Research Progress in Biotransformation of Plant and Phytochemicals by Aspergillus: Active Metabolites and Industrial Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

• Main Research Question
  The article addresses a central and well-defined question: How can Aspergillus species be leveraged to biotransform plant-derived phytochemicals, thereby enhancing their bioactivity, bioavailability, and industrial applicability? The authors examine specifically how Aspergillus niger, A. oryzae, and A. nidulans utilize their enzymatic machinery to convert complex plant compounds into high-value metabolites with applications across food, pharmaceutical, and cosmetic sectors.
• Originality and Relevance
  The topic is both relevant and addresses a meaningful gap in the literature. While biotransformation by Aspergillus is not entirely novel, this review provides a systematic integration of recent advances (2020–2025) that have previously been dispersed across the literature. The manuscript successfully bridges three dimensions typically treated separately: enzymatic mechanisms, specific phytochemical classes, and industrial-scale applications. Furthermore, it addresses a practically significant challenge—valorization of agro-industrial waste through fermentation—which aligns with circular economy principles. Yes, the article fills a specific gap by consolidating fragmented knowledge into a comprehensive framework accessible to researchers and practitioners.
• Contribution to the Field
  The article makes several valuable contributions: (1) it systematically integrates data on multiple Aspergillus species and phytochemical classes within a unified framework; (2) Table 1 consolidates representative transformations with cross-referenced citations, facilitating rapid comparison; (3) it extends beyond fundamental science to address industrial-scale challenges, including solid-state fermentation optimization, strain stability, and downstream purification; (4) it proposes concrete future strategies, such as CRISPR/Cas9 applications, AI-assisted process monitoring, and hybrid bioprocessing approaches; and (5) it incorporates recent literature through 2025, capturing cutting-edge developments in the field.
• Consistency of Conclusions
  The conclusions are consistent with the evidence and arguments presented throughout the manuscript. Section 5 appropriately synthesizes the main findings: advantages of biotransformation, industrial applications, core challenges, and proposed solutions. The conclusions adequately address the central research question by confirming Aspergillus as a viable tool for phytochemical transformation. Yes, the conclusions are well-aligned with the evidence presented and directly address the principal research question.
• Reference Appropriateness
  The 158 references constitute an adequate foundation for a comprehensive review. The bibliography includes both foundational works and recent publications (2020–2025), encompasses multiple perspectives (molecular biology, bioprocess engineering, industrial applications), and appropriately represents both English-language and Chinese-language literature. The references are appropriate and well-suited to support the arguments presented.
• Tables and Figures
  Table 1 is well-organized with four clear columns (substrate, transformed component, strain, reference) and effectively covers the three primary Aspergillus species. The tables and figures are appropriate and effectively support the manuscript's narrative.

Author Response

Dear Reviewer,

We greatly appreciate you reviewing our manuscript in your busy schedule. Thank you for your thorough and in-depth review. Your positive feedback on the research design, originality, academic merits, coherent conclusions, references and graphics has been a great encouragement to our team.

You clearly identified the core strength of this review: we consolidated fragmented literature from 2020–2025 into a three-dimensional framework covering enzymatic mechanisms, phytochemical categories and industrial-scale applications. You also affirmed its practical value for circular economy and high-value valorization of agro-food by-products, which strengthens the academic credibility of this work.

We have carefully proofread and revised the full text according to your suggestions to guarantee top academic quality for publication. The item-by-item responses to your evaluations are shown below.

 

Comments 1：Main Research Question
The article addresses a central and well-defined question: How can Aspergillus species be leveraged to biotransform plant-derived phytochemicals, thereby enhancing their bioactivity, bioavailability, and industrial applicability? The authors examine specifically how Aspergillus niger, A. oryzae, and A. nidulans utilize their enzymatic machinery to convert complex plant compounds into high-value metabolites with applications across food, pharmaceutical, and cosmetic sectors.

Response 1:Thank you greatly for your positive comments on the clarity of the core research questions in this review. Accurately elucidating the key enzymatic mechanisms and metabolic pathways underlying the biotransformation of phytochemicals by Aspergillus fungi — particularly Aspergillus niger, A. oryzae and A. nidulans — serves as the core foundation of this work. Your recognition has further strengthened our confidence to deepen the discussion on this research topic.

Throughout the Introduction and all main chapters of the manuscript, we have conducted in-depth discussions focused on the extracellular and intracellular enzyme systems of these three dominant Aspergillus species. We have fully retained the inherent logical chain of enzymatic mechanisms — high-value metabolites — diverse industrial applications and made no compromises to this core framework, ensuring the entire manuscript maintains a highly concentrated research focus.

 

Comments 2：Originality and Relevance
The topic is both relevant and addresses a meaningful gap in the literature. While biotransformation by Aspergillus is not entirely novel, this review provides a systematic integration of recent advances (2020–2025) that have previously been dispersed across the literature. The manuscript successfully bridges three dimensions typically treated separately: enzymatic mechanisms, specific phytochemical classes, and industrial-scale applications. Furthermore, it addresses a practically significant challenge—valorization of agro-industrial waste through fermentation—which aligns with circular economy principles. Yes, the article fills a specific gap by consolidating fragmented knowledge into a comprehensive framework accessible to researchers and practitioners.

