Metagenomics and metagenome-assembled genomes mining of health benefits in jalebi batter, a naturally fermented cereal-based food of India.

Jalebi is one of the oldest Indian traditional fermented wheat-based confectioneries. Since jalebi is prepared by natural fermentation, diverse microbial community is expected to play bio-functional activities. Due to limited studies, information on microbial community structure in jalebi is unknown. Hence, the present study is aimed to profile the microbial community in jalebi by shotgun metagenomics and also to predict putative probiotic and functional genes by metagenome-assembled genome (MAG). Bacteria were the most abundant domain (91.91%) under which Bacillota was the most abundant phylum (82%). The most abundant species was Lapidilactobacillus dextrinicus followed by several species of lactic acid bacteria, acetic acid bacteria including few yeasts. Lap. dextrinicus was also significantly abundant in jalebi when compared to similar fermented wheat-based sourdough. Additionally, Lap. bayanensis, Pediococcus stilesii, and yeast- Candida glabrata, Gluconobacter japonicus, Pichia kudriavzevii, Wickerhamomyces anomalus were only detected in jalebi, which are not detected in sourdough. Few viruses and archaea were detected with < 1 % abundance. In silico screening of genes from the abundant species was mined using both KEGG and EggNOG database for putative health beneficial attributes. Circular genomes of five high-quality MAGs, identified as Lapidilactobacillus dextrinicus, Enterococcus hirae, Pediococcus stilesii, Acetobacter indonesiensis and Acetobacter cibinongensis, were constructed separately and putative genes were mapped and annotated. The CRISPR/Cas gene clusters in the genomes of four MAGs except Acetobacter cibinongensis were detected. MAGs also showed several secondary metabolites. Since, the identified MAGs have different putative genes for bio-functional properties, this may pave the way to selectively culture the uncultivated putative microbes for jalebi production. We believe this is the first report on metagenomic and MAGs of jalebi.

Genome Analysis of Potential Probiotic Levilactobacillus brevis AcCh91 Isolated from Indian Home-Made Fermented Milk Product (Chhurpi).

Consumption of naturally fermented milk (NFM) products is the dietary culture in India. The mountainous people of Arunachal Pradesh in India prepare the assorted artisanal home-made NFM products from cow and yak milk. Previously, we isolated and identified 76 strains of lactic acid bacteria (LAB) from NFM products of Arunachal Pradesh, viz. mar, chhurpi and churkam. We hypothesized that some of these LAB strains may possess probiotic potentials; hence, we investigated the probiotic potentials of these strains. On the basis of in vitro and genetic screening for probiotic attributes including haemolytic ability, 20 LAB strains were selected out of 76 strains, for further analysis. Using in silico analysis, viz. multivariate heatmap and PCA (principal component analysis) biplot, Levilactobacillus brevis AcCh91 was selected as the most promising probiotic strain, which was further characterized by the whole-genome analysis. Lev. brevis AcCh91 showed the highest survival rate of 93.38% in low pH and 86.68 ± 2.69% in low bile and the highest hydrophobicity average of 86.34 ± 5.53%. This strain also showed auto-aggregation and co-aggregation with antimicrobial properties against the pathogens, showed ability to produce beta-galactosidase and cholesterol reduction property and, most importantly, produced GABA, an important psychobiotic element. Genomic analysis of Lev. brevis AcCh91 showed the presence of genes corresponding to GABA, vitamins, amino acids, cholesterol reduction, immunomodulation, bioactive peptides and antioxidant activity. The absence of antimicrobial-resistant genes and virulence factors was observed. Hence, genome analysis supports the probiotic potentials of Lev. brevis AcCh91, which may be further investigated to understand its health-promoting properties.

Metagenome-assembled genomes for biomarkers of bio-functionalities in Laal dahi, an Indian ethnic fermented milk product.

