Effects of Lactobacillus plantarum and Cellulase on Mixed Silages of Amaranthus hypochondriacus and Cornmeal: Fermentation Characteristics, Nutritional Value, and Aerobic Stability

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments and suggestions for authors (will be shown to authors): The experimental article “The Effects of Lactobacillus Plantarum and Cellulase on Mixed Silages of Amaranthus Hypochondriacus and Corn Meal: Fermentation Characteristics, Nutritional Value and Aerobic Stability” is devoted to the creation of new types of feed and ways of using resources in the agricultural industry, which contributes to the sustainable development of livestock and poultry farming. This article presents a large amount of research, materials and methods are described in great detail. A comparison of the obtained data with the literature is presented. After eliminating some of the comments listed, the article can be published. Notes and recommendations: 1. The 85 mixing ratios (w/w) of amaranth and corn meal ingredients were 69:31, 76:24, and 84:16. Why exactly these ratios of amaranth and corn flour were chosen. 2. Discuss the data in Table 1 in the article and compare the data obtained with the literature. There is a lot of data presented in the table, but there is no discussion of it. What are the advantages of amaranth compared to corn flour? 3. Many abbreviations have been introduced, making the text difficult to read.

Author Response

Point 1:The 85 mixing ratios (w/w) of amaranth and corn meal ingredients were 69:31, 76:24, and 84:16. Why exactly these ratios of amaranth and corn flour were chosen.

Response 1: We very much agree with your suggestion. In order to regulate the moisture content of mixed silage of amaranth and corn meal to 60%, 65% and 70%. the 23rd reference cited in lines 327-328 has the indication that silage is best fermented at a moisture content of about 65%. Therefore, we chose the above three moisture contents for silage preparation.

Point 2: Discuss the data in Table 1 in the article and compare the data obtained with the literature. There is a lot of data presented in the table, but there is no discussion of it. What are the advantages of amaranth compared to corn flour?

Response 2: We strongly endorse this suggestion of yours. This study was mainly done on mixed silage of seed amaranth and corn meal. There will be no comparison between seed amaranth and corn meal because corn meal is a secondary ingredient and seed amaranth is the main ingredient. Corn meal was added mainly to regulate the moisture of the seeded amaranth silage.

Point 3: Many abbreviations have been introduced, making the text difficult to read.

Response 3: Thanks for the suggestion, some of the abbreviations have been replaced with full names.I hope this helps in understanding the content of this thesis.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper, titled “The Effects of Lactobacillus Plantarum and Cellulase on Mixed Silages of Amaranthus Hypochondriacus and Corn Meal: Fermentation Characteristics, Nutritional Value and Aerobic Stability,” addresses an important and timely topic. I found the subject matter of the article fascinating and read the manuscript with great interest. The paper aligns well with the scope of the journal. However, I believe that in its current form, it has several shortcomings.

The abstract provides a concise overview of the study. However, it could be improved by clearly stating the key findings and their implications for the field. For instance, specific numerical results should be highlighted to underline the significance of the findings.

While the introduction sets the stage for the research, it would benefit from a more detailed discussion on the importance of amaranth as a feed resource and the challenges associated with its high moisture and protein content. Additionally, the introduction should more clearly articulate the novelty of this study compared to previous research.

Is not clear how the poultry have a role in the paper.

Line 40 I suggest citing 10.1080/1828051X.2021.1884005 as a novel and substitute protein source, and 10.1080/1828051X.2021.1916408 for starch source.

The methods section is thorough but could use more clarity in describing the experimental design. Specifically, the rationale for choosing the mixing ratios and moisture content levels should be elaborated. Additionally, a clearer explanation of how the lactic acid bacteria and cellulase were applied would enhance reproducibility.

While the statistical analysis appears sound, the manuscript would benefit from a more detailed explanation of the statistical methods used. This includes a brief description of the statistical tests applied and the criteria for significance. The statistical methods used need a reference, I suggest 10.3389/fvets.2024.1332207 for tukey test.

The results are presented in a detailed manner. However, some tables and figures could be simplified for better readability. For instance, combining certain results into summary tables could help in understanding the overall trends without getting lost in the details.

