PONE-D-21-07582

Polydextrose with and without Bifidobacterium animalis ssp. lactis 420 drives the prevalence of Akkermansia and improves Liver Health in a Multi-compartmental obesogenic Mice Study.

PLOS ONE

Dear Dr. Yde,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Nikolaos Perakakis

Academic Editor

PLOS ONE

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"This study was funded by IFF Health & Biosciences. CCY was financially supported by the Innovation Fund Denmark (Project No. 4228-00010B)."

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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

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

Reviewer #1: 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

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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

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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: I am thankful for the opportunity to review this manuscript. Apparently, I would like to declare that the conceptual design of this work is very similar to what my team and I recently published in Scientific Reports (https://www.nature.com/articles/s41598-021-85449-2). I believe I was selected as a reviewer because of my relevant work. Hence, please allow me to provide comments with regard to it.

The major differences were that the present work investigated the effect of PDX and B. animalis ssp. lactis 420 on a few outcomes and made a conclusion about liver health improvement whereas mine assessed the comprehensive effects of L. acidophilus LA5 on weight reduction, regional fat accumulation, lipidemia, liver injury (liver weight, lipid compositions, and liver enzyme), gut permeability defect, endotoxemia, and serum cytokines; using both in vitro and in vivo evidences.

Interestingly, both B. animalis lactis 420 and L. acidophilus LA5 contributed to the fat reduction through the Akkermansia promotion mechanism. Although at least two probiotics show this potential, many other probiotics may have insufficient evidence so the Short Title and the Conclusions should be more specific. (ie. PDX and B. animalis lactis improves liver health and change levels of Akkermansia in obese mice).

It is essential to demonstrate and/or discuss how the PDX and B. animalis lactis 'move' to liver and other organs. In our L. acidophilus LA5 study, we demonstrated the gut leakage mechanism. Also, potential toxicity, inflammation, and injury of the PDX and B. animalis lactis on the liver should also be included.

Please discuss whether PDX could have a similar or different effect with probiotics other than B. animalis lactis.

The design of this study implicitly suggested the preventive effect of B. animalis lactis 420 but not treatment effect. Please mention this limitation in the Discussion. Also, the improvement of liver health was assessed without in vitro evidence, which should be addressed as another limitation.

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Reviewer #1: Yes: Krit Pongpirul

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PONE-D-21-07582R1Polydextrose with and without Bifidobacterium animalis ssp. lactis 420 drives the prevalence of Akkermansia and improves Liver Health in a Multi-compartmental obesogenic Mice Study.PLOS ONE

Dear Dr. Christian Clement Yde,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it is an interesting article and 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.

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ACADEMIC EDITOR: Thank you for submitting your article in PLOS one. I apologize for the delay which was due to difficulties in the recruitment of reviewers in the summer period. Your manuscript has been carefully reviewed from two reviewers and they both agree that this is a very interesting study and a solid work. Before accepting the manuscript for publication, we would like you though to try to address the remaining comments of the reviewers, especially of reviewer 2. Reviewer 2 has also committed to review the revised form of the manuscript in a timely manner.

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

Please submit your revised manuscript within 4 weeks. 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,

Nikolaos Perakakis

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 submitting your article in PLOS one. I apologize for the delay which was due to difficulties in the recruitment of reviewers in the summer period. Your manuscript has been carefully reviewed from two reviewers and they both agree that this is a very interesting study and a solid work. Before accepting the manuscript for publication, we would like you though to try to address the remaining comments of the reviewers, especially of reviewer 2. Reviewer 2 has also committed to review the revised form of the manuscript in a timely manner.

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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: (No Response)

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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: Yes

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

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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

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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: Thank you very much for the revised manuscript. All of my comments have been addressed satisfactorily.

Nonetheless, there are some minor formatting issues to be corrected. For example, the 'author' of the first reference should be 'OECD', not '2019 O'.

Reviewer #2: Clement Yde et al. present an impressive assessment of murine metabolomics and metagenomics following five (4+control) different treatment combinations of prebiotic and probiotic elements alongside a high-fat diet. The study is well-written and conveys an interesting message, whereas its methods section comprehensively describes all implemented modalities. The authors have provided extensive evaluations of murine microbiota both at treatment initiation and study endpoint, showcasing relative similarities in diversity indices upon initiation, which is important given the numerous relevant confounding factors (cage, bedding, basal animal conditions etc.) The metabolomic arm could be ideally supplemented with the inclusion of biopsy images from tissues obtained and the analysis and discussion of liver histology and/or immunohistochemistry pertaining to non-alcoholic fatty liver disease.

In its current form the manuscript is well delineated and the reviewer believes the authors should follow the suggestions below. However, if available, the inclusion of data on murine body composition and plasma concentrations of insulin, liver enzymes, triglycerides, conventionally-measured cholesterol fractions etc would be of great scientific interest, since it could ascertain the precise pathobiological fingerprint of all described interventions that reflect the altered metabolomic profiles and their correlation with microbiota.

Additionally, some further suggestions are in order to improve overall readability and quality.

• Consider describing the full dietary energy composition of the murine HFD (total calories, %carbs, %protein, %fat)

• Was there any attrition / mice deaths before study completion?

• Gut microbiome constitutes a primary driver of hepatic steatosis and inflammation, both components of non-alcoholic fatty liver disease, whereas relevant interventions through probiotic administration are being discussed across the literature as potential therapeutic strategies (Meroni et al., Nutrients . 2019 Nov 4;11(11):2642; Delzenne et al., Nat Med 2018; 24: 906–907; Le Roy et al., Gut 2013; 62(12): 1787-94) Consider expanding your discussion and thus further abridging the two main areas of your study (microbiota + metabolomics) towards this direction, and to address the functional significance and potential long-term clinical relevance of your findings regarding liver metabolomics, especially considering weight gain differences between human and murine models as described in your already published work, already cited (Ref. 11), and the similarities or differences of metabolomic findings observed herein to recently published evidence on murine NAFLD therapeutics regarding liraglutide (especially relevant owing to the known PDX-induced increase of GLP-1) /elafibranor/empagliflozin administration in HFD-fed mice with HFD-induced non-acoholic steatohepatitis (Perakakis et al., Liver Intl 2021 Aug;41(8):1853-1866).

• Consider providing an additional image, embedded in Figure 5, in the form of a time scale graph/diagram showcasing the study design, randomization and number of total mice per group at study end, and measurements per week

• Figure 1: Consider including all between-group significances in a small table included within the image, rather than in the figure legend

Minor:

• If possible, consider increasing the DPI of your tiff figures to ensure that all legends are easily readable in their final form, because owing to the small fonts, it can be hard for a curious reader to discern them, especially in the components of figure 2 (A-E)

• Optionally, consider including supplementary figures alongside their respective legends in a separate PDF file to facilitate readability and continuity rather than including captions separately and uploading a zip file with images only;

• Consider carefully screening the text for minor English language mistakes, especially adverb utilization, tense discrepancies etc.

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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: Assoc. Prof. Dr. Krit Pongpirul, MD, MPH, PhD.

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.

Polydextrose with and without Bifidobacterium animalis ssp. lactis 420 drives the prevalence of Akkermansia and improves Liver Health in a Multi-compartmental obesogenic Mice Study.

PONE-D-21-07582R2

Dear Dr. Clement Yde,

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,

Nikolaos Perakakis

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: All comments have been addressed

**********

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 #2: Yes

**********

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

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 #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 #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 #2: (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 #2: No