Metagenomic Analysis of Bacterial Diversity in Traditional Fermented Foods Reveals Food-Specific Dominance of Specific Bacterial Taxa

Round 1

Reviewer 1 Report

The authors present a first of a kind metagenomic sequencing study of traditional foods in India. This descriptive paper discusses the macro content of food and also the major genera of bacteria detected during fermentation.

The data is relatively sparse and lacks controls, but represents a first look into these locally important traditional foods with potential health benefits to be discovered.

The present manuscript is a substantially improved version and previous comments have been addressed adequately. Some minor comments remain:

• In Conclusions, is using FBS, FSB, FPF acronyms necessary, or could this be removed: "in FBS, FSB, and FPF samples" since from this sentence its not clear if genera are respective to sample or similarly behaving in all samples
• Please simplify/rephrase last sentence in Conclusions
• Figure 3, please add to the caption or related text the depth at which the samples were rarefied

Author Response

Response to Reviewer 1 Comments

Comments and Suggestions for Authors

The authors present a first-of-a-kind metagenomics sequencing study of traditional foods in India. This descriptive paper discusses the macro content of food and also the major genera of bacteria detected during fermentation.

The data is relatively sparse and lacks controls, but represents the first look into these locally important traditional foods with potential health benefits to be discovered.

The present manuscript is a substantially improved version and previous comments have been addressed adequately. Some minor comments remain:

Dear Reviewer 1,

We would like to thank you for your valuable comments and suggestions. We have carefully revised the manuscript as per your suggestions. Please see below a response to the comments provided by you.

Point 1: In Conclusions, is using FBS, FSB, FPF acronyms necessary, or could this be removed: "in FBS, FSB, and FPF samples" since from this sentence it's not clear if genera are respective to sample or similarly behaving in all samples

Response1: Thank you for taking the time to assess our manuscript and for giving valuable suggestions. We have revised the Conclusions. Acronyms “FBS, FBS, FPF” were removed with the samples name “fermented bamboo shoots (Tuaither), fermented soybean (Bekang), and fermented pork fat (Sa-um)” Also the sentence is rephrased (page no: 15-16, line no: 439-442, highlighted in yellow)

Point 2: Please simplify/rephrase the last sentence in Conclusions

Response 2: Thank you for your comment. The last sentence in Conclusions have been rephrased (page no: 16, line no: 450-453, highlighted in yellow)

Point 3: Figure 3, please add to the caption or related text the depth at which the samples were rarefied

Response 3: Thank you for your suggestion. Figure caption has been added in figure 3 with the depth at which the samples were rarefied (page no: 07, line no: 245-247, highlighted in yellow).

Reviewer 2 Report

In this study, the authors evaluate the microbial diversity and the nutritional composition of traditional fermented food North-East India. The manuscript and the scientific work are well structured and explained. My comments are in the PDF file, marked in yellow along with the text. 

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

In this study, the authors evaluate the microbial diversity and the nutritional composition of traditional fermented food in North-East India. The manuscript and the scientific work are well structured and explained. My comments are in the PDF file, marked in yellow along with the text.

Dear Reviewer 2,

We would like to thank you for your valuable comments and suggestions. We have carefully revised the manuscript as per your suggestions. Please see below a response to the comments provided by you.

Point 1: Keywords must be rethought. They can be more specific and representative of the work performed. For example, I would start by removing the words: "Ethnic population" and "Northeast India".

Response1:Thank you for taking the time to assess our manuscript and for giving valuable suggestions. Keywords like “Ethnic population" and "North-east India" have been removed and new keywords like “Metagenomics” “Bacterial diversity” “Health benefits” are added. (page no: 02, line no: 48-49).

 Point 2: Repeated information, I would suggest a possible removal.

Response 2: Thank you for your comment. As per your suggestion, the repeated information has been removed and the sentence has been revised (page no: 2-3, line no: 95-100)

Point 3: It was not 3 successive days of fermentation but 3 distinct days during fermentation.

Response 3: Thank you for your suggestion. The sentence has been corrected (page no: 3 line no: 104-105)

 Point 4: Make the sentence clearer for what is intended mention.

