Oxidative, Inflammatory, and Constipation Stress Modulation by a Heteropolysaccharide from Lacticaseibacillus rhamnosus CRL75

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Major Comments:

Title & Abstract: The title is informative but somewhat lengthy. Consider simplifying it while retaining the key terms. The abstract summarizes the study well but should explicitly state the primary findings along with their statistical significance.

Introduction: The introduction provides a solid background but should better emphasize the novelty of the study. Some references are outdated or redundant; consider integrating more recent literature to strengthen the context.

Materials and Methods:
- **Fermentation and EPS Isolation:** Clearly specify the statistical methods used for analyzing microbial growth and fermentation kinetics.
- **EPS Characterization:** While FTIR and Raman spectroscopy are suitable for elucidating molecular structures, complementary techniques such as NMR or MS would enhance the findings.
- **Biological Assays:** The study claims antioxidant activity but does not include a positive control for comparison (e.g., ascorbic acid). Including a standard compound for benchmarking is recommended.
- **Laxative Activity in Mice:** Provide more details regarding ethical considerations and approvals for animal studies.

Results and Discussion: Some sections lack indicators of statistical significance. For instance, when discussing the antioxidant capacity of EPS, it is unclear whether the differences between doses are statistically significant. The interpretation of FTIR and Raman results is well explained, but additional discussion on validating linkage types (e.g., confirming glycosidic bonds via methylation analysis) is necessary. While the in vivo results regarding laxative properties are promising, further discussion on potential mechanisms of action is needed.

Figures and Tables: Some figure legends require further explanation, particularly those related to spectroscopy results. Ensure that all tables are uniformly formatted and have clear headers.

Language and Style: The manuscript contains grammatical inconsistencies. A thorough review of the English language is recommended to improve clarity and readability. Additionally, some sections are overly detailed; consider consolidating information for conciseness.

Minor Comments:
- Line 10: Define "LAB" upon its first mention.
- Line 35: Clarify whether "extended viscosity" refers to an increase or a prolonged effect.
- Line 145: Specify the equipment and software used for spectral analysis.
- Line 421: Ensure consistent citation formatting.
- Check for any missing or misplaced citations in the discussion section.

Comments on the Quality of English Language

as above

Author Response

Major Comments:

1. Title & Abstract: The title is informative but somewhat lengthy. Consider simplifying it while retaining the key terms. The abstract summarizes the study well but should explicitly state the primary findings along with their statistical significance.

R1. We appreciate your suggestion regarding the conciseness of the title. In response, we have revised the previous title (Oxidative, inflammatory, and constipation stress responses of a partially characterized heteropolysaccharide isolated from fermented milk by Lacticaseibacillus paracasei subsp. paracasei CRL75) and changed it to: “Oxidative, Inflammatory, and Constipation Stress Modulation by a Heteropolysaccharide from Lacticaseibacillus paracasei CRL75” (see lines 2–4). This version supports the key terms while improving clarity and readability. Moreover, we appreciate your insightful feedback regarding the abstract. In response, we have revised it to explicitly state the primary findings and their statistical significance. This modification enhances clarity and ensures that the key results are clearly conveyed to the reader (see lines 27–44). Thank you for your valuable suggestions, which have helped improve the manuscript.

Introduction: The introduction provides a solid background but should better emphasize the novelty of the study. Some references are outdated or redundant; consider integrating more recent literature to strengthen the context.

R2. In response to your suggestion, we have revised the introduction to better emphasize the novelty of our study. Additionally, we have updated the references by replacing outdated or redundant sources and have integrated a recent article to enhance the context of the study. Thank you again for your time and valuable feedback.

a). Reference included in the Introduction section of the revised manuscript version:

Juraskova, D.; Ribeiro, S.C.; Silva, C.C.G. Exopolysaccharides Produced by Lactic Acid Bacteria: From Biosynthesis to Health-Promoting Properties. Foods 2022, 11. doi:10.3390/foods11020156. (see lines 709–710)

b). References deleted in the Introduction section of the previous manuscript version.

Hassan, A.N. ADSA Foundation Scholar Award: Possibilities and challenges of exopolysaccharide-producing lactic cultures in dairy foods. J. Dairy Sci. 2008, 91, 1282-1298, doi:10.3168/jds.2007-0558.

Leroy, F.; De Vuyst, L. Advances in production and simplified methods for recovery and quantification of exopolysaccharides for applications in food and health. J. Dairy Sci. 2016, 99, 3229-3238, doi:10.3168/jds.2015-9936.

