Influence of Inoculating Saccharomyces cerevisiae and Levilactobacillus brevis on the Quality of Fermented Large Yellow Croaker (Larimichthys crocea): Biogenic Amines, Volatile Components, and Microbial Communities Changes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The correct name of the used strain is Levilactobacillus, not Levilevilactobacillus.  Line 19. Please also avoid grammatically untypical structures like: positively correlation. This should be better „should be “positive correlation”

In the introduction, the flow is uneven; at times it reads like a literature dump rather than a progressive argument. The novelty statement could be stronger: emphasize why this work adds more than prior LAB/yeast inoculation studies. Too many references are clustered without sufficient critical analysis of prior studies.

Some methodological descriptions are verbose but miss critical details (especially sequencing depth/coverage, rarefaction thresholds for diversity analysis).

Results section sometimes blends interpretation with reporting; should separate observations from discussion (for example line 220 following; line 249 following; line 274 following). If the authors decide to seperate results and discussion instead of presenting it combined, the separation needs to be strict.

Some claims are overgeneralized without sufficient statistical basis—for example, yeast "promote" LAB growth (line 375).

Please revise Figure 6 in the way, that organism names are written in italic.

Overstates conclusions—claims of “improve the flavor and safety” are supported, but mechanistic understanding remains preliminary. The authors should acknowledge limitations: short fermentation, limited replicates, strain specificity.

Figures and tables caption are informative but too long and at times redundant

Overall: The work is incrementally novel rather than groundbreaking. It extends prior research to a new substrate (croaker) and combines microbial ecology with safety and flavor profiling. However, because similar approaches and outcomes have been published, the authors must highlight species specificity, industrial relevance, and comprehensive multi-parameter assessment as your unique contributions. Papers which are already published in similar topics are for example: https://doi.org/10.3390/foods11182756; https://doi.org/10.3390/foods13203297; doi:10.1111/ijfs.17392

 

Comments on the Quality of English Language

Please also avoid grammatically untypical structures like: positively correlation. This should be better „should be “positive correlation”

Author Response

Dear Reviewers,

Thank you for your contributions to the improvement of this manuscript (foods-3905670), entitled "Influence of Inoculation Saccharomyces cerevisiae and Levilactobacillus brevis on the Quality of Fermented Large Yellow Croaker (Larimichthys Crocea): Biogenic Amines, Volatile Components, and Microbial Communities changes." In light of the

Reviewers' comments and suggestions, we respond to your comments as follows:

Reviewer1:

Comments 1: The correct name of the used strain is Levilactobacillus, not Levilevilactobacillus. Line 19. Please also avoid grammatically untypical structures like: positively correlation. This should be better, should be “positive correlation".

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have:

• Corrected the genus name to “Levilactobacillus” throughout the manuscript, please check line 3, 19.
• Replaced “positively correlation" with “positive correlation" please check line 29.

Comments 2: In the introduction, the flow is uneven; at times it reads like a literature dump rather than a progressive argument. The novelty statement could be stronger: emphasize why this work adds more than prior LAB/yeast inoculation studies. Too many references are clustered without sufficient critical analysis of prior studies.

Response 2: Thank you for this suggestion. We have restructured the Introduction to improve logical flow and critical analysis, please check line 68-85.

Comments 3: Some methodological descriptions are verbose but miss critical details (especially sequencing depth/coverage, rarefaction thresholds for diversity analysis).

Response 3: Thank you for this suggestion. We have:

• Added sequence processing and analysis information, please check line 185-187.
• Streamlined DNA extraction procedure, please check line 171-175.

Comments 4: "Results section sometimes blends interpretation with reporting; should separate observations from discussion... If the authors decide to separate results and discussion, the separation needs to be strict."

Response 4: Thank you for your comment. We have restructured the text to distinguish observations from discussion, please check line 239-241, line 269-270, line 296-297.

Comments 5: "Some claims are overgeneralized without sufficient statistical basis—for example, yeast 'promote' LAB growth (line 375)."

Response 5: We have toned down overgeneralized claims, please check line 392-395.

