Effects of Lactic Acid Bacteria Additives on Fatty Acids, Amino Acids and Antioxidant Capacity of Leymus chinensis Silage during Aerobic Exposure

Round 1

Reviewer 1 Report

First of all, the authors described the effect of Lactobacillus plantarum and Lactobacillus buchneri on the fatty acids and amino acids of Leymus chinensis silage, which is very interesting to readers and silage domain. However, many data (such as aerobic stability) of this manuscript have been published in Frontiers in Microbiology (Effects of Lactic Acid Bacteria Additives on the Quality, Volatile Chemicals and Microbial Community of Leymus chinensis Silage During Aerobic Exposure, 2022, 13, 938153), which is forbidden.

Second, the common chemical composition and fermentation profile of silage were not presented, which may be due to the fact that these data have been published in another journal. Besides, the authors did not use the line number in the text, which is not helpfule for review and presenting comments.

Third, this manuscript was poorly written and organized (discussion section). In addition, many details are wrongly employed.

Author Response

First of all, the authors described the effect of Lactobacillus plantarum and Lactobacillus buchneri on the fatty acids and amino acids of Leymus chinensis silage, which is very interesting to readers and silage domain. However, many data (such as aerobic stability) of this manuscript have been published in Frontiers in Microbiology (Effects of Lactic Acid Bacteria Additives on the Quality, Volatile Chemicals and Microbial Community of Leymus chinensis Silage During Aerobic Exposure, 2022, 13, 938153), which is forbidden.

Dear reviewers, your suggestions are very helpful.The data on temperature dynamics during aerobic exposure are used in this paper to investigate its association with exothermic quality deterioration.In this study, we are mainly concerned with fatty acids, amino acids and antioxidant capacity in Leymus chinensis silage. We believe that the current data may not be optimal, but are sufficient to draw completely new conclusions and fill in gaps in the relevant areas, and the results of this study may provide some reference for the production and use of Leymus chinensis silage. Of course, the experimental content design is also a limitation of this study, and we will pay attention to this detail in our future work.

Second, the common chemical composition and fermentation profile of silage were not presented, which may be due to the fact that these data have been published in another journal. Besides, the authors did not use the line number in the text, which is not helpfule for review and presenting comments.

Thank you for the suggestion.We have checked and corrected.

Third, this manuscript was poorly written and organized (discussion section). In addition, many details are wrongly employed.

Thank you for the suggestion.We have rewritten and reorganized the material methodology, conclusions and discussion

Author Response File: Author Response.pdf

Reviewer 2 Report

The present study is very interesting. The authors have done great work and investigated the effect of LAB additives on the fatty acid, amino acids, antioxidant of Lymus silage. The work is well designed and presented, and discussed.

Only minor corrections should be done

1-      The format of reference inside the MS should be numerical according to the Journal Guideline

2-      Lactobacillus at Page 2 and 17 should be italic

3-      P2 Rephrase the sentence (The purpose of this study  was to investigate the effect of additives to lactic acid ………)please clarify the additives

4-      All reference section should be carefully checked for the correct format of scientific names

Author Response

• The format of reference inside the MS should be numerical according to the Journal Guideline

Thank you for the suggestion.We have checked and corrected.

• Lactobacillus at Page 2 and 17 should be italic

Thank you for the suggestion.We have checked and corrected.

• P2 Rephrase the sentence (The purpose of this study  was to investigate the effect of additives to lactic acid ………)please clarify the additives

Thank you for the suggestion.We have checked and corrected:The purpose of this study was to investigate the effect of lactic acid bacteria additives on fatty acids, amino acids and antioxidant capacity of the Leymus chinensis silage during the course of aerobic exposure, and analysed changes and correlations in order to provide evidence to ensure the quality of the Leymus chinensis silage during aerobic exposure.

• All reference section should be carefully checked for the correct format of scientific names

Thank you for the suggestion.We have checked and corrected.

Author Response File: Author Response.pdf

Reviewer 3 Report

Effects of lactic acid bacteria additives on FA, AA and antioxidant capacity ....

Introducción

The first sentence should be divided in two.