The discussion should be expanded to better connect the findings with practical implications. Specifically, how can the improved fermentation quality and aerobic stability achieved with Lactobacillus plantarum and cellulase be utilized in large-scale feed production? Additionally, a comparison with other similar studies would help position this work within the broader context of silage research.

I recommend incorporating a discussion paragraph highlighting the significance of educating future veterinarians, technicians, and farmers about the issues addressed in the paper. Emphasizing the importance of effective teaching methods in shaping knowledgeable students and proficient veterinarians would add depth to the paper's implications. It is advisable to refer to recent publications on veterinary education to provide up-to-date insights into best practices in preparing future professionals to address the challenges discussed in the paper. Please see: 10.1016/j.jevs.2023.104537 and 10.3390/ani13223503.

The conclusion effectively summarizes the main findings, but it should also suggest future research directions. Identifying potential limitations of the study and proposing how these could be addressed in future research would provide a more comprehensive closure.

Author Response

Dear reviewer, thank you for your encouraging and warm comments and suggestions, all of your suggestions are very important, and they all have important guiding significance for our future research work. Based on this we have revised and (we think) strengthened our paper.

Point 1: The abstract provides a concise overview of the study. However, it could be improved by clearly stating the key findings and their implications for the field. For instance, specific numerical results should be highlighted to underline the significance of the findings.

Response 1: We fully appreciate your suggestion.Following your suggestion, specific numerical results have now been added to the abstract and the importance of the results of this experiment has been emphasized. They are now labeled in yellow.(PDF:30,33-35)

Point 2: While the introduction sets the stage for the research, it would benefit from a more detailed discussion on the importance of amaranth as a feed resource and the challenges associated with its high moisture and protein content. Additionally, the introduction should more clearly articulate the novelty of this study compared to previous research.Is not clear how the poultry have a role in the paper.

Response 2: We strongly endorse this suggestion.New literature has been cited in the introduction section, which has been highlighted in yellow font.(PDF:42-43) It has also been compared with previous studies to further emphasize the novelty of this study.(PDF:79-81)

Point 3:Line 40 I suggest citing 10.1080/1828051X.2021.1884005 as a novel and substitute protein source, and 10.1080/1828051X.2021.1916408 for starch source.

Response 3: We very much recognize this piece of your advice.  10.1080/1828051X.2021.1884005 has been cited and yellowed in the article. (PDF:42-43) However, since this article mainly discusses seed amaranth as a novel plant-based protein feed resource, it does not discuss starch sources. Therefore the article 10.1080/1828051X.2021.1916408 was not cited.

Point 4:The methods section is thorough but could use more clarity in describing the experimental design. Specifically, the rationale for choosing the mixing ratios and moisture content levels should be elaborated. Additionally, a clearer explanation of how the lactic acid bacteria and cellulase were applied would enhance reproducibility.

Response 4: We fully appreciate your suggestion. The 25rd reference cited in lines 365-366 has the indication that silage ferments best at around 65% moisture. Therefore, three moisture contents, 60%, 65%, and 70%, were selected for silage conditioning. The moisture content was regulated by the mixing ratio of seed amaranth and maize meal, therefore three different mixing ratios were selected as mentioned above. It has been recapitulated how lactic acid bacteria and cellulase are used.(PDF:100-102,104-107) And labeled yellow in the article.

Point 5:While the statistical analysis appears sound, the manuscript would benefit from a more detailed explanation of the statistical methods used. This includes a brief description of the statistical tests applied and the criteria for significance. The statistical methods used need a reference, I suggest 10.3389/fvets.2024.1332207 for tukey test.

Response 5: Thank you for your suggestion, we have read the article you presented in detail and cited it in the Materials and Methods section of this article.

Point 6:The results are presented in a detailed manner. However, some tables and figures could be simplified for better readability. For instance, combining certain results into summary tables could help in understanding the overall trends without getting lost in the details.

Response 6: We very much recognize this piece of your advice.We have now standardized all the major categories of silage quality into whole tables, for example, Table 2 standardizes the indicators of fermentation quality, Table 3 standardizes chemical composition and energy, and Table 4 standardizes in vitro digestibility.

