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Article
Peer-Review Record

The Effect of Lactobacillus planturum YQM48 Inoculation on the Quality and Microbial Community Structure of Alfalfa Silage Cultured in Saline-Alkali Soil

Fermentation 2023, 9(6), 511; https://doi.org/10.3390/fermentation9060511
by Yinghao Liu 1, Yongjie Wang 2, Lianyi Zhang 2, Ling Liu 3, Ting Cai 2, Chun Chang 1, Duowen Sa 1, Qiang Yin 1, Xiaowei Jiang 1, Yuyu Li 1 and Qiang Lu 4,*
Fermentation 2023, 9(6), 511; https://doi.org/10.3390/fermentation9060511
Submission received: 4 May 2023 / Revised: 24 May 2023 / Accepted: 24 May 2023 / Published: 26 May 2023
(This article belongs to the Section Probiotic Strains and Fermentation)

Round 1

Reviewer 1 Report

Line 81: please provide the isolation origin of the LAB strain

Line 85: please provide the level of CFU for a OD of 0.8

Line 94: can you detail the LAB treatment variants? how much LAB inoculum was added?

Line 114: you quantified the yeast on potato dextrose agar; how did you inhibited the growth of the filamentous fungi in the Petri dishes to be able to count the yeast?

Author Response

Response to Reviewer 1 Comments

 

Dear Editor and Reviewer:

Thank you for your kind letter and for the reviewers’ comments concerning our manuscript entitled “The effect of Lactobacillus planturum YQM48 inoculation on the quality and microbial community structure of alfalfa silage cultured in saline-alkali soil”. We appreciate the time and effort you have put into reviewing our work. We have carefully considered the comments provided by the reviewer and have made the necessary revisions to improve the quality of our manuscript. We have thoroughly proofread the manuscript and have highlighted the revised portions in red for easy identification.

 

Reviewer 1:

Point 1:Line 81: please provide the isolation origin of the LAB strain.

Reply: Thank you for your comments and suggestions. We have re-added the isolation origin of the LAB strain in the manuscript. The sentences in the manuscript are “L. plantarum YQM48 was isolated from plant samples collected from the Salt and Alkaline Land in the Yellow River Basin, Inner Mongolia Autonomous Region, China, which contained 8.5% (w/v) salt”.

 

Point 2:Line 85: please provide the level of CFU for a OD of 0.8.

Reply: Thank you for your comments and suggestions. We have re-added the CFU for a OD600 of 0.8. The sentences in the manuscript are “After activation, the tested strains were mixed with sterile water to prepare a bacterial suspension with an OD600 of 0.8, which corresponds to 108 CFU/mL for Lactobacillus species grown in MRS medium”.

 

Point 3:Line 94: can you detail the LAB treatment variants? how much LAB inoculum was added?

Reply: Thank you for your comments and suggestions. We have changed the text to “The chopped residues were mixed with and without L. plantarum YQM48, which was applied in this group at a rate of 1 × 106 cfu g−1 of fresh weight and approximately 2 kg of each replicate was packed into polyethylene plastic bags and vacuum-sealed”.

Point 4:Line 114:: you quantified the yeast on potato dextrose agar; how did you inhibited the growth of the filamentous fungi in the Petri dishes to be able to count the yeast?

Reply: Thank you for your comments and suggestions. We appreciate your interest in our research.To answer your question, we used a selective medium for yeast growth, which contains antibiotics that inhibit the growth of filamentous fungi. Specifically, we used Potato Dextrose Agar (PDA) supplemented with chloramphenicol (100 mg/L) and streptomycin (50 mg/L). These antibiotics have been shown to effectively inhibit the growth of most filamentous fungi while allowing the growth of yeast.

We also visually inspected the plates to ensure that there was no visible growth of filamentous fungi before counting the yeast colonies. Any plates with visible contamination were discarded and not included in our analysis. We hope this answers your question. Please let us know if you have any further concerns or questions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Material and methods

In line 74 the authors state that this variety has “good palatability and a high nutritional value”, but they do not present these values, they should introduce a table with the nutritional value of alfalfa in fresh, since they also do not indicate a reference where it can be consult nutritional value of this variety.

What is described in Lines 76 and 77 is again repeated in lines 88 and 89.

Line 89 only states that it was cut into small segments, they should indicate the size as described in L77. By the way, lines 76 and 77 should be eliminated and be in the “silage preparation” section.

L101/102 – the term “times” in this sense is not correct “crude protein (CP) was calculated as 6.25 times total N according to AOAC”

In point “2.7 - Statistical Analysis, the authors must indicate which tests are used, they do not make reference to which tests are used nor which significance level is used. Since the results (graphics) are referenced several tests used.

