Application of UHPLC-ESI-MS/MS-Based Metabonomic Techniques to Analyze the Cordyceps cicadae Metabolic Profile Changes to the CO(NH2)2 Response Mechanism in the Process of Ergosterol Synthesis
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsJournal Fermentation (ISSN 2311-5637)
Manuscript ID fermentation-3388451
Type Article
Title: Application of UHPLC-MS/MS-based on metabonomic techniques to analyze the Cordyceps cicadae metabolic profile changes on urea response mechanism in the process of ergosterol synthesis.
1- Abstract: Improve the writing of the first paragraphs of the abstract. Regarding previous studies, the phrase is not understood.
2- Introduction
Paragraph: Furthermore, ……in the presence of
CO(NH2)2, and the alteration ……synthesis.
Associate the word urea with its formula
Paragraph: Furthermore, ……in the presence of
urea (CO(NH2)2), and the alteration ……synthesis.
3- Introduction
. The aims of this study is to explain the mechanism to increase of ergosterol yield in the process of C. cicadae
Write the aim in accordance with the title of the paper, it should mention the word urea
4- 2.1. Microorganism and culture conditions: Include a reference number or voucher for the strain and mention the origin in that Laboratory. It is an isolate, or it comes from ATCC or whatever.
5- 2.2. The preparation of C. cicadae samples
The biomass of C. cicadae was prepared according to a method previously described with minor modifications [22] (Su et al., 2021).
Include the minor changes you are referring to and delete (Su et al., 2021);
6- 2.4. UHPLC-MS/MS analysis
6.1- Include in the paragraph the following acronym or the one you consider appropriate or correct: (UHPLC-ESI-OT-MS-MS)
“The analytical UHPLC-MS/MS consisted of a Vanquish UHPLC system (ThermoFisher, Germany) coupled with an Orbitrap Q Exactive HF-X mass spectrometer(Thermo Fisher, Germany) as the detector and equipped with an electrospray source in both positive and negative ion modes”
6.2. Include the analytical quality of water, methanol and FA (write full name). Also the company and country of origin of the solvents and reagents used in this analysis.
7- 3.1. Effect of CO(NH2)2 on the contents of intracellular and extracellular ergosterol
Include in footnote the statistic or significance, if it was applied in these trials
The following final paragraph of the manuscript should be moved to a Conclusion section
In summary, Biomass metabonomic analysis using UPLC-Q-TOF/MS presented a useful tool in identifying ………Therefore, the strategy of urea supplementation was expected to be an effective way to
improve ergosterol synthesis in the fermentation of C. cicadae.
8- Check if the acronym mentioned UPLC-Q-TOF/MS, which refers to quadrupole time-of-flight mass spectrometry and in the UHPLC analysis section, mentions an Orbitrap for which the appropriate acronym is (UHPLC-ESI-OT-MS-MS) or the one you consider appropriate or correct
Once the suggestions have been considered, the paper should be considered for acceptance.
Author Response
Question 1- Abstract: Improve the writing of the first paragraphs of the abstract. Regarding previous studies, the phrase is not understood.
Response: “Regarding previous studies, the phrase is not understood” has been changed into “To improve the yield of ergosterol, in the previous study, we have used the transcriptome technology to explore the difference in gene expression of Cordyceps cicadae by urea in the process of synthesizing ergosterol.”
Question 2- Introduction : Paragraph: Furthermore, ……in the presence of urea (CO(NH2)2), and the alteration ……synthesis.
Response: we have changed “urea” in the sentence “in a previous study, we have proved that urea is the most suitable nitrogen source for ergosterol synthesis by C. cicadae [22]” have changed into “CO(NH2)2 (urea)”
Question 3- Introduction
The aims of this study is to explain the mechanism to increase of ergosterol yield in the process of C. cicadae. Write the aim in accordance with the title of the paper, it should mention the word urea
Response: The last section of introduction has been changed into “In order to further increse the ergosterol field in the fermentation process of C. cicadae, it is particularly important to reveal the metabolic regulation mechanism of ergosterol synthesized by C. cicadae. According to the general knowledge of microbial metabolic regulation, microbial metabolic regulation is mainly carried out at four levels including cell membrane permeability regulation, regional regulation of metabolism, flow regulation of metabolism and rate regulation of metabolism. Thus, the aim the present study is to use UPLC-MS technology with PCA and OPLS-DA and automated processing software platform to systematically screen the key biomarkers in the synthesis process of ergosterol, and revealed the mechanism of urea increasing ergosterol synthesis from four levels of metabolic regulation，to lay foundations to increase of ergosterol yield in the fermentation process of C. cicadae.“
Question 4- 2.1. Microorganism and culture conditions: Include a reference number or voucher for the strain and mention the origin in that Laboratory. It is an isolate, or it comes from ATCC or whatever.
