Optimizing Ergothioneine Biosynthesis and Antioxidant Activity in Agaricus spp. Through Amino Acid Supplementation and Yeast–Peptone Mixtures

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The submitted article aimed to optimize culture media for Ergo production and antioxidant activity in four Agaricus strains by supplementing them with specific amino acid precursors. Also, it examines the impact of yeast extract and peptone, as organic nitrogen sources, on Ergo production and antioxidant activity in the same four Agaricus strains. The suggestions for the authors are listed below:

Page 1, remove from the abstract titles 1Background, 2 Methods, 3 Results and 4 Conclusions, they are just guidelines how to organize abstract.

The introduction is clear, however, instead of just saying "a notable gap," be more specific. What aspects of culture media optimization are lacking? Also, explicitly state why enhancing Ergo production in this widely consumed mushroom is crucial.

In the Material and Methods section, some details are missing.

If possible, add the supplier and purity of the chemicals used.

Explain why the fermentation time was 25 days for the amino acid supplementation experiment and 15 days for the nitrogen source optimization experiment. Is there a specific reason for this difference?

For the Soxhlet extraction with methanol, consider adding the ratio of mushroom powder to methanol used.

In the results section, include standard deviation values in all tables. For the optimization, consider using Standard Score analysis.

In the discussion section, ensure each paragraph directly references specific findings from your study.

Author Response

Answer: Thank you for your insightful comment. We have carefully considered your feedback and have revised lines 97–110 to provide a clearer and more detailed discussion. Specifically, we have strengthened our explanation of the existing gaps in the literature by explicitly identifying the key areas where culture media optimization remains insufficiently explored. In particular, we now highlight the need for systematic research on the precise modulation of the carbon-to-nitrogen ratio, trace element supplementation, and pH regulation—each of which plays a fundamental role in secondary metabolite biosynthesis in fungi. Additionally, we emphasize the lack of studies investigating the interactive effects of these parameters on Ergo production, a crucial factor in achieving optimal metabolic efficiency. Furthermore, we have reinforced our discussion on the significance of enhancing Ergo production, not only due to its well-documented pharmacological benefits but also because of its growing commercial demand in pharmaceutical and nutraceutical industries. These revisions ensure that our introduction is both comprehensive and aligned with the highest academic standards.

Comment 3:  In the Material and Methods section, some details are missing.

If possible, add the supplier and purity of the chemicals used.

Explain why the fermentation time was 25 days for the amino acid supplementation experiment and 15 days for the nitrogen source optimization experiment. Is there a specific reason for this difference?

For the Soxhlet extraction with methanol, consider adding the ratio of mushroom powder to methanol used.

Answer: Thank you for your valuable comments. We have carefully addressed each of your points to enhance the clarity and reproducibility of our methodology.

Supplier and Purity of Chemicals: We have now provided the supplier details and purity specifications of all chemicals used in the study to ensure transparency and reproducibility (Lines 141, 148-152).

Fermentation Time Differences (25 Days vs. 15 Days): The difference in fermentation duration between the amino acid supplementation experiment (25 days) and the nitrogen source optimization experiment (15 days) was based on preliminary trials and existing literature (lines 164–167; 178–182). The amino acid supplementation experiment required an extended fermentation period to allow for sufficient assimilation and metabolic conversion of amino acids into secondary metabolites, particularly Ergo. In contrast, the nitrogen source optimization experiment was conducted over a shorter duration (15 days) as nitrogen metabolism in submerged fungal cultures typically reaches a plateau within this timeframe, and prolonged fermentation could lead to nitrogen depletion or metabolic shifts unfavorable for Ergo biosynthesis. These timeframes were determined through optimization studies to balance maximum yield with metabolic efficiency.

Soxhlet Extraction Ratio: We have now specified the exact ratio of mushroom powder to methanol used in the Soxhlet extraction method to ensure methodological precision and reproducibility. This information is incorporated in the revised section (lines 195–196).

Comment 4: In the results section, include standard deviation values in all tables. For the optimization, consider using Standard Score analysis.

Answer: Thank you for your comment. We have now addressed this issue in in all tables.

Comment 5:  In the discussion section, ensure each paragraph directly references specific findings from your study.