Response 4: Thank you for your comment. The sentence has been rephrased (page no: 4, line no: 129-130)

Point 5: Enter a reference or references.

Response 5: Thank you for your suggestion. As per your suggestion, references have been added (page no: 5, line no: 194)

Point 6: Authors should, whenever possible, increase the text sizes in all figures throughout the manuscript.

Response 6: Thank you for your suggestion. We have tried to increase the font size, but most of the figures are software-based and not possible to do manually. However, the figure size has been increased to see the cleartext.

Point 7: Make the sentence clearer for what is intended mention.

Response 7:Thank you for your suggestion. The sentence has been rephrased (page no: 6, line no: 238-240)

 Point 8: I would put this part of the text at the beginning of the sentence to facilitate the interpretation.

Response 8: Thank you for your suggestion. As per your suggestion, the sentence has been revised (page no: 7, line no: 251)

 Point 9: Correct the word

Response 9: Thank you for your suggestion. The word has been corrected (page no: 7, line no: 260)

 Point 10: The order is not correct.

Response 10: Thank you for your suggestion. The order has been corrected by revising the sentence (page no. 9, line no: 280-282)

 Point 11: Please enter values.

Response 11: Thank you for your suggestion. The sentence has been revised and values are added (page no: 10, line no: 303-305)

 Point 12: Although not the focus of the work. Is this a concern? What implications does it have in terms of food security?

Response 12: Thank you for your suggestion. The sentence has been revised by removing some information (page no: 10, line no: 318-321)

 Point 13: Correct the word.

Response 13: Thank you for your suggestion. The word has been corrected (page no: 10, line no:326)

 Point 14: Information in duplicate

Response 14:Thank you for your suggestion. The sentence has been removed (page no: 15-16, line no: 439-442)

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Dake et al., Describe the taxonomic composition of microorganisms in the preparation of traditional fermented foods widely consumed by the local people of Mizoram state in northwest India. These are products such as Fermented bamboo shoot, fermented soybean and fermented pork fats. The taxonomic composition was determined based on the analysis of several thousand V3-V4 gene fragments of the 16S rRNA gene fragment for each sample obtained using high-throughput sequencing methods on a MiSeq instrument (Illumina). The manuscript presents in detail the results of bioinformatic analysis of the data obtained. The main novelty of the work is only a set of new data on the sequence of 16S rRNA gene fragments. After getting acquainted with the work, there are a number of fundamental remarks:
1. The work lacks as such a formulation of the scientific problem. Technical work on the collection of product samples, total DNA extraction, amplification of the V3-V4 region and bioinformatic analysis was performed. I would like to highlight a solution to a scientific problem: for example, the search for key microorganisms involved in the preparation of the investigated products; study of the dynamics of changes in the composition of microorganisms during the preparation process and how this is related to the stage of product readiness, etc.
2. There are no microbiological data in the work, for example, an estimate of the number of microorganisms in the studied samples, microscopy, isolation of pure cultures and an assessment of their pathogenicity. Correcting these results with tacosnomic profiling data can significantly improve performance.
3. In the work there is no assessment of the statistical significance of the data obtained.
4. All received data of the V3-V4 region sequence are not made publicly available.
Thus, the presented materials do not meet the high requirements and standards for articles published in the journal "Microorganisms". I do not recommend the presented manuscript for publication in the journal.

Author Response

Dear Reviewer 1,

We would like to thank you for your valuable comments and suggestions, which helped to improve the manuscript. We have carefully revised the manuscript as per your and other suggestions. Please see below a response to the comments provided by you.

Point 1: The work lacks as such a formulation of the scientific problem. Technical work on the collection of product samples, total DNA extraction, amplification of the V3-V4 region and bioinformatic analysis was performed. I would like to highlight a solution to a scientific problem: for example, the search for key microorganisms involved in the preparation of the investigated products; study of the dynamics of changes in the composition of microorganisms during the preparation process and how this is related to the stage of product readiness, etc.