Response: Thank you. We have added reference number (bio-33088) after C. cicadae.
Question 5- 2.2. The preparation of C. cicadae samples
The biomass of C. cicadae was prepared according to a method previously described with minor modifications [22] (Su et al., 2021). Include the minor changes you are referring to and delete (Su et al., 2021);
Response: Thank you, we have deleted (Su et al., 2021) in revised version.
Question 6- 2.4. UHPLC-MS/MS analysis
6.1- Include in the paragraph the following acronym or the one you consider appropriate or correct: (UHPLC-ESI-OT-MS-MS)
“The analytical UHPLC-MS/MS consisted of a Vanquish UHPLC system (ThermoFisher, Germany) coupled with an Orbitrap Q Exactive HF-X mass spectrometer(Thermo Fisher, Germany) as the detector and equipped with an electrospray source in both positive and negative ion modes”
Response: Thank you and your reviewer. We have changed UHPLC-MS/MS into UHPLC-ESI-MS/MS.
6.2. Include the analytical quality of water, methanol and FA (write full name). Also the company and country of origin of the solvents and reagents used in this analysis.
Response: Thank you. We have supplied the information of reagents in first paragraph of “material and methods”
Question 7- 3.1. Effect of CO(NH2)2 on the contents of intracellular and extracellular ergosterol Include in footnote the statistic or significance, if it was applied in these trials.
Response: Thank you very much. These are our careless. In the revised version, all “CO(NH2)2” have changed into “CO(NH2)2”
The following final paragraph of the manuscript should be moved to a Conclusion section
In summary, Biomass metabonomic analysis using UPLC-Q-TOF/MS presented a useful tool in identifying ………Therefore, the strategy of urea supplementation was expected to be an effective way to improve ergosterol synthesis in the fermentation of C. cicadae.
Response: Thank you and your reviewer for a good idea. We have move the final paragraph to a “Conclusion section”
Question 8- Check if the acronym mentioned UPLC-Q-TOF/MS, which refers to quadrupole time-of-flight mass spectrometry and in the UHPLC analysis section, mentions an Orbitrap for which the appropriate acronym is (UHPLC-ESI-OT-MS-MS) or the one you consider appropriate or correct
Response: Thank you and your reviewer. We have changed UHPLC-MS/MS into UHPLC-ESI-MS/MS.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe article provides a detailed and insightful exploration of how urea influences fermentation and ergosterol synthesis in Cordyceps cicadae. By leveraging metabonomics, the study sheds light on the intricate biochemical and physiological mechanisms impacted by urea supplementation, offering a novel perspective on optimizing medium composition for fungal fermentation.
The abstract is well-written and has sufficient information.
The introduction is very naive and short, with limited information. Authors must add recent references for the literature review. The objective of the study is unclear. I recommend adding the hypothesis in a separate paragraph using the latest references and then in the objective.
The methodology is fine, I recommend splitting the heading 2.5 into two different headings.
The results and discussion are fine. I recommend using the hypothesis (from the introduction section) to declare whether the authors achieved their hypothesis or not.
Add the conclusion headings separately from the discussion and must add take-home message for better understanding to reviewers.
Author Response
Question 9-: The article provides a detailed and insightful exploration of how urea influences fermentation and ergosterol synthesis in Cordyceps cicadae. By leveraging metabonomics, the study sheds light on the intricate biochemical and physiological mechanisms impacted by urea supplementation, offering a novel perspective on optimizing medium composition for fungal fermentation.
Response: Thank you and reviewer.
Question 10-The abstract is well-written and has sufficient information.
