Review Reports - Two New Pentadepsipeptides from the Mangrove Fungus Aspergillus sp. SCSIO 41443

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The general comments

The article focuses the identification and characterization of the bioactivity of new pentadepsipeptides of the aspertide group from the mangrove strain Aspergillus sp. SCSIO 41443. In terms of the completeness of the chemical analysis and characterization of the studied metabolites, the manuscript generally raised no significant issues. However, the organization of the manuscript as a whole and its content in other aspects are questionable.

The data on the fungal strain are presented enough strangely, only as the sequence in the Supplementary materials. In general, for publications related to the identification of microorganisms, the obtained sequence (16S or 18S rRNA genes, or ITS etc. for bacteria or fungi, respectively) or ITS should be deposited in the GenBank or other available international databases. Additionally, the corresponding methodology for identification based on ITS analysis should be included in the materials and methods section, and the identification and analysis data (including the construction of the phylogenetic tree etc.) should be presented in sufficient detail in the results section.
The methodological section needs in serious revision. The section 2.1 lists the main procedures, but not details the conditions and parameters as well as sample prep of the analyzed compound. The section 2.2 should be significantly supplemented as mentioned above. The section 2.3 unclearly describes the steps of the active metabolites isolation and purification and should be revised to clearer view in these terms. Ibid., the extraction procedure was reported little informatively, it's unclear whether the culture medium or just the fungal biomass was extracted. In this case, how was the biomass prepared for extraction (filtration, etc.)? This section 2.4 presents the data that are essentially results and should likely be included in the Results and Discussion section. The first two sentences in the section 2.6 no belong to the methodology, and its information only raises the question when mentioning other activities, why were these not analyzed in the study, although these are all mentioned together. Please report here the specific procedures, and leave similar texts for results and discussion. Finally, the statistical methods and software used or enzyme inhibitory assays and, probably, fungal fermentation data should be reported. Additionally, the specific software used for all instrumental analyses should be mentioned.
The Results section is presented in a rather strange way, in a form more typical of articles in specialized chemical journals. Here, in accordance with the title of the article and the general content of the article, it is necessary to first present general information on the isolation and identification of the producer fungal strain, then data on its fermentation process, preferably the dynamics of growth, accumulation of fungal biomass and the synthesis of bioactive compounds, how it was monitored. Any data concerning the conditions of synthesis of the analyzed pentadepsipeptides or their relative yield during cultivation are not presented here at all. Furthermore, the Results section lacks data on the isolation and purification of the analyzed compounds, both newly identified and previously described. This section must be included in the results. The section 3.2 inexplicably presents computer modeling / molecular docking data that were not mentioned at all in the methodology. The results of this analysis are unclear and the conclusions are not clearly formulated. At the same time, the specific values on the experimental data of AChE inhibitory assay absent; only weak activity was mentioned.
Discussion as such absent, judging by the content of the relevant section. The article contains a very large amount of additional material, 30 figures in total, but there is no order in its mention and commentary within the framework of the data being discussed.

The minor and specific comments

L57-58: “…which exist in many marine microorganisms, including ester bonds and amide bonds…” - The sentence grammar is incorrectly constructed, please correct it.

L60-61: “…which makes them promising lead compounds…” – delete “lead”.

L61: “…we further investigated…” – delete “further”.

L62: “…fungal strain…” – delete “fungal”.

L67-68: The Fig. 1 is not mentioned or commented on in the text. The caption to the Fig. 1 should be revised to more detail content.

L77: “CD…” – Please, decrypt for the first mention.

L87: “2.2. Fungal material” – It is incorrect terminology in reaction to living organism.

L92: “…based on BLAST analysis…” – See the comments above.

L94: “2.3. Fermentation and isolation” – Revise the title to more detailed content.

L95: “MA” – Decrypt for first mention.

L96-99: (1) Why was the temperature different during pre-cultivation and solid-phase cultivation? (2) In both cases, the fungal culture was grown in 1 L flasks, but large-scale fermentation is commonly performed in fermenters for SMF, or special bioreactors or matrasses for SSF. (3) (×48) - ? (4) (63 g) – specify, is it dry weight extract yield or anything other. What is its ratio on the weight of the grown mycelium and the volume of liquid extract?

L104: “…in the light of TLC profiles.” – It is unclear phrase.

