Effects of Different Cutting Stages and Additives on the Fermentation Quality and Microbial Community of Sudangrass (Sorghum sudanense Stapf.) Silages
, Rui Dong, Yulong Zheng * and Chao Chen *Round 1
Reviewer 1 Report
In this manuscript, the authors sought to investigate the effects of various cutting phases and additives on sudangrass in order to identify the ideal cutting stage and silage additives for sudangrass in order to improve silage quality and fiber degradation and thus boost livestock feed utilization. Overall, the manuscript presents valuable insights. However, there are several sections where the meaning is not clear. To enhance the clarity and readability of the text, I recommend revising the English language throughout the manuscript. This will ensure that the intended message is effectively communicated to the readers. Also, there are several concerns that need to be addressed to be fit for publication as follows:
1- Line 24: The conclusion section should be rewritten, as in the current form, it is a concise sentence. The conclusion should answer the aim of the study.
1- Line 50-52: The hypothesis of the study should be stated at the end of the Introduction section.
2- Line 69: add to the references list.
3- Lines 76-80: The objective of the study is not clear please rewrite.
4- Line 86: “The cutting heights of the sudangrass samples were 1.8 m and 2.0 m” The authors had to justify on what basis they selected these cutting heights.
5- Lines 89-90: it is recommended to include more details about the production process of ferulic acid esterase from Lactobacillus plantarum.
6- Throughout the manuscript, the writing style should be formal from the third-person perspective. Do not use “we” (e.g. in lines 50, 73, 101, … etc) or “our” (e.g. in lines 72, 77, 90… etc ).
7- Lines 123-124: “using the method described above” Which method were you referring to? A similar issue is found in many parts of the M&Ms section (e.g., lines 126, 127, 130, ....etc). Please elaborate.
8- Line 124: If a reference is made to AOAC, the specific procedure identification number must be mentioned.
9- Line 125-126: “The neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) contents were analyzed by the method reported above [34]”. Specify If you add α amylase or not, and was their content expressed as exclusive or inclusive of residual ash?, please refer in the M&Ms.
10- Line 157: “…lignocellulose (NDF and ADF)…” the lignocellulose is content not NDF + ADF. Please revise the intended meaning.
11- Throughout the manuscript, Latin names should be written in italic format (e.g. lines 420, 474…. etc).
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Author Response
The English language: Thank you very much for your reminding. We have revised the language throughout the manuscript once again by the highly qualified native English speaking editors at AJE company.
Point 1: Line 24: The conclusion section should be rewritten, as in the current form, it is a concise sentence. The conclusion should answer the aim of the study.
Response 1: Thank you very much for your suggestion. We have rewritten the conclusion section.
“The results suggested that FAE-producing L. plantarum, cellulase and xylanase had synergistic effects on sudangrass silages, especially at the S2 stage, which can serve as an efficient method for ensiling.”
Point 2: Line 50-52: The hypothesis of the study should be stated at the end of the Introduction section.
Response 2: Thank you very much for your suggestion. We have stated the hypothesis at the end of the introduction section.
“In summary, whether adding FAE-producing LAB, cellulase and xylanase to sudangrass silage at different cutting stages should have different effects on the fermentation and microbial community of silages.’
Point 3: Line 69: add to the references list.
Response 3: Thank you very much for your suggestion. We have added it to the references list.
“Mkabayi et al. (2020) found that xylanase interacts with FAE and could achieve a better degradation rate” revised to “A previous study also found that xylanase interacts with FAE and could achieve a better degradation rate[26]”
Point 4: Lines 76-80: The objective of the study is not clear please rewrite.
Response 4: Thank you very much for your suggestion. We have rewritten it.
“Therefore, investigating the fermentation characteristics, fiber degradation and bacterial community of the two growth stages of sudangrass silages by adding the compound additive would lay a foundation for research on sudangrass silage production.”
Point 5: Line 86: “The cutting heights of the sudangrass samples were 1.8 m and 2.0 m” The authors had to justify on what basis they selected these cutting heights.
Response 5: Thank you very much for your suggestion. In a previous study, sudangrass were randomly collected at the vegetative stage for silage making, and the sudangrass height were 1.7 to 2.0 m (Wang et al., 2022). Therefore, we intend to investigate the effect of different cutting height at the vegetative stage on sudangrass silage. And the average height were 1.8 m and 2.0 m of two fields at the harvest day. Therefore, the cutting heights of the sudangrass samples were 1.8 m and 2.0 m.
