Review Reports

Round 1

Reviewer 1 Report

In my opinion, the paper is well written and suitable for pubblication in the current form.

Author Response

We greatly thank the editor and reviewers for the helpful comments on our manuscript. We answered all questions and comments and modified the text following the reviewers’ suggestions. All changes were written in red in the text.

In my opinion, the paper is well written and suitable for publication in the current form.

Response) Thank you very much for your positive comment on our manuscript.

Reviewer 2 Report

Dear authors,

I have read your article with interest and I can highly recommend its publication in present form except following comments:

• In abstract:

  I do not understand following sentence: “Genome sequencing analysis showed that post plasma treatment, approximately 0.0016-0.0017% variations in the sequence of A. oryzae genomic DNA”

• I can polemize with your final statement "Plasma jet treatment induced mutational variations in the genomic DNA of A. oryzae and there is a possibility that these genetic changes might affect the enzyme secretion." I agree that plasma makes variations in DNA, however high  majority of this mutations are negative and it is highly unprobable they affect the enzyme production. Much more probable explanation is the epigenetics or the environmental behavior. The plasma treatment induce other metabolic pathways, i.e. the mould in this displeasing environment may try to grow more quickly and speed up the reproduction or produce other metobolites to neutralize the unpleasant molecules or particles.

Author Response

We greatly thank the editor and reviewers for the helpful comments on our manuscript. We answered all questions and comments and modified the text following the reviewers’ suggestions. All changes were written in red in the text.

I have read your article with interest and I can highly recommend its publication in present form except following comments:

1. In abstract: I do not understand following sentence: “Genome sequencing analysis showed that post plasma treatment, approximately 0.0016-0.0017% variations in the sequence of A. oryzae genomic DNA”

Response) Thank the reviewer for pointing out this. The sentence was not clearly stated. We changed it to a new sentence to make it clearer “Genome sequencing analysis showed approximately 0.0016-0.0017% variations (changes in 596–655 base pairs out of total 37,912,014 base pairs) in the genomic DNA sequence of A. oryzae after plasma treatment.”.

Revised area: line 20-22

2. I can polemize with your final statement "Plasma jet treatment induced mutational variations in the genomic DNA of A. oryzae and there is a possibility that these genetic changes might affect the enzyme secretion." I agree that plasma makes variations in DNA, however high majority of this mutations are negative and it is highly unprobable they affect the enzyme production. Much more probable explanation is the epigenetics or the environmental behavior. The plasma treatment induce other metabolic pathways, i.e. the mould in this displeasing environment may try to grow more quickly and speed up the reproduction or produce other metobolites to neutralize the unpleasant molecules or particles.

Response) We thank the reviewer for the comment. We agree with the reviewer’s opinion. Our description of this part was not clear. We added several sentences in the discussion part addressing the epigenetic change and added a new reference [37]. In addition, we modified sentences in conclusions.

Revised area: line 388-392, 402-404

Reviewer 3 Report

The paper “Influence of non-thermal atmospheric pressure plasma jet on extracellular activity of α-amylase in Aspergillus oryzae” provides novel and interesting data on positive effects of plasma on microorganisms, namely, the changes induced in germination and enzyme secretion of A. oryzae, the fungus widely applied in food industry due to strong potential for enzyme production. Importantly, the effects were tested and reproduced under the scaled-up condition.

In general, the manuscript is clearly written and logically well-structured, the obtained findings are well presented. The manuscript is of high scientific value, informative and worth publishing. Language is correct, length is proper and quality of graphs is sufficient, the used literary sources are adequate to the given issue. I would recommend only minor corrections:

 

Remarks and suggestions for improvement:

 

