Review Reports - Dietary Administration Effects of Microbial Exopolysaccharide from <i>Bacillus subtilis</i> P1 on Growth Performance and Immunity in Nile Tilapia (<i>Oreochromis niloticus</i>)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I believe that the work cannot yet be published in the form presented and needs to be corrected.

summary

Clearly state the objective of your work.

introduction

what is the aim of your work ?

what is the hypothesis of your work?

Materials and methods

As your work is divided into two experiments, however from point 2.1 to 2.6.4 your methodology is mixed up and not understood.

1. first describe the place where the first experiment was carried out.

2. environmental conditions in which the fish were kept.

3. how many fish of the 5 species did you use?

4. were the fish mixed or did you make groups by species?

5. what is the origin of the EPS you used for the inoculation?

6. did you inoculate all the selected fish?

7. line 175-181. i consider that the statistical part goes in a section at the end of the experiment.

8. line 232-234 how many plates did you use for inoculation?

9. line 248. how many samples did you use?

10. line 279. how many fish per treatment did you use?

11. check your statistical analyses in detail because you are applying only t-tests, however for some of your results you need to perform repeated mean analysis with respect to time, for example the results presented in table 3.

results

I think you should reduce your paragraphs, they are too big and difficult to understand.

line 384-386. I think these lines go under materials and methods, and you never mention that you used 1 different strain in your methodology.

I think you should put the standard error of the mean in your results, as well as your P value.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions and corrections have been highlighted in red color in the re-submitted files.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "Dietary administration effects of Microbial Exopolysaccharide from Bacillus subtilis P1 on Biological activities, Growth Performance and Immunity in Nile Tilapia (Oreochromis niloticus)" presents results of an original study of exopolysaccharide (EPS) analysed with modern methods of molecular biology and microbiology.

The main question addressed by the research is biochemical and biological characteristics of inulin-like EPS produced by Bacillus subtilis, and evaluation of its influence the growth parameters and immunity of aquaculture fish Nile Tilapia.

All results obtained by the authors are original and relevant for environmental microbiology. The paper addresses the specific gap in the field of properties and biological activities of inulin-like EPS produced by probiotic strains of Bacillus subtilis. The study adds new data on the structure, functional properties of inulin-like microbial EPS, and stimulation of fish growth and immunity.

All main questions posed and mentioned above were addressed with modern techniques of DNA sequencing, Gel Permeation Chromatography, Fourier transform infrared (FTIR) spectroscopy, biochemical and immunological examination, experimental infection.

The authors have described comprehensively current state of research in use of EPS for aquaculture, and have provided all necessary and appropriate references.

Unfortunately, there are some unclarities that require major and minor corrections.

- The title does not meet the manuscript contents and should be corrected, because “Biological activities … in Nile Tilapia” were really not studied by the authors. Instead, Biological activities of the isolated EPS have been studied.

- Line 26: “EPS cytotoxicity” should be exchanged by “Low EPS cytotoxicity”.

- Line 29: The abbreviations “WG, %SGR, and ADG” should be deciphered.

- Line 32: “challenged with” should be exchanged by “infected by”.

- Line 33: “in the T-group within two weeks”. Unfortunately, meaning of “two weeks” is unclear, because results of experimental infection have not been described in the manuscript. It is a crucial point, so this chapter describing results of the experimental infection is necessary.

- Line 35: “responses” should be exchanged by “response”.

- The manuscript contains a lot of giant and really too long sentences, which are so complicated and unclear. They should be divided into few shorter and clear statements. For example, lines 70-79, 86-90, 507-514, 523-531, 704-709, 775-783 are too long, unclear and should be rephrased and divided into shorter statements.

- Line 85: “physiochemical” should be exchanged by “physiochemical properties”.

- Line 87: “EPS immunological activity were” should be exchanged by “EPS activity was”.

- Line 86-90: the statement is too long and unclear and should be rephrased.

- Lines 369-374 and 385-387 repeat the same phrases in “Materials and methods” Chapter, and should be removed.