Response 2:We sincerely appreciate the reviewer for insightfully pointing out the original contributions of this review regarding the three-dimensional linkage (mechanisms, classification and applications), as well as the research on circular economy and high-value utilization of agricultural and food by-products. Systematically collating scattered up-to-date literature has always been the original intention of compiling this manuscript.

In the revised version, we have further refined and supplemented the latest process data published between 2020 and 2025 concerning Solid-State Fermentation (SSF) for the bioconversion of agro-residues, including citrus peels, sugarcane bagasse and tea residues, in Section 3 (Biotransformation of specific phytochemicals) and Section 5 (Challenges and future perspectives). The multi-dimensional integrated framework has been fully retained in the revised manuscript, aiming to provide a clear reference for both academic researchers and industrial practitioners.

 

Comments 3：Contribution to the Field
The article makes several valuable contributions: (1) it systematically integrates data on multiple Aspergillus species and phytochemical classes within a unified framework; (2) Table 1 consolidates representative transformations with cross-referenced citations, facilitating rapid comparison; (3) it extends beyond fundamental science to address industrial-scale challenges, including solid-state fermentation optimization, strain stability, and downstream purification; (4) it proposes concrete future strategies, such as CRISPR/Cas9 applications, AI-assisted process monitoring, and hybrid bioprocessing approaches; and (5) it incorporates recent literature through 2025, capturing cutting-edge developments in the field.

Response 3:Thank you for your concise overview of the five key academic contributions of this review. We put considerable work into prospectively analyzing industrial constraints (strain stability, downstream purification) and emerging technologies including CRISPR/Cas9 editing and AI-based monitoring, and we appreciate your positive feedback.

The full manuscript has been thoroughly revised and refined, especially Section 4 on industrial challenges and Section 5 on future strategies. We have kept the latest research on CRISPR/Cas9-mediated Aspergillus engineering, AI-driven fermentation control and literature updated to 2025 accurate and highlighted, to maintain the review’s leading role in this field.

 

Comments 4：Consistency of Conclusions
The conclusions are consistent with the evidence and arguments presented throughout the manuscript. Section 5 appropriately synthesizes the main findings: advantages of biotransformation, industrial applications, core challenges, and proposed solutions. The conclusions adequately address the central research question by confirming Aspergillus as a viable tool for phytochemical transformation. Yes, the conclusions are well-aligned with the evidence presented and directly address the principal research question.

Response 4:We sincerely appreciate the reviewer’s high praise for the closed logical loop of Section 5 (Conclusions). As a basic principle of academic writing, we always ensure that the conclusions are free from overstatement and digression, and perfectly consistent with the data and arguments presented in previous sections.

We have fully retained the original structure and reasoning of Section 5, and no changes have been made to the core conclusions. Strictly based on the foregoing enzymatic evidence and industrial cases, the conclusions clearly outline the development roadmap for Aspergillus-mediated biotransformation, and directly address the core issue of improving the biological activity and application value of phytochemicals.

 

Comments 5：Reference Appropriateness
The 158 references constitute an adequate foundation for a comprehensive review. The bibliography includes both foundational works and recent publications (2020–2025), encompasses multiple perspectives (molecular biology, bioprocess engineering, industrial applications), and appropriately represents both English-language and Chinese-language literature. The references are appropriate and well-suited to support the arguments presented.

Response 5:We greatly appreciate the reviewer for recognizing the comprehensiveness, timeliness and global coverage of our literature retrieval. While preparing this review, we focused on balancing classic foundational literature with the latest interdisciplinary advances published over the past five years (2020–2025), so as to deliver a global research perspective.

To live up to your positive comments, we have conducted a one-by-one format check for all references. We confirm that all citations feature accurate authors, journal titles, publication year, volume, issue and DOI, and all in-text citations are correctly positioned. These efforts fully maintain the academic rigor of this review, which acts as a cornerstone reference for the discipline.

 

Comments 6：Tables and Figures
Table 1 is well-organized with four clear columns (substrate, transformed component, strain, reference) and effectively covers the three primary Aspergillus species. The tables and figures are appropriate and effectively support the manuscript's narrative.

Response 6:We are pleased to receive the reviewer’s positive comments on the design and functionality of Table 1. This table was originally constructed to allow readers to make horizontal comparisons across different species and substrates at a glance, so as to improve the reading efficiency of this data-intensive review.

We have fully retained the existing four-column structure of Table 1, namely Substrate, Transformed component, Strain and Reference. In addition, we have meticulously proofread all biological names (e.g., Latin names such as A. niger formatted in italics) and corresponding reference numbers in the table. These efforts ensure the information is presented accurately, neatly and efficiently.

 

Once again, we deeply appreciate your thorough and encouraging review, which has been instrumental in helping us present this research at its highest possible quality. We hope that our responses have satisfactorily addressed your comments and that the manuscript is now suitable for publication. Please do not hesitate to contact us if any further modifications or clarifications are required.

Sincerely,

Kuntao Xu

On behalf of all co-authors

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review: Research Progress in Biotransformation of Plant and Phytochemicals by Aspergillus: Active Metabolites and Industrial Applications

The review is strong and well-structured, with clear scientific relevance and good topical focus. The scope is appropriate for a review paper, and the contribution is positioned convincingly within existing literature.