Answer: Thank you for your insightful comment. We appreciate your suggestion to ensure that each paragraph in the discussion section directly references specific findings from our study. Upon reviewing our discussion, we found that our analysis is indeed closely tied to our results. However, to further clarify this connection, we have highlighted key sections where our findings are explicitly referenced. These highlighted portions emphasize the direct link between our experimental results and the interpretations presented in the discussion.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript titled "Optimizing Ergothioneine Biosynthesis and Antioxidant Activity in Agaricus spp. through Amino Acid Supplementation and Yeast-Peptone Mixtures" investigates cost-effective strategies to enhance ergothioneine production in Agaricus mushrooms, a potent antioxidant with significant health benefits.

This study demonstrates that supplementing cultures with 0.5 mM amino acids + 2 g/L yeast extract + 2 g/L peptone optimally enhances ergothioneine biosynthesis, antioxidant activity, and fungal growth. While Agaricus bitorquisexhibited strong metabolic potential, Agaricus bisporus (white) responded more effectively to optimized conditions, offering a scalable alternative for commercial production.These findings provide valuable insights for optimizing antioxidant-rich mushroom cultivation for nutraceutical and functional food applications.

Best regards.

However, several critical aspects require attention to improve the manuscript's clarity, coherence, and overall impact:

Introduction

• Strengths: Provides a solid background on the significance of ergothioneine, its antioxidant potential, and its dietary sources.
• Criticisms:
• The discussion on Agaricus species’ nutritional importance is somewhat generic and could be better contextualized within the study's objectives.
• The transition to submerged fermentation needs more clarity—why this method is preferable over traditional cultivation should be elaborated.
• Some citations (e.g., references 5 and 12) need stronger contextual integration.

Materials and Methods

• Strengths: Detailed experimental design with clear methodological descriptions.
• Criticisms:
• The selection of amino acid concentrations (0.5, 1, 2 mM) should be justified based on prior studies or preliminary trials.
• The description of yeast-peptone supplementation lacks biochemical reasoning—why were these specific concentrations (2+2 g/L, etc.) chosen?
• HPLC analysis: More details on calibration curves, detection limits, and validation protocols would improve transparency.
• Statistical analyses need more details—mentioning post-hoc tests or confidence intervals would strengthen the methodology.

Results

• Strengths: Well-organized presentation of data, effective use of tables.
• Criticisms:
• The impact of amino acid supplementation on fungal growth needs further discussion—what metabolic trade-offs are involved?
• Some statistical results are unclear—were p-values or confidence intervals reported?
• In Table 1, the letter-based statistical grouping is difficult to follow—consider using asterisks or footnotes for better readability.
• The findings on yeast-peptone supplementation would benefit from a mechanistic explanation—how does this combination enhance ergothioneine synthesis?

Discussion

• Strengths: The study is well-supported by literature, linking findings to previous research.
• Criticisms:
• The metabolic pathways of ergothioneine synthesis should be more explicitly discussed—what enzymes or genes might be involved?
• The trade-off between growth rate and ergothioneine production should be examined in more detail—is there a metabolic bottleneck?
• The discussion of yeast extract’s role in enhancing fungal metabolism is somewhat superficial—consider citing studies on yeast-derived growth factors.
• The comparison between A. bisporus and A. bitorquis lacks depth—what specific genetic or metabolic differences contribute to their differing responses?

Conclusions

• Strengths: Effectively summarizes key findings and practical applications.
• Criticisms:
• The industrial relevance of the study could be better emphasized—how can this research be scaled for commercial production?
• The recommendation for metabolic engineering or CRISPR-based modifications is intriguing but should be framed with more caution—are there known genetic modifications that enhance ergothioneine production?
• Consider rewording to make it more impactful—how does this study advance the field beyond prior research?

Minor Issues & Formatting

• Figures & Tables: Consider adding graphical abstracts or summary diagrams to illustrate key findings.
• Grammar & Style:
• Some sections are redundant (e.g., repeated explanations of antioxidant activity mechanisms).
• Scientific terminology is mostly appropriate but could be more precise and concise in places.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Answers to the queries from Reviewer 2:

Answer: We sincerely appreciate the reviewer’s valuable feedback. The necessary revisions have been made to clarify the rationale behind the selection of amino acid and nitrogen source concentrations, as well as to enhance the transparency of the statistical analyses.