- Line 518-519: The phrase “According to the literatures,” requires corresponding references.

- Line 546: “was confirm” should be exchanged by “was confirmed”.

- Line 557: “The EPS-producer” should be exchanged by “Species of the EPS-producer”.

- Line 562: “was further confirmation” should be exchanged by “further confirm”.

- Line 657-658: the statement “Although a slight increase in hemolysis activity increasing the EPS concentration” is unclear and should be rephrased.

- Line 662: “revealing differing” should be exchanged by “and revealed different”.

- Lines 692-694: the statement “Additionally, for immunological parameters such as NBT and lysozyme values were significantly increased (p < 0.05) in EPS-supplemented group which reached about OD630 of 0.44±0.02 and 6.88±0.16 U/min, respectively (Table 4).” is unclear, repeats data from Table 4, and should be rephrased. For example: “Additionally, immunological parameters such as NBT and lysozyme increased significantly (p < 0.05) in EPS-supplemented group compared to control group (Table 4).”

- Lines 723-731: the statements “Additionally, Ghafarifarsan et al. (2020) evaluated the effects of dietary inulin on growth performance and immune factors as well as innate immune responses of Oncorhynchus mykiss (rainbow trout) fry challenged with A. hydrophila and found that the inulin affected significantly increased the WG, FCR, SGR, and protein content while for humoral immune responses showed that the inulin supplements significantly increased lysozyme and complement activities in addition total IgM was improved [63]. For the relative level of protection (RLP) after challenge infection the fish with A. veronii for 6 week, about 50% RLP was obtained in the EPS supplemented groups which was lower than that of commercial inulin (33.33-35.71%RLP)[2]” are unclear and should be rephrased. For example: “Additionally, Ghafarifarsan et al. (2020) evaluated the effects of dietary inulin on growth performance and immune factors as well as innate immune response of Oncorhynchus mykiss (rainbow trout) fry challenged with A. hydrophila. They have found that inulin supplementation to diet increased significantly WG, FCR, SGR, and protein content as well as components of the humoral immune response such as lysozyme and complement activities, and total IgM [63]. Relative level of protection (RLP), about 50%, in the six-week-old fish feed EPS after challenge infection with A. veronii was higher than RLP in fish feed the commercial inulin (33.33-35.71%) [2]”

- Line 733: “agent” should be exchanged by “effects” or “activities”.

- Line 748: “was” should be exchanged by “were”.

- Line 752: “statistically” should be exchanged by “significant”.

- Line 753: the statement “that was implied that the EPS feeding had no negative adverse on fish health” is unclear and should be rephrased.

- Lines 753-756: the statement is unclear and should be rephrased.

- Lines 788-797: The conclusions are not in a good agreement with the results and should be improved.

Comments on the Quality of English Language

The manuscript contains some errors as well as a lot of too long, unclear, and improper sentences, which should be improved.

Author Response

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I consider that it is not yet ready for publication, as it needs to be corrected.

1. materials and methods

the statistical analysis of the second experiment is not adequate, if you do repeated means with respect to time, plus interactions (time by treatment) instead of just a T-test your results will be better expressed.

2. results.

check all your figures, because in some of them your data is not clear, e.g. figure 1, the numbers are overlapping.

3. check all your tables and put the standard error of the mean, standard deviation and the p-value obtained.

4. check all the abbreviations that appear in your tables and indicate their meaning, e.g. table 2.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections were highlighted in red color in the re-submitted files

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have described comprehensively current state of research in use of EPS for aquaculture, and have provided all necessary and appropriate references.

In the revised version of the manuscript all incorrect statements and unclarities have been addressed, so the manuscript is recommended to be published.

Comments on the Quality of English Language

The authors have corrected a lot of errors in the manuscript. However, thorough proofreading is necessary.

Author Response

Thank you very much for taking the time to review this manuscript and giving valuable comments. Please find the detailed responses below and the corresponding revisions/corrections were highlighted in red color in the re-submitted files.

Author Response File: Author Response.docx