This study provides practical information on: “Exploring Lactobacillus plantarum on fermentation quality, gas emissions, and in vitro digestibility of different varieties of litchi leaves silage”.

Important comment:

It is not enough to conduct an experiment in one year on perennial trees to obtain sufficient results that can be recommended.

No information was mentioned about the orchard from which the samples were taken (details are provided in the commentary in Materials and Methods).

There is no statistical analysis that shows the differences between the varieties according to the different treatments.

Important note: Is the word "litchi" which was written in this study, or "lychee" as it appeared in many references?? Please make sure of this and adopt the correct spelling.

In Abstract:

· Shorten the abstract to be about 200 words as follows:

o Summarize lines 14-19 with just two lines.

o Not elaborating on explaining the details of all the results and writing the important ones only.

Keywords: Add the word silage to the keywords.

In Introduction:

Add to the introduction a paragraph on the use of litchi leaves as fodder for animals, whether in the study area or similar areas, because it must be palatable by animals before making silage from them.

In Materials and Methods:

· It was stated in the paragraph on Silage preparation, line 88: that Litchi leaves were collected from the South China Agricultural University: Were the leaves collected directly from the trees or is it fallen leaves?

· From which part of the tree were the leaves collected? and what is meant here is the age of the leaves

· Nothing was mentioned or described about the trees from which the leaves were collected (the age, the number of trees from which the leaves were taken, the method of breeding, the agricultural processes of the trees, especially fertilization, etc.) All of this will affect the quality of the resulting silage.

· It is known that trees pruning residues are the branches with the leaves, which is what the study objective was based on, but we find here that only the leaves were collected from the trees.

· On the other hand, is the month (September) in which the leaves were taken is the same month in which the trees are usually pruned?? It is known that pruning is not in September.

· Silage is prepared by placing it in the dark place or in opaque packages or bags. This was not mentioned in the study! And in all cases, at room temperature, i.e. at a moderate temperature.

· As it is known and found in many references: the silage process lasts from 6 to 8 weeks, it must be clarified and justified why silage samples were taken out after only 30 days?

· The methods by which the components of the nutritional value (protein, fiber, etc.) are estimated should be mentioned. It is not sufficient to write: the estimate was made according to Wang et al. (2019) because the mentioned reference is a study only on silage and is not an approved method as one of the standard methods!!

· The same note for the estimation of the in vitro dry matter digestibility and gas production: mention the standard reference method that is used internationally.

· In Statistical analyses: It cannot be said that 5% and 1% significance levels were used at the same time, it must be determined in which of the coefficients each of them was used in the comparison of averages.

In Results and Discussion

· Why was the nutritional value of litchi leaves not evaluated before silage? This is important for comparison before and after silage. And if table 1 expresses the nutritional value before silage, how did the lactic acid bacteria do?

· Put the meaning of all the abbreviations under all the tables.

· How was it concluded that there are no significant differences between cultivars in crude protein content? Because the results of each category were placed in a separate table.

· Why wasn't a table set out to show the differences between the varieties in all the treatments and values?

· A statistical analysis showing the differences between the four varieties of litchi in each treatment should be carried out separately.

Discussion:

· Normally the pH of silage should not be more than 4.7 while in the current study it was greater than that. Please provide a justification for this in the discussion of the results.

· At the end of each paragraph in a discussion, a conclusion must be reached that is not necessarily consistent with the results of this study, so please pay attention to that and add it if it is not present.

Conclusion:

This mean, how will farmers apply it? How will the lychee leaves be collected? When and how is the silage done?

The manuscript needs medium proofreading.

Author Response

Dear reviewr:

Thank you very much for your comments and professional advice. These opinions help to improve academic rigor of our article. Based on your suggestion and request, we have made modifications on the manuscript. In addition, we uploaded the file of the revised manuscript. Revisions in the text are shown using yellow highlight. The responses to your comments are prssented following.

Important comment:

1:It is not enough to conduct an experiment in one year on perennial trees to obtain sufficient results that can be recommended.

