Possible Involvement of NAMPT in the Anti-Obesity Effect of Oral Administration of Fermented Rice with Lactobacillus kefiranofaciens (Rice Kefiran) in C57BL/6J Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the anti-obesity effects of Rice Kefiran (RK) in high-fat diet–induced obese mice, focusing on NAMPT and NAD⁺ metabolism. The topic is relevant and contributes to the growing interest in functional foods with metabolic benefits. The study is clearly written, the experimental design is appropriate, and the data are generally well presented. However, some aspects related to mechanistic interpretation and data discussion require further clarification.

Major Comments

1. The discussion could be strengthened by expanding on the mechanistic role of NAMPT and NAD⁺/NADH regulation, which represents the main novelty of the study.
2. The section discussing the potential anti-aging effects of RK (lines 209–215) appears speculative and is not fully supported by the presented data. It would be preferable to tone down this part or indicate clearly that it is a hypothesis for future investigation.
3. In Figure 6, the gene expression data for FAS show that Kef10 significantly reduced FAS expression, whereas Kef50 did not. The authors should discuss possible reasons why the higher Kef50 dose failed to show this effect.

Minor Comments

1. The manuscript alternates between “Kef,” “RK,” and “Rice Kefiran.” Please unify the terminology throughout the text.
2. In the Abstract, the phrase “attenuate obesity” could be replaced with “ameliorate obesity-related metabolic disturbances” for greater precision and clarity.

Author Response

Comment 1: The discussion could be strengthened by expanding on the mechanistic role of NAMPT and NAD⁺/NADH regulation, which represents the main novelty of the study.

Response 1: Thank you for this insightful suggestion. We have expanded the mechanistic discussion of NAMPT and NAD⁺/NADH regulation in the Discussion section (lines 275-284), (yellow-highlighted).

Comments 2: The section discussing the potential anti-aging effects of RK (lines 209–215) appears speculative and is not fully supported by the presented data. It would be preferable to tone down this part or indicate clearly that it is a hypothesis for future investigation.

Response 2: I concur that the present results do not support claims of RK's anti-aging potential. Nevertheless, they suggest a hypothesis that RK may influence aging-associated pathways, warranting targeted future studies. This perspective is already outlined in the Discussion (lines 287–290). 

Comments 3: In Figure 6, the gene expression data for FAS show that Kef10 significantly reduced FAS expression, whereas Kef50 did not. The authors should discuss possible reasons why the higher Kef50 dose failed to show this effect.

Response 3:

Comments 4: The manuscript alternates between “Kef,” “RK,” and “Rice Kefiran.” Please unify the terminology throughout the text.

Response 4: Thank you for addressing this point. I have already changed it (yellow-highlighted in the text)

Comments 5: In the Abstract, the phrase “attenuate obesity” could be replaced with “ameliorate obesity-related metabolic disturbances” for greater precision and clarity.

Response 5: Done (yellow-highlighted)

Reviewer 2 Report

Comments and Suggestions for Authors

Title. OK

Abstract. Please include numeric results to highlight the main findings. Genes are indicated as proteins, Please revise throughout the manuscript.

Introduction. OK.

Results. Reconsider adjusting the size of figures for a better interpretation. 

Discussion. How did authors define that the selected dosages were 'appropiate preclinical dosages'. Authors stated that NAMPT was restored; however, higher levels were observed than the healthy control. This result must be further explained. Further discussion must be included regarding the possible mechanisms by which this polysaccharide can modulate these pathways. 
Materials and methods. Did authors verified the content of kefiran? Did authors use the verified kefiran content to adjust the dosage? Authors must justify the low number of rats per group (n=5). What other components are found in the RK? did authors exclusively administrated kefiran or RK? Authors must include the primers used for qPCR. Why authors assumed that all variables were parametric?

 

Author Response

Comment 1 (Abstract): Please include numeric results to highlight the main findings. 