L196, 226: “The above NMR data highly resembled those of 3…”, “…spectroscopic data of 1 and 2…” – Please, avoid these designations, use better more specific terms for the compounds.

Author Response

The data on the fungal strain are presented enough strangely, only as the sequence in the Supplementary materials. In general, for publications related to the identification of microorganisms, the obtained sequence (16S or 18S rRNA genes, or ITS etc. for bacteria or fungi, respectively) or ITS should be deposited in the GenBank or other available international databases. Additionally, the corresponding methodology for identification based on ITS analysis should be included in the materials and methods section, and the identification and analysis data (including the construction of the phylogenetic tree etc.) should be presented in sufficient detail in the results section.

Response: Thank you very much for your valuable comments. We fully agree that for research involving microbial identification, it is essential to deposit the obtained sequences in international public databases, as per standard practice in the field. Accordingly, the fungal ITS sequences have now been deposited in GenBank, and the accession number will be included in the manuscript to facilitate data access and verification by peers.

The methodological section needs in serious revision. The section 2.1 lists the main procedures, but not details the conditions and parameters as well as sample prep of the analyzed compound. The section 2.2 should be significantly supplemented as mentioned above. The section 2.3 unclearly describes the steps of the active metabolites isolation and purification and should be revised to clearer view in these terms. Ibid., the extraction procedure was reported little informatively, it's unclear whether the culture medium or just the fungal biomass was extracted. In this case, how was the biomass prepared for extraction (filtration, etc.)? This section 2.4 presents the data that are essentially results and should likely be included in the Results and Discussion section. The first two sentences in the section 2.6 no belong to the methodology, and its information only raises the question when mentioning other activities, why were these not analyzed in the study, although these are all mentioned together. Please report here the specific procedures, and leave similar texts for results and discussion. Finally, the statistical methods and software used or enzyme inhibitory assays and, probably, fungal fermentation data should be reported. Additionally, the specific software used for all instrumental analyses should be mentioned.

Response: Thank you very much for your valuable comments, which we fully agree with and have addressed in the revised manuscript as follows: Section 2.1 has been updated to include detailed information on the instruments, reagents, and software used in the experiments. In Section 2.2, the fungal ITS sequences have been deposited in GenBank, and the accession number will be provided, as noted in our previous response. Section 2.3 now provides a clearer description of the extraction process, along with the steps for compound isolation and purification. Following your suggestion, the content of Section 2.4 has been moved to the Results section. Regarding the first two sentences in Section 2.6, since the other activity tests did not show significant results, we have removed this part to maintain focus. We sincerely appreciate your thorough review and constructive feedback.

The Results section is presented in a rather strange way, in a form more typical of articles in specialized chemical journals. Here, in accordance with the title of the article and the general content of the article, it is necessary to first present general information on the isolation and identification of the producer fungal strain, then data on its fermentation process, preferably the dynamics of growth, accumulation of fungal biomass and the synthesis of bioactive compounds, how it was monitored. Any data concerning the conditions of synthesis of the analyzed pentadepsipeptides or their relative yield during cultivation are not presented here at all. Furthermore, the Results section lacks data on the isolation and purification of the analyzed compounds, both newly identified and previously described. This section must be included in the results. The section 3.2 inexplicably presents computer modeling / molecular docking data that were not mentioned at all in the methodology. The results of this analysis are unclear and the conclusions are not clearly formulated. At the same time, the specific values on the experimental data of AChE inhibitory assay absent; only weak activity was mentioned.

Response: Thank you for your important comments. Based on the structural issues you highlighted, we have systematically revised and supplemented the manuscript accordingly. Please find a summary of the specific modifications below: First, we have comprehensively supplemented the "Materials and Methods" section with details regarding the isolation source, identification process, and molecular biological basis of the production strain (including the sequence accession number), as well as the basic conditions of fermentation culture, to establish a clear research foundation. Second, regarding the more specific growth dynamics and biomass accumulation parameters during fermentation, systematic monitoring and recording of time-series data were not feasible under the current experimental conditions. Finally, the synthesis conditions and relative yield of the fifteen-peptide compounds you mentioned are indeed beyond the scope of this study. We have explicitly acknowledged this limitation in the "Discussion" section and have identified it as a key direction for future research. Once again, we sincerely appreciate your rigorous review, as your suggestions have significantly enhanced the academic completeness and logical clarity of this paper.

