Performance of Different Fibers in the Extraction Parameters of Volatile Compounds Present in Alecrim-Do-Campo (Baccharis dracunculifolia)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Despite the relevance of the study, the work must be continued and the article seriously revised. Specifically: Plants of the Asteracea family are not monoecious or dioecious; their flowers are always hermaphrodite. The authors need to understand the intricacies of botany and taxonomy. More information should be gathered on the plant's use in folk medicine in Latin American countries. It is important to review all published modern and other works devoted to the comprehensive study of this plant species. This will significantly expand the list of cited literature. Careful attention should be paid to the presentation of the material in English. International databases are available for identifying components; therefore, the identification of isolated (discovered) components must be taken very seriously. I wish the authors success in their serious revision of the article.

Comments on the Quality of English Language

Not only the authors themselves, but also the editorial staff, always provide the text to a native English speaker for proofreading and verification before publishing in the journal. This is to correct and clarify specific terms.

Author Response

Dear Editor and Reviewer,

We thank you for your careful evaluation of our manuscript submitted to Metabolites and for the constructive comments and suggestions provided. We have revised the manuscript accordingly and believe that these changes have strengthened the quality, clarity, and scientific rigor of the work.

Below, we respond to each reviewer’s comment in detail. For clarity, the reviewers’ comments are reproduced and followed by our point-by-point responses. All modifications made to the manuscript are indicated using the highlight function in the revised version. Where comments did not result in textual changes, we provide a rationale to clarify the choices made in the original submission.

We appreciate the time and effort devoted to the review of our work and hope that our revisions adequately address all concerns raised.

 

Reviewer 1

We thank the Reviewer for the careful evaluation of our manuscript and for the constructive and detailed comments, which helped us to improve both the scientific content and the presentation of the work. The manuscript has been substantially revised in response to these remarks. Our point-by-point responses are provided below.

Comment 1. Despite the relevance of the study, the work must be continued and the article seriously revised. Specifically: Plants of the Asteraceae family are not monoecious or dioecious; their flowers are always hermaphrodite. The authors need to understand the intricacies of botany and taxonomy.

Response. We acknowledge this correction and agree with the Reviewer. All references to flower sex have been removed from the manuscript to avoid inaccurate botanical characterization, and the text has been revised accordingly to ensure taxonomic correctness.

Comment 2. More information should be gathered on the plant's use in folk medicine in Latin American countries. It is important to review all published modern and other works devoted to the comprehensive study of this plant species. This will significantly expand the list of cited literature.

Response. In response to this suggestion, we have expanded the Introduction by adding a new paragraph that discusses the documented uses of the plant in folk medicine across Latin American countries. In parallel, we conducted a more comprehensive review of the relevant literature and incorporated additional primary and review sources to provide a broader and more balanced contextual framework.

Comment 3. Careful attention should be paid to the presentation of the material in English. International databases are available for identifying components; therefore, the identification of isolated (discovered) components must be taken very seriously.

Response. The manuscript has been thoroughly revised to improve clarity, grammar, and overall quality of the English language. Additionally, we strengthened the rigor of compound identification by carefully re-evaluating the data using internationally recognized databases and clearly reflecting this process in the revised text.

We appreciate the Reviewer’s insightful comments and believe that the revisions made have significantly improved the manuscript.

We thank the Reviewer for the insightful comments and believe that the revisions made in response have improved the methodological transparency, clarity, and overall quality of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Title manuscript: Optimization of extraction parameters of volatile compounds present in alecrim-do-campo (Baccharis dracunculifolia) submitted to Metabolites by Lucas Silveira Garcia et al.

 Through this study, the authors address an important topic in the field of agroindustrial and medicinal plant research, focusing on Baccharis dracunculifolia, commonly known as alecrim-do-campo. Their investigation is innovative and represents a good support to improve the quality control and traceability of products derived from alecrim-do-campo.  For that, to achieve these interesting findings, the authors used a robust and appropriate methodology. The choice of headspace solid-phase microextraction (HS-SPME) is well-suited as the extraction method for volatile compounds.  The three different semipolar fibers (PDMS/DVB, DVB/CAR/PDMS, and CAR/PDMS) employed in this study empower the selection of which fiber is used to extract more volatile compounds. In addition, the use of a 2³ factorial design reflects a good approach to optimizing extraction conditions by combining the mass of sample, time of extraction and temperature. The obtained results are encouraging and represent a footprint in product quality assurance derived from Baccharis dracunculifolia.

