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Kinema is a popular sticky fermented soybean food of the Eastern Himalayan regions of North East India, east Nepal, and south Bhutan. We hypothesized that some dominant bacteria in kinema may contribute to the formation of targeted and non-targeted metabolites for health benefits; hence, we studied the microbiome–metabolite mining of kinema. A total of 1,394,094,912 bp with an average of 464,698,304 ± 120,720,392 bp was generated from kinema metagenome, which resulted in the identification of 47 phyla, 331 families, 709 genera, and 1,560 species. Bacteria (97.78%) were the most abundant domain with the remaining domains of viruses, eukaryote, and archaea. Firmicutes (93.36%) was the most abundant phylum with 280 species of Bacillus, among which Bacillus subtilis was the most dominant species in kinema followed by B. glycinifermentans, B. cereus, B. licheniformis, B. thermoamylovorans, B. coagulans, B. circulans, B. paralicheniformis, and Brevibacillus borstelensis. Predictive metabolic pathways revealed the abundance of genes associated with metabolism (60.66%), resulting in 216 sub-pathways. A total of 361 metabolites were identified by metabolomic analysis (liquid chromatography-mass spectrophotometry, LC-MS). The presence of metabolites, such as chrysin, swainsonine, and 3-hydroxy-L-kynurenine (anticancer activity) and benzimidazole (antimicrobial, anticancer, and anti-HIV activities), and compounds with immunomodulatory effects in kinema supports its therapeutic potential. The correlation between the abundant species of Bacillus and primary and secondary metabolites was constructed with a bivariate result. This study proves that Bacillus spp. contribute to the formation of many targeted and untargeted metabolites in kinema for health-promoting benefits.

Pe poke is a naturally fermented sticky soybean food of Myanmar. The present study was aimed to profile the whole microbial community structure and their predictive gene functionality of pe poke samples prepared in different fermentation periods viz. 3 day (3ds), 4 days (4ds), 5 days (5ds) and sun-dried sample (Sds). The pH of samples was 7.6 to 8.7, microbial load was 2.1–3.9 x 108 cfu/g with dynamic viscosity of 4.0±1.0 to 8.0±1.0cP. Metataxonomic profile of pe poke samples showed different domains viz. bacteria (99.08%), viruses (0.65%), eukaryota (0.08%), archaea (0.03%) and unclassified sequences (0.16%). Firmicutes (63.78%) was the most abundant phylum followed by Proteobacteria (29.54%) and Bacteroidetes (5.44%). Bacillus thermoamylovorans was significantly abundant in 3ds and 4ds (p<0.05); Ignatzschineria larvae was significantly abundant in 5ds (p<0.05), whereas, Bacillus subtilis was significantly abundant in Sds (p <0.05). A total of 172 species of Bacillus was detected. In minor abundance, the existence of bacteriophages, archaea, and eukaryotes were also detected. Alpha diversity analysis showed the highest Simpson’s diversity index in Sds comparable to other samples. Similarly, a non-parametric Shannon’s diversity index was also highest in Sds. Good’s coverage of 0.99 was observed in all samples. Beta diversity analysis using PCoA showed no significant clustering. Several species were shared between samples and many species were unique to each sample. In KEGG database, a total number of 33 super-pathways and 173 metabolic sub-pathways were annotated from the metagenomic Open Reading Frames. Predictive functional features of pe poke metagenome revealed the genes for the synthesis and metabolism of wide range of bioactive compounds including various essential amino acids, different vitamins, and enzymes. Spearman’s correlation was inferred between the abundant species and functional features.

Spontaneously fermented soybean foods with sticky-textured and umami-flavor are popular delicacies of multi-ethnic communities of the Eastern Himalayas. Even though species of Bacillus have been reported earlier as pre-dominant bacteria, we hypothesized that some lactic acid bacteria (LAB) present in these unique soy-based foods may exhibit probiotic properties. Hence, the present study is aimed to evaluate some probiotic attributes of LAB. A total of 352 bacterial isolates from spontaneously fermented soybean foods of the Eastern Himalayas viz., kinema, grep-chhurpi, peha, peron namsing and peruñyaan were preliminarily screened for survival in low pH, bile salt tolerance, and cell surface hydrophobicity. Finally, eight probiotic LAB were selected and identified, based on the 16S rRNA gene sequencing, as Pediococcus acidilactici Ki20 and Enterococcus faecium Kn19 (isolated from kinema), E. faecalis Gc21 (grep-chhurpi), P. acidilactici Ph32 (peha), E. faecium Pn11 and E. faecalis Pn37 (peron namsing), E. lactis Py03, and E. lactis Py08 (peruñyaan). In vitro probiotic attributes, E. faecium Kn19 (73.67 ± 1.05) and P. acidilactici Ph32 (79.71 ± 0.13%) recorded higher survival ability in acid and bile salt test, respectively. Furthermore, attachment ability of isolates to hydrocarbons showed ≥80% adhesion property with E. faecalis Gc21 (90.50 ± 10.14%) marked the highest degree of hydrophobicity, and P. acidilactici Ki20 showed the higher auto-aggregation and co-aggregation property. LAB strains were able to produce antibacterial activity against pathogenic bacteria. Genetic screening revealed the presence of genes responsible for acid tolerance (groEl, clpL), bile salt tolerance (apf, bsh), adhesion (msa, mub1), and bacteriocin producing genes for pediocin (pedA, pedB) and enterocin (entA, entB). The present study highlighted the probiotic potentials of LAB strains isolated from Himalayan naturally fermented soybean foods that may be developed as a starter or co-starter culture for controlled and optimized fermentation of soybeans.