Point 7:The discussion should be expanded to better connect the findings with practical implications. Specifically, how can the improved fermentation quality and aerobic stability achieved with Lactobacillus plantarum and cellulase be utilized in large-scale feed production? Additionally, a comparison with other similar studies would help position this work within the broader context of silage research.I recommend incorporating a discussion paragraph highlighting the significance of educating future veterinarians, technicians, and farmers about the issues addressed in the paper. Emphasizing the importance of effective teaching methods in shaping knowledgeable students and proficient veterinarians would add depth to the paper's implications. It is advisable to refer to recent publications on veterinary education to provide up-to-date insights into best practices in preparing future professionals to address the challenges discussed in the paper. Please see: 10.1016/j.jevs.2023.104537 and 10.3390/ani13223503.

Response 7: We very much recognize this piece of your advice. We have now added a new paragraph to the discussion based on your comments and cited the literature you introduced. Thank you for your suggestion to deepen the discussion of our paper.(PDF:488-499)

Point 8:The conclusion effectively summarizes the main findings, but it should also suggest future research directions. Identifying potential limitations of the study and proposing how these could be addressed in future research would provide a more comprehensive closure.

Response 8: We very much recognize this suggestion of yours.We conclude our conclusions by pointing out the potential limitation that the experiment requires further adaptive experiments to assess the effect of mixed silage of amaranth and cornmeal on rumen growth performance and propose a solution.(PDF:509-510)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The present study examined the impact of adding lactobacillus and cellulase to silage production using Amaranthus and corn meal as silage materials. However, the current manuscript has omitted some crucial data.

General comment:

1.     The data in Table 3 only shows the silage composition at the endpoint. The silage composition at the ensilage date should also be presented.

2.     How about microbial count data for silage products? The microbial counts of the silage substrates are important? This manuscript showed a lot of discussion about the effect of microbes, but no future microbial results of silage products were presented in tables or figures.

3.     This study combined Amaranthus and cornmeal to create three different moisture-containing silage. The author adjusted the moisture using cornmeal instead of adding water. Why?

4.     Why did the author choose cellulase supplementation instead of amylase or directly adding molasses if they suggested that cellulase benefited WSC release during silage?

Specific comment:

L29

The full name of “AA” in the abstract should be shown.

L93

How to decide the lactic acid bacteria supplementation level? The moisture of silages was different in W1 to W3 treatments.

L95

How to define the “U” of the cellulase activity? The substrate may affect the cellulase degradation product.

L121

“…to the methods reported by Van et al. [18].”

Please check the reference number again.

L173-175

If the ambient and silage temperatures were recorded at 1-hr intervals, the temperature curve during the aerobic stability test should be shown in the result.

L193

How to measure the “buffering capacity” of amaranth and corn meal? No related method was listed in the material and method section.

L238 Table 2

"The concentration of acetic acid was similar to that of lactic acid. However, it is important to note that the pKa of acetic acid is 4.74, which should be discussed by the author in the corresponding section.

The high concentration of acetic acid in silage products might contribute to improved aerobic stability. Nevertheless, it's worth mentioning that the results of aerobic stability in Figure 1 were inconsistent." 

L248-257

How about the loss of nutrient composition during ensilage? Without the nutrient composition data before the ensilage of each treatment, the fermentation loss could not be calculated.

If the author attempts to enhance WSC in silage by adding cellulase, the WSC change during ensilage should be assayed.

L298 Table4

The NDF% and ADF% of silage products in each treatment differed, which may affect the efficiency of lactic acid bacteria or cellulase supplementation. These effects should be considered in the discussion about in vitro digestion.

L311 Figure 1

Was the y-axis wrong? It should be confirmed. 

L327-331

Without the silage microbial data, present results did not support this discussion.

L349-353

These assays should be done if the nitrite and nitrate affected the fermentation result.

L372-373

The moisture content should be considered at the same time. The WSC of each treatment also affects the additive function.

L416-419

In this study, the ivDMD and ivNDFD of the W2 group < W1 group, but the silage NDF% of W2 > W1. How do we explain this result?

