is not completely known, and yet it advanced from culture-based studies to the advent of next-generation DNA sequencing, with the generation of a myriad of data that need bioinformatic approaches to be properly analyzed. Although the majority of metagenomic studies have been based on short reads (operational taxonomic units), it is also important to analyze entire genomes to determine more precisely possible ecological roles of different species. Metagenome-assembled genomes (MAGs) are very useful for this purpose; here, MAGs from cocoa fermentation microbiomes are described, and the possible implications of their phenotypic and metabolic potentials are discussed.

Does Quorum Sensing play a role in microbial shifts along spontaneous fermentation of cocoa beans? An in silico perspective.

Cocoa fermentation is a spontaneous process shaped by a variable microbial ecosystem which is assembled due to cross-feeding relationship among yeasts and bacteria, resulting in a synchronized microbial succession started by yeasts, followed by lactic acid bacteria (LAB) and finalized by acetic acid bacteria (AAB). Several studies have indicated the effect of microbial interactions in food ecosystems highlighting the importance of quorum sensing (QS) in bacterial adaptation in harsh environments modulating several phenotypes such as biofilm formation, tolerance to acid stress, bacteriocin production, competence, morphological modifications, motility, among others. However, antagonic interactions also occur, and can be marked by Quorum Quenching (QQ) activity, negatively impacting QS regulated phenotypes. Our current knowledge regarding microbial cocoa composition and functioning is based on culture-based analysis and culture-independent PCR-based methods. Therefore, we set out to investigate the application of metagenomics analysis on a classical spontaneous cocoa fermentation in order to describe: (I) the microbial taxonomic composition; (II) the functional potential of the cocoa microbiome; (III) the microbiome putative QS potential; and (IV) the microbiome QQ potential. Both aims III and IV are related to the expression of effectors that may confer advantageous traits along fermentation which can explain their dominance in specific time zones during the entire process. We have observed a bacterial succession shaped by yeasts and filamentous fungi and then Enterobacteriaceales, LAB and AAB, as well as a diverse genetic metabolic potential related to proteins and carbohydrates metabolism associated to the yeast Saccharomyces cerevisiae and members of the Enterobacteriaceales order and LAB and AAB groups. In addition, in silico evidences of interspecific QS arsenal were found in members of the genera Enterobacter, Lactobacillus, Bacillus and Pantoea, while inferences of intraspecific QS potential were found in the members of the genera Bacillus, Enterobacter, Komagataeibacter, Lactobacillus and Pantoea. In addition, a QQ potential was detected in Lactobacillus and in AAB members. These findings indicate that QS and QQ may modulate bacterial dominance in different time points during fermentation, along with cross-feeding, being responsible for their maintenance in a large time range.

In vitro protective effect of lactic acid bacteria on Listeria monocytogenes adhesion and invasion of Caco-2 cells.

The adhesion of Listeria monocytogenes to intestinal endothelial cells is a crucial step in the infection process, which is not well understood. In this study, we evaluated the potential ability of bacteriocin-producing Enterococcus faecium, Leuconostoc mesenteroides and Lactobacillus sakei strains to prevent the adhesion and invasion of eukaryotic cells by ten different L. monocytogenes isolates. The results showed that E. faecium 130 co-cultured with L. monocytogenes was the most effective in preventing infection of Caco-2 cells, as the vast majority of isolates showed significantly lower adhesion counts and invasion rates below the quantification limit of the method (<30 cfu/plate). L. sakei 1 was the least effective strain in preventing L. monocytogenes infection; only one isolate presented a lower adhesion rate and two isolates reduced the invasion rate of Caco-2 cells. Fluorescence in situ hybridisation (FISH) assay was shown to be an effective tool to illustrate and identify species in co-culture with L. monocytogenes during the adhesion process to Caco-2 cells.

Improved adsorption-desorption extraction applied to the partial characterization of the antilisterial bacteriocin produced by carnobacterium maltaromaticum C2

Bacteriocins are ribosomally produced peptides useful for food biopreservation. An improved adsorption-desorption process is proposed for the partial purification of the bacteriocin produced by the fish isolate Carnobacterium maltaromaticum C2. Analyzis of extract by SDS-PAGE indicated this method may offer an alternative to improve the yield of purification of bacteriocins.

Improved adsorption-desorption extraction applied to the partial characterization of the antilisterial bacteriocin produced by Carnobacterium maltaromaticum C2.

