Review Reports - Comparative Optimization of Microwave and Ultrasound-Assisted Extraction of Bioactive Compounds and Proteins from <i>Craterellus cornucopioides</i>

Round 1

Reviewer 1 Report

The manuscript presents a sound innovative methodology using multiple parameters for evaluations, focusing on the different bioactive group of compounds including phenol, flavonoids etc that are part a broader concept of ecofriendly techniques to counter plant and human diseases. The results are clearly presented with high quality figures, however these issues need to be addressed. Here is my report for author and editor.

There is no profiling of biologically active compounds by using different techniques like HPLC/LC-MS that can be attributed for specific activity. How author can link to biological activity without proper profiling?
The manuscript include the screening against few pathogens while does not have any MIC and mechanisms that should be included in discussion.
There are no test that extracts are stable in lab or field conditions or how this study can be practically implemented?
The RSM models clear, but author did not report ANOVA p-values for all responses. Similarly, there is no multi-response optimization like desirability function.
There is repetition in manuscript like fungi roles in introduction so improve it to make the manuscript upto the standard of the journal.
The author should define acronyms like RSM) early in the manuscript.

7.The author can add error bars to all graphs.

References are solid but author can cite more on fungal UAE comparisons.
Cite (https://www.sciencedirect.com/science/article/pii/S0753332222002165) in methodology section UHPLC-MS to discuss bioactive/antioxidant assessment. Cite (https://www.nature.com/articles/s41467-025-61519-1) in discussion to discuss the ultrasound for eradication of fungi and link the discussion with UAE antimicrobial enhancement. Similarly cite (https://www.mdpi.com/1422-0067/27/4/2016) in discussion section to include the ultrasound-responsive nanomaterials for cell death and expand it to advanced UAE applications in bio actives.

Recommendation

Minor Revision. The manuscript is technically sound and these minor revisions will enhance the impact manuscript for J. Fungi.

There is no profiling of biologically active compounds by using different techniques like HPLC/LC-MS that can be attributed for specific activity. How author can link to biological activity without proper profiling?
The manuscript include the screening against few pathogens while does not have any MIC and mechanisms that should be included in discussion.
There are no test that extracts are stable in lab or field conditions or how this study can be practically implemented?
The RSM models clear, but author did not report ANOVA p-values for all responses. Similarly, there is no multi-response optimization like desirability function.
There is repetition in manuscript like fungi roles in introduction so improve it to make the manuscript upto the standard of the journal.
The author should define acronyms like RSM) early in the manuscript.

7.The author can add error bars to all graphs.

References are solid but author can cite more on fungal UAE comparisons.
Cite (https://www.sciencedirect.com/science/article/pii/S0753332222002165) in methodology section UHPLC-MS to discuss bioactive/antioxidant assessment. Cite (https://www.nature.com/articles/s41467-025-61519-1) in discussion to discuss the ultrasound for eradication of fungi and link the discussion with UAE antimicrobial enhancement. Similarly cite (https://www.mdpi.com/1422-0067/27/4/2016) in discussion section to include the ultrasound-responsive nanomaterials for cell death and expand it to advanced UAE applications in bio actives.

Recommendation

Minor Revision. The manuscript is technically sound and these minor revisions will enhance the impact manuscript for J. Fungi.

Author Response

Comment 1: There is no profiling of biologically active compounds by using different techniques like HPLC/LC-MS that can be attributed for specific activity. How author can link to biological activity without proper profiling?

Response 1: We agree that individual profiling provides deeper insights. However, the focus of this study was the comparative optimization of extraction efficiency for major bioactive groups using RSM. To address the reviewer’s concern, we have expanded the discussion in to link the observed biological activity with specific classes of compounds known to be dominant in C. cornucopioides based on literature. Furthermore, we have cited the suggested reference in the methodology section to discuss the importance of bioactive assessment through advanced techniques.

Comment 2: The manuscript include the screening against few pathogens while does not have any MIC and mechanisms that should be included in discussion.

Response 2: In this screening phase, we focused on the presence/absence of inhibitory effects. Following the reviewer's suggestion, we have significantly expanded the discussion regarding the antimicrobial mechanisms. We have now included a discussion on how UAE enhances antimicrobial activity by disrupting fungal cell structures, citing the suggested papers to link our findings with advanced ultrasound applications and cell death mechanisms.

Comment 3: There are no test that extracts are stable in lab or field conditions or how this study can be practically implemented?

Response 3: This is a valuable point. While long-term stability was not the primary focus, we have added a statement in the Conclusion regarding the practical implementation

Comment 4: The RSM models clear, but author did not report ANOVA p-values for all responses. Similarly, there is no multi-response optimization like desirability function.