Author Response

Dear reviewer, thank you for your encouraging and warm comments and suggestions, all of your suggestions are very important, and they all have important guiding significance for our future research work. Based on this we have revised and (we think) strengthened our paper.

Point 1:    The data in Table 3 only shows the silage composition at the endpoint. The silage composition at the ensilage date should also be presented.

Response 1: We fully appreciate your suggestion. We carried out measurements of amaranth and cornmeal composition before silage and also of amaranth and cornmeal composition at the end of silage, but our aim was not to look at changes in the silage process and therefore did not measure silage composition in silage.

Point 2:    How about microbial count data for silage products? The microbial counts of the silage substrates are important? This manuscript showed a lot of discussion about the effect of microbes, but no future microbial results of silage products were presented in tables or figures.

Response 2: We strongly endorse this suggestion. Microbial count data for silage products are presented in Table 1, which lists the species and number of microorganisms that mainly influence the silage fermentation profile of seed amaranth and cornmeal mixtures prior to silage. The number of microorganisms in the silage substrate is an important factor influencing the quality and success of silage fermentation. However, due to the equipment we did not carry out measurements of microorganisms in the silage product.

Point 3:     This study combined Amaranthus and cornmeal to create three different moisture-containing silage. The author adjusted the moisture using cornmeal instead of adding water. Why?

Response 3:  We very much recognize this piece of your advice. Amaranth is characterized by high moisture and low soluble carbohydrate content. Therefore, it is difficult to preserve amaranth directly by traditional silage methods. The soluble carbohydrate content of amaranth was determined to be only 50.46 g kg-1 DM, which only meets the requirement of 50 g kg-1 soluble carbohydrate [9]. The addition of 10% cornmeal has been reported to improve fermentation quality and apparent digestibility of silage [10]. In addition, cornmeal, characterized by its high content of soluble carbohydrates (WSC) and dry matter, is a good mixed silage adjuvant that can directly add to the fermentation substrate, make up for the lack of fermentable carbohydrates in amaranth, reduce the moisture content, and improve the success of fermented silage. However, to the best of our knowledge, few studies have tested the effects of mixing amaranth silage with corn meal for storage. This is described in detail in lines 62-66.

Point 4:    Why did the author choose cellulase supplementation instead of amylase or directly adding molasses if they suggested that cellulase benefited WSC release during silage?

Response 3:  Thank you for your questions and suggestions. This thesis mainly provides theoretical support for practical production, if we choose to supplement amylase or directly add molasses will make the cost higher. At the same time adding cellulase can degrade the cellulase in forage and improve the utilization of forage, so we chose cellulase.

Specific comment:

L29

The full name of “AA” in the abstract should be shown.

Response :  I apologize for not noticing this before, it has now been modified.

L93

How to decide the lactic acid bacteria supplementation level? The moisture of silages was different in W1 to W3 treatments.

Response : We use the purchased finished lactobacillus preparation (Chikusou-1 (Lactobacillus 96 plantarum) obtained by Snow Brand Seed Co. Ltd. (Tokyo, Japan) strictly, so we can be assured that the amount of lactobacilli added is determined. Yes, W1 contains 60% water and W3 contains 70% water, so silage moisture is different.

L95

How to define the “U” of the cellulase activity? The substrate may affect the cellulase degradation product.

Response : Cellulase activity units are defined as the amount of enzyme required to catalyze the hydrolysis of sodium carboxymethylcellulose to produce 1 μmol of reducing sugar per minute at 55°C and pH 5.0 as one enzyme activity unit U. Thus the substrate does not affect the degradation products of cellulose. 

L121

“…to the methods reported by Van et al. [18].”

Please check the reference number again.

Response : Thank you for your suggestion. We have now rechecked all references.

L173-175

If the ambient and silage temperatures were recorded at 1-hr intervals, the temperature curve during the aerobic stability test should be shown in the result.

Response : Thank you for your suggestion, we have added three line graphs in response to your suggestions. Shows the effect of additives on temperature changes after exposure to aerobic conditions for three moisture contents of mixed silage of amaranth and corn meal. Shows temperature profiles during aerobic stability tests.

