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.

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.

Metagenomics and metabolomics of Toddy, an Indian fermented date palm beverage.

Toddy is a popular fermented palm beverage of India. No scientific information on shotgun metagenomics and metabolomics are available on toddy of India till date. Hence, we choose the fermented date palm beverage, locally called khejur toddy, of West Bengal and Jharkhand states of India, to profile microbial community, their targeted and untargeted metabolites to study the putative bio-functional genes corresponding to regulatory metabolic pathways. Shotgun-based metataxonomic analyses revealed the existence of all domains where bacteria were the most abundant domain (94.48%) followed by eukaryotes (3.38%), viruses (1.53%) and archaea (0.61%). Overall, 54 phyla, 363 families, 1087 genera and 1885 species were observed and identified. Bacillota (49.3%) was the most abundant bacterial phylum. At species level, several species of bacteria and yeasts were detected in toddy samples which included Leuconostoc mesenteroides,Leuconostoc citreum,Lactobacillus helveticus,Lactiplantibacillus plantarum,Lactococcus lactis, Acetobacter malorum, Gluconobacter japonicus, Gluconacetobacter liquefaciens, Fructobacillus durionis, Zymomonas mobilis and yeastsSaccharomyces cerevisiae, Hanseniaspora uvarumandHanseniaspora guilliermondii. Toddy metagenome was also compared with metagenome of pulque, the Mexican fermented fresh sap ofAgave, which was retrieved from NCBI database, and also with metagenomic data of some amplicon-based previous studies on toddy and African fermented palm drink for similarity, dissimilarity and uniqueness among them. Predictive biosynthesis of ethanol, acetic acid, butanoate, linalool, staurosporine, prodigiosin, folic acid, riboflavin, etc. were annotated by KEGG/COG database. Clustered regularly interspaced short palindromic repeats (CRISPR) analysis detected 23 arrays (average length 23.69 bp ± 4.28). Comprehensive Antibiotic Resistance Database (CARD) analysis did not show the presence of any momentous antibiotic resistance gene among the major microbial members. Metabolomics analysis detected many primary and secondary metabolites. We believe this is the first report on complete shotgun metagenomics, and metabolomics of fermented palm drink of India as well as Eastern India.

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.

Diversity of yeasts in Indian fermented foods and alcoholic beverages.

Ethnic Indian people have been domesticating beneficial microorganisms (bacteria, yeasts, and moulds) by their wisdom of ethno-microbiological knowledge for production of flavoured and socio-culturally preferred fermented foods and alcoholic beverages for more than 8000 years. The purpose of this review is to collate the available literatures of diversity of Saccharomyces and non-Saccharomyces species associated with Indian fermented foods and alcoholic beverages. A colossal diversity of enzyme- and alcohol-producing yeasts under the phylum Ascomycota has been reported from Indian fermented foods and alcoholic beverages. The distributions of yeast species show 13.5% of Saccharomyces cerevisiae and 86.5% of some non-Saccharomyces spp. in Indian fermented foods and alcoholic beverages, based on the reported literatures available till date. There is a research gap on prospect of yeasts research in India. Hence, we suggest that validation of traditional knowledge of domestication of functional yeasts needs to be studied to develop the functional genomics platforms for Saccharomyces and non-Saccharomyces spp. in Indian fermented foods and alcoholic beverages.

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.

Novel pathways in bacteriocin synthesis by lactic acid bacteria with special reference to ethnic fermented foods.

Ethnic fermented foods are known for their unique aroma, flavour, taste, texture and other sensory properties preferred by every ethnic community in this world culturally as parts of their eatables. Some beneficial microorganisms associated with fermented foods have several functional properties and health-promoting benefits. Bacteriocins are the secondary metabolites produced by the microorganisms mostly lactic acid bacteria present in the fermented foods which can act as lantibiotics against the pathogen bacteria. Several studies have been conducted regarding the isolation and characterization of potent strains as well as their association with different types of bacteriocins. Collective information regarding the gene organizations responsible for the potent effect of bacteriocins as lantibiotics, mode of action on pathogen bacterial cells is not yet available. This review focuses on the gene organizations, pathways include for bacteriocin and their mode of action for various classes of bacteriocins produced by lactic acid bacteria in some ethnic fermented foods.

Probiotic properties of yeasts in traditional fermented foods and beverages.

The interest in potentiality and functionality of probiotic yeasts from fermented foods has increased drastically over the years. In many fermented foods and beverages, lactic acid bacteria and yeasts exist synergistically by stimulating their growth and survival. Probiotic strains of lactic acid bacteria are more widely studied than potential probiotic yeasts. Saccharomyces cerevisiae variety boulardii is the only commercialized probiotic yeast, which are extensively studied. This review article provides information on the presence of potential probiotic yeasts in some traditional fermented foods and beverages.

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.

In vitro and genetic screening of probiotic properties of lactic acid bacteria isolated from naturally fermented cow-milk and yak-milk products of Sikkim, India.

