Degradation of Ochratoxin A by a UV-Mutated Aspergillus niger Strain
, Yongli Ye 1, Yang Yang 1, Jian Yu 1, Meng Wang 3, Yi Zheng 4 and Xiulan Sun 1,*Round 1
Reviewer 1 Report
The study is interesting and methodologically well done, but a major revision of the English language and improving redaction are needed.
The title “Study and application of ochratoxin A by UV-mutated Aspergillus niger” is confusing, I suggest changing it for the actual objective of the study. Could be “Degradation of ochratoxin A by a UV-mutated Aspergillus niger strain”
Please make a major revision of the English language: example in the abstract:
“Ochratoxin A (OTA), IS a mycotoxin THAT can contaminate a wide range of crops such as grains
and grapes”.
“In this study, a novel FUNGAL mutant strain (FS-UV-21)…”
Please improve the redaction, the abstract is not clear. The same for the introduction.
In introduction
“Furthermore, there is a lack of understanding of the degradation products of OTA and their biotransformation in degrading strain”. Please explain what you mean by “degradation products”
Meaning of “actual samples”
In conclusion
Is needed clarification about the metabolites generated by the FS-UV-21 degradation (e.g. OTα). Are they safe or not? The description of the cytotoxicity of OTA is well known.
Author Response
We would like to thank the reviewers and editor for the time they spent and their helpful comments on our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our review article. We have studied comments carefully and have made correction and marked up using the “Track Changes” function. We hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the editors’ and reviewer’s comments are as following.
The study is interesting and methodologically well done, but a major revision of the English language and improving redaction are needed.
Comments and suggestions:
-The title “Study and application of ochratoxin A by UV-mutated Aspergillus niger” is confusing, I suggest changing it for the actual objective of the study. Could be “Degradation of ochratoxin A by a UV-mutated Aspergillus niger strain”
Author respond: Yes, we agree.
Thank you for your comment. After reflection and reconsideration, we have taken your suggestion and improved the title. The new title is: Degradation of ochratoxin A by a UV-mutated Aspergillus niger strain.
-Abstract: Please make a major revision of the English language: example in the abstract:
“Ochratoxin A (OTA), IS a mycotoxin THAT can contaminate a wide range of crops such as grains and grapes”.
“In this study, a novel FUNGAL mutant strain (FS-UV-21)…”
Please improve the redaction, the abstract is not clear. The same for the introduction.
Author respond: Yes, we agree.
Thanks for your recommendation and comments which were highly insightful and enabled us to greatly improve the quality of our manuscript. We have revised the whole manuscript carefully according to suggestion. And sorry for our improper expression confused you. All the English issues have been revised throughout under the help of native English speaker in the manuscript. The corresponding modifications in red color in the manuscript were also added. For example:
In line 3-17: Abstract: Ochratoxin A (OTA), is a mycotoxin that can contaminate a wide range of crops such as grains and grapes. In this study, a novel fungal mutant strain (FS-UV-21) with a high OTA degradation rate (74.5%) was obtained from Aspergillus niger irradiated with ultraviolet light (15 W for 20 min). The effect of pH, temperature and inoculation concentration on the degradation of OTA by FS-UV-21 were investigated, and the results revealed that the detoxification effect was optimal (89.4%) at a pH of 8 and a temperature of 30 ℃. Ultra-performance liquid chromatography-tandem mass spectrometry was used to characterize the degraded products of OTA, and the main degraded product was ochratoxin α. Triple quadrupole-linear ion trap-mass spectrometry combined with LightSight software was used to analyze the biotransformation pathway of OTA in FS-UV-21, to trace the degraded products, and to identify the main metabolite, P1 (C19H18ClNO6，m/z 404). After the FS-UV-21 strain was treated with OTA, the HepG2 cellular toxicity of the degradation products was significantly reduced. For the real sample, FS-UV-21 was used to remove OTA from wheat bran contaminated by mycotoxins through fermentation, resulting in the degradation of 59.8% of OTA in wheat bran. Therefore, FS-UV-21 can be applied to the degradation of OTA in agricultural products and food.
In line 27-29: Therefore, OTA contamination of foods and feed should be carefully managed 10. However, many preventive measures have been taken to avoid the OTA contamination 11, many grains are still tainted by the mycotoxin.
In line 45: Furthermore, there is a lack of understanding of the degradation products of OTA and their biotransformation by degrading strains 10, 24.