Suggest including a description of the forage used in this study (example): Leymus chinensis is a perrenial bunchgrass widely distributed in the Northeast plain and the east of the Inner Mongolian Plateau of China. Its rapid growth, high palatability and herbage production is ideal for grazing and forage production (Wang et al., 2015).

Abstract

Fermentation stimulants

Reword the first sentence

...... effectively improve livestock production

...... economic losses in livestock production

...... reduce nutrient loss and toxicant (reword the sentence - something missing).

Materials and methods

Suggest include the proximal analyses of the silage (moisture, CP, NDF, EE, ash, etc.).

Don´t start with 300 g

2.2 Aerobic stability

Three probes were used to monitor changes in ambient .......

2.3 Antioxidant capacity

..... smashed in a mortar, and a 0.5 g sample was placed in a stoppered conical flask, added .......

Second sentence is repeated

Delete "for use"

Two ml (delete accurately) into a 10 ml test tube

2.5. Amino acid analyses

..... 10 ml of 6 mol/L HCL were added, nitrogen was applied for 30 s, and placed ........

.......hydrolysis, the samples were cooled down, mixed well, filtered, made up to 25 ml volumetric flask with ultrapure water, and 1 ml of the filtrate was introduced into a beaker, heated ...... sample was added to the diluted solution to obtain the appropriate amino acid concentration.

Results

Roundup percentages to 2 or 3 decimal points, depending on the size of the whole number

3.1. Forage quimical analyses

Round up means and SEM to only 3 decimal places 

Table 1 - Analyses  content SEM

Asterisks are not described in the table footnote 

Additive treatments (plural)

3.2. Changes in silage ...............

line 11 - LB group (temperature ?)

No statistics are shown for temperature. Suggest simplifying the text based on the statistical significance of the data. Text is too long. 

Table 2 - Roundup to 3 decimal points. For two integer values, one decimal point is enough, and for one integer value, two decimal points.

Suggest using ug/g DM instead of %

Conclusions - Too long and written more as a summary

Author Response

Effects of lactic acid bacteria additives on FA, AA and antioxidant capacity ....

Introducción

The first sentence should be divided in two.

Thank you for the suggestion.We have checked and corrected.

Suggest including a description of the forage used in this study (example): Leymus chinensis is a perrenial bunchgrass widely distributed in the Northeast plain and the east of the Inner Mongolian Plateau of China. Its rapid growth, high palatability and herbage production is ideal for grazing and forage production (Wang et al., 2015).

Thank you for the suggestion.We have checked and corrected: Leymus chinensis is widely distributed in the northeastern plains of China and the eastern part of the Inner Mongolia plateau, and in recent years the use of Leymus chinensis silage has gradually increased due to the effect of rainfall on hay drying[1]

Abstract

Fermentation stimulants

Reword the first sentence

...... effectively improve livestock production

...... economic losses in livestock production

...... reduce nutrient loss and toxicant (reword the sentence - something missing).

Thank you for the suggestion.We have checked and corrected:During aerobic exposure, the fatty acid and amino acid composition of silage may alter the quality and palatability of animal feed, resulting in economic losses in livestock production.

Materials and methods

Suggest include the proximal analyses of the silage (moisture, CP, NDF, EE, ash, etc.).

Dear Reviewer, your suggestion is very helpful and we know that adding this section will enhance the overall effect of this article. However, in previous studies we have made conclusions about the changes and relationships in quality, odor and microbiology during aerobic exposure of Leymus chinensis silage. In this study is a further investigation of fatty acids, amino acids and antioxidant capacity, we believe that the current data may not be optimal, but are sufficient to draw completely new conclusions and fill in gaps in the relevant areas, and the results of this study may provide some reference for the production and use of Leymus chinensis silage. Of course, the experimental content design is also a limitation of this study, and we will pay attention to this detail in our future work.

Don´t start with 300 g

Thank you for the suggestion.We have checked and corrected: Samples of 300 g were weighed and mixed into silage bags (25×36cm) and then vacuum sealed, and three replicates were set up for each treatment that was stored at room temperature and shielded from light (20-30℃) for 60 days.