Response 1: Thank you for taking the time to assess our manuscript and for giving valuable suggestions. We agree with you regarding this aspect of the study. However, this is the first study has been conducted on these traditional fermented foods prepared by ethnic people of Mizoram from ancient time. To date, no comprehensive studies were reported for such fermented products. These fermented foods are prepared using the methods developed by these people and are generally based on the household fermentation process that occurs spontaneously without the addition of any inoculums cultures. Since, no previous microbiological data were available, at first we thought to study the bacterial diversity and composition of the fermented food products in order to understand the dominant microbial groups that are present in these foods. Since these fermented foods are consumed from the 3rd day to 7 days of fermentation, we have processed samples at three different dates (3rd, 5th, and 7th day) of fermentation. In this study, we have obtained interesting results about the dominance of bacterial genera. At the genus level, food-specific dominance of bacterial genera was observed indicating the process and raw material of fermented foods has an influence on the microbial composition of these foods. A recent study on other fermented foods from the NE region also showed the food-specific enrichment of bacterial genera in the fermented samples (Jani and Sharma, LWT, 2021, 47, 111578). Therefore, these are interesting results that open up for future studies to see the dynamics of changes in the composition of microorganisms during the fermentation process and relating it to the stage of product readiness. Also, other studies such as isolation and characterization of properties of bacteria that are involved in fermentation can further be studied. Therefore, we request you to give your consideration for this study, as it is the first time we have revealed the bacterial diversity of such traditional fermented foods that are important among the ethnic population of North-East India.

Point 2: There are no microbiological data in the work, for example, an estimate of the number of microorganisms in the studied samples, microscopy, isolation of pure cultures and an assessment of their pathogenicity. Correcting these results with tacosnomic profiling data can significantly improve performance.

Response 2: Thank you for this suggestion. This study was conducted to determine the taxonomic composition of bacteria based on V3-V4 gene fragments of the 16S rRNA gene-based amplicons sequencing. The result revealed that bacterial diversity showed variation in the relative abundances at the genus level across three different fermented food samples. Although, there was not much variation observed during the fermentation process like samples were analyzed on the 3rd, 5th, and 7th day of fermentation. The interesting aspect of the study was the food-specific bacterial dominance, which opens up various interesting things to study in these fermented samples. As these are prepared using methods developed from ancient times, the microbiological studies could definitely help to understand the fermentation process, and how these people can improve the process to ensure its healthiness for the consumption. As, this is being the first study that has determine the bacterial composition and diversity of these fermented food products that has to open up a new aspect to study using microbial cultural techniques. Like isolation of microbial species and characterization for their benefits as well pathogenic properties that will be helpful for understating the nature of such fermented foods. We are very much interested in such aspects as suggested by you and in the future, we will definitely work on such aspects for these fermented food products using a larger sample size. However, being the first study, it does a novelty and we request you to kindly give your consideration for the present study.

Point 3: In the work there is no assessment of the statistical significance of the data obtained.

Response 3: Thank you for your comment. We partially agree with your comment. As only data for proximate compositional analysis was not with the statistical significance, however, we have analyzed the microbiological data using statistical significance. Since we have pooled different subsets of samples and analyzed them only once for proximate compositional analysis, but we were more concerned about the microbial diversity analysis. We used the proximate data as the supportive data for microbiological studies. The microbial data were processed using statistical significance. The processed the data that included data integrity check, data filtering, and data normalization by total sum scaling (TSS) method and using other statistical methods in QIIME, Microbiome Analyst, STAMP, and PAST software.

Point 4: All received data of the V3-V4 region sequence are not made publicly available.

Thus, the presented materials do not meet the high requirements and standards for articles published in the journal "Microorganisms". I do not recommend the presented manuscript for publication in the journal.

Response 4: Thank you for your suggestion. The data obtained from the study has been analyzed and incorporated in the tables and figures and also as supplementary files. The V3-V4 data is under the process of submission and soon we will submit the data in the database that will be available for the public.

Reviewer 2 Report

This study examines the microbial diversity and composition of three traditional fermented foods from India. Samples from three different dates during the fermentation process were analyzed and compared.
The study is likely of interest to food microbiologists and could yield insights into beneficial and harmful content in these foods.