Response 4: We have now explicitly reported the p-values for all individual responses directly in the text and ensured they are clearly presented in Tables. Furthermore, we have performed a multi-response optimization using the Desirability Function D, which is now described in the new Section.

Comment 5: There is repetition in manuscript like fungi roles in introduction so improve it to make the manuscript upto the standard of the journal.

Response 5: The Introduction has been thoroughly revised and condensed. Redundant descriptions of general fungal roles were removed, and the text now focuses more concisely on the specific value of C. cornucopioides and the extraction techniques used.

Comment 6: The author should define acronyms like RSM) early in the manuscript.

Response: All acronyms are now defined at their first mention in the Introduction and Abstract.

Comment 7: The author can add error bars to all graphs.

Response 7: We have ensured that error bars are clearly visible on all graphs.

Comments 8 and 9: References are solid but author can cite more on fungal UAE comparisons. Cite (https://www.sciencedirect.com/science/article/pii/S0753332222002165) in methodology section UHPLC-MS to discuss bioactive/antioxidant assessment. Cite (https://www.nature.com/articles/s41467-025-61519-1) in discussion to discuss the ultrasound for eradication of fungi and link the discussion with UAE antimicrobial enhancement. Similarly cite (https://www.mdpi.com/1422-0067/27/4/2016) in discussion section to include the ultrasound-responsive nanomaterials for cell death and expand it to advanced UAE applications in bio actives.

Response: As requested, we have incorporated these references.

Reviewer 2 Report

The authors achieved interesting and significant results. The study is well structured and addresses a topical issue in the field of biotechnology. The main question concerned the search for the most effective methods and protocols for extracting biologically active compounds from Craterellus cornucopioides fruit bodies. The complexity of this work and the various research strategies that were applied to successfully develop effective ultrasonic extraction with water allowed the authors to confirm that this method of extracting biologically active substances is more suitable. This makes an important contribution to the understanding of the extraction process of various compounds with high biological potential of natural origin, in particular from mushroom fruit bodies.

Overall, the topic is of practical relevance, and the manuscript cites an body of literature, reflecting the major research trajectories and recent developments in this field. The manuscript is clearly structured and the material is presented in a logical and consistent way. The introduction clearly and scientifically demonstrates the relevance and feasibility of the study. The experimental design is appropriately planned. Research methodology is presented clearly and understandably. A significant amount of material has been obtained by the authors, which is well presented and graphically illustrated. Obtained material is easy to understand and comprehend.

The concluding remarks are well-founded and summarize the work done. I also recommend adding a description or direction for potential next steps in the research.

However, the article has one significant drawback: the authors failed to present their own results on antimicrobial activity.

Comments:

The title of the article mentions biologically active components, so I don't see the point in singling out protein.

The article also studied antimicrobial activity, so it would be advisable to add it to the keywords.

The manuscript contains a subsection on the study of antimicrobial activity in the section on materials and methods, but the results section does not contain any results of the studies conducted. Although there is a subsection 3.5. Evaluation of antimicrobial activity, it contains an introductory part and highlights certain literary aspects of the evaluation of antimicrobial activity. Please take note of this. And add any missing results, tables, or relevant drawings and photographs from the studies conducted.

Author Response

Comment 1: The title of the article mentions biologically active components, so I don't see the point in singling out protein.

Response 1: We appreciate the reviewer's comment. However, we decided to keep "proteins" in the title to emphasize the high protein recovery achieved in this study, which is a specific focus of our research.

Comment 2: The article also studied antimicrobial activity, so it would be advisable to add it to the keywords.

Response 2: As suggested, "antimicrobial activity" has been added to the keywords.

Comment 3: The manuscript contains a subsection on the study of antimicrobial activity in the section on materials and methods, but the results section does not contain any results of the studies conducted. Although there is a subsection 3.5. Evaluation of antimicrobial activity, it contains an introductory part and highlights certain literary aspects of the evaluation of antimicrobial activity. Please take note of this. And add any missing results, tables, or relevant drawings and photographs from the studies conducted.

Response 3: We sincerely apologize for the initial absence of our experimental results in Section 3.5. We have now fully revised this section to include the results of our comprehensive screening against 8 pathogens using disk diffusion, agar well diffusion, and turbidimetric assays. While the results were negative at the tested concentrations, we have provided a thorough scientific discussion to explain this finding.

Reviewer 3 Report

The author only conducted a rough determination of active components and a simple in vitro antioxidant analysis on different extracts of Craterellus cornucopioides. This study lacks qualitative and quantitative analysis of the extracts as well as reliable in vitro experiments, such as cell experiments. Currently, the health benefits and molecular mechanisms of Craterellus cornucopioides have been extensively reported and verified through in vivo and in vitro experiments. Overall, this manuscript lacks innovation and is difficult to arouse the author's interest.