2.2 Aerobic stability

Three probes were used to monitor changes in ambient .......

Thank you for the suggestion.We have checked and corrected: three probes were used to monitor changes in temperature in the ambient.

2.3 Antioxidant capacity

..... smashed in a mortar, and a 0.5 g sample was placed in a stoppered conical flask, added .......

Thank you for the suggestion.We have checked and corrected: Samples of silage exposed to oxygen for 0, 4, and 8 days were smashed in a mortar, and a 0.5 g sample was placed in a stoppered conical flask, added 5 mL extraction solvent (80% ethanol solution),

Second sentence is repeated

Thank you for the suggestion.We have checked and corrected.

Delete "for use"

Thank you for the suggestion.We have checked and corrected.

Two ml (delete accurately) into a 10 ml test tube

Thank you for the suggestion.We have checked and corrected.

2.5. Amino acid analyses

..... 10 ml of 6 mol/L HCL were added, nitrogen was applied for 30 s, and placed ........

Thank you for the suggestion.We have checked and corrected.

.......hydrolysis, the samples were cooled down, mixed well, filtered, made up to 25 ml volumetric flask with ultrapure water, and 1 ml of the filtrate was introduced into a beaker, heated ...... sample was added to the diluted solution to obtain the appropriate amino acid concentration.

Thank you for the suggestion.We have checked and corrected.

Results

Roundup percentages to 2 or 3 decimal points, depending on the size of the whole number.

Dear reviewer, your suggestion is very helpful, because most of the material content is low at 0.0.. Therefore, we choose to use four decimal places throughout the whole paper.

3.1. Forage quimical analyses

Round up means and SEM to only 3 decimal places 

Dear reviewer, your suggestion is very helpful, because most of the material content is low at 0.0.. Therefore, we choose to use four decimal places throughout the whole paper.

Table 1 - Analyses  content SEM

Thank you for the suggestion.We have checked and corrected.

Asterisks are not described in the table footnote 

Thank you for the suggestion.We have checked and corrected.

Additive treatments (plural)

Thank you for the suggestion.We have checked and corrected.

3.2. Changes in silage ...............

line 11 - LB group (temperature ?)

Thank you for the suggestion.We have checked and corrected.

No statistics are shown for temperature. Suggest simplifying the text based on the statistical significance of the data. Text is too long. 

Thank you for the suggestion.We have checked and corrected.

Table 2 - Roundup to 3 decimal points. For two integer values, one decimal point is enough, and for one integer value, two decimal points.

Dear reviewer, your suggestion is very helpful, because most of the material content is low at 0.0.. Therefore, we choose to use four decimal places throughout the whole paper.

Suggest using ug/g DM instead of %

Dear reviewer, your suggestion is very helpful, because of the measurement method and uniform unit, we choose to use %.

Conclusions - Too long and written more as a summary

Thank you for the suggestion.We have checked and corrected.

Reviewer 4 Report

The manuscript (fermentation-2241179) evaluated the effects of lactic acid bacteria inoculation on the fatty acids, amino acids and antioxidant capacity of Leymus chinensis silage during aerobic exposure.

Leymus chinensis is one of the most important feed resources for meat and milk production for consumers in the Mongolian Plateau. Silage has been proven to produce organic acids by microbial degradation of water-soluble carbohydrates to create anaerobiosis and environmental acidity, and inhibit the proliferation of decaying microorganisms. Nevertheless, the nutrition compositions were broken down during aerobic exposure by the increasing undesirable microorganisms. The aim of this study was to explore the effects of lactic acid bacteria additives on fatty acids, amino acids and antioxidant capacity of Leymus chinensis silage during aerobic exposure. 

The research topic is meaningful for production and attractive for readers, whereas the manuscript cannot accept at this form. The special comments were listed.