Response: We are very much appreciate the time and effort you have put into your comments. We agree with your comment. However, we have refered to the relevant references and found that most studies of woody feed on perennial tress is also conduct an experiment in one year. These are relevant references:

[1]:He L, Lv H, Xing Y, Chen X, Zhang Q. Intrinsic tannins affect ensiling characteristics and proteolysis of Neolamarckia cadamba leaf silage by largely altering bacterial community. Bioresour Technol. 2020 Sep;311:123496.

[2] Hao Y, Huang S, Liu G, Zhang J, Liu G, Cao Z, Wang Y, Wang W, Li S. Effects of Different Parts on the Chemical Composition, Silage Fermentation Profile, In Vitro and In Situ Digestibility of Paper Mulberry. Animals (Basel). 2021 Feb 5;11(2):413.

[3] Wen Z, Chen Y, Wu L, Tian H, Zhu N, Guo Y, Deng M, Liu J, Sun B. Effects of Broussonetia papyrifera silage on rumen fermentation parameters and microbes of Holstein heifers. AMB Express. 2022 May 25;12(1):62.

Of course, regarding the concerns of the reviewer is very worth considering for us that we will conduct experiments to improve this issue in the next study. Thank you!

2:No information was mentioned about the orchard from which the samples were taken (details are provided in the commentary in Materials and Methods).

Response: Thank reviwer for this valuable feedback. We have added sentences to introduce the specific information about litchi leaves in the section of “Materials and Methods”.Thank you! Line 85-94.

3:There is no statistical analysis that shows the differences between the varieties according to the different treatments.

Response: As reviewer suggested that it is important to show the differences between the varieties according to the different treatments.However, we want to provide some explanation. In recent years, there have been more and more varieties of litchi. From Table 1, we found that there is not much change in nutritional composition among different varieties. To fully recycle and utilization of discarded litchi leaves, We think that the focus of the experiment is whether there are significant differences in the fermentation quality and in vitro fermentation characteristics of litchi leaves with or without Lactiplantibacillus plantarum.

4:Important note: Is the word "litchi" which was written in this study, or "lychee" as it appeared in many references?? Please make sure of this and adopt the correct spelling.

Response: Thank reviwer for this valuable feedback. We check the word, which is right. "lychee" is often used by chinese, but "litchi" is generally used internationally. Thank you!

In Abstract(1-3)

1:Shorten the abstract to be about 200 words as follows:

2:Summarize lines 14-19 with just two lines.

3:Not elaborating on explaining the details of all the results and writing the important ones only.

Response(1-3): Thank reviwer for this valuable feedback. We have shortened the abstract to less than 200 words. We also revised and summarized lines 14-19 in just two lines. In addition, we have revised the results in the abstract section.Line 14-28.

Keywords

1:Add the word silage to the keywords.

Response: Thank reviwer for this valuable feedback. We revised and added the word ”silage” to the keywords.Line 29.

In Introduction:

1:Add to the introduction a paragraph on the use of litchi leaves as fodder for animals, whether in the study area or similar areas, because it must be palatable by animals before making silage from them.

Response:We have carefully considered the suggestion of reviewer. As we said, litchi leaves silage is the same as tea leaves silage. somes studies showed that litchi leaves are often used in traditional medicine and tea infusions. But there is little research directly applying litchi leaves as silage. Therefore, it is difficult to add a paragraph on the use of litchi leaves as fodder for animals, whether in the study area or similar areas. But we will carefully consider the comments of the reviewer. In next study, we will add the application of lichi leaves in feeding ruminants.

In Materials and Methods:

1:It was stated in the paragraph on Silage preparation, line 88: that Litchi leaves were collected from the South China Agricultural University: Were the leaves collected directly from the trees or is it fallen leaves?

2:From which part of the tree were the leaves collected? and what is meant here is the age of the leaves

3:Nothing was mentioned or described about the trees from which the leaves were collected (the age, the number of trees from which the leaves were taken, the method of breeding, the agricultural processes of the trees, especially fertilization, etc.) All of this will affect the quality of the resulting silage.

