PONE-D-19-15766

A novel nutritional supplement containing amino acids and chromium decreases postprandial glucose response in a randomized, double-blind, placebo-controlled study

PLOS ONE

Dear Dr Ostman,

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 reviewers raised several important points related to the design of the study, and suggestions for improving the level of detail and clarity in which you described methods of reporting the statistical analyses. Please carefully consider these points in your revision. There are some differences in opinion about the validity of some measures of glucose, and while I would appreciate you considering these, you may of course, disagree.

We would appreciate receiving your revised manuscript by Dec 23 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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

Please include the following items when submitting your revised manuscript:

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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Russell J de Souza, ScD, RD

Academic Editor

PLOS ONE

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'The study was funded in full by Aventure AB/Double Good AB, Sweden.'

We note that one or more of the authors are employed by a commercial company: starScience GmbH & Aventure AB.

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Additional Editor Comments (if provided):

We note that in the demographic classification used, Asians, Hispanics and Aboriginal are included in the"North White Americans" group. As these populations show different risk levels for diabetes and CVDs, please clarify why it was chosen to group them together. 

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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: Partly

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: No

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: This is a well conducted and reported study. There were a number of issues related to the statistical design and analysis which I would recommend that the authors report differently, with reasons:

(1) The primary outcome should be explained a bit more clearly. The authors should, in a brief paragraph, describe how the iAUC is obtained. Where they cite methods of calculating it (e.g the trapezoid rule), they should explain why they have chosen this method instead of others.

(2) The study has several secondary outcomes. The authors should describe any considerations they have made to deal with multiple testing.

(3) The sample size calculation is poorly described. The authors say that it was calculated based on an expected effect size of 35.8mmol/L/min; however, this is not an effect size (a standardised difference). The authors should say what the expected mean value of the outcome was expected to be in the control group, along with the standard deviation and the difference in the mean outcome value expected due to the intervention, with justification.

(4) In the analysis of the outcome, the authors used a mixed model repeated measures ANCOVA with the pre-treatment value as a covariate. When using this approach, the effect of the pre-treatment value should be fixed to zero - the authors do not appear to have done this.

(5) In Table 1 the authors have presented a comparison of the characteristics of participants included in the ITT vs. PP populations. While this is useful information, what the authors should have presented in this table is a comparison of the same characteristics of individuals in the intervention group to those in the placebo group.

(6) In Table 2, the authors should report the mean value of the outcome and the standard errors (not standard deviation - SDs are reported for description (e.g. baseline tables), but SEs are reported for inference). They should also report the difference in outcome and the confidence intervals for the difference along with the p-value. Please see the report of other clinical trials, e.g. (https://journals.plos.org/plosmedicine/article/figure?id=10.1371/journal.pmed.1001518.t003) to see how this should be done.

(7) Instead of cite the treatment effect as a percent reduction in serum glucose, the authors should report the absolute reduction in serum glucose, along with the 95% confidence intervals and p-value. If the authors want to express this as a relative reduction, this should be done in the discussion, or as an alternative (but not replacement for the absolute reductions). If reporting the relative reductions, the confidence intervals for these reductions should also be reported.

(8) The authors should avoid the +/- designation when reporting numerical results as it is very misleading and actually quite meaningless in many of the contexts it has been used. For example, in the abstract, it is difficult to tell whether a decrease in glucose iAUC-120min 49.5 +/- 47 refers to decreases varying from 2.5 to 96.5 or whether 47 is the IQR. Similarly in Table 1, mean age in the ITT population is reported as 34.6 +/- 10.4 with a footnote indicating that the +/- are SD; however, what does a mean +/- the SD really mean in practical terms? This should not be reported in this manner. Instead, the abstract should be reporting the mean reductions and their 95% confidence intervals; this has a very clear statistical meaning. When reporting the table of characteristics where it is typical to report means and their SDs, the authors should simply report the mean followed by the SD in brackets and indicate so in the footnote, e.g. 34.6 (10.4). It may be common to use the +/- designation in the literature, but because of the inconsistent way in which this is done (some use it to mean ranges, others IQRs, yet others SDs, mostly without clear indication), it is best to avoid this. See https://journals.plos.org/plosmedicine/article/figure?id=10.1371/journal.pmed.1001594.t001 for an example of how to report means SDs and ranges for descriptive purposes.