In line 62: (iii) to study the ability of the strain to degrade different concentrations of OTA and to optimize the factors.
In line 76: …, and the results showed that the degradation rate of OTA of mutant strain no. 21 (FS-UV-21) was the highest (74.5%) and significantly higher than that of the wild type strain FS10 (55.6%).
In line 114: From OTA concentrations ranging from 0.1 to 5.0 μg/mL, the degradation rates were greater than 60%.
In line 162-163: The OTA MS/MS spectrum is shown in Fig. 4C, with precursor ion 402.0065[M-H]-. The MS/MS spectrum of peak A is shown in Fig. 4D, with precursor ion 255.0062[M-H]-.
In line 167: Structural elucidation was carried out using MS Finder, and OTα had the highest score (3.4410).
In line 189-191: Therefore, P1 is the most likely transformation product of OTA in the FS-UV-21 strain.
In line 195-196: It is also possible that these metabolites are not related to the formation of OTα, but are novel metabolites of OTA generated in A. niger FS-UV-21 strains.
In line 293: The A. niger mutant strain was obtained by UV irradiation as described by Sun et al. 35.
-In introduction: “Furthermore, there is a lack of understanding of the degradation products of OTA and their biotransformation in degrading strain”. Please explain what you mean by “degradation products”
Author respond: Yes, we agree.
Thank you for your suggestions. The “degradation products” refer to other substances formed by OTA decomposing or producing other chemical reactions under the action of strains or enzymes. There are many experiments that have demonstrated that OTα is a degradation product of OTA, but there are still many products that biodegrade OTA that are not clear, so I raise this question in the introduction.
-Meaning of “actual samples”
Author respond: Yes, we agree.
Thank you for your suggestions. Most of the existing degradation studies are carried out in the culture medium system without combining the data of the “actual samples”. So “actual samples” refer to food matrices such as wheat, peanuts, corn, etc. “Actual” is not the best choice of word, we have used “real”. we have replaced through whole the text. As follow:
In line 14: For the real sample, FS-UV-21 was used to remove OTA from wheat bran contaminated by mycotoxins through fermentation.
In line 46: Most studies have not used real samples, …
In line 48: …but also to define the mechanism of detoxification and to use real samples.
-In conclusion: Is needed clarification about the metabolites generated by the FS-UV-21 degradation (e.g. OTα). Are they safe or not? The description of the cytotoxicity of OTA is well known.
Author respond: Yes, we agree.
Thank you for your suggestions. Thank you for your comment. According to your questions, we have made improvements and modifications. As follow:
In line 247-268: Our study shows that the A. niger FS-UV-21 strain we identified has a good degradation effect on OTA, and the highest degradation rate was 89.36% after optimizing the conditions. UPLC-q-ToF identification confirmed that OTA degradation product degraded by A. niger FS-UV-21 was OTα (C11H8ClO5). Among the reported degradation products, OTα is considered to be non-toxic or less toxic [52,53]. Q-Trap-MS detection combined with LightSight software analysis identified three related biotransformation metabolites, in which P1 (C19H18ClNO6) was likely to be a new metabolite of OTA generated by demethylation and oxidation in the FS-UV-21 strain. The results of this experiment need to be further confirmed. HepG2 cytotoxicity experiments showed that OTA not only significantly affected the morphology of HepG2 cells, but also led to decreased cell viability or even death. The toxicity of OTA to HepG2 cells was significantly reduced after FS-UV-21 treatment, which is beneficial to the safe application of FS-UV-21 in agricultural products. The application of FS-UV-21 in the fermentation of wheat bran could effectively reduce the content of OTA. However, the application of A. niger to degrade OTA in real samples is still in its infancy, and it cannot be directly used for OTA degradation in food. During the fermentation of wheat bran or other real samples, FS-UV-21 will improve or reduce the quality of the samples, whether the processed samples are safe as feed, and whether they will cause harm to the environment, all of which need to be verified by experiments. Therefore, the biotransformation process of OTA needs to be further explored by nuclear magnetic resonance technology 33, and safety assessment methods are needed to determine the toxicity of the degradation products, such as better cytotoxicity assays and animal models of toxicity.
Reviewer 2 Report
In this research different mutant strains were obtained from Aspergillus niger irradiated with ultraviolet light to assess their degradation ability of the mycotoxin ochratoxin A (OTA). The mutant strain FS-UV-21 were selected due to its high OTA degradation rate and the pH, temperature and spore concentration were optimized to maximize the OTA degradation. Moreover, OTA degradation products were identify by UPLC-q-TOF and their cytotoxicity were assessed. Although the research is not highly novel, the results can be considered of interest for Toxins journal. However, several problems/doubts should be solved before obtaining a suitable manuscript to be published:
- Figure 1C. Virtually, the degradation rate of OTA by the mutant strains FS-UV-21, FS-UV-1 or FS-UV-5 is quite similar. Why was only one strain selected for further studies? Perhaps the following studies would yield even better results for these other mutant strains. This issue should clarify in the manuscript.
- Was the quantification of the degradation products performed? In this regard, the authors should provide the data. Otherwise, the term “analysis” should be replaced by “identification” of degradation products.
Author Response
We would like to thank the reviewers and editor for the time they spent and their helpful comments on our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our review article. We have studied comments carefully and have made correction and marked up using the “Track Changes” function. We hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the editors’ and reviewer’s comments are as following.
In this research different mutant strains were obtained from Aspergillus niger irradiated with ultraviolet light to assess their degradation ability of the mycotoxin ochratoxin A (OTA). The mutant strain FS-UV-21 were selected due to its high OTA degradation rate and the pH, temperature and spore concentration were optimized to maximize the OTA degradation. Moreover, OTA degradation products were identify by UPLC-q-TOF and their cytotoxicity were assessed. Although the research is not highly novel, the results can be considered of interest for Toxins journal. However, several problems/doubts should be solved before obtaining a suitable manuscript to be published.
Thanks for your recommendation and comments which were highly insightful and enabled us to greatly improve the quality of our manuscript. We have revised the whole manuscript carefully according to suggestion.
-Figure 1C. Virtually, the degradation rate of OTA by the mutant strains FS-UV-21, FS-UV-1 or FS-UV-5 is quite similar. Why was only one strain selected for further studies? Perhaps the following studies would yield even better results for these other mutant strains. This issue should clarify in the manuscript.
Author respond: Yes, we agree.
Your suggestions are of great help to the improvement of our manuscript. We have made corresponding modifications and answered the above questions in detail.
In the pre-experiment, we selected strains with higher degradation rates for passage experiments (FS-UV-1, FS-UV-5 and FS-UV-21), and found that although the degradation rates of strains FS-UV-1 and FS-UV-5 were comparable, the passage stability was not very good, so the other two were not specifically analyzed in the paper. The following discussion has been added:
In line 76-78: Although strains FS-UV-1 and FS-UV-5 had a similar degradation rate to FS-UV-21, they showed poor passage stability in the passage experiment, so FS-UV-21 was selected for subsequent experiments.
-Was the quantification of the degradation products performed? In this regard, the authors should provide the data. Otherwise, the term “analysis” should be replaced by “identification” of degradation products.
Author respond: Yes, we agree.
Thank you for your comment and apologize for our carelessness. We did not quantify degradation products, so we have modified the term "analysis", as follows:
In line 142: 2.4. Identification of OTA degradation products
In line 249-250: UPLC-q-ToF identification confirmed that OTA degradation product degraded by A. niger FS-UV-21 was OTα (C11H8ClO5).
In line 345: 4.8. Extraction and identification of OTA degradation products
Author Response File: 
Author Response.pdf
Reviewer 3 Report
The presented study entitled “Study and application of ochratoxin A by UV-mutated Aspergillus niger”, investigates OTA detoxification using a novel isolated mutant strain. The novelty of the study is based on the screening and identification of a novel mutant strain of A. niger, and the investigation of its degradation products. Finally, the optimized factors for OTA detoxification were tested in an OTA spiked wheat bran sample. In general, the study is well structured and executed.
The following suggestions should be considered by the authors:
- Title: In my opinion, the title of the paper could be improved, do not indicate the main topics of the study.
- Line 10: “…and the main degraded product was ochratoxin α, which exhibited low toxicity”, in the study cytotoxicity evaluation of the degradation products was carried out in group, not specifically with ochratoxin α. Please, correct this sentence to make more precise.
- Line 13: “For the actual sample,…” “Actual” is not the best choice of word. I suggest use “real”. Please, replace through whole the text.
- Line 15: “Therefore, FS-UV-21 has broad application prospects in the degradation of OTA in agricultural products and foods”, this sentence is nonspecific and idealistic.
