Physiological, Genetic, and Fermentative Traits of Oenococcus oeni Isolates from Spontaneous Malolactic Fermentation in Koshu Wine

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The core of malolactic fermentation is malolase (MLE), and its activity directly determines the success or failure of MLF. Understanding its mechanism is helpful for screening superior strains or optimizing fermentation processes, which is particularly crucial in low-ph wines such as Koshu. The study is a valuable contribution but requires deeper mechanistic data (enzyme kinetics, multi-timepoint genomics) and broader physiological profiling to justify industrial potential. Moderate revisions needed before final acceptance.

1. ppm better use mg/L 
2. The study only examines mleA and cfa expression on Day 2, which is insufficient to capture dynamic changes during MLF. A time-course analysis (e.g., Days 0, 2, 5, 7) should be conducted to track gene expression trends alongside malolactic enzyme (MLE) activity measurements. Additional genomic or transcriptomic data (e.g., whole-genome sequencing, stress-related gene networks) would provide deeper insights.
3. Environmental factors (e.g., pH, ethanol, SO₂) influence gene regulation and enzyme function, yet no quantitative enzyme activity data is provided. HPLC or spectrophotometric assays for MLE activity under varying conditions would strengthen mechanistic insights.
4. The study focuses narrowly on MLF-related traits (pH/ethanol tolerance, carbon utilization). For industrial applicability, additional physiological tests are needed:Nitrogen source utilization (e.g., arginine metabolism, which affects MLF efficiency)；Metabolic byproducts such as acetic acid, diacetyl, biogenic amines; growth kinetics (lag phase, doubling time under stress). Without this data, the strains’ suitability for commercial use remains speculative.
5. Error bars are missing in Figures 2–4.
6. The authors note that MLF did not occur in stainless steel tanks but was active in barrels. This critical observation lacks explanation.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Misa Otoguro and colleagues describes research and the results related with the use of different Oenococcus strains isolated from Koshu grapes to promote the malacotic fermentation.

The research is interesting and relevant.

After a careful analysis, I have only a few comments/suggestions for this manuscript.

Line 32: I suggest removing “wine microbiology” as a keyword.

Line 91-101: Authors used a previous methodology already developed and published. If yeas, I suggest introducing the reference.

Line 156-162: For he HPLC methodology/conditions used, a reference is missing.

Line 200. “p” italic form.

Line 212: Introduce the SO₂ concentration added.

For figures 2, 3 and 4, to compare the values (for example L-malic acid concentration in figure 2) at different pH levels, should be important to introduce a statistical analysis using ANOVA.

Line 298: Total or free SO₂ values ?

Line 301: similar previous comments.

Line 326-330: But these differences were statistical different?

Table 3: A legend as footnote for “+” and “-“ is missing.

Line 396: “high ethanol”, Authors should be careful with this designation, as 13% ethanol cannot be considered high in wines today.

Line 415-420: Very interesting data that should be deepened in future research.

Line 443-445: This is a negative point. However, authors should compare with commercial LAB.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Please refer to the attached document for detailed comments.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors revised the manuscript and responded to the reviewer's comments.  I still suggest current manuscript could be further improved and strengthen with more genomic and related data. 

Author Response

Thank you very much for taking the time to review this manuscript again. Please find the detailed responses below and the corresponding revision in track changes in the re-submitted files.

Comments 1: The authors revised the manuscript and responded to the reviewer's comments.  I still suggest current manuscript could be further improved and strengthen with more genomic and related data.

Response 1:　Thank you for pointing this out. We fully agree that such information would provide deeper insights into the genetic basis of the observed phenotypes and strengthen the scientific value of our study. However, due to current time constraints and the scope of the present work, we were unable to conduct related genomic analyses for the isolated strains.

We consider this an important direction for future research and plan to perform comprehensive genomic and functional analyses to further characterize these indigenous Oenococcus oeni strains. We have added a statement to this effect in the revised manuscript (Discussion section, line 434).

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“Furthermore, whole-genome sequencing and comparative genomics could provide deeper insights into the genetic determinants of stress tolerance and the unique phenotypes observed in the Koshu-derived O. oeni strains. While these genomic analyses were beyond the scope of the present study due to time constraints, we recognize their importance and intend to address them in future investigations.”

Reviewer 3 Report

Comments and Suggestions for Authors

The author has made remarkable revisions to the manuscript to make it eligible for publication.

Author Response

Thank you very much for taking the time to review this manuscript again. Please find the detailed responses below and the corresponding revisions in track changes in the re-submitted files.

Comments 1: The author has made remarkable revisions to the manuscript to make it eligible for publication.

Response 1: Thank you for pointing this out. We have added a statement to this effect in the revised manuscript (Discussion section, line 434) about genomic analysis for O. oeni.

 

“Furthermore, whole-genome sequencing and comparative genomics could provide deeper insights into the genetic determinants of stress tolerance and the unique phenotypes observed in the Koshu-derived O. oeni strains. While these genomic analyses were beyond the scope of the present study due to time constraints, we recognize their importance and intend to address them in future investigations.”