I will kindly recommend some clarification in technical areas, such as:

Abstract

The abstract communicates the scope, novelty, examples, applications, and future directions effectively. From critical perspective, while precision is good, the abstract has some overly long sentences, which may affect readability. I kindly suggest to consider a stronger statement explaining:

• why Aspergillus-based biotransformation is particularly important,
• what gap this review fills compared with previous reviews.

Currently, the novelty is implied rather than explicit.

 

Introduction

The Introduction is scientifically solid, logically organized, and it successfully establishes the importance of plant bioactives, identifies limitations of conventional extraction technologies, and positions Aspergillus-mediated biotransformation as a promising alternative. The Introduction explains:

• why plant bioactives matter,
• why extraction is problematic,
• why Aspergillus is useful.

However, it does not clearly identify:

• what previous reviews failed to cover,
• what specific gap this review addresses.

 

Biotransformation of plants by Aspergillus and its core advantages

This section demonstrates substantial scholarly effort and extensive literature collection, particularly in compiling representative examples of Aspergillus-mediated plant biotransformations. The scientific scope is broad and relevant, and the section effectively highlights enzymatic mechanisms, metabolic diversity, and industrial applications.

Although enzymes are mentioned frequently, mechanistic analysis remains superficial.

For example: β-glucosidase, cellulase, hydroxylase, and demethylase are named, but:

• catalytic specificity,
• pathway regulation,
• gene clusters,
• metabolic flux,
• cytochrome P450 systems,
• cofactor dependence

are rarely discussed. For a higher-level review, more mechanistic integration is needed.

Table 1 significantly improves manuscript value; however, I kindly suggest to add columns for:

• transformation type,
• enzyme involved,
• conversion yield,
• industrial relevance.

This would greatly enhance analytical usefulness.

 

Classification of active ingredients in biotransformation systems and multi-domain functional mechanisms

This section represents the strongest and most mature part of the manuscript so far in terms of scientific breadth, thematic organization, mechanistic integration, and industrial relevance. Compared with the previous sections, it demonstrates a substantially improved structure and stronger synthesis between biochemical mechanisms and applied biotechnology.

The review effectively describes each compound class independently, but there is insufficient comparative synthesis. For example:

• Why are flavonoids easier to biotransform than terpenoids?
• Which enzyme systems dominate in each category?
• Which pathways are most industrially scalable?
• Which metabolite classes face the greatest purification challenges?

Adding comparative insights would substantially elevate scholarly depth.

Additionally, incorporating a dedicated discussion on safety aspects would substantially strengthen the manuscript, particularly regarding mycotoxin risks, regulatory challenges, and food safety considerations associated with Aspergillus-mediated biotransformation systems.

 

Core challenges in the industrial application of biotransformation

This section is one of the most academically mature and publication-ready parts of the manuscript. Compared with earlier sections, it demonstrates substantially improved scientific synthesis, stronger critical analysis, clearer thematic organization, and a more balanced review tone. Importantly, the section moves beyond descriptive literature compilation and engages with genuine industrial biotechnology challenges, which significantly elevates the scholarly quality of the review.

 

Conclusions and Perspectives

This “Conclusions and Perspectives” section is scientifically comprehensive, forward-looking, and well aligned with the overall scope of the review. It successfully summarizes the core findings of the manuscript while identifying major technological bottlenecks and future research directions.

The conclusion should more clearly articulate:

• what this review contributes conceptually,
• why Aspergillus biotransformation is strategically important,
• how the field is evolving.

That type of synthesis would substantially strengthen the conclusion.

A major conceptual limitation of the manuscript is the insufficient discussion of safety and regulatory considerations associated with Aspergillus-mediated biotransformation systems. In view of the strong industrial orientation of the review, the Conclusions and Perspectives section would benefit from a dedicated discussion addressing key issues such as potential mycotoxin contamination, regulatory approval requirements, limitations associated with GRAS status, biosafety assessment of engineered strains, and compliance with food and pharmaceutical quality standards. Incorporating these aspects would significantly strengthen the translational relevance and industrial applicability of the review.

 

 

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We would like to express our sincere gratitude for your highly constructive and insightful comments on our manuscript, "Research Progress in Biotransformation of Plant and Phytochemicals by Aspergillus: Active Metabolites and Industrial Applications". We deeply appreciate the time and effort you have dedicated to reviewing our work. Your valuable feedback has been instrumental in enhancing the scientific depth, logical structure, and translational relevance of our review.

We have carefully studied your suggestions and made comprehensive revisions to the manuscript (highlighted in red in the revised document). Below, please find our point-by-point responses to your comments.

 

Comments 1:Abstract

The abstract communicates the scope, novelty, examples, applications, and future directions

effectively. From critical perspective, while precision is good, the abstract has some overly long

sentences, which may affect readability. I kindly suggest to consider a stronger statement

explaining: why Aspergillus-based biotransformation is particularly important, what gap this review fills compared with previous reviews. Currently, the novelty is implied rather than explicit.

Response 1:Thank you for this excellent suggestion. We have carefully reviewed and revised the abstract to improve its overall readability and flow. To explicitly highlight our novelty and the strategic importance of Aspergillus, we have incorporated stronger statements to clarify the exact gap this review fills.

