PONE-D-21-17352

Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortium

PLOS ONE

Dear Dr. higashinakagawa,

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Stefan Wölfl, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The manuscript in its present form is not yet suited and needs some minor improvments, that are substantial, before the manuscript can be accepted for publication.

There are several point mentioned and addressed by the reviewers that need to be corrected in a revised version. I also recommend careful language editing before resubmission.

For details on the required changes please carefully address all points raised be the reviewers. Because it may not be clear from the comment from reviewer one, I like to add that the authors should add and discusse more carefully the state of the art regarding possible mechanisms.

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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

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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

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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: The work Higashinakagawa et al. describes stimulatory effects of an extract from culture of Effective Microorganisms (ECEM) and a health drink containing ECEM (EM XGOLD) on cellular activity of model organism Dictyostelium discoideum. Higashinakagawa et al. show that inorganic material such as magnesium and potassium containing microparticles can be active substances of ECEM. However, no statement about possible mechanisms of this effect is included. Authors only discuss few papers regarding the biological effect of inorganic materials and write that this subject is currently under their investgation.

This simple manuscript is clearly written. The methodological data are well documented by nice micrographs, graphs and by statistic evaluation and I do not have not any major reservations. I believe this work will interest many workers in the field of cell biology and action of inorganic compounds on cellular functions as well as in nutrition supplements.

Reviewer #2: Higashinakagawa et al, Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortium.

Higashinakagawa et al reported a very interesting and intriguing study and explain how simple elements have shown significant biological activity, namely, as they called the Effective Microorganisms (EM) on life cycle of Dictyostelium discoideum. They carried out systematic and careful studies of such EM･XGOLD, which is a well-known and popular health drink in Japan. This study is a continuation from their report in 2019 that “The life cycle of Dictyostelium discoideum is accelerated via MAP kinase cascade by a culture extract produced by a synthetic microbial consortium.”

They used various methods including i) Liquid-liquid partition, ii) column chromatography, iii) electron microscopy, iv) elemental analysis using Energy Dispersive X-ray (EDX) Spectroscopy, v) acid treatment and alkali pH titrations to figure out the major component of the biological activity. Through their careful observations and detailed studies, they conclude that the Mg, K-containing microparticle biological effect could be due to MgCO3. Although intriguing, their careful data and systematic analysis justify their conclusion. They may have solved one of long-standing puzzles in the popular health drink in Japan.

Some minor points:

1) They use the term EM for Effective Microorganisms. This abbreviation is not necessary since there are only 2 words. EM commonly refers to Electron Microscopy. It is rather confusion to read EM but it refers to something totally different. The legendary Francis Crick advised this reviewer 30 years ago to avoid use all unnecessary abbreviations and acronyms, so the readers can understand the articles better and avoid confusions.

2) Please write out ECEM on line 83, page 5.

3) Please write out CMC (carbon microcoils) on line 238, page 9. CMC commonly refers to critical micelle concentration.

4) Please add more figure legend to Figure 5, provide more information about the microparticle, why the sizes cross a wide range. Do they all have similar elemental contents?

5) On lines 183-184, they wrote “The result was exactly as shown in Fig. 6”. Please replace the word exactly with “similar” since the results in Figure are not exactly, but similar. For example, in Panel A, E20 is ~33% and in Panel B, the E20 is ~38%. They are similar but not exactly identical.

6) On line 221, page 9, they wrote “we have no idea regarding the functional role and the state of being of potassium……”. Please change to “in the current study, we did not focus on the study of potassium”.

After they make these corrections and changes, this reviewer highly recommends publication in PLoS ONE.

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Reviewer #1: No

Reviewer #2: Yes: Shuguang Zhang

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Attachments

Attachment

Submitted filename: Referee report for PONE 6-2021.docx

PONE-D-21-17352R1Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortiumPLOS ONE

Dear Dr. higashinakagawa,

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.

Please submit your revised manuscript by Nov 20 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Stefan Wölfl, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Thank you for the submission of the revised version. As the reviewers point out most of their major concerns have been addressed in the revision. There is however still one aspect that I do not fully understand at that makes the manuscript difficult to understand and unclear regarding the Intension of the work. The work is solely used on the trademark registered EM consortium of microorganisms from EM research organization. This is a commercial product and registered trademark. This needs to be clear in the manuscript and it will be necessary to stay potential conflicts of interest regarding the impact the work will have on the commercialization of EM XGold or EM(r).

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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: 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: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: 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

**********

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

**********

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: (No Response)

Reviewer #2: The authors have addressed the reviewer's concerns and suggestions. They have made corrections and revised the manuscript. This reviewer now recommends publication of this very interesting paper.

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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: No

Reviewer #2: No

[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.

Mg, K-containing microparticle: a possible active principle of a culture extract produced by a microbial consortium

PONE-D-21-17352R2

Dear Dr. higashinakagawa,

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.

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Kind regards,

Stefan Wölfl, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: