PONE-D-20-34408

Enhanced Antioxidant Activity of Chenopodium formosanum K oidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions

PLOS ONE

Dear Dr. Cheng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Authors should pay more attention to the aims of the study (Introduction section) and how they present results in the Results section. Calculation methods of antioxidant assays may be questionable. Some statistical significance might be presented in an inappropriate manner, as noted in the Reviewers' reports. Labels in Fig 1A and Fig 1B are not visible. Can they stay in an oblique way, as in Fig 1C? Figure 3 should be prepared according to Submission Guidelines specified for figures: https://journals.plos.org/plosone/s/figures.

Language needs considerable improvement.

Please submit your revised manuscript by Jan 21 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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"This project was funded by the Ministry of Science and Technology, Taiwan (MOST

 109-2628-E-002-007-MY3 and MOST 107-2320-B-255-001-MY3). The funding

grant of BMRPD42 for this study was also provided in part by research grants from

the Chang Gung Memorial Hospital, Chiayi, Taiwan."

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Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"This project was funded by the Ministry of Science and Technology, Taiwan (MOST 109-2628-E-002-007-MY3 and MOST 107-2320-B-255-001-MY3). HC Kuo received grant of MOST 107-2320-B-255-001-MY3. KC Cheng received grant of MOST 109-2628-E-002-007-MY3. URL: https://www.most.gov.tw/?l=en . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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?

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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: Overall, the manuscript provides a good information of the subject matter. The experimental designs and analyses are standard and appropriate for the study. However, there are some concerns that need to be addressed by the authors.

1. The authors may wish to send the manuscript for a professional proof-read. There are some grammatical errors and inconsistency of sentences. Many of the scientific names of organisms throughout the manuscript are not in italic.

2. Introduction:

i) The sentence 'Lactic acid bacteria (LAB) are responsible for a group of famous strains of probiotics' (page 4, line 62-63) is somewhat misleading.

ii) The motivation/cause for this study is not clearly explained. The authors may wish to further explain the reason why they wanted to enhance the antioxidant activity and also why they selected LAB in the fermentation process. And perhaps explain why they selected ABTS and DPPH assays as the anti-oxidant assays for this study.

3. Materials and methods:

i) Page 7, line 143 - ABTS full name is repetitive.

ii) Page 9, line 193 - stated here that the authors used SAS software. But on page 13, line 300 - the authors stated that they used Minitab. Which statistical software did they actually use? Why did the authors used two different statistical software?

4. Results and discussion: In my opinion, this section is not sufficiently elaborated.

i) Page 12, line 262 - secrets? or secretes?

ii) Page 12, line 267-268 - at this stage, why did the authors left out the DPPH-radical scavenging evaluation?

iii) Page 12, line 271-274 - this statement is redundant. Similar meaning has been stated in line 259-263. The authors should provide another explanation along with appropriate references.

iv) Page 13, line 284 - I wonder why at this stage the authors expressed the results of ABTS scavenging activity in Ic50 but in % at the earlier stage of the study?

v) Page 15, line 336-337 - the sentence 'in the case of quinoa fermentation.....' - please provide reference for this statement.

vi) Page 16, line 367-374 - I think that comparison to yogurt fermentation is inappropriate. In my opinion, it is better if the authors compare their results with other studies on cereal or grains fermentation.

5. Conclusions : It is stated in line 384 that the RSM was applied to evaluate the interaction among the three factors. But I could not find the discussion on the interaction of factors anywhere in the manuscript. I can see in Table 3 that the interaction between temperature and agitation speed is significant. The authors should elaborate in the Results and discussion section.

6. Table 4: Are the superscript letters indicating significance of the data are stated correctly? Please check.

Reviewer #2: Reviewer

The research article entitled “Enhanced Antioxidant Activity of Chenopodium formosanum Koidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions” submitted in your esteemed journal is a good work where the authors have tried to improve the antioxidant activity by lactic acid bacterial and also optimized the fermentation conditions.

Minor Comments: -

What is the method of preparation of bacterial cell suspensions used for fermentation? Also describe about the starter culture for fermentation in material and method section.

In DPPH section what is 1 in the calculation of scavenging activity (%)?

Author cite only one reference for both DPPH and ABTS assay but author used two different calculation method for both DPPH as well as ABTS. Be specific about the citations as well as the calculation.

