The Exploration of Cordyceps militaris Extract as a Postharvest Preservative for Flammulina filiformis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study presents a robust and well-structured approach to evaluating the efficacy of Cordyceps militaris extract (CME) as a postharvest preservative for Flammulina filiformis. The authors successfully combined physiological, biochemical, and transcriptomic analyses to investigate the effects of the treatment over time. 

The manuscript is well written, the objectives are clear, and the methodology is appropriate for the research question. However, some changes can improve the readability of the article and make it more robust, as shown below.

• L48: Standardize the use of the term "postharvest" (without hyphen) throughout the manuscript.
• L161–164: Justify the browning index formula or provide a literature reference.
• L215–219: Clearly specify the number of statistical replicates for all analyses.
• L215–219: Specify the statistical test adopted to indicate the difference between treatments.
• L235: In Figure 1A, the specific epithet "coli" (in E. coli) should be written with a lowercase letter.
• L249: In Table 1, write the name of the bacteria correctly: Escherichia coli.
• L279: In scientific names in Table 4, "sp." should not be italicized.
• L563–564: Do not separate the value from the percentage symbol.
• L601–603: Figure 7 should be presented in the Discussion section instead of the Conclusion.
• L621–710: Review all references so that scientific names are written correctly (genus in capital letters and specific epithet in lower case letters, such as Lentinula edodes)

Author Response

Comments 1: L48: Standardize the use of the term "postharvest" (without hyphen) throughout the manuscript.

Response 1: Thank you for pointing this out. We deleted all hyphens within “post-harvest” throughout the manuscript (L48, 341, 351).

 

Comments 2: L161–164: Justify the browning index formula or provide a literature reference.

Response 2: We provided a relevant literature reference for support (L162 [12]).

 

Comments 3: L215–219: Clearly specify the number of statistical replicates for all analyses.

Response 3: We stated the number of statistical replicates used in all analyses within the revised manuscript (L225-226).

 

Comments 4: L215–219: Specify the statistical test adopted to indicate the difference between treatments.

Response 4: Thank you for reminding. We specified the adopted statistical test in the revised manuscript (L223-224).

 

Comments 5: L235: In Figure 1A, the specific epithet "coli" (in E. coli) should be written with a lowercase letter.

Response 5: Thank you for your careful review. We corrected the writing of “E. coli” in Figure 1A (L242).

 

Comments 6: L249: In Table 1, write the name of the bacteria correctly: Escherichia coli.

Response 6: Thanks. We rewrote the name as “Escherichia coli” in Table 1 (L256).

 

Comments 7: L279: In scientific names in Table 4, "sp." should not be italicized.

Response 7: Thanks. We revised Table 4, ensuring that “sp.” was no longer italicized (L286).

 

Comments 8: L563–564: Do not separate the value from the percentage symbol.

Response 8: Thanks. We removed all spaces between the value and the percentage symbol (L42, the part of L563-564 was moved to L294).

 

Comments 9: L601–603: Figure 7 should be presented in the Discussion section instead of the Conclusion.

Response 9: Agree. We moved Figure 7 to the new Results and Discussion section (L588).

 

Comments 10: L621–710: Review all references so that scientific names are written correctly (genus in capital letters and specific epithet in lower case letters, such as Lentinula edodes)

Response 10: Thank you for your thorough review of the references. We corrected all scientific names in references (L641-729).

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This manuscript presents a comprehensive and well-structured investigation into the use of Cordyceps militaris extract (CME) as a natural preservative for Flammulina filiformis (enoki mushroom). The authors convincingly demonstrate the antimicrobial and antioxidative efficacy of CME through a combination of phenotypic assessments and molecular analyses. The study is timely and relevant, addressing pressing needs in the domains of postharvest biology, sustainable food preservation, and the valorization of natural bioactive compounds. Overall, the work makes a significant contribution to the scientific literature.

However, several sections—particularly those detailing the transcriptomic and quantitative real-time PCR (qRT-PCR) analyses—would benefit from greater methodological transparency and interpretative depth to fully support the conclusions drawn.

Please find all my suggestions and comments provided directly within the manuscript file, along with an accompanying supplementary table for clarity.

Comments for author File: Comments.pdf

Author Response

Comments 1: Lines 172-174: Please state whether you used the manufacturer's protocol when performing these tests or if you introduced any modifications. If so, please specify them.

Response 1: Thanks. We followed the manufacturer's protocol for all tests here without modification. All procedures were performed according to the standardized kit instructions to ensure reproducibility as clarified in Lines 173-175.

 

Comments 2: Lines 177-178: Please state which protocol did you use for total RNA extraction; did you use liquid N?

Response 2: Thanks. The extraction of total RNA was performed following the protocol of the manufacturer, and liquid N₂ was used to grind the samples. All above was stated in Lines 180-182.

 

Comments 3: Lines 179-182: Please describe the methodology in more detail, or refer to specific protocols.

