Effect of Lentilactobacillus buchneri on Chemical and Microbial Compositions of Herba Leonuri (Leonurus japonicus Houtt.)-Contained Alfalfa Silage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled “Effect of Lentilactobacillus buchneri on chemical and microbial compositions of herba leonuri (Leonurus japonicus Houtt.)-contained alfalfa silage” presents an interesting study on the use of Lentilactobacillus buchneri as an inoculant in alfalfa silage mixed with Leonurus japonicus (LJH). Here are some comments and suggestions that could enhance the clarity and rigor of the manuscript:

Strengths:

1. Importance of Research: To improve animal feed quality and expand our knowledge of silage fermentation, it is essential to look into how lactic acid bacterial inoculants affect silage quality, particularly when antimicrobial components are present.

2. Methodology: It is praiseworthy and rational to utilize vacuum-sealed polyethylene bags for anaerobic circumstances and to choose the right ratios for mixing alfalfa and LJH.

3. Results Presentation: The text clearly compares the Lentilactobacillus buchneri treatment to the control (CK), pointing out notable alterations in the microbial and chemical makeup.

 

Areas for Improvement:

1. Abstract Clarity: The first sentence could benefit from clearer phrasing. Consider rephrasing to clarify the context, such as, "Lactic acid bacteria (LAB) inoculants are commonly used in silage production, yet their effects on silage containing antimicrobial components, such as those found in Leonurus japonicus, remain less explored.”
2. Statistical Analysis: The manuscript mentions significant differences (P < 0.05) but does not provide detailed methods for statistical analysis. Including specific statistical tests and methods used for data analysis would enhance the rigor of the findings.
3. Discussion Section: The obtained results are well discussed.
4. Future Directions: Including suggestions for further research could improve the manuscript. For example, investigating the mechanisms behind LJH’s impact on LAB or exploring different LAB strains could be beneficial.
5. Conclusions: The conclusion might be strengthened by summarizing the broader implications of using LJH in livestock feed and its potential economic benefits for farmers, as well as its environmental impact.

Technical Corrections:

• Ensure that all scientific names are italicized consistently throughout the manuscript.
• The sentence structure could be improved in some areas for clarity. For example, the phrase “as result from increased (P < 0.05) bacterial dominance” should read “as a result of increased (P < 0.05) bacterial dominance.”
• The use of specific terms such as “bacterial richness indices” could be made clearer by briefly defining them upon first mention for readers less familiar with the terminology.

Overall Impression:

This manuscript presents a valuable contribution to the literature on silage production and the impact of specific LAB on microbial and chemical compositions. Addressing the areas for improvement will enhance its clarity, rigor, and impact. I look forward to seeing the final version of the manuscript.

 

 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Good

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1: Abstract Clarity: The first sentence could benefit from clearer phrasing. Consider rephrasing to clarify the context, such as, "Lactic acid bacteria (LAB) inoculants are commonly used in silage production, yet their effects on silage containing antimicrobial components, such as those found in Leonurus japonicus, remain less explored.”

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we revised the first sentence of the abstract to read  "Lactic acid bacteria (LAB) inoculants are commonly used in silage production, yet their effects on silage containing antimicrobial components, such as those found in Leonurus japonicus, remain less explored.”we have revised the appropriate parts of the text and the revised parts are shown in red.

Comments 2: Statistical Analysis: The manuscript mentions significant differences (P < 0.05) but does not provide detailed methods for statistical analysis. Including specific statistical tests and methods used for data analysis would enhance the rigor of the findings.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we revised the first sentence of the abstract to read “ANOVA analyses of variations in chemical composition, fermentation characteristics and bacterial community indices during storage were performed using SPSS (version 26.0; IBM Corp., Armonk, NY, USA) and Duncan's multiple comparisons were performed on so samples. When the probability level was less than 0.05 (P < 0.05), differences were considered statistically significant. The main effects were not discussed if an interaction occurred between the treatment and storage period.”we have revised the appropriate parts of the text and the revised parts are shown in red.

Comments 3: Discussion Section: The obtained results are well discussed.