Response 1: Thank you for your comment. I already added numeric results in the abstract section (yellow-highlighted).

Comment 2 (Abstract): Genes are indicated as proteins. Please revise throughout the manuscript.

Response 2: Done

Comment 3 (Results): Reconsider adjusting the size of figures for a better interpretation

Response 3: We have revised all figures by increasing their size to improve readability.

Comment 4 (Discussion): How did the authors define that the selected dosages were "appropriate preclinical dosages"

Response 4: We thank the reviewer for raising this important point. The selected RK doses (10 and 50 mg/kg) were based on our previous dose-ranging studies in which RK was evaluated at doses between 10 and 100 mg/kg. Although higher doses, including 100 mg/kg, also showed beneficial metabolic effects, to enhance translational relevance and future clinical applicability, we focused on the lowest effective doses rather than higher-dose interventions. This rationale has now been clarified in the Discussion section (lines 270-275).

Comment 5 (Discussion): The authors stated that NAMPT was restored; however, higher levels were observed than in the healthy control. This result must be further explained.

Response 5: We thank the reviewer for this insightful comment. We agree that NAMPT levels in RK-treated mice exceeded those of healthy controls. We have revised this point in the Discussion section to explain that RK induces an elevation of NAMPT beyond baseline, likely reflecting an adaptive metabolic response aimed at enhancing NAD⁺ biosynthesis and restoring redox balance. Importantly, hepatic NAD⁺/NADH ratios were normalized toward control levels, supporting a regulated metabolic adaptation rather than dysregulation.

Comment 6 (Discussion):  Further discussion must be included regarding the possible mechanisms by which this polysaccharide can modulate these pathways

Response 6: We appreciate the reviewer's valuable suggestion. We have expanded the Discussion to include a mechanistic explanation of how kefiran, as a polysaccharide, may indirectly modulate metabolic pathways. Specifically, we discuss potential roles of gut microbiota–derived metabolites, inflammatory modulation, and bile acid signaling in mediating the observed effects on NAMPT–NAD⁺ metabolism, lipid metabolism, and glucose homeostasis.

Comment 7 (Materials and Methods): Did the authors verify the content of kefiran? Did the authors use the verified kefiran content to adjust the dosage?

Response 7: We appreciate the reviewer's comment. The kefiran content was verified and confirmed to meet the stated specification (≥5 mg/g). The choice of doses was based on the total RK preparation, as the aim of this study was to evaluate RK as a functional food rather than isolated kefiran. This clarification has been added to the Materials and Methods section (lines 367-370).

Comment 8 (Materials and Methods): Authors must justify the low number of rats per group (n=5)

Response 8: Actually, I am working on a non-toxic sample (the ratio of mortality is almost zero). The second reason is the recommendation of the "3Rs Principle" to minimize the use of animals in research.

Comment 9 (Materials and Methods): What other components are found in the RK?

Response 9: Already added to the “materials and methods” section (lines 370-374).

Comment 10 (Materials and Methods): Did the authors exclusively administer kefiran or RK?

Response 10: Thank you for your question. In this study, animals were administered with RK, not pure kefiran.

Comment 11 (Materials and Methods): Authors must include the primers used for qPCR

Response 11: We thank the reviewer for this suggestion. Primer sequences used for qPCR analysis have now been included in the Materials and Methods section (lines 418-428, yellow-highlighted).

Comment 12 (Materials and Methods): Why authors assume that all variables were parametric?

Response 12: We appreciate the reviewer's important comment. All datasets were evaluated for normality and homogeneity of variance before analysis and met the assumptions for parametric testing. Therefore, unpaired two-tailed Student’s t-tests were used for statistical comparisons.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors

The article entitled: Possible involvement of NAMPT in the anti-obesity effect of oral administration of fermented rice with Lactobacillus kefiranofaciens (Rice Kefiran) in C57BL/6J mice find the manuscript very interesting and important however, I suggest some changes for its acceptance:   

This manuscript examines obesity and its potential treatment through natural products, emphasizing their therapeutic mechanisms and recent supporting evidence. However, several scientific, methodological, and analytical aspects require clarification or improvement to strengthen the study.

