Lactic Acid Bacteria-Fermented Diet Containing Bacterial Extracellular Vesicles Inhibited Pathogenic Bacteria in Striped Beakfish (Oplegnathus fasciatus)
, Yu-Sheng Wu 2, Wei-Hsuan Hsu 3,* and Fan-Hua Nan 1,*Round 1
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsThe article “Application of lactic acid bacteria-fermented diet contained 2 bacterial extracellular vesicles improved intestinal flora and inhibit pathogenic bacteria in Oplegnathus fasciatus ” by Bao-Hong et al., describes the effects of a diet enriched with a probiotic extracellular vescicles on intestinal flora and pathogen inibition in striped beakfish
The topic of the Article is interesting and the data are well presented.
I would suggest only some minor corrections.
In particular:
Title – I would suggest: Lactic acid bacteria-fermented diet containing 2 bacterial extracellular vesicles improves intestinal flora and inhibit pathogenic bacteria in Striped beakfish (Oplegnathus fasciatus)
Line 72: where this fish is often…
Line 482: the declaration of animal welfare approval should be reported also here
Author Response
In particular:
Title – I would suggest: Lactic acid bacteria-fermented diet containing 2 bacterial extracellular vesicles improves intestinal flora and inhibit pathogenic bacteria in Striped beakfish (Oplegnathus fasciatus)
ANS: Thank you for your suggestion. We have proofed the topic title according to reviewer’s comment.
Line 72: where this fish is often…
ANS: Thank you for your suggestion. We have proofed the sentence according to reviewer’s comment (line 72).
Line 482: the declaration of animal welfare approval should be reported also here
ANS: Thank you for your suggestion. We have reported the approval number according to reviewer’s comment (line 482).
Reviewer 2 Report (New Reviewer)
Comments and Suggestions for AuthorsThe manuscript described the application of lactic acid bacteria-fermented diet on the improvement of intestinal flora and inhibition of pathogenic bacteria in Oplegnathus fasciatus. The work is interesting but some major issues are needed to address:
1. There‘s no direct evidence indicated that extracellular vesicles could impact intestinal flora, so the title and corresponding description should be modifued;
2. Please mention why fermented the feed for 1, 3, or 5 days, and the concentration of lactic acid bacteria applied for fermentation should be mentionedï¼›
3. Please mention why use different bacteria for analysis in section 2.5 and 2.8ï¼›
4. Is Acinetobacter baumannii (17106) isolated from fish？
5. The isolation of small RNA and microRNA content should be mentioned;
6. Please explain why extracellular vesicles can inhibit the growth of bacteria?
Comments on the Quality of English LanguageMinor editing of English language required. Such as line 21-22, line 27, line 31-32,
line 72-73, line 377-378, line 439, etc.
Author Response
Comments and Suggestions for Authors
The manuscript described the application of lactic acid bacteria-fermented diet on the improvement of intestinal flora and inhibition of pathogenic bacteria in Oplegnathus fasciatus. The work is interesting but some major issues are needed to address:
- There‘s no direct evidence indicated that extracellular vesicles could impact intestinal flora, so the title and corresponding description should be modified;
ANS: Thank you for your suggestion. We have proofed the topic title according to reviewer’s comment.
- Please mention why fermented the feed for 1, 3, or 5 days, and the concentration of lactic acid bacteria applied for fermentation should be mentionedï¼›
ANS: Thank you for your suggestion. As shown in Figure 1B, the bacterial number of L. reuteri on feed stuff after 1, 3, and 5 days of fermentation has been determined (line 209). This study originally expected that the number of L. reuteri should increase with the extension of fermentation days. However, in the actual bacterial number analysis results, it was found that after 3 days of fermentation, the number of L. reuteri reached a high level, and after 5 days of fermentation, the number of L. reuteri was decreased. Therefore, this study only showed fermentation results for 1, 3, and 5 days fermentation.
- Please mention why use different bacteria for analysis in section 2.5 and 2.8ï¼›
ANS: Thank you for your suggestion. Escherichia coli is a commonly used standard bacterium for testing the bactericidal activity of immune cells [Reference 25/26], so in section 2.5, Escherichia coli was used to explore. After we completed the animal experiments, we found that Acinetobacter baumannii in the intestines would be inhibited by sample treatment. Therefore, in order to confirm the antibacterial activity of extracellular vesicles against Acinetobacter baumannii, we further analyzed in vitro experiments to explore the antibacterial potential of extracellular vesicles (section 2.8)
- Is Acinetobacter baumannii (17106) isolated from fish？
ANS: Thank you for your question. This strain is commonly used as a standard strain for drug-resistant bacteria and is not isolated from fish.
- The isolation of small RNA and microRNA content should be mentioned;
ANS: We have provided detail in line 170-173.
- Please explain why extracellular vesicles can inhibit the growth of bacteria?
ANS: Relevant test information has been explained (lines 453-466).
Comments on the Quality of English Language
Minor editing of English language required. Such as line 21-22, line 27, line 31-32,
line 72-73, line 377-378, line 439, etc.
ANS: Thank you for your suggestion. We have proofed these sentences.
Round 2
Reviewer 2 Report (New Reviewer)
Comments and Suggestions for AuthorsThe manuscript has been modified well and should be considered to publish.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have only added the sheet with the response to the reviewers without having included the manuscript
Reviewer 2 Report
Comments and Suggestions for AuthorsThe article “Application of lactic acid bacteria-fermented diet contained bacterial extracellular vesicles improved intestinal flora and inhibit pathogenic bacteria in Oplegnathus fasciatus ” by Bao-Hong et al., described an interesting trial on fishes.
I would like to suggest some minor revisions:
Title
Try to reduce the lenght of the title, if possible
Abstract
Avoid separation in sections.
Line 18: lactic acid bacteria-fermented feed … please clarify in Methods how the diet was prepared
Material and Methods
Line 92: A commercial diet was used ? please give more details on the diet, includindg a table with nutritional values.
Why this diet can be defined “fermented” ? decribe better this process please
Line 98: Please give the same details requested below for the control diet.
This species of fish is commercially breed in taiwan for food production ? It is very interesting for an european reader, please give more details
Line 110: How often is the weight collected?
Line 142: Blood collection. Do you have a ethical permit? Is it necessary in you country? Please, in the declarations at the end of the manuscript give more details about the ethical permits or legislation in your country
Describe how fish were killed for intestine sampling.
Lines 158-173: this part was performed in vivo or in vitro ?
Line 167: Agilent Bioanalyzer.. please indicate city state
Comments for author File: 
Comments.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis paper investigates the effects of feeding Oplegnathus fasciatus with lactic acid-fermented feed.
Why are the results of the analysis of the gut microbiota limited to the control group and the 3-days group? This needs further clarification.
It is assumed that one fish was selected from each tank for gut microbiota analysis and DNA extraction. However, the paper lacks details on how these fish were chosen.
The sample size (n=4) for the comparison of the four groups is small and should be increased for more robust statistical analysis.
The use of Duncan's test for multiple comparisons is not recommended. Alternate methods should be considered.
The paper lacks a description of the statistical methods used for gut microbiota data analysis.
Due to these issues with experimental methods and statistical analysis, which significantly impact the results, it would be difficult to publish this paper in this journal.