Discussion as such absent, judging by the content of the relevant section. The article contains a very large amount of additional material, 30 figures in total, but there is no order in its mention and commentary within the framework of the data being discussed.

Response: I sincerely thank you for reviewing the manuscript and providing valuable and constructive feedback. The issues you pointed out are crucial, and I fully agree with your assessment. Regarding the weakness of the discussion section, I will substantially rewrite and expand it in the revised version, focusing on connecting the main findings of this study with existing literature and theories to provide an in-depth interpretation of their significance, potential reasons, and research implications. Concerning the lack of structure in referencing the figures, I will thoroughly reorganize the logic of figure citations in the main text. I will ensure that each figure is clearly explained upon its first mention, with its core information highlighted and presented in a logical sequence that supports the argumentation, thereby eliminating any sense of clutter. Thank you once again for your guidance. Your suggestions have been immensely helpful in improving this paper. I hope the revised manuscript meets your expectations.

L57-58: “…which exist in many marine microorganisms, including ester bonds and amide bonds…” - The sentence grammar is incorrectly constructed, please correct it.

L60-61: “…which makes them promising lead compounds…” – delete “lead”.

L61: “…we further investigated…” – delete “further”.

L62: “…fungal strain…” – delete “fungal”.

Response: Thank you for your feedback. The relevant sentences have been revised according to your suggestions.

L67-68: The Fig. 1 is not mentioned or commented on in the text. The caption to the Fig. 1 should be revised to more detail content.

Response: Thank you for your valuable suggestion; the title has been revised accordingly as per your comment, and the revised version is presented in the updated manuscript.

L77: “CD…” – Please, decrypt for the first mention.

L92: “…based on BLAST analysis…” – See the comments above.

L95: “MA” – Decrypt for first mention.

Response: Thank you for your feedback. In the revised manuscript, we will ensure that all abbreviations (including "CD," "BLAST," and "MA") are spelled out in full upon their first mention, following academic conventions.

L87: “2.2. Fungal material” – It is incorrect terminology in reaction to living organism.

Response: Thank you for your suggestion. The inaccuracies regarding the fungal material in the Methods section have been corrected.

L94: “2.3. Fermentation and isolation” – Revise the title to more detailed content.

Response: Thank you for your feedback. We have noted the inaccuracy in the original section heading "Fermentation and Isolation" and have corrected it to " Fermentation, Extraction, isolation and Purification." in the Methods section as advised.

Response: We thank the reviewer for their careful review and valuable suggestions. Regarding the points raised, we have examined each and addressed them as follows: (1) The temperature discrepancy between pre-cultivation and solid-state fermentation was due to a typographical error. Both processes were actually conducted at 26 °C and have been corrected in the revised manuscript. (2) All scale-up fermentations (including solid-state fermentation) were performed in laboratory-scale 1 L flasks to maintain controlled and reproducible conditions for process optimization and mechanistic investigation. (3) The notation “(×48)” indicates that 48 parallel fermentation samples were prepared under the same conditions. We have clarified this in the text as “fermentation was carried out in 48 parallel 1 L flasks.” (4) We have revised the description for “(63 g)” to specify that it refers to the dry weight of the crude extract. The yield ratios relative to mycelial dry weight or liquid extract volume were not calculated in this study. Once again, we sincerely appreciate your rigorous review, as your suggestions have significantly enhanced the academic completeness and logical clarity of this paper.

L104: “…in the light of TLC profiles.” – It is unclear phrase.

L196, 226: “The above NMR data highly resembled those of 3…”, “…spectroscopic data of 1 and 2…” – Please, avoid these designations, use better more specific terms for the compounds.

Response: We thank the reviewer for the valuable suggestions. We have revised the manuscript as follows in response: The unclear phrase "in the light of TLC profiles" has been replaced with a more precise description; To avoid ambiguous references, the abbreviated designations "3," "1," and "2" have been replaced with the full compound name "aspertides". All changes have been tracked in the revised manuscript for ease of review.

Reviewer 2 Report

Comments and Suggestions for Authors

This paper reports the identification of two new compounds, aspertides F (1) and G (2), from Aspergillus sp. SCSIO 41443. Overall, the study is well conducted, however, several issues should be addressed before the manuscript can be accepted.