A total of 272 isolates of lactic acid bacteria (LAB) were isolated from 22 samples of naturally fermented milk products of Sikkim in India viz. dahi, soft-variety chhurpi, hard-variety chhurpi, mohi and philu, out of which, 68 LAB isolates were randomly grouped on the basis of phenotypic characteristics, and were identified by 16S rRNA gene sequence analysis. Leuconostoc mesenteroides was the most dominant genus, followed by Leuc. mesenteroides subsp. jonggajibkimchii, Lactococcus lactis subsp. cremoris, Lc. lactis, Lc. lactis subsp. hordniae, Lc. lactis subsp. tructae, Enterococcus faecalis, E. italicus and E. pseudoavium. LAB strains were tested for probiotics attributes by in vitro and genetic screening, based on marker genes. LAB strains showed tolerance to pH 3.0, bile salt, resistance to lysozyme and ß-galactosidase activity. Enterococcus faecalis YS4-11 and YS4-14 and Lactococcus lactis subsp. cremoris SC3 showed more than 85% of hydrophobicity. Genes clp L and tdc encoding for low pH tolerance, agu A and Ir1516 encoding for bile tolerance, LBA1446 gene encoding for BSH activity, map A, apf, mub 1 and msa encoding for mucosal binding property were detected. Gene mesY for bacteriocin production was detected only in Leuconostoc spp. Based on the in vitro and genetic screening of probiotic attributes, Leuc. mesenteroides; Leuc. mesenteroides subsp. jonggajibkimchii and Lc. lactis subsp. cremoris were tentatively selected for possible probiotic candidates.

"Ethno-microbiology" of ethnic Indian fermented foods and alcoholic beverages.

The concept of "ethno-microbiology" is to understand the indigenous knowledge of the Indian people for production of culturally and organoleptically acceptable fermented foods by natural fermentation. About 1000 types of common, uncommon, rare, exotic and artisan fermented foods and beverages are prepared and consumed in different geographical regions by multi-ethnic communities in India. Indian fermented foods are mostly acidic and some are alkaline, along with various types of alcoholic beverages. A colossal diversity of microorganisms comprising bacteria mostly belongs to phylum Firmicutes, filamentous moulds and enzyme- and alcohol-producing yeasts under phyla Ascomycota and Mucoromycota, and few bacteriophages and archaea have been reported from Indian fermented foods. Some microorganisms associated with fermented foods have functionalities and health promoting benefits. "Ethno-microbiology" of ethnic Indian people has exhibited the proper utilisation of substrates either singly or in combination such as fermented cereal-legume mixture (idli, dosa and dhokla) in South and West India, sticky fermented soybean food (kinema and related foods), fermented perishable leafy vegetable (gundruk and related foods), fermented bamboo shoots (soibum and related foods) and fermented fish (ngari and others) in North East India, and fermented meat and sausage-like products in the Indian Himalayas, fermented coconut beverage (toddy) in coastal regions, and various types of naturally fermented milk products (dahi and related products) in different regions of India. This review has also highlighted the "ethno-microbiology" knowledge of the people involving the consortia of essential microorganisms in traditionally prepared amylolytic starters for production of cereal-based alcoholic beverages. The novelty of this review is the interpretation of ethno-microbiological knowledge innovated by ethnic Indian people on the use of beneficial microorganisms for food fermentation to obtain the desired fermented food products for consumption.

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.

Helicobacter pylori in Human Stomach: The Inconsistencies in Clinical Outcomes and the Probable Causes.

Pathogenic potentials of the gastric pathogen, Helicobacter pylori, have been proposed, evaluated, and confirmed by many laboratories for nearly 4 decades since its serendipitous discovery in 1983 by Barry James Marshall and John Robin Warren. Helicobacter pylori is the first bacterium to be categorized as a definite carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO). Half of the world's population carries H. pylori, which may be responsible for severe gastric diseases like peptic ulcer and gastric cancer. These two gastric diseases take more than a million lives every year. However, the role of H. pylori as sole pathogen in gastric diseases is heavily debated and remained controversial. It is still not convincingly understood, why most (80-90%) H. pylori infected individuals remain asymptomatic, while some (10-20%) develop such severe gastric diseases. Moreover, several reports indicated that colonization of H. pylori has positive and negative associations with several other gastrointestinal (GI) and non-GI diseases. In this review, we have discussed the state of the art knowledge on "H. pylori factors" and several "other factors," which have been claimed to have links with severe gastric and duodenal diseases. We conclude that H. pylori infection alone does not satisfy the "necessary and sufficient" condition for developing aggressive clinical outcomes. Rather, the cumulative effect of a number of factors like the virulence proteins of H. pylori, local geography and climate, genetic background and immunity of the host, gastric and intestinal microbiota, and dietary habit and history of medicine usage together determine whether the H. pylori infected person will remain asymptomatic or will develop one of the severe gastric diseases.