Specifically, we revised the text to emphasize that while existing literature primarily focuses on isolated compounds or general fungal processes, this review fills a critical gap by systematically linking specific Aspergillus enzymatic systems to the "enzymatic hydrolysis-transformation-synthesis" closed-loop framework, which is essential for industrial-scale applications. (Please see the revised Abstract section)

 

Comments 2:Introduction

The Introduction is scientifically solid, logically organized, and it successfully establishes the

importance of plant bioactives, identifies limitations of conventional extraction technologies, and

positions Aspergillus-mediated biotransformation as a promising alternative. The Introduction

explains: why plant bioactives matter, why extraction is problematic, why Aspergillus is useful.

However, it does not clearly identify: what previous reviews failed to cover, what specific gap this review addresses.

Response 2:We completely agree with your assessment. To make our conceptual contribution explicit early in the paper, we have expanded the Introduction to clearly state the limitations of previous reviews and the exact gap we are addressing. We added text explaining that previous literature has often neglected the genus-wide metabolic versatility of Aspergillus across diverse phytochemical classes. We explicitly state that our review bridges microscopic enzymatic mechanisms with macroscopic industrial challenges by incorporating the "enzymatic hydrolysis-transformation-synthesis" closed-loop framework. (Please see the revised Introduction section)

 

Comments 3:Biotransformation of plants by Aspergillus and its core advantages

This section demonstrates substantial scholarly effort and extensive literature collection, particularly in compiling representative examples of Aspergillus-mediated plant biotransformations. The scientific scope is broad and relevant, and the section effectively highlights enzymatic mechanisms, metabolic diversity, and industrial applications. Although enzymes are mentioned frequently, mechanistic analysis remains superficial. For example: β-glucosidase, cellulase, hydroxylase, and demethylase are named, but: catalytic specificity, pathway regulation, gene clusters, metabolic flux, cytochrome P450 systems, cofactor dependence are rarely discussed. For a higher-level review, more mechanistic integration is needed.

Response 3: We appreciate your highly insightful observation. To elevate the scholarly depth of the review, we have significantly enriched the mechanistic analysis within this section. We have added detailed discussions on:

Catalytic Specificity: Detailing how specific amino acid residues in Aspergillus β-glucosidases achieve highly regioselective deglycosylation.

Metabolic Flux & Gene Clusters: Explaining how central carbon metabolism is redirected to balance product synthesis and biomass, and how global regulators and specific gene clusters tightly control enzyme expression.

Cytochrome P450 Systems & Cofactor Dependence: Expanding on the structural modification of terpenoids and alkaloids, emphasizing the strict requirement of NADPH/NADH regeneration for efficient metabolic flux. (Please see the revised Section 2.1)

 

Comments 4:Table 1 significantly improves manuscript value; however, I kindly suggest to add columns for: transformation type, enzyme involved, conversion yield, industrial relevance. This would greatly enhance analytical usefulness.

Response 4: We sincerely thank you for this constructive suggestion to enhance the analytical utility of Table 1. We have given this request careful consideration during our revision.

After thorough deliberation, we have decided to maintain the original structure of Table 1 for the following reasons, which we believe preserve the integrity and clarity of the review:

Alignment with the Paper’s Core Scope: Our review specifically focuses on the biotransformation performance of three core Aspergillus species (A. niger, A. nidulans, and A. oryzae). The current table is designed to provide a concise and direct summary that maps perfectly to the subsections dedicated to these three species. We believe that adding additional columns would fragment this cohesive structure and distract readers from the comparative overview we aim to present.

Data Consistency and Reliability: In compiling this table, we observed that the primary literature is highly heterogeneous. Many studies, particularly those focusing on the valorization of agro-industrial wastes, do not consistently report specific conversion yields, reaction types, or precise reaction times. Including these columns would result in a table with significant data gaps, which might undermine the reliability and professional presentation of the work.

Readability and Focus: To ensure the table remains highly readable and useful as a quick reference guide, we have prioritized clarity and consistent formatting. We believe that the current structure strikes the optimal balance between providing a representative overview and maintaining the focus on our primary analysis.

We hope you understand that this decision was made in the interest of maintaining the highest standards of scientific clarity and internal consistency for our readers.

 

 

Comments 5:Classification of active ingredients in biotransformation systems and multi-domain functional mechanisms

This section represents the strongest and most mature part of the manuscript so far in terms of

scientific breadth, thematic organization, mechanistic integration, and industrial relevance. Compared with the previous sections, it demonstrates a substantially improved structure and stronger synthesis between biochemical mechanisms and applied biotechnology. The review effectively describes each compound class independently, but there is insufficient comparative synthesis. For example: Why are flavonoids easier to biotransform than terpenoids? Which enzyme systems dominate in each category? Which pathways are most industrially scalable? Which metabolite classes face the greatest purification challenges? Adding comparative insights would substantially elevate scholarly depth. Additionally, incorporating a dedicated discussion on safety aspects would substantially strengthen the manuscript, particularly regarding mycotoxin risks, regulatory challenges, and food safety considerations associated with Aspergillus-mediated biotransformation systems.

Response 5:These are brilliant points. We have introduced comparative synthesis throughout this section. For example, we explicitly compared flavonoids and terpenoids, noting that flavonoid biotransformation (relying primarily on glycoside hydrolases) faces lower steric hindrance and lacks the strict cofactor dependence seen in the P450-mediated oxidation of terpenoids, making them more amenable to industrial scale-up.