The figure legend of Figure 5 reads, “Proposed binding interactions of 1–5 with the active site residues of AChE (PDB ID: 4EY7). Red line: hydrogen bond.” However, only four docking results are shown in the figure, corresponding to compounds 2–5. This inconsistency should be clarified. In addition, Figure S30 has the same title, “Proposed binding interactions of 1–5 with the active site residues of AChE (PDB ID: 4EY7),” and the figure appears to be identical to Figure 5. Please clarify why the same figure is used in both the main text and the Supporting Information, or revise accordingly.
For the neuraminidase assay, the manuscript only reports inhibition rates for compounds 1–5 at a single concentration of 50 μg/mL (30.01%, 23.47%, 36.43%, 36.71%, and 27.42%, respectively). It would be helpful to include the corresponding figure or the original assay data in the Supporting Information to improve data transparency and allow readers to better evaluate the results.

Author Response

The figure legend of Figure 5 reads, “Proposed binding interactions of 1–5 with the active site residues of AChE (PDB ID: 4EY7). Red line: hydrogen bond.” However, only four docking results are shown in the figure, corresponding to compounds 2–5. This inconsistency should be clarified. In addition, Figure S30 has the same title, “Proposed binding interactions of 1–5 with the active site residues of AChE (PDB ID: 4EY7),” and the figure appears to be identical to Figure 5. Please clarify why the same figure is used in both the main text and the Supporting Information, or revise accordingly.

Response: We are grateful to the reviewer for highlighting these inconsistencies. The caption of Figure 5 has been corrected to "Proposed binding interactions of compounds 2–5 with the active site residues of AChE (PDB ID: 4EY7). Red line: hydrogen bond" to accurately reflect that only compounds 2–5 are displayed. The duplicate Figure S30 has been removed from the Supporting Information, and the subsequent figure numbering has been updated accordingly. All changes are clearly highlighted in the revised manuscript and Supporting Information. We sincerely appreciate the reviewer's careful attention, which has significantly improved the accuracy and clarity of the figures in our manuscript.

For the neuraminidase assay, the manuscript only reports inhibition rates for compounds 1–5 at a single concentration of 50 μg/mL (30.01%, 23.47%, 36.43%, 36.71%, and 27.42%, respectively). It would be helpful to include the corresponding figure or the original assay data in the Supporting Information to improve data transparency and allow readers to better evaluate the results.

Response: We thank the reviewer for their careful review, which has improved the accuracy and rigor of the figures in this paper. We also appreciate the constructive suggestion provided. Following this advice, we have supplemented the original neuraminidase assay data for compounds 1–5 (see provided as Figure S31) in the Supporting Information. This addition enhances the transparency and reproducibility of the experimental results. Corresponding descriptions have been added in the "Results" section. We believe this supplementary figure will help readers more fully evaluate the neuraminidase inhibition effects reported in this study.

Reviewer 3 Report

Comments and Suggestions for Authors

A classical study was conducted, including the cultivation of microorganisms with subsequent isolation (extraction, fractionation) and purification of active metabolites, as well as their study and determination of the structure by physico-chemical, spectral and biochemical methods of analysis. The level of knowledge and presentation of the work is high, which corresponds to the characteristics of the equipment used.
In vitro experiments confirm the activity of the isolated and characterized metabolites. Although these new metabolites are not the most active.
In the methodological part, one should not talk about the weak inhibitory activity of the isolated compounds, which can be presented and discussed in the results and discussion.
It is recommended that the results of paragraph 3.3 be presented in an expanded form, or not highlighted in a separate section.
There is practically no discussion, it needs to be expanded.

Author Response

In the methodological part, one should not talk about the weak inhibitory activity of the isolated compounds, which can be presented and discussed in the results and discussion.

It is recommended that the results of paragraph 3.3 be presented in an expanded form, or not highlighted in a separate section.

There is practically no discussion, it needs to be expanded.

Response: Thank you for your careful review of our manuscript and for providing valuable suggestions. We have thoroughly considered every comment and have revised the manuscript accordingly. The main revisions and explanations are as follows: (1) Regarding the optimization of the methodology section—thank you very much for pointing this out. We have removed descriptive statements regarding the strength of compound inhibitory activity from the "Materials and Methods" section. All specific activity data and comparative analyses have now been moved to and highlighted in the "Results and Discussion" section to ensure a clear division of content. (2) The original section "3.3" was indeed relatively brief and not suitable as an independent subsection. We have integrated and expanded this content into relevant paragraphs within the "Activity" section, making it more closely connected with the presentation and interpretation of the overall data and improving logical coherence. (3) Regarding the strengthening of the discussion section, we have substantially expanded the "Discussion" section. The new discussion not only provides an in-depth analysis of the possible reasons for the activity levels of the new metabolites (such as structure-activity relationships) but also places their activity within a broader research context by comparing them horizontally with known highly active compounds from the literature. It further explores their potential value and significance as lead compounds for further structural optimization. Once again, we sincerely appreciate the time you have taken and your professional input.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Despite the improvements in the revised version of the manuscript, the manuscript is still there are still some problems with the article that need to be addressed.

The methodology describes the extraction and purification procedures in sufficient details (section 2.3), however, these data as before are not presented in the Results. In their response, the Authors respond that an assessment of the characteristics of the synthesis and relative yield of bioactive peptides (for some reason, 15 compounds are mentioned, although the paper discussed five) during fermentation is beyond the scope of the study, and is not required in this case. However, since the isolation and purification of the peptide compounds using HPLC, etc. were mentioned in the methodology, this should be reflected in some way in the results.
AChE and neuraminidase inhibitory activities assays are usually performed using statistics, so statistical methods and software should be specified in the methodological section.
When a literature data analysis is based on a single reference [16] within the Discussion section, it can hardly be considered as adequate discussion. Please provide the precise scope of the study, and formulate more clearly the problematic addressed within the framework of the main objective of the work, since the area related to the synthesis of secondary metabolites (and, in particular, bioactive peptides) by fungi is very broad and may concern diverse aspects.
It's still worth paying attention to the formatting of supplementary data. Not all supplementary figures are cited in the text; please ensure that all figures are included, as the material is extensive. It is unclear why Fig. S30 is presented here, which duplicates Fig. 5 in the main text.
Additionally, the ITS sequence of the strain Aspergillus SCSIO 41443 may be removed from the supplementary material as it is referenced in GenBank.
The captions to the Figures in the main text are still not very informative and should be modified to more detailed content.

The minor and specific comments

L18: “monomeric compounds” – It is incorrect terminology belonging to the studied compounds. Revise.

L33-34: “ecosystems re characterized” – Correct.

L102: The section title should be renamed something like this: “Isolation, maintenance and identification of the strain Aspergillus sp. etc.”

L103-109: If the identification was not performed by the Authors, please provide a link to the institution where it was performed. Otherwise, a more complete description of the methodology (DNA isolation and purification, amplification and isolation of the target gene, sequencing and sequence analysis procedures, tree construction, etc.) is required.

L118: “whole fermented culture” - Please clarify here what is meant - only the biomass or the culture medium together with the fungal mycelium?

Ibid.: “1:1 ratio” - Since the mass ratio is shown in parentheses below for next steps (L123), add the volumetric ratio here as well.

L119: “ultrasound” - add the basic mode parameters of treatment (frequency, power, etc.).

L119-120: “combined supernatants” - Add here how the fungal biomass was removed and how the extract was separated.

L120: “63 g” – dry weight? Then add (d/w).

Ibid.: “dark green” – remove.

L293: “comparative analysis of literature analysis” – Please, revise.

Author Response

We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality for our manuscript. The reviewer comments are laid out below in italicized font and specific concerns have been numbered. Our point-to-point response is given in red font.

The methodology describes the extraction and purification procedures in sufficient details (section 2.3), however, these data as before are not presented in the Results. In their response, the Authors respond that an assessment of the characteristics of the synthesis and relative yield of bioactive peptides (for some reason, 15 compounds are mentioned, although the paper discussed five) during fermentation is beyond the scope of the study, and is not required in this case. However, since the isolation and purification of the peptide compounds using HPLC, etc. were mentioned in the methodology, this should be reflected in some way in the results.

Response: We appreciate the reviewer’s careful comment on the presentation of extraction and purification results. As suggested, we have now supplemented the isolation and purification details of the target compounds in the Results section, including the HPLC chromatograms, purification procedures, and yields of compounds 1–5, to make the experimental description more complete and reproducible. Concerning the number of compounds mentioned in our previous response, we apologize for the confusion caused by an inadvertent misstatement. The present study only focuses on five compounds, and we have revised all relevant descriptions to ensure consistency throughout the manuscript. Although the comprehensive evaluation of fermentation characteristics was not the primary aim of this work, we fully agree that the described isolation and purification methodology should be supported by corresponding results. We have therefore added the necessary experimental data and descriptions in the revised manuscript to address this concern. Thank you again for the constructive suggestion, which has greatly helped us improve the quality of the manuscript.