- Lines 26-28: “Therefore, OTA…tainted by the mycotoxin”, this sentence is too long. Sentences excessively long are difficult to understand.
- Line 41: “For instance, detoxification efficiency is affected by environmental conditions”, this paragraph has not been properly justified with a reference.
- Line 43: Please, replace “in” with “by”.
- Line 58: Please, replace “conditions” with “factors”.
- Line 59: “to study the ability of the strain to remove multiple mycotoxins at the same time”, unfortunately this goal was not studied.
- Line 60: Why the authors choose wheat bran for the study? It would be interesting to include in the introduction section, the reasons of selecting this product for check OTA detoxification.
- Line 61: “…and to evaluate the possibility of applying the strain to agricultural products and food”, this purpose was not really investigated, only was studied using one agricultural product (wheat bran).
- Line 73: Please, complete this sentence as follows “…and significantly higher than the wild type strain FS10 (55.6%)”.
- Line 80: “MEA” and “CYA” media were not described in the text. Please, insert their descriptions or a reference in material and methods section.
- Lines 86 and 87: Please, specify which “liquid medium” was used.
- Line 110: Please change “progradation rate” by “degradation rate”.
- Lines 141-143: Please, modify this sentence as follows “A comparison of the HPLC chromatograms of OTA before (Fig. 4A) and OTA after exposure to the FS-UV-21 strain (Fig 4B) revealed that…”
- Lines 158 and 159: Please, change “MSMS” by “MS/MS”.
- Line 163: Please, correct this sentence as follows “Structural elucidation was carried out using MS Finder,…”
- Line 186: Please, insert a full stop after “strain”.
- Line 191: This sentence would be more precise as follows “…but are novel metabolites of OTA generated by A. niger FS-UV-21 strains”
- Lines 196, 200, 214: The use of different terms: “cell viability”, “cell survival rate”, “cell inhibition” and “cell growth inhibition rate” could induce to misunderstand.
- Line 196: Please, discuss better about “apoptosis” data in the text.
- Line 203: Please, acronyms would be defined with the first time when it occurs in the text.
- Line 236: Please, revise the verb in this sentence.
- Please, improve the discussion paragraphs; there is little information on comparison of results with other’s authors.
- Lines 241-258: Authors must rewrite the conclusions emphasizing the current findings and revise these sentences in relation with the results: “…and inhibit toxin production…” and “The research results provide an effective means for the control of toxins in agricultural products such as wheat bran”. Such generalization has not been properly justified.
- Line 277: Insert the reference number at the end of the sentence “…by Sun et al.36”.
- Line 286: Please, check the temperature units through the text, thermal values must be separated from the corresponding units: “28 ºC” not “28ºC”.
- Line 364: Please, provide the meaning of “DMEM”
- Line 366: Please, provide the meaning of “DMSO”
- Supplementary materials, page 1: please, change “r/min” by “rpm” and should be used consistently throughout all the text.
- Supplementary materials, table S2: please, modify the caption as “Chemical information of metabolic degradation products of OTA”.
- Supplementary materials, table S3: please, modify the caption as “Gradient elution procedure at LC-MS/MS analysis”.
- Supplementary materials, figure S1: please, complete the caption as “Phylogenetic tree of A. niger FS-UV-21 strain”.
- References: please, check the format of the references according to the journal´s instructions.
- Reference 18 is duplicated.
- Figure 1: please, provide at the caption, the “liquid medium” used. Change the X and Y-axes labels units as “survival rate (%)”, “degradation rate (%)” and “UV exposure time (min)”.
- Figures 2 and 3: Change the X and Y-axes label units as “time (h)”, “degradation rate (%)”, “Temperature (ºC)”, “Concentration (µg/mL)” and “Spore suspension concentration (CFU/mL)”.
- Figure 6: likewise, units in brackets.
- Table 1: Complete the table caption as follows “OTA degradation in wheat bran by A. niger FS-UV-21 fermentation (µg/mL ± SD)”.
Author Response
We would like to thank the reviewers and editor for the time they spent and their helpful comments on our manuscript. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our review article. We have studied comments carefully and have made correction and marked up using the “Track Changes” function. We hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the editors’ and reviewer’s comments are as following.
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Corrections were made as requested
Reviewer 3 Report
The authors have satisfactorily resolved all the points questioned, so I consider that it is ready for possible publication.