Novel Food Supplement Containing a Combination of Postbiotics and Plant-Derived Compounds Regulates Epithelial Barrier Integrity and Immune Response in Human Enterocytes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Epithelial barrier and immune response play a key role in the pathogenesis of allergy. This article reveals that the product of postbiotics and plant-derived compounds significantly strengthens both the physical and immunological components of the gut barrier, especially exerts synergistic action on intestinal epithelial cells by reinforcing tight junction integrity, stimulating mucus production and antimicrobial peptide secretion, and promoting epithelial maturation. These results not only highlight the potential of this formulation as a supportive strategy to restore mucosal homeostasis and tolerance in allergic diseases, but also provides a new idea for the research and development of functional foods. This article is clear, scientific, logical, well-organized, and systematic, and provides information on food allergy and epithelial barrier as well as immune response. However, there are still many paragraphs to improve the quality of the paper:

1. Abstract: The summary can be compressed and must strictly follow the Foods format.
2. Introduction: Supplement with Perilla frutescens containing quercetin and suggesting the reference of crops with high quercetin content, such as onion, approach of functional food to prevent and treat allergic diseases.The literature should be supplemented if butyrate has anti-allergic properties.
3. Materials and Methods: “1 Study design+2.2 Study product”， The wording seems imprecise. Could you consider a more dynamic approach, such as 'materials and products'? Briefly describe the sources of heat inactivated LGG, butyrate, quercetin and perilla extract? This section would be more comprehensible if supplemented with a method roadmap.
4. Results: This section has only 3 curves or bars, making the results appear sparse. We recommend adding necessary tables and images. 5. Figures and Tables cannot be headings. The unprocessed control (NT) appears to be poorly defined.
5. Discussion: Adding subheadings is required in discussions. The results should correspond to the section headings in Materials and Methods and Discussion.
6. References: Write strictly in the format of Foods.

Author Response

Reviewer #1

Epithelial barrier and immune response play a key role in the pathogenesis of allergy. This article reveals that the product of postbiotics and plant-derived compounds significantly strengthens both the physical and immunological components of the gut barrier, especially exerts synergistic action on intestinal epithelial cells by reinforcing tight junction integrity, stimulating mucus production and antimicrobial peptide secretion, and promoting epithelial maturation. These results not only highlight the potential of this formulation as a supportive strategy to restore mucosal homeostasis and tolerance in allergic diseases, but also provides a new idea for the research and development of functional foods. This article is clear, scientific, logical, well-organized, and systematic, and provides information on food allergy and epithelial barrier as well as immune response. However, there are still many paragraphs to improve the quality of the paper:

We thank the Reviewer for this overall positive evaluation of our study.

We have revised the wording and structure of the Materials and Methods section to improve clarity and readability. Specifically, we refined the section headings to better reflect the experimental data and to provide clearer description of the origin and sourcing of each component of the study product, including heat-inactivated Lacticaseibacillus rhamnosus GG, Butyrate, Quercetin, and Perilla frutescens extract. In addition, we introduced a concise methodological roadmap summarizing the experimental workflow, thereby facilitating comprehension of the study design and results.

 

1. Results: This section has only 3 curves or bars, making the results appear sparse. We recommend adding necessary tables and images. 5. Figures and Tables cannot be headings. The unprocessed control (NT) appears to be poorly defined.

We thank the Reviewer for this constructive observation. We have expanded the Results section by adding supplementary tables summarizing quantitative outcomes across all experimental endpoints. We believe that these modificatons could allow a clearer overview of the magnitude and consistency of the observed effects without introducing redundant graphical elements. The revised presentation better reflects the robustness of the experimental dataset while maintaining clarity.

 

1. Discussion: Adding subheadings is required in discussions. The results should correspond to the section headings in Materials and Methods and Discussion.

We thank the Reviewer for this important structural suggestion. In response, we have revised the Discussion by introducing clear subheadings that mirror the organization of the Materials and Methods and Results sections. This harmonization ensures a consistent flow across sections and facilitates direct comparison between experimental procedures, outcomes, and their interpretation. The revised structure improves clarity and readability, in line with Foods editorial guidelines.

 

1. References: Write strictly in the format of Foods.

The references section was mofified according to this suggestion.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates a multi-component food supplement combining postbiotics and plant-derived compounds and evaluates its effects on epithelial barrier integrity and innate immune markers using a Caco-2 cell model.

The manuscript does not fully comply with Foods journal standards in terms of structure.

Figures, The authors should improve overall figure resolution.

Overall, the authors’ conclusions are not sufficiently supported by rigorous experimental evidence, and the manuscript lacks the depth of scientific validation.

Line 71, The rationale for combining postbiotics and plant-derived compounds is descriptive rather than hypothesis-driven, lacking clear mechanistic justification for expected synergy.

Line 148, Tight junction proteins were evaluated only at the mRNA level, without protein-level or localization validation.

Line 263, The absence of component-specific controls or synergy assessment models makes this claim scientifically unsupported.

Line 324, Limitations of the model were acknowleged only briefly and were not sufficiently integrated to the interpretation of the result.

Line 313, The authors infer pathway activation based on previously published literature rather than direct experimental measurements, and no comparative analyses were provided to support additive or synergistic interactions.

Author Response

Reviewer #2

 

1. This manuscript investigates a multi-component food supplement combining postbiotics and plant-derived compounds and evaluates its effects on epithelial barrier integrity and innate immune markers using a Caco-2 cell model.
2. The manuscript does not fully comply with Foodsjournal standards in terms of structure.
3. Figures, The authors should improve overall figure resolution.
   
   Overall, the authors’ conclusions are not sufficiently supported by rigorous experimental evidence, and the manuscript lacks the depth of scientific validation.