General remarks:

1. It would have been valuable to include replicates to see how stable the results are, across vendors and seasons for example. Also microbiome of ingredients before fermentation would be interesting. However, this first study of these foods is still valuable.
2. It was unclear if the food samples were collected from the same vendor, same container etc., some more details on the sampling are needed.
3. It was unclear if diversity results in Figure 3 were using the rarefied data and at which depth. If the data were not rarefied to common depth across all samples, the results should be re-computed with a common depth.
4. The network analysis was not explained in Methods and it was unclear what value it adds. Please clarify these results.

Specific comments on the presentation:

1. Please explain "proximate composition analysis" in Abstract/Intro
2. Ranges in abstract (e.g. 94% - 82.72%) are in decreasing order sometimes, why?
3. The use of the word "significant" should be avoided as statistically significant findings were not presented (and difficult to have with only 9 samples)
4. I would change the order in the Introduction to start it  from line 76 with the general introduction, and merge lines 54-75 into the latter part of Introduction to make it more compact and give first a general overview
5. Figure 1, confusing scale/units, Mizoram appears ~0.3KM wide, is KM thousand miles or kilometers? needs to be clarified
6. Some of the methods and databases are cited in the Methods section, citations should be consistently added for all.
7. Were the measurements in Table 1 measured only once? Would it be possible to do this on a few subsets of each sample to have an idea of the variance across sampled material (or better yet across several samples from the same ingredient)
8. Lines 211 and 214 are contradictory w.r.t. pH
9. Line 227, does this correlate with loss of water or some other components?
10. Line 232, is this before quality filtering? What % of reads were lost in filtering? Also what % were unannotated?
11. Figure 2, any quantitative measure to determine if the curves are plateauing? Visually they are still somewhat increasing
12. Figure 4, what is OD1, and only the top 4 are visible in the figure so other labels could be removed
13. Line 313, does OTUs here refer to "sequenced reads" i.e. half the reads were assigned to OTUs labeled Staphylococcus
14. Figure 8, the color scheme is confusing -- either find a way to use the same colors for genera across plots, or perhaps this data can be included as a suppl. table
15. Page 19 is quite verbose, is there a way to summarize the main findings without writing up all the observations -- since there is scarce data, this sounds somewhat like over-interpretation
16. Figure 9, 10 could be panels A and B in one figure, the quality of the image appears somewhat poor

Author Response

Response to Reviewer 2 Comments

Dear Reviewer 2,

We would like to thank you for your valuable comments and suggestions, which have helped to improve the manuscript. We have carefully revised the manuscript as per your suggestions. Please find below a response to the comments provided by you. (Changes incorporated in the revised manuscript are highlighted in yellow)

General remarks comments

Point 1: It would have been valuable to include replicates to see how stable the results are, across vendors and seasons for example. Also microbiome of ingredients before fermentation would be interesting. However, this first study of these foods is still valuable.

Response 1: Thank you for taking the time to assess our manuscript and for giving valuable suggestions. We agree with you that adding more samples from vendors and across seasons as well as including raw material microbiome would be giving more stable and conclusive results. However, we would like to highlight that fermented foods selected in the study are traditional and prepared by spontaneous fermentation process. No previous reports were available about the microbial data for these foods. Therefore, for the first time, we tried to reveal its microbial composition. As the process is natural, we were more concerned about the dominant microbial groups that are involved in fermentation and how their abundance changes during the fermentation process that usually ends after the 7^th day of fermentation. We obtained interesting results that are useful to set future studies for microbial isolation and characterization for their beneficial as well pathogenic properties. Since these three fermented foods (Tuaither, Bekang, and Sa-um) are more famous and consumed by local people on daily basis. Usually, they start their consumption from the 3rd day of fermentation and consume till 7 days of fermentation. Therefore, we selected these foods at three different stages, in order to understand the bacterial composition and changes that occur in their abundances during the fermentation process from the third day to the seventh day. In future, we will definitely work on the aspects suggested by you.

Point 2: It was unclear if the food samples were collected from the same vendor, same container etc., some more details on the sampling are needed.