In general, this article makes a meaningful contribution by giving the advancing knowledge on the efficient extraction of bioactive volatiles from B. dracunculifolia   

Despite the strong potential of this manuscript, some aspects require clarification and improvement before it can be considered for publication in Metabolites. Therefore, a major revision is recommended to address the points outlined below and further strengthen the clarity, methodological transparency, and scientific impact of the study.

Remark: I insist that the authors must correct the document because more issues are not acceptable in the final article after acceptance and publication.

Overall comments:

• I mentioned that some comments are listed directly on the manuscript: See the pdf format.
• Minor grammatical and stylistic issues are present throughout the manuscript. I recommend the English language revision.
• Some sentences are long and could be simplified for better understanding and readability.
• From pages 4 to 11, please reorganize your paragraphs; the reader cannot understand with type of fiber you describe. For that, I recommend starting with the fiber of CAR/PDMS, followed by PDMS/DVB, and DVB/CAR/PDMS. For each one, you give a chromatogram followed by its Response surface plots.
• How did you choose the specific ranges of time, temperature, and sample mass? Should be briefly justified, preferably with references.
• Do you think that the use of a 2³ factorial design with central-point replicates is appropriate? If yes, can you give more details about this design regarding experimental reproducibility and variability?
• Why in this study, there is no statistical analysis. I think more details are required.
• The authors should report p-values or confidence levels for significant and non-significant factors to strengthen the interpretation.
• Figure numbering and captions should be carefully checked for consistency. The figures are misplaced in the text.
• Ensure to give the full terms at the beginning, such as volatile organic compounds and after mention just the abbreviation VOCs. Avoid putting several terms for the same abbreviation, such as “volatile compounds,” in the titles of figures, and sometimes “volatiles” throughout the text.
• In the discussion, page 14, the result of identification highlights the predominance of terpenoids. However, stronger links should be made between the observed volatile profile and known biological or ecological roles of dracunculifolia. Could you provide some literature references to enrich this part by showing the potential industrial and pharmacological implications of this species?
• On the same page, I highlight that the optimized extraction conditions should be discussed more explicitly.
• I recommend increasing resolution and clarifying axis labels of some response surface plots.

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Minor grammatical and stylistic issues are present throughout the manuscript. I recommend the English language revision.

Author Response

Dear Editor and Reviewer,

We thank you for your careful evaluation of our manuscript submitted to Metabolites and for the constructive comments and suggestions provided. We have revised the manuscript accordingly and believe that these changes have strengthened the quality, clarity, and scientific rigor of the work.

Below, we respond to each reviewer’s comment in detail. For clarity, the reviewers’ comments are reproduced and followed by our point-by-point responses. All modifications made to the manuscript are indicated using the highlight function in the revised version. Where comments did not result in textual changes, we provide a rationale to clarify the choices made in the original submission.

We appreciate the time and effort devoted to the review of our work and hope that our revisions adequately address all concerns raised.

 

Reviewer 2

We thank the Reviewer for the thorough, detailed, and constructive assessment of our manuscript, as well as for recognizing the relevance of the topic, the robustness of the methodology, and the potential contribution of this work to quality control and traceability of products derived from Baccharis dracunculifolia. We also appreciate the Reviewer’s emphasis on rigor and clarity, which guided a careful and comprehensive revision of the manuscript. Our point-by-point responses are provided below.

General comments.
We acknowledge the Reviewer’s remark regarding the need for a carefully corrected final version. All comments and annotations provided directly in the PDF file were addressed in detail. The manuscript underwent an extensive English-language revision, stylistic corrections, and sentence restructuring to improve readability and coherence throughout.

Comment 1. Minor grammatical and stylistic issues are present throughout the manuscript. Some sentences are long and could be simplified for better understanding and readability.

Response. This point is fully acknowledged. All suggested corrections in the annotated PDF were incorporated, and additional language revision was carried out to simplify sentence structure and improve overall readability.

Comment 2. From pages 4 to 11, please reorganize your paragraphs; the reader cannot understand which type of fiber you describe. I recommend starting with CAR/PDMS, followed by PDMS/DVB, and DVB/CAR/PDMS, presenting for each one a chromatogram followed by its response surface plots.

Response. We appreciate this recommendation and carefully considered it during revision. The section in question was intended as an overview summarizing the global results across fibers. Detailed discussions for each fiber, including chromatograms and response surface analyses, are presented in the subsequent sections in a structured and fiber-specific manner. To improve clarity, we revised the introductory paragraphs of the Results section to better guide the reader through this organization and clarify the rationale behind the presentation.