4:It is known that trees pruning residues are the branches with the leaves, which is what the study objective was based on, but we find here that only the leaves were collected from the trees.

Response (1-4): Thank reviwer for this valuable feedback. We have added sentences to introduce the specific information about litchi leaves in the section of “Materials and Methods”.Line 85-94.

5:On the other hand, is the month (September) in which the leaves were taken is the same month in which the trees are usually pruned?? It is known that pruning is not in September.

Response: We sincerely thank the reviewer for this valuable feedback. We have carefully considered the question of reviewer. Therefore, we consulted some references and asked pruners, and found that litchi leaves pruning is usually done in March to April or September to October of the year.

6:Silage is prepared by placing it in the dark place or in opaque packages or bags. This was not mentioned in the study! And in all cases, at room temperature, i.e. at a moderate temperature.

Response: Thank reviwer for this valuable feedback. We have added the special description about them. Thank you! Line 95-101.

7:As it is known and found in many references: the silage process lasts from 6 to 8 weeks, it must be clarified and justified why silage samples were taken out after only 30 days?

Response:We sincerely thank your valuable feedback. We read some reference and found that silge can reach good fermentation quality after 30 ensiling. These are some of references that we have referenced:

[1]Zhang Q, Zou X, Wu S, Wu N, Chen X, Zhou W. Effects of Pyroligneous Acid on Diversity and Dynamics of Antibiotic Resistance Genes in Alfalfa Silage. Microbiol Spectr. 2022 Aug 31;10(4):e0155422.

[2] Ítavo LCV, Dos Santos GT, Ítavo CCBF, Dias AM, da Silva Zornitta C, Gurgel ALC, Dos Santos Difante G. Characterization and in vitro evaluations of Baru pulp from almond production waste ensiled with different additives. Trop Anim Health Prod. 2022 Aug 10;54(5):255.

[3] Dai T, Dong D, Wang J, Yin X, Zong C, Jia Y, Shao T. Effects of wet brewers grains on fermentation quality and in vitro ruminal digestibility of mixed silage prepared with corn stalk, sweet potato peel and dried apple pomace in southeast China. J Anim Physiol Anim Nutr (Berl). 2023 Mar;107(2):340-349.

8:The methods by which the components of the nutritional value (protein, fiber, etc.) are estimated should be mentioned. It is not sufficient to write: the estimate was made according to Wang et al. (2019) because the mentioned reference is a study only on silage and is not an approved method as one of the standard methods!!

Response:Thank reviwer for this valuable feedback. We have added the specific description about a methodology for evaluating. In addition, We checked the method, which is widely used as the standard methods.Line 116-121.

9:The same note for the estimation of the in vitro dry matter digestibility and gas production: mention the standard reference method that is used internationally.

Response: Thank reviwer for this valuable feedback. We have revised it. Thank you! Line 127-135.

10:In Statistical analyses: It cannot be said that 5% and 1% significance levels were used at the same time, it must be determined in which of the coefficients each of them was used in the comparison of averages.

Response: Thank reviwer for this valuable feedback. We have revised it. Line 137-139.Thank you!

In Results and Discussion

1:Why was the nutritional value of litchi leaves not evaluated before silage? This is important for comparison before and after silage. And if table 1 expresses the nutritional value before silage, how did the lactic acid bacteria do?

Response: Thank reviwer for this valuable feedback. We analyzed the nutritional components(neutral detergent fibre、acid detergent fibre、crude protein) of Silage before and after silage, and found that there was no significant difference between them. Therefore, we did not display it in the same table. 20 g sample was mixed and fully shaken with 180 mL of sterile normal saline. The supernatant was gradiently diluted from 10-1 to 10-6. Serial dilutions of 1 mL were respectively inoculated in Man, Rogosa, Sharpe (MRS) agar a to culture LAB under the temperature of 30℃ for 2 days. Then we counted the number of lactic acid bacteria in culture medium

2:Put the meaning of all the abbreviations under all the tables.