Response 2: Thank you for your comment. Yes, samples were collected from the same vendor and that were processed in the same container. We discussed with the vendor, asked them to prepare the fermented foods as per their procedure using the same raw material (i.e. sufficient raw material was taken to produce the fermented product in the same container). After raw material processing, they pack the food in small packets on the third day, 5th day, and 7days and provided it to us. So, the raw material and container used for each of the fermented food samples are the same. We have added more in the revised manuscript on sample collection (page no: 3-4, line no: 131-136).

Point 3: It was unclear if diversity results in Figure 3 were using the rarefied data and at which depth. If the data were not rarefied to common depth across all samples, the results should be re-computed with a common depth.

Response 3: Thank you for your comment. We have represented the results using rarefied data. As there was variability in reads among the samples, we processed the data that included data integrity check, data filtering, and data normalization by total sum scaling (TSS) method before the diversity estimation in MicrobiomeAnalyst. The data was normalized at the depth of the minimum library size (sample having the least reads: FBS-3D; 93,857).

Point 4: The network analysis was not explained in Methods and it was unclear what value it adds. Please clarify these results.

Response 4: Thank you for your comment. We have included the co-occurrence network analysis performed using the SparCC method in the method section (page no: 6, line no: 200-203 and the page no: 18, line no: 471-474, 484-485). It was performed to reveal the complex co-operative interaction among the bacterial communities across the fermented samples.

 Response to Specific comments

 Point 1: Please explain "proximate composition analysis" in Abstract/Intro

Response 1: Thank you for your comment, as per your suggestion we have included the parameters that has been analysed in the proximate composition analysis in the abstract (page no: 1, line no: 36-37 and 46-48) and introduction (page no: 3, line no: 113-117).

 Point 2: Ranges in abstract (e.g. 94% - 82.72%) are in decreasing order sometimes, why?

Response 2: Thank you for your comment. The results were written in the range from lowest to highest percentage observed among the three types of samples. We have corrected the order of the range (increasing) for the dominant bacterial phyla in the revised abstract (page no: 1, line no: 40)

 Point 3: The use of the word "significant" should be avoided as statistically significant findings were not presented (and difficult to have with only 9 samples)

Response 3: Thank you for your comment, as per your suggestion, we have removed the word "significant" in the abstract (page no: 1, line no: 41).

Point 4: I would change the order in the Introduction to start it from line 76 with the general introduction, and merge lines 54-75 into the latter part of Introduction to make it more compact and give first a general overview

Response 4: Thank you for your comment. As per your suggestion, we have changed the order in the introduction section. Line numbers 76-104 are at the start of the introduction, line numbers 54-75 at the latter part of the introduction as suggested (page no: 2, line no: 56-83).

Point 5: Figure 1, confusing scale/units, Mizoram appears ~0.3KM wide, is KM thousand miles or kilometers? needs to be clarified

Response 5: Thank you for your suggestion. We have corrected the figure 1 with new scale (page no: 4, line no: 141-144)

Point 6: Some of the methods and databases are cited in the Methods section, citations should be consistently added for all.

Response 6: Thank you for your suggestion. We have included appropriate citation for the methods and databases used in the method section (page no: 5-6, line no: 182, 188, and 192).

Point 7: Were the measurements in Table 1 measured only once? Would it be possible to do this on a few subsets of each sample to have an idea of the variance across sampled material (or better yet across several samples from the same ingredient)

Response 7: Thank you for your comment. Yes, the measurements in table 1 are measured only once. However, we have pooled various subsets of the raw material obtained from the vendor and then analyzed it. Since the same samples that were obtained, would not be available, so it would not be possible to perform with different subsets. Moreover, we were concerned only about the variation in the proximate composition across different fermented samples (as their raw material is very different, we were expecting, pork fat will be with the highest fat content, soybean samples with being with highest protein content). Furthermore, we would like see the impact of proximate content on the microbiological composition.

Point 8: Lines 211 and 214 are contradictory w.r.t. pH

Response 8: Thank you for your comment, as per your suggestion; we have corrected the sentences (page no: 7, line no: 218-221). Among three food types of food samples (Tuaither, Sa-um, and Bekang), Tuaither, Sa-um food samples showed acidic pH (4.04: average value for three different day wise samples). But the Bekang food showed 7.93 average pH values, which is different from the values obtained for Tuaither and Sa-um).