Comment 3. How did you choose the specific ranges of time, temperature, and sample mass? This should be briefly justified, preferably with references.

Response. We agree that this justification should be explicit. The selection of experimental ranges was based on previously published HS-SPME optimization studies, particularly those reported in Molecules. We clarified this point in the revised manuscript and explicitly cited the work of Ramos et al. (2022)¹, which served as a methodological reference for defining these parameters.

1. Ramos, A. L. C. C., Nogueira, L. A., Silva, M. R., do Carmo Mazzinghy, A. C., Mariano, A. P. X., de Albuquerque Rodrigues, T. N., de Paula, A. C. C. F. F., de Melo, A. C., Augusti, R., de Araújo, R. L. B., Lacerda, I. C. A., & Meloelo, J. O. F. (2022). Chemical approach to the optimization of conditions using HS-SPME/GC-MS for characterization of volatile compounds in Eugenia brasil-iensis fruit. Molecules, 27(15), 4955

Comment 4. Do you think that the use of a 2³ factorial design with central-point replicates is appropriate? If yes, can you give more details about this design regarding experimental reproducibility and variability?

Response. We appreciate this question. The 2³ factorial design with central-point replicates was selected because it is a well-established approach for evaluating main effects and interactions while allowing assessment of experimental variability and curvature. Similar designs have been successfully applied in related studies, including García et al. (2019)¹ and Ramos et al. (2022)². We clarified this rationale in the revised manuscript and expanded the methodological description to better emphasize reproducibility and the role of central points in assessing experimental consistency.

1. García, Y. M., Rufini, J., Campos, M. P., Guedes, M. N., Augusti, R., & Melo, J. O. (2019). SPME fiber evaluation for volatile organic compounds extraction from acerola. Journal of the Brazilian Chemical Society, 30, 247-255.
2. Ramos, A. L. C. C., Nogueira, L. A., Silva, M. R., do Carmo Mazzinghy, A. C., Mariano, A. P. X., de Albuquerque Rodrigues, T. N., de Paula, A. C. C. F. F., de Melo, A. C., Augusti, R., de Araújo, R. L. B., Lacerda, I. C. A., & Meloelo, J. O. F. (2022). Chemical approach to the optimization of conditions using HS-SPME/GC-MS for characterization of volatile compounds in Eugenia brasil-iensis fruit. Molecules, 27(15), 4955

Comment 5. Why is there no statistical analysis? More details are required.

Response. Statistical analysis is indeed an integral part of the study. The evaluation of factor significance and interactions is presented through Pareto charts, which are included throughout the Results section. To avoid ambiguity, we revised the text to explicitly state this and to clarify how these charts were used to interpret statistical relevance.

Comment 6. The authors should report p-values or confidence levels for significant and non-significant factors.

Response. We acknowledge this suggestion. While individual p-values are not explicitly listed, significance thresholds are clearly defined and visually represented in the Pareto charts using a significance level of p = 0.05. We clarified this point in the revised manuscript to ensure transparency in the interpretation of statistical significance.

Comment 7. Figure numbering and captions should be carefully checked for consistency. The figures are misplaced in the text.

Response. This issue is acknowledged. All figures were renumbered where necessary, captions were revised for consistency, and figure placement was corrected to align properly with the corresponding sections of the text.

Comment 8. Ensure to give the full terms at the beginning, such as volatile organic compounds, and then use abbreviations consistently. Avoid using multiple terms for the same abbreviation.

Response. We agree with this recommendation. Full terms are now provided at first mention, abbreviations are used consistently thereafter, and redundant terminology has been eliminated throughout the manuscript, including figure titles and captions.

 

We are grateful to the Reviewer for the detailed and rigorous feedback. We believe that the revisions made in response to these comments have substantially improved the clarity, methodological transparency, and overall quality of the manuscript.

 

We thank the Reviewer for the insightful comments and believe that the revisions made in response have improved the methodological transparency, clarity, and overall quality of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors evaluate the efficiency of different extraction conditions for volatile compounds using headspace solid-phase microextraction (HS-SPME) in alecrim-do-campo, aiming to support the identification of biochemical markers for this species. The topic is relevant; however, several points need to be addressed to improve the scientific quality and clarity of the manuscript.