1. Possibly, some words are missing in the summary - the sentence on lines 22-24: Genome sequencing analysis showed that post plasma treatment, approximately 0.0016-0.0017% variations in the sequence of A. oryzae genomic DNA. I recommend to reformulate this sentence making information more clear about the size and importance of the induced changes.
2. The referencing should be adjusted according to the instructions to Applied sciences journal authors: https://www.mdpi.com/journal/applsci/instructions: “In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3].”
3. It is indicated in the section 2.3 (lines 101-102): “The average germination percentage was calculated from three replicate plates per experiment and the experiment itself was repeated three times.” Why the legend to Figure 2 gives different numbers - “Each value is the mean of 6-9 replicate measurements” ?
4. It is not indicated whether SDs or SEMs are shown on columns in all figures.
5. The last sentence in the Conclusions section (Plasma jet treatment induced mutational variations in the genomic DNA of oryzae and there is a possibility that these genetic changes might affect the enzyme secretion) seems to contradict with the statement in the end of discussion: “Since most of the genes found to be mutated upon plasma treatment in our study were uncharacterized, it is not clear whether the increased activity of α-amylase results from mutations or not”. Thus, the version “Plasma jet treatment induced mutational variations in the genomic DNA of A. oryzae but it remains to be elucidated whether these genetic changes might affect the enzyme secretion” could sound more reliable.

Author Response

We greatly thank the editor and reviewers for the helpful comments on our manuscript. We answered all questions and comments and modified the text following the reviewers’ suggestions. All changes were written in red in the text.

1. Possibly, some words are missing in the summary - the sentence on lines 22-24: Genome sequencing analysis showed that post plasma treatment, approximately 0.0016-0.0017% variations in the sequence of A. oryzae genomic DNA. I recommend to reformulate this sentence making information more clear about the size and importance of the induced changes.

Response) We appreciate the reviewer's comment. We revised the sentence in the abstract as the reviewer suggested.

Revised area: line 20-22

2. The referencing should be adjusted according to the instructions to Applied sciences journal authors: https://www.mdpi.com/journal/applsci/instructions: “In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3].” 

Response) As the reviewer suggested, we revised the format of references in the text according to the instructions of Applied sciences. All changes were indicated in red color.

3. It is indicated in the section 2.3 (lines 101-102): “The average germination percentage was calculated from three replicate plates per experiment and the experiment itself was repeated three times.” Why the legend to Figure 2 gives different numbers - “Each value is the mean of 6-9 replicate measurements” ?

Response) Thank you for the comment. We are sorry for some mistakes. For germination measurement, we used three replicate plates per experiment and the experiment itself was repeated two times. We revised it in the text of section 2.3 (line 106).

For germination measurement (figure 2a and b), we did two repeated experiments (measurement from total 6 replicate plates). For dry weight measurement (figure 2c), we did three repeated experiments (3 replicate plates per experiment, total 9 replicate plates). We added a sentence explaining this (line 111-112). Taken together, the mean value of figure 2a and b is from 6 replicates whereas the mean value of figure 2c from 9 replicates measurements. This was why we described as “6-9 replicates” in figure 2 legend. 

Revised area: line 106, 111-112

 

4. It is not indicated whether SDs or SEMs are shown on columns in all figures.

Response) We showed the SDs in data. We modified sentences in figure 2-7 legends. “Each value is indicated as the mean and standard deviation of x replicate measurements.

Revised area: line 198-199, 213-214, 258, 273, 275-276, 286

5. The last sentence in the Conclusions section (Plasma jet treatment induced mutational variations in the genomic DNA of oryzae and there is a possibility that these genetic changes might affect the enzyme secretion) seems to contradict with the statement in the end of discussion: “Since most of the genes found to be mutated upon plasma treatment in our study were uncharacterized, it is not clear whether the increased activity of α-amylase results from mutations or not”. Thus, the version “Plasma jet treatment induced mutational variations in the genomic DNA of A. oryzae but it remains to be elucidated whether these genetic changes might affect the enzyme secretion” could sound more reliable.

Response) We thank the reviewer for the comment. We agree with the reviewer’s opinion. Our description of this part was not clear. We revised it according to the reviewer’s suggestion.

Revised area: line 402-404