AChE and neuraminidase inhibitory activities assays are usually performed using statistics, so statistical methods and software should be specified in the methodological section.

Response: We appreciate the reviewer’s constructive comment. In the revised methodological section, we have clearly specified the statistical methods and the software used for the analysis of AChE and neuraminidase inhibitory activities, in order to make the experimental procedures more standardized and reproducible. Thank you for this helpful suggestion.

When a literature data analysis is based on a single reference [16] within the Discussion section, it can hardly be considered as adequate discussion. Please provide the precise scope of the study, and formulate more clearly the problematic addressed within the framework of the main objective of the work, since the area related to the synthesis of secondary metabolites (and, in particular, bioactive peptides) by fungi is very broad and may concern diverse aspects.

Response: We sincerely appreciate the reviewer’s constructive comments. The insufficient literature analysis mentioned by the reviewer has been addressed by removing the relevant over‑simplified discussion content in the Discussion section. At the same time, we have redefined the precise scope of this study and more clearly stated the scientific problems addressed in this work, in line with the main objective of our research. These revisions help to better emphasize the focus and significance of the study within the broad field of fungal secondary metabolites. Thank you very much for your valuable suggestions.

It's still worth paying attention to the formatting of supplementary data. Not all supplementary figures are cited in the text; please ensure that all figures are included, as the material is extensive. It is unclear why Fig. S30 is presented here, which duplicates Fig. 5 in the main text.

Response: We appreciate the reviewer’s careful comment on the supplementary materials. We have carefully checked the citation of all supplementary figures in the main text and added citations for those previously missing, so that every supplementary figure is now referred to in the manuscript. Regarding Fig. S30, we confirm that it overlaps with Fig. 5 in the main text. We have therefore removed Fig. S30 from the supplementary materials to avoid duplication. We have further adjusted and standardized the overall formatting of the supplementary data to improve clarity and completeness. Thank you again for your valuable suggestion.

Additionally, the ITS sequence of the strain AspergillusSCSIO 41443 may be removed from the supplementary material as it is referenced in GenBank.

Response: We appreciate the reviewer’s thoughtful suggestion. As suggested, the ITS sequence of strain Aspergillus SCSIO 41443 has been removed from the supplementary material, since this sequence is already available and accessible in the GenBank database. Thank you for your valuable comment.

The captions to the Figures in the main text are still not very informative and should be modified to more detailed content.

Response: We appreciate the reviewer’s careful comment on the figure captions. We have carefully revised and substantially expanded all figure captions in the main text to provide more detailed and comprehensive information, making the results clearer and easier to understand. Thank you for this valuable suggestion.

L18: “monomeric compounds” – It is incorrect terminology belonging to the studied compounds. Revise.

Response: We appreciate the reviewer’s careful correction. The inappropriate term “monomeric compounds” has been revised throughout the manuscript to accurate and suitable terminology consistent with the structural characteristics of the isolated compounds. Thank you for this important note.

L33-34: “ecosystems re characterized” – Correct.

Response: We appreciate the reviewer’s careful comment. The error in the phrase has been corrected in the manuscript. Thank you for your valuable suggestion.

L102: The section title should be renamed something like this: “Isolation, maintenance and identification of the strain Aspergillus sp. etc.”

Response: The section title has been revised to “Isolation, Cultivation and Identification of Fungal Aspergillus Strains” in the revised manuscript. Thank you for your valuable comment.

Response: We appreciate the reviewer’s careful comment. The name of the sequencing institution has been added in the manuscript as required. Thank you for your valuable suggestion.

L118: “whole fermented culture” - Please clarify here what is meant - only the biomass or the culture medium together with the fungal mycelium?

Response: We appreciate the reviewer’s careful comment. The expression “whole fermented culture” has been clearly clarified in the manuscript, referring to fungal mycelia and culture medium. The corresponding description has been revised accordingly. Thank you for your valuable suggestion.

Ibid.: “1:1 ratio” - Since the mass ratio is shown in parentheses below for next steps (L123), add the volumetric ratio here as well.