Response: We were really sorry for our careless mistakes. Thank you for your reminder. We revised and put the meaning of all the abbreviations under all the tables.

3:How was it concluded that there are no significant differences between cultivars in crude protein content? Because the results of each category were placed in a separate table.

Response: Thank reviwer for this valuable feedback. We carefully checked the part of “Results and Discussion”, and found that we did not compare the differences in crude protein content between different varieties. But we reanalyzed the crude protein content between different varieties of raw materials, and they did not show significant differences.

4:Why wasn't a table set out to show the differences between the varieties in all the treatments and values?

Response: Thank reviwer for this valuable feedback. As reviewer suggested that showed the differences between the varieties in all the treatments and values. We believe that there is no comparability between the composition of the substances contained in litchi leaves and their fermentation effects due to different varieties. We preliminarily believe that fermenting litchi leaves as feed is more worthwhile to explore in order to understand the fermentation quality of each variety and the fermentation effect after adding lactic acid bacteria.

5:A statistical analysis showing the differences between the four varieties of litchi in each treatment should be carried out separately.

Response: As reviewer suggested that it is important to show the differences between the four varieties of litchi in each treatment.However, we want to provide some explanation. In recent years, there have been more and more varieties of litchi. From Table 1, we found that there is not much change in nutritional composition among different varieties. To fully recycle and utilization of discarded litchi leaves, We think that the focus of the experiment is whether there are significant differences in the fermentation quality and in vitro fermentation characteristics of litchi leaves with or without Lactiplantibacillus plantarum.

Discussion:

1:Normally the pH of silage should not be more than 4.7 while in the current study it was greater than that. Please provide a justification for this in the discussion of the results.

Response: Thank reviwer for this valuable feedback. We have revised it. Thank you! Line 258-259.

2:At the end of each paragraph in a discussion, a conclusion must be reached that is not necessarily consistent with the results of this study, so please pay attention to that and add it if it is not present.

Response: We are very sorry that we cannot understand your meaning very well

Conclusion:

1:This mean, how will farmers apply it? How will the lychee leaves be collected? When and how is the silage do’ne?

Response:Thank reviwer for this valuable feedback. We want to provide three directions for future application on litchi leaves silage. As fellows:

1: We should analyze the types and content of active ingredients in common varieties litchi leaves of market, and clarify the substances with obvious antibacterial effects in litchi leaves.

2: We want to feed the ruminant with litchi leaf silage, analyze the growth performance and palatability of ruminant, and further evaluate the suitability of litchi leaves as silage.

3: Producing of litchi leaves is mixed silage or extracted effective components as a feed additive to improve the utilization of litchi leaves.

About collection:

Due to our limited understanding of the harvesting machines in the market, we are unable to evaluate the collection method of litchi leaves.

As the production of silage is not limited by time and weather, we only need to consider the pruning time of litchi trees in March-April (or September-October) every year.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

The reviewed manuscript „ Exploring Lactobacillus plantarum on fermentation quality, gas emissions, and in vitro digestibility of different varieties of litchi leaves silage” contains the results of an interestingly planned experiment to investigate the feasibility of litchi leaves as silage material. Authors determined the fermentation quality of four varieties of litchi leaves ensiled with or without Lactobacillus plantarum. The in vitro dry matter digestibility and gas production was also determined. It was conformed that addition of Lactobacillus plantarum can improve the fermentation quality and in vitro digestion characteristics of litchi leaves silage.

Both the introduction to the research problem and the project assumptions, and the methodology were presented in a clear and essential manner, sufficient for the needs of the article submitted for review. I find this manuscript valuable and interesting and due to its cognitive value it should be published in Fermentation journal. However, I have few minor comments on it that should be included to improve its clarity.

Introduction. I propose to add information about the properties of Lactobacilus plantarum, for example, in line 78, as a strain of LAB popularly used as a bacterial additive for ensiling various types of raw materials intended to feeding.