Diversity of beneficial microorganisms and their functionalities in community-specific ethnic fermented foods of the Eastern Himalayas.

The Eastern Himalayan regions of India, Nepal and Bhutan have more than 200 varieties of unsurpassed ethnic fermented foods and alcoholic beverages, which are lesser known outside the world. However, these ethnic foods are region- and community-specific, unique and some are exotic and rare, which include fermented vegetables, bamboo shoots, soybeans, cereals, milk (cow and yak), meats, fishes, and cereal-based alcoholic beverages and drinks. Ethnic communities living in the Eastern Himalayas have invented the indigenous knowledge of utilization of unseen microorganisms present in and around the environment for preservation and fermentation of perishable plant or animal substrates to obtain organoleptically desirable and culturally acceptable ethnic fermented food and alcoholic beverages. Some ethnic fermented products and traditionally prepared dry starters for production of alcoholic beverages of North Eastern states of India and Nepal were scientifically studied and reported till date, and however, limited publications are available on microbiological and nutritional aspects of ethnic fermented foods of Bhutan except on few products. Most of the beneficial microorganisms isolated from some ethnic fermented foods of the EH are listed in microbial food cultures (MFC) safe inventory. This study is aimed to review the updates on the beneficial importance of abundant microbiota and health-promoting benefits and functionalities of some ethnic fermented foods of the Eastern Himalayan regions of North East India, Nepal and Bhutan.

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%).

Changes in microbial communities and their predictive functionalities during fermentation of toddy, an alcoholic beverage of India.

Toddy is a traditional mild-alcoholic drink of India, which is produced from fresh palm saps by natural fermentation. We studied the successional changes in bacterial and fungal communities during the natural fermentation (0 h-96 h) of toddy. During fermentation, alcohol content of the fermenting saps increased significantly from 0.6 %±0.15 to 5.6 %±0.02, pH decreased from 6.33 %±0.02-3.93 ± 0.01, volatile and titratable acidity acidity (g/100 mL) increased from 0.17 ± 0.02 (0 h) to 0.48 ± 0.02 (96 h) and 1.30 ± 0.005 (0 h) to 2.47 ± 0.005 (96 h), respectively. Total sugar content and ˚BRIX also decreased during the fermentation. Firmicutes (78.25 %) was the most abundant phylum followed by Proteobacteria (21.57 %). Leuconostoc was the most abundant genus in the early stages of fermentation. However, Lactobacillus and Gluconoacetobacter were found abundant with increase in pH during the later phases of fermentation (72 h-96 h). Ascomycota (99.02 %) was the most abundant fungal phylum. Hanseniaspora was the abundant yeast in the initial stages of fermentation, whereas the population of Saccharomyces increased significantly after 24 h of fermentation. Torulaspora, Lachancea and Starmerella showed their heterogeneous distribution throughout the fermentation. Computational analysis of metagenomes based on KEGG and MetaCyc databases showed different predictive functional profiles such as folate biosynthesis, glutathione metabolism, terpenoids biosynthesis and biosynthesis of amino acids with significant differences between the fresh palm saps and fermenting saps during toddy fermentation.

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.

Probiotic properties of lactic acid bacteria isolated from traditionally prepared dry starters of the Eastern Himalayas.

The Himalayan people prepare dry and oval to round-shaped starter cultures to ferment cereals into mild-alcoholic beverages, which contain lactic acid bacteria (LAB) as one of the essential microbiota. There is no report on probiotic characters of LAB isolated from dry starters. Hence, we screened the probiotic and some functional properties of 37 LAB strains isolated from dry starters of the Eastern Himalayas viz. marcha, phab, paa, pee and phut. About 38% of the LAB strains showed high survival rate (> 50%) at pH 3 and 0.3% bile salts. Enterococcus durans BPB21 and SMB7 showed the highest hydrophobicity percentage of 98%. E. durans DMB4 and SMB7 showed maximum cholesterol assimilation activity. About 65% of the LAB strains showed the ability to produce ß galactosidase. Majority of the strains showed phytase activity, whereas none of the strain showed amylase activity. About 86% of LAB strains showed an optimum tolerance of 10% ethanol concentration. Genetic screening of some probiotic and functional marker genes have also been analysed. The occurrence of clp L gene, agu A gene (survival of gastrointestinal tract conditions), apf, mub1 and map A gene (adhesion genes) was higher compared to other genes. The occurrence of bsh gene (bile salt tolerance) was detected in Pediococcus pentosaceus SMB13-1 and Enterococcus faecium BPB11. Gene ped B for pediocin with amplicon size of 375 bp was detected in E. durans DMB13 and Pediococcus acidilactici AKB3. Detection of nutritional marker gene rib A and fol P in some strains showed the potential ability to synthesize riboflavin and folic acid. LAB with probiotic and functional properties may be explored for food industry in future.