L193

How to measure the “buffering capacity” of amaranth and corn meal? No related method was listed in the material and method section.

Response : This is described in the penultimate line of the first paragraph of the Chemical Composition, Energetics, and In Vitro Degradability Analysis section of the Materials and Methods section (PDF:135-136) . Buffering capacity (BC) was measured using the method of Playne and McDonald.

L238 Table 2

"The concentration of acetic acid was similar to that of lactic acid. However, it is important to note that the pKa of acetic acid is 4.74, which should be discussed by the author in the corresponding section.

The high concentration of acetic acid in silage products might contribute to improved aerobic stability. Nevertheless, it's worth mentioning that the results of aerobic stability in Figure 1 were inconsistent." 

Response :  We very much recognize this piece of your advice. We have added new references in the discussion paragraph to argue our findings. Although high concentrations of acetic acid contribute to aerobic stability, it is not the only factor affecting aerobic stability. In this experiment, the aerobic stability of mixed silage of amaranth and corn meal decreased with increasing water content (Fig. 4). However, acetic acid content did not decrease with increasing moisture content. This may be due to the fact that a moist environment is more favorable for the growth of microorganisms such as yeasts and acetic acid bacteria, and acid-tolerant yeasts can also survive in silage [45].Hao et al.'s [46] study on a total mixed ration also showed a higher growth rate of yeasts in the high-moisture treatment.

L248-257

How about the loss of nutrient composition during ensilage? Without the nutrient composition data before the ensilage of each treatment, the fermentation loss could not be calculated. If the author attempts to enhance WSC in silage by adding cellulase, the WSC change during ensilage should be assayed.L298 Table 4 The NDF% and ADF% of silage products in each treatment differed, which may affect the efficiency of lactic acid bacteria or cellulase supplementation. These effects should be considered in the discussion about in vitro digestion.

Response : We strongly endorse this suggestion of yours. Table 1 showed the data on nutrient composition before silage. Therefore it is possible to calculate the fermentation losses. We measured the pre- and post-fermentation metrics, but unfortunately did not measure the metrics during fermentation. Table 4 Differences in NDF% and ADF% of silage products among treatments, which may affect the efficiency of lactic acid bacteria or cellulase supplementation. With the addition of lactic acid bacteria and cellulase we focused on the differences at the same moisture content, under these conditions the NDF and ADF contents are essentially the same and therefore do not affect the efficiency of lactic acid bacteria or cellulase supplementation.

L311 Figure 1

Was the y-axis wrong? It should be confirmed. 

Response : We fully appreciate your suggestion. Yes, the vertical coordinate should be aerobic stability (h), which we have changed.

L327-331

Without the silage microbial data, present results did not support this discussion.

Response : We very much recognize this piece of your advice. This content is quoted from previous experimental conclusions, and there are measurements of silage microorganisms in the cited literature. We therefore hypothesize that the present results support this conclusion.

L349-353

These assays should be done if the nitrite and nitrate affected the fermentation result.

Response : This content is a discussion of the experimental results appearing in this paper citing the conclusions of previous experiments. Therefore no testing for nitrite and nitrate was performed.

L372-373

The moisture content should be considered at the same time. The WSC of each treatment also affects the additive function.

Response : We are combining the effects of lactic acid bacteria and cellulase at each moisture content, not a single comparison. Therefore we have taken into account that the WSC of each treatment also affects the functionality of the additives.

L416-419

In this study, the ivDMD and ivNDFD of the W2 group < W1 group, but the silage NDF% of W2 > W1. How do we explain this result?

Response : Lines 455-457 We hypothesized that in vitro digestibility was negatively correlated with the content of NDF and ADF based on the available experimental results and previous references, and our conclusions were the same as those of Bao et al.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript comprehensively investigated the influence of adding lactic acid-producing bacteria and cellulase on the silage quality of amaranth and corn meal at different mixed ratios. The results are generally interesting. However, the quality of the manuscript must be improved with the comments followed:

1. Abstract: this part should be re-written. First, the background and importance of carrying out the current study is missing. Second, the results should be presented in a condense manner. The current format makes the reviewer confused. In addition, there was an error in Line 20-21 for DMD. Last, what is the result of the mixed ratio between amaranth and corn meal?