Reviewer #2: Aim of the study was that to evaluate postprandial responses in serum glucose and insulin levels in a cohort of overweight subjects of both genders, treated with a supplement consisting in 2.6 g of amino acids (L-Leucine, L-Threonine, L-Lysine, L-Isoleucine and L-Valine) and 250 mcg of chromium picolinate vs. placebo. In the intention-to-treat-analysis (n = 60), the supplement significantly decreased glucose levels (as iAUC0-120 min, iAUC0-180 min and Cmax), without affecting those of insulin. The treatment was well tolerated.

The study is well designed and the results are adequately presented. Due to the paucity of biochemical parameters (serum glucose and insulin), the manuscript should be submitted as a rapid communication in a journal of nutrition sciences. Furthermore, some major and minor issues should be solved.

Comments

1. In the Introduction the rationale has not been clearly reported. In particular, there is no information regarding the reasons for which the Authors chose the specific ingredients of the supplement. For instance, the gastroenteropharmacological properties of the selected amino acids (branched-chain amino acids?) should be mentioned.

2. Few biochemical parameters have been measured. Only serum levels of glucose and insulin. To understand the biochemical or pharmacological mechanisms underlying the hypoglycemic effect of the supplement, the Authors should evaluate circulating levels of some gastrointestinal peptides, particularly incretins such as GLP-1, which exert insulinotropic effects and reduce gastric empting. See the article by Rigamonti et al., Nutrients, 2019;11(2). pii: E247. doi: 10.3390/nu11020247.

3. The results referring to glycaemia measured with glucometer, which is defined an “imprecise” method by the Authors (pag. 17 – line 81), should be removed, because they do not add anything.

4. The Authors should explain the differences in results obtained in the per-protocol analysis vs in the intention-to-treat analysis.

5. In the Abstract the Authors should state that subjects were overweight and of both genders. Not only in the conclusions.

6. The Authors should report the phase of the menstrual cycle during which the experimental tests were performed in female subjects.

7. In the final part of the Discussion, when reporting limitations of the study, the Authors state that fiber content in bread used in the two centers is different. An appropriate statistical analysis should rule out the potential “interfering” effect of this factor.

8. Numbers of females/males should be reported in the section of Participants (pag. 4).

9. The exact doses of the single amino acids should be indicated. Not simply 2.6 g of amino acids (L-Leucine, L-Threonine, L-Lysine, L-Isoleucine and L-Valine).

10. Pag. 5: protein/fat should be replaced with proteins/fats.

11. Sensitivity and intra-/inter-assay coefficients of variation of the analytical methods for glucose and insulin should be indicated.

12. Pag. 6; the safety endpoints were evaluated at the three visits. Please, specify these time points.

13. Fig. 1 is of low resolution and difficult to read. Please, improve the quality of this figure.

14. Pag. 14 – Line 1: “Lead” should be “led”.

15. Pag. 14 – Line 16; what are the “other subgroups”? Please, specify.

16. Pag. 14 – Line 19: the Authors state that adverse events were categorized as possibly related to supplement or placebo. What system of categorization (or algorithm) was used?

17. Pag. 15 – line 33: where was the acronym IGT (impaired glucose tolerance) abbreviated?

18. Pag. 16 – lines 65-68: the Authors discuss the results by citing “unpublished” results. This is scientifically not appropriate.

Reviewer #3: This clinical trial was designed to evaluate the efficacy of a nutritional supplement to lower blood glucose levels after a meal in health individuals. The study was well-conducted and the data were clearly described. I only have few comments, but that should be included in the revised manuscript:

1) A conflicting result is regarding the lack of changes when blood glucose levels were evaluated using glucometers, compared to enzymatic methods that determined serum glucose. The authors argued that regular glucometers used in the present study are imprecise, but I personally think that this argument is not necessarily valid. Anyway, I think that the authors should include the information of the lack of differences in blood glucose levels when using glucometers in the Abstract. Thus, the readers can be informed that the improvement in glucose homeostasis was verified by one method of analysis but was not confirmed by data obtained using glucometers.

2) The adverse events should be described in Results. They are only briefly mentioned without explaining what they refer to.

3) Although I agree that this is not required for this clinical trial, I recommend that the authors measure GI-secreted hormones (e.g., GLP-1, PYY, GIP or ghrelin) during their trials to try to determine whether the supplement could have its effects via humoral mechanisms.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Jose Donato Junior

[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 to be viewed.]

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PONE-D-19-15766R1

A novel nutritional supplement containing amino acids and chromium decreases postprandial glucose response in a randomized, double-blind, placebo-controlled study

PLOS ONE

Dear Dr Ostman,

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 respond to the Reviewer's comments including explanation regarding patent-protected information.

