A Feed Additive Containing Encapsulated 6-Phytase within Recombinant Yarrowia lipolytica Cells Produced by Cultivation on Fat-Containing Waste

Round 1

Reviewer 1 Report

There is no logic in the introduction, what is the purpose of this study?

The results are not sufficiently clear and concise, and the discussion does not adequately compare this study with others.

There are still many details that need to be amended.

 

1. P2, L78: First appearance of lipolytica,what is the full name?
2. P3, L98:g/l should be g/L.
3. P3, L105: mlshould be mL.
4. P4, L182-183 andP5, L205-226: for descriptions and explanations like this, please put them in the discussion.
5. P6-7, It is recommended that the four diagrams in Figure 1 be combined.
6. Starting from Figure 2, the page number is chaotic.
7. It is suggested to delete Figure 3.
8. The references are very confusing, with some article and journal titles in all lower case and some in all upper case, and some journals in all caps and some in lower case.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript “A feed additive containing encapsulated 6-phytase within re-2 combinant Yarrowia lipolytica cells produced by cultivation on 3 fat-containing waste” by Maria А. Danilova et al. is devoted to the consideration encapsulated phytase. Exogenous phytases c are used on an industrial scale as an effective means to reduce the harm caused to the environment by industrial animal husbandry, as well as to increase the profitability of poultry and pig farming. These possibilities are related to the ability of phytase to release phosphorus from phytate contained in plant feeds. However, enzymes derived from yeast are extremely sensitive to the effects of temperature, and encapsulation will reduce this effect. The manuscript can presents an interest to the readers of Applied Sciences, however, now it is chaotic and requires serious revision.

1. This study does not have a clearly formulated goal, and the relevance of the work carried out is also in doubt. As far as I understand, the authors have not used encapsulation technology for the first time. Are there really no similar additives in the agricultural industry (enzymes encapsulated in Yarrowia lipolytica)? What is the advantage of the study of this group of authors?
2. The text of the manuscript lacks one of the key sections of any experimental/review article - the conclusion, thanks to which the reader could get information about the main theses of this study.  
3. The Discussion section mostly includes a review of the literature data that does not show the mechanisms and processes studied in this work, and represents only a set of known data. The authors should think about writing a review article on this topic, but in this manuscript, the “Discussion” section should be modified. Moreover, the use of table 3 will be more appropriate in the section “Introduction”, but not in the “Discussion” section.
4. The authors say that encapsulation of the enzyme increases its thermal stability. Experimental data concerning the thermal stability of the system created by the authors should be presented.
5. Lines 205-210. The authors point out: "Biological trials of feed phytase preparations are usually carried out on broilers and pigs” and in such studies “a complex analysis of the physiological activity of phytase preparation is carried out” in particular, on “carcass parameters; relative weights of visceral organs; meat quality parameters; phosphorous content in excreta; length of the bones; relative yield of the carcass and the meat; physical, mechanical and chemical properties of the tibia; and morphology of the jejunum”. Has an autopsy of animals been performed for pathomorphological examination of internal organs and bones? Without such an analysis (autopsy), the mice could gain weight by depositing only adipose tissue.
6. The placement of the graphs shown in Figure 1 is inconvenient for perception. Moreover, it is unclear why individual graphs are built for each diet. Since the same controls were used for all four graphs, perhaps it is necessary to combine the results of the remaining experimental groups on one graph?
7. In the caption to Figure 1, the authors need to sign the number of mice in each group.
8. Were the mice kept in individual cages? If not, how many mice were in the cage? It is known that mice have their own hierarchy in the cage, and a mouse that is stressed by the attacks of the leader can eat less food and also gain weight. Was this data taken into account?
9. It is unclear for what purpose the authors present two essentially identical parts of the table, differing only in the absence and presence of specific p values? One part is enough.
10. Table 2, row 7, column 2. The average value is less than the error of the average - the authors should pay attention. Is this a misprint?
11. Authors should combine the values of the average and the errors of the average in one column.
12. The authors should decrypt the use of the “*" symbols in the table caption and by what criterion reliability was determined. In addition, authors should pay attention to the use of symbols indicating the levels of statistical significance of the results. So, one asterisk (*) always decrypts asp ≤ 0.05; two asterisks (**) p ≤ 0.01 and three asterisks (***) p ≤ 0.001. However, the authors use the sign of only one asterisk in all cases.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors,

The manuscript that you presented needed major corrections. 

Line 130: I think you should present rotation in rpm?

Line 148: How do you know that the size was 0.9 mm, it depends on the used machine or you measure it?

Line 171: Please describe what mean ADG?

