Effects of Wilting and Exogenous Lactic Acid Bacteria on the Fermentation Quality and Microbial Community of Plantago lanceolata Silage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work is very timely, innovative and the theme is very important for the thematic area in question.

All suggestions and few corrections are noted in the body of the manuscript that is attached.

Comments for author File: Comments.pdf

Author Response

Comments 1: Inform in this abstract where the research was conducted and also inform the evaluation result.

Response 1: Thank you for pointing this out. We have written this part and highlighted it in yellow in the manuscript. It can be found in the manuscript – page1, and line 16.

 

Comments 2: Keywords should not repeat words given in the title of the work. It is necessary to mark this change.  

Response 2: Thank you very much for pointing out these issues. We have reorganized this part of the content. It can be found in the manuscript – page 1, and line 32.

 

Comments 3: Line 65–It is important to include a paragraph about the economic importance of this plant species: This is the plant that is being evaluated. Complement with data from the literature about productivity and other characteristics if appropriate.

Response 3: Thank you very much for pointing this out. This improves the readability of our manuscripts. We have re-sorted and supplemented this content. These changes can be found in the Introduction – page 1, and line 35-78.

 

Comments 4: Line 77–81. Does this paragraph at the end of the Introduction correspond to the general objective of the research? Inform. Remember that the conclusions generated in this research correspond to an answer to this objective.

Response 4: Thank you for pointing this out. We agree and rewrite this content. These changes can be found in the Introduction – page 2, paragraph 2, and line 78-83.

 

Comments 5: Line 109. Explain why 60 days was established? And specific reason?

Response 5: Thank you for pointing this out. Because in reading the references of related research, most of the fermentation time of this material is 60, and good silage quality is obtained. And in this experiment, 60 days after the beginning of the winter, the temperature is relatively cool season bag helps the quality of silage.

 

Comments 6: Line 187. What is the multiple test involved in comparing means? LSD, Tukey or Duncan?

Response 6: Thank you for pointing this out. Duncan test was used to compare the mean. I added this supplement to the method– page 4, paragraph 2, and line 162.

 

Comments 7: Line 201. Compare with literature results. Important for the study.

Response 7: Thank you for your advice. We have supplemented this. These changes can be found in the manuscript – page 6, paragraph 1, and line 228 .

 

Comments 8: Line 256-258. Explain the reason for this oscillation. Increase and decrease.

Response 8: Thank you for pointing this out. We have supplemented this. These changes can be found in the manuscript – page 7, paragraph 1, and line 288-293.

 

Comments 9: Line 272 and Line 291. Add the magnitude of these correlations whether they were high or low.

Response 9: Thank you for pointing out our problem, we all agree to modify. The changed contents have been highlighted in yellow in the manuscripts – page 8, paragraph 1, and line 308-333.

 

Comments 10: Line 314-317. Add whether these correlations were high or low, regardless of whether they were positively or negatively correlated. Compare this statement with data from the literature for comparison purposes.

Response 10: Thank you for pointing out our problem, we all agree to modify. The changed contents have been highlighted in yellow in the manuscripts – page 9, paragraph 1, and line 358-366.

 

Comments 11: Line 349-351. Compare this statement with data from the literature for comparison purposes.

Response 11: Thank you for pointing this out. I have modified this part and compared the literature. These changes can be found in the yellow prominent part of the manuscript – page 11, paragraph 1, and line 398-401.

 

Comments 12: Line 357. Great conclusions. They just need to be rearranged. There is no need to explain the conclusions. They become results. Conclusions should be stated in short, to the point sentences.

Response 12: Thank you for pointing this out. Your opinion has improved the quality of our manuscripts. We all agree and rewrite the conclusion. These changes can be found in the yellow prominent part of the manuscript – page 12, and line 407.

Reviewer 2 Report

Comments and Suggestions for Authors

This paper presents an interesting research, with practical applications, about the effects of wilting and exogenous lactic acid bacteria on the quality of Plantago lanceolata silage. The authors made a comparative study that showed that the combined treatment by wilting and the addition of L. brucei represents an improved method of treating the raw material to obtain Plantago silage.