Duboc, P.; Mollet, B. Applications of exopolysaccharides in the dairy industry. Int. Dairy J. 2001, 11, 759-768. doi:10.1016/S0958-6946(01)00119-4

Materials and Methods:

- **Fermentation and EPS Isolation:** Clearly specify the statistical methods used for analyzing microbial growth and fermentation kinetics.

R3. Thank you for your observation. We have modified the "Materials and Methods" section by adding the requested information (see lines 106–126).

- **EPS Characterization:** While FTIR and Raman spectroscopy are suitable for elucidating molecular structures, complementary techniques such as NMR or MS would enhance the findings.

R4. Thank you for your valuable suggestion. You are absolutely right that techniques such as NMR or MS would provide further insights into the molecular structure of the EPS. However, this study focuses on a “partial characterization” (see lines 135–189) of the biopolymer, including monomeric composition, molecular weight analysis, vibrational spectroscopy, hydrodynamic properties, and thermal behavior. In fact, for this reason, the title of the work was previously called: "Oxidative, inflammatory, and constipation stress responses of a partially characterized heteropolysaccharide isolated from fermented milk by Lacticaseibacillus paracasei subsp. paracasei CRL75").

However, we plan to perform methylation analysis (validation of glycosidic linkage types via GC chromatography), 1D (¹H and ¹³C) and 2D NMR analyses (HMBC, HSQC, TOCSY, and DOCSY) at the Department of Cellular Engineering and Biocatalysis in collaboration with Dr. Agustín López Munguía’s team at the National Autonomous University of Mexico (UNAM) between June and August of this year. The results obtained from these structural characterization studies, along with insights into the molecular mechanisms involved in EPS75 production, will be disseminated in a future publication in this journal. We appreciate your constructive feedback and hope this clarification addresses your concern.

- **Biological Assays:** The study claims antioxidant activity but does not include a positive control for comparison (e.g., ascorbic acid). Including a standard compound for benchmarking is recommended.

R5. In the original manuscript (ID: fermentation-3524985), we did include this information (see lines 188 and 189). Specifically, we used ascorbic acid (Vc) as the standard reagent, and all results were expressed in terms of ascorbic acid equivalents (Vc eq.). In revised version this paragraph includes lines 196-198.

- **Laxative Activity in Mice:** Provide more details regarding ethical considerations and approvals for animal studies.

R6. In the original manuscript (ID: fermentation-3524985), we included this information in the "Institutional Review Board Statement" section (lines 662–665). However, in the revised version of the manuscript, we have also incorporated this information in the relevant section of Materials and Methods (lines 275-278).

Results and Discussion: Some sections lack indicators of statistical significance. For instance, when discussing the antioxidant capacity of EPS, it is unclear whether the differences between doses are statistically significant.

R7. Thank you for your suggestion. We would like to clarify that the current version of the manuscript includes graphs regarding the antioxidant capacity of EPS75 (Figure 4), along with the corresponding statistical analysis and discussion (see Figure 4).

The interpretation of FTIR and Raman results is well explained, but additional discussion on validating linkage types (e.g., confirming glycosidic bonds via methylation analysis) is necessary.

R8. Please consider the response R4 to analyze this topic.

While the in vivo results regarding laxative properties are promising, further discussion on potential mechanisms of action is needed.

R9. We appreciate your perspective on the need for further discussion regarding the potential mechanisms of action. While our current study focuses on evaluating the laxative properties, we acknowledge the importance of exploring the underlying mechanisms. In future research, we plan to investigate different aspects related to constipation-induced stress, including the determination of gene expression through Reverse Transcription and Quantitative PCR (RT-qPCR) in mouse colon tissues, immunohistochemistry analysis of interstitial cells of Cajal (ICC), and blood biochemistry analysis. These approaches will provide a more comprehensive understanding of the physiological and molecular mechanisms involved.

Figures and Tables: Some figure legends require further explanation, particularly those related to spectroscopy results. Ensure that all tables are uniformly formatted and have clear headers.

R10. We appreciate your attention to detail and your suggestions for improvement. We have carefully reviewed and revised all figure legends and table headers throughout the manuscript.

Language and Style: The manuscript contains grammatical inconsistencies. A thorough review of the English language is recommended to improve clarity and readability. Additionally, some sections are overly detailed; consider consolidating information for conciseness.

R11. Thank you for your feedback. In the revised version of the manuscript, we have made the text more concise and have improved grammar and fluency to enhance clarity and readability.

