Exploration of Microbial Community Diversity and Bioactive Substances during Fermentation of Mulberry Jiaosu, an Edible Naturally Fermented Mulberry Product

Round 1

Reviewer 1 Report

• A brief summary- The study presents and discusses the fluctuation in microbial diversity during the laboratory fermentation of mulberry Jiaosu as well as in the content of bioactive microbial metabolites and substrates. As a novelty and the strength of the study authors highlight the use of next-generation sequencing for the microbial diversity analysis in this specific product. Indeed, the study in a short and concise manner describes a wealth of data on this interesting, potentially probiotic product. All methods used by the authors seem adequate. 
• General concept comments- In the opinion of the reviewer, the article requires some changes, such as highlighting the impact of obtained results, enriching the discussion, and correcting grammar and typos throughout the manuscript. Nevertheless, the topic itself is certainly interesting and the study was well designed, so overall the manuscript is worth publication after some changes, in regards to style as well as certain substantive issues (mentioned in specific comments), are made.
• Specific comments 
• Title- Would be clearer to the international readers if you had added a few words on what Jiaosu is.
• Abstract- Is missing a brief explanation of the fermentation process. Not all interested readers will be familiar with it. Please outline in the first sentence if the product is fermented by wild strains and where it is manufactured.

Sentence in lines 21-22 please rephrase so it does not start with a number (similarly through the text).

Please write what kind of influence Zygosaccharomyces has on bioactives.

• Introduction- here you should definitely explain the term Jiaosu and add more detailed information on where the mulberry is cultured, because this will have an impact on the biodiversity of microbiota in the product. Furthermore, it would be of interest to the readers (such as myself) to find out how the Jiaosu production process looks like. Is it uniform through the production plants? How this may impact the biodiversity of the microorganisms in the fermented product?

Sentence in lines 48-50- what do you mean by the instability of product quality, please explain.

Sentence in lines 65-67- it would be better to either split this in two sentences as chemical and molecular biology analyses are separate topics or refrain from focusing on the methods (although NGS you should mention for coherence with the previous paragraph) and instead focus on the aim of your study.

• Materials and methods- section 2.2- please explain where was the commodity purchased and whether during the fermentation you have tried to mimic the fermentation conditions used industrially/ traditionally (especially in relation to the fermentation temperature).

Section 2.3- line 87- change detection to determination, further in this section add a reference or describe the method you have used for the acid-base titration

Section 2.4-line 91- change title to determination of organic acid content (similar change to be made to titles of sections 2.5 and 2.6), further in this section, please mention the make and model of the used instruments including the UV detector (similarly in section 2.5 and 2.6, or consider merging into one section on HPLC analyses).

• Results and discussion- lines 161-173- as you do not give table S1 in the main text, please explain what organic acids were in the highest concentration at the beginning and at the end of the fermentation process. Similar comment to amino acids (results in lines 174-182), were these top 3 at the end of fermentation also the most concentrated at its beginning? In relation to amino acids, please also comment on the content of protein and free amino acids in the fruits themselves.

Lines 183-194- please also state what polyphenols were most abundant at the beginning of the fermentation process

Line 205- PCA is more commonly used to abbreviate Principal Component Analysis, please use PCoA instead

Sections 3.1, 3.2, and 3.5 have very scarce discussion included- I am sure that authors have expected reported results based on the literature data and the experience, please explain how your results compared to these expectations and was your Jiaosu sample similar to commercial/ traditional products, were the microbial metabolites attributed to certain genera in similar products?

Lines 219-234- it would be neater to first describe the results and then discuss them as the content seems confusing. Also, you do not really give any information on the sensory characteristics of your product, hence only related information should be included in the discussion.

Lines 274—291- if you are talking about genera being correlated with the metabolites or other substances, please explain what you mean by correlated (statistically significant correlation or some cut-off point for the correlation coefficient value), then please wherever you mention the number of correlating genera, also list these genera because all information are in the supplementary files. 

Line 303- change on the whole to overall

• Conclusions- Please include recommendations for further research and some information on the impact of your results- shortly state the importance of the bioactive substances and focus on those that increased during the fermentation process. Then according to this information outline which microbes would be most important based on your results. To avoid repetitions consider formatting the rest of the text in this section.
• Figures- 1 and 2 are well thought, but the lines are not very clear, please improve the quality for the print version. Why do you report all results per volume except for amino acids? Are amino acids calculated for the fruit content or the beverage weight? Please explain in the caption.

Figures 3-5- very small font, it cannot be read

• Figure captions- please mention the number of replicates for reported results (Figure 1, 2 and 3)
• References- the list contains current literature, most citations from the last 5 years, but authors could use some more references for the discussion of these numerous results.
• Supplementary material- Since the fermentation trends are an important element to show, I would rather present data in Tables S1-4 as line graphs with up to 4 data series on each. The information on some figures (S2b and S3) cannot be read, please make the font and the figures larger. In addition, it is not clear why the authors decided to show in the main paper data for fungi in Figures 3 and 5 and did not include data for bacteria. Consider replacing Figure 3b with S2b and Figure 5b with S3a. This way you can make the font larger and more readable in the manuscript while in the supplement you should enlarge figures to enable readability of the font as well.

There are many typos and grammar issues through your manuscript, but in majority these do not impact the meaning of your sentences greatly. In contrary cases I have advised changes in Comments and Suggestions for Authors part of the review.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Exploration of microbial community diversity and bioactive substances during fermentation of mulberry Jiaosu” explores changes of bioactive substances and microbial diversity during fermentation for 80 days of mulberry Jiaosu. The manuscript is rather interesting from the scientific point of view, however, there are some changes that needs to be made. The biggest concern is English language. There are a lot of grammar and spelling mistakes, therefore English language should be looked through the whole manuscript. In the current form the writing is not acceptable. Also, in the methods part for the detection of organic acids description, gradient of the mobile phase should be provided and time of the analysis too. Likewise, the name of the high performance liquid chromatography manufacturer should be provided. The same applies for the detection of amino acids part.

There are a lot of grammar and spelling mistakes, therefore English language should be looked through the whole manuscript. In the current form the writing is not acceptable.

Author Response

Please see the attachment.

Author Response File: Author Response.docx