Major comments

1. In line 49, p-coumaric acid: the letter p should be written in italic.
2. In line 56, before discussing headspace microextraction, the authors should briefly introduce the differences between HS-SPME and conventional extraction methods.
3. At the end of the Introduction, the objective and aim of the study should be clearly and explicitly stated.
4. Please specify the period of plant collection and provide the voucher specimen number.
5. The identification of volatile organic compounds should be based on a comparison between the calculated Kovats indices and values reported in the literature, not solely on the NIST library.
6. Figure 1b is of low resolution and should be improved.
7. The title of Figure 2 should be revised, and a caption explaining the meaning of a, b, and c should be added.
8. The Kovats index must be included in Table 3 to justify the identification of volatile compounds.
9. Please add the area of each volatile compound in Table 3, as well as the total area for each chemical family.
10. In line 262, Baccharis species should be written in italic.
11. The Results section is not sufficiently discussed. The authors should compare their findings with previously published literature.
12. The Conclusion should be rewritten and should clearly mention that the dominant volatile compounds were terpenoids.
13. References 2 and 9 should be rechecked for accuracy.
14. In the References section, the terms in silico, in vitro, Baccharis dracunculifolia, and Baccharis species should be written in italic.

 

Author Response

Dear Editor and Reviewer,

We thank you for your careful evaluation of our manuscript submitted to Metabolites and for the constructive comments and suggestions provided. We have revised the manuscript accordingly and believe that these changes have strengthened the quality, clarity, and scientific rigor of the work.

Below, we respond to each reviewer’s comment in detail. For clarity, the reviewers’ comments are reproduced and followed by our point-by-point responses. All modifications made to the manuscript are indicated using the highlight function in the revised version. Where comments did not result in textual changes, we provide a rationale to clarify the choices made in the original submission.

We appreciate the time and effort devoted to the review of our work and hope that our revisions adequately address all concerns raised.

 

Reviewer 3

We thank the Reviewer for the careful reading of our manuscript and for the detailed and constructive comments aimed at improving its scientific quality and clarity. We have revised the manuscript extensively in response to these remarks. Our point-by-point responses are provided below.

Major Comments

Comment 1. In line 49, p-coumaric acid: the letter p should be written in italic.

Response. This correction is acknowledged and has been implemented in the revised manuscript.

Comment 2. In line 56, before discussing headspace microextraction, the authors should briefly introduce the differences between HS-SPME and conventional extraction methods.

Response. We agree with this suggestion. A concise section has been added to the Introduction paragraph to briefly contrast HS-SPME with conventional extraction techniques, highlighting its advantages for volatile compound analysis.

Comment 3. At the end of the Introduction, the objective and aim of the study should be clearly and explicitly stated.

Response. The objective of the study is explicitly stated at the end of the Introduction. To further improve clarity, this section was reviewed and slightly revised to ensure that the study aims are clearly and unambiguously presented.

Comment 4. Please specify the period of plant collection and provide the voucher specimen number.

Response. The plant material was collected continuously throughout the year. No voucher specimen was deposited in a herbarium, as taxonomic authentication was not the primary objective of this study, which focused on analytical optimization of HS-SPME conditions. We acknowledge the importance of voucher specimens in botanical and taxonomic studies and have clarified this point in the revised manuscript to avoid ambiguity.

Comment 5. The identification of volatile organic compounds should be based on a comparison between the calculated Kovats indices and values reported in the literature, not solely on the NIST library.

Response. We appreciate this important comment. In the present study, compound identification was intentionally described as tentative, based primarily on mass spectral matching with the NIST library. The focus of the work was the optimization and comparative evaluation of extraction conditions rather than definitive structural identification of volatile compounds. We have clarified this limitation in the manuscript and emphasized that the reported compounds should be interpreted as tentatively identified.

Comment 6. Figure 1b is of low resolution and should be improved.

Response. This issue has been addressed. Figure 1b has been replaced with a higher-resolution image in the revised manuscript.

Comment 7. The title of Figure 2 should be revised, and a caption explaining the meaning of a, b, and c should be added.

Response. The figure title and caption were revised accordingly, and the meaning of labels a, b, and c is now clearly explained.

Comment 8. The Kovats index must be included in Table 3 to justify the identification of volatile compounds. Please add the area of each volatile compound in Table 3, as well as the total area for each chemical family.