Observations

Line 89 - "(1×1010 cfu/g)" - superscript

Table 1 – "Water Soluble Carbohydrate"

Line 102 – it is not clear in what form the culture of L. brucei is added, if it is liquid or in powder form. It is not clear if this preparation is added after mixing with water, and in what proportion.

- The initial characteristics of the raw material (Plantago) should be described more clearly, the differences from DM% or other properties of the raw material after wilting should be highlighted. What is the initial ash content? This should remain constant for the material in the bags throughout fermentation. How do the authors explain the change in the percentage of ash (minerals) in table 2 (it should be mentioned if the ash was determined in certain fractions of PS, or other explanations). Otherwise, for the sample as a whole, the ash does not change over time.

- If there is data on the microbial load of the raw material before the initial moment, these should be shown. Was there an initial analysis of the number or even of the type of microorganisms in the raw material?

- What modifications of the plant material through the wilting treatment could have an effect on the PS properties? (Loss of water, modification of plant tissue structure, others).

- It is not clear how the number of L. brucei cells changed during the experiment, how many cells are found in the final PS product? Perhaps it would have been interesting to follow the evolution of this LAB over time.

- Lactobacillus – the name of the genus must be written in italics.

- Line 227 - "Lactobacillus was the dominant bacterial genera" - genus

- Line 364 – the expression "wilting treatment inhibited the activity of sugar-fermenting bacteria by reducing the production of wastewater" is not clear, it should be explained.

Author Response

Comments 1: Line 89 - "(1×1010 cfu/g)" - superscript

Response 1: Thank you for pointing this out. I have modified this and modified it to “1×1010 cfu/g”. This change can be found in the revised manuscript – page 2, paragraph 4, and line 89.   Comments 2: Table 1 – "Water Soluble Carbohydrate" Response 2: Thank you for pointing this out. I have modified this in Table 1 . This change can be found in the revised manuscript – page 2, paragraph 5, and line 92.   Comments 3: Line 102 – it is not clear in what form the culture of L. brucei is added, if it is liquid or in powder form. It is not clear if this preparation is added after mixing with water, and in what proportion. Response 3: Thank you for pointing this out. Your comments help us greatly optimize the content of the manuscript. I have added to this. “L. brucei was diluted with deionized water to a concentration of 0.0025 g/ml. LB-PS group and WLB-PS group were sprayed with 10 ml of additives respectively. During the spraying process, the grass samples were thoroughly mixed to ensure even distribution of the additives. The same volume of deionized water was added to the C-PS and W-PS groups.” This change can be found in the revised manuscript – page 3, paragraph 1, and line 105-109. I have highlighted this part in yellow.   Comments 4: - The initial characteristics of the raw material (Plantago) should be described more clearly, the differences from DM% or other properties of the raw material after wilting should be highlighted. What is the initial ash content? This should remain constant for the material in the bags throughout fermentation. How do the authors explain the change in the percentage of ash (minerals) in table 2 (it should be mentioned if the ash was determined in certain fractions of PS, or other explanations). Otherwise, for the sample as a whole, the ash does not change over time. Response 4: Thank you for your valuable comments, we are very recognition of this. Since our samples after wilting treatment, the raw material characteristics only changed in the dry matter content. Therefore, we only list the raw material characteristics before wilting in Table 1. The water content of the raw material after wilting can be found in – page 3, paragraph 1, and line 100. The change of crude ash content raised by you has attracted great attention from our team. In order to verify the accuracy of the data, we have carried out many times of experimental verification. However, the data results are still consistent with those in the manuscript. The initial crude ash content is 12.15 % DM, which we have listed in Table 1. Regarding the change of crude ash percentage, we speculated that it may be caused by the change of DM in silage samples. The loss of dry matter resulted in an increase in ash % DM. We have modified this speculation in the manuscript. The revised manuscript this change can be found – page 4, paragraph 3, and line 180-186.   Comments 5: - If there is data on the microbial load of the raw material before the initial moment, these should be shown. Was there an initial analysis of the number or even of the type of microorganisms in the raw material? It is not clear how the number of L. brucei cells changed during the experiment, how many cells are found in the final PS product? Perhaps it would have been interesting to follow the evolution of this LAB over time. Response 5: Thank you very much for your valuable comments. Due to the limitations of the experimental settings, we are unable to supplement the relevant data for the time being. However, this has attracted our great attention, and we will pay attention to this content in subsequent research. Comments 6: - What modifications of the plant material through the wilting treatment could have an effect on the PS properties? (Loss of water, modification of plant tissue structure, others). Response 6: Thank you for pointing this out. The wilting treatment can reduce the moisture content of the material, inhibit the fermentation of harmful microorganisms, and preserve the nutritional components of the raw material to a large extent, thereby improving the silage quality. The relevant background information has been put into the Introduction – page 2, paragraph 2, and line 59-65. Comments 7: - Lactobacillus – the name of the genus must be written in italics.  - Line 227 - "Lactobacillus was the dominant bacterial genera" - genus Response 7: Thank you for pointing this out. This increases the readability of our manuscripts. I have modified this. The revised manuscript this change can be found – page 6, paragraph 3, and line 249.   Comments 8: -- Line 364 – the expression "wilting treatment inhibited the activity of sugar-fermenting bacteria by reducing the production of wastewater" is not clear, it should be explained. Response 8: Thank you very much for pointing this out. After consideration, we found that this sentence is not clear enough, and the content should appear in the results and discussions, not suitable for the conclusion. Therefore, I have rewritten the conclusion part.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

