Fungal Biodegradation of Procyanidin in Submerged Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled Fungal biodegradation study of procyanidin in submerged fermentation” have demonstrated significant results including estimation of biodegradation procyanidin (PC) by A. niger GH1. In this study, the degradation or biotransformation of procyanidin was evaluated.

In my opinion, the topic of the study is important and current, but it needs a slight correction. Authors should correct manuscript according to the suggestion.

General comments:

Although Aspergillus sp. are known for their efficiency in biodegradation/biotransformation of various compounds, some strains produce aflatoxins (mycotoxin) as secondary metabolites adding another problem to the already existing environmental problem. On the other hand, it is known that the ability to synthesize mycotoxins is strain-dependent and is conditioned by certain environmental factors. I wonder, if the Authors performed tests confirming the safety of Aspergillus niger GH1 and excluded the production of aflatoxin?.

Moreover, aim of the study should be more clearly marked and described. There is no clearly defined aim of the research. On the one hand, the Authors emphasize that PC is a compound present in coffee and has health-promoting properties, on the other hand, the methods of its removal are important, but why? Please justify. If PC degradation products are important, their possible use should be highlighted.

Another controversial area is the enzyme that is synthesized by A. niger GH1 and used for biodegradation/biotransformation of PC. The Authors do not know what enzyme it is („potential procy- anidin-degrading enzyme”), they do not name this enzyme, it is not known from which group the enzyme is and what catalytic properties it has.

 

Major issus:

Line 61-73: this fragment fits better into the discussion

Line 133: It should be „10⁷”

Line 200-209: please describe the enzymes synthesized by microorganisms that are involved in the biodegradation of PC

Line 230: please use the more scientific term PC removal rather than „disappearance”, please check in the whole text

 

Author Response

Response to comments made by reviewer 1:

Comment 1: 
The manuscript entitled Fungal biodegradation study of procyanidin in submerged fermentation” have demonstrated significant results including estimation of biodegradation procyanidin (PC) by A. niger GH1. In this study, the degradation or biotransformation of procyanidin was evaluated. In my opinion, the topic of the study is important and current, but it needs a slight correction. Authors should correct manuscript according to the suggestion.
Response 1: 
First of all, we want to thank the reviewer for pointing this out. 

Comment 2: 
Although Aspergillus sp. are known for their efficiency in biodegradation/biotransformation of various compounds, some strains produce aflatoxins (mycotoxin) as secondary metabolites adding another problem to the already existing environmental problem. On the other hand, it is known that the ability to synthesize mycotoxins is strain-dependent and is conditioned by certain environmental factors. I wonder, if the Authors performed tests confirming the safety of Aspergillus niger GH1 and excluded the production of aflatoxin?.
Response 2: 
We agree with this comment related to the ability of the fungi to synthesize mycotoxins is strain-dependent and is conditioned by certain environmental factors.   Aspergillus niger GH1 is a non-toxigenic fungal strain. We have evaluated the production of aflatoxin of this strain in several substrates fermented under solid state conditions. Although we did not evaluate the production of aflatoxins about this culture system, we have as the reference of the use of condensed tannin rich substrates (coffee pulp, apple pomace, pecan nut shells, etc). For us, to select a non-toxigenic fungus, in this case GH1 among 15 prominent fungal strains (https://doi.org/10.1139/cjm-2014-0163) belonging to Aspergillus niger was decisive previous to deposit the microorganism in the collection of University of Minho, Portugal (MUM 02.48) 

Comment 3: 
Moreover, aim of the study should be more clearly marked and described. There is no clearly defined aim of the research. On the one hand, the Authors emphasize that PC is a compound present in coffee and has health-promoting properties, on the other hand, the methods of its removal are important, but why? Please justify. If PC degradation products are important, their possible use should be highlighted.
Response 3: 
The aim of the research was rewritten in order to avoid any confusion. 
For us, the study of the degradation of condensed tannins or procyanidins is essential to provide technological alternatives that avoid the problems of pollution generated by the accumulation of agro-industrial waste rich in this type of compounds. We have highlighted this in the document. In addition to this, we have discovered that the degradation of this type of compounds allows the generation of oligomeric intermediates or procyanidins that have anti-neurodegenerative potential, a study that we are carrying out at the moment. 

Comment 4: 
Another controversial area is the enzyme that is synthesized by A. niger GH1 and used for biodegradation/biotransformation of PC. The Authors do not know what enzyme it is („potential procy- anidin-degrading enzyme”), they do not name this enzyme, it is not known from which group the enzyme is and what catalytic properties it has.
Response 4: 
Indeed, the reviewer is right, and we have decided not to name it because during this study it was shown that this enzyme was synthesized. Today we know that it is a dio-oxygenase enzyme and it will be reported in the paper that reveals the metabolic pathway of fungal degradation of procyanidins that has been prepared for another article due to the amount of experimental work involved.