“The cutting heights of the sudangrass samples were 1.8 m and 2.0 m according to a previous study [5].”
Point 6: Lines 89-90: it is recommended to include more details about the production process of ferulic acid esterase from Lactobacillus plantarum.
Response 6: Thank you very much for your suggestion. We have added the details about the production process of ferulic acid esterase from Lactobacillus plantarum.
“The added FAE-producing L. plantarum was screened from rumen fluid previously, which was screened from a total of 110 LAB strains using Man Rogosa Sharpe (MRS) agar. Ethyl ferulate (1% w/v in dimethyl formamide) was added to the agar without glucose (pH 6.5) and incubated at 37 ℃ for 48 h. The LAB producing FAE were confirmed by visualization of a ring of clearance around the colonies on the plates.’
Point 7: Throughout the manuscript, the writing style should be formal from the third-person perspective. Do not use “we” (e.g. in lines 50, 73, 101, … etc) or “our” (e.g. in lines 72, 77, 90… etc ).
Response 7: Thank you very much for your suggestion. We have revised these throughout the manuscript.
“we”
We have deleted “Therefore, we hypothesized that different cutting stages would have different effects on sudangrass silage fermentation and the microbial community, clarifying it can lay a theoretical foundation for forage production.”
We have deleted “Hence, we explored the effects of FAE-producing LAB, cellulase and xylanase on the quality and lignocellulose degradation of sudangrass silages.”
“Then, we sprayed the solution evenly for sample treatment” revised to “Then, the solution was sprayed evenly for sample treatment”
“which we also detected in another study” revised to “which were also detected in another study”
“our”
We have deleted “The primary aim of our study was to compare”
“The added FAE-producing L. plantarum was screened previously by our research team” revised to “The added FAE-producing L. plantarum was screened from rumen fluid previously”
“This result was consistent with the results of our previous studies “revised to “This result was consistent with the results of previous studies “
“In our study, the D × A × S interaction affected” revised to “In this study, the D × A × S interaction affected”
“which was consistent with the results of our previous study “ revised to “which was consistent with the results of a previous study “
“Based on the results of our research” revised to “Based on the results of this research”
Point 8: Lines 123-124: “using the method described above” Which method were you referring to? A similar issue is found in many parts of the M&Ms section (e.g., lines 126, 127, 130, ....etc). Please elaborate.
Response 8: Thank you very much for your suggestion. We have elaborated it.
“The crude protein (CP) content was determined by a Kjeldahl nitrogen analyzer (Kjeltec 8400, FOSS, Sweden) using the Kjeldahl method [32].”
“The neutral detergent fiber (NDF, assayed without alpha-amylase), acid detergent fiber (ADF) and acid detergent lignin (ADL) contents were analyzed by the method reported by Van Soest et al [34]”
“Acid-insoluble ash was determined by incineration for 3 h at 550 ℃ according to the method described by Bergero et al. [35].”
“The number of microbial populations was determined by the plate counting method according to Li et al. [29]. “
Point 9: Line 124: If a reference is made to AOAC, the specific procedure identification number must be mentioned.
Response 9: Thank you very much for your suggestion. We have added the specific procedure identification number.
“AOAC. Official methods of analysis, 16th edition. Volume 1. Association of Official Analytical Chemists, Washington, DC. 1995.”
Point 10: Line 125-126: “The neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) contents were analyzed by the method reported above [34]”. Specify If you add α amylase or not, and was their content expressed as exclusive or inclusive of residual ash?, please refer in the M&Ms.
Respo nse 10: Thank you very much for your suggestion. We have revised it.
“The neutral detergent fiber (NDF, assayed without alpha-amylase ),”
Point 11: Line 157: “…lignocellulose (NDF and ADF)…” the lignocellulose is content not NDF + ADF. Please revise the intended meaning.
Point 11: Thank you very much for your suggestion.
The degradation rates of lignocellulose (NDF and ADF) during the ensiling process were calculated according to the following equation:
Reviewer 2 Report
Abstract - The structure of the Abstract is not in accordance with the instructions for authors. instructions for authors. The whole text should be written following. Revomver (1) Background, (2) Methods, etc....