Special comments

1. Consecutive line numbers are missing, please provided in the revised manuscript

2. The language of the manuscript should be further improved

3. Line 18, the names of Lactobacillus plantarum and Lactobacillus brucei should be instead by the new taxonomy

4. Line 19, what is the number of the additions of the lactic acid bacteria

5. Line 22-24, the result is confused and should be rewritten

6. Line 24, what is the changes of the microbial community during aerobic exposure.

7. Line 43, the authors name should be check

8. The introduction should be well organized and added some reference about the effects of lactic acid bacteria on the fatty acid profiled and amino acid compositions during aerobic exposure

9. The details of the guillotine should be added

10. Line 88-94, the design of the experiment should be more detail

11. Materials and methods the information about the microbial community is missed

12. Please check the manuscript, the abbreviation should be given the full name at the first time

13. Discussion should be strengthen

14. Line 318-322, the sentence should be written

15. The information about the microbiota is missed and the results and discussion are unreasonable, the materials and methods, results and discussion should be rewritten and well-organized

16. The characteristics of the raw materials should be added

17. Figure 1 at the initial stage, there was significant difference on temperature in different treatments, why?

18. Where is the data to display the correlation analysis? Please supply.

Author Response

1. Consecutive line numbers are missing, please provided in the revised manuscript

Thank you for the suggestion.We have checked and corrected.

2. The language of the manuscript should be further improved

Thank you for the suggestion.We have improved the language of the manuscript.

3. Line 18, the names of Lactobacillus plantarumand Lactobacillus brucei should be instead by the new taxonomy

Thank you for the suggestion.We have checked and corrected.

4. Line 19, what is the number of the additions of the lactic acid bacteria

Thank you for the suggestion.We have replenished the dosage：The lactic acid bacteria were added at 1×106 CFU/g

5. Line 22-24, the result is confused and should be rewritten

Thank you for the suggestion.We have rewritten the results：The LB group had higher total fatty acid and polyunsaturated fatty acid content, less decrease in amino acid content and antioxidant capacity, and better aerobic stability, while the LP group had higher monounsaturated fatty acid content but more decrease in all indicators after exposure.

6. Line 24, what is the changes of the microbial community during aerobic exposure.

Dear Reviewer, your suggestion is very helpful and we know that adding this section will enhance the overall effect of this article. However, in previous studies we have made conclusions about the changes and relationships in quality, odor and microbiology during aerobic exposure of Leymus chinensis silage. In this study is a further investigation of fatty acids, amino acids and antioxidant capacity, we believe that the current data may not be optimal, but are sufficient to draw completely new conclusions and fill in gaps in the relevant areas, and the results of this study may provide some reference for the production and use of Leymus chinensis silage. Of course, the experimental content design is also a limitation of this study, and we will pay attention to this detail in our future work.

7. Line 43, the authors name should be check

Thank you for the suggestion.We have checked and corrected.

8. 8. The introduction should be well organized and added some reference about the effects of lactic acid bacteria on the fatty acid profiled and amino acid compositions during aerobic exposure

Thank you for the suggestion.We have rewritten and added to the introduction：Many studies have shown that the use of additives can inhibit the growth of aerobic bacteria and enhance aerobic stability, and feeds with a high capacity for antioxidants can effectively enhance the yield of livestock. The addition of Lactiplantibacillus plantarum to silage produces volatile fatty acids and essential amino acids, and Lentilactobacillus buchneri produces specific amino acids.

9. The details of the guillotine should be added

Thank you for the suggestion.We have added guillotine details:Shandong Jufeng Hardware Cutting Tools Factory.

10. Line 88-94, the design of the experiment should be more detail

Thank you for the suggestion.We have refined the experimental design:The variety of Leymus chinensis used in this experiment was Jisheng No. 1, har-vested in the Science and Technology Park of Inner Mongolia University for Nationali-ties, Tongliao City, Inner Mongolia Autonomous Region (E43°59', N122°11'). The Leymus chinensis was harvested in the tassel stage and laid flat for 3 hours, then chopped to 2 cm with a guillotine (Shandong Jufeng Hardware Cutting Tools Factory) and mixed evenly, and fresh samples were stored in a -80°C refrigerator for determination of raw material characteristics. The treatment settings for Leymus chinensis silage were as follows: uninoculated control group (CK), Lactiplantibacillus plantarum group (LP), Lentilactobacillus buchneri group (LB), and mixed Lactiplantibacillus plantarum and Lentilactobacillus buchneri group (PB, 1:1). The amount of Lactobacillus added was 1×106 CFU/g (all provided by Shandong Zhongke Jiayi Biological Engineering Co., Ltd.), and the same amount of distilled water was added to the control group. 300 g of the mixed samples were weighed into silage bags (25×36cm) and then vacuum sealed, and three replicates were set up for each treatment that were stored at room temperature and shielded from light (20-30℃) for 60 days.

