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.

Dahi and datshi are common naturally fermented milk (NFM) products of Bhutan. Population of lactic acid bacteria (LAB) in dahi (pH 3.7) and datshi (pH 5.2) was 1.4 × 107 and 3.9 × 108 cfu/ml, respectively. Based on 16S rRNA gene sequencing isolates of LAB from dahi and datshi were identified as Enterococcus faecalis, E. faecium, Lactococcus lactis subsp. lactis. LAB strains were tested for some technological properties. All LAB strains except E. faecalis CH2:17 caused coagulation of milk at both 30°C for 48 h. Only E. faecium DH4:05 strain was resistant to pH 3. No significant difference (P > 0.05) of viable counts was observed in MRS broth with and without lysozyme. All LAB strains grew well in 0.3% bile showing their ability to tolerate bile salt. None of the LAB strains showed >70% hydrophobicity. This study, being the first of its microbiological analysis of the NFM of Bhutan, has opened up to an extent of research work that gives a new insight to the products.

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Background: Naturally fermented milk (NFM) products are popular food delicacies in Indian states of Sikkim and Arunachal Pradesh. Bacterial communities in these NFM products of India were previously analysed by high-throughput sequence method. However, predictive gene functionality of NFM products of India has not been studied. In this study, raw sequences of NFM products of Sikkim and Arunachal Pradesh were accessed from NCBI database server. PICRUSt2 and Piphillin tools were applied to study microbial functional gene prediction in combination with the commonly used error-corrected denoising programs like DADA2 and Deblur.Results: Significant functional hits were observed from the Piphillin analysis which included some interesting pathways including GABAergic synapse, glutamatergic synapse and serotonergic synapse, which are known to be probiotic-related, among others that are absent in PICRUSt2 analysis. This study also showed the negative correlation of lactic acid bacteria (LAB) members (Lactococcus, Lactobacillus, Leuconostoc) with most of the disease-related functions, which were on the other hand, positively correlated with unwanted contaminants like Staphylococcus, Bacillus and Pseudomonas.Conclusion: The study explored the potential of microbial functional gene prediction using PICRUSt2 and Piphillin software, and indicated the significance of the presence of LAB in these NFM products of India. Since most LAB members are known to be potential health-promoting bacteria, their negative correlation to many of the disease-related functions also indicates their role in combatting unwanted potential contaminants.