Bacteriocins are ribosomally produced peptides useful for food biopreservation. An improved adsorption-desorption process is proposed for the partial purification of the bacteriocin produced by the fish isolate Carnobacterium maltaromaticum C2. Analyzis of extract by SDS-PAGE indicated this method may offer an alternative to improve the yield of purification of bacteriocins.

Improved treatment of vulvovaginal candidiasis with fluconazole plus probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14.

AIMS: To determine the ability of probiotic lactobacilli to improve the treatment of vulvovaginal candidiasis (VVC) using a randomized, double-blind and placebo-controlled trial. METHODS AND RESULTS: Fifty-five women diagnosed with VVC by vaginal discharge positive for Candida spp. (according to culture method) associated with at least one of the symptoms (itching and burning vaginal feeling, dyspareunia and dysuria), were treated with single dose of fluconazole (150 mg) supplemented every morning for the following 4 weeks with two placebo or two probiotic capsules (containing Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14). At 4 weeks, the probiotic treated group showed significantly less vaginal discharge associated with any of the above mentioned symptoms (10.3%vs 34.6%; P = 0.03) and lower presence of yeast detected by culture (10.3%vs 38.5%; P = 0.014). CONCLUSION: This study has shown that probiotic lactobacilli can increase the effectiveness of an anti-fungal pharmaceutical agent in curing disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel finding of probiotic lactobacilli augmenting the cure rate of yeast vaginitis, not only offers an alternative approach to a highly prevalent condition that adversely affects the quality of life of women around the world, but also raises the question of how this combination works.

Quantitative evaluation of Listeria monocytogenes in fresh and processed surubim fish (Pseudoplatystoma sp) / Avaliação quantitativa de Listeria monocytogenes em surubim (Pseudoplatystoma sp) fresco e processado

L. monocytogenes is a foodborne psychrotrophic bacterial pathogen of special importance for minimally processed foods. In this work, it was enumerated in samples of surubim fish by MPN technique. The population of L. monocytogenes was estimated as < 0.012 MPN/cm² in fresh and < 0.03 MPN/g in minimally processed fish.

L. monocytogenes é um patógeno psicrotrófico transmitido por alimentos, de importância especial para alimentos minimamente processados. Neste trabalho, a bactéria foi enumerada em amostras de peixe surubim utilizando-se a técnica do NMP. A população de L. monocytogenes foi estimada como < 0.012 NMP/cm² do peixe fresco e < 0.03 NMP/g do peixe minimamente processado.

Quantitative evaluation of Listeria monocytogenes in fresh and processed surubim fish (Pseudoplatystoma sp).

L. monocytogenes is a foodborne psychrotrophic bacterial pathogen of special importance for minimally processed foods. In this work, it was enumerated in samples of surubim fish by MPN technique. The population of L. monocytogenes was estimated as < 0.012 MPN/cm(2) in fresh and < 0.03 MPN/g in minimally processed fish.

Antilisterial activity of lactic acid bacteria inoculated on cooked ham.

This study was conducted to evaluate the ability of Lactobacillus sakei 1, a bacteriocin-producing (bac(+)) lactic acid bacterium (LAB), isolated from Brazilian fresh pork sausage to inhibit two Listeria monocytogenes strains (serotypes 4b and 1/2a) on cooked, sliced vacuum-packaged ham. L. sakei ATCC 15521 was used as a non-bacteriocin producer (bac(-)). L. monocytogenes (ca. 2 logCFU/mL) and LAB (ca. 6 logCFU/ml) were inoculated on the sterilized ham, vacuum-sealed and incubated at 8°C for 10 days. A treatment with the bacteriocin Chrisin (UI/ml) was included. Both L. monocytogenes strains were significantly inhibited in the presence of either bac(+) and bac(-) LAB in comparison to the control (L. monocytogenes alone). Using a bacteriocinogenic strain of LAB did not offer an additional barrier to listerial growth in the studied meat system. The application of Chrisin did not affect at all the growth of L. monocytogenes.

Identification of meat isolated bacteriocin-producing lactic acid bacteria using biotyping and ribotyping

Três cepas de bactérias láticas previamente isoladas a partir de produtos cárneos foram caracterizadas comparando-se resultados de testes bioquímicos e ribotipagem. Os resultados obtidos foram concordantes para Lactobacillus sake 1 e Lactobacillus curvatus 5. A cepa 16 foi identificada como Lactobacillus curvatus por testes bioquímicos e como Lactobacillus sake por ribotipagem, demonstrando a diferença entre as técnicas utilizadas. Recomenda-se, no momento, a técnica de ribotipagem para a identificaçäo de bactérias láticas isoladas de carnes