Introduction - This is good, but overuses coordinating conjunctions.
coordinating conjunctions. For example, in the introduction you have used the word "Therefore" in 3 sentences in a row the authors used 3 coordinating conjunctions (However, Therefore, etc.). coordinating conjunctions (However, Therefore, However). The text of the introduction should be improved.
Materials and methods - The authors indicate that they reduced the moisture content of the the moisture content of the plants to 70%, which method was used, in greenhouse? There is no indication of how long it took. They should give more details of how the bags of silage were stored (with or without light, whether during fermentation they were turned over or fermentation, were they turned over or did they always remain the same, etc...). In the statistical analysis it is necessary to test the normality of the samples. On which parameters was ANOVA used? Why did they use a one-way ANOVA and did not use the t-student statistical test?
Results and Discussion - The authors report that probably the differences found in LAB between S1 and S2 are due to the increase in mature tissues, which release some nutrients, citing a study using cane citation of a study using sugar cane. There are no studies that this theory, but with grasses?
In Table 1 if no Enterobacteriaceae were detected in S1 and S2, it does not need to be in the table.
The authors justified the reason why DM decreases when the when the CX treatment of silages is added. What is the reason that when CX is added together with LP, the DM increases, surpassing the results obtained in the in the CK group? Is there any interaction that blocks the action of CX?
In Table 2, the meaning of the capital and small letters is missing in the legend uppercase and lowercase letters next to the values.
According to Oshima and McDonald (1978); AFRC (1987) and Henderson (1993), for silage to be considered of good quality, the N N-NH3/NT should reach a maximum of 8 to 11 per cent. The N/NH3 values are high, only one of the values is 9% (30d LP+CX) for grass silages.
What is the reason for this? Were there problems with the conservation of the forage?
The pH levels for most of the trials are below the expected values for this type of expected values for this type of forage. The pH should vary between 4.3 and 4.7, in your study it is possible to observe average values of 3.78. You should justify why this acidification of the silage has occurred and present solutions to improve this situation in the future.
Conclusion - It is simple and answers the initial problem. However, the decrease in pH, for me, is excessive, since the silage of the control group is within normal control group is within normal parameters. In this case the decrease in pH is not beneficial, it should not be included with the other parameters such as NDF and ADF.
Author Response
Point 1: Abstract - The structure of the Abstract is not in accordance with the instructions for authors. instructions for authors. The whole text should be written following. Revomver (1) Background, (2) Methods, etc....
Response 1: Thank you very much for your suggestion. We have revised the abstract according your comments.
Point 2: Introduction - This is good, but overuses coordinating conjunctions.
coordinating conjunctions. For example, in the introduction you have used the word "Therefore" in 3 sentences in a row the authors used 3 coordinating conjunctions (However, Therefore, etc.). coordinating conjunctions (However, Therefore, However). The text of the introduction should be improved.
Response 2: Thank you very much for your suggestion. We have revised it according your comments.
We have deleted “Therefore, we hypothesized that different cutting stages would have different effects on sudangrass silage fermentation and the microbial community, clarifying it can lay a theoretical foundation for forage production.”
“Therefore, researchers have utilized ferulic acid esterase (FAE) to destroy the ester bonds between lignin and structural polysaccharides to improve the degradability and nutritional value of feed” revised to “To improve the degradability and nutritional value of feed, researchers have utilized ferulic acid esterase (FAE) to destroy the ester bonds between lignin and structural polysaccharides”
“Therefore, whether the use of hemicellulase can achieve a better effect on silage is unclear. “ revised to “Whether the use of hemicellulase can achieve a better effect on silage is unclear. “
“However, previous research found that hemicellulase (xylanases and acetyl xylan esterases) and ferulic acid esterases have functional synergies” revised to “Previous research found that hemicellulases (xylanases and acetyl xylan esterases) and ferulic acid esterases have functional synergies”
“Moreover, Mkabayi et al. (2020) found that xylanase interacts with FAE and could achieve a better degradation rate” revised to “A previous study also found that xylanase interacts with FAE and could achieve a better degradation rate”
We have deleted “Hence, we explored the effects of FAE-producing LAB, cellulase and xylanase on the quality and lignocellulose degradation of sudangrass silages.”