Point 9: Line 227, does this correlate with loss of water or some other components?

Response 9: Thank you for your comment. We have corrected the sentence, as the increase was not significant (page no: 7, line no: 234-236). Also, it does not seem to be correlated with the loss of water, since the moisture content was not much decreased from the 3^rd day to 5^th day and so on.

Point 10: Line 232, is this before quality filtering? What % of reads were lost in filtering? Also what % were unannotated?

Response 10: Thank you for your comment. No sir, this is actually after quality filtering. Around 10% of reads were lost during quality filtration and around 2-3 % were remained unannotated in each sample.

Point 11: Figure 2, any quantitative measure to determine if the curves are plateauing? Visually they are still somewhat increasing

Response 11: Thank you for your comment. Rarefaction curves are a representation of the species richness for a given number of individual samples. The curve looks like started to plateau, although not much but at least suggest a good representation of the microbial community with having good sampling depth.

Point 12: Figure 4, what is OD1, and only the top 4 are visible in the figure so other labels could be removed.

Response 12: Thank you for your suggestion. OD1 is the Candidate Phylum OD1 bacteria (OD1), also known as the Parcubacteria. However, as per your suggestion, we have removed other phyla in the figure 4, and only the top five phyla were shown in the revised figure 4 (page no: 10, line no: 288-291) as per your suggestion.

Point 13: Line 313, does OTUs here refer to "sequenced reads" i.e. half the reads were assigned to OTUs labeled Staphylococcus.

Response 13: Thank you for your suggestion. We have corrected the sentence (page no: 12, line no: 313-315). Around a half of the sequence reads were assigned to OTUs that belong to Staphylococcus in fermented soybeans on the third day of fermentation.

Point 14: Figure 8, the color scheme is confusing -- either find a way to use the same colors for genera across plots, or perhaps this data can be included as a suppl. Table

Response 14: Thank you for your suggestion. We have removed the figure 8 (page no: 15, line no: 395-397) and this data is included in the supplementary table 1 (Table S1) as per your suggestion.

Point 15: Page 19 is quite verbose, is there a way to summarize the main findings without writing up all the observations -- since there is scarce data, this sounds somewhat like over-interpretation

Response 15: Thank you for your suggestion. We have revised the conclusion as suggested based on the results and observations and removed the over-interpretation about the finding of the study (page no: 19, line no: 492-520).

Point 16: Figure 9, 10 could be panels A and B in one figure, the quality of the image appears somewhat poor

Response 16: Thank you for your suggestion. Both the figures 9 and 10 are clubbed in one figure with panel A and B (page no: 17, line no: 457-461).

Reviewer 3 Report

Reviewed article was well written although some minor errors in naming genera (Aceneto..into Acinetobacter), as well as references should be formated according to journal`s guidance for authors. 

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3 Comments

Dear Reviewer 3,

We would like to thank you for your valuable comment for our manuscript. We have carefully revised the manuscript as per your and other reviewer suggestions. Please see below a response to the comments provided by you. (changes incorporated in the revised manuscript are highlighted in red)

Point 1: Reviewed article was well written although some minor errors in naming genera (Aceneto..into Acinetobacter), as well as references should be formatted according to journal`s guidance for authors.

Response 1: Thank you for taking the time to assess our manuscript and for giving valuable suggestions. As per your suggestion, we have corrected the genus name Acenetobacter into Acinetobacter (page no: 18, line no: 475).

Also all the references are formatted according to journal`s guidance for authors (page no: 20-23, line no: 538-666).

Round 2

Reviewer 1 Report

In the work of Deck and others, interesting data were obtained on the composition of microorganisms in fermented foods of Northeastern India. These data are of some interest. And in the submitted version of the manuscript, this aspect of the description has improved. In my opinion, less to the very formulation of the work there are questions that, in my opinion, are important for evaluating and comparing the results obtained with other works.
1. The article does not substantiate the analyzed points of the analysis of the composition of microbial communities. Is it somehow related to creation or some other function? It would be interesting to compare the starting point and how the community changed by the time the microbial community was ready.
2. There are no biological repetitions for each point.
3. It is not clear why there is a section with the analysis of microorganism interaction networks in the results section. It is not possible to carry out an analysis between microorganisms only on the basis of a fragment of 16S rRNA genes. Therefore, all these data are of a controversial nature and, in my opinion, it is logical to place them in the discussion section.
In my opinion, the manuscript still needs to be finalized.

