PONE-D-20-24290

Effect of physicochemical parameters on the stability and activity of garlic alliinase and its use for in situ allicin synthesis

PLOS ONE

Dear Dr. Kašpar,

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Pradeep Kumar

Academic Editor

PLOS ONE

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4. Please provide the source (name and location of company, market) of the garlic used in the study.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Authors,

Green chemistry, also called sustainable chemistry, is a rapidly growing field of chemistry that is based on the principles of reducing the impact of chemical production on the environment and human health. Your article “Effect of physicochemical parameters on the stability and activity of garlic alliinase and its use for in situ allicin synthesis” and idea of using allinase to form in situ the bactericidal agent allicin is consistent with the principles of green chemistry. The manuscript is well written and understandable. In my opinion, the article fully complies with the PLOS ONE editorial criteria. Much attention is paid to the technical aspects of the effects of various physicochemical parameters on allinase activity. This work may have an impact on the relevant field of science.

However, below you can find my minor suggestions for improving the manuscript.

1) The main limitation of the work is that you use a system of coupled reactions to analyze the effect of physicochemical parameters on allinase activity. In the first stage of the reaction, pyruvic acid is formed, which is then reduced by LDH in the presence of NADH. The analyzed physicochemical parameters can affect the activity of the second enzyme (LDH). Thus, the reported effects, for example of additives and pH, can be related to the effect on both alliinase and the second enzyme. However, LDH is known to be a stable enzyme. I believe this limitation should be noted in the discussion. It is necessary to indicate works that confirm the stability of this enzyme.

2) I was surprised by the lack of a “Statistical Analysis” section. Please add this section and include the following information. How is the data presented (Mean ± SD?)? What statistical criteria were used to compare different groups?

3) Lines 111-112. The characterization of the substrate used is very important for biochemical research. Please indicate the synthesis method (as well as the reference), describe the purity of the obtained alliin. Please interpret the abbreviation “UCT”.

4) Lines 133-135. In the sentence “The characterization of the enzyme was carried out by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) run under reducing conditions (in the presence of β-ME)” the word “run” may be redundant.

5) “1.4 Enzyme assay”. I assume that you used the previously developed method for determining of alliinase activity. Please provide a reference to the method. Could this be the method of Selby and Collin 1976? Lines 150-153: Indicate the concentration of alliinase used. Please indicate how the Michaelis – Menten kinetic constants are calculated. Which program was used? Have you used the non-linear approximation by the least square method in the Origin program?

6) Lines 265-266. Please indicate the concentration of the alliinase. Could you explain the choice of this enzyme concentration? I think you know that for the Michaelis - Menten kinetic constants analysis it is necessary to use the enzyme concentration corresponding to the linear region of the dependence of the initial reaction rate on the enzyme concentration. Have you conducted such experiments? It is necessary to provide this data.

The Vmax value depends on the enzyme concentration. Please calculate kcat values and compare with previously known data for alliinase.

7) Figure 4 A. Please indicate in the caption to the figure, what was taken as 100%? In Figure 5, you indicated that.

8) Line 367. “statistically relevant relationship” Please indicate what you mean. What statistical criterion was used?

9) Figure 6 A,B. What is the initial reference value? Please indicate in the caption to the figure or in the text, what was taken as 100%? I may be wrong, but you take five initial reference values for each of the five value pairs (0, 1, 2, 3, 24h). I propose to recalculate the data and take the initial reference value of the activity in 0 hours of incubation with no additives in all cases. Then the graph will be clearer.

10) Figure 7 A. Please indicate in the caption to the figure or in the text, what was taken as 100%?

11) Figure 8. “Note: …” This sentence can be transferred to the methods, or you can add a similar “Note:…” to each figure, if applicable.

12) Figure 9 A: Indicate that the measurements were taken at a fixed concentration of alliin. Figure 9 B: Indicate that measurements were taken at a fixed enzyme concentration. Please indicate the appropriate concentration in the text or caption.

13) My comment on lines 466-468. The absence of a plateau may be associated with a low concentration of alliinase, or its partial destruction by bacterial proteases. If you analyzed the dependence of the initial reaction rate on the enzyme concentration (see my comment 6), this would be more understandable.

14) Figure 10 and 11. On the right side of the pictures, what was taken as 100%?

You have done a great and interesting work.

Good luck with your further experiments.

Best regards

Reviewer #2: Manuscript evaluation: PONE-D-20-24290

Effect of physicochemical parameters on the stability and activity of garlic alliinase and its use for in situ allicin synthesis

General Comment

In this manuscript, authors described studies about the effect of physicochemical parameters on the stability and activity of garlic alliinase and its use for in situ allicin synthesis. Authors tried to provides a comprehensive insight into the alliinase stability and activity in the solution and lyophilized state as well as the influence of commonly used buffers, and some additives, on the enzyme activity and stability. While the manuscript contains massive data and results, only some interesting results, mainly the effect of additives on alliinase stability and Storage stability of lyophilized literature could be considered as novel. All others data were vastly reported in the literature. Hence I consider that the manuscript is not sufficiently original and should be rejected at its actual form. If the authors could identify and strength studies and results not previously reported, manuscript could be reconsidered for publication.

Reviewer #3: 1. The manuscript needs revision with respect to the typos, grammar and language

2. 1.1 Chemicals - This section needs to be precise. Name of all chemicals not needed

3. Line 119. T Mallika to be written as Mallika

4. Line 125 Cheesecloth- cheese cloth

5. Minutes to be written as min

6. Line 136 give details of molecular mass markers used

7. BSA- Expand

8. 150. Maximum reaction rate - revise at maximum velocity.

9. Line 161 and 164 Temperature range is confusing. at Line 161 it 20 C and at Line 164 it is -20 C

10. room temperature (25 C) only mention 25 C

11. Give the details of lyophilization

12. Line 194. Table 25 and 37 C- write as 25 C and 37 C

13. 1.6. What is the source of these microorganisms? were these human pathogens?

14. Statistical analysis is missing

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Reviewer #1: Yes: Evgeny Ermakov

Reviewer #2: No

Reviewer #3: Yes: R Z Sayyed

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Effect of physicochemical parameters on the stability and activity of garlic alliinase and its use for in situ allicin synthesis

PONE-D-20-24290R1

Dear Dr. Ondřej Kašpar,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Pradeep Kumar

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The decision of manuscript acceptance based on two positive response from the reviewer. 

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Authors,

It seems to me that in this form your manuscript can be approved for publication in Plos One. In my opinion, the article fully complies with the PLOS ONE editorial criteria. After revision, the article became more understandable and readable. I am satisfied with the responses of the authors. I have no objection to the manuscript.

Good luck with your further experiments.

Best regards

Reviewer #2: While the revised manuscript seems to be enhanced according to the comments of others reviewers, I did not really see changes relating to my initial comment. Indeed I had advised to authors to strength studies and results not previously reported. Although I respect the opinion of others reviewer, but in line of my first opinion, my decision is reject.

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Evgeny Ermakov

Reviewer #2: No

Reviewer #3: Yes: R. Z. Sayyed