A single dietary factor, daily consumption of a fermented beverage, can modulate the gut bacteria and fecal metabolites within the same ethnic community

ABSTRACT In this study, the impact of traditional rice-based fermented alcoholic beverages (two types of Apong drink: Poro and Nogin) on the gut microbiome and health of the Mishing community in India was examined. Two groups (n = 71 in each group, 58 females and 84 males) that consumed one of these beverages were compared to a control group (n = 24, all males) that did not consume either beverage. Gut microbial composition was analyzed by sequencing 16S rRNA of fecal metagenomes and analyzing untargeted fecal metabolites, and short-chain fatty acids (SCFAs). We also collected data on anthropometric measures and serum biochemical markers. Our results showed that Apong drinkers had higher blood pressure, but lower blood glucose and total protein levels than other non-drinkers. Also, gut microbiome composition was found to be affected by the choice of Apong, with Apong drinkers having a more diverse and distinct microbiome compared to non-drinkers. Apong drink type or being a drinker or not explained even a higher variation of fecal metabolome composition than microbiome composition and Apong drinkers had lower levels of the SCFA isovaleric acid than non-drinkers. Overall, this study shows that a single dietary factor can significantly impact the gut microbiome of a community and highlights the potential role of traditional fermented beverages in modulating gut bacteria. IMPORTANCE Our study investigated how a traditional drink called Apong, made from fermented rice, affects the gut and health of the Mishing community in India. We compared two groups of people who drink Apong to a group of people who do not drink it. To accomplish this, we studied the gut bacteria, fecal metabolites, and blood samples of the participants. It was found that the people who drank Apong had higher blood pressure but lower blood sugar and protein levels than people who did not drink it. We also found that the gut microbiome composition of people who drank Apong was different from those who did not drink it. Moreover, people who drank Apong had lower levels of isovaleric acid in their feces. Overall, this study shows that a traditional drink like Apong can affect the gut bacteria of a community.

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Reviewer comments:
Reviewer #1 (Comments for the Author): The authors have done an excellent job of addressing all previously provided remarks on the manuscript, including to statistical analyses and descriptions.
Reviewer #3 (Comments for the Author): The paper has made several improvements from the previous version and has provided much greater detail on the shaping of the gut microbiome with introduction of a fermented alcoholic beverage.however the following questions need further clarification 1. 50 % of the data being assigned as chimeric is too high to qualify for acceptance without clarification.Is this because the default parameters were used?were the primers removed and checked for their removal via cutadapt pipeline?at what stage does the quality drop (phread<30) for the averae of all forward and reverse reads and then for the merged forward and reverse reads.This information should also be integrated into the manuscript.2. if there are 216 correlations between gut microbiota and fecal metabolome then why is the supplementary only restricted top 30? 3.In the supplementary table 2 under the column labelled metabolites and under row number 30: metabolite "Holdemanella" is mentioned.please clarify and correct the table.Kindly double check the analysis given the nature of error in the table 4.lines 304 to 308: discussion of impact of apong in modulating blood pressure via the gut microbiome.how is this connection made.the cited literature talks about a multitude of long chain fatty acids that could be a potential implicator of CAD, however those metabolites are not seen or mentioned in this study...maybe further teasing apart can be done in the discussion section regarding the associations between microbial players and clinical conditions.Minor comments: 1. being on a vegetarian diet might alter the blood albumin level and must be mentioned in the study 2. line 173:"We agglomerated gut microbes at the family level" however the results have been modified according to previous comments to genus level.this correction has to be made in the text appropriately 3. the discussion section mentions the correlation between apong and fecal carboxylic acid but again does not shed light as to what this could mean?have other studies reported this?

Reviewer #3
The paper has made several improvements from the previous version and has provided much greater detail on the shaping of the gut microbiome with introduction of a fermented alcoholic beverage.however the following questions need further clarification Q1.50 % of the data being assigned as chimeric is too high to qualify for acceptance without clarification.Is this because the default parameters were used?were the primers removed and checked for their removal via cutadapt pipeline?at what stage does the quality drop (phread<30) for the average of all forward and reverse reads and then for the merged forward and reverse reads.This information should also be integrated into the manuscript.
Response: Thank you for the valuable suggestion.We removed the primers using the -forwardPrimer and -reversePrimer options in the LotuS2 pipeline (https://doi.org/10.1186/s40168-022-01365-1)and the pipeline reported in their detailed log file that primer removal was successful.LotuS2 pipeline uses a strict filtering criterion.24,049,359 of reads were classified as "High-quality" and "Medium quality", and 4,033,740 as low by the pipeline.We believe that relatively high proportion of chimeric reads (10,805,289 accounting for 44.43%) stemming from a chemistry-related problem occurred during sequencing.However, although we had to filter an important number of reads, our samples were deeply sequenced, therefore we still ended up with high number of reads: 24,049,359 reads (median: 89916 reads/sample).(Please see Line 415-418 for further information).

Old manuscript
New 4.lines 304 to 308: discussion of impact of apong in modulating blood pressure via the gut microbiome.how is this connection made.the cited literature talks about a multitude of long chain fatty acids that could be a potential implicator of CAD, however those metabolites are not seen or mentioned in this study...maybe further teasing apart can be done in the discussion section regarding the associations between microbial players and clinical conditions.
Response: Thank you for the valuable comment.We would like to clarify that the association between the gut microbes and the biomarkers (serum and anthropometric) were established through correlation studies.Moreover, in the discussion section we have drawn comparisons to studies those have similar findings.However, establishing a causal relationship of biomarkers and the gut microbes was beyond the scope of this study, which was mainly focused on understanding the role of a dietary factor.Nevertheless, as suggested we have expanded the discussion on how key gut bacterial members might play a role in the regulation of blood pressure.

The manuscript has been modified as below
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-23 (A single dietary factor, daily consumption of a fermented beverage, can modulate the gut bacteria and faecal metabolites within the same ethnic community) Dear Dr. Mojibur Rohman R Khan: Thank you for submitting your paper to mSystems.The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.
Thank you for the valuable comment.Although we obtained a total of 216 correlations, we had set a cut off limit at rho ≥ 0.7 to include only the strong correlations.Therefore, correlations having a strength of rho ≥ 0.7 were represented in the supplementary table 2. Accordingly, this has been changed in the text of the manuscript.
3. In the supplementary table 2 under the column labelled metabolites and under row number 30: metabolite "Holdemanella" is mentioned.pleaseclarify and correct the table.Kindly double check the analysis given the nature of error in the tableResponse: Thank you for the valuable comment.We apologize for the mistake in the table.The table has now been corrected.
Apong drinkers had similar dietary habits.Both Nogin and Poro drinkers consume occasionally meat.Although we observed significant difference in the serum globulin levels among the groups, it was within the permissible limit.We are pleased to report that our study did not reveal any adverse impacts of Apong on these vital organs.Q2. line 173:"We agglomerated gut microbes at the family level" however the results have been modified according to previous comments to genus level.this correction has to be made in the text appropriately Response: We apologize for this.Following your suggestions now we have corrected the family level to genus level at appropriate place.
assessing biochemical parameters, such as serum globulin levels, was to gain insights into the functioning of vital organs like the liver and kidneys.We specifically aimed to determine if long-term consumption of Apong had any detrimental effects on these essential organs.
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