Response: Thank you and reviewer for your evaluation
Question 11-: The introduction is very naive and short, with limited information. Authors must add recent references for the literature review. The objective of the study is unclear. I recommend adding the hypothesis in a separate paragraph using the latest references and then in the objective.
Response：Thank you for a good idea. We add the sentence : ” According to the general knowledge of microbial metabolic regulation, we speculated that the supplementation of CO(NH2)2 increased ergosterol synthesis from four levels including cell membrane permeability regulation, metabolic regionalization, metabolic fluxes direction and metabolic rate. Thus, the aim the present study is to use UHPLC-ESI-MS/MS technology with PCA and OPLS-DA and automated processing software platform to systematically screen the key biomarkers in the synthesis process of ergosterol to our hypothesis and to lay foundations to further control fermentation condition and increase of ergosterol yield in the fermentation process of C. cicadae.”
Question 12-: The methodology is fine, I recommend splitting the heading 2.5 into two different headings.
Response: the heading have been split into ”Data processing” and “Differential metabolites identification”
Question 13-:The results and discussion are fine. I recommend using the hypothesis (from the introduction section) to declare whether the authors achieved their hypothesis or not.
Response: Thank you and your reviewer. The conclusion was changed into “In summary, biomass metabonomic analysis using UHPLC-ESI-MS/MS presented a useful tool in identifying physiological changes caused by a component in the medium. Based on biomarkers including isocitric acid and α-ketoglutaric acid, glycerol 3-phosphate and dihydroxyacetone phosphate, trehalose, Choline, carnitine, propionyl carnitine, acetyl-L-carnitine, abscisic acid, folinic acid and Cafestol, we proved the our speculation that CO(NH2)2 supplementation significantly promoted the growth and ergosterol synthesis of C. cicadae by increasing cell membrane permeability, changing the metabolic regional and the metabolic flux direction and regulating metabolic rate. Therefore, the strategy of CO(NH2)2 supplementation was expected to be an effective way to improve ergosterol synthesis in the fermentation of C. cicadae.”
Question 14-: Add the conclusion headings separately from the discussion and must add take-home message for better understanding to reviewers.
Response: We have added the conclusion headings
Reviewer 3 Report
Comments and Suggestions for AuthorsThank you for the opportunity to review your work.
The English needs to be revised.
The lines are not numbered, so I do not write where the typos and spelling mistakes are. Lower case needs to be corrected, and write “in vivo” in italics.
Last sentence in the Introduction section needs to be rewritten.
Figure 1 could be improved.
Figure 6 and some parts of the Discussion section should be part of the Results section.
In the section 4.1. you wrote “In the present study, 6 phospho gluconate dehydrogenase was inhibited, the synthesis of NADPH was decreased, the ergosterol systhesis was decreased”. Typo “synthesis” Did you mean study from Wang et al. 2012?
Missing references in the discussion section.
Author Response
Question 15-: The English needs to be revised.
Response: Thank you. The manuscript has been revised by English experts
Question 16-: The lines are not numbered, so I do not write where the typos and spelling mistakes are. Lower case needs to be corrected, and write “in vivo” in italics. Last sentence in the Introduction section needs to be rewritten.
Response: Thank you and your reviewer for such well suggestion. The line number has been addedLast sentence in the Introduction section has been re-written.
Question 17-: Figure 1 could be improved.
Response: Thank you. We have adjusted Figure 1.
Question 18-: Figure 6 and some parts of the Discussion section should be part of the Results section.
Response: Thank you. “To gain insights into the metabolic mechanism in response to CO(NH2)2 and to provide more accurate information of ergosterol synthesis, these different metabolites obtained from the volcano map were made into a heatmap according to their VIP values (Figure 6). They could be divided into seven groups as follows: carbohydrate, amino acid, lipid, nucleotides, vitamins, hormones, and other secondary metabolisms.” has been moved to the Results.
Question 19-: In the section 4.1. you wrote “In the present study, 6 phospho gluconate dehydrogenase was inhibited, the synthesis of NADPH was decreased, the ergosterol systhesis was decreased”. Typo “synthesis” Did you mean study from Wang et al. 2012?
Missing references in the discussion section.
Response: Thank you. The systhesis has been changed into “synthesis”
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have made all the suggested changes.
Comments on the Quality of English LanguageFine