1. All data in this study lacked statistical analysis. 2. This study lacks in vitro cell experiments to validate its bioactive effect. 3. The experiment lacks chemical identification and analysis of the Craterellus cornucopioides extract.

Author Response

Comment 1: The author only conducted a rough determination of active components and a simple in vitro antioxidant analysis on different extracts of Craterellus cornucopioides. This study lacks qualitative and quantitative analysis of the extracts as well as reliable in vitro experiments, such as cell experiments. Currently, the health benefits and molecular mechanisms of Craterellus cornucopioides have been extensively reported and verified through in vivo and in vitro experiments. Overall, this manuscript lacks innovation and is difficult to arouse the author's interest.

Response 1:

Response 1: We thank the reviewer for the critical observation. However, we would like to clarify that the primary focus of this study is process engineering and optimization using Response Surface Methodology (RSM). The innovation lies in the systematic comparison of two intensified "green" extraction techniques (MAE and UAE) to establish an energy-efficient and food-safe protocol for C. cornucopioides. Such optimization is a mandatory prerequisite for any standardized industrial application or further biological testing. We have addressed the specific concerns below.

Comment 2: All data in this study lacked statistical analysis

Response 2: We respectfully disagree with the statement that statistical analysis was lacking. The core of this research is based on Response Surface Methodology (RSM), a sophisticated statistical tool used for process optimization. The reliability of our models was rigorously confirmed through ANOVA, where all R² values exceeded 0.97, indicating an excellent fit. However, to address the reviewer's concern regarding the visual representation of comparative data, we have implemented the following improvements: We have now performed a post-hoc multiple comparison test for all 16 experimental points presented in Figure 2 to evaluate the significance of differences between individual extracts. Statistical significance is now explicitly indicated on all bar graphs (Figures 2a, 2b, and 2c) using lowercase letters. The figure captions have been updated to include these statistical details, providing a clearer and more comprehensive interpretation of the results.

Comment 3: This study lacks in vitro cell experiments to validate its bioactive effect

Response 3: We acknowledge the importance of cell-based assays in validating bioactive effects at a molecular level. However, the scope of this manuscript is specifically centered on the technical optimization of extraction parameters to maximize the yield of antioxidants and proteins. Establishing a standardized extraction protocol is a fundamental first step.

Comment 4: The experiment lacks chemical identification and analysis of the Craterellus cornucopioides extract.

Response 4: We appreciate the reviewer's comment regarding the chemical profile. In the manuscript, we have already cited several key studies (e.g., Adamska & Felisiak, 2024; Bumbu et al., 2024; Turfan et al., 2018) that provide a comprehensive qualitative and quantitative identification of the bioactive compounds in C. cornucopioides. These cited works confirm that the mushroom is rich in specific phenolic acids, flavonoids, and polysaccharides. Given that our study was strictly focused on process engineering and the optimization of extraction yields using RSM, we relied on these established chemical profiles to contextualize our results.

Reviewer 4 Report

Please see the attachment.

Comments for author File: Comments.pdf

Author Response

Comment 1: Linoleic acid is a PUFA, not a MUFA.

Response1: We thank the reviewer for this accurate observation. The text has been revised to correctly classify oleic acid as the primary MUFA and linoleic acid as a PUFA.

Comment 2: Compare energy supply for UAE/MAE vs traditional methods.

Response 2: A qualitative comparison has been added to the Discussion.

Reviewer Comment 3: Finalize the sentence on line 76.

Response 3: The sentence has been completed.

Comment 4: Explanation of the 99.18% dry matter content.

Response 4: We have clarified in Section 2.4. that 99.18% refers to the standardized dry matter content of the mushroom powder, determined by drying the samples to a constant weight at 105 °C.

Comment 5: Definition of "Reagent C" in the Lowry assay.

Response 5: Following the reviewer's suggestion, we have explicitly defined the chemical composition and preparation of Reagent C in the protein determination subsection.

Comment 6: Sequence of steps in the Turbidimetric assay.

Response 6: The sequence has been corrected in Section 2.4.6.3. to reflect the chronological order of the experiment.

We have revised Figure 2 according to the reviewer’s suggestion. The x-axis now uses more compact labeling ("1 2 3 4") with grouped notations ("MAE water", "UAE 30% EtOH", etc.) to avoid duplication.