Furthermore, we heavily integrated discussions on safety aspects and mycotoxin risks. We highlighted the necessity of rigorous monitoring during solid-state fermentation and discussed how the Aspergillus oryzae genome possesses mutated or transcriptionally silent aflatoxin biosynthetic gene clusters, conferring a critical safety advantage over toxigenic relatives. (Please see the revised Section 3.1 and Section 3.3)

 

Comments 6:Core challenges in the industrial application of biotransformation

This section is one of the most academically mature and publication-ready parts of the manuscript. Compared with earlier sections, it demonstrates substantially improved scientific synthesis, stronger critical analysis, clearer thematic organization, and a more balanced review tone. Importantly, the section moves beyond descriptive literature compilation and engages with genuine industrial biotechnology challenges, which significantly elevates the scholarly quality of the review.

Response 6: We are truly honored and encouraged by your positive assessment of the section regarding the "Core challenges in the industrial application of biotransformation". We are delighted that our efforts to move beyond descriptive literature and engage with genuine industrial biotechnology challenges have resonated with your perspective. Your recognition of the improved scientific synthesis, critical analysis, and thematic organization in this section serves as a great motivation for us to uphold this high standard of scholarly quality throughout the entire review. We sincerely appreciate your constructive feedback, which has been invaluable in helping us elevate the manuscript to its current level.

 

Comments 7:Conclusions and Perspectives

This “Conclusions and Perspectives” section is scientifically comprehensive, forward-looking, and well aligned with the overall scope of the review. It successfully summarizes the core findings of the manuscript while identifying major technological bottlenecks and future research directions. The conclusion should more clearly articulate: what this review contributes conceptually, why Aspergillus biotransformation is strategically important, how the field is evolving. That type of synthesis would substantially strengthen the conclusion.A major conceptual limitation of the manuscript is the insufficient discussion of safety and regulatory considerations associated with Aspergillus-mediated biotransformation systems. In view of the strong industrial orientation of the review, the Conclusions and Perspectives section would benefit from a dedicated discussion addressing key issues such as potential mycotoxin contamination, regulatory approval requirements, limitations associated with GRAS status, biosafety assessment of engineered strains, and compliance with food and pharmaceutical quality standards. Incorporating these aspects would significantly strengthen the translational relevance and industrial applicability of the review.

Response 7: Thank you for pushing us to strengthen our concluding thoughts. We have thoroughly rewritten the "Conclusions and Perspectives" section to better reflect the manuscript's conceptual weight:

Conceptual Contribution & Field Evolution: We articulated the strategic importance of the "hydrolysis-transformation-synthesis" framework and mapped the field's evolutionary trajectory toward precision green biomanufacturing driven by multi-omics and genome editing.

Safety and Regulatory Constraints: We added a dedicated, in-depth discussion specifically addressing the limitations of the GRAS status under varying industrial conditions. We heavily emphasized the absolute necessity of rigorous biosafety assessments for engineered strains, adherence to stringent food/pharmaceutical quality standards, and the critical importance of mitigating mycotoxin contamination risks for successful commercialization. (Please see the revised Section 5)

 

We hope that these revisions comprehensively address your concerns and meet the high standards of the journal. We thank you once again for your constructive guidance, which has undoubtedly resulted in a much stronger manuscript.

Sincerely,

Kuntao Xu

On behalf of all co-authors

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for submitting your review manuscrip, the topic is highly relevant to the scope of Fermentation, particularly in the context of sustainable fermentation technologies, microbial biotechnology, and the development of high-value bioactive compounds. Your manuscript demonstrates substantial effort in compiling recent literature and provides a broad overview of the potential industrial applications of Aspergillus mediated biotransformation in the food, pharmaceutical, cosmetic, and agricultural sectors. The emphasis on sustainability and waste valorization is particularly appreciated and aligns well with current research priorities.

That said, there are several important areas where the manuscript could be significantly strengthened to meet the scientific rigor and critical depth expected by Fermentation. I respectfully encourage the authors to consider the following points during revision:

Clarify the methodology for literature selection

It would be beneficial to describe how the literature included in this review was identified. Please clarify whether this is a narrative review and specify the databases, keywords, timeframe, and selection criteria used to gather references. This would improve transparency and methodological rigor.

Expand discussion on biosafety concerns

The manuscript frequently highlights the industrial applicability of Aspergillus species; however, not all species are universally regarded as safe. A more balanced discussion regarding mycotoxin production, strain-specific safety concerns, regulatory requirements, and quality control measures is strongly recommended, particularly for food and pharmaceutical applications.

Provide stronger quantitative comparisons

The manuscript frequently suggests that microbial biotransformation is superior to conventional extraction methods; however, these statements should be supported by more quantitative comparisons involving yield, efficiency, cost/effectiveness, scalability, and environmental impact. A comparative summary table may be particularly useful.

Reduce redundancy across sections

Certain examples (such as polydatin-to-resveratrol and ginsenoside conversions) are repeated multiple times throughout the manuscript. Reducing repetitive content would improve readability and allow space for deeper analysis of additional case studies.

Revise figures and tables for clarity

Table 1 contains useful information but may be improved through better categorization and formatting. Additionally, Figure 1 could provide greater mechanistic detail to better support the discussion presented in the text.

Comments on the Quality of English Language

The manuscript would benefit from careful English language editing to improve grammar, sentence structure, and overall clarity. Addressing these issues will significantly improve readability and professionalism.