The study presents an interesting concept, but it contains several inconsistencies that prevent me from recommending it for publication. Below are my detailed observations:

Introduction: The authors used the "Agronomy" journal template instead of the correct "Fermentation" template. The introduction focuses on advocating the use of Chinese medicinal plants in animal feed, with a specific mention of aquaculture, but lacks clarity regarding the primary target species. This raises the question: what is the main focus of the study? Furthermore, it is questionable whether ensiling is the most appropriate method for preserving medicinal plants, considering they are typically used in small doses, whereas silage is used in large volumes for ruminants. Additionally, it is important to consider whether all compounds in Plantago are preserved during the ensiling process. The introduction needs a clearly defined hypothesis and objective at the end.

Materials and Methods:

• The palatability of the Tonic cultivar for cattle is understandable, but its suitability for poultry is questionable, given that poultry predominantly consume grains and that the plant's NDF (neutral detergent fiber) content is high. It is necessary to clarify the type of animal for which the research was conducted.
• In line 89, the number of CFU/g needs to be corrected.
• In Table 1, the correct sequence of analyses should be: dry matter, ash, protein, WSC, fat, NDF, ADF. I recommend organizing these parameters in a table with two columns and seven rows for better clarity.
• The chemical analysis presents a serious error: NDF, which includes cellulose, hemicellulose, and lignin, cannot be lower than ADF, which includes only cellulose and lignin. It is recommended to repeat this analysis.
• The description of the experimental methods is insufficient and lacks essential details, making it impossible to verify the quality of the research. It is necessary to clarify which Plantago species/cultivar was used, how the plant dehydration was monitored, how the plants were chopped, and how the inoculant was prepared and applied. Additionally, the methodology for weighing dehydrated and non-dehydrated treatments needs to be revised.
• The description of the chemical analyses is also incomplete, with insufficient information on how crude fat was determined.
• The data analysis section needs to be more detailed, specifying the statistical models used, the normality and homogeneity of variance tests applied, and the method of mean comparison employed.
• It is crucial that silage studies include consecutive harvests to assess the quality of treatments/additives, considering the annual variations in plants due to climatic instabilities. The absence of temporal repetition prevents validation of the treatments.

Results:

• The NDF and ADF analyses need to be revisited, as the results presented are inconsistent, especially considering that the difference between NDF and ADF (level of hemicellulose) is less than 1%, seems unrealistic.
• The ash content is high, at 20%, which is incompatible with a plant of high nutritional value.
• The sum of ash, protein, fat, fiber, WSC, and fatty acids exceeds 100%, indicating serious errors in chemical analyses.
• Fatty acid production was high, but the pH values do not reflect this acidity, which requires an explanation.
• It is necessary to clarify why treatments with higher acid production had higher residual WSC levels, contrary to what would be expected.
• The initial and final WSC levels being very close in the W-PS and WLB-PS treatments need to be explained.
• Due to all the inconsistencies in the analyses, the conclusions cannot be validated in their current form.

