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Peer-Review Record

Fermentation Properties and Bacterial Community Composition of Mixed Silage of Mulberry Leaves and Smooth Bromegrass with and without Lactobacillus plantarum Inoculation

Fermentation 2023, 9(3), 279; https://doi.org/10.3390/fermentation9030279
by Weihan Yang 1,†, Fengyuan Yang 2,†, Changsong Feng 3, Shanshan Zhao 1, Xueying Zhang 1 and Yanping Wang 1,*
Reviewer 1: Anonymous
Reviewer 2:
Reviewer 3: Anonymous
Fermentation 2023, 9(3), 279; https://doi.org/10.3390/fermentation9030279
Submission received: 16 February 2023 / Revised: 9 March 2023 / Accepted: 10 March 2023 / Published: 13 March 2023
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)

Round 1

Reviewer 1 Report

The work Fermentation properties and bacterial community composition 2 of mixed silage of mulberry leaves and smooth bromegrass 3 with and without Lactobacillus plantarum inoculation is a work that presents the use of lactobacillus plantarum to improve the fermentation profile of leaves and grasses. The work has serious weaknesses, such as some information, for example: what is the hypothesis of using moringa leaves? Furthermore, the manuscript requires some important measures such as the quantification of losses and the dynamics when the silage was exposed to air. The introduction does not provide a clear and objective hypothesis. But the work has scientific merit to be published on fermentation.

Introduction

-The introduction needs to be improved. It needs to be direct and objective, a review of what the ensiling technique is is not necessary, the introduction is quite debatable, which makes the authors deviate from the objective of the study.

Material and methods

-Is the strain an isolate from the authors' laboratory? This must be mentioned

-Insert the statistical model used in the analyzes

Results and discussion

-I am not satisfied with the discussion and description of results for microbial populations. The inoculant controlled the events. This needs to be described.

-The authors must explain the reasons for ensiling this component with 14.79% DM

-What is the optimal level of epiphytic LAB? The authors talk about the results but do not cite a reference.

-As a suggestion, authors should better explore their main findings. In an attempt to validate the use of Plantarum.

-I missed the data on gas and effluent losses.

-Did the authors assess aerobic stability? 

Conclusion

-The completion needs to be improved. authors must report the reason for recommending the 20%. The description of the results are quite confusing. Authors need to improve the entire manuscript

Author Response

Response to Reviewer 1 Comments

We gratefully thank your comments and carefully check on the manuscript.

Below the comments are response point by point.

 

Point 1: The introduction needs to be improved. It needs to be direct and objective, a review of what the ensiling technique is is not necessary, the introduction is quite debatable, which makes the authors deviate from the objective of the study.

Response 1: Mulberry leaves could be served as an optimum protein supplement in the diets of terrestrial farm animals, but seasonal production of mulberry might hinder its continuous and extensive application in feed rations. An effective ensiling strategy would aid in its extened application. The main purpose of this study is to evaluate the fermentation properties and bacterial community composition of mulberry leaves when ensiled with smooth bromegrass at different ratios with or without L. plantarum inoculation, and determine the optimum ratio of the mixture for good fermentation quality. The introduction has been altered accordingly, and the review of what the ensiling technique is has been deleted.

 

Point 2: -Is the strain an isolate from the authors' laboratory? This must be mentioned

Response 2: We have added “L. plantarum A345 is analfalfa epiphytic strain isolated from Shanxi, Beijing, China, and is stored in Henan key laboratory of ion beam bioengineering.”in L77-78.

 

Point 3: -Insert the statistical model used in the analyzes

Response 3: The statistical analyses part has been modified as following:

Effects of ratio of mulberry leaves (R), L. plantarum inoculation (I), and their inter-actions (R*I) on fermentation properties and alpha diversity indices were analyzed us-ing general liner model in IBM SPSS Statistics for Windows, version 21.0. (IBM Corpo-ration, Armonk, NY, USA). Alpha diversity indices including observed species, Chao 1 and Shannon indices were calculated using mothur (version 1.9.0 https:// mothur.org/ wiki/ mothur_v.1.9.0/) [29]. Principal coordinate analyses (PCoA) based on weighted unifrac distance matrix and Linear discriminant analysis effect size (LEfSe) was per-formed using python (version 2.7.14; https://www.python.org/). Correlations of fermentation properties with bacterial community were analyzed using two-tailed Spearman correlations in IBM SPSS Statistics for Windows, version 21.0. Significant differences between means were identified by Duncan’s multiple range tests in which a probability (P) value of <0.05 was designated as significant.

 

Point 4: -I am not satisfied with the discussion and description of results for microbial populations. The inoculant controlled the events. This needs to be described.