We thank the Reviewer for these suggestions. We respectfully acknowledge the concern regarding the strength of the conclusions and the depth of validation. In response, we have carefully revised both the Discussion and Conclusions to better align our interpretations with the scope and limitations of the experimental model used. Specifically, we have tempered causal claims, clarified that our findings derive from a preclinical in vitro setting, and strengthened the mechanistic interpretation by explicitly linking each conclusion to the corresponding experimental evidence. Furthermore, we expanded the Discussion to contextualize our results within the existing literature and to clearly delineate the translational implications and limitations of the study. These revisions enhance the scientific rigor and ensure that the conclusions are fully supported by the presented data.

 

4. Line 71, The rationale for combining postbiotics and plant-derived compounds is descriptive rather than hypothesis-driven, lacking clear mechanistic justification for expected synergy.

We thank the Reviewer for this insightful comment. In response, we have revised the Introduction to explicitly frame the combination of postbiotics and plant-derived compounds within a hypothesis-driven, mechanistic rationale. Specifically, we now clarify that the formulation was designed to target complementary and converging epithelial pathways involved in barrier integrity and innate immune defense, including tight junction regulation, epithelial differentiation, redox-sensitive signaling, and antimicrobial peptide production. By articulating the expected mechanistic synergy between microbiota-derived postbiotics and polyphenol-rich plant compounds, the revised text strengthens the biological justification underlying the combined formulation.

 

5. Line 148, Tight junction proteins were evaluated only at the mRNA level, without protein-level or localization validation.

We thank the Reviewer for this important observation. We acknowledge that tight junction proteins were assessed at the transcriptional level and that protein expression or localization was not directly evaluated in the present study. However, mRNA analysis was intentionally combined with functional readouts of epithelial barrier integrity, including transepithelial electrical resistance measurements and paracellular permeability assessment, which provide established functional correlates of tight junction assembly. In the revised manuscript, we have clarified this methodological choice and explicitly acknowledged this limitation in the Discussion, while tempering conclusions accordingly and indicating that future studies will address protein-level and localization analyses.

 

 

 

6. Line 263, The absence of component-specific controls or synergy assessment models makes this claim scientifically unsupported.

We thank the Reviewer for this important and well-founded comment. We acknowledge that the present study did not include component-specific control conditions or formal synergy assessment models. Accordingly, we have revised the manuscript to avoid overinterpretation of synergistic effects. The term “synergistic” is now used in a mechanistic and descriptive context, referring to the coordinated modulation of complementary epithelial pathways by the combined formulation, rather than to a quantitatively demonstrated pharmacological synergy. In addition, we have clarified this point in the Discussion and tempered the Conclusions to ensure that all claims are fully supported by the experimental design and data presented.

 

7. Line 324, Limitations of the model were acknowleged only briefly and were not sufficiently integrated to the interpretation of the result.

We thank the Reviewer for this important comment. In response, we have expanded and restructured the Discussion to more explicitly integrate the limitations of the in vitro epithelial model into the interpretation of the results. Rather than confining limitations to a brief statement, we now contextualize each major finding within the boundaries of the Caco-2 model, clarifying which biological aspects can be reliably inferred and which require further validation in more complex systems. This revised approach strengthens the interpretative framework and ensures that conclusions remain fully aligned with the experimental model.

 

8. Line 313, The authors infer pathway activation based on previously published literature rather than direct experimental measurements, and no comparative analyses were provided to support additive or synergistic interactions.

We thank the Reviewer for this important and well-taken comment. We acknowledge that pathway activation was inferred based on established literature rather than directly measured in the present study, and that comparative analyses required to demonstrate additive or synergistic interactions were not performed. In response, we have revised the Discussion to clearly distinguish between experimentally observed outcomes and mechanistic interpretations derived from prior evidence. In addition, we have removed any implication of additive or synergistic interactions not directly supported by the experimental design, reframing these statements as hypothesis-generating interpretations. These revisions ensure that all mechanistic considerations remain fully aligned with the data presented.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors of the manuscript "Novel food supplement containing a combination of postbiotics and plant-derived compounds regulates epithelial barrier integrity and immune response in human enterocytes" presented research involving the use of postbiotics in modeling intestinal epithelial integrity. The results of the presented study highlight the possibility of restoring homeostasis and mucosal tolerance in allergic diseases through the use of appropriately selected postbiotics. The manuscript is well-developed, but requires some minor revisions. The scope of the proposed changes is included in the attached file.

Comments for author File: Comments.pdf

Author Response

Thank you for your positive feedback and for your insightful comments on our manuscript.

We have carefully addressed all the minor revisions requested in the attached file. We believe these changes have further strengthened the clarity and quality of our work.

Please find the revised manuscript attached, with all changes highlighted.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author is requested to take the comments from the preliminary reviewers (1, 2, 6, etc.) seriously, with particular attention to the abstract, which should be formatted according to the latest academic standards. 

Author Response

We would like to thank the reviewer for his valuable comments and suggestions, which helped us improve the quality of our manuscript. We have made the requested changes.

Reviewer 2 Report

Comments and Suggestions for Authors

Despite the authors’ revision, the manuscript does not adequately address the important concerns regarding insufficient experimental depth, interpretative overreach, and methodological limitations. The conclusions remain broader than what can be reliably supported by the experimental framework.

In addition, the manuscript still does not comply with the journal’s formatting requirements, as the abstract continues to use a structured format with section headings.

Given these issues, the manuscript would require substantial reworking beyond the scope of a revision. I therefore recommend rejection.

Author Response

We sincerely thank the reviewer for their critical assessment and for the opportunity to further refine our manuscript. We carefully considered concerns regarding experimental depth, interpretive scope, and formatting. We recognize that, as this is a preliminary in vitro study, its results must be presented with due caution. We have implemented the work based on their criticisms.