Introduction

The manuscript states that it explores the “causes and treatment of obesity”. However, based on the content, the emphasis seems to be on evaluating the effects of natural treatments rather than on investigating the causes of obesity. This should be aligned more clearly in the introduction.

I recommend including a more detailed metabolic context regarding the NAD+ hypothesis, lipid metabolism regulation, and appetite control. These aspects are central to the proposed mechanism of action.

The review should incorporate key mechanisms through which the gut microbiota contributes to the development of obesity, such as energy harvest, modulation of inflammation, SCFA signaling, metabolic endotoxemia, and bile acid metabolism.

Line 230: The bacterial name L. kefiranofaciens should be italicized.

Animals: missing details

Please include: Age of the mice,  Sex of the animals (were they males?)

 Diet descriptions.

Line 237: briefly describe the composition of the standard chow diet. Specify: the exact diet given to each group (control, HFD, RK-treated groups, etc. The percentage of fat used in the high-fat diet (HFD).

Rationale for diet choice: Explain why a high-carbohydrate diet was not included, considering that the study focuses on metabolic pathways related to sugar metabolism.

Monitoring of animals: Clarify why animals were not weighed weekly, as this is standard practice in obesity studies.

Describe the euthanasia procedure used, according to AVMA or institutional guidelines.

Serum biochemical analysis: Provide detailed information on:

Techniques used, equipment and kits, specific methodology for measuring the serum parameters.

PCR primers: Clarify: Which primers were used, how they were selected, whether they had been previously reported, what target gene or region they amplified, expected amplicon length.

NAD+/NADH measurement

The method for determining hepatic NAD+/NADH levels is not described in Materials and Methods. Please include the technique, kit, or assay used.

Statistical approach: It is unclear whether data were tested for normal distribution. This step is essential to justify the choice of statistical tests. Given that the study includes four groups, Student’s t-test is not appropriate. A one-way ANOVA (for normally distributed data) or Kruskal–Wallis test (for non-parametric data) should be used. After ANOVA or Kruskal–Wallis, an appropriate post hoc test must be performed to determine which groups differ. This will strengthen the reliability of the results. Using multiple t-tests increases the probability of Type II error and inflates false positives. This should be addressed by applying proper global statistical tests.

Figure 1 – Body weight and adipose tissue. Statistical analysis must be replaced by ANOVA or Kruskal–Wallis with appropriate post hoc tests. Clarify how total adipose tissue mass was measured, and whether the method is standardized in mice. Specify if subcutaneous and visceral fat depots were quantified separately.

Figure 2. Oral glucose tolerance test. The statistical test is inappropriate. Replace Student’s t-test with ANOVA or Kruskal–Wallis depending on data normality.

Figure 3. Serum lipid parameters. The statistical tests must be corrected as noted above.

Figure 4. Serum NAMPT levels. Again, ANOVA or Kruskal–Wallis should replace Student’s t-test.

Figure 5. Hepatic NAD+/NADH. The statistical test is not described. Correct this and ensure the method is described in Materials and Methods.

Figure 6. The statistical method used for this figure is not specified. Please include it.

The discussion section will need to be revised after statistical analyses are corrected, as the interpretation of significance levels and group differences may change.

Comments for author File: Comments.pdf

Author Response

Comment 1: The manuscript states that it explores the “causes and treatment of obesity”. However, based on the content, the emphasis seems to be on evaluating the effects of natural treatments rather than on investigating the causes of obesity. This should be aligned more clearly in the introduction.

Response 1: We appreciate the reviewer's helpful comment. We agree that the original wording overstated the manuscript's scope. The Introduction has been revised to clarify that the study focuses on evaluating the therapeutic and mechanistic effects of natural products, rather than investigating the causes of obesity.