Response 4: Effects of the inoculant on microbial population has been discussed as “This indicated that the inoculant performed potential effects to stabilize the bacterial community in silage” basis on the PCoA analyses. Results of microbial population in the non-inoculated and inoculated silage were discussed seperately in L270-312. Effects of the inoculant on microbial population was futher discussed as “Notably, while co-ensiled with smooth bromegrass and L. planturum inoculation both improved fermentation quality of mulberry leaves silage, the two strategies failed to perform a stack effect in this study. Li et al. (2018) [13] reported improvement in fermentation quality by L. planturum inoculation in bur clover and annul ryegrass mixed silage. In this study, however, proportion of Lactobacillus and LA accumulation de-creased (P <0.05) in the inoculated ML80 silage comparing with the non-inoculated group. One potential explanation is that the inoculant had poor adaption to the envi-ronmental conditions with the Incorporation with smooth bromegrass[18]. It might fail to perform a synergistic effect with the epiphytic LAB of smooth bromegrass, which led to poorer acidification in silage compared with the ML100 group, and finally resulted in growth of undesirable microbes such as Enterobacter and Clostridium. Screening of adapted LAB inoculant is necessary to further improve fermentation quality of the mixed silage” in L313-324.

 

Point 5: - The authors must explain the reasons for ensiling this component with 14.79% DM

Response 5: The raw materials were harvested and directly chopped without wilting in this study. Through this way, we tried to imitate the ensiling situation during rainy season. The relevant content has been modified as “The DM content of fresh smooth bromegrass was low compared with previous studies (31.7% and 33.9%, respectively) [15][35]. This might be due to the rainy weather condition during the harvest season in our study. Low DM content of the raw material was also reported in previous studies in stylo (22.5%), bur clover (16.15%), and annual ryegrass (18.88%) [13][36]” in L162-166.

 

Point 6: - What is the optimal level of epiphytic LAB? The authors talk about the results but do not cite a reference.

Response 6: The optimal level of epiphytic LAB was above 105. A reference was cited as “17. Oliveira, A.S.; Weinberg, Z.G.; Ogunade, I.M.; Cervantes, A.A.P.; Arriola, K.G.; Jiang, Y.; Kim, D.; Li, X.J.; Goncalves, M.C.M.; Vyas, D.; Adesogan, A.T. Meta-analysis of effects of inoculation with homofermentative and facultative heterofermentative lactic acid bacteria on silage fermentation, aerobic stability, and the performance of dairy cows. J. Dairy Sci. 2017, 100: 4587-4603. https://doi.org/ 10.3168/jds.2016-11815” in L149.

 

Point 7: - As a suggestion, authors should better explore their main findings. In an attempt to validate the use of Plantarum.

Response 7: The use of L. planturum was validated and discussed as “Notably, while co-ensiled with smooth bromegrass and L. planturum inoculation both improved fermentation quality of mulberry leaves silage, the two strategies failed to perform a stack effect in this study. Li et al. (2018) [13] reported improvement in fermentation quality by L. planturum inoculation in bur clover and annul ryegrass mixed silage. In this study, however, proportion of Lactobacillus and LA accumulation de-creased (P <0.05) in the inoculated ML80 silage comparing with the non-inoculated group. One potential explanation is that the inoculant had poor adaption to the envi-ronmental conditions with the Incorporation with smooth bromegrass[18]. It might fail to perform a synergistic effect with the epiphytic LAB of smooth bromegrass, which led to poorer acidification in silage compared with the ML100 group, and finally resulted in growth of undesirable microbes such as Enterobacter and Clostridium. Screening of adapted LAB inoculant is necessary to further improve fermentation quality of the mixed silage” in L313-324.

 

Point 8: -I missed the data on gas and effluent losses.

Response 8: The DM loss has been evaluated, and no significant DM loss was observed. The relevant content has been added as “Significant DM loss was not observed in silages (P >0.05)” in L184-185.

 

Point 9: -Did the authors assess aerobic stability?

Response 9: It’s a pity that the aerobic stability of silage was not assessed in this study. stretch film ensilage is the common application in our area for ensiling with the scale of a single bale less than 800 kg. And the silage was generally consumed on the day unpacking. We’ll involve the aerobic stability assess in our further study, as it’s an important property of silage.

 

Point 10: -The completion needs to be improved. authors must report the reason for recommending the 20%. The description of the results are quite confusing. Authors need to improve the entire manuscript

Response 10: The conclusion part has been modified as “Mulberry leaves ensiled with L. planturum inoculation achieved optimum fermen-tation quality after 60-d ensiling. L. planturum inoculation, thus, could be a potential strategy for mulberry leaves ensiling. Combined ensiling with smooth bromegrass could also aid in improving silage quality of mulberry leaves, with the optimum ratio of mulberry leaves being 80%. But the two strategies failed to perform a synergistic effect.”

 

In all, we thank again for your careful check and impresive comments on this MS. Hope our revisions and response could help to improve the quality of this MS.

Reviewer 2 Report

The manuscript is interesting and correctly written. It is of practical importance in ensiling forage for ruminant nutrition. In some parts, the work should be supplemented and expressed more clearly.  The instructions for authors should be followed, the results and discussion section should be presented separatelly.

Some specifics are written bellow:

36: Is the measurement unit for biomass yield 25-30 tons od dry matter per ha per year, please, state it clear 

71-74: What was the size of the experimental area? 