Discussion and Conclusion:

• The discussion is absent and needs to be developed to contextualize the results with the existing literature.
• The conclusion should be reformulated to accurately reflect the results obtained and their correct interpretations.

I hope these observations help improve the quality of the work.

Sincerely.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Comments 1: Introduction: The authors used the "Agronomy" journal template instead of the correct "Fermentation" template. The introduction focuses on advocating the use of Chinese medicinal plants in animal feed, with a specific mention of aquaculture, but lacks clarity regarding the primary target species. This raises the question: what is the main focus of the study? Furthermore, it is questionable whether ensiling is the most appropriate method for preserving medicinal plants, considering they are typically used in small doses, whereas silage is used in large volumes for ruminants. Additionally, it is important to consider whether all compounds in Plantago are preserved during the ensiling process. The introduction needs a clearly defined hypothesis and objective at the end.

Response 1: Thank you very much for pointing out these issues. This improves the quality of our manuscripts. We all agree on this content. After our thinking, this part of the content has been completely rewritten. Also changed to the correct template, all the changes in the content with yellow highlighted.

 

Comments 2:The palatability of the Tonic cultivar for cattle is understandable, but its suitability for poultry is questionable, given that poultry predominantly consume grains and that the plant's NDF (neutral detergent fiber) content is high. It is necessary to clarify the type of animal for which the research was conducted.

Response 2: Thank you for pointing this out. We agree with your point of view, and have modified this section, in the Introduction has been clearly indicated for livestock.

 

Comments 3: In line 89, the number of CFU/g needs to be corrected. In Table 1, the correct sequence of analyses should be: dry matter, ash, protein, WSC, fat, NDF, ADF. I recommend organizing these parameters in a table with two columns and seven rows for better clarity.

Response 3: Thank you for pointing this out. I 've changed this to “1×10¹⁰ cfu/g”. We also agree with the proposal for the Table 1 format and have completed the changes.

 

Comments 4: The chemical analysis presents a serious error: NDF, which includes cellulose, hemicellulose, and lignin, cannot be lower than ADF, which includes only cellulose and lignin. It is recommended to repeat this analysis.

Response 4: Thank you for your valuable comments, we are very recognition of this. In order to verify the accuracy of the data, we have carried out many times of experimental verification. And the samples were sent to more authoritative institutions for testing. We have revised this result and listed it in Table 1. It may be because our own analysts are not skilled enough to cause human error, or the accuracy of the equipment may lead to the emergence of this problem. We are very grateful to you for pointing out that this is of great inspiration and help to us.

 

Comments 5: The description of the experimental methods is insufficient and lacks essential details, making it impossible to verify the quality of the research. It is necessary to clarify which Plantago species/cultivar was used, how the plant dehydration was monitored, how the plants were chopped, and how the inoculant was prepared and applied. Additionally, the methodology for weighing dehydrated and non-dehydrated treatments needs to be revised.

Response 5: Thank you for pointing this out. We have modified and supplemented this part. The variety we use is “P. lanceolata cultivar Tonic was bred by the PGG Wrightson Seeds Corporate (New Zealand), and introduced by the Guizhou Extension Station of Grassland Technology to plant on the farm in Shangsi Town, Dushan County, Guizhou Province.” This part can be found in the manuscript – page 2, paragraph 3, and line 86-88. About monitoring plant dehydration, because we are linked to the actual production from mowing to transportation, and then to the consumption in the process of bundling. Then the wilting state was determined by the senses. The relevant content has been added to the manuscript – page 3, paragraph 1, and line 101. We use a sterile cutting machine to cut the material to 2cm. The preparation and use of inoculants have been added to the manuscript– page 3, paragraph 1, and line 105-109. The weighing method has also been modified in the manuscript – page 3, paragraph 3, and line 118-120.

 

Comments 6: The description of the chemical analyses is also incomplete, with insufficient information on how crude fat was determined.

Response 6: Thank you for pointing this out. Determination of crude fat I 've added to the manuscript – page 3, paragraph 3, and line 121.

 

Comments 7: It is crucial that silage studies include consecutive harvests to assess the quality of treatments/additives, considering the annual variations in plants due to climatic instabilities. The absence of temporal repetition prevents validation of the treatments.

Response 7: Thank you very much for your valuable comments, we fully agree. However, because our experiment is in the preliminary exploration, the experimental settings are limited, and the exploration of this part of the content will be completed in the next study.