Comment 2: I recommend including a more detailed metabolic context regarding the NAD+ hypothesis, lipid metabolism regulation, and appetite control. These aspects are central to the proposed mechanism of action. The review should incorporate key mechanisms through which the gut microbiota contributes to the development of obesity, such as energy harvest, modulation of inflammation, SCFA signaling, metabolic endotoxemia, and bile acid metabolism.

Response 2: Thank you for this comment. To strengthen the mechanistic framework of the study, we have added a dedicated paragraph in the Introduction providing a more detailed metabolic context linking NAD⁺ metabolism, lipid regulation, and appetite control to the proposed mechanism of action.

Comment 3: Line 230: The bacterial name L. kefiranofaciens should be italicized.

Response 3: done

Comment 4: Please include: Age of the mice,  Sex of the animals (were they males?)

Response 4: I already mentioned the age (5 weeks old) and the sex (male) in the materials and methods section. Thank you

Comment 5: Line 237: Briefly describe the composition of the standard chow diet. Specify: the exact diet given to each group (control, HFD, RK-treated groups, etc. The percentage of fat used in the high-fat diet (HFD).

Response 5: We thank the reviewer for this comment. A brief description of the normal chow diet and the percentage of fat used in HFD has now been added to the Materials and Methods section.

Comment 6: Rationale for diet choice: Explain why a high-carbohydrate diet was not included, considering that the study focuses on metabolic pathways related to sugar metabolism.

Response 6: Thank you for this comment. The present study was designed to investigate the anti-obesity effects of RK in a well-established model of diet-induced obesity characterized by excess adiposity, dyslipidemia, and impaired NAD⁺ metabolism. High-fat diets are widely used and validated for inducing obesity-associated metabolic dysfunctions, including alterations in lipid handling and insulin sensitivity.

Comment 7: Monitoring of animals: Clarify why animals were not weighed weekly, as this is standard practice in obesity studies.

Response 7: We appreciate the reviewer's comment. Body weight was monitored twice per week throughout the experimental period. This frequency was selected for two reasons: (i) to identify the onset of overweight status in HFD-fed mice and determine the appropriate timing for dietary intervention, and (ii) because body weight measurements were performed concomitantly with routine food replacement, thereby minimizing additional handling and stress. This clarification has now been added to the Materials and Methods section.

Comment 8: Describe the euthanasia procedure used, according to AVMA or institutional guidelines.

Response 8: Thank you for this comment. The euthanasia procedure has now been explicitly described in the Materials and Methods section in accordance with the institutional animal care regulations.

Comment 9: Serum biochemical analysis: Provide detailed information on: Techniques used, equipment and kits, specific methodology for measuring the serum parameters.

Response 9: Thank you for this comment. Detailed information regarding the serum biochemical analyses, including the assay kits and methodology used, has now been added to the Materials and Methods section.

Comment 10: PCR primers: Clarify: Which primers were used, how they were selected, whether they had been previously reported, what target gene or region they amplified, and expected amplicon length.

Response 10: Actually, a similar concern was raised by another reviewer and has been fully addressed in the revised manuscript. Thank you

Comment 11: NAD+/NADH measurement; the method for determining hepatic NAD+/NADH levels is not described in Materials and Methods. Please include the technique, kit, or assay used.

Response 11: We thank the reviewer for this comment. The method used to measure hepatic NAD⁺ and NADH levels has now been clearly described in the Materials and Methods section. Specifically, hepatic NAD⁺/NADH levels were determined using a commercially available NAD/NADH Assay Kit-WST (Dojindo Molecular Technologies, Japan) according to the manufacturer’s instructions.

Comment 12: Figure 1 – Body weight and adipose tissue. Statistical analysis must be replaced by ANOVA or Kruskal–Wallis with appropriate post hoc tests. Clarify how total adipose tissue mass was measured, and whether the method is standardized in mice. Specify if subcutaneous and visceral fat depots were quantified separately.