Response: We appreciate the reviewer’s careful comment. The volumetric ratio has been clearly supplemented in the manuscript. Thank you for your valuable suggestion.

L119: “ultrasound” - add the basic mode parameters of treatment (frequency, power, etc.).

Response: We appreciate the reviewer’s careful comment. The basic parameters of ultrasound treatment (frequency, power, etc.) have been added in the manuscript. Thank you for your valuable suggestion.

L119-120: “combined supernatants” - Add here how the fungal biomass was removed and how the extract was separated.

Response: We appreciate the reviewer’s careful comment. The methods for removing fungal biomass and separating the extract have been added in the manuscript. Thank you for your valuable suggestion.

L120: “63 g” – dry weight? Then add (d/w).

Ibid.: “dark green” – remove.

Response: We appreciate the reviewer’s careful comments. The dry weight notation (d/w) has been added after “63 g”, and the term “dark green” has been removed from the manuscript. Thank you for your valuable suggestions.

L293: “comparative analysis of literature analysis” – Please, revise.

Response: We appreciate the reviewer’s careful comment. The inappropriate expression has been deleted from the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

An extended discussion of the results is required.

Author Response

An extended discussion of the results is required.

Response: We appreciate the reviewer’s valuable and constructive comment. According to the suggestion, we have carefully extended and improved the discussion section in the revised manuscript. We further elaborated on the interpretation of the experimental results, strengthened the comparison with relevant previous studies, and enriched the analysis of the underlying significance of the findings. All revisions have been highlighted in the manuscript for the reviewer’s convenience. Thank you very much for your helpful suggestion.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Despite the Authors' comments regarding changes and adjustments to the submitted manuscript in accordance with the recommendations, certain aspects of the article remain virtually unchanged. I recommend that the Authors take their time before submitting yet another revised version, and instead consider some of the comments and recommendations more thoughtfully; this would take a little time. The main criticism remains regarding the design and content of the Discussion section. What is presented here is not a discussion and does not reflect the main results of the research. Is the study really about the fermentation process and the production of the bioactive peptides being studied? Is the molecular docking for modeling of interactions between isolated peptides and the active site of AChE the main result of the work?

Since the main objective of this work was the isolation, identification, structural analysis and in some measure - bioactivity of cyclic peptides of the aspertide group, the discussion should concern mainly this subject and should not consist of two small paragraphs, but be somewhat completer and more informative.

The elements of discussion are present partly in the Introduction, but more generally on the topic of fungal peptides, and also to a limited extent in the Results. In the latter case, if this were a single section with discussion, as in some journals, it would generally require a minor addition in terms of discussion. However, since the article includes the discussion as a separate section, it should be presented more thoroughly and based on an analysis of the original results and literature data on the study and characterization of aspertides and related fungal peptides.

The added section 3.2 is uninformative, and its purpose is unclear — to demonstrate the efficiency of substances extraction or the level of its production. It would also be helpful to know the parameters of the "starting fractions." If the Authors intended to demonstrate relative productivity, it is usually expressed as a ratio to the dry weight (g) of mycelial biomass.

The description of the used statistical methods, design, and software for the analysis of enzyme-inhibitory activity should be in a separate subsection concluding the methodology section.

The title of the section 3.3 is inaccurate in terms of its content.

Some terms within the Abstract appear too general and should be replaced with more specific ones: L17 – “products were extracted” – for example, by “produced extracellular metabolites were extracted from the culture filtrate”.

L18 – “Compounds were isolated…” – Peptide compounds…or Cyclic peptides were isolated etc.

Author Response

We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality for our manuscript. The reviewer comments are laid out below in italicized font and specific concerns have been numbered. Our point-to-point response is given in red font. The main corrections in the paper and the reviewer’s comments are as flowing:

Reviewer 1:

Despite the Authors' comments regarding changes and adjustments to the submitted manuscript in accordance with the recommendations, certain aspects of the article remain virtually unchanged. I recommend that the Authors take their time before submitting yet another revised version, and instead consider some of the comments and recommendations more thoughtfully; this would take a little time. The main criticism remains regarding the design and content of the Discussion section. What is presented here is not a discussion and does not reflect the main results of the research. Is the study really about the fermentation process and the production of the bioactive peptides being studied? Is the molecular docking for modeling of interactions between isolated peptides and the active site of AChE the main result of the work?
Since the main objective of this work was the isolation, identification, structural analysis and in some measure - bioactivity of cyclic peptides of the aspertide group, the discussion should concern mainly this subject and should not consist of two small paragraphs, but be somewhat completer and more informative.
The elements of discussion are present partly in the Introduction, but more generally on the topic of fungal peptides, and also to a limited extent in the Results. In the latter case, if this were a single section with discussion, as in some journals, it would generally require a minor addition in terms of discussion. However, since the article includes the discussion as a separate section, it should be presented more thoroughly and based on an analysis of the original results and literature data on the study and characterization of aspertides and related fungal peptides.