Response. We respectfully note that, as the volatile compounds were only tentatively identified, the inclusion of Kovats indices and absolute or relative peak areas could suggest a level of identification confidence and quantitative interpretation beyond the scope of the present study. Our analytical strategy focused on comparative evaluation of extraction performance and optimization trends across experimental conditions rather than exhaustive compound identification or semi-quantitative profiling. To avoid overinterpretation of tentative identifications, we chose not to include Kovats indices or compound areas.

Comment 9. In line 262, Baccharis species should be written in italic.

Response. This correction has been implemented.

Comment 10. The Results section is not sufficiently discussed. The authors should compare their findings with previously published literature.

Response. We agree with this assessment. A dedicated Discussion section has been added to the manuscript, in which the results are interpreted in the context of previously published studies on Baccharis species and HS-SPME analysis of volatile compounds.

Comment 11. The Conclusion should be rewritten and should clearly mention that the dominant volatile compounds were terpenoids.

Response. This suggestion has been fully incorporated. The Conclusion was revised to clearly state that terpenoids constitute the dominant class of volatile compounds identified in this study.

We are grateful to the Reviewer for the insightful comments and believe that the revisions undertaken have significantly improved the scientific rigor, clarity, and overall presentation of the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript titled "Optimization of extraction parameters of volatile compounds present in alecrim-do- campo (Baccharis dracunculifolia)" presents interesting results on the analysis of volatile compounds from a plant of agro-industrial interest. However, the experimental design used is not ideal for the study's objective, and the statistical analysis may not be appropriate.

In section 2.3, what ionization method was used?
In section 2.4, the title is incorrect.
2^3 factorial designs are not typically used for optimization. Please justify the choice of experimental design.

How many replicates were performed for the different treatments?

Why was the number of compounds used as the response variable?

Figure 1 needs improvement.
Figure 2 is cited on line 155, but this figure does not show the information described in the text.

The title of Figure 2 is incorrect.
Figures 3, 5, and 6 show the response surfaces of the effect of mass, extraction time, and extraction temperature on the number of compounds identified. However, statistical analysis shows that most of these effects are not significant, so these graphs are not useful.
Table 2 is missing.

Author Response

Dear Editor and Reviewer,

We thank you for your careful evaluation of our manuscript submitted to Metabolites and for the constructive comments and suggestions provided. We have revised the manuscript accordingly and believe that these changes have strengthened the quality, clarity, and scientific rigor of the work.

Below, we respond to each reviewer’s comment in detail. For clarity, the reviewers’ comments are reproduced and followed by our point-by-point responses. All modifications made to the manuscript are indicated using the highlight function in the revised version. Where comments did not result in textual changes, we provide a rationale to clarify the choices made in the original submission.

We appreciate the time and effort devoted to the review of our work and hope that our revisions adequately address all concerns raised.

Reviewer 4

We thank the Reviewer for the careful assessment of our manuscript and for the constructive comments regarding the experimental design, statistical analysis, and presentation of results. We have revised the manuscript accordingly and provide our point-by-point responses below.

Comment 1. In Section 2.3, what ionization method was used?

Response. We acknowledge that this information was inadvertently omitted in the original version. The ionization method employed in this study has now been clearly specified in Section 2.3 of the revised manuscript.

Comment 2. In Section 2.4, the title is incorrect.

Response. This issue has been acknowledged and corrected in the revised manuscript.

Comment 3. 2³ factorial designs are not typically used for optimization. Please justify the choice of experimental design.

Response. We appreciate this comment and agree that factorial designs are often associated with screening rather than full optimization. In the present study, the 2³ factorial design with central-point replicates was chosen as a pragmatic and well-established approach to evaluate the main effects and interactions of key HS-SPME parameters (sample mass, extraction time, and temperature) across different fiber coatings. This strategy allowed us to efficiently identify trends, assess factor significance, and compare extraction performance among fibers under controlled conditions. Similar designs have been successfully applied in HS-SPME–GC–MS studies focused on extraction parameter optimization and method development, particularly when the objective is to establish optimal operational ranges rather than to build predictive quadratic models.

Comment 4. How many replicates were performed for the different treatments?

Response. Three replicates were performed for each treatment, as stated in the first line of Section 2.4 of the Materials and Methods. This information has been retained and highlighted for clarity.

Comment 5. Why was the number of compounds used as the response variable?

Response. The number of tentatively identified volatile compounds was selected as the response variable because it provides a direct and practical metric of extraction efficiency in comparative HS-SPME studies. In the context of response surface methodology applied to method optimization, maximizing the diversity of extracted volatiles is a commonly used criterion, particularly when the objective is to enhance chemical coverage rather than quantitative determination of individual analytes. This approach is especially relevant when comparing fiber performance and extraction conditions for complex plant matrices.