Response 12: We appreciate the reviewer's important comment. Data distribution was assessed using the Shapiro–Wilk test and confirmed to be normally distributed. Accordingly, statistical analysis was revised using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. This approach avoids the inflation of Type I error associated with multiple t-tests. The Results section, figures, and statistical descriptions have been updated accordingly.

Comment 13: Figure 2. Oral glucose tolerance test. The statistical test is inappropriate. Replace Student’s t-test with ANOVA or Kruskal–Wallis depending on data normality.

Response 13: We have applied the same revisions requested in the previous comment to Figure 1 as well.

Comment 14: Figure 3. Serum lipid parameters. The statistical tests must be corrected as noted above.

Response 14: We have applied the same revisions requested in the previous comment to Figures 1 and 2 as well.

Comment 15: Figure 4. Serum NAMPT levels. Again, ANOVA or Kruskal–Wallis should replace Student’s t-test.

Response 15: Done as per previous comments.

Comment 16: Figure 5. Hepatic NAD+/NADH. The statistical test is not described. Correct this and ensure the method is described in Materials and Methods.

Response 16: I agree with you about this issue. In accordance with the suggestion, we have revised the statistical analysis and now use one-way ANOVA followed by Tukey’s post‑hoc test for all multi-group comparisons. The corresponding results, figure legends, and Methods section have been updated accordingly.

Comment 17: Figure 6. The statistical method used for this figure is not specified. Please include it.

Response 17: Thank you for pointing this out. The statistical method used for this figure has now been clearly specified in the figure legend and in the Statistical Analysis section. Briefly, data were analyzed using one-way ANOVA followed by Tukey’s post‑hoc test (mean ± SEM).

Comment 18: The discussion section will need to be revised after statistical analyses are corrected, as the interpretation of significance levels and group differences may change.

Response 18: We appreciate the reviewer's important comment. Following the correction of the statistical analyses, we thoroughly revised the Discussion section to ensure that all interpretations accurately reflect the updated significance levels and group differences. The revised text now aligns with the corrected ANOVA and post‑hoc results, and all statements regarding treatment effects have been updated accordingly.

Reviewer 4 Report

Comments and Suggestions for Authors

Major Comments

1. While the Methods section mentions that food intake was measured, this crucial data is missing from the Results, making it impossible to distinguish metabolic weight loss from simple caloric restriction. The authors must present cumulative and daily energy intake data to rule out satiety or palatability issues as primary drivers. If reduced intake is observed, the "metabolic" claims regarding NAMPT involvement must be tempered accordingly.
2. The central claim regarding NAMPT involvement is plausible but currently relies on correlative evidence (mRNA and serum levels) rather than direct mechanistic proof. To substantiate this mechanism and align with the journal's scope, the authors should validate protein levels via Western Blot and measure NAMPT enzymatic activity or downstream markers (e.g., SIRT targets) in relevant tissues. Furthermore, in the absence of inhibitor or knockout studies, the manuscript must temper causal language, framing the findings as associations rather than definitive mediation.
3. While microbiota analysis is a strength, the reporting lacks statistical rigor and methodological transparency. The authors must provide technical details on the bioinformatics pipeline and clearly state how they controlled for multiple comparisons (e.g., False Discovery Rate) in their taxa and pathway analyses. Finally, correlation data between microbial changes and metabolic phenotypes must include adjusted p-values and correlation coefficients (r) for clarity.
4. The study claims significant reductions in adipose tissue mass and improved liver health, but provides no visual histological evidence to support these morphological changes. To confirm the anti-obesity effects, H&E staining of adipose tissue is required to visualize changes in adipocyte hypertrophy. Furthermore, to validate the anti-steatotic effects, H&E or Oil Red O staining of liver sections is essential to demonstrate the reduction in lipid accumulation.
5. The small sample size of n=5 per group is the minimum for metabolic studies and requires justification for statistical power. The authors must expand the statistical methods section to specify the exact tests used, including whether they checked for assumptions like normality and homogeneity of variance. Finally, all figure legends must clearly state whether error bars represent Standard Error of the Mean (SEM) or Standard Deviation (SD).
6. Minor Comments

