Positive modulation of a new reconstructed human gut microbiota by Maitake extract helpfully boosts the intestinal environment in vitro

Abstract

The human gut is a complex environment where the microbiota and its metabolites play a crucial role in the maintenance of a healthy state. The aim of the present work is the reconstruction of a new in vitro minimal human gut microbiota resembling the microbe-microbe networking comprising the principal phyla (Bacillota, Bacteroidota, Pseudomonadota, and Actinomycetota), to comprehend the intestinal ecosystem complexity. In the reductionist model, we mimicked the administration of Maitake extract as prebiotic and a probiotic formulation (three strains belonging to Lactobacillus and Bifidobacterium genera), evaluating the modulation of strain levels, the release of beneficial metabolites, and their health-promoting effects on human cell lines of the intestinal environment. The administration of Maitake and the selected probiotic strains generated a positive modulation of the in vitro bacterial community by qPCR analyses, evidencing the prominence of beneficial strains (Lactiplantibacillus plantarum and Bifidobacterium animalis subsp. lactis) after 48 hours. The bacterial community growths were associated with the production of metabolites over time through GC-MSD analyses such as lactate, butyrate, and propionate. Their effects on the host were evaluated on cell lines of the intestinal epithelium and the immune system, evidencing positive antioxidant (upregulation of SOD1 and NQO1 genes in HT-29 cell line) and anti-inflammatory effects (production of IL-10 from all the PBMCs). Therefore, the results highlighted a positive modulation induced by the synergic activities of probiotics and Maitake, inducing a tolerogenic microenvironment.

Fig 1. Schematic representation of the extraction method for polysaccharides from the fungus Grifola frondosa (Maitake).

The image was created using Canva and Biorender.

Fig 2. Growth levels of individual bacterial strains on Maitake extract.

The growth is presented as the mean value of OD_600nm ± SE in the presence of 2% Maitake extract and the CTR medium. Statistical differences were calculated using t-Student’s test: * p—value <0.05, ** < 0.01.

Fig 3. Growth curves of probiotics, minimal core, and the whole community in batch fermentation.

The figure depicts the growth curve (mean value of OD_600nm ± SE during time) of only the probiotics, only the minimal core strains and the whole in vitro gut microbiota in the presence of 2% Maitake extract in batch fermentation.

Fig 4. Single strain level modulation.

The figure represents the bacterial counts/mL of, the probiotics (A), the minimal core strains (B), and the whole community (C) obtained after qPCR analyses with species-specific primers during the growth on 2% Maitake extract. Statistical differences were calculated using t-Student’s test: * p—value < 0.05.

Fig 5. Analyses of bacterial metabolites by GC-MSD after Maitake extract fermentation.

In (A) is reported the chromatogram regarding the metabolites produced by the minimal core strains, in (B) the metabolites released by the probiotics, and in (C) the chromatogram of the whole community.

Fig 6. Antioxidant response of HT-29 cell line.

After the treatment of the cells with a fixed concentration of the different metabolites and the Maitake extract alone for 48 hours, the expression of specific genes was verified via qPCR analyses. The heatmap (A) reports the modulation considering the fold change with respect to a reference gene. In (B) is highlighted the increment of SOD1 and NQO1 genes with respect to the RPL32 gene as mean value ± SD. The statistical differences are calculated by parametric ANOVA test.

Fig 7. Anti-inflammatory response of PBMCs.

The production of anti-inflammatory IL-10 was evaluated through ELISA tests (A) in the presence of a fixed concentration of the different bacterial metabolites or Maitake extract alone. Quantities are expressed as mean values ± SD. The statistical differences were calculated by ANOVA. In (B) are reported the results of the FACS analyses as cells producing IL-10 with respect to FSC-A.