Laal dahi is a sweetened and soft pudding-like fermented milk product of the Eastern regions of India, which has not been studied for its microbial community structures and health promoting functionality in terms of 'omics' approaches. We applied metagenomic and metagenomes-assembled genomes (MAGs) tools to decipher the biomarkers for genes encoding for different health promoting functionalities in laal dahi. Abundance of bacterial domains was observed with negligible presence of eukaryotes and viruses. Bacillota was the most abundant phylum with different bacterial species viz., Enterococcus italicus, Lactococcus raffinolactis, Lactobacillus helveticus, Bifidobacterium mongoliense, Hafnia alvei, Lactococcus lactis, Acetobacter okinawensis, Streptococcus thermophilus, Thermus thermophilus, Leuconostoc citreum, Leuconostoc pseudomesenteroides, Acetobacter orientalis, Lactobacillus gallinarum, Lactococcus chungangensis and Lactobacillus delbrueckii. Comparison of laal dahi microbiome with that of similar fermented milk products was also carried out after retrieving the metagenomic datasets from public databases. Significant abundance of Lb. helveticus, E. italicus, Lc. raffinolactis and Lc. lactis in laal dahi. Interestingly, Bifidobacterium mongoliense, Lb. gallinarum, Lc. chungangensis and Acetobacter okinawensis were only detected in laal dahi but Streptococcus infantarius, Lacticaseibacillus rhamnosus and Lb. johnsonii were absent. Reconstruction of putative single environment-specific genomes from metagenomes in addition to subsampling of the abundant species resulted in five high-quality MAGs identified as Lactobacillus delbrueckii, Lactobacillus helveticus, Lactococcus chungangensis, Lactococcus lactis and Streptococcus thermophilus. All MAGs showed the presence of various genes with several putative functions corresponding to different probiotic and prebiotic functions, short-chain fatty acids production, immunomodulation, antitumor genes, essential amino acid and vitamin biosynthesis. Genes for γ-Aminobutyric acid (GABA) production were only detected in MAG of Lactococcus lactis. Gene clusters for secondary metabolites (antimicrobial peptides) were detected in all MAGs except Lc. chungangensis. Additionally, detection of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) elements was observed only in Lactobacillus delbrueckii and Streptococcus thermophilus. Annotation of several genes with potential health beneficial properties in all five MAGs may support the need to explore the culturability of these MAGs for future use in controlled fermentation of functional dairy products.

High-throughput sequence analysis of bacterial communities and their predictive functionalities in traditionally preserved fish products of Sikkim, India.

Traditionally preserved fish products viz. suka ko maccha, a smoked fish product, sidra and sukuti, sun-dried fish products are commonly consumed in Sikkim state in India. Bacterial communities in these fish products were analysed by high-throughput sequence (HTS) method supported by bioinformatics tool. Metataxonomic of the overall bacterial communities in samples revealed the abundance of phylum Firmicutes followed by Proteobacteria. Psychrobacter was abundant genus in all traditionally preserved fish products of Sikkim, followed by Bacillus, Staphylococcus, Serratia, Clostridium, Enterobacter, Pseudomonas, Rummeliibacillus, Enterococcus, Photobacterium, Myroides, Peptostreptococcus, Plesiomonas and Achromobacter. Product-wise distribution showed that Bacillus was abundant in suka ko maacha and sidra samples, whereas Psychrobacter was abundant in sukuti samples. Unique genus to each product was observed on the basis of analysis of shared operational-taxonomic-unit (OTU) contents, Alpha diversity indices showed significantly differences among the samples, and also showed maximum coverage as per Good's coverage (0.99). Beta diversity showed clustering of bacterial compositions between suka ko maacha and sidra, whereas sukuti showed scattering pattern among the other samples, indicating a diverse population in suka ko maacha and sidra samples. Non-parametric analysis of abundant genera and predictive functionalities showed the complex bacterial inter-dependencies with predictive functionalities mostly in metabolism (79.88%).

Bacterial community in naturally fermented milk products of Arunachal Pradesh and Sikkim of India analysed by high-throughput amplicon sequencing.

Naturally fermented milk (NFM) products are popular ethnic fermented foods in Arunachal Pradesh and Sikkim states of India. The present study is the first to have documented the bacterial community in 54 samples of NFM products viz. chhurpi, churkam, dahi and gheu/mar by high-throughput Illumina amplicon sequencing. Metagenomic investigation showed that Firmicutes (Streptococcaceae, Lactobacillaceae) and Proteobacteria (Acetobacteraceae) were the two predominant members of the bacterial communities in these products. Lactococcus lactis and Lactobacillus helveticus were the predominant lactic acid bacteria while Acetobacter spp. and Gluconobacter spp. were the predominant acetic acid bacteria present in these products.