Response 3: Thank you for pointing this out.We would like to express our sincere gratitude for taking the time to review our manuscript and provide constructive feedback. We appreciate your positive remarks regarding the thoroughness of the discussion section.To further enhance the quality of our paper, we have taken this opportunity to revisit the discussion section with a critical eye. Specifically, we have:Clarified the implications of our research in broader scientific contexts, making explicit connections to ongoing debates within the field.Strengthened the argumentation by refining logical flow and ensuring each point is clearly supported by data presented in the results section.We believe these enhancements will contribute to a more robust and compelling narrative that resonates with both experts and readers unfamiliar with the subject matter. Your comments were instrumental in guiding these improvements, for which we are truly thankful.we have revised the appropriate parts of the text and the revised parts are shown in red.

Comments 4: Future Directions: Including suggestions for further research could improve the manuscript. For example, investigating the mechanisms behind LJH’s impact on LAB or exploring different LAB strains could be beneficial.

Response 4: Thank you for pointing this out.We sincerely appreciate your constructive feedback and the valuable suggestions you've provided for our manuscript. We agree that exploring the underlying mechanisms of LJH's influence on LAB and studying its effects on different LAB strains would enrich the scientific community's understanding of this phenomenon.The revised section reads as follows:“While this study provides initial insights into the role of LJH in shaping LAB populations during alfalfa fermentation, further investigations are warranted to elucidate the complex mechanisms at play. Future research should focus on identifying the specific biochemical pathways through which LJH influences LAB, as well as exploring strain-specific responses to better understand the genetic determinants involved. Moreover, optimizing the application of LJH to maximize antimicrobial benefits and studying the effects of environmental factors on its performance will be crucial for practical applications in the food and agricultural industries.”we have revised the appropriate parts of the text and the revised parts are shown in red.

Comments 5: Conclusions: The conclusion might be strengthened by summarizing the broader implications of using LJH in livestock feed and its potential economic benefits for farmers, as well as its environmental impact.

Response 5:Thank you for pointing this out.We greatly appreciate your insightful comment about expanding the conclusions to encompass the wider implications of incorporating LJH into livestock feed, including economic benefits for farmers and environmental considerations. In light of your feedback, we have revised the conclusions to highlight these important dimensions.Below is the revised concluding section:“In summary, our findings demonstrate the effectiveness of LJH in modulating the bacterial ecology in alfalfa-based feeds during fermentation, providing a safer, more sustainable alternative to conventional practices. This novel approach not only enhances feed quality but also offers substantial economic benefits to farmers by potentially decreasing reliance on costly chemicals and antibiotics. Moreover, the use of LJH aligns with growing consumer demands for environmentally friendly and antibiotic-free products, presenting a promising avenue for the livestock industry to adopt greener practices without compromising productivity. Further research is warranted to fully explore the long-term effects on livestock health and environmental sustainability.”we have revised the appropriate parts of the text and the revised parts are shown in red.

Comments 6:Ensure that all scientific names are italicized consistently throughout the manuscript.

Response 6:Thank you for pointing this out.We appreciate your diligence in ensuring the accuracy and consistency of our manuscript. You correctly identified that scientific names should be italicized throughout the text. We have meticulously reviewed the entire manuscript and have now italicized all scientific names, including those within the abstract, main text, tables, and figures, to conform to standard biological nomenclature conventions.Thank you for bringing this oversight to our attention. We are committed to maintaining high standards of scientific writing and presentation, and your input has been invaluable in helping us achieve this.

Comments 7:The sentence structure could be improved in some areas for clarity. For example, the phrase “as result from increased (P < 0.05) bacterial dominance” should read “as a result of increased (P < 0.05) bacterial dominance.”

Response 7:Thank you for pointing this out.We extend our thanks for your meticulous review and for highlighting opportunities to refine our manuscript's clarity. Your observation regarding the sentence structure—"as result from increased (P < 0.05) bacterial dominance"—is noted and appreciated. We agree that revising this phrase will enhance readability and adherence to grammatical norms.The sentence has been corrected to read, “as a result of increased (P < 0.05) bacterial dominance,” ensuring proper preposition use and grammatical coherence. This adjustment contributes to clearer communication of the intended outcomes of our study's analysis.we have revised the appropriate parts of the text and the revised parts are shown in red.

Comments 8:The use of specific terms such as “bacterial richness indices” could be made clearer by briefly defining them upon first mention for readers less familiar with the terminology.