L 143 Ensiling is an effective method to preserve plant nutrients. I propose to move this sentence to introduction section, for example to line 71.

In the M&M chapter, the Authors provided a methodology for evaluating the protein fractions (crude protein, true protein, and non-protein nitrogen), NDF, ADF and WSC contents, but I did not find such results for pickles. Can the Authors refer to this fact?

L 165 Please add: “differ significantly” in under this table and the following ones.

L 219. Sentence: As an aside, corn is the most widely planted silage material in the world, and its protein content typically is lower than 9% DM – is true but corn is not a protein feed but is grown as a crop to provide energy in animal rations.

L 249. „It might because that Tongzai had active substances with antibacterial effect”. Please provide appropriate reference.

Finally, could the Authors provide directions for further research on litchi leaves silage?

Please read the text carefully and correct the so-called typos, replace uppercase letters with lowercase letters in the middle of the sentences and standardize the font size.

Author Response

Dear reviewr:

1:Introduction. I propose to add information about the properties of Lactobacilus plantarum, for example, in line 78, as a strain of LAB popularly used as a bacterial additive for ensiling various types of raw materials intended to feeding.

Response: We are grateful for the suggestion. We have added the information about the properties of Lactobacilus plantarum in introduction section.Line 71-75. Thank you!

2:Line 143 Ensiling is an effective method to preserve plant nutrients. I propose to move this sentence to introduction section, for example to line 71.

Response: We are grateful for the suggestion. We have moved the sentence to introduction section. Line 64-67. Line114-121 Thank you!

3:In the M&M chapter, the Authors provided a methodology for evaluating the protein fractions (crude protein, true protein, and non-protein nitrogen), NDF, ADF and WSC contents, but I did not find such results for pickles. Can the Authors refer to this fact?

Response:Thank reviwer for this valuable feedback. We have added the specific description about a methodology for evaluating.line 116-121.Thank you!

4:Line165 Please add: “differ significantly” in under this table and the following ones.

Response:Thank reviwer for this valuable feedback. We have added “differ significantly” in under this table and the following ones.Thank you!

5:Line 219. Sentence:As an aside, corn is the most widely planted silage material in the world, and its protein content typically is lower than 9% DM– is true but corn is not a protein feed but is grown as a crop to provide energy in animal rations.

Response:Thank reviwer for this valuable feedback. We have added a sentence to introduce the high protein feed (alfalf). We just want to express that although the protein content of litchi leaves is not high, compared to general plant feed, its protein content also relatively good. Line239-241.Thank you!

6:Line 249. “It might because that Tongzai had active substances with antibacterial effect”. Please provide appropriate reference.

Response:Thank reviwer for this valable feedback. We feel sorry that we did not provide

appropriate reference about it. At present, there are not the relevent studies to analysis the active substances of “Tongzai” litchi leaves. However, this speculation seems reasonable. Because “Tongzai” litchi leaves showed well bacteriostastic activity. In the next study, we will detect the active ingredients of lychee leaves to verify our view.

7:Finally, could the Authors provide directions for further research on litchi leaves silage?

Response:Thank reviwer for this valuable feedback. We want to provide three directions for future application on litchi leaves silage. As fellows:

1: We should analyze the types and content of active ingredients in common varieties litchi leaves of market, and clarify the substances with obvious antibacterial effects in litchi leaves.

2: We want to feed the ruminant with litchi leaf silage, analyze the growth performance and palatability of ruminant, and further evaluate the suitability of litchi leaves as silage.

3: Producing of litchi leaves is mixed silage or extracted effective components as a feed additive to improve the utilization of litchi leaves.

8:Please read the text carefully and correct the so-called typos, replace uppercase letters with lowercase letters in the middle of the sentences and standardize the font size.