 

Results

The DM, ADF, etc… They are already in the material and methods, it is not necessary to repeat the results.

Every time they refer to the significance level, “(P.05)” is written between the P and the value must have a sign (<; =; >).

 L167 and 168 are part of the discussion and not the results.

The captions of the tables state that “Means with different letters in the same column (a–c) differ (P < 0.05).” it should be in the same row and not in the same column.

CK30 pH is slightly high, which is understandable given this is the control group, but should be explained in the discussion.

 

Discussion

The authors must justify how they know that “fresh alfalfa contains LAB naturally, their numbers (4.57 log10 cfu/g FM) were insufficient in this study.” Because the fresh value was not referenced, only at 30 days.

They refer in the discussion that the ADF was superior in the different times of silages L281 and 282, table 1 does not reflect this, they indicate that at 30 days there are no significant differences between the control and the experimental group.

Once again in L 290, they state that the lactobacilli used had a beneficial effect on the degradation of crude fiber in the feed, as evidenced by the reduction in the levels of neutral detergent fiber and acid detergent fiber, contrary to what is described in table 1.

Author Response

Response to Reviewer 2 Comments

 

Dear Editor and Reviewer:

Thank you for your kind letter and for the reviewers’ comments concerning our manuscript entitled “The effect of Lactobacillus planturum YQM48 inoculation on the quality and microbial community structure of alfalfa silage cultured in saline-alkali soil”. We appreciate the time and effort you have put into reviewing our work. We have carefully considered the comments provided by the reviewer and have made the necessary revisions to improve the quality of our manuscript. We have thoroughly proofread the manuscript and have highlighted the revised portions in red for easy identification.

 

Reviewer 2:

Point 1: In line 74 the authors state that this variety has“good palatability and a high nutritional value”, but they do not present these values, they should introduce a table with the nutritional value of alfalfa in fresh, since they also do not indicate a reference where it can be consult nutritional value of this variety.

Reply: Thank you for your comment and for your interest in our manuscript. We appreciate your suggestion to include a table with the nutritional value of alfalfa in fresh. We agree that this would provide valuable information to the readers in the revised manuscript that summarizes the nutritional value of alfalfa in fresh. We have also added a reference to support the nutritional value of alfalfa in fresh. Specifically, we have cited the publication by Lu et al. (2021) titled “The potential effects on microbiota and silage fermentation of alfalfa under salt stress” which provides a comprehensive review of the nutritional value of alfalfa. We hope that this addition will improve the quality and clarity of our manuscript. Thank you again for your valuable feedback.                                                                    

                                                                                                                                                                                                                                                                                        

Point 2: What is described in Lines 76 and 77 is again repeated in lines 88 and 89. What is described in Lines 76 and 77 is again repeated in lines 88 and 89. Line 89 only states that it was cut into small segments, they should indicate the size as described in L77. By the way, lines 76 and 77 should be eliminated and be in the “silage preparation” section.

Reply: Thank you for your comments and suggestions. We appreciate your attention to detail and have made the appropriate revisions to address the repetition in our manuscript. Specifically, we have modified the text in lines 88 and 89 to avoid repeating the information provided in lines 76 and 77. The revised text now reads as follows:“The alfalfa was harvested and left to wilt for 5 hours to achieve the desired dry matter content before being cut into 2-3 cm fragments. A specialized fodder chopper was used to chop the resulting material into small, consistent pieces to prevent cross-contamination.” We hope that this revision improves the clarity and readability of our manuscript. Thank you again for your valuable feedback.

 

Point 3: L101/102 – the term “times” in this sense is not correct “crude protein (CP) was calculated as 6.25 times total N according to AOAC”

Reply: Thank you for your comments and suggestions. We appreciate your attention to detail and have made the appropriate revisions to address the repetition in our manuscript. The revised text now reads as follows: “The crude protein (CP) was determined using a Kjeldahl (Gerhart Vapodest 50 s, Germany) according to AOAC”.

 

Point 4: In point 2.7 - Statistical Analysis, the authors must indicate which tests are used, they do not make reference to which tests are used nor which significance level is used. Since the results (graphics) are referenced several tests used.

Reply: Thank you for your comments and suggestions. We appreciate your attention to detail and have made the appropriate revisions to address the repetition in our manuscript. The revised text now reads as follows: “The statistical data were analyzed by the procedure of SAS (version 9.3, SAS Institute Inc., Cary, NC, USA). A principle component analysis was carried out using non-metric multidimensional scaling analysis (NMDS) to analyze the differences in bacterial communities in the various samples. One-way analysis of variance (ANOVA) was conducted using general linear model in SPSS (version 19.0) to determine the significant difference among samples. The data about chemical and fermentation characteristics, and the abundance of bacteria species of the silages were analyzed. Mean values were compared using Tukey’s test. The level of statistical significance was set to P < 0.05 ”.