 

Comments 8: The NDF and ADF analyses need to be revisited, as the results presented are inconsistent, especially considering that the difference between NDF and ADF (level of hemicellulose) is less than 1%, seems unrealistic.

Response 8: Thank you for your valuable comments, we are very recognition of this. In order to verify the accuracy of the data, we have carried out many times of experimental verification. And the samples were sent to more authoritative institutions for testing. We have revised this result. It may be because our own analysts are not skilled enough to cause human error, or the accuracy of the equipment may lead to the emergence of this problem. We are very grateful to you for pointing out that this is of great inspiration and help to us.

 

Comments 9: The ash content is high, at 20%, which is incompatible with a plant of high nutritional value.

Response 9: Thank you for pointing this out. We agree with this. Considering that ash is rich in minerals, the definition of high nutrition is wrong. We have deleted this unreasonable content.

 

Comments 10: The sum of ash, protein, fat, fiber, WSC, and fatty acids exceeds 100%, indicating serious errors in chemical analyses.

Response 10: Thank you for pointing this out. Neutral detergent fiber including cellulose, hemicellulose, lignin, acid detergent fiber including cellulose and lignin, two have repeated content, so will be more than 100 %.

 

Comments 11: Fatty acid production was high, but the pH values do not reflect this acidity, which requires an explanation.

Response 11: We agree with this view and have added this content to the manuscript – page 5, paragraph 1, and line 206. After reading the literature and the high content of ash in our study. It may be that the minerals in the ash are buffer substances, so they resist the change of PH.

 

Comments 12: It is necessary to clarify why treatments with higher acid production had higher residual WSC levels, contrary to what would be expected.

Response 12: Thank you for pointing this out. It may be due to the high water content in the non-wilting treatment group, resulting in the consumption of WSC by poor microbial proliferation and fermentation. The wilting treatment group increased the dry matter content by controlling the water content, inhibited the growth of harmful bacteria, reduced the WSC loss, and increased the WSC % DM content. We have added this to the manuscript – page 4, paragraph 3, and line 179-183.

 

Comments 13: The initial and final WSC levels being very close in the W-PS and WLB-PS treatments need to be explained.

Response 13: Thank you for suggesting this, we have added this content to our manuscript– page 5, paragraph 1, and line 186-190. This result may be that the pH in the W-PS group is too high to inhibit butyric acid fermentation, and the nutrients in the PS are converted to butyric acid, while the WLB-PS group converts WSC into lactic acid because of LAB.

 

Comments 14: The discussion is absent and needs to be developed to contextualize the results with the existing literature.

Response 14: Thank you for your point, which increases the readability of our manuscripts. I have added this section to the results and discussions and highlighted it in yellow.

 

Comments 15: The conclusion should be reformulated to accurately reflect the results obtained and their correct interpretations.

Response 15: We agree with you and rewrite the conclusion. These modifications can be found in the manuscript conclusion – page 12, paragraph 1, and line 408-418.

Reviewer 4 Report

Comments and Suggestions for Authors

The experimental article "Effects of wilting and exogenous lactic acid bacteria on the fermentation quality and microbial community of Plantago lanceolata silage" is devoted to the study of the effects of wilting and exogenous lactic acid bacteria treatments on the chemical composition, fermentation quality, and microbial community composition of Plantago lanceolata silage (PS). The relevance of the topic is due to the need to develop safe, effective and environmentally friendly feed additives to replace antibiotics. The content of the article corresponds to the Fermentation edition. The authors have a good command of the terminology and are knowledgeable about this topic, so it will not be difficult for them to eliminate a number of comments for the publication of the article.

Recommendations:

1. It is recommended to enhance the relevance of the work in the introduction by including links to more recent publications (2023-2024) on this topic.

2. Lines 159-184. A large number of abbreviations, which complicates the perception of information. Please correct, if possible.

3. When discussing the chemical composition and fermentation characteristics of the samples, I would like to see explanations from the authors as to why this is so, and not just a comparison of the values ​​from the table.

4. In conclusion, I would like to see the scientific novelty of the results obtained.

5. Quite a high percentage of coincidence. Needs to be reduced to at least 15%.

Author Response

Comments 1: It is recommended to enhance the relevance of the work in the introduction by including links to more recent publications (2023-2024) on this topic.