76:How much of the plant material was taken? How the forage was chopped?

77-78: How the forage was mixed together? 

86: How the samlpe was taken?

95: What type of owen drier was used? Was it fan circulated?

97: The instruments used for CP dand for fibre determination?

Author Response

Response to Reviewer 2 Comments

We gratefully thank your comments and carefully check on the manuscript.

Below the comments are response point by point.

 

Point 1: The instructions for authors should be followed, the results and discussion section should be presented separately.

Response 1: We have re-checked the instructions for authors of this jounal (https://www.mdpi.com/journal/fermentation/instructions), and its allowed to combine the results and discussion part. The original description was as following:

Discussion: Authors should discuss the results and how they can be interpreted in perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible and limitations of the work highlighted. Future research directions may also be mentioned. This section may be combined with Results.

 

Point 2: 36: Is the measurement unit for biomass yield 25-30 tons od dry matter per ha per year, please, state it clear

Response 2: The measurement unit for biomass yield 25-30 tons od fresh matter per ha per year. The relevant content has been modified as “Its leaves are rich in protein (15−35 % dry matter (DM)), minerals and vitamins, with biomass yield of approximately 25−30 tons od fresh matter /ha/year [2,3]” in L34-36.

 

Point 3: 71-74: What was the size of the experimental area?

Response 3: The relevant content has been modified as “Mulberry leaves were harvested on 11th June 2020 from an experimental field of Henan Academy of Agricultural Sciences in Lankao, Henan, China (34.82°N, 114.82°E) with the experimental area being 0.3 ha. Smooth bromegrass was harvested on 11th June 2020 from an experimental field of Henan Academy of Agricultural Sciences in Zhengzhou, Henan, China (34.76°N, 113.65°E) with the experimental area being 0.2 ha” in L65-69.

 

Point 4: 76: How much of the plant material was taken? How the forage was chopped?

Response 4: The relevant content has been modified as “The raw materials (approximately 15 kg of mulberry leaves and 5 kg of smooth brome-grass) were immediately taken to the laboratory and chopped to an approximate length of 2 cm by a handy cutter. Chopped smooth bromegrass was manually mixed with mulberry leaves at ratios of 0% (ML100), 10% (ML90), 20% (ML80), 30% (ML70), 40% (ML60) on a fresh matter (FM) basis” in L69-74.

 

Point 5: - 77-78: How the forage was mixed together?

Response 5: We’re sorry that the original description of how the forage was treated and packed might be misleading. The relevant content has been modified as “Six kg of each homogenous mixture was equally divided into 6 parts. Approximately 1 kg for each of three replicates of the homogenous mixture was treated with the fol-lowing: (1) distilled water control (CK); (2) 1 × 106 colony forming units (cfu)/g of L. plantarum A345 (LP). L. plantarum A345 is an alfalfa epiphytic strain isolated from Shanxi, Beijing, China, and is stored in Henan key laboratory of ion beam bioengineering. Treated samples were then vacuum-packed separately with vacuum-sealed polyeth-ylene plastic bags (dimensions: 200 mm × 300 mm, Dongda, Zhengzhou, China)” in L74-80.

 

Point 6: 86: How the samlpe was taken?

Response 6: The relevant content has been modified as “A sample of 10 g was taken from each bag of silage after manually and homoge-neously mixed within the bag” in L83-84.

 

Point 7: 95: What type of owen drier was used? Was it fan circulated?

Response 7: The relevant content has been modified as “A sample of approximately 150 g was taken from each bag for determination of oven DM content at 65 °C for 48 h in a drying oven (FX101-0, Shanghai Shuli Yiqi YIbiao Co. Ltd., Shanghai, China)” in L92-94.

 

Point 8: 97: The instruments used for CP dand for fibre determination?

Response 8: The relevant content has been modified as “CP concentration was calculated according to the Kjeldahl method [20] using a Kjeldahl nitrogen analyzer (K1160, Hanon, Shanghai, China). The NDF and acid detergent fiber (ADF) concentration was calculated according to Van Soest et al. (1991) [21] using an Ankom 200 Fiber Analyzer System (Ankom Technology Corp., Fairport, NY, USA).” in L96-100.

 

In all, we thank again for your careful check and impresive comments on this MS. Hope our revisions and response could help to improve the quality of this MS.

Reviewer 3 Report

The authors evaluated the fermentation properties and bacterial community composition of mulberry leaves when ensiled with smooth bromegrass at different ratios with or without L. plantarum inoculation, and determine the optimum ratio of the mixture for good fermentation quality. They found that L. planturum inoculation and combined ensiling with smooth bromegrass proportion being 20% could both aid in improving silage quality of mulberry leaves. Mulberry leaves ensiled with L. planturum inoculation achieved optimum fermentation quality after 60-d ensiling. The overall Ms is well designed, presented and discussed.

Author Response

Response to Reviewer 3 Comments

We gratefully thank your comments and carefully check on the manuscript.

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