Comment 6. Figure 1 needs improvement.

Response. This point is acknowledged. Figure 1 has been revised to improve its quality and clarity.

Comment 7. Figure 2 is cited on line 155, but this figure does not show the information described in the text.

Response. This inconsistency has been corrected. The figure citation and content were revised to accurately reflect the information discussed in the text.

Comment 8. The title of Figure 2 is incorrect.

Response. The title of Figure 2 has been corrected accordingly.

Comment 9. Figures 3, 5, and 6 show response surfaces, but statistical analysis indicates that most effects are not significant, so these graphs are not useful.

Response. We acknowledge the Reviewer’s observation regarding statistical significance. While some main effects and interactions are not statistically significant at the chosen confidence level, the response surface plots remain useful as a visual tool to illustrate trends, interaction patterns, and comparative behavior across experimental conditions and fiber types. These plots support interpretation of the Pareto analyses and provide an intuitive understanding of how the factors jointly influence extraction performance, even when effects are subtle.

Comment 10. Table 2 is missing.

Response. Table 2 is present in the manuscript and appears at line 159 in the revised version. We reviewed the layout to ensure that the table is clearly visible and properly referenced in the text.

 

We thank the Reviewer for the insightful comments and believe that the revisions made in response have improved the methodological transparency, clarity, and overall quality of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I believe that the authors, having made a little effort, themselves saw that the text of the article became more interesting, more informative, and for a new reader, more understandable and accessible.

Comments on the Quality of English Language

Not only the authors themselves, but also the editorial staff, always provide the text to a native English speaker for proofreading and verification before publishing in the journal. This is to correct and clarify specific terms.

Yes, I confirm that it is always necessary for all non-English speaking authors to have their text checked by a native speaker of scientific English.

Author Response

We thank the reviewer for the careful evaluation of the manuscript and for the comments regarding the quality of the English language.

We confirm that the entire manuscript has been thoroughly revised to improve clarity, readability, and overall presentation. In addition, the text was professionally edited and proofread by a native English speaker with experience in scientific writing, ensuring appropriate use of terminology and compliance with the journal’s editorial standards.

We believe that these revisions have significantly enhanced the quality, accessibility, and comprehensibility of the manuscript for an international audience.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant topic and presents valuable findings. After careful revision addressing the comments and suggestions raised, I agree with its publication.

Comments on the Quality of English Language

Minor grammatical and stylistic issues are present throughout the manuscript. I recommend the English language revision.

Author Response

We thank the reviewer for the careful evaluation of the manuscript and for the comments regarding the quality of the English language.

We confirm that the entire manuscript has been thoroughly revised to improve clarity, readability, and overall presentation. In addition, the text was professionally edited and proofread by a native English speaker with experience in scientific writing, ensuring appropriate use of terminology and compliance with the journal’s editorial standards.

We believe that these revisions have significantly enhanced the quality, accessibility, and comprehensibility of the manuscript for an international audience.

Reviewer 3 Report

Comments and Suggestions for Authors

The Authors replied all questions and made the corrections requested.
The manuscript can be accepted for publication in its current form.

Author Response

We thank the reviewer for the careful evaluation of the manuscript and for the comments regarding the quality of the English language.

We confirm that the entire manuscript has been thoroughly revised to improve clarity, readability, and overall presentation. In addition, the text was professionally edited and proofread by a native English speaker with experience in scientific writing, ensuring appropriate use of terminology and compliance with the journal’s editorial standards.

We believe that these revisions have significantly enhanced the quality, accessibility, and comprehensibility of the manuscript for an international audience.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors addressed all the comments. However, the experimental design used is not an optimization design. Furthermore, figures 5, 6, and 7 are unnecessary. I suggest changing the title to reflect better the manuscript's content and removing Figures 5, 6, and 7.

Author Response

We thank the reviewer for the careful evaluation of the manuscript and for the comments regarding the quality of the English language.

We confirm that the entire manuscript has been thoroughly revised to improve clarity, readability, and overall presentation. In addition, the text was professionally edited and proofread by a native English speaker with experience in scientific writing, ensuring appropriate use of terminology and compliance with the journal’s editorial standards.

We believe that these revisions have significantly enhanced the quality, accessibility, and comprehensibility of the manuscript for an international audience.