We would appreciate receiving your revised manuscript by Apr 27 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Maciej S. Buchowski

Academic Editor

PLOS ONE

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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: (No Response)

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: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

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: No

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: This manuscript is vastly improved since the previous round of peer review, and I commend the authors for taking on board most of the reviewers' suggestions. There is still one major issue which the authors have not addressed, and a few minor ones which have arisen since the last round.

Major issues:

- in my comment (6) in the previous round, I made a recommendation on how authors should report means and SDs in the descriptive tables, and group means and SEs in the inference tables, along with effects as between-group differences with their 95% confidence intervals and p-values. The authors have done most of this, but have not reported the 95% confidence intervals for the differences/effects; this is very important and should be done. Even where authors have decided to report effects in relative terms (e.g. 24.7% reduction in venous serum iAUC), a 95% confidence interval for this effect should be reported - although I would generally recommend that you stick to absolute differences in the results and perhaps keep relative differences to the discussion, because your analytical method does not estimate relative differences. Reporting SEs and SDs is not necessary in the abstract, but the 95% confidence intervals are crucial.

- the authors report some rank transformation technique to calculate p-values due to non-normality of 'values' (I'm assuming you mean outcome measures here). Normality of outcomes is not a requisite to report p-values from an ANOVA/regression; it is only the normality of residuals that is assumed.

Other points:

Abstract

- If you choose to report the SEs in the abstract despite the comment above, please indicate what quantities are in the brackets (in addition to specifying the confidence intervals). Also, consider rephrasing all through to refer to 'differences' rather than 'reductions'/'decreases' in quantities. For example, you could say: "... the supplement resulted in decreased venous blood glucose compared to placebo, mean (SE) of 69.3 (7.2) versus 52.2 (6.4) respectively, a difference of 17.1 mmol/L (95%CI X to Y, p-value x.xxx)". Please report the exact p-values unless they are less than 0.001.

- the following phrase is completely inappropriate and should be removed: "... missed statistical significance". It implies that statistical significance is some magical target to be hit or missed which is not the case. You either find evidence of a difference, based on a statistically significant finding, or you don't find that evidence; but phraseology such as this suggesting that you aimed to find a difference but missed it is very poor statistical reporting.

Methods

- p5 line 94: do you mean "... a total of 2.6g of five amino acids (L-leucine..."

- p6, 'Outcome measures': the iAUC-0,120min could still be better described than has been done so far. Does it represent a change in plasma glucose concentration up to 120 minutes after ingestion of the meal/product? If so, describe it as such (or similar terms). Same for the other iAUCs.

- p8 second paragraph of 'statistical analysis' - there appears to be an incomplete sentence beginning "Although simple, the trapezoidal rule..."

- p8 line 178: you mean "complete observations on 57 individuals", not complete datasets.

- p8 line 180: the paragraph on page 9 from line 184 should be moved to the sentence that ends with "imputation." on line 180. After that, the next sentence should begin with p-values, not probabilities, which has a very different meaning in this context.

- p9 line 187 - see comment above about normality.

- Table 2 - see comment above about reporting differences with their 95% confidence intervals and p-values

- p14 line 8 - see comment above about "missing statistical significance", which is a very serious and important point about statistical reporting.

Reviewer #2: The Authors have minimally responded to the Referee’s comments.

As no new data have been added in the revised version, the Referee again suggests the Authors to submit the manuscript as a rapid communication to a journal dealing with nutrition and related topics.

The Referee does not hide the disappoint due to the Authors’ refusal to report the amounts of amino acids present in the supplement, which the Authors declare to be protected by a patent.

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.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: 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 to be viewed.]

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A novel nutritional supplement containing amino acids and chromium decreases postprandial glucose response in a randomized, double-blind, placebo-controlled study

PONE-D-19-15766R2

Dear Dr. Ostman,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Maciej S. Buchowski

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

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

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

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: (No Response)

Reviewer #2: No

Reviewer #3: (No Response)

**********

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: (No Response)

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

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: Line 135 - the sentence that has been added since the last review does not add any clarity, if anything it makes the description harder to understand. I was hoping for a more 'lay-appropriate' explanation for what the incremental area-under-the-curve represents. I had previously tempted the authors to add clarity by asking them "Does it represent a change in plasma glucose concentration over 120 minutes post-ingestion of the product?" I was hoping that in answering this one way or another, the authors would have been able to explain what this outcome measure represents. This is a missed opportunity, however, I am not inclined to hold up the publication of this excellent piece of research on this account!

Reviewer #2: (No Response)

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: No

Reviewer #2: No

Reviewer #3: Yes: Jose Donato Jr.