Justification for Amino Acid Concentrations: The selection of amino acid concentrations (0.5, 1, and 2 mM) has been explicitly justified in the revised manuscript, drawing on a comprehensive literature review and prior studies, as referenced in the updated text (lines 161–162).

Rationale for Yeast Extract and Peptone Supplementation: The justification for yeast extract and peptone supplementation, including their roles as nitrogen sources and growth factors essential for fungal metabolism and secondary metabolite biosynthesis, has been elaborated in Lines 115–126. The chosen concentrations (2+2 g/L, etc.) align with standard practices in fungal cultivation and were selected based on prior research (Lines 174-178).

HPLC analysis: We have now provided additional details regarding the HPLC analysis. Specifically, we have included comprehensive information on the calibration curves, detection limits, and validation protocols to enhance transparency. This includes a detailed description of the calibration standards used, the linearity range, the limits of detection (LOD) and quantification (LOQ), as well as the validation parameters such as accuracy, precision, and reproducibility. These updates aim to ensure clarity and robustness in our analytical methodology (Lines 254-258).

Clarification of Statistical Analyses: We have clarified that mean comparisons were conducted using the LSD test at a 0.05 probability level (Lines 262-265).

Comment 3: The impact of amino acid supplementation on fungal growth needs further discussion—what metabolic trade-offs are involved?

Some statistical results are unclear—were p-values or confidence intervals reported?

In Table 1, the letter-based statistical grouping is difficult to follow—consider using asterisks or footnotes for better readability.

The findings on yeast-peptone supplementation would benefit from a mechanistic explanation—how does this combination enhance ergothioneine synthesis?

Answer: Thank you for your insightful comment. The observed reduction in fungal growth upon amino acid supplementation is a result of metabolic trade-offs between primary and secondary metabolism. Ergo biosynthesis requires significant metabolic energy and precursor molecules, leading to a shift in resource allocation from growth-related processes to secondary metabolite production. Additionally, elevated Ergo levels may trigger feedback inhibition mechanisms that restrict metabolic flux toward fungal proliferation. Furthermore, amino acid supplementation likely modulates oxidative stress response pathways and TOR signaling, both of which are crucial regulators of fungal growth. The alteration in nutrient assimilation dynamics, particularly the carbon-to-nitrogen balance, may also contribute to growth suppression by shifting metabolic priorities. These findings highlight the intricate balance fungi maintain between growth and secondary metabolite biosynthesis, emphasizing the need for careful optimization of amino acid supplementation strategies (Lines 370- 390).

Lines 262–265 of the manuscript provide details on the statistical analysis conducted. Due to space constraints and to maintain clarity, we did not include a full analysis of variance (ANOVA) in the main text. However, as stated in the Materials and Methods section, means comparison was performed with a significance threshold of p < 0.05. This ensures the robustness and reliability of our findings. Confidence intervals were not explicitly reported, but the statistical framework employed is designed to account for variability and significance testing.

We have now added asterisks to improve the clarity and understanding of the letter-based groupings in the mean comparison tables.

In addition, lines 434–446 of the manuscript provide a mechanistic explanation of how yeast-peptone supplementation enhances ergothioneine (Ergo) synthesis

Comment 4: The metabolic pathways of ergothioneine synthesis should be more explicitly discussed—what enzymes or genes might be involved? The trade-off between growth rate and ergothioneine production should be examined in more detail—is there a metabolic bottleneck?

The discussion of yeast extract’s role in enhancing fungal metabolism is somewhat superficial—consider citing studies on yeast-derived growth factors. The comparison between A. bisporus and A. bitorquis lacks depth—what specific genetic or metabolic differences contribute to their differing responses?

Answer: Thank you for this insightful comment.

Metabolic Pathways of Ergothioneine Biosynthesis: This section has been expanded to provide a more detailed explanation of the biosynthetic pathways of ergothioneine, with a particular focus on the key enzymes and genes involved. The revisions include a more precise introduction of the enzymes participating in this pathway and their roles in converting precursor amino acids into ergothioneine (Lines 360-369).

Balance Between Growth Rate and Ergothioneine Production: To provide a deeper analysis of this balance, the metabolic constraints arising from intracellular resource competition have been addressed. Additionally, key metabolic pathways and enzymatic regulations influencing ergothioneine production have been examined, and optimization strategies for culture conditions to minimize these interferences have been proposed (Lines 380–407).