Table 1: Please change "," for ".", and give all results with two decimal places. % in the first column should be in "[]". I suggest changing "nutritional fact" for " Nutritional parameter" or something else. 

Figure 1:  Please change the x-axis "days" to "time [days]". what do these deviations in the figure refer to? If to the weight (I think so) then they should be in a different direction.

Table 2: Should the results be up to three decimal places, or is this the accuracy of the test method? 

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The introduction is too wordy and the discussion is inadequate, so it presents a top-heavy presentation. The reference management software recommended by journals is not just apply it mechanically.

Author Response

Dear Reviewer,

Thank you so much for the criticism. We reduced the introduction (description of PhyB and PhyD groups of the phytases is abandoned since they are not practically used for feeding). Therefore, references 19-25 are removed. We added two paragraphs in the top of the Discussion section. We hope that they explain why we choose the swine blood as a substrate for cultivation of the OPP producing strain. We would like to strengthen the conclusion that the biological activity of the microencapsulated OPP is much higher than one of the Ladozym Proxi control preparation. In our opinion this effect compensates low yield of the non-secretory enzyme in comparison to the modern secreted producer strains based on micellar fungi. All changes in this are highlighted in blue.

The list of references was reformatted manually.

Two formal changes are done on our initiative:

1. Affiliation of the first author Maria A. Danilova in IBCP is abandoned; the single affiliation in VIGG RAS remains in the title of the manuscript.
2. Acknowledgments section is added. It includes a text “The study was supported with a research grant 204-21/223 since Nov. 12, 2021 by Russian ministry of science and higher education”.

 

On behalf of the author team,

Sincerely Alexei Shevelev

Reviewer 2 Report

The authors addressed all the comments appropriately. The manuscript is now suitable for publication.

Author Response

Dear Reviewer,

We reduced the introduction (description of PhyB and PhyD groups of the phytases is abandoned since they are not practically used for feeding). Therefore, references 19-25 are removed. We added two paragraphs in the top of the Discussion section. We hope that they explain why we choose the swine blood as a substrate for cultivation of the OPP producing strain. We would like to strengthen the conclusion that the biological activity of the microencapsulated OPP is much higher than one of the Ladozym Proxi control preparation. In our opinion this effect compensates low yield of the non-secretory enzyme in comparison to the modern secreted producer strains based on micellar fungi. All changes in this are highlighted in blue.

The list of references was reformatted manually.

Two formal changes are done on our initiative:

1. Affiliation of the first author Maria A. Danilova in IBCP is abandoned; the single affiliation in VIGG RAS remains in the title of the manuscript.
2. Acknowledgments section is added. It includes a text “The study was supported with a research grant 204-21/223 since Nov. 12, 2021 by Russian ministry of science and higher education”.

 

On behalf of the author team,

Sincerely Alexei Shevelev

Reviewer 3 Report

Dear Authors, 

Taking into account the corrections in the text, I suggest editors to accept the article.

Author Response

Dear Reviewer,

We reduced the introduction (description of PhyB and PhyD groups of the phytases is abandoned since they are not practically used for feeding). Therefore, references 19-25 are removed. We added two paragraphs in the top of the Discussion section. We hope that they explain why we choose the swine blood as a substrate for cultivation of the OPP producing strain. We would like to strengthen the conclusion that the biological activity of the microencapsulated OPP is much higher than one of the Ladozym Proxi control preparation. In our opinion this effect compensates low yield of the non-secretory enzyme in comparison to the modern secreted producer strains based on micellar fungi. All changes in this are highlighted in blue.

The list of references was reformatted manually.

Two formal changes are done on our initiative:

1. Affiliation of the first author Maria A. Danilova in IBCP is abandoned; the single affiliation in VIGG RAS remains in the title of the manuscript.
2. Acknowledgments section is added. It includes a text “The study was supported with a research grant 204-21/223 since Nov. 12, 2021 by Russian ministry of science and higher education”.

 

On behalf of the author team,

Sincerely Alexei Shevelev

Round 3

Reviewer 1 Report

Very important: please delete Table 1 and carefully condense the abstract and introduction sections.

It is important to keep the abstract concise and the introduction organised to highlight the purpose of the research.

Author Response

Dear Reviewer,

Thank you so much for the criticism. We reduced the summary: two sentences with explanation of shortcoming of the secreting producers of the phytases are removed. The introduction is also reduced: Table 1 is removed. Therefore, references 20 appearing for the first in the Discussion changed the number.

Numeration of the Tables is changed along the whole text.

 

On behalf of the author team,

Sincerely Alexei Shevelev