Response 8:Thank you for pointing this out.We are deeply appreciative of your thorough reading and insightful feedback aimed at enhancing our manuscript's accessibility. Your suggestion regarding the clarification of "bacterial richness indices" is well-taken. Indeed, ensuring that technical terms are comprehensible to non-specialist readers is vital for broadening the reach and impact of our research.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I am sending a detailed review in the attachment.

Comments for author File: Comments.pdf

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments:The article entitled "The effect of Lentilactobacillus buchneri on the chemical and microbiological composition of alfalfa silage containing herba leonuri (Leonurus japonicus Houtt.)" is very well written in terms of content and language. The work concerns the effect of bacteria on the chemical and microbiological composition of alfalfa silage. The aim of the authors' work was to demonstrate the effect of Lentilactobacillus buchneri on the chemical and microbiological composition of alfalfa silage containing herba leonuri. The pre-experimental studies showed that L-treated silage had high levels of water-soluble carbohydrates (4.98% dry matter (DM)) and acid detergent fiber (27.88% DM). Compared to CK treatment, L treatment increased the acetic acid content of silage, resulting in increased (P < 0.05) bacterial dominance and decreased (P < 0.05) bacterial richness indices (e.g. Pielou's E, Shannon and Simpson) during the pre-storage period. However, these changes gradually decreased with increasing storage length. L treatment changed the bacterial community structure of silage, increasing the dominance of Lactobacillus and decreasing the relative abundance of Enterococcus and Weissella. The article is quite short but very concise and informative. It fits into 13 typewritten pages and is divided into 5 chapters: introduction, material and methods, results, discussion and conclusions. Each of the listed chapters is written correctly and does not raise any objections. The only thing that I think would be beneficial for the reader is to place graphs and tables in the text of the work. My second comment is that I would recommend placing more current literature. Items 38-41 are not literature, so please remove them. To sum up, the article can be published in the journal Fermentation in its current form, but after the minor corrections listed above.

Response:Thank you for pointing this out.We appreciate the opportunity to address the comments provided by the reviewer. We are delighted to learn that our manuscript is considered well-written and informative. Below, we address the specific points raised:

1.Graphs and Tables Placement:

   We agree with the reviewer's suggestion to enhance the manuscript's clarity by integrating graphs and tables directly into the text. This will improve the flow and readability for the readers. We have revised the layout accordingly, ensuring that all figures and tables are now embedded at the most relevant points within the text.

2. Incorporation of Current Literature:

   The reviewer recommended updating the reference list to include more current literature. We have conducted a thorough literature search and added several recent studies that are pertinent to our research. Items 38-41, which were not literature references, have been removed as suggested.

3. Removal of Non-Literature Items:

   We have reviewed the reference list and removed any non-literature items as instructed. This has been done to ensure that the reference section accurately reflects scholarly sources.We are grateful for your support and look forward to your confirmation that these changes are satisfactory for publication.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper by Mingjie Zhang et al aims to assess the effect of Lentilactobacillus buchneri on chemical and microbial

compositions of herba leonuri -contained alfalfa silage. 

While the work scientifically sounds, some issues should be addressed  before it can be considered for publication. 

 

Major

This is very difficult to understand whether the introduction of either herba leonuri or Lentilactobacillus buchneri significantly affect the quality of silage. For that 1) a control sample is strictly required 2) statistical analysis should be performed for data shown in Tables 1 and 2. To make the work more clear for the general reader who is not an expert in the field, authors are recommended to make some figure showing several most important integral characteristics and explain what do they mean, i.e should they increase or decrease.

 

Why there is a drop im Chao and OTUs on 15th day in L-treated silage?

 

What is the day of fermentation for which metagenomics has been made?

 

Minor

Tables should be placed after first mention

 

Figures lack captions

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1:This is very difficult to understand whether the introduction of either herba leonuri or Lentilactobacillus buchneri significantly affect the quality of silage. For that 1) a control sample is strictly required 2) statistical analysis should be performed for data shown in Tables 1 and 2. To make the work more clear for the general reader who is not an expert in the field, authors are recommended to make some figure showing several most important integral characteristics and explain what do they mean, i.e should they increase or decrease.

Response 1:Thank you for pointing this out.We are grateful for the valuable feedback provided by the reviewer, which has helped us to identify areas where our manuscript can be improved for clarity and rigor.We have performed comprehensive statistical analyses on the data presented in Tables 1 and 2. These analyses include ANOVA (to compare means among treatments)  at a significance level of P < 0.05. The results of the statistical analyses are now included in the revised manuscript, providing a clearer understanding of the significance of our findings.Thank you once again for the opportunity to enhance our work.