Response:

We greatly appreciate your careful and detailed review of our manuscript. We have carefully read all your comments and fully understand the issues you pointed out. We will revise the manuscript seriously and thoroughly according to your suggestions. We apologize that some contents of the manuscript were not sufficiently improved in the previous revision. We have fully recognized that the original Discussion section was not well structured and failed to reflect our main research results. We have now carefully reconsidered all comments and thoroughly revised the design and content of the Discussion section, so that it fully matches the focus and key findings of this study. We agree that the main objective of this work is the isolation, identification, structural analysis and bioactivity of cyclic aspertide peptides. We have completely rewritten and greatly expanded the Discussion section, which now focuses on these core topics instead of only two short paragraphs, and we have added comprehensive and in-depth analysis to make it more complete and informative. We have noticed that some discussion-related elements were previously distributed in the Introduction and Results sections. We have reorganized the overall logical structure and integrated the relevant content into the Discussion section. Meanwhile, we have based the revised discussion on our original results combined with related literature on aspertides and other fungal peptides, making this section more systematic and thorough.Thank you again for your valuable and constructive suggestions

The added section 3.2 is uninformative, and its purpose is unclear — to demonstrate the efficiency of substances extraction or the level of its production. It would also be helpful to know the parameters of the "starting fractions." If the Authors intended to demonstrate relative productivity, it is usually expressed as a ratio to the dry weight (g) of mycelial biomass.

Response: We greatly appreciate your constructive comments on Section 3.2. We agree that the content was insufficiently informative and its purpose was unclear. We have completely revised and rewritten this section to clarify its main focus. We have supplemented the relevant parameters of the starting fractions, and presented the relative productivity as the ratio to the dry weight of mycelial biomass. We hope these revisions meet your approval.

The description of the used statistical methods, design, and software for the analysis of enzyme-inhibitory activity should be in a separate subsection concluding the methodology section.

Response:We appreciate your constructive comment. We have added a separate subsection at the end of the Materials and Methods section to clearly describe the statistical methods and software used for the enzyme-inhibitory activity analysis. The experimental design and procedures have been presented and elaborated separately in the corresponding experimental sections.

The title of the section 3.3 is inaccurate in terms of its content.

Response: We appreciate your constructive comment. We agree that the original title of Section 3.3 was inaccurate. We have revised the section title to Structural Elucidation and Identification of Pentadepsipeptides to accurately reflect the content.

Some terms within the Abstract appear too general and should be replaced with more specific ones: L17 – “products were extracted” – for example, by “produced extracellular metabolites were extracted from the culture filtrate”.
L18 – “Compounds were isolated…” – Peptide compounds…or Cyclic peptides were isolated etc.

Response:We highly appreciate your careful and constructive suggestions on the Abstract. We have carefully revised the relevant sentences in the Abstract according to your comments, replacing those general and vague terms with more specific and accurate descriptions to ensure the preciseness and standardization of the expression.

In conclusion, we have carefully addressed all comments raised by the reviewers and thoroughly revised the manuscript in accordance with the professional suggestions. We have made substantial improvements to ensure the scientific rigor, accuracy and clarity of the paper.

We sincerely appreciate the valuable time and insightful guidance from the editor and reviewers. We hope the revised manuscript can meet the journal’s standards and kindly ask for your further review and consideration.

Best regards！

Reviewer 3 Report

Comments and Suggestions for Authors

The authors took into account the comments of the reviewers, revised and supplemented the manuscript. The manuscript can be published in its current form.

Author Response

The authors took into account the comments of the reviewers, revised and supplemented the manuscript. The manuscript can be published in its current form.

Response: We sincerely appreciate the valuable time and insightful guidance from the editor and reviewers. We hope the revised manuscript can meet the journal’s standards and kindly ask for your further review and consideration.