Development of a Lactic Bacteria Starter for Amaranth Silage and Investigation of Its Influence on Silage Quality

Round 1

Reviewer 1 Report

The present study strive to develop a lactic acid bacteria starter for Amaranth silage and also investigated its influence on silage quality. However, there are several substantial confounding going on which are mentioned below.

Line 29, please write the scientific names of fodder crops.

Please provide more background related to influence of lactic acid bacteria on silage quality.

Please use italic font while writing bacterial species names; L51-54.

L49, from where did you isolated lactic acid bacteria strains? Please provide source.

L58, why did you grow lactic acid bacteria for only 16 hours?

L106-107; unclear, generally silage samples are dried to determine the chemical composition; while ensiled samples (without drying) are used for organic acid determination.

Please give detailed method for organic acid determination.

Statistical analysis is unclear; did you perform one-way analysis of variance?

Figures 2, 3, 4; please show the significance difference among treatments

Figures are not attractive; please use GraphPad prism or other good software to draw figures.

Table 3,4; unclear, why did you use comma in the middle of values such as 15,6±0,2? Please revise it and also provide the significance among treatments.

Please discuss your results with consent of previous studies.

Moderate improvement is needed.

Author Response

1. Thank you for your comment. Comment corrected
2. Supplemented the Discussion on the effect of LAB on the quality of silage in paragraph 3.3
3. Thank you for your comment. Comment corrected
4. Provided in the Materials and methods, paragraph 3.1
5. To determine antibacterial activity against test cultures LAB strains were grown for 16 h, by which time they synthesized bacteriocins
6. Corrected in the Materials and methods, paragraph 2.5
7. Indicate what the method for determining organic acids is based on and provide a link to English-language literature.
8. Yes, one-way analysis of variance was performed
9. Supplemented the tables with P-values. In the discussion of the results, we indicated the significance of the differences between different treatments of amaranth
10. Tried to improve figures
11. Thank you for your comment. Comment corrected
12. Corrected the Discussion of the results, taking into account the comments

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

evaluating new starter cultures for ensiling valuable plants is of increasing interest for years. Thus, the purpose of the study is justified. Nevertheless, the article has serious flaws:

- the abstract is not informative enough;

- there is a mistake in interpretation, bacteriocins are not antibiotics, so you did not evaluated antibiotic activity of strains;

- are you sure that there is no other studies on nutritional value of amaranth and its silage? You did not discussed your results in chapter 3.2. at all;

- please write names of microorganisms italics;

- some methods are described not clear e.g. chapter 2.1;

- you do not describe why production of bacteriocins was so important. Does amaranth is predistined for infections with undesirable microorganims? You did not measured them in silage.

- why did you use only one criterium for LABs selection? what about lactic acid synthesis or biomass yield etc.;

- I have no idea on what basis did you selected three starter cultures. Moreover, two of them you did not use in silage experiments at all. What was the point of presenting some results related to them;

- are you sure that "Baktosil" contained living LAB cells, becouse there is no difference in number of cells between variant 3 and 4;

- you did not provide post-hoc tests after ANOVA, so you cannot tell whether there were any significant differences between groups;

- conclusions give not as much interesting points as it could be.

English of the manuscript is simple and in my opinion need to be improved and more sophisticated. Some words are wrongly used like ferment (line 10), seed material (line 71), these are not scientific definitions.

Author Response

1. Thank you for your comment. Comment corrected
2. We agree that bacteriocins exhibit antibacterial activity, corrected in text
3. Added new references on amaranth and its silage. Corrected the Discussion of the results, taking into account the comments
4. Thank you for your comment. Comment corrected
5. Thank you for your comment. Rewrote paragraph 2.1
6. Completed the discussion of the results in paragraph 3.2. Bacteriocins exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli, which, when ensiled, along with other undesirable microorganisms, can enter the silage and multiply intensively in plant sap. We did not determine the content of these microorganisms specifically; we monitored the dynamics of changes in the content of LAB, ammonifying bacteria, and yeast.
7. Selection was initially made from LAB strains with good biomass yield and lactic acid production. Since amaranth is a hard-to-silage raw material, it was necessary to select strains that could have a positive effect on the fermentation process, not only due to the synthesis of lactic acid.
8. Three experimental starter cultures were made from LAB strains with antibacterial activity, which differed most in terms of acid formation and optimal growth temperatures. The idea was that strains of different properties in the composition of starter cultures would be included in the ensiling process sequentially, which would increase the efficiency of fermentation. Of these 3 starter cultures, 2 were selected (in one, antagonism between strains was observed), which were used for ensiling amaranth in comparison with the industrial starter culture "Baktosil" and without treatment.
9. Baktosil starter contained live bacteria, but it turned out to be ineffective for amaranth ensiling, so there were few differences from the control silage without starter.
10. Tables 3 and 4 present P-values. It is difficult to indicate the significance of differences in the figures. They are calculated, if necessary, we can provide a separate file.
11. Conclusion corrected

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors have substantially improved the manuscript in revised version and addressed all the raised concerns, therefore, I recommend its publication. 

Minor improvement is need.

Reviewer 2 Report

Thank you very much for providing necessary corrections.