In section 2.5 author mentioned Wu et al (2020). What is the number of this citation in final list of references???

In section 2.7 line space is different as compared to the other text. Kindly follow the uniformity in whole MS.

The figures are not clearly visible.

At line no 310 author highlights the bracket with red colour. Correct it accordingly.

From section 3.3 it showed that fermentation decrease the carbohydrate, protein and fat content. Kindly explain? What is the reason behind this?

In table 4 correct the word TPC instead of TCP.

Correct the MS with a uniform pattern according to the journal guidelines and also formatting it accordingly.

“After reviewing the manuscript, in my opinion that once the corrections will amended by the author than it will be fit and accepted for publication in your esteemed journal”.

Reviewer #3: The paper describes the utilization of RSM in the optimization of fermentation parameters in order to maximize antioxidant activity. The work is scientifically interesting and sound.

It is good that the authors point out that the increase in antioxidant capacity may be partly related to the enhanced extractability of phenolic compounds. It needs to be remembered that human gut is able to effiicently "extract" and utilize also phenolic compounds that - due to non-extractability in sample preparation - may not be observed when total phenolics/antixoidant capacity is measured.

English needs to be slightly improved.

Some clarification is needed in the following:

1) What is the difference between "Fermented" and "Fermented-RSM" in Table 4?

2) What is "quinoa" mentioned in Table 5? Reference to literature source may be needed. Does "Djulis" denote the nonfermented samples?

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Reviewer #1: No

Reviewer #2: Yes: DR. Pardeep Kumar

Reviewer #3: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-34408R1

Enhanced Antioxidant Activity of Chenopodium formosanum K oidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions

PLOS ONE

Dear Dr. Cheng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The general impression is that the authors have put insufficient effort in revising the manuscript. The provided answers to the reviewers' comments are not properly addresses:

R: "And perhaps explain why they selected ABTS and DPPH assays as the anti-oxidant assays for this study."

A: "The reason why we choose ABTS and DPPH assays is because djulis is rich of phenolic compounds."

There are numerous antioxidant assays, which differ in their ability to scavenge particular radicals. The authors have to better explain why they have chosen the stated two. It should be discussed in the manuscript too.

R: "Results and discussion: In my opinion, this section is not sufficiently elaborated."

The authors provided no answer. It is a general remark; however, the Discussion section should be considerably supplemented in several parts where the reviewers' have made their notes.

R: "Conclusions : It is stated in line 384 that the RSM was applied to evaluate the interaction among the three factors. But I could not find the discussion on the interaction of factors anywhere in the manuscript. I can see in Table 3 that the interaction between temperature and agitation speed is significant. The authors should elaborate in the Results and discussion section."

A: "Page 14, lines 323-324: The interaction between temperature and agitation speed can be observed from the results (p < 0.05)."

I cannot accept the provided statement as the elaboration on the concern raised by the reviewer.

A: "We thank the reviewer for the comment, the data presented in % was in comparison to the control based on the equation: DPPH scavenging activity (%) = [1—(A1—A2) / A0] × 100, A0 = DPPH (without samples), A1 = Sample + DPPH, and A2 = Sample (without DPPH). We used this method to screen the optimal LAB for djulis fermentation. When the optimal fermentation protocol was evaluated, IC50 was expressed to provide absolute number to the audience."

This reply should stand in the main text. If two out of three reviewers raised the concern on different presentation of scavenging activities, it is expected readers will also do.

The language must be further polished. Some examples:

L46-47: "...can significantly increase the phenolic compounds," - phenolic compounds cannot be increased. Its amount can instead.

L137 and elsewhere:  "scavenging activity of the djulis" - No article is used when a non-count noun is generic or nonspecific.

L244: "T test" it is rather "t- test"

L363: no contractions such as "it's" are allowed.

Table titles and figure captions: please do not write djulis capitalized.

Figures 3 and 4 should be submitted in the .tiff format. Figure S1 is not a raw image since has been edited.

Significance letters in three histograms representing Figure 1 might be questionable. Please provide statistical working sheet with the raw data as Supporting information.

==============================

Please submit your revised manuscript by Mar 13 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Enhanced Antioxidant Activity of Chenopodium formosanum K oidz. by Lactic Acid Bacteria: Optimization of Fermentation Conditions

PONE-D-20-34408R2

Dear Dr. Cheng,

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.

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Kind regards,

Branislav T. Šiler, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments: