Microbiome and Volatile Metabolic Profile of Acetic Acid Fermentation Using Multiple Starters for Traditional Grain Vinegar

Round 1

Reviewer 1 Report

Line77-78 this sentense is misexpressed

Line91 AAB adjusted to OD₆₆₀=0.5?

line95 the purpose to supplemented with 5% alcohol

• Line100-108 what is post-inoculation ？

• Line181 The isolates underwent an induction period until day 3 of fermentation?
• Table 2, 3 ,4 : the significance difference was incorrectly labeled
• Line233  How is fermentation efficiency defined?
• Line246-253 the logic is confused
• Line 257 why was 27 days chosen as the fermentation end point?
• Line 265-271 Result description confusion
• Line285-286 the  growth increased 7–fold? wrong expression
• Line363-364 wrong expression
• Line370-371  wrong expression
• Line387-400 Description is too long
• Line 409-410 adjusted to 3.6
• Line421-440 Description is too long
• Conclusion should be in brief and result oriented
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• The font size of the full-test figure needs to be adjusted
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Author Response

Response letter to the comments from the reviewer #1

◇ Journal: Fermentation

◇ Manuscript ID: fermentation-2332667

◇ Title: Microbiome and Volatile Metabolic Profile of Acetic Acid Fermentation using Multiple Starters for Traditional Grain Vinegar

Dear Editor,

We would like to express our gratitude to the reviewers for their opinions and for a fair evaluation of this thesis. The reviewers' comments and their responses are attached as follows. The appropriate changes made in the revised manuscript are indicated in red. In addition, the manuscript was edited in American style by a professional English proofreading company.

1. Does the introduction provide sufficient background and include all relevant references? : Must be improved

→ As suggested, we have revised and added an introduction of the necessity of multiple starters and examples of its use in the wine industry in the Introduction section of the manuscript (lines 46–55).

2. Are all the cited references relevant to the research? : Can be improved

→ We believe that in our manuscript, we have used appropriate references to provide evidence to support our claims. Several references have been added in the Introduction and Discussion sections [4, 11].

3. Is the research design appropriate? : Can be improved

→ Following the advice of the reviewer, several items have been added or deleted to aid the reader’s understanding.

4. Are the methods adequately described? : Can be improved

→ Details that may confuse the reader have been specifically modified.

5. Are the results clearly presented? : Can be improved

→ Please see the responses to the comments below.

6. Are the conclusions supported by the results? : Can be improved

→ Please see the responses to the comments below.

Comments and Suggestions for Authors

1. Line77-78 this sentense is misexpressed

→ The sentence on lines 87-88 was modified for clarity.

2. Line91 AAB adjusted to OD660=0.5?

→ This means that the number of bacteria was constantly adjusted using the absorbance (line 101–102).

3. line95 the purpose to supplemented with 5% alcohol

→ Acetic acid bacteria are known to produce acetic acid by consuming ethanol (line 207–208), and the efficiency of acetic acid production decreases due to stress at ethanol concentrations above 10%. Therefore, ethanol concentrations of 5% and 8% were set.

4. Line100-108 what is post-inoculation?

→ In post–inoculation, the following strain was inoculated on day 0 of fermentation, and the former strain was inoculated on day 12 of fermentation. If strains A and B were used, in the pre-inoculation, strain A was inoculated on day 0 of fermentation and strain B was inoculated on day 12. In post-inoculation, in contrast to pre-inoculation, strain B was inoculated on day 0, and strain A was inoculated on day 12. This example was added to the manuscript (lines 117–122).

5. Line181 The isolates underwent an induction period until day 3 of fermentation?

→ Acetic acid bacteria produce acetic acid by consuming ethanol while growing. Therefore, the result of not showing an increase in acidity and growth rate for 3 days after inoculation can be interpreted as an isolate undergoing an induction period in which it adapts to the environment for growth. This has been corrected in the manuscript (lines 196–199).

6. Table 2, 3, 4: the significance difference was incorrectly labeled

→ Significant differences between the main text and supplementary tables were extensively revised following the reviewer’s meticulous advice. A column is displayed for each parameter.

7. Line233 How is fermentation efficiency defined?

→ The fermentation efficiency is defined on line 130.

8. Line246-253 the logic is confused

→ Section 3.2 describes the selection of multiple acetic acid bacteria candidates based on the characteristics of a single acetic acid bacteria previously identified. Some modifications have been made to aid the reader's understanding (line 266-271).

9. Line 257 why was 27 days chosen as the fermentation end point?

→ When the acetic acid bacteria consume all the ethanol present, the produced acetic acid is volatilized and the titratable acidity decreases. In this study, it was confirmed that titratable acidity decreased after 27 days of fermentation. Therefore, day 27 was set as the end point of fermentation.

10. Line 265-271 Result description confusion / Line285-286 the growth increased 7–fold? wrong expression

→ We agree that this information can be confusing to the reader, and we have removed the growth information to focus on increasing titratable acidity.

11. Line363-364 wrong expression

→ Since quantitative observation of volatile compounds is difficult in heat maps, we focused on temperature and strain-specific profiling (line 379–380).

12. Line370-371 wrong expression

→ In reference 48, the author explained the mechanism of conversion of ethanol to acetaldehyde to acetic acid (lines 386–388).

13. Line387-400 Description is too long

→ Non-essential sentences have been deleted (line 403–414). However, since we wanted to present the difference in volatile components according to fermentation temperature and strain, the description based on temperature was retained.

14. Line 409-410 adjusted to 3.6

→ Thank you for your meticulous observation; this has been modified to 3.6.

15. Line421-440 Description is too long

→ Some lengthy sentences were removed (lines 435–488).

16. The font size of the full-test figure needs to be adjusted

→ To improve readability, the font size of the figure has been increased.

The comments made by the reviewer have been corrected. Thank you for your detailed comments which have helped improve the completeness of this manuscript. We believe that these modifications have strengthened the manuscript and hope that the revised manuscript is suitable for publication in Fermentation.

Sincerely,

Soo-Hwan Yeo

Reviewer 2 Report

In the manuscript entitled "Microbiome and Volatile Metabolic Profile of Acetic Acid Fermentation using Multiple Starters for Traditional Grain Vinegar" the authors evaluate the effect of multiple acetic acid bacteria on the quality of traditional vinegar. The use of fermented acetic acid starter can help standardize the taste and quality of the final product and control the diversity of the microorganism community. It is proposed to apply several acetic acid bacteria with acid-producing and flavor-producing abilities to prepare traditional vinegar.

For my part, this article could be published after making some  changes.

Paragraphs with examples can be added in the introduction for a better understanding.

Materials and methods are properly exemplified and can be replicated.

The results and discussions are well organized and easy to follow.

Table 2 and 4 can be presented horizontally to be more readable.

Tables 2, 3 and 4 are presented adequately, but I recommend improving the discussion of the results in these tables.

I recommend improving the discussions in sections 3.2, 3.3, 3.4., 3.5. It is not enough to present the results.

Author Response

Response letter to the comments from the reviewer #2

◇ Journal: Fermentation

◇ Manuscript ID: fermentation-2332667

◇ Title: Microbiome and Volatile Metabolic Profile of Acetic Acid Fermentation using Multiple Starters for Traditional Grain Vinegar

Dear Editor,

We would like to express our gratitude to the reviewers for providing their opinions through a fair evaluation of this manuscript. The reviewers’ comments and their responses are attached as follows. The changes made in the revised manuscript are indicated in red. In addition, the manuscript was edited in American style by a professional English proofreading company.

1. Does the introduction provide sufficient background and include all relevant references? : Can be improved

→ Please see the responses to the comments below.

2. Are all the cited references relevant to the research? : Yes

→ (No instructions)

3. Is the research design appropriate? : Yes

→ (No instructions)

4. Are the methods adequately described? : Yes

→ (No instructions)

5. Are the results clearly presented? : Can be improved

→ Please see the responses to the comments below.

6. Are the conclusions supported by the results? : Yes

→ (No instructions)

Comments and Suggestions for Authors

1. Paragraphs with examples can be added in the introduction for a better understanding.

→ As suggested, we revised and added an introduction regarding the necessity of multiple starters and examples of its use in the wine industry in the Introduction section of the manuscript (lines 46–55).

2. Table 2 and 4 can be presented horizontally to be more readable.

→ The tables have been presented horizontally to improve readability.

3. Tables 2, 3 and 4 are presented adequately, but I recommend improving the discussion of the results in these tables

→ We have added some suggestions to support our argument (lines 214–216, 237–239, 257–259).

4. I recommend improving the discussions in sections 3.2, 3.3, 3.4., 3.5. It is not enough to present the results.

→ We added a discussion to sections 3.5 and 3.6 in addition to the changes made in response to comment 2.

According to the comments made by the member, the manuscript has been corrected. Thank you for your detailed comments to improve the completeness of this manuscript. We believe that these modifications have strengthened the manuscript and hope that the revised manuscript is suitable for publication in Fermentation.

Sincerely,

Soo-Hwan Yeo

Round 2

Reviewer 1 Report

line 104 1% for multiple AAB in a liquid medium,  need to elaborate

line 112 how to control the inoculation rate of 1:1, 1:2 and 2:1

line 117-122 what is the significance of pre-inoculation and post-inoculation?

Line 209 Conclusion needs to be verified

line 281 how to control the inoculation rate of ABCD? based on inoculation volume?

line 371 it seems that 20 ℃ was a better choice than 30 ℃ 

Author Response

Dear Reviewer

Thanks for the report for the new review.
We have amended the text based on the reviewer's response.
We hope our answers fit the reviewer's intent.

line 104 1% for multiple AAB in a liquid medium,  need to elaborate

• The sentence was added in line 104.

line 112 how to control the inoculation rate of 1:1, 1:2 and 2:1

line 281 how to control the inoculation rate of ABCD? based on inoculation volume?

• I prepared 500 mL of medium in a 1 L Erlenmeyer flask. In order to inoculate 1% of multi AAB in 500 mL of medium, 1.25 mL of A-D strains were prepared and mixed to prepare 5 mL, which was then inoculated into the medium. Relevant information has been corrected in lines 103 and 114.

line 117-122 what is the significance of pre-inoculation and post-inoculation?

• Pre- and post-inoculation are just names. It is only a difference depending on which strain of multiple AAB is inoculated first (day 0) and which strain is inoculated later (day 12).

Line 209 Conclusion needs to be verified

• Since AAB increase acidity by consuming ethanol in AAF, the fact that acidity does not increase at 20 ℃ can be interpreted as the result of the growth of microorganisms other than AAB (line 210).

line 371 it seems that 20 ℃ was a better choice than 30 ℃ 

• On the 18th day of fermentation, 20 ℃ was better than 30 ℃, but I focused on the 27th, the end of fermentation. To avoid misunderstanding, the end of fermentation is mentioned in the manuscript (line 372).

Thank you and Best Regards,

Haram Kong

Reviewer 2 Report

The authors responded to all my comments

Author Response

Dear Reviewer,

Thank you for getting back to me and the report for review.
In Round 1, I made major revisions to the manuscript according to the reviewer's advice, and in Round 2, I edited a few words.
Many thanks for your message.

Thank you and best regards,
Haram Kong