Overall, this manuscript addresses an important and promising area of fermentation biotechnology and has clear potential to make a meaningful contribution to the field. With substantial revision focused on improving critical analysis, scientific depth, and clarity, the manuscript could become a valuable resource for readers of Fermentation.

Author Response

Dear Reviewer,

Thank you very much for your thoughtful and encouraging feedback on our manuscript, "Research Progress in Biotransformation of Plant and Phytochemicals by Aspergillus: Active Metabolites and Industrial Applications". We truly appreciate the time and effort the reviewers spent providing such constructive criticism. Your insights have been instrumental in helping us refine the depth, rigor, and clarity of our work.

We have taken your comments very seriously and have completed a comprehensive revision of the manuscript. Below, we provide a detailed, point-by-point response regarding the improvements made:

 

Comments 1:Clarify the methodology for literature selection

It would be beneficial to describe how the literature included in this review was identified. Please clarify whether this is a narrative review and specify the databases, keywords, timeframe, and selection criteria used to gather references. This would improve transparency and methodological rigor.

Response 1: Thank you for this important suggestion to improve methodological transparency.We have revised the "Introduction" section to explicitly define this work as a narrative review. To ensure methodological rigor and transparency, we have added a detailed description of our search strategy:

We conducted a systematic search across major electronic databases, including PubMed and Web of Science, spanning the period from 2020 to 2025.

The search strategy employed specific combinations of keywords, including “Aspergillus,” “biotransformation,” “phytochemicals,” “polyphenols,” “saponins,” and “industrial fermentation”.

We applied stringent inclusion criteria, selecting only peer-reviewed primary research articles and high-impact professional reviews that provided clearly defined enzymatic mechanisms or robust quantitative industrial performance metrics.

 

Comments 2:Expand discussion on biosafety concerns

The manuscript frequently highlights the industrial applicability of Aspergillus species; however, not all species are universally regarded as safe. A more balanced discussion regarding mycotoxin production, strain-specific safety concerns, regulatory requirements, and quality control measures is strongly recommended, particularly for food and pharmaceutical applications.

Response 2: We fully agree that biosafety is a critical issue for industrial applications of Aspergillus. We have significantly strengthened the discussion on biosafety concerns, strain-specific safety profiles, regulatory requirements and quality control measures in the following sections of the revised manuscript:

Section 2.2.3 (Aspergillus oryzae) : We expanded the discussion on the genetic basis of A. oryzae safety, highlighting that despite high homology with toxigenic A. flavus, the aflatoxin biosynthetic gene clusters in A. oryzae have undergone natural mutations or remain transcriptionally silent. We also added details on the aflatoxin degradation capacity of A. oryzae NRRL 3483 (degrading >95% AFB1) and its safety validation in food applications.

Section 3.1 (Polysaccharides) : We added a dedicated paragraph addressing mycotoxin contamination risks during SSF, emphasizing the need for rigorous monitoring of metabolic products and strict compliance with food-grade purity standards even for GRAS strains.

Section 5 (Conclusions and Perspectives) : We included a comprehensive discussion on regulatory compliance, clarifying the limitations of GRAS status under specific industrial conditions, and highlighting that rigorous biosafety assessments of genetically engineered strains are fundamental prerequisites for commercialization.

We also explicitly noted that not all Aspergillus species are universally recognized as safe, and strain-specific safety evaluations are mandatory for all industrial applications.

 

Comments 3:Provide stronger quantitative comparisons

The manuscript frequently suggests that microbial biotransformation is superior to conventional extraction methods; however, these statements should be supported by more quantitative comparisons involving yield, efficiency, cost/effectiveness, scalability, and environmental impact. A comparative summary table may be particularly useful.

Response 3: Thank you for this excellent suggestion. We have added Table 1 at the beginning of Section 2.1of the revised manuscript, which provides a direct quantitative comparison of biotransformation efficiency between Aspergillus platforms and conventional extraction methods. The table includes key parameters such as conversion type, platform/strain, enzymes involved, yield/conversion rate, reaction time, and production advantages, with corresponding references.

This table clearly demonstrates the quantitative advantages of Aspergillus-mediated biotransformation: Conversion rates of 80%–94.4% within 4–48 hours for Aspergillus platforms, compared to only 4%–54% for conventional extraction methods Up to 35-fold increase in bioactive components when using agricultural waste substrates. Significant reduction in processing time and elimination of solvent residue risks.

We have also integrated additional quantitative data throughout the text to support our claims about the superiority of microbial biotransformation over conventional methods

 

Comments 4:Reduce redundancy across sections

Certain examples (such as polydatin-to-resveratrol and ginsenoside conversions) are repeated multiple times throughout the manuscript. Reducing repetitive content would improve readability and allow space for deeper analysis of additional case studies.

Response 4: We have carefully reviewed the entire manuscript and systematically removed redundant content. Specifically, for the repeatedly mentioned polydatin-to-resveratrol and ginsenoside Rb1-to-compound K conversions:

We integrated the polydatin-to-resveratrol case into Section 2.2.1 (A. niger) and Section 2.2.3 (A. oryzae), with each section focusing on the unique enzymatic characteristics of the respective strain (β-glucosidase from A. niger M85 vs piceid-β-d-glucosidase from A. oryzae) rather than repeating the same basic information.

We consolidated the ginsenoside Rb1-to-CK conversion into Section 3.2 (Saponins), where we provided a detailed analysis of the C20-III degradation pathway and the regioselective mechanism of β-glucosidases.

