PONE-D-25-63339 Hydrolyzed corn starch with maltotetraose for skin defense through NRF2 pathway activation in human keratinocytes PLOS One

Dear Dr. Hagawa,

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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: No

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

Reviewer #1: Yes

Reviewer #2: Yes

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: No

Reviewer #3: No

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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: No

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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: Comments to the authors:

1. Repetition of the phrase “hydrolyzed corn starch containing maltotetraose” affects flow and should be reduced in the Abstract section.

2. No pigmentation-related markers, melanocyte models, or aging-associated endpoints are mentioned.

3. In line 32, the phrase “novel functionality” is vague and should be replaced with more precise scientific language.

4. Typographical error identified: “maltotetoraose” should be corrected to “maltotetraose” in line 33.

5. In line 56, the statement that NRF2 is a “promising target in cosmetic applications” is plausible but under-argued. Clarify why moderate NRF2 activation is beneficial and safe in cosmetic formulations.

6. “Raw and functional cosmetic ingredient” is vague. Consider replacing with “naturally derived functional cosmetic ingredient” in line 57.

7. The assertion that MTO has “superior skin-moisturizing properties” based on hydroxyl group content alone is chemically simplistic. Provide experimental or literature-based evidence supporting superior humectancy, or rephrase to a more cautious claim (e.g., “potentially enhanced moisturizing capacity”) in line 60.

8. In line 184, the term “robes” appears to be a typographical error and should be corrected to “probes”. The phrase “data not shown” is not acceptable for a primary result in a manuscript. Transparency requires that the underlying data be accessible.

9. Reporting only the number of selected probes (n = 73) without presenting any supporting data (e.g., fold changes, adjusted p-values, or gene identities) is insufficient. At a minimum, a supplementary table should be provided.

10. The discussion appropriately outlines the NRF2–ARE antioxidant pathway and its relevance to skin oxidative stress. However, the conclusion that hydrolyzed corn starch mitigates wrinkles, sagging, and pigmentation is speculative and not directly supported by the presented data.

11. Justify this statement: the anti-inflammatory effect is inferred solely from literature citing NRF2-mediated suppression of IL-6 and IL-1β. Since no inflammatory markers were measured in this study, this conclusion should be framed as a potential implication, not as an observed effect in lines 265-268.

Reviewer #2: It’s an interesting piece of work which should be corroborated with human trials as early as possible.

1. Please provide latest references for the second paragraph of the Introduction section.

2. If possible please provide a schematic to explain the pathways during presence and absence of oxidative stress.

3. Is there previously available literature that supports hydrolyzed corn starch or MTO f as a potent cosmetic ingredient?

4. The data of 73 probes can be provided as supplementary information?

5. Need more clarity on the sample size and availability of the statistical analysis data.

Reviewer #3: Comments to the authors

The author investigated the potential of hydrolyzed corn starch containing maltotetraose in skin defense through nuclear factor erythroid 2- related factor 2 (NRF2) pathway activation in human keratinocytes exposed to oxidative stress. They have checked the hydrolyzed corn starch on antioxidant-responsive genes, including HMOX1, GPX2, and NQO1, which are known targets of NRF2 human keratinocytes cells. The manuscript lacks rigor in several key aspects, including statistical analysis, experimental depth, novelty, and the discussion. Therefore, I recommend rejection of the manuscript in its current form.

1. The bar graphs are not properly labeled on the x-axis, and statistical significance is not indicated.

2. The authors claim that maltotetraose is a promising ingredient for cosmetic applications with potential roles in improving skin health, reducing pigmentation, and combating aging. However, no experimental data are provided to support claims related to pigmentation reduction or anti-aging effects. Moreover, the experiments were conducted exclusively in keratinocytes. The role of dermal fibroblasts, which are critical for maintaining skin elasticity and extracellular matrix integrity, has not been addressed.

3. The discussion section requires substantial improvement to better interpret the findings and place them in the context of existing literature.

4. The authors evaluated NRF2-associated antioxidant response genes, including HMOX1, GPX2, and NQO1. However, the effects of maltotetraose on other antioxidant systems, such as PRDX1, as well as overall enzymatic and non-enzymatic antioxidant status, were not investigated.

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Reviewer #1: No

Reviewer #2: Yes: Dr. Gauri Awasthi

Reviewer #3: Yes: Agilan Balupillai

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Hydrolyzed corn starch with maltotetraose for skin defense through NRF2 pathway activation in human keratinocytes

PONE-D-25-63339R1

Dear Dr. Hagawa,

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,

Ahmed E. Abdel Moneim

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

Reviewer #4: 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 #2: Yes

Reviewer #4: Yes

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

Reviewer #2: Yes

Reviewer #4: Yes

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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

Reviewer #4: 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 #2: Yes

Reviewer #4: 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 #2: The authors have addressed all the comments according to the authors satisfaction including the schematic figure and data relevant to the study hence the manuscript should be considered for publication.

Reviewer #4: An important point to be considered for future studies shall be the qualitative analysis of the corn starch hydrolysate, particularly for contents of maltotetraose and other relevant components, as the compositions can vary from batch to batch.

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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: Yes: Dr. Gauri Awasthi

Reviewer #4: Yes: Muhammad Waheed Akhtar

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