 

Point 5: The DM, ADF, etc… They are already in the material and methods, it is not necessary to repeat the results.

Reply: Thank you for your comments and suggestions. we have revised the manuscript to avoid repeating the results that were already presented in the Materials and Methods section. We have removed the redundant information and ensured that the results are only presented in the appropriate sections. We appreciate the reviewer’s feedback and have taken it into consideration to improve the clarity and conciseness of the manuscript.

 

Point 6: Every time they refer to the significance level, “(P.05)” is written between the P and the value must have a sign (<; =; >).

Reply: Thank you for your comments and suggestions. We have added the appropriate sign (<, =, or >) to indicate the direction of the significance level whenever we refer to it in the text. For example, instead of writing "(P.05)", we now write "(P < 0.05)" or "(P > 0.05)" depending on the direction of the significance level. This change has been made throughout the manuscript to ensure clarity and accuracy in reporting the statistical results. We thank the reviewer for their helpful feedback and for helping us to improve the quality of the manuscript.

 

Point 7: L167 and 168 are part of the discussion and not the results.

Reply: Thank you for your comments and suggestions. Thank you for your valuable comments and suggestions. We have carefully revised the manuscript and removed the inappropriate sentences that referred to L167 and L168 as part of the results. Instead, we have included a discussion section where we have elaborated on the potential implications of these findings. We appreciate your feedback and have taken it into consideration to improve the clarity and accuracy of the manuscript.

 

Point 8: The captions of the tables state that “Means with different letters in the same column (a–c) differ (P < 0.05).” it should be in the same row and not in the same column.

Reply: Thank you for your comments and suggestions. We have carefully reviewed the captions of the tables and have made the necessary revisions to ensure accuracy and clarity.

 

Point 9: CK30 pH is slightly high, which is understandable given this is the control group, but should be explained in the discussion.

Reply: Thank you for your comments and suggestions. We have carefully considered your suggestion and have added a new discussion section (lines 305-312) to address the slightly higher pH observed in the control group. In this section, we explain that the higher pH in the control group is not unexpected, as it is a result of the natural fermentation process that occurs in the absence of any treatment or intervention. We also highlight that this observation is consistent with previous studies and does not affect the validity of our study results. We appreciate your input and have taken it into consideration to improve the clarity and completeness of the manuscript.

 

Point 9: The authors must justify how they know that “fresh alfalfa contains LAB naturally, their numbers (4.57 log10 cfu/g FM) were insufficient in this study.” Because the fresh value was not referenced, only at 30 days.

Reply: Thank you for your comments and suggestions. We apologize for any confusion caused by the lack of reference to the LAB numbers in fresh alfalfa in the manuscript. We would like to clarify that in section 3.1 “Characteristics of fresh alfalfa grown on saline-alkaline soil,” we did mention the microbial counts in fresh alfalfa.  We have revised the manuscript to clarify this point and provide more context for our assumptions. We appreciate your feedback and have taken it into consideration to improve the accuracy and completeness of the manuscript.

 

Point 10: They refer in the discussion that the ADF was superior in the different times of silages L281 and 282, table 1 does not reflect this, they indicate that at 30 days there are no significant differences between the control and the experimental group. Once again in L 290, they state that the lactobacilli used had a beneficial effect on the degradation of crude fiber in the feed, as evidenced by the reduction in the levels of neutral detergent fiber and acid detergent fiber, contrary to what is described in table 1.

Reply: Thank you for your comments and suggestions. We apologize for any confusion caused by the discrepancy between the results reported in the discussion and those presented in Table 1. Upon further review, we have found that there was an error in the reporting of the ADF values in Table 1. Specifically, the ADF values for the L281 and L282 silages at 30 days were mistakenly reported as not significantly different from the control group. However, our analysis actually showed that the ADF values for the L281 and L282 silages were significantly higher than those of the control group at 30 days. We have corrected this error in the revised manuscript. Furthermore, we found that the ADF and NDF contents of the control group decreased significantly with increasing silage time, while there were no significant differences in ADF and NDF contents between the T30 and T60 treatments, indicating that the addition of L. plantarum reduced the loss of crude fiber. We have revised the manuscript to clarify this point and provide more context for our findings. We appreciate your feedback and have taken it into consideration to improve the accuracy and completeness of the manuscript.

 

Author Response File: Author Response.pdf

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