The Effect of a Glutathione (GSH)-Containing Cryo-Protectant on the Viability of Probiotic Cells Using a Freeze-Drying Process

Round 1

Reviewer 1 Report

·        The submission is a valuable short communication on how glutathione is revisited as probiotic cryo-protectant during freeze-drying process

·        Please provide in the Introduction some information on why there is a need to find other freeze-drying cryo-protectants for the probiotics, as long as there are several solutions already;

·        Line 60-61: in the uploaded PDF the microorganisms names are not written in Italic format; please apply changes. Same issues appear in other different sections of the manuscript (like lines 147-153; 170-173; 188-190; 220).

·        Line 78: please provide the freeze-drying conditions while using the equipment

·        Line 123: “well” is the correct word? What is the meaning here?

·        Line 213; can you explain what do you mean by “the cells emerged”?

·        Line 227: is there a need to have twice the word “coating”?

·        Can you make an assumption on how cost efficient versus benefits can bring such coating solution on industrial level?

Author Response

13 February, 2023

Article ID: Fermentation-2220352

 

Dear reviewer in fermentation

 

We are very grateful for the comments of reviewers to improve the quality of the article.

This manuscript has been revised based on the comments in the review.

Thanks for your attentive comments.

Below is a summary of the answers to each comment.

 

Reviewer 1.

1. The submission is a valuable short communication on how glutathione is revisited as probiotic cryo-protectant during freeze-drying process

+ Please provide in the Introduction some information on why there is a need to find other freeze-drying cryo-protectants for the probiotics, as long as there are several solutions already;

Reply:

Some information was added in the introduction part in line.

“However, some protectants may cause undesirable effects such as whey and skimmed milk might cause adverse effects to hosts due to lactose intolerance and the allergy to milk protein, and some are metabolized by Lactobacillus with the production of acids which have a negative effect on the survival rate.” (page 1, line 40-44)

Thank you for your comment.

 

2.  Line 60-61: in the uploaded PDF the microorganisms names are not written in Italic format; please apply changes. Same issues appear in other different sections of the manuscript (like lines 147-153; 170-173; 188-190; 220).

Reply:

The microorganisms names were written in Italic format in all of manuscript.

 

3. Line 78: Please provide the freeze-drying conditions while using the equipment

Reply:

Corrected as advised. Thank you for your comment.

“The cell mixture was pre-frozen at −80 °C and freeze-dried (drying at different steps -30 °C, -20 °C, -10 °C, 0 °C, 10 °C, 20 °C, 30 °C and vacuum 0.5 mbar) for 24 h using a freeze dryer (Ilshin Biobased Co., Dongducheon, Korea).” (page 2, line 77-79).

 

4. Line 123: “well” is the correct word? What is the meaning here?

Reply:

Thank you for your comment

“well” is the correct word, it comes from “12-well plates” which is a material for cell culture.

 

5. Line 213; can you explain what do you mean by “the cells emerged”?

Reply:

We changed “the cells emerged” to “the cells were covered” in lines 253-254

 

6. Line 227: is there a need to have twice the word “coating”?

Reply:

The word “coating” has been deleted from manuscript

 

7. Can you make an assumption on how cost efficient versus benefits can bring such coating solution on industrial level?

Reply:

The GSH is now produced on an industrial scale at an affordable price. Therefore, it can replace other traditional materials without much concern about the price.

“Furthermore, the GSH is now produced on an industrial scale at an affordable price.” (Page 8, line 273-274)

Reviewer 2 Report

The manuscript contain interesting results, nevertheless the Results and Discussions section must be improved since, even though, the results are well presented and described, they are poorly discussed. In addition to the above, please attend to the observations included in the attached file.

Comments for author File: Comments.pdf

Author Response

13 February, 2023

Article ID: Fermentation-2220352

 

Dear reviewer in fermentation

 

We are very grateful for the comments of reviewers to improve the quality of the article.

This manuscript has been revised based on the comments in the review.

Thanks for your attentive comments.

Below is a summary of the answers to each comment.

 

Reviewer 2.

1. The manuscript contain interesting results, nevertheless the Results and Discussions section must be improved since, even though, the results are well presented and described, they are poorly discussed. In addition to the above, please attend to the observations included in the attached file.

Reply:

Thank you for your comment

Some discussions have been added to manuscript

“A loss of viability in accelerated storage conditions may explain by the oxidation of membrane lipid and protein denaturation resulting in macromolecular degradation of probiotic cells, which occurs faster at high temperatures.” (Page 5-6, 211-214)

“Additionally, Kim et al. presented that the collagen- peptide-contained protectant prevents membrane disruption, and maintains the structural integrity of the cell membrane barrier [26].”

(Page 7, 256-258)

 

2. Line 13, 60 and table 1 Lactiplantibacillus Instead of Lactoplantibacillus

Reply:

Thank you for your comment

We changed “Lactoplantibacillus” to “Lactiplantibacillus” in all of manuscript

 

3. Line 36 sugar alcohols instead of sugar alcohol

Reply:

We changed “sugar alcohol” to “sugar alcohols” in line 36

 

4. Line 37 gel compounds instead of gel compound

Reply:

We changed “gel compound” to “gel compounds” in line 37

 

5. Line 54-57 The information exposed in these lines is more suitable to be described in the methodology section. I do not consider of importance to be included in the introduction section. I leave it to your consideration.

Reply:

We deleted these sentences from the introduction part.

 

6. Line 67 I would prefer to abbreviate B. animalis ssp. Lactis instead of Bi. animalis ssp. Lactis

I leave it to your consideration. If the change is done, please apply to the full manuscript. If it is a format according to the journal instructions, ignore my suggestion.

Reply:

Thank you for your comment

We changed “Bi. animalis ssp. lactis” to “B. animalis ssp. lactis” in all of manuscript

 

7. Line 83 I would prefer to abbreviate L. lactis instead of Lc. lactis

I leave it to your consideration. If the change is done, please apply to the full manuscript. If it is a format according to the journal instructions, ignore my suggestion.

Reply:

We changed “Lc. lactis” to “L. lactis” in all of manuscript

 

8. Line 94 is it gastrointestinal (GI) tract? Please check

Reply:

We changed “gut and intestinal” to “gastrointestinal (GI)” in line 95

 

9. Line 141-142 I would change “between the results” by “among treatments” I leave it to your consideration

Reply:

Thank you for your comment

We changed “between the results” to “among treatments” in line 144-145

 

10. Line 147-150 the paragraph must be rewrite since the main idea is lost in a long sentence.

Thank you for your comment

the sentence has been rewritten in line.

“Viability was determined after the freeze-drying process. The viability comparison was carried out between the non-coated cells, coating with basic protectants containing skim milk, sucrose (SK), and coating with protectants containing GSH.” (Page 4, lines 149-151)

 

11. Line 163 and 209 among means within each strain instead of between means

Reply:

We changed “between means” to “among means within each strain” in all of manuscript

 

12. Line 171 I did not find what does GIT mean

Reply:

We changed “GIT” to “gastrointestinal tract” in line 176

 

13. Line 227 Check coating repeated

Reply:

Corrected as advised. The word “coating” has been deleted from the manuscript.

Thank you for your comment.

Round 2

Reviewer 2 Report

The manuscript was significantly improved. I would have liked that the discussions were deeper. Nevertheless i agree with the publication of this paper.