2. Introduction: (1) the contribution and novelty of the current study is not clear. The authors mentioned that few studies were found to report relative results. However, why the mix of amaranth and corn meal is important for feed supply? Why is corn meal, not other carbohydrate-rich materials? This should be clearly stated.

(2) Line 53: why high content of protein and water make amaranth hard to store？Line 63-64: re-write.

3. 2.1 Experimental design: (1) It is difficult to follow the detailed experimental design, can you add a table or a schematic diagram to show the different?

(2) Line 85-87: Why do the authors chose the mixing ratio. In the abstract, the authors mentioned that the water content is adjusted to 60, 65 and 70%. But here it seems that the water content is because of the mixing ratio?

(3) Line 95: What is the addition concentration of LA bacterial and cellulase for mixed additives? If they are the same with the individual ones, this means the addition in the mixed ones had one more additive than the individual ones? Line 118: change to "chemical composition analysis"

4. Results:

(1) Line 196-199: 1) the buffering data is missing in line 196 for the corn meal. 2) the microbial of corn meal is missing.

(2) Table 1: the caption of the table should be "Characteristics of amaranth and corn meal." This should also be applied to other tables.

(3) Line 221-222: There is a description error for the BA in W1.

(4) Line 242: SEM: Is this the error for all the figures or just the average?

(5) Line 262: it should be changed to "... However, It remained unchanged without the addition of L"

(6) Line 351-352: "..., which was low, indicating that..." should be changed to "...; the low BA content indicated that ..."

(7) Line 423: change people to human beings

5. Conclusions: this part should add the mix ratio results as it is primary factor on the quality change during silage. Change "at the same time" to "In addition"

6. The English must be polished for the whole manuscript. Moreover, tons of format errors were found throughout the manuscript. 

Comments on the Quality of English Language

The English must be polished for the whole manuscript. Moreover, tons of format errors were found throughout the manuscript. 

Author Response

Dear reviewer, thank you for your encouraging and warm comments and suggestions, all of your suggestions are very important, and they all have important guiding significance for our future research work. Based on this we have revised and (we think) strengthened our paper.

Point 1: Abstract: this part should be re-written. First, the background and importance of carrying out the current study is missing. Second, the results should be presented in a condense manner. The current format makes the reviewer confused. In addition, there was an error in Line 20-21 for DMD. Last, what is the result of the mixed ratio between amaranth and corn meal?

Response 1: Thanks to your suggestion, we currently present the background and importance of this study at the beginning and end of the abstract section of the manuscript, respectively, in addition to adding numerical results in the experimental results section to increase credibility. The effects of sequential water content and additives are discussed according to the introduction first. The DMD content of lines 20-21 has been removed. The mixing ratios of amaranth and maize meal are described in lines 15-16, with mass ratios of 69:31, 76:24 and 84:16 for amaranth and maize meal to facilitate the adjustment of silage moisture content to 60% (W1), 65% (W2) and 70% (W3), respectively.

Point 2:

Introduction: (1) the contribution and novelty of the current study is not clear. The authors mentioned that few studies were found to report relative results. However, why the mix of amaranth and corn meal is important for feed supply? Why is corn meal, not other carbohydrate-rich materials? This should be clearly stated.

Response 2.1: The introduction section recites the literature for clarification, highlighting our novelty. (PDF:42-43,79-81)Corn meal was chosen because lines 66-70 describe the potential advantages of corn meal. In addition to being rich in carbohydrates it can improve the fermentation quality and apparent digestibility of silage.

• Line 53: why high content of protein and water make amaranth hard to store？Line 63-64: re-write.

Response 2.2: We strongly endorse this suggestion of yours. This is due to previous research showing that high moisture, high protein forage silage alone is prone to silage failure. Thank you for your comments, which have now been rewritten and highlighted in yellow.(PDF:63-65)

Point 3: 

Experimental design: (1) It is difficult to follow the detailed experimental design, can you add a table or a schematic diagram to show the different?

