Lactobacillus lactis and Pediococcus pentosaceus-driven reprogramming of gut microbiome and metabolome ameliorates the progression of non-alcoholic fatty liver disease.

BACKGROUND: Although microbioa-based therapies have shown putative effects on the treatment of non-alcoholic fatty liver disease (NAFLD), it is not clear how microbiota-derived metabolites contribute to the prevention of NAFLD. We explored the metabolomic signature of Lactobacillus lactis and Pediococcus pentosaceus in NAFLD mice and its association in NAFLD patients. METHODS: We used Western diet-induced NAFLD mice, and L. lactis and P. pentosaceus were administered to animals in the drinking water at a concentration of 109 CFU/g for 8 weeks. NAFLD severity was determined based on liver/body weight, pathology and biochemistry markers. Caecal samples were collected for the metagenomics by 16S rRNA sequencing. Metabolite profiles were obtained from caecum, liver and serum. Human stool samples (healthy control [n = 22] and NAFLD patients [n = 23]) were collected to investigate clinical reproducibility for microbiota-derived metabolites signature and metabolomics biomarker. RESULTS: L. lactis and P. pentosaceus supplementation effectively normalized weight ratio, NAFLD activity score, biochemical markers, cytokines and gut-tight junction. While faecal microbiota varied according to the different treatments, key metabolic features including short chain fatty acids (SCFAs), bile acids (BAs) and tryptophan metabolites were analogously restored by both probiotic supplementations. The protective effects of indole compounds were validated with in vitro and in vivo models, including anti-inflammatory effects. The metabolomic signatures were replicated in NAFLD patients, accompanied by the comparable levels of Firmicutes/Bacteroidetes ratio, which was significantly higher (4.3) compared with control (0.6). Besides, the consequent biomarker panel with six stool metabolites (indole, BAs, and SCFAs) showed 0.922 (area under the curve) in the diagnosis of NAFLD. CONCLUSIONS: NAFLD progression was robustly associated with metabolic dys-regulations in the SCFAs, bile acid and indole compounds, and NAFLD can be accurately diagnosed using the metabolites. L. lactis and P. pentosaceus ameliorate NAFLD progression by modulating gut metagenomic and metabolic environment, particularly tryptophan pathway, of the gut-liver axis.

Evaluation of probiotic characteristics and whole genome analysis of Pediococcus pentosaceus MR001 for use as probiotic bacteria in shrimp aquaculture.

The development of non-antibiotic and environmentally friendly agents is a key consideration for health management in shrimp aquaculture. In this study, the probiotic potential in shrimp aquaculture of Pediococcus pentosaceus MR001, isolated from Macrobrachium rosenbergii, was investigated by means of feeding trial and genetic characterization. In the feeding trial, dietary supplementation with P. pentosaceus MR001 significantly increased weight gain and digestive enzyme activity (p < 0.05) in shrimp, Litopenaeus vannamei. The intestinal histology showed that shrimp given the probiotic diet had healthier guts than the control group. Also, the immune gene expression and the survival rate in the treatment group were significantly increased when compared with the control group. The genetic characteristics of P. pentosaceus strain MR001 were explored by performing whole-genome sequencing (WGS) using the HiSeq 2500 platform and PacBio system, revealing the complete circular genome of 1,804,896 bp. We also identified 1789 coding genes and subsequently characterized genes related to the biosynthesis of bacteriocins, stress resistance, and bile tolerance. Our findings suggest that insights in the functional and genetic characteristics of P. pentosaceus strain MR001 could provide opportunities for applications of such strain in shrimp diet supplementation.

In silico analyses of the genomes of three new bacteriocin-producing bacteria isolated from animal's faeces.

Here, we have analysed and explored the genome sequences of three newly isolated bacteria that were recently characterised for their probiotic activities and ability to produce bacteriocins. These strains, isolated from faeces of animals living in captivity at the zoological garden of Lille (France), are Escherichia coli ICVB443, Enterococcus faecalis ICVB501 and Pediococcus pentosaceus ICVB491. Their genomes have been analysed and compared to those of their pathogenic or probiotic counterparts. The genome analyses of E. coli ICVB443 and Ent. faecalis ICVB501 displayed similarities to those of probiotics E. coli 1917 Nissle, and Ent. faecalis Symbioflor 1, respectively. Furthermore, E. coli ICVB443 shares at least 89 genes with the enteroaggregative E. coli 55989 (EAEC), and Ent. faecalis ICVB501 shares at least 315 genes with the pathogenic Ent. faecalis V583 strain. Unlike Ped. pentosaceus ICVB491, which is devoid of virulence genes, E. coli ICVB443 and Ent. faecalis ICVB501 both carry genes encoding virulence factors on their genomes. Of note, the bioinformatics analysis of these two genomes located the bsh gene, which codes for bile salt hydrolase (BSH). The presence of BSH is of major importance, as it can help to increase the viability of these two strains in the gastrointestinal tract (GIT). The genome analysis of Ped. pentosaceus ICVB491 confirmed its GRAS status (Generally Recognised As Safe), as no genomic virulence factor determinant was found.