Comments 6: "Please revise Figure 6 so organism names are written in italic."

Response 6: Thank you. We have italicized all genus names in Figure 6.

Comments 7: "Overstates conclusions... The authors should acknowledge limitations: short fermentation, limited replicates, strain specificity."

Response 7: Thank you for this suggestion. We have revised the conclusions, please check line 476-480.

Comments 8: "Figures and tables captions are informative but too long and at times redundant."

Response 8: We have shortened all captions, please check line 221-223, line 329-330.

Comments 9: "Overall: Highlight species specificity, industrial relevance, and comprehensive multi-parameter assessment as unique contributions."

Response 9: Thank you for your comments. We have revised the conclusions, please check line 82-84, line 88-90, line 92-95.

Comments 10: The English could be improved to more clearly express the research.

Response 10: The manuscript has undergone professional language editing by LanSci, and a certificate is attached.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents an interesting subject, and the working plan was well designed and executed. However, the authors did not thoroughly explore the results, and the manuscript text requires substantial improvements.

 

Methodology

 

The methods codified as national standards methods (GBXXX) must be described in the manuscript text. Those methods are not widely known.

The methodology of CG analysis needs to be revised. It is not possible to carry out VFC with the column at 60 ^oC

The purpose of DNA extraction is unclear to the reader in the materials and methods section.

Figure 3 needs improvement. It is not possible to read the information; the letter is too small. The same comment applies to Figure 5.

 

Results

The results are only presented and commented on without a deeper analysis and explanation for all results. Discussion must be added and supported in this section, which should be named Results and Discussion.

The manuscript only presented the results of microbial population and volatile compounds determined in separate analyses. The experiments carried out do not allow for the correlations presented in Figure 6. Isolated analyses alone (i.e., a list of volatile compounds and a list of microbial populations, separately) do not provide the necessary foundation for constructing or interpreting this correlation heatmap. The fact that "Acetone exists" and "Aeromonas exists"). This figure lacks a biochemical foundation: Correlation only makes sense if there is a biological basis, which was not found in the manuscript text. Its purpose is to correlate the amount of a microorganism with the amount of a chemical compound it is known to produce. It is microbial biochemistry that dictates the "organism → compound" relationship.

Author Response

Dear Reviewers,

We are deeply appreciative of your efforts in helping to refine our manuscript (foods-3905670), whose title is "Influence of Inoculation Saccharomyces cerevisiae and Levilactobacillus brevis on the Quality of Fermented Large Yellow Croaker (Larimichthys Crocea): Biogenic Amines, Volatile Components and Microbial Communities changes" Having considered the Reviewers' comments and suggestions, we respond to your comments as follows.

Reviewer2:

Comments 1:"The methods codified as national standards methods (GBXXX) must be described in the manuscript text."

Response 1: We have supplemented the experimental methods and added additional reference literature, please check line 138-141, line 143-155.

Comments 2:"The methodology of GC analysis needs to be revised. It is not possible to carry out VFC with the column at 60 °C."

Response 2: Based on our assessment, the column temperature setting is considered appropriate. Multiple studies indicate that that GC-IMS instruments of the same model as ours consistently employ a column temperature of 60°C for sample analysis, as seen in: https://doi.org/10.3390/foods14030431; https://doi.org/10.1016/j.lwt.2021.111585; https://doi.org/10.1016/j.fochx.2024.101126.

Comments 3:"The purpose of DNA extraction is unclear."

Response 3: We have clarified the DNA extraction purpose, please check line 170-171.

Comments 4:"Figure 3 needs improvement. It is not possible to read the information; the letter is too small. The same comment applies to Figure 5."

Response 4: We have redesigned Figures 3 and 5 with enlarged fonts/legends and higher resolution.

Comments 5:"The results are only presented and commented on without a deeper analysis and explanation for all results. Discussion must be added and supported in this section, which should be named Results and Discussion."

Response 5: We have implemented revisions throughout the manuscript and expanded the discussion, please check line 213-216, line 270-273, line 305-307, line 385-388, line 451-455.

