Whole genome sequencing of the poly-γ-glutamic acid-producing novel Bacillus subtilis Tamang strain, isolated from spontaneously fermented kinema.

Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain's Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.

Lactic acid bacteria in some Indian fermented foods and their predictive functional profiles.

Lactic acid bacteria (LAB) were isolated from naturally fermented foods of India, viz., sidra, a dried fish product; kinema, a naturally fermented sticky soybean food; and dahi, a naturally fermented milk product. Five strains of LAB, based on 16S rRNA gene sequence, were identified: Lactococcus lactis FS2 (from sidra), Lc. lactis C2D (dahi), Lc. lactis SP2C4 (kinema), Lactiplantibacillus plantarum DHCU70 (=Lactobacillus plantarum) (from dahi), and Lactiplantibacillus plantarum KP1 (kinema). The PICRUSt2 software, a bioinformatic tool, was applied to infer the raw sequences obtained from LAB strains mapped against KEGG database for predictive functionality. Functional features of LAB strains showed genes associated with metabolism (36.47%), environmental information processing (31.42%), genetic information processing (9.83%), and the unclassified (22.28%). KEGG database also showed abundant genes related to predictive membrane transport (29.25%) and carbohydrate metabolism (11.91%). This study may help in understanding the health-promoting benefits of the culturable LAB strains in fermented foods.

Microbiome and metabolome in home-made fermented soybean foods of India revealed by metagenome-assembled genomes and metabolomics.

Grep-chhurpi, peha, peron namsing and peruñyaan are lesser-known home-made fermented soybean foods prepared by the native people of Arunachal Pradesh in India. Present work aims to study the microbiome, their functional annotations, metabolites and recovery of metagenome-assembled genomes (MAGs) in these four fermented soybean foods. Metagenomes revealed the dominance of bacteria (97.80 %) with minor traces of viruses, eukaryotes and archaea. Bacillota is the most abundant phylum with Bacillus subtilis as the abundant species. Metagenome also revealed the abundance of lactic acid bacteria such as Enterococcus casseliflavus, Enterococcus faecium, Mammaliicoccus sciuri and Staphylococcus saprophyticus in all samples. B. subtilis was the major species found in all products. Predictive metabolic pathways showed the abundance of genes associated with metabolisms. Metabolomics analysis revealed both targeted and untargeted metabolites, which suggested their role in flavour development and therapeutic properties. High-quality MAGs, identified as B. subtilis, Enterococcus faecalis, Pediococcus acidilactici and B. velezensis, showed the presence of several biomarkers corresponding to various bio-functional properties. Gene clusters of secondary metabolites (antimicrobial peptides) and CRISPR-Cas systems were detected in all MAGs. This present work also provides key elements related to the cultivability of identified species of MAGs for future use as starter cultures in fermented soybean food product development. Additionally, comparison of microbiome and metabolites of grep-chhurpi, peron namsing and peruñyaan with that of other fermented soybean foods of Asia revealed a distinct difference.

Metagenomic-Metabolomic Mining of Kinema, a Naturally Fermented Soybean Food of the Eastern Himalayas.

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.

Shotgun metagenomics of Cheonggukjang, a fermented soybean food of Korea: Community structure, predictive functionalities and amino acids profile.

Cheonggukjang is a naturally fermented soybean food of Korea. The present study was aimed to reveal the whole microbial community structure of naturally fermented cheonggukjang along with the prediction of microbial functional profiles by shotgun metagenomic sequence analysis. Metataxonomic profile of cheonggukjang samples showed different domains viz. bacteria (95.83%), virus (2.26%), unclassified (1.84%), eukaryotes (0.05%) and archaea (0.005%). Overall, 44 phyla, 286 families, 722 genera and 1437 species were identified. Firmicutes was the most abundant phylum (98.04%) followed by Proteobacteria (1.49%), Deinococcus-Thermus (0.14%). Bacillus thermoamylovorans was the most abundant species in cheonggukjang followed by Bacillus licheniformis, Bacillus glycinifermentans, Bacillus subtilis, Bacillus paralicheniformis, Bacillus amyloliquifaciens, Brevibacillus borstelensis, Brevibacillus sonorensis Brevibacillus, Acinetobacter, Carnobacterium, Paenibacillus, Cronobacter Enterococcus, Enterobacter, Terriglobus, Psychrobacter and Virgibacillus. A colossal diversity of the genus Bacillus was detected with 150 species. Functional analysis of cheonggukjang metagenome revealed the genes for the synthesis and metabolism of wide range of bioactive compounds including, various essential amino acids, conjugated amino acids, different vitamins, flavonoids, and enzymes. Amino acid profiles obtained from KEGG annotation in cheonggukjang were validated with experimental result of amino acid profiles.

Shotgun sequence-based metataxonomic and predictive functional profiles of Pe poke, a naturally fermented soybean food of Myanmar.

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.

Bacterial and fungal communities and their predictive functional profiles in kinema, a naturally fermented soybean food of India, Nepal and Bhutan.

Bacterial and fungal communities in kinema, a naturally fermented soybean food of the Eastern Himalayan regions of India, Nepal and Bhutan were profiled by high-throughout sequence analysis. Firmicutes (78.4%) was the most abundant phylum in kinema, followed by Proteobacteria (14.76%) and other phyla. Twenty seven species of Bacillus were detected, among which Bacillus subtilis (28.70%) was the most abundant bacterium, followed by B. licheniformis, B. thermoamylovorans, B. cereus, Ignatzschineria larvae, Corynebacterium casei, B. sonorensis, Proteus vulgaris, Brevibacillus borstelensis, Thermoactinomyces vulgaris, Lactobacillus fermentum and Ignatzschineria indica. Ascomycota was the most abundant fungal phylum in kinema. Wallemia canadensis, Penicillium spp., Aspergillus spp., Exobasidium spp., Arthrocladium spp., Aspergillus penicillioides, Mortierella spp., Rhizopus arrhizus and Mucor circinelloides, were major moulds, and Pichia sporocuriosa, Trichosporon spp., Saccharomycopsis malanga and Rhodotorula cycloclastica were abundant yeasts in kinema. We detected 277 species of bacteria among which, 99.09% were culturable and 0.91% were unculturable; and 80 fungal species among which, 33.72% were culturable and 66.28% were unculturable. Several unique bacterial genera to each country were observed, whereas no unique fungal genus was observed in kinema. Maximum coverage of sequencing depth was observed in all samples. Based on PCA plot, close relation was observed between samples of India and Nepal, whereas samples of Bhutan was clearly distinctive. Predictive functional features of bacterial and fungi related to metabolisms were inferred by the KEGG Orthology and MetaCyc databases, respectively.