Response 3: Thanks. We added a relevant reference to specify the protocol (Line 185, Reference[14]).

 

Comments 4: Lines 204-205: Please specify which fungal genes were the targets of the analysis and provide the rationale for their selection.

Response 4: Thanks. The fungal genes chosen for qPCR were listed in Table S1, and we specified the reason for choosing in Lines 207-212.

 

Comments 5: Lines 207-210: Please specify the protocol, including the PCR reaction setup, temperature conditions, number of cycles, etc.

Response 5: Thanks. We added the qPCR reaction setup and conditions in Table S2 and Table S3 (Lines 214-215).

 

Comments 6: Line 273: Internal transcribed spacer (ITS) region and the 18S ribosomal RNA gene are used as biomarkers for studying fungal communities. ITS region is commonly used as a DNA marker for identifying fungal species, not bacterial? Please explain your statement more precisely.

Response 6: Thank you for highlighting this point. We acknowledge the need for clarification regarding molecular markers. The ITS region was exclusively used as the fungal DNA barcode in our study. In the manuscript, there was a misunderstanding in the description, and we incorrectly mentioned the use of the ITS region in relation to bacterial identification. We have now removed any association between ITS and bacterial characterization, and explicitly stated that ITS sequencing was solely applied to fungal community profiling (Line 123, Lines 281-283).

 

Comments 7: Lines 273-274: The 16S rRNA gene is commonly used to assess the diversity of bacterial populations. Could you please clarify how bacterial diversity was determined in your study? Was it based solely on sequencing the ITS region? If so, please provide a reference where bacterial diversity is assessed using this approach.

Response 7: Thank you for pointing this out. To clarify, bacterial diversity in our study was indeed assessed using the 16S rRNA gene, not the ITS region. We acknowledge the confusion caused by the mention of the ITS region in relation to bacterial diversity. The ITS region was exclusively used for the fungal diversity analysis, while bacterial diversity was determined using the 16S rRNA gene. We have corrected this in the manuscript to ensure clarity and accuracy in the description (Line 123, Lines 281-283).

 

Comments 8: Line 274: The ITS region is mainly used for fungal diversity studies, while 18S rRNA is mainly used for high-resolution taxonomic studies of fungi. Did you use both regions for sequencing and fungi determination, and based on which region did you assess bacterial diversity?

Response 8: Thank you for raising this point. We sincerely apologize for the confusion regarding molecular marker usage in our original manuscript. To clarify, we used the ITS region for fungal diversity analysis and the 16S rRNA gene for bacterial diversity assessment. The 18S rRNA gene was not used in our study, whose accidental inclusion resulted from an editing error during manuscript editing. The manuscript has been corrected to specify these regions and their respective roles in fungal and bacterial identification (Line 123, Lines 281-283).

 

Comments 9: Line 452: Please refer to the principal component analysis (PCA) in the text, so that it can be more easily identified and followed in the Results section.

Response 9: Thank you very much for your suggestion. We added a relevant reference to clarify the use of PCA (Lines 475-476, Reference [33]).

 

Comments 10: Line 474&498: What about the results regarding B0 and G0?

Response 10: We appreciate your inquiry about the B0 and G0 groups. In our experimental design, B0 and G0 samples were primarily utilized for normalization purposes in RNA-seq data analysis, so they were intentionally excluded from downstream comparative analyses, as clarified in Lines 469-470.

 

Comments 11: Figure 5 & 6: I suggest using the same color for each specific category (BP, CC, MF) across all graphs, if possible, to facilitate easier interpretation and comparison of the results.

Response 11: We sincerely thank you for the constructive suggestion on Figures 5 and 6. We fully agree that consistent color coding across graphs can improve cross-comparison, and we acknowledge the potential value of this suggestion for future studies. In the current design, the color assignments (red, blue, green) within each subplot were intentionally mapped to a hierarchy of statistical significance rather than to fixed categories (BP, CC, MF). This approach was adopted because the primary analytical goal of these figures is to emphasize relative significance levels within each experimental condition, rather than direct category-to-category comparisons across subplots. Therefore, the original design was maintained.

 

Comments 12: Line 561: I recommend reconsidering the structure of the manuscript by merging the Results and Discussion sections.

Response 12: Thank you for your recommendation. After careful consideration, we merged the Results and Discussion sections to improve the manuscript’s flow and ensure a more coherent presentation of the findings (Lines 289-299, Lines 446-458, Lines 585-600).

 

Comments 13: It is not typical for the conclusion to refer to figures; such references should instead be placed in the Results and Discussion sections. Additionally, please provide a detailed description of the schematic presentation of the mode of action (MOA) for CME, and include a proper citation for the source of the image if it is not an original creation.

Response 13: Thank you for your valuable feedback. We have made the necessary revisions as suggested. We moved Figure 7 to the section of Results and Discussion. Additionally, a detailed description of the schematic presentation of MOA for CME has been added in the figure legend (Lines 602-609), along with the source of the images in the figure(Lines 609-610).