Response: We were really sorry for our careless mistakes. Thank you for your reminder. We checked and revised these errors.Line105、137、150、158、167、228、229、231、249、268.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments for author File: Comments.pdf

Author Response

Dear reviewr:

1:line 46-47: Please clarify if you are describing only leaves or leaves and stems. Because you mentioned pruning and I believe pruning include also stems

Response: We are grateful for the suggestion. To be more clearly, we have added more detailed interpretation regareding the using fresh material.Line 85-94.

2: line 83: After 30 ?

[1]Zhang Q, Zou X, Wu S, Wu N, Chen X, Zhou W. Effects of Pyroligneous Acid on Diversity and Dynamics of Antibiotic Resistance Genes in Alfalfa Silage. Microbiol Spectr. 2022 Aug 31;10(4):e0155422.

3:line 91:Define the size of small pieces.

Response: Thank reviwer for this valuable feedback. We have added the specific size of small pieces. Thank you! Line95

4:Line 95: Define room temperature.

Response: Thank reviwer for this valuable feedback. We have added the specific range of room temperature. Thank you! Line101.

5:Line 110 :Crushed? Do you mean grinding? Please provide the size.

Response: Thank reviwer for this valuable feedback. We have added the specific size of DM sample. Thank you! Line114-116.

6:Line 117: Describe the diet of the cpws and provide the ethical permission of using animals

Response: Thank reviwer for this valuable feedback. We have added a sentence to introduce the diet of the cows and provide the ethical permission of using animals. Line123-126 and Line341-343. Thank you!

7:Line 123: Werecompleted change to were completed

Response: We were really sorry for our careless mistakes. Thank you for your reminder. We have changed “werecompleted” to “ were completed”. Line 137. Thank you!

8:Table: All tables need to be improved. To be better presented. Some of the numbers are overlapping.

Response: Thank reviwer for this valuable feedback. We had adjusted all the tables to be better presented. Thank you very much for your reminder.

9:Line 216-218: Litchi leaves are poor in CP compared to alfalfa. This statement is relevant to the discussion. Your product might be defined as haylage and silage according to the pH values and DM.

Response: Thank reviwer for this valuable feedback. We have added a sentence to introduce the relevant statement to discussion. Line 239-241. Thank you!

10:Line 225-258: Ruminants utilize N-NH3 in the rumen. Please omit this statement.

Response:Thank reviwer for this valuable feedback. We have added a sentence to introduce it. Line 279-286. Thank you!

11:Line 271: “it might prefer rapidly degradable DM fractions” not clear statement. Please re-write to be better understood.

Response:Thank reviwer for this valuable feedback. We tried to rewrite this section. Line 298-300. Thank you!

12:Line 273-275: “On the one hand, Gas production means that nutrients in silage are not well absorbed by ruminants, on the other hand, The main components of gas production were H2, CO2, and CH4”. Not clear

Response:Thank reviwer for this valuable feedback. We tried to rewrite this section. Line 302-305.Thank you!

13:Line 273-280: This is not related to in vivo. You did comparative in vitro gas production. Gas production is parallel to your IVDMD parameters. Please either omit or try to rephrase to fit your application.

Response:Thank reviwer for this valuable feedback. We tried to rewrite this section. Line 302-305. Thank you!

Line 281-286: Omit this

Response:Thank reviwer for this valuable feedback. We have deleted the relevant introduction.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

However, please pay attention and make the following adjustments:

- In results: Point (1) It is good that the components of the nutritional value of lychee leaves were estimated before and after the silage, but although there were no significant differences, it should be mentioned in the body of the text.

- In discussion: The point (2) The comment is intended: in the first report is that at the end of each paragraph in the discussion a conclusion must be reached.

Thanks

Minor editing of English language required

Author Response

Thank you again for your professional suggestions. We have revised our manuscript again, according to your advices. We uploaded the file of the revised manuscript. Revisions in the text are shown using yellow highlight.

Reviewer 3 Report

Authors responded to comments and improved their MS

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Exploring Lactobacillus plantarum on Fermentation Quality, Gas Emissions, and In Vitro Digestibility of Different Varieties of Litchi Leaves Silage

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