• The authors should provide a clearer justification for the chosen 10 mg/kg and 50 mg/kg doses, timing, and route of administration, citing dose-finding studies or relevant literature.
• The manuscript must explicitly state the full ethical approval details (committee name and number) and expand the methods to ensure reproducibility of the Rice Kefiran preparation, including its purity and key compositional details.
• The Results section should be restructured with clearer subsections that conclude with concise summaries. The authors should also calculate and present the Area Under the Curve (AUC) for the OGTT data for better quantitative summary, and ensure all figures have standardized, legible labels and units.
• The Data Availability statement needs an update to clarify where raw sequencing data and primary numerical data will be deposited. Finally, the authors must verify and state that the starting body weights were not significantly different between the groups at Week 0.

Author Response

Comment 1: While the Methods section mentions that food intake was measured, this crucial data is missing from the Results, making it impossible to distinguish metabolic weight loss from simple caloric restrictions. The authors must present cumulative and daily energy intake data to rule out satiety or palatability issues as primary drivers. If reduced intake is observed, the "metabolic" claims regarding NAMPT involvement must be tempered accordingly.

Response 1: We thank the reviewer for highlighting this important point. We agree that food intake is essential for distinguishing metabolic effects from potential reductions in caloric consumption. Daily and cumulative food intake data have now been added to the Results section (new Figure 2). Our analysis shows that no significant differences in daily or cumulative food intake were observed between the HFD, RK10, and RK50 groups throughout the 4-week intervention (one-way ANOVA, p > 0.05 for all comparisons). RK administration did not reduce food consumption, indicating that the attenuation of body weight gain cannot be attributed to satiety effects or reduced palatability of the diet. These findings support the interpretation that the anti‑obesity effects of RK arise from metabolic modulation rather than caloric restriction.

Comment 2: The central claim regarding NAMPT involvement is plausible but currently relies on correlative evidence (mRNA and serum levels) rather than direct mechanistic proof. To substantiate this mechanism and align with the journal's scope, the authors should validate protein levels via Western Blot and measure NAMPT enzymatic activity or downstream markers (e.g., SIRT targets) in relevant tissues. Furthermore, in the absence of inhibitor or knockout studies, the manuscript must temper causal language, framing the findings as associations rather than definitive mediation.

Response 2: We appreciate the reviewer’s insightful comments regarding the mechanistic interpretation of NAMPT involvement. We fully agree that our current evidence-based on serum NAMPT concentrations, hepatic NAD⁺/NADH ratios, and Nampt mRNA expression-supports an association but does not establish direct causality. At the reviewer’s suggestion, we have carefully revised the manuscript to ensure that all statements regarding NAMPT are framed as associations rather than definitive mechanistic mediation. Specifically, we now describe NAMPT upregulation as a potential contributor to the metabolic effects of RK, rather than a proven causal driver. Regarding additional mechanistic experiments, we agree that Western blot analysis of NAMPT protein levels, enzymatic activity assays, and downstream SIRT1 target measurements (e.g., PGC‑1α acetylation) would provide deeper mechanistic insight. However, these analyses require additional tissue processing and experimental workflows that extend beyond the scope of the current study and were not included in the original experimental design. We have therefore acknowledged this limitation explicitly in the Discussion and highlighted these approaches as important directions for future research.

Comment 3: While microbiota analysis is a strength, the reporting lacks statistical rigor and methodological transparency. The authors must provide technical details on the bioinformatics pipeline and clearly state how they controlled for multiple comparisons (e.g., False Discovery Rate) in their taxa and pathway analyses. Finally, correlation data between microbial changes and metabolic phenotypes must include adjusted p-values and correlation coefficients (r) for clarity.