Optimization of the yield of bacteriocin-like substance (BLIS) produced by Pediococcus pentosaceus and its application as food bioconservative / Otimização do rendimento de substância semelhante a bacteriocina (BLIS) produzido por Pediococcus pentosaceus e sua aplicação como bioconservante de alimentos

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria (LABs), which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. However, when this bacteriocin has not been completely characterized regarding its amino acid and the nucleotide sequences of the corresponding gene, the qualified term bacteriocin-like inhibitory substance (BLIS) is recommended. In order to increase the antimicrobial activity of bacteriocins, the ability of probiotics LABs, such as Pediococcus pentosaceus, to ferment different carbon and nitrogen sources has been studied. For the development of an improved culture medium, carbon and nitrogen sources must be considered as nutrients responsible for cell growth and bacteriocin production. The best condition, after 48 h of cultivation, for growth (3.420 g/L) and for BLIS production by Pediococcus pentosaceus ATCC 43200 was in Man, Rogosa and Sharp (MRS) culture medium supplemented with 1.5% peptone, initial pH 6.0 and under the following culture conditions: anaerobiosis, 30oC and agitation of 200 rpm. Compared with control (MRS without supplement), the growth of Pediococcus was significantly lower (1.995 g/L) as well as it reduced significantly its generation time from 2.05 h (control) to 1.28 h (MRS supplemented), a reduction of approximately 62.5%. Moreover, addiction of peptone to MRS medium promoted reduction of 4 h to the Pediococcus exponential phase onset. Regarding BLIS antimicrobial activity, addition of nitrogen source to MRS medium was also quite significant. Through the agar diffusion method, BLIS showed inhibition halos between 12.50 and 19.50 mm against LABs strains (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 and Carnobacterium piscicola CECT 4020). Against Listeria strains (Listeria innocua NCTC 111288 and Listeria seeligeri NCTC 11289), their antimicrobial activity was better detected in liquid medium assay, evaluating the minimal inhibitory concentration of 50%. BLIS was able to inhibit 60 and 100% of L. seeligeri and L. innocua, respectively, as well as, diluted 1x (v/v) in water was able to inhibit 100% growth of both Listeria. BLIS 17 showed also good results as food preservative when applied in ready-to-eat pork ham artificially contaminated with L. seeligeri in vacuum-package at 4oC during shelf life of 10 days. BLIS was able to maintain low Listeria multiplication, lower samples weight loss, low lipid peroxidation and good color parameters during samples storage. Results demonstrated the importance of optimizing the culture medium to increase microbial mass, to produce and to improve the activity of this antimicrobial molecule. Moreover, results also suggest the possible application of BLIS as a natural food preservative

Bacteriocinas são peptídeos produzidos por várias espécies de bactérias, especialmente bactérias ácido-láticas (BALs) e apresentam um amplo espectro de ação contra bactérias deteriorantes e patógenos de origem alimentar. Entretanto, quando estas bacteriocinas não foram completamente caracterizadas quanto a sequência de seus nucleotídeos e do seu gene correspondente, é recomendada a denominação de substância semelhante a bacteriocina (BLIS). Para aumentar a atividade antimicrobiana de bacteriocinas, a habilidade de BALs probióticas, como Pediococcus pentosaceus, em fermentar diferentes fontes de carbono e nitrogênio tem sido estudado. Para o desenvolvimento de um meio de cultura melhorado, fontes de carbono e nitrogênio devem ser consideradas como nutrientes responsáveis pelo crescimento celular e pela produção de bacteriocina. A melhor condição, após 48 h de cultivo, para o crescimento (3,420 g/L) e para a produção de BLIS por P. pentosaceus ATCC 43200 foi em meio de cultivo Man, Rogosa e Sharp (MRS) suplementado com 1,5% de peptona, pH inicial 6,0 e sob as seguintes condições de cultivo: anaerobiose, 30oC e agitação de 200 rpm. Comparado ao controle (MRS sem suplementação), o crescimento de Pediococcus foi significativamente menor (1,995 g/L) assim, como também, reduziu significativamente o tempo de geração de 2,05 h (controle) para 1,28 h (MRS suplementado), uma redução de aproximadamente 62,5%. Além disso, a adição de peptona ao meio MRS promoveu redução de 4 h para o início da fase exponencial de Pediococcus. Quanto a atividade antimicrobiana de BLIS, a adição de fonte de nitrogênio ao meio MRS também foi bastante significativa. Através do método ágar difusão, BLIS apresentou halos de inibição entre 12,50 a 19,50 mm contra cepas de BALs (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 e Carnobacterium piscicola CECT 4020). Contra cepas de Listeria (Listeria innocua NCTC 11288 e Listeria seeligeri NCTC 11289), a sua atividade inibitória foi melhor detectada em meio líquido, através da determinação da concentração mínima inibitória de 50%. BLIS sem diluição foi capaz de inibir 60 e 100% de L. seeligeri e L. innocua, 15 respectivamente, assim como, diluído 1x (v/v) em água foi capaz de inibir 100% o crescimento de ambas Listeria. BLIS também apresentou bons resultados como conservante de alimento quando aplicado em presunto contaminado artificialmente com L. seeligeri e armazenado a 4oC a vácuo por 10 dias. BLIS foi capaz de manter baixa a multiplicação de Listeria, menor perda de peso das amostras, baixa peroxidação lipídica e bons parâmetros de cor durante o armazenamento das amostras. Os resultados demonstraram a importância de se otimizar meio de cultivo tanto para o aumento da massa microbiana como para a produção e melhoramento da atividade desta molécula antimicrobiana. Além disso, os resultados também sugerem a possível aplicação de BLIS como conservante natural de alimentos