Response 1: Thank you for pointing this out. We have rewritten this part and highlighted it in yellow in the manuscript.

 

Comments 2: Lines 159-184. A large number of abbreviations, which complicates the perception of information. Please correct, if possible.

Response 2: Thank you very much for pointing out these issues. We have reorganized this part of the content. It can be found in the manuscript – page 4, paragraph 3, and line 168-208.

 

Comments 3: When discussing the chemical composition and fermentation characteristics of the samples, I would like to see explanations from the authors as to why this is so, and not just a comparison of the values from the table.

Response 3: Thank you for your questions, which improve the readability of our manuscripts. We agree and modify and supplement this content. It can be found in the manuscript – page 4, paragraph 3, and line 168.

 

Comments 4: In conclusion, I would like to see the scientific novelty of the results obtained.

Response 4: Thank you for pointing this out. We have re-summarized the conclusion part. It can be found in the manuscript – page 12, paragraph 1, and line 408.

 

Comments 5: Quite a high percentage of coincidence. Needs to be reduced to at least 15%.

Response 5: Thank you very much for pointing out these issues. We have re-written and re-reduced the content of the high-repetition parts.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

The article has improved a lot but there are still some points that need to be clarified. Below are my detailed observations:

Introduction:

Lines 49-50: The range (0.91 g/cow.day to 1.08 g/cow.day) seems incorrect. It should be expressed as “kg/cow/day.” Please standardize the units throughout the manuscript. The correct format would be either "kg/cow/day" or "kg.cow⁻¹.day⁻¹". Avoid mixing unit formats as done here.

Citations within the text are not consistent. Sometimes, citations appear in the required numerical format [1], while other times, the author’s name is mentioned (e.g., "Duru et al." in line 67, "Bariro et al." in line 71). Please standardize citation formatting as per the journal's guidelines.

Materials and Methods:

Please provide the bibliographic reference for the method used to determine water-soluble carbohydrates.

Results:

The values presented in Table 2 are more coherent, but I recommend adding the fractions for better clarity. For example:

C-PS: CP (16.52%) + CF (3.55%) + CA (18.54%) + NDF (60.88%) + WSC (2.47%) = 101.96%. It is wrong

Obs: This calculation does not account for fatty acids or other carbohydrates present in the plant. The authors should further clarify these results.

Discussion

The authors seem to have misunderstood Comment 13 from the previous review. Comment 13: "The initial and final WSC levels being very close in the W-PS and WLB-PS treatments need to be explained."

The WSC content in the plant prior to ensiling (Table 1) was 12.23% of DM. After ensiling, the WSC levels in the W-PS and WLB silages were 10.21% and 10.68%, respectively. This indicates that only about 2% DM of WSC was consumed, yet large amounts of organic acids were produced. In contrast, PS consumed 10% DM of WSC to produce fewer volatile fatty acids. How is this discrepancy possible? Stating that PS led to greater butyric acid production is incorrect, as the levels of butyric acid across all treatments were insignificant (ranging from 0.02 to 0.05% DM). The authors should reconsider their explanation.

Conclusion: It must be clearly stated that these are preliminary results. The experiment needs to be repeated in order to provide a more consistent conclusion about the use of the inoculant in combination with plant dehydration.

Comments on the Quality of English Language

Minor English language editing is required throughout the manuscript.

Author Response

Comments 1: Lines 49-50: The range (0.91 g/cow.day to 1.08 g/cow.day) seems incorrect. It should be expressed as “kg/cow/day.” Please standardize the units throughout the manuscript. The correct format would be either "kg/cow/day" or "kg.cow⁻¹.day⁻¹". Avoid mixing unit formats as done here.

Response 1: Thank you for pointing this out. We have amended this to "kg/cow/day". These changes can be found in the manuscript– page 2, paragraph 1, and line 48-49.

 

Comments 2: Citations within the text are not consistent. Sometimes, citations appear in the required numerical format [1], while other times, the author’s name is mentioned (e.g., "Duru et al." in line 67, "Bariro et al." in line 71). Please standardize citation formatting as per the journal's guidelines. 