Minor Comments:

- Line 10: Define "LAB" upon its first mention.

R12. In the original manuscript (ID: fermentation-3524985), we explained this information in the "Abstract" (line 28) and "Introduction" section (line 50).

- Line 35: Clarify whether "extended viscosity" refers to an increase or a prolonged effect.

R13. The terminology has been corrected in the revised manuscript (see line 33)

- Line 145: Specify the equipment and software used for spectral analysis.

R14. Both requests were added to the current manuscript version (see lines 154–159).

“- Line 421: Ensure consistent citation formatting.” and “- Check for any missing or misplaced citations in the discussion section.”

R15. Thanks for the suggestion. We have taken all the necessary precautions to verify citations and bibliographic references correctly, following the journal guidelines.

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer Comments

1. In the Methods section, please include additional details regarding the sugar samples collected. Ensure that you provide sufficient information to allow for the replication of the analyses.
2. Please include a reference for LAB strain produces capsular.
3. In Figure 4, the labels on the x-axis and y-axis are unclear. Please ensure they are clearly defined and legible.
4. In line 67, does the term " other fermented milk drinks" indicate that an unidentified component, distinct from polysaccharides, and proteins, plays a significant role in the EPS75?
5. In line 567: What's specific enzymes here? More description is needed.?
6. Why extracellular polysaccharides (EPS75) responsible for the ropy phenotype. Should explain this.

Comments on the Quality of English Language

Reviewer Comments

1. In the Methods section, please include additional details regarding the sugar samples collected. Ensure that you provide sufficient information to allow for the replication of the analyses.
2. Please include a reference for LAB strain produces capsular.
3. In Figure 4, the labels on the x-axis and y-axis are unclear. Please ensure they are clearly defined and legible.
4. In line 67, does the term " other fermented milk drinks" indicate that an unidentified component, distinct from polysaccharides, and proteins, plays a significant role in the EPS75?
5. In line 567: What's specific enzymes here? More description is needed.?
6. Why extracellular polysaccharides (EPS75) responsible for the ropy phenotype. Should explain this.

Author Response

Reviewer Comments

In the Methods section, please include additional details regarding the sugar samples collected. Ensure that you provide sufficient information to allow for the replication of the analyses.

R1. Thank you for your valuable suggestion. We have carefully addressed your comment and included additional details regarding the sugar sample preparation in the Materials and Methods section. This information is now available in the current version of the manuscript to ensure the reproducibility of the analyses (see lines 143–151).

Please include a reference for LAB strain produces capsular.

R2. In the original manuscript (ID: fermentation-3524985) we provided this information in the "Introduction" (lines 51–58) and "Materials and Methods" sections (lines 118–121).

In Figure 4, the labels on the x-axis and y-axis are unclear. Please ensure they are clearly defined and legible.

R3. We have carefully considered your comment about the clarity of the x-axis and y-axis labels in Figure 4. In the revised version of the manuscript, we have improved the figure to ensure that all labels are clearly defined and more legible. Additionally, we have included an inset with a statistical analysis of significant differences in the results. We hope that this updated version meets the publication requirements.

In line 67, does the term " other fermented milk drinks" indicate that an unidentified component, distinct from polysaccharides, and proteins, plays a significant role in the EPS75?

R4. We have revised the wording to clarify this point and avoid any unintended dichotomy. The updated version of the manuscript now reflects this modification (see lines 66–68).

In line 567: What's specific enzymes here? More description is needed.?

R5. Thank you for your comment. We provide further details on this topic in the current manuscript version (see lines 586-589).

Why extracellular polysaccharides (EPS75) responsible for the ropy phenotype. Should explain this.

R6. In the previous version of the manuscript (ID: fermentation-3524985) this explanation was already provided in the Discussion section, specifically between lines 518 and 521. However, in the updated version, we have refined the content to improve clarity and coherence. We appreciate your valuable feedback and careful review (see lines 535–541).

Comments on the Quality of English Language

R7. Please consider the response R11 expressed to Reviewer 1 for analysis of this topic.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all previous reviewer comments, and the manuscript is now clearly written, well-structured, and scientifically sound. The revised version demonstrates significant improvements in clarity, methodological detail, and data presentation. The study provides robust characterization of a heteropolysaccharide (EPS75) from Lacticaseibacillus paracasei CRL75, with convincing evidence of its antioxidant, anti-inflammatory, and laxative activities, including in vivo validation. The novelty and relevance to functional food development are clear, and the supplementary materials adequately support the main findings. I therefore recommend that the manuscript be accepted.