Response 3.1: Thank you for your suggestion. This section has now been re-narrated. so that it is more accessible and clearer.(PDF:92-95,99-101,104,106)

(2) Line 85-87: Why do the authors chose the mixing ratio. In the abstract, the authors mentioned that the water content is adjusted to 60, 65 and 70%. But here it seems that the water content is because of the mixing ratio?

Response 3.2: Thank you very much for your suggestions and questions. Yes, the mass ratios of amaranth and cornmeal were 69:31, 76:24, and 84:16 in order to facilitate the adjustment of silage moisture content to 60% (W1), 65% (W2), and 70% (W3), respectively. We are currently revising the abstract and the description of the experimental methodology to make it clearer that we performed the mixing ratios for the purpose of moisture adjustment.

• Line 95: What is the addition concentration of LA bacterial and cellulase for mixed additives? If they are the same with the individual ones, this means the addition in the mixed ones had one more additive than the individual ones? Line 118: change to "chemical composition analysis"

Response 3.3: Thank you for your question. Yes, the additive blend was added with both lactic acid bacteria and cellulase.The dosage of lactic acid bacteria was 4.7 × 106 colony-forming units (cfu) per gram of fresh weight (FW).Cellulase was added at a concentration of 4.2 × 103 U g-1 FW. This does not mean that the additive mixture added one more additive than the individual additives. This is because we want to discuss whether adding both lactobacilli and cellulase is better than adding them individually, so the dosages are consistent.

Row 118: Change to “chemical composition analysis”. Thank you for the suggestion, it has now been revised.

Point 4: Results:

• Line 196-199: 1) the buffering data is missing in line 196 for the corn meal. 2) the microbial of corn meal is missing.

Response 4.1: We fully appreciate your suggestion.Corn meal buffer data has been added. As a result of our measurements, the cornmeal we used indicated that there were no microorganisms attached and therefore the microbial content was ND. It is not the case that the microorganisms in the cornmeal were not measured.

• Table 1: the caption of the table should be "Characteristics of amaranth and corn meal." This should also be applied to other tables.

Response 4.2: We strongly agree with your suggestion.We have modified the title of Table 1. However, we downloaded published papers from your journal for reference and found that everyone's table headings on the fermentation section summarize what the tables measure, so we did not revise Tables 2-4. Thank you for your understanding.

• Line 221-222: There is a description error for the BA in W1.

Response 4.3: Thank you for your careful review and corrections. We have checked again. Without the addition of E, it refers to two groups, U and L groups. In these two groups the BA content of W1 is 0.152 is the lowest value among the groups.

• Line 242: SEM: Is this the error for all the figures or just the average?

Response 4.4: SEM is a measure used in statistics to measure the deviation of a sample statistic from an estimate of an overall parameter. It represents the uncertainty or margin of error between a sample statistic (e.g., sample mean) and an overall parameter (e.g., overall mean).

• Line 262: it should be changed to "... However, It remained unchanged without the addition of L"

Response 4.5: Thank you for your suggestions, which have now been revised in line with your comments.

• Line 351-352: "..., which was low, indicating that..." should be changed to "...; the low BA content indicated that ..."

Response 4.6: Thank you for your suggestions, which have now been revised in line with your comments.

• Line 423: change people to human beings

Response 4.7: Thank you for your suggestions, which have now been revised in line with your comments.

Point 5: Conclusions: this part should add the mix ratio results as it is primary factor on the quality change during silage. Change "at the same time" to "In addition"

Response 5: We fully appreciate your suggestion. We have now added mixing ratios to the conclusions section and revised them as per your comments.(PDF:504-505)

Point 6:The English must be polished for the whole manuscript. Moreover, tons of format errors were found throughout the manuscript. 

Response 6: Thank you for your comments. We have now touched up the manuscript in English and highlighted the touches in red. Formatting issues have also been checked and corrected.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

after the revision round the paper improved a lot

Reviewer 3 Report

Comments and Suggestions for Authors

The present data was sufficient to support the conclusion and the discussion. The response also provided clear explain for the question.

Reviewer 4 Report

Comments and Suggestions for Authors

The comments are well addressed except the schematic flow of the experiments.

Comments on the Quality of English Language

The sentence should be more condensed.