Response 3: We thank the reviewer for these constructive comments. We agree that microbiota studies often require specialized statistical frameworks, including multiple‑comparison correction and advanced differential‑abundance tools. However, in the present study, the microbiota analysis was intended as a supportive descriptive component, rather than a primary mechanistic endpoint. For this reason, we limited our statistical testing to one-way ANOVA, consistent with the rest of the manuscript, and did not perform high-dimensional differential‑abundance analyses that require FDR correction.

Comment 4: The study claims significant reductions in adipose tissue mass and improved liver health, but provides no visual histological evidence to support these morphological changes. To confirm the anti-obesity effects, H&E staining of adipose tissue is required to visualize changes in adipocyte hypertrophy. Furthermore, to validate the anti-steatotic effects, H&E or Oil Red O staining of liver sections is essential to demonstrate the reduction in lipid accumulation.

Response 4: We thank the reviewer for this important comment. We agree that histological analyses such as H&E staining of adipose tissue and H&E or Oil Red O staining of liver sections provide valuable morphological confirmation of adipocyte hypertrophy and hepatic lipid accumulation. However, these analyses were not included in the original experimental design, and the available tissue samples were fully allocated to biochemical and molecular assays. As a result, additional histological staining cannot be performed at this stage. In addition, we have added a statement to the Discussion acknowledging this limitation and noting that future studies incorporating adipose and hepatic histology will be essential to visually confirm the observed metabolic improvements.

Comment 5: The small sample size of n=5 per group is the minimum for metabolic studies and requires justification for statistical power. The authors must expand the statistical methods section to specify the exact tests used, including whether they checked for assumptions like normality and homogeneity of variance. Finally, all figure legends must clearly state whether error bars represent Standard Error of the Mean (SEM) or Standard Deviation (SD).

Response 5: This concern was also noted by another reviewer and has been comprehensively addressed in the revised manuscript.

Comment 6: The authors should provide a clearer justification for the chosen 10 mg/kg and 50 mg/kg doses, timing, and route of administration, citing dose-finding studies or relevant literature.

Response 6: A comparable issue was identified by another reviewer, and we have thoroughly addressed it in the revised manuscript.

Comment 7: The manuscript must explicitly state the full ethical approval details (committee name and number) and expand the methods to ensure reproducibility of the Rice Kefiran preparation, including its purity and key compositional details

Response 7: Actually, a similar concern was raised by another reviewer and has been fully addressed in the revised manuscript. Thank you

Comment 8: The Results section should be restructured with clearer subsections that conclude with concise summaries. The authors should also calculate and present the Area Under the Curve (AUC) for the OGTT data for a better quantitative summary, and ensure all figures have standardized, legible labels and units.

Response 8: Thank you for this insightful comment. We have restructured the Results section by adding clearer subsections and brief concluding statements to improve readability and logical flow. We also calculated the area under the curve (AUC) for the OGTT using the trapezoidal method and incorporated these quantitative data into both the Results and the corresponding figure. In addition, all figures were reviewed and updated to ensure consistent, legible labeling and standardized units throughout. These revisions strengthen the clarity and presentation of our findings.

Comment 9: The Data Availability statement needs an update to clarify where raw sequencing data and primary numerical data will be deposited. Finally, the authors must verify and state that the starting body weights were not significantly different between the groups at Week 0.

Response 9: We thank the reviewer for this helpful comment. We have modified the Data Availability statement to clearly specify the repositories where both the raw sequencing data and the primary numerical datasets will be deposited upon acceptance. In addition, we verified the baseline body weights at Week 0 and confirmed that there were no significant differences among the experimental groups. This information has now been added to the Results section for clarity.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been revised in accordance with the reviewers’ comments.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors included all recommendations suggested.