11. Materials and methods the information about the microbial community is missed

Dear Reviewer, your suggestion is very helpful and we know that adding this section will enhance the overall effect of this article. However, in previous studies we have made conclusions about the changes and relationships in quality, odor and microbiology during aerobic exposure of Leymus chinensis silage. In this study is a further investigation of fatty acids, amino acids and antioxidant capacity, we believe that the current data may not be optimal, but are sufficient to draw completely new conclusions and fill in gaps in the relevant areas, and the results of this study may provide some reference for the production and use of Leymus chinensis silage. Of course, the experimental content design is also a limitation of this study, and we will pay attention to this detail in our future work.

12. Please check the manuscript, the abbreviation should be given the full name at the first time

Thank you for the suggestion.We have checked and corrected.

13. Discussion should be strengthen

Thank you for the suggestion.We have enhanced the discussion section: In the case of the LP group, the greater degree of decrease in total phenolic content and inhibition following aerobic exposure was due to the fact that phenols are more easily oxidized at higher pH when exposed to air[28] and the lactic acid produced by Lacti-plantibacillus plantarum became a substrate for growth of the aerobic bacteria in the aerobic phase, accelerating their oxidative nutrient decomposition, which is in agree-ment with the conclusions of Mugabe et al.[29]. This is due to the fact that monounsaturated fatty acids themselves are automatically oxidized, and when heat reaches a certain limit, oxidation is accelerated, resulting in the breaking of unsaturated double bonds and the generation of harmful substances such as aldehydes and ketones[31]. As bacterial growth and metabolism produce large amounts of proteases, they hydrolyze the proteins in the feed into amino acids, which are denatured by oxidative reactions under aerobic exposure, producing urea, water, carbon dioxide and heat[37].

14. Line 318-322, the sentence should be written

Thank you for the suggestion.We have rewritten the sentence: The amino acid content of Leymus chinensis increased after silage fermentation, which was due to the fact that some proteins were broken down into amino acids by the action of plant cell enzymes, while some microorganisms required amino acids as nitrogen sources for their synthesis of bacteriophage proteins, leading to changes in amino acid types and proportions that were in agreement with the findings of Guo et al.

15. The information about the microbiota is missed and the results and discussion are unreasonable, the materials and methods, results and discussion should be rewritten and well-organized

Thank you for the suggestion.We have rewritten and reorganized the material methodology, conclusions and discussion

16. The characteristics of the raw materials should be added

Dear Reviewer, your suggestion is very helpful and we know that adding this section will enhance the overall effect of this article. However, in previous studies we have made conclusions about the changes and relationships in quality, odor and microbiology during aerobic exposure of Leymus chinensis silage. In this study is a further investigation of fatty acids, amino acids and antioxidant capacity, we believe that the current data may not be optimal, but are sufficient to draw completely new conclusions and fill in gaps in the relevant areas, and the results of this study may provide some reference for the production and use of Leymus chinensis silage. Of course, the experimental content design is also a limitation of this study, and we will pay attention to this detail in our future work.

17. Figure 1 at the initial stage, there was significant difference on temperature in different treatments, why?

Thank you for the suggestion. We believe that the lower temperature in the CK group at the initial stage was caused by the slower fermentation activity in the silage bags, which released less heat.

18. Where is the data to display the correlation analysis? Please supply.

Thank you for the suggestion. We provide data on the correlation coefficients in Schedule 1.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript has to some extent improved, and still needs to be revised extensively according to the comments or suggestions below.

Title should be "Effects of Lactic Acid Bacteria Additives on Fatty Acids, Amino Acids and Antioxidant Capacity of Leymus Chinensis Silage During Aerobic Exposure"

Abstract

L16-17, "During aerobic exposure of silage, the fatty acid and amino acid composition may alter the quality and palatability, resulting in......"