Comment 7: Figure 2. Abscissa axis, that contains the comparative antioxidant capacity values for the mushroom extracts, seems reasonable to be marked with more compact signs to avoid duplication between, e.g., "aqueous MAE" and "MW1MW2MW3MW4". It could be presented in the form "1 2 3 4" under the columns and "MAE by water" ("UAE by ethanol (30%)", where necessary) as the notation combining a given group in the picture field. The Figure caption should be terminated by "The samples 1–4 correspond to those in Table 1".

Response 7:

We have revised Figure 2 according to the reviewer’s suggestion.

Reviewer 5 Report

Manuscript entitled Comparative Optimization of Microwave and Ultrasound-Assisted Extraction of Bioactive Compounds and Proteins from Craterellus cornucopioides presents a well-designed and methodologically sound study focused on optimizing the extraction of bioactive compounds and proteins. The experimental design, particularly the application of Response Surface Methodology to evaluate key extraction parameters, is appropriate and allows for a comprehensive assessment of the extraction efficiency of both microwave- and ultrasound-assisted techniques. The study is clearly written and logically structured, with results that are well presented and supported by a coherent discussion. Overall, the findings provide valuable insights into green extraction approaches and contribute meaningfully to the field of mushroom bioactives and sustainable extraction technologies.

Specific comments are addressed in pdf file attached.

Comments for author File: Comments.pdf

Author Response

As suggested by the reviewer, we have thoroughly revised Section 4.2. and performed the requested deletions of certain parts to improve the manuscript. All these changes are clearly visible in the revised manuscript.

Round 2

Reviewer 3 Report

The author has responded to and revised the reviewer's comments one by one. However, the revisions are not thorough, and additional revisions are still needed to enhance the quality. In addition, the experimental design of this paper still has deficiencies.

Regarding comment 2, the author has added a multiple comparison test and improved the statistical presentation of the data. This revision is appropriate.
Regarding comment 3, considering that there have been numerous reports on the activity research of Craterellus cornucopioides. if this article only focuses on the extraction rate, it is necessary to add a limitation in the appropriate place stating the lack of further experimental exploration of biological activity.
Regarding comment 4, the author's avoidance of specific characterization of the extract in this study, citing previously reported literature, is unacceptable. Different extraction processes inevitably lead to differences in chemical composition. Therefore, chemical characterization is indispensable.

Author Response

Comment 3: ...it is necessary to add a limitation in the appropriate place stating the lack of further experimental exploration of biological activity.

Comment 4: ...author's avoidance of specific characterization of the extract in this study, citing previously reported literature, is unacceptable... chemical characterization is indispensable.

We sincerely thank the Reviewer for these critical insights. We fully acknowledge that the chemical composition of extracts is inevitably influenced by the extraction methodology and that a comprehensive biological characterization is essential for a complete study.

To address these concerns thoroughly, we have revised the manuscript and structured our response as follows:

Refinement of Chemical Characterization (Comment 4):

We have moved away from general literature citations regarding the composition of C. cornucopioides. Instead, we have emphasized in the revised Results and Discussion that the quantification of total phenols, total flavonoids, total proteins, and glutathione conducted in this study represents the specific quantitative chemical characterization of the extracts produced by our unique MAE and UAE protocols. These parameters were selected as standardized chemical markers to monitor the influence of extraction variables within the Response Surface Methodology (RSM).

Clarification of Research Scope and Phase-based Methodology (Comments 3 & 4):

As suggested by the Editor in the previous round, we have clarified that the primary objective of this specific paper is process engineering and optimization. This study serves as the foundational phase of the project "Evaluation of alternative sources of bioactive compounds and proteins (BioAlter)" (https://www.croris.hr/projekti/projekt/16428?lang=en). To ensure scientific rigor, the project is structured into consecutive specialized phases:

The Current Phase (presented here): Focuses on the technological optimization of green extraction methods and initial bioactive screening.

The First Subsequent Phase: Will focus on the antiglycation properties of the optimized extracts using HSA models. This phase will include detailed HPLC-MS profiling to correlate individual metabolites with their observed therapeutic potential.

The Second Subsequent Phase: Will investigate the incorporation of these extracts into active bio-packaging systems.

Addition of "Study Limitations and Future Directions" (Comment 3):

As evidenced in the revised Conclusion, we have explicitly added a section addressing the study's limitations. We acknowledge the absence of in-depth in vitro assays and molecule-specific profiling in this paper, explaining that these are integrated into the next specialized phases of the BioAlter project. This ensures full transparency regarding the scope and provides a clear scientific context for the absence of HPLC-MS data in this specific technological study.

We believe that this phased approach, now clearly outlined in the manuscript, justifies the current scope while ensuring that a more granular chemical and biological characterization is conducted in the appropriate research context.

Round 3

Reviewer 3 Report

No further comments.