Role of Yeast Extract in Enhancing Fungal Metabolism: This section has been revised to elaborate on the role of compounds present in yeast extract, including amino acids, peptides, and vitamins, in regulating fungal metabolism. Furthermore, references to recent studies demonstrating the impact of yeast-derived growth factors on the enhanced production of secondary metabolites have been incorporated (Lines 432–446).

Comparison Between A. bisporus and A. bitorquis: To enhance the depth of comparison between these two species, additional explanations regarding genetic and metabolic differences related to ergothioneine production have been included. In this regard, the expression of key genes involved in the ergothioneine biosynthetic pathway and the regulatory mechanisms influencing metabolic responses under different conditions have been highlighted (Lines 482–508).

Comment 5: The industrial relevance of the study could be better emphasized—how can this research be scaled for commercial production? The recommendation for metabolic engineering or CRISPR-based modifications is intriguing but should be framed with more caution—are there known genetic modifications that enhance ergothioneine production? Consider rewording to make it more impactful—how does this study advance the field beyond prior research?

Answer: Thank you for your valuable feedback. In response to your comments, we have explicitly emphasized the industrial relevance of our findings by discussing the scalability of optimized amino acid supplementation for commercial production. Additionally, we have framed the discussion on metabolic engineering and CRISPR-based modifications with greater caution, acknowledging the limited studies on genetic enhancements for Ergo production in Agaricus species. Lastly, we have refined our wording to better highlight how this study advances the field by integrating genetic, environmental, and nutritional factors to optimize fungal bioactive compound production. These revisions can be found in the In the Results section, lines 519 to 531.

Comment 6:  Minor Issues & Formatting

Figures & Tables: Consider adding graphical abstracts or summary diagrams to illustrate key findings.

Grammar & Style: Some sections are redundant (e.g., repeated explanations of antioxidant activity mechanisms).

Scientific terminology is mostly appropriate but could be more precise and concise in places.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Answer: We appreciate the reviewer’s insightful suggestions and have addressed the minor issues accordingly:

graphical abstract is now provided.

Grammar & Style: Redundant explanations, particularly regarding antioxidant activity mechanisms, have been streamlined to improve conciseness and avoid repetition. Additionally, the scientific terminology has been refined for greater precision and clarity.

English Language Quality: The manuscript has been carefully revised to enhance readability and ensure that the research findings are conveyed more clearly and effectively.

We sincerely appreciate the reviewer’s valuable feedback, which has helped improve the clarity and presentation of our study.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Article
Optimizing Ergothioneine Biosynthesis and Antioxidant Activity in Agaricus spp. through Amino Acid Supplementation and Yeast-Peptone Mixtures

A brief summary
The authors attempted to answer interesting and important questions from an application point of view. It seems, however, that only part of these questions have been answered in the presented study.

Broad comments
1. The aim of this study was to determine if and how the supplementation of 3 amino acids plus yeast extract and peptone would affect ergothioneine levels in 4 species of Agaricus mushrooms.

2. The work is innovative and interesting in that it provides information on the possibility of increasing the content of valuable metabolites in food products.

3. Since the title refers to antioxidant activity, perhaps it would be appropriate to test this parameter with a larger number of poplar tests and try to relate the results more closely to ergothioneine and polyphenol content.

4. Some notes on methodology:
a. Please add information on the freeze-drying paraneters (pressure, tempetature), the model of grinder used to shredding the dried samples and the size of the particles obtained.
b. How concentrated was the extract? To dryness, to a certain volume?
c. If a method was used that allowed simultaneous determination of polyphenols and ergothioneine, why were these polyphenols not determined and total phenolic content had to be determined? Which specific polyphenols are present in these mushrooms?
d. Please list the manufacturers of the reagents and standard used.

5. In conclusion, the influence of experimental factors on ergothioneine levels in individual mushrooms aspecies and their growth is very well described. What is missing is a summary of the antioxidant activity, as mentioned in the title of the paper, and content of polyphenolic compounds, which was also declared to be investigated.

6. Literature items are cited in accordance with the scope of the work.

7. Additional comments and suggestions can be found below.

Specific comments

Lines 14, 18, 28 and 36 However, it seems that the inclusion of the titles of the different parts of the summary is probably not necessary.

Tables 3 and 4 Please arrange the table headings and data so that they are legible. 

Author Response

Answers to the queries from Reviewer 3:

Answer: We appreciate your insightful suggestion regarding the assessment of antioxidant activity. In our study, we have evaluated this parameter using two well-established antioxidant assays: DPPH and FRAP, which provide complementary insights into the radical-scavenging ability and reducing power of the samples. These methods were selected based on their reliability and widespread use in antioxidant research.

Furthermore, we have discussed the relationship between ergothioneine and polyphenol content in the context of antioxidant activity in the manuscript. Please refer to lines 408–425 for a detailed discussion. We believe these analyses sufficiently capture the antioxidant potential of the samples while maintaining the study's focus. However, we acknowledge that incorporating additional assays could further enrich the findings and will consider this aspect in future research. Thank you for your valuable feedback.

Comment 4:  Some notes on methodology:

1. Please add information on the freeze-drying paraneters (pressure, tempetature), the model of grinder used to shredding the dried samples and the size of the particles obtained.
2. How concentrated was the extract? To dryness, to a certain volume?
3. If a method was used that allowed simultaneous determination of polyphenols and ergothioneine, why were these polyphenols not determined and total phenolic content had to be determined? Which specific polyphenols are present in these mushrooms?
4. Please list the manufacturers of the reagents and standard used.

Answer: Thank you for your insightful comments and valuable suggestions, which have helped improve the clarity and rigor of our manuscript. Below are our detailed responses to your queries:

a & b) The requested details regarding freeze-drying parameters, grinding process, and extract concentration have been provided in lines 193–198 of the revised manuscript.

1. c) In this study, total phenolic content (TPC) was determined instead of individual polyphenols due to the primary focus on ergothioneine quantification and the need for a standardized assessment of overall antioxidant capacity across different Agaricus strains. While analytical methods enabling the simultaneous determination of polyphenols and ergothioneine exist, the selected approach was specifically optimized for ergothioneine quantification, with TPC serving as a complementary measure of the overall phenolic contribution.

Moreover, individual polyphenol profiling was beyond the scope of this study, as the primary objective was to evaluate ergothioneine production under different cultivation conditions. However, prior research has identified phenolic acids (e.g., gallic acid, p-hydroxybenzoic acid, and protocatechuic acid) and flavonoids (e.g., quercetin and catechin) as predominant polyphenols in Agaricus species. Future investigations may focus on detailed polyphenol characterization to provide deeper insights into their interactions with ergothioneine biosynthesis.

1. d) The necessary details regarding the manufacturers of reagents and standards have been incorporated into the Materials and Methods section (Lines 141, 148-152).

We sincerely appreciate your constructive feedback, which has helped refine the manuscript. Thank you for your time and effort in reviewing our work.

 Comment 5: In conclusion, the influence of experimental factors on ergothioneine levels in individual mushrooms species and their growth is very well described. What is missing is a summary of the antioxidant activity, as mentioned in the title of the paper, and content of polyphenolic compounds, which was also declared to be investigated.

Answer: Thank you for your valuable feedback. We appreciate your thorough evaluation of our work. In response to your comment, we have refined the conclusion (Lines 532–539) to explicitly summarize the findings on antioxidant activity and polyphenolic content. We believe these revisions improve both the clarity and comprehensiveness of the conclusion.

Comment 6:  Literature items are cited in accordance with the scope of the work.

Answer: Thank you for your valuable feedback. We have carefully selected and cited literature relevant to the scope of our research to ensure a well-supported discussion, and we appreciate your acknowledgment of this aspect.

Comment 7:  Additional comments and suggestions can be found below.

Specific comments

Lines 14, 18, 28 and 36 However, it seems that the inclusion of the titles of the different parts of the summary is probably not necessary.

Tables 3 and 4 Please arrange the table headings and data so that they are legible.

Answer: Thank you for your detailed feedback. We have revised the summary and removed the unnecessary section titles for improved readability. Additionally, we have adjusted the formatting of Tables 3 and 4 to ensure clarity and legibility of the headings and data.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors addressed all my comments in a constructive manner and made significant improvements to the manuscript. I believe that the article has benefited greatly.