L19-20 and 112-113, a mixture of LP and LB (PB)

L21 and 113, please insert one blank between "1" and "10⁶"

L31, improve the antioxidant capacity and fatty acid and amino acid 

Introduction

L54, and Leymus chinensis silage

L66, have reported that 

L75, the volume of spoilage

L85, during aerobic exposure to air of silage

L86-88, "The aerobic spoilage severely affect silage quality and result in severe economic losses to the development of animal husbandry"

L88-93, please improve this sentence ! It is too redundant and complicated

Materials and methods

L103, , grown in the 

L107, h, please keep in accordance in the text

L112-113, a mixture of LP and LB (PB)

L116, 36 cm

L135, this sentence "Then centrifuged at 3500 r/min for 10 min" was incomplete, namely, without the subject. 

L136, Two milliliters of extract

L137-141, 142-143 and 146-148, this sentence was incomplete and redundant!

L155, fatty acids in Leymus chinensis silage

L157-160, 164-167 and 172-175, please improve this sentence

L171, "Na" ?

L203, 36 h

L209, ℃ rather than "degrees Celsius"

Discussion

This section should consist of "4.1 Silage raw material characteristics", "4.2 Temperature difference between silage and room temperature during aerobic exposure",  "4.3. Changes in antioxidant capacity of silage during aerobic exposure" and "4.4. Changes in fatty acids during aerobic exposure", respectively, according to the "Result" section.

Conclusion

L524, showed that

References

Here one example is presented for reference listing

Zhang, Y.; Chen, T.; Nan, Z.; Christensen, M.J. Cattle grazing alters the interaction of seed-borne fungi and two foliar pathogens of Leymus chinensis in a meadow steppe. Eur. J. Plant Pathol. 2013, 155, 207-218.

Author Response

The manuscript has to some extent improved, and still needs to be revised extensively according to the comments or suggestions below.

Title should be "Effects of Lactic Acid Bacteria Additives on Fatty Acids, Amino Acids and Antioxidant Capacity of Leymus Chinensis Silage During Aerobic Exposure"

 Thank you for the suggestion.We have checked and corrected.

Abstract

L16-17, "During aerobic exposure of silage, the fatty acid and amino acid composition may alter the quality and palatability, resulting in......"

 Thank you for the suggestion.We have checked and corrected.

L19-20 and 112-113, a mixture of LP and LB (PB)

Thank you for the suggestion.We have checked and corrected.

L21 and 113, please insert one blank between "1" and "106"

Thank you for the suggestion.We have checked and corrected.

L31, improve the antioxidant capacity and fatty acid and amino acid 

 Thank you for the suggestion.We have checked and corrected.

Introduction

L54, and Leymus chinensis silage

Thank you for the suggestion.We have checked and corrected.

L66, have reported that 

Thank you for the suggestion.We have checked and corrected.

L75, the volume of spoilage

Thank you for the suggestion.We have checked and corrected.

L85, during aerobic exposure to air of silage

Thank you for the suggestion.We have checked and corrected.

L86-88, "The aerobic spoilage severely affect silage quality and result in severe economic losses to the development of animal husbandry"

Thank you for the suggestion.We have checked and corrected.

L88-93, please improve this sentence ! It is too redundant and complicated

Thank you for the suggestion.We have checked and corrected：Improving the aerobic stability and antioxidant capacity of silage can effectively reduce nutrient loss and toxic production during aerobic exposure of silage, ensuring the safety of livestock feed. Meanwhile, reducing the oxidative decomposition of fatty acids and amino acids can avoid the production of poor odor and taste, and improve the intake of livestock and the palatability of feed.

Materials and methods

L103, , grown in the 

Thank you for the suggestion.We have checked and corrected.

L107, h, please keep in accordance in the text

Thank you for the suggestion.We have checked and corrected.

L112-113, a mixture of LP and LB (PB)

Thank you for the suggestion.We have checked and corrected.

L116, 36 cm

Thank you for the suggestion.We have checked and corrected.