Author Response

Comments and Suggestions for Authors

In the work of Decka and others, interesting data were obtained on the composition of microorganisms in fermented foods of Northeastern India. These data are of some interest. And in the submitted version of the manuscript, this aspect of the description has improved. In my opinion, less to the very formulation of the work, there are questions that, in my opinion, are important for evaluating and comparing the results obtained with other works.

Dear Reviewer 1,

We would like to thank you for your valuable comments and suggestions. We have carefully revised the manuscript as per your suggestions. Please see below a response to the comments provided by you.

Point 1: The article does not substantiate the analyzed points of the analysis of the composition of microbial communities. Is it somehow related to the creation or some other function? It would be interesting to compare the starting point and how the community changed by the time the microbial community was ready.

Response1:Thank you for taking the time to assess our manuscript and for giving valuable suggestions. We agree with you regarding this aspect of the study. However, this is the first report on the microbial diversity of traditional fermented foods prepared by ethnic people of Mizoram. The fermented foods are prepared using a household fermentation process that involved sometimes various constituents and raw products. We aimed to analyze the microbial composition of the ready for consumption food products that were concern more about the effect of consumption of such foods on the health of the local peoples. A few previous studies showed that traditional fermented foods showed the presence of pathogens that can cause health consequences (Keisam et al. 2019, International Journal of Food Microbiology, 296, 21-30; Biswas et al. 2019, Journal of global antimicrobial resistance, 17, 79-83). Furthermore, since the fermented food is ready for consumption from the 3rd day after processing and consumed till the 7th day of fermentation. Therefore, we have selected three sampling times, starting (3rd day, middle: 5th day, and last: 7th day) for analysis. Since various types of raw food constituents are used and processed using a traditional method, it is difficult to correlate the Microbiome of fermented food with the starting material. Various studies on such types of fermented foods reported the microbial composition of the foods that were sampled when they were ready for consumption (Xie et al. 2020, LWT, 129, 109450; Leech et al. 2020, MSystems, 5(6):e00522-20; Jani and Sharma, LWT, 2021, 47, 111578, Tan et al. Food Control. 2021, 121:107641, etc.). Your suggestion is very important for us, we will work on this aspect in the future. Since, this is the first study describing the microbial diversity data of these fermented food products that will be valuable for the attention of the researcher to explore more and understand the microbiological aspect of such traditional foods in such remote areas. So, we request you to give your consideration for this study.

 Point 2: There are no biological repetitions for each point.

Response 2: Thank you for your comment. We agree with you regarding this aspect of the study. Sampling has been done randomly in triplicates (three packets were collected) but from the same vendor and same container. The processed food from three packets was pooled together for each food type and considered as one sample. The sampling details are corrected in the manuscript (Page no: 3 line no: 123-124; page no: 4, line no: 133-137) (highlighted in red). Since, for the first time, we have conducted a study on these food samples in this area, we have included samples only from one vendor that has processed the food in the same container to avoid the variation. Your suggestion is important to analyse the biological replicate for getting more significant results. However, the present study has revealed valuable data that can be useful in getting the attention of the researcher to explore more and understand the microbiological aspect of such traditional foods of such remote areas. So, we request you to give your consideration for this study.

Point 3: It is not clear why there is a section with the analysis of microorganism interaction networks in the results section. It is not possible to carry out an analysis between microorganisms only on the basis of a fragment of 16S rRNA genes. Therefore, all these data are of a controversial nature and, in my opinion, it is logical to place them in the discussion section.

Response 3:Thank you for your suggestion, using interaction network analysis, we were looking for a co-occurrence pattern of microbial taxa among these three different types of food samples. Network inference is possible by calculating the Spearman's rank correlations between OTUs of each sample set. However, as per your suggestion, we have removed this data from the method and result section of the manuscript. (Page no: 5, line no: 199; page no: 17, line no: 464).