Comments 6:"The experiments do not allow for the correlations in Figure 6... Correlation only makes sense with a biological basis."

Response 6: Thank you for your insightful comments regarding Figure 6. We respectfully clarify the rationale behind our correlation analysis as follows:

The heatmap in Figure 6 aims to identify potential relationships between microbial genus and volatile compounds in an untargeted exploratory context – a common first step in complex systems (e.g., fermented foods, environmental microbiomes). Such analyses do not imply direct biochemical causation but serve to prioritize hypotheses for future mechanistic validation，for example：

Jiang et al. used Pearson correlations between 16S rDNA data and GC-IMS volatiles without prior biochemical evidence to shortlist candidate interactions. (https://doi.org/10.1016/j.fochx.2024.101126).

Xu et al. explicitly stated: Correlation networks identified putative microbial contributors to metabolite dynamics. (https://doi.org/10.3390/foods13203297)

We have also discussed the results of the correlation analysis in the context of mechanistic evidence from the literature, please check line 440-441, line 445-447, line 451-455.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This revised version represents a clear improvement over earlier drafts. The manuscript is now well structured, scientifically sound, and easier to follow. The writing has become more fluent. Figures and tables are logically presented, and methodological details are thorough enough for reproducibility.

 

While the introduction is concise, it could briefly highlight what gap in industrial-scale fermentation this study specifically fills

The studys discussion nicely links microbial activity to metabolite changes, but a short paragraph comparing findings with other fermented fish systems (e.g., Suanyu, Zhayu) in terms of sensory outcomes would strengthen contextualization.

The conclusion is strong overall, but could mention potential for starter culture standardization or application to pilot-scale fermentation explicitly.

Author Response

Comments 1: While the introduction is concise, it could briefly highlight what gap in industrial-scale fermentation this study specifically fills

Response 1: Thank you for this suggestion. We have revised the introduction, please check line 91-94.

Comments 2: The studys discussion nicely links microbial activity to metabolite changes, but a short paragraph comparing findings with other fermented fish systems (e.g., Suanyu, Zhayu) in terms of sensory outcomes would strengthen contextualization.

Response 2: In terms of sensory outcomes , we have compared them with other fermented fish products, please check line 241-242, 259-262, 307-313, 386-387, 453-454, 460-462.

Comments 3: The conclusion is strong overall, but could mention potential for starter culture standardization or application to pilot-scale fermentation explicitly.

Response 3: Thank you for this suggestion. We have revised the conclusion, please check line 500-502.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Although the manuscript was improved, I still have concerns regarding the heat map and its interpretation. 

Heat maps allow the visualization of patterns and the generation of hypotheses, but do not establish direct causal relationships. They show correlations that may lack a direct biological basis and can be confounded by uncontrolled factors, leading to unsupported associations. Rigorous interpretation requires biological knowledge, functional validation, and integration with pathway analysis, biological network modeling, and experimental confirmation. Heat maps represent a correlational picture affected by temporal and environmental variations and complex microorganism-metabolite interactions.

Some correlations are metabolically plausible. Others, such as the strong correlation between Lactobacillus and aldehydes (heptanal) or secondary ketones (acetone), are indirect. Lactobacillus does not produce heptanal or acetone. These correlations indicate indirect ecological or temporal relationships influenced by factors such as fermentation stage or activity of other microorganisms and enzymes. Thus, correlations on heat maps should be viewed with caution, as they do not account for microbial metabolic contribution. Mentioning these limitations and potential confounding variables is essential to avoid misinterpretation and unreliable conclusions. Thus, the manuscript text must mention the limitations of using heat maps without confirmation of a biological basis.

Author Response

Comments : Limitations of heatmap correlation analysis.

Response : We appreciate this critical insight. The following revisions were implemented:

• Enhanced mechanistic analysis of microbe-metabolite relationships, please check line 447-456.
• Explicit limitations of heatmaps, please check line 474-476.
• Added future validation workflows, please check line 476-479.

Author Response File: Author Response.pdf