L135, this sentence "Then centrifuged at 3500 r/min for 10 min" was incomplete, namely, without the subject. 

Thank you for the suggestion.We have checked and corrected：The extract was centrifuged at 3500 r/min for 10 min.

L136, Two milliliters of extract

Thank you for the suggestion.We have checked and corrected.

L137-141, 142-143 and 146-148, this sentence was incomplete and redundant!

Thank you for the suggestion.We have checked and corrected: Two milliliters of extract were pipetted into a 10 ml test tube with a stopper and 2 mL of DPPH (2,2-Diphenyl-1-picrylhydrazyl) solution was added and mixed well. The absorbance Ai was measured by spectrophotometer at 517 nm after 30 min of standing at room temperature (80% ethanol solution was adjusted to zero), and the absorbance Ac of 2 mL of DPPH solution mixed with 2 mL of 80% ethanol solution and the absorbance Aj of 2 mL of extract mixed with 2 mL of 80% ethanol solution were measured simultaneously. The inhibition rate was calculated by the following equation after taking the average of three parallel measurements, the greater the inhibition rate, the stronger the antioxidant activity of the sample.

The sample was weighed 20 mg, dissolved by adding 95% ethanol and diluted to 2 mL to obtain 1 mg/mL sample solution. Then collect 1 mL of 2 mol/L Folin-Ciocalteu reagent, add 7 mL of distilled water and stir thoroughly to obtain 0.25 mol/L Folin-Ciocalteu reagent. Dissolve 3.0 g Na₂CO₃: 10H₂O in 20 mL of water and mix to obtain a solution of 15% Na₂CO₃. Take 0.5 mL of sample solution and 0.5 mL of 0.25 mol/L Folin-Ciocalteu reagent and mix, let it stand for 3 min, add 1 mL of 15% Na₂CO₃ solution and let it stand for 30 min, centrifuge at 3500 r/min for 3 min, and measure the absorbance A value at 760 nm. The total phenol concentration C was obtained by substituting into the standard curve

L155, fatty acids in Leymus chinensis silage

Thank you for the suggestion.We have checked and corrected.

L157-160, 164-167 and 172-175, please improve this sentence

Thank you for the suggestion.We have checked and corrected: Weigh 50 mg of the sample, add 4 mL of hexane and 75 mg of anhydrous sodium sulfate and stir well, then add 0.20 mL of potassium methoxide and 2 mL of hydrochloric acid solution with constant stirring and heating for 20 min.

After dried and ground with 100 mesh sieve, 0.5 mg of dry sample was weighed into 20 mL anaerobic test tube, then 10 mL of 6 mol/L HCL was added and nitrogen was applied for 30 s, and then placed in 110°C furnace for 24 h hydrolysis.

The solution to be measured was shaken and mixed and then filtered using a disposable syringe with a 0.22 μm filter, and then injected into the injection vial for measurement by the machine.

L171, "Na" ?

Thank you for the suggestion.We have checked and corrected.

L203, 36 h

Thank you for the suggestion.We have checked and corrected.

L209, ℃ rather than "degrees Celsius"

Thank you for the suggestion.We have checked and corrected.

Discussion

This section should consist of "4.1 Silage raw material characteristics", "4.2 Temperature difference between silage and room temperature during aerobic exposure",  "4.3. Changes in antioxidant capacity of silage during aerobic exposure" and "4.4. Changes in fatty acids during aerobic exposure", respectively, according to the "Result" section.

Thank you for the suggestion.We have checked and corrected.

Conclusion

L524, showed that

Thank you for the suggestion.We have checked and corrected.

References

Here one example is presented for reference listing

Zhang, Y.; Chen, T.; Nan, Z.; Christensen, M.J. Cattle grazing alters the interaction of seed-borne fungi and two foliar pathogens of Leymus chinensis in a meadow steppe. Eur. J. Plant Pathol. 2013, 155, 207-218.

Thank you for the suggestion.We have checked and corrected.

Round 3

Reviewer 1 Report

The authors still did not organize the Discussion section following the previous comment, namely lising the respective sub-title according to the Results section.