To enhance the depth of the review, we added extensive new content on:

The role of cytochrome P450 monooxygenase systems in the structural modification of terpenoids and alkaloids (Section 2.1)

Metabolic flux regulation and cofactor regeneration requirements in large-scale fermentation (Section 2.1)

Transcriptional regulation of secondary metabolic gene clusters in Aspergillus (Section 2.2.2, )

These additions have significantly improved the scientific depth of the manuscript while eliminating unnecessary repetition.

 

Comments 5: Revise figures and tables for clarity

Table 1 contains useful information but may be improved through better categorization and formatting. Additionally, Figure 1 could provide greater mechanistic detail to better support the discussion presented in the text.

Response 5:We greatly appreciate your suggestions for improving the figures and tables. After careful consideration, we have decided to retain the original versions of Table 1 and Figure 1 for the following reasons:

Regarding Table 1(The table 2 of the revised manuscript)

We did consider re-categorizing and reformatting the table during the initial drafting process. However, we found that many of the included studies have incomplete data:

Some studies did not report the specific enzymes involved in the biotransformation.

Several studies using agricultural waste substrates focused on fold increases in bioactivity rather than absolute yields.

A few studies did not provide precise reaction time data.

Adding incomplete or estimated data would compromise the scientific accuracy of the table. The current structure of Table 2 is concise, easy to read, and closely aligns with the textual discussion in Section 2.2. Our review specifically focuses on the biotransformation performance of three core Aspergillus species (A. niger, A. nidulans, and A. oryzae). The current table is designed to provide a concise and direct summary that maps perfectly to the subsections dedicated to these three species. We believe that adding additional columns would fragment this cohesive structure and distract readers from the comparative overview we aim to present.

We believe this structure best serves the purpose of the table.

Regarding Figure 1

We originally planned to add details such as specific enzymes, reaction types, and conversion yields to Figure 1. However, Figure 1 is designed as a conceptual framework diagram to illustrate the "enzymatic hydrolysis-transformation-synthesis" closed-loop system. The central core module is already content-dense, and adding additional details would create a significant imbalance between the central and side modules, severely reducing the readability and clarity of the diagram. The primary purpose of Figure 1 is to provide a simple, intuitive overview of the biotransformation process, and adding excessive technical details would defeat this purpose.

We sincerely hope you can understand our decision to retain the original versions of Table 1 and Figure 1.

 

Comments 6:Comments on the Quality of English Language

The manuscript would benefit from careful English language editing to improve grammar, sentence structure, and overall clarity. Addressing these issues will significantly improve readability and professionalism.

Response 6: In response to your suggestion, we have meticulously polished the entire manuscript. We have corrected grammatical inconsistencies, refined sentence structures for greater precision, and enhanced the overall professional tone to ensure the review meets the high standards of Fermentation.

 

Once again, we thank you for your valuable time and constructive comments, which have significantly improved the quality of our manuscript. We believe the revised version now addresses all your concerns and is suitable for publication in Fermentation. Please do not hesitate to contact us if you have any further questions or require additional revisions.

Sincerely,

Kuntao Xu

On behalf of all co-authors

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I express my satisfaction with the improvements made by the authors and the resolution of my suggestions. Furthermore, I commend the authors for their effort and demonstrated scientific contribution.

Author Response

We would like to express our deepest gratitude to the reviewer for the highly positive evaluation. It is very rewarding to hear that our revisions have resolved your concerns. We truly appreciate your commendation of our efforts; your insightful feedback has been instrumental in guiding our improvements and strengthening the overall scientific value of this paper.

Reviewer 3 Report

Comments and Suggestions for Authors

Based on a comparison between the original version (v1) and the revised manuscript (v2), several of the major concerns previously identified have been partially or substantially addressed. However, some important issues remain and should be further improved before the manuscript can fully satisfy the scientific rigor and review standards expected by Fermentation.

The revised version demonstrates a clear effort by the authors to address several concerns raised during the previous review round. The manuscript has been expanded from approximately 25 to 30 pages and now includes additional discussion regarding literature selection methodology, mechanistic aspects of Aspergillus mediated biotransformation, industrial scalability challenges, biosafety considerations, and quantitative comparisons with conventional extraction methods.

The revised manuscript now includes comparative performance data and references describing conversion efficiencies, processing times, and biotransformation yields. Additionally, Table 1 provides a more structured comparison of biotransformation performance indicators.

The manuscript has improved linguistically; however, numerous sentences remain excessively long and occasionally repetitive. Professional English language editing is still recommended prior to publication.

The manuscript generally conforms to the structure expected for a review article in Fermentation.

Before acceptance, the authors should:

• Ensure consistency of terminology throughout the manuscript.
• Verify that all abbreviations are defined at first use.
• Confirm that all figures and tables are self-explanatory and adequately referenced in the text.
• Review statements related to GRAS status and biosafety for scientific accuracy.

Comments on the Quality of English Language

To meet the editorial standards of Fermentation, I recommend a final round of professional english language editing to improve sentence structure, eliminate redundancy, ensure consistency in terminology, and enhance overall precision and fluency. Such revisions would strengthen the manuscript's clarity and facilitate communication of its scientific contributions to an international audience.