Major issus:
Line 61-73: this fragment fits better into the discussion
Done
Line 133: It should be „107”
Done
Line 200-209: please describe the enzymes synthesized by microorganisms that are involved in the biodegradation of PC
Done
Line 230: please use the more scientific term PC removal rather than „disappearance”, please check in the whole text
Done

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates fungal degradation of procyanidins from coffee pulp using Aspergillus niger GH1. Procyanidin C1 was degraded, yielding novel compounds, including a monomer such as catechin or epicatechin. The findings confirm A. niger GH1's potential for procyanidin biotransformation and provide insights into biodegradation pathways. Below are major and minor comments and suggestions for improving the manuscript:

Title:

Remove the word "study" for a more concise and impactful title.

Abstract:

Briefly highlight the potential of coffee pulp as a promising by-product for valorisation, focusing on its abundance and current underutilisation.

Add a novelty statement at the end of the abstract.

Keywords:

Include "extracellular enzyme" as one of the keywords.

Introduction:

Line 35: Provide examples of fungal enzymes such as laccase, peroxidase, dioxygenase, and cytochrome P450, which are involved in biodegradation/biotransformation. Cite the following references: https://link.springer.com/article/10.1007/s11274-023-03737-7, https://doi.org/10.1016/B978-0-12-820524-2.00013-4.

Line 69: Avoid capitalising each word in "High-Performance Liquid Chromatography." Line 87: Remove redundant mention of "GRAS."

Results and Discussion:

Figure 1: Add explanations for ET1, ET2, and ET3 in the figure legend.

Figure 2: Verify the time units; if they are in hours, correct them. Include error bars in all graphs for clarity and statistical relevance.

Figure 3: Add MS data to the figure or supplementary materials. The chromatograms (A and B) appear similar, with differences only in peak shifts. Address this visually or in the text. The catechin/epicatechin peak is minimal, suggesting limited procyanidin degradation. Discuss this limitation in the text. Explain how the m/z of 289 was linked to catechin or epicatechin. Include comparisons of MS fragments from standard catechin or epicatechin to confirm identification.

Suggestion: Instead of using extracellular extracts, directly incubate procyanidin with live A. niger cultures and then perform HPLC-MS. This approach may yield more significant degradation products, as enzyme activity could be affected during extraction.

Line 252: Mention monooxygenases (cytochrome P450) as key enzymes in degrading aromatic xenobiotics. Cite this study: https://doi.org/10.1016/j.enzmictec.2022.110102.

Expand on the significance of newly formed compounds and their potential applications.

Discuss limitations such as enzyme stability and scalability, proposing future research directions to address these challenges.

Conclusion:

The conclusion summarises the findings well but should emphasise practical applications, such as the role of this process in sustainable bioprocessing or industrial waste management.

Author Response

Comment 1:
This study investigates fungal degradation of procyanidins from coffee pulp using Aspergillus niger GH1. Procyanidin C1 was degraded, yielding novel compounds, including a monomer such as catechin or epicatechin. The findings confirm A. niger GH1's potential for procyanidin biotransformation and provide insights into biodegradation pathways. Below are major and minor comments and suggestions for improving the manuscript:
Response 1: 
Authors thank all the comments and suggestions made to improve our contribution

Title:
Remove the word "study" for a more concise and impactful title.
Done

Abstract:
Briefly highlight the potential of coffee pulp as a promising by-product for valorisation, focusing on its abundance and current underutilisation. Add a novelty statement at the end of the abstract.
Done

Keywords:
Include "extracellular enzyme" as one of the keywords.
Done

Introduction:
Line 35: Provide examples of fungal enzymes such as laccase, peroxidase, dioxygenase, and cytochrome P450, which are involved in biodegradation/biotransformation. Cite the following references: https://link.springer.com/article/10.1007/s11274-023-03737-7, https://doi.org/10.1016/B978-0-12-820524-2.00013-4.
Done, but included in discussion section

Line 69: Avoid capitalising each word in "High-Performance Liquid Chromatography." Line 87: Remove redundant mention of "GRAS."
Done

Results and Discussion:
Figure 1: Add explanations for ET1, ET2, and ET3 in the figure legend.
Done

Figure 2: Verify the time units; if they are in hours, correct them. Include error bars in all graphs for clarity and statistical relevance.
It is correct

Figure 3: Add MS data to the figure or supplementary materials. The chromatograms (A and B) appear similar, with differences only in peak shifts. Address this visually or in the text. The catechin/epicatechin peak is minimal, suggesting limited procyanidin degradation. Discuss this limitation in the text. Explain how the m/z of 289 was linked to catechin or epicatechin. Include comparisons of MS fragments from standard catechin or epicatechin to confirm identification.
Explanation was included

Suggestion: Instead of using extracellular extracts, directly incubate procyanidin with live A. niger cultures and then perform HPLC-MS. This approach may yield more significant degradation products, as enzyme activity could be affected during extraction.
Ok, check the blue highlighted section

Line 252: Mention monooxygenases (cytochrome P450) as key enzymes in degrading aromatic xenobiotics. Cite this study: https://doi.org/10.1016/j.enzmictec.2022.110102.
Expand on the significance of newly formed compounds and their potential applications.
Discuss limitations such as enzyme stability and scalability, proposing future research directions to address these challenges.
Ok

Conclusion:
The conclusion summarises the findings well but should emphasise practical applications, such as the role of this process in sustainable bioprocessing or industrial waste management.
Ok, check the blue highlighted section

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

All the responses are well clear and satisfactory.

Good luck.