Point 3: Materials and methods - The authors indicate that they reduced the moisture content of the the moisture content of the plants to 70%, which method was used, in greenhouse? There is no indication of how long it took. They should give more details of how the bags of silage were stored (with or without light, whether during fermentation they were turned over or fermentation, were they turned over or did they always remain the same, etc...). In the statistical analysis it is necessary to test the normality of the samples. On which parameters was ANOVA used? Why did they use a one-way ANOVA and did not use the t-student statistical test?
Response 3: Thank you very much for your suggestion. We have added the detail message and give the reason.
Wilt: “The grass was wilted for approximately 5 hours until the moisture content was approximately 70% by natural air-drying”
Storage：“A total of 48 bags were stored in a carton and placed in the storage room (27 ± 1 ℃) without turn over.’
Statistical analysis: We have tested the normality of the samples. The parameters of ANOVA was Duncan’s multiple range test for significance, and when P<0.05, the difference was considered statistically significant.
T-student statistical testis applicable to two experimental groups and one control group, but we have eight groups.
Point 4: Results and Discussion - The authors report that probably the differences found in LAB between S1 and S2 are due to the increase in mature tissues, which release some nutrients, citing a study using cane citation of a study using sugar cane. There are no studies that this theory, but with grasses?
Response 4: Thank you very much for your reminding. The abundance of epiphytic LAB increased with the delay in the cutting stage, we have found in the previous studies about the king grass silage and Italian ryegrass silage [14,15],and so on. But these research have not given the reason. Therefore, we cited the reason of epiphytic LAB number increased with the delay in the cutting stage of sugar cane. We would appreciate you giving us an example of grass.
Point 5: In Table 1 if no Enterobacteriaceae were detected in S1 and S2, it does not need to be in the table.
Response 5: Thank you very much for your suggestion. We have deleted in Table 1.
Point 6: The authors justified the reason why DM decreases when the CX treatment of silages is added. What is the reason that when CX is added together with LP, the DM increases, surpassing the results obtained in the in the CK group? Is there any interaction that blocks the action of CX?
Response 6: Thank you very much for your reminding.
Because a large amount of homofermentative LAB (FAE-producing L. plantarum ) adding to the silages, celulase and xylanase in synergy with FAE-producing L. plantarum degraded the sudangrass and provided more WSC for LAB, and then rapidly reduced pH, which resulted in more DM being preserved.
And there might have no interaction that blocks the action of CX, just the homofermentative LAB (FAE-producing L. plantarum ) in synergy with CX. However, more substrate was consumed by the heterolactic fermentation of epiphytic LAB in the groups of CX.
“The reason might be the synergistic effects of cellulase, xylanase and FAE-producing L. plantarum (homofermentative LAB), which degraded the sudangrass and provided more WSC for LAB, and then rapidly reduced the pH of the silages, resulting in more DM being preserved [19, 39]”
Point 7: In Table 2, the meaning of the capital and small letters is missing in the legend uppercase and lowercase letters next to the values.
Response 7: Thank you very much for your suggestion. We have added it.
“Different capital letters show that the same additive and stage treatment have significant differences in different ensiling times (P<0.05). Different lowercase letters show that the same ensiling time treatment was significantly different in different additives and stage (P<0.05)”
Point 8: According to Oshima and McDonald (1978); AFRC (1987) and Henderson (1993), for silage to be considered of good quality, the N N-NH3/NT should reach a maximum of 8 to 11 per cent. The N/NH3 values are high, only one of the values is 9% (30d LP+CX) for grass silages.
What is the reason for this? Were there problems with the conservation of the forage?
Response 8: Thank you very much for your reminding. But the units of NH3-N is g.kg−1 TN, and the maximum of NH3-N is 2.7% TN, which is below to the 8 to 11 per cent.
Point 9: The pH levels for most of the trials are below the expected values for this type of expected values for this type of forage. The pH should vary between 4.3 and 4.7, in your study it is possible to observe average values of 3.78. You should justify why this acidification of the silage has occurred and present solutions to improve this situation in the future.
Response 9: Thank you very much for your reminding. As far as I know, a lot of studies suggested that the quality of silage can only be guaranteed when the pH is less than 4.2. Moreover, the result of many researches had lower pH which below 4.0. For example, Li et al.(2019) found that adding ferulic acid esterase-producing lactic acid bacteria and cellulase in corn stalk silage had lower pH( 3.94, AC+Lp). Guan et al.(2018) found that the lowest pH of corn silage was 3.71. LI et al.(2020) found that the lowest pH of Pennisetum sinese silage was 3.79. Therefore, we thought the lower pH in our study is acceptable.
The reason of the result of us and these previous studies might the addition of LAB and cellulose degrading enzyme. If we want improve the lower pH of sudangrass silages, The addition amount of lactic acid bacteria and degrading enzymes can be reduced.
Li, F.H. et al. Ferulic Acid Esterase-Producing Lactic Acid Bacteria and Cellulase Pretreatments of Corn Stalk Silage at Two Different Temperatures: Ensiling Characteristics, Carbohydrates Composition and Enzymatic Saccharification. Bioresource Technology 2019, 282, 211-221. http://doi.org/10.1016/j.biortech.2019.03.022.
Guan, H. et al. Microbial communities and natural fermentation of corn silages prepared with farm bunker-silo in Southwest China. Bioresource Technology 2018, 265,282-290. https:// doi.org/10.1016/j.biortech.2018.06.018.
Li, F.H. et al. Pretreatment of Pennisetum sinese silages with ferulic acid esteraseproducing lactic acid bacteria and cellulase at two dry matter contents:Fermentation characteristics, carbohydrates composition and enzymaticsaccharification. Bioresource Technology 2020, 295 122261. https://doi.org/10.1016/j.biortech.2019.122261.
Point 10: Conclusion - It is simple and answers the initial problem. However, the decrease in pH, for me, is excessive, since the silage of the control group is within normal control group is within normal parameters. In this case the decrease in pH is not beneficial, it should not be included with the other parameters such as NDF and ADF.
Response 10: Thank you very much for your reminding. Based on the answer to the previous question, we thought the lower pH in our study is acceptable.
Round 2
Reviewer 1 Report
The authors adequately responded to all comments and performed all required modifications.
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Author Response
Thank you very much for your reminding about the language of manuscript. We have revised the language throughout the manuscript by the highly qualified native English speaking editors at AJE company. You can find the AJE Editing Certificate in the attachment. After the revise of AJE, we have reviewed the whole manuscript, and several sentences be revised as follows.
“Therefore, investigating the fermentation characteristics, fiber degradation and bacterial community of the two growth stages of sudangrass silages by adding the compound additive would lay a foundation for research on sudangrass silage production.” revised to “To verify this hypothesis, the silage quality, fiber degradation and microbial community were explored to evaluate the optimal cutting stages and additives. This study would lays a research foundation for sudangrass silage production.”
“adding FAE-producing LAB and cellulase to corn stalk silages resulted in improved quality and a higher degradation rate” revised to “adding FAE-producing LAB and cellulase to corn stalk silages resulted in higher quality and degradation rate”
”The amount of LP added was 1×106 cfu/g fresh matter (FM), and the amount of both cellulase and xylanase added was 25 U/g FM, according to the previous research [18,19]”
“Compared with the S1 stage, a higher abundance of LAB was found at the S2 stage of sudangrass growth (P<0.01)” revised to “Compared with the S1 stage, a higher abundance of LAB was found at the S2 stage of sudangrass (P<0.01)”
“Previous studies have reported that FAE combined with xylanase and FAE combined with cellulase showed improved degradation ability [26,39].” revised to “Previous studies have reported that FAE combined with xylanase or cellulase showed improved degradation ability [26,39]”
“positively correlated with the WSC content (P<0.05), “
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The last paragraph of the introduction is still a bit confusing, because it summarises the introduction to justify the aim of the work.
Author Response
The last paragraph of the introduction is still a bit confusing, because it summarises the introduction to justify the aim of the work.
Response : Thank you very much for your reminding. We have revised the last paragraph of the introduction according your comments. But we are concerned that we may have misunderstood your question. We would appreciate you giving us specific comments if we have not made the necessary modifications according to your wishes.
“In summary, whether adding FAE-producing LAB, cellulase and xylanase to sudangrass silage at different cutting stages should have different effects on the silages. To verify this hypothesis, the silage quality, fiber degradation and microbial community were explored to evaluate the optimal cutting stages and additives. This study would lays a research foundation for sudangrass silage production.”