Response 2: Thank you very much for pointing out these errors. We fully agree with you and have revised it. These changes can be found in the manuscript– page 2, paragraph 2, and line 67-70.

 

Comments 3: Please provide the bibliographic reference for the method used to determine water-soluble carbohydrates.

Response 3: Thank you for pointing this out. We have added references to the measurement methods of WSC to the manuscript. It can be found in the manuscript – page 3, paragraph 3, and line 124-125.

 

Comments 4: The values presented in Table 2 are more coherent, but I recommend adding the fractions for better clarity. For example:

C-PS: CP (16.52%) + CF (3.55%) + CA (18.54%) + NDF (60.88%) + WSC (2.47%) = 101.96%. It is wrong

Obs: This calculation does not account for fatty acids or other carbohydrates present in the plant. The authors should further clarify these results.

Response 4: Thank you very much for raising this point. Our team is very concerned about this. After reviewing the literature and carefully considering the discussion, it is speculated that the reason for this result may be due to the common part of NDF and CA.

First, DM = CP + CA + EE + CF + NFE ( Nitrogen-free extract )=100%.  

Secondly, CF includes cellulose, hemicellulose and lignin. NDF includes cellulose, hemicellulose, lignin and silicate. Because NDF contains the composition of CF, and CA contains the same silicate as NDF, the sum of them will be greater than 100 %.

 

Comments 5: The authors seem to have misunderstood Comment 13 from the previous review. Comment 13: "The initial and final WSC levels being very close in the W-PS and WLB-PS treatments need to be explained."

The WSC content in the plant prior to ensiling (Table 1) was 12.23% of DM. After ensiling, the WSC levels in the W-PS and WLB silages were 10.21% and 10.68%, respectively. This indicates that only about 2% DM of WSC was consumed, yet large amounts of organic acids were produced. In contrast, PS consumed 10% DM of WSC to produce fewer volatile fatty acids. How is this discrepancy possible? Stating that PS led to greater butyric acid production is incorrect, as the levels of butyric acid across all treatments were insignificant (ranging from 0.02 to 0.05% DM). The authors should reconsider their explanation.

Response 5: Thank you very much for your clear guidance which is of great help to my research. After literature query and data observation and analysis, it is speculated that the reasons for this phenomenon may be as follows :

1. Due to the existence of a certain amount of oxygen in the raw materials at the initial stage of silage fermentation, aerobic microorganisms and plant materials are active in a high-moisture environment, consuming WSC and generating heat through respiration. The wilting treatment reduced the metabolic activities of plants and microorganisms, and reduced the consumption of WSC by respiration. In our previous observation, the surface temperature of the PS group was higher than that of the wilting treatment group.
2. By analyzing the microbial composition and relative abundance of the PS group, the relative abundance of aerobic bacteria (Stenotrophomonas rhizophila) and bad microorganisms (Clostridium lutlcellarliand Clostridium tyrobutyricum) that can grow and reproduce in high water environment in the PS group accounted for a large proportion. These bacteria consumed a large amount of WSC for reproduction in the early stage of silage fermentation. After wilting treatment, the bacterial species in the WPS group and the WLB group changed and the diversity was lower than that in the PS group, in which the relative abundance of lactic acid bacteria accounted for a large proportion. It may be that some harmful bacteria are killed by ultraviolet light under the sun exposure, thereby reducing the competition with lactic acid bacteria to consume WSC. It may be that after wilting treatment, the water content of WPS group and WLB group was reduced, and the relative concentration of WSC was increased, which was conducive to the rapid consumption of a certain amount of WSC by lactic acid bacteria to produce a large amount of organic acids.

Based on the above reasons, the WSC changes in our study should be correct. However, it was still many shortcomings in this study, due to the limitation of the experiment. We will explore that in the future study.

 

Comments 6: It must be clearly stated that these are preliminary results. The experiment needs to be repeated in order to provide a more consistent conclusion about the use of the inoculant in combination with plant dehydration.

Response 6: Thank you for pointing this out. This enhances the rigor of the content of our manuscript. We have added this to the conclusion of the manuscript– page 12, paragraph 1, and line 416-419. Due to time constraints and financial problems, we are currently unable to carry out repetitive experiments. However, we will deepen the discussion and verification of this content in the next research.