The Response of Mucosal Colonic Microbiota to Probiotic and Dietary Intervention In Vitro

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study employed the M-SHIME^® and L-SHIME^® models to systematically investigate the differential effects of dietary macronutrient intake and probiotic supplementation on simulated intestinal mucosal and luminal microbiota, and attempted to correlate the in vitro findings with results from human research. The study demonstrates a robust experimental design and logical structure, providing valuable in vitro model data for understanding the dynamics of mucosal microbiota. However, several issues related to data accuracy, clarity, and scientific rigor need to be addressed. The specific suggestions are as follows:

• In the Introduction, it is recommended to rephrase the research objectives to highlight the main thread of the study.
• Line 167-168: Please provide additional details regarding the specific strain identification, purchase information, and strain characteristics of Lactobacillus rhamnosusGG (LGG).
• In Section 2.1, please provide additional details on the key operational parameters for running the L-SHIME® and M-SHIME® modes, such as pH and retention time.
• In Methods (lines 217-218), generative artificial intelligence was used to assist in the development of the R script. Please provide the final validated complete R script.
• In the Results section, the Latin names of bacteriain Fig. 2, 5, and 7 should be italicized, please double check and revise.
• In Section 2.3 (lines 177-178), the pooling of DNA isolates from multiple independent bioreactors for sequencing masks the variation among biological replicates. This results in ineffective error estimation for subsequent statistical inference and significantly influences the reliability of the conclusions.
• In Result 3.2 (lines 242-245), several outlier samples were identified and excluded. However, the methodology section did not specify the criteria for their identification or the procedure for their exclusion. Please provide a detailed supplement.
• Regarding the F/B ratio in Fig. 4 (Results 3.2), data on the relative abundance of the phyla Firmicutes and Bacteroidetes are lacking. Please provide these data.
• The statement that "These differences were all statistically significant" is inconsistent with the data presented in Fig. 4. Please correct.
• In the discussion section, the findings of this study were based on a fat-deprived M-SHIME® system (line 532-533: ...none of the nutrients in fat-deprived M-SHIME® feed...). However, this critical information was not explicitly described in the Materials and Methods section.
• The discussion and conclusion section are lengthy and lacks focus. It is recommended to reorganize and streamline this section by merging overlapping points and focusing on the core conclusions.
• The reference format is inconsistent. Specifically, the URL in reference 26 is too long. Please correct.

 

Author Response

We have given a detailed response to the Reviewer's comments in the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well-designed, methodologically sound in vitro study that leverages the M-SHIME® model to address an important and underexplored topic: dietary and probiotic modulation of mucosa-associated microbiota. The manuscript is comprehensive, technically robust, and thoughtfully interpreted in the Discussion.

Decision: Minor Revision

 

Abstract

The conclusion currently emphasizes model reliability but could more clearly state why mucosal microbiota stability matters biologically. I recommend adding the following sentence “These findings are relevant for understanding diet-microbiome interactions at the mucosal interface, which is critically involved in intestinal immune regulation.”

 

Introduction

1. The Introduction focuses heavily on methodological justification, but the clinical and pathological relevance (e.g., IBD, barrier dysfunction) is underdeveloped. For the paragraph discussing mucosal proximity to the epithelial barrier (lines 64–67), I recommend adding the following sentence “Given the central role of mucosa-associated microbiota in intestinal immune regulation and inflammatory bowel disease pathogenesis, understanding how diet modulates this niche is of particular clinical relevance”  https://doi.org/10.3390/immuno4040026

 

2. For the paragraph introducing dietary modulation of mucosal microbiota (lines 86–93), This paragraph discussing dietary interventions in terms of macronutrients, but dietary additives and non-nutritive compounds are not considered. I recommend to briefly discussing the effect of non-nutritive compounds and the authors could use this reference to support this addition https://doi.org/10.3390/diseases13040115.

 

Materials and Methods

1. Clarify why a single donor was chosen and how this affects generalizability.
2. The exclusion of outliers (M3, M4, M9, L3, L4, and L10) is justified statistically, but the biological rationale should be briefly acknowledged.

 

Discussion

The Discussion would benefit from a short subsection explicitly addressing limitations, including: Single donor, In vitro constraints, and Absence of host immune components

Conclusion

1. The conclusion could more explicitly articulate why mucosal stability matters for human health.
2. Consider adding a forward-looking statement regarding application to disease-oriented research (e.g., IBD, metabolic disorders).

 

Minor Comments

1. Please ensure consistent use of terminology throughout the manuscript when referring to mucosal-associated microbiota versus mucus-associated microbiota, as both terms appear in different sections.
2. Abbreviations should be defined at first mention in the main text (e.g., MPG, F/B ratio, LGG) and used consistently thereafter.

Author Response

We have given a detailed response to the Reviewer's comments in the attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The study presents a well-designed and relevant approach to the analysis of the mucosal gut microbiota, and the use of the M-SHIME® and L-SHIME® models is appropriate for the proposed objectives. The results are interesting and, for the most part, well supported. However, some clarifications and adjustments are needed to improve the clarity and rigor of the manuscript.
Recommendations for authors:
- a more detailed description of the statistical methods used and the significance criteria would be useful, to facilitate the evaluation of the results;
- claims regarding the superior stability of the mucosal microbiota and the reliability of the in vitro models should be formulated more cautiously and contextualized in relation to the limitations of the study;
- although concordance with previous human studies is mentioned, a more explicit discussion of the differences and similarities between the in vitro and in vivo results would be beneficial;
- a clearer definition of the terms “mucosal” and “luminal” is recommended when first used, to avoid ambiguity for readers unfamiliar with SHIME® models.

Author Response

We have given a detailed response to the Reviewer's comments in the attached file.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Regarding this manuscript, I have the following comments:

1. It was suggested that M-SHIME is a standard in vitro model. Please elaborate more on this model to further emphasize this point.
2. In figure 2, the authors showed the relative abundance of microbes under each condition for M-SHIME. Where is the result for L-SHIME?
3. what are the authors' hypothesis on the outliers in Figure 3?
4. Could the authors elaborate more on what the arrows represent in Fig 6?
5. what are the authors' rationale that the confidence intervals for luminal data are larger than those of mucusal? Does this mean that probiotics mainly affect luminal microbiome?

Author Response

We have given a detailed response to the Reviewer's comments in the attached file.

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

This study used advanced in vitro gut models (M-SHIME® and L-SHIME®) to compare how mucosal and luminal intestinal microbiota respond to dietary macronutrient changes and probiotic supplementation. The results showed that mucosal microbiota responded differently and was more stable than luminal microbiota, with specific bacterial genera correlating positively with macronutrient—especially protein—intake.

The findings highlight the importance of studying mucosal microbiota separately from luminal communities and support the value of sophisticated in vitro models for investigating gut microbial dynamics that are difficult to assess in humans.

Upon thorough evaluation, I did not identify any remarks or concerns with the paper.

In my opinion it can be published in the present form.

 

Author Response

Thank you very much for your kind comment and for the time taken to read and evaluate our manuscript.