Author Response

Dear Reviewer,

We would like to express our sincere gratitude for your highly constructive and insightful comments on our manuscript submitted to Fermentation. We are pleased to note that you found our previous revisions regarding the methodology, mechanistic aspects, scalability, and quantitative comparisons to be a clear effort in addressing the initial concerns.

We have carefully studied your latest recommendations and conducted a comprehensive and meticulous revision of the manuscript. Please note that all modifications, structural adjustments, and new additions in the revised manuscript have been highlighted in green for your convenience.

Below is a point-by-point response detailing how we have addressed each of your comments to meet the scientific rigor and editorial standards of the journal.

Comment 1: Ensure consistency of terminology throughout the manuscript.

Response:

We thank the reviewer for pointing this out. We have conducted a thorough check to ensure the consistency of all professional terminologies throughout the text.

A major fix involved standardizing the terminology for the ginseng metabolite. It is now properly introduced as "ginsenoside compound K" in the Abstract, defined with its abbreviation as "compound K (CK)" upon its first mention in the main text (Section 1), and consistently referred to as "CK" or "ginsenoside CK" in the subsequent sections (e.g., Section 3.2).

Comment 2: Verify that all abbreviations are defined at first use.

Response:

We have strictly verified and updated the manuscript to ensure that all abbreviations are explicitly defined upon their first appearance. The specific additions include:

PHB and CDW: Defined as "polyhydroxybutyrate (PHB)" and "cell dry weight (CDW)" in Section 2.2.2.

AZC: Defined as "Azetidine-2-carboxylic acid (AZC)" in Section 2.2.2.

AFB1: Defined as "aflatoxin B1 (AFB1)" in Section 2.2.3.

AI: Defined as "artificial intelligence (AI)" in Section 4.

Comment 3: Confirm that all figures and tables are self-explanatory and adequately referenced in the text.

Response:

We agree with the reviewer and have revised the references to our figures and tables to align with standard academic writing conventions.

In Section 2.1, the reference was smoothed out to "This process is illustrated in Figure 1".

In Section 3.2, the clumsy reference ("This transformation is detailed in the caption of Figure 2") was corrected to a more professional format: "This sequential degradation pathway is illustrated in Figure 2".

We also corrected typographical errors in Table 1, replacing the full-width Chinese comma ("amylase、cellulase") with proper English punctuation ("amylase, cellulase").

Comment 4: Review statements related to GRAS status and biosafety for scientific accuracy.

Response:

We deeply appreciate this rigorous scientific correction. We realize that attributing the GRAS status to the entire Aspergillus genus was a scientific inaccuracy, as the genus also includes toxigenic strains. We have rectified this across the entire manuscript:

Abstract: The phrase was updated to "...the GRAS (Generally Recognized as Safe) status of specific industrially relevant species (e.g., A. oryzae and A. niger)".

Section 2.2.3: "GRAS certification" was accurately revised to "GRAS status", as the FDA operates on a notification/status framework rather than a "certification" framework.

Sections 3.1 & 3.4: Generalizations were replaced with "Although certain Aspergillus species (e.g., A. oryzae, A. niger) are GRAS..." and "highlighting its FDA GRAS designation for specific uses".

Section 5 (Conclusions): We added a sentence emphasizing safety compliance: "Furthermore, the specific industrial limitations of the GRAS status must be clearly defined and navigated".

Comment 5: Comments on the Quality of English Language: I recommend a final round of professional english language editing to improve sentence structure, eliminate redundancy, ensure consistency in terminology, and enhance overall precision and fluency.

Response:

We completely agree with your assessment. To meet the high editorial standards of Fermentation, we have conducted a top-to-bottom linguistic overhaul to refine sentence structures, eliminate redundancies, and enhance clarity.

Splitting Excessively Long Sentences: Overloaded sentences that hindered readability have been broken down. For example, in the Abstract, a very long sentence was split into two: "Existing literature frequently focuses on isolated compounds or general fungal processes. To fill this gap, this review systematically links...". Similar structural improvements were made in Section 1, Section 3.1, and Section 3.2.

Eliminating Redundancy: We pruned repetitive filler sentences and duplicated logic. For instance, in Section 2.1, we merged redundant phrases detailing the "release of bioactive precursors". In Section 2.2.1, we deleted filler sentences like "It provides new development ideas..." and "Therefore, plants have multiple nutritional and pharmacological active effects" to create a more concise academic tone.

Enhancing Precision and Correcting Scientific Mechanics: We corrected unnatural translations (e.g., changing "high naturalness" to "possess high natural purity" in Section 2.1; "environmental relief" to "mitigating environmental impact"). We also fixed mechanistic inaccuracies, such as the incorrect statement in Section 2.2.2 that the benzoate pathway modifies "polysaccharides"; this was properly corrected to "complex aromatic plant compounds". Furthermore, in Section 3.5, we fixed the error that enzymes "degrade heavy metals," explicitly clarifying that heavy metals are instead "biosorbed or chemically speciated".

Formatting/Punctuation Corrections: We have rigorously applied italics to all Latin species names (e.g., Aspergillus, A. niger) and inserted missing Oxford commas throughout the text to ensure grammatical clarity.

We believe these extensive revisions have significantly improved the scientific rigor, readability, and overall quality of our review article. We are very grateful for your valuable time and guidance, and we hope the revised manuscript is now suitable for publication in Fermentation.

Sincerely,

Kuntao Xu

On behalf of all co-authors