Comments 2:Why there is a drop im Chao and OTUs on 15th day in L-treated silage?

Response 2:Thank you for pointing this out.We appreciate the attention to detail in your review of our manuscript. The observation of a drop in Chao indices and operational taxonomic units (OTUs) on the 15th day in the L-treated silage is indeed a point of interest and requires clarification. Here is our explanation:The decrease in Chao indices and OTUs observed on the 15th day in the L-treated silage is primarily attributed to the fermentation process initiated by Lentilactobacillus buchneri. This bacterium, as a lactic acid bacterium, rapidly metabolizes readily available substrates in the silage, leading to a decrease in the diversity of the bacterial community as the more competitive and fast-fermenting lactobacilli outcompete other less dominant bacteria.Thank you for your thorough review and for bringing this point to our attention. We are committed to ensuring that our findings are accurately and comprehensively presented.

Comments 3:What is the day of fermentation for which metagenomics has been made?

Response 3:Thank you for pointing this out.Thank you for your detailed review and the specific question regarding the timing of our metagenomic analysis. In general, fermentation is completed within 30 days. This time point was chosen to capture the stabilisation phase of microbial community structure and function after the early stages of active fermentation. At this stage, the chemical and microbiological composition of the silage reaches a relatively stable state, making it an ideal time for metagenomic analysis to accurately reflect the established microbial community and its functional potential. We are very sorry that we did not use metagenomic analysis in our study, your valuable comments we can explore the study in the future study.Thank you for your thorough review and for giving us the opportunity to improve the clarity of the manuscript. Please let us know if you need more information or have any other questions.

Comments 4:Tables should be placed after first mention

Response 4:Thank you for pointing this out.We would like to thank you for the constructive feedback on our manuscript. We understand the importance of enhancing the readability and flow of information for our readers. In response to your suggestion regarding table placement, we have revised the manuscript to ensure that all tables are now located immediately after their first mention in the text. 

Comments 5:Figures lack captions

Response 5:Thank you for pointing this out.We appreciate your meticulous review of our manuscript and the feedback provided. We acknowledge the oversight in not including captions for the figures, which are essential for clarity and understanding. To rectify this, we have now added detailed captions to each figure in the manuscript.we have revised the appropriate parts of the text and the revised parts are shown in red.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No further comments are required.

Comments on the Quality of English Language

Good

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1: No further comments are required.

Response 1: Thank you for pointing this out. We are pleased to note that no further comments were required from your end following our recent revisions. We appreciate the thorough evaluation and helpful guidance you provided throughout the peer-review process. Your input was invaluable in refining our manuscript to better serve both the scientific community and interested readers.We remain at your service should any additional queries arise in the future, and we are eager to see our manuscript progress through the final stages towards publication. Thank you for your diligence and expertise, which have greatly contributed to the enhancement of our work.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have significantly improved the paper. 

Only one minor correction is required. Please describe in Table captions what different letters regarding statistical differences mean, is this various tresholds? Or leave only one letter (a) if only one treshold (0.05) has been used. 

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1:Authors have significantly improved the paper. Only one minor correction is required. Please describe in Table captions what different letters regarding statistical differences mean, is this various tresholds? Or leave only one letter (a) if only one treshold (0.05) has been used.

Response 1:Thank you for pointing this out.We are delighted to hear that our efforts have substantially improved the paper and are appreciative of your positive feedback. Regarding the suggested minor correction, we have taken the following steps:We have revised the table captions to explicitly define the letters indicating statistical differences.Our aim is to ensure that readers can easily interpret the significance levels indicated by these annotations. The updated captions now read “Means with different superscripts in the same column (a-c) are significantly different from each other (P < 0.05).”, which we believe addresses the concern raised while maintaining clarity and precision. The revised manuscript has been uploaded for your review. We trust that this amendment meets your expectations and contributes to the overall accuracy and readability of our work.Should you need any further modifications or have additional concerns, please do not hesitate to inform us. We are fully dedicated to delivering a high-quality manuscript for publication.we have revised the appropriate parts of the text and the revised parts are shown in red.

Author Response File: Author Response.pdf