The Impact of Formulation and Freeze Drying on the Properties and Performance of Freeze-Dried Limosilactobacillus reuteri R2LC

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript titled “Impact of formulation and freeze drying on the properties and performance of freeze-dried Limosilactobacillus reuteri R2LC” (No. applmicrobiol-2707371) determined the biological and physicochemical properties of freeze-dried Limosilactobacillus reuteri R2LC under different cytoprotectants (trehalose and sucrose), bacteria concentration and freeze-drying parameters. The experiments designed for this study were appropriately performed with some data. However, there several drawbacks in this manuscript should be pointed out. Firstly, the title of this manuscript covers impact of formulation of freeze-dried Limosilactobacillus reuteri R2LC, but it is hard to find how the cytoprotectants trehalose and sucrose protect strain R2LC cells from injury. Authors used SEM to observe the structure of the freeze-dried bacterial cells embedded by trehalose and sucrose, but they did not support any evidence that illuminated the mixture’s structure. Secondly, Figure 1 specifies how to design these experiments, but the manuscript’ structure was not well-organized because authors did not provide a clear route to make this manuscript more coherent. Thirdly, several sections, including the parts Results and Conclusion, were generally organized to be more like a textbook rather than a research article. Overall, authors have to make a major revision to their manuscript before resubmitting to the Journal Applied Microbiology.

Specific points are issued as follows.

1/Line 12--Line18, it is too much words for introducing the probiotics and freeze-drying topics, please simply it.

2/For the part Abstract, it should highlight some symbol data and measuring methods rather than display lots of conclusion, and thus needs to be redrafted for directly indicating the main purpose of this study.

3/The keyword “Live Biotherapeutics” is important for this manuscript? And the key word “Limosilactobacillus reuteri R2LC” should be written in italic.

4/Line 38-60, too much words with no sense description because of irrelevant information (for example live biotherapeutic products), and should be redrafted to directly indicate the importance of protectants and the effect of freeze-drying on lactic acid bacteria.

5/Line 88, what means “interesting”? please more detail.

6/For the sections 2.6 and 2.7, References should be added.

7/Line 201, the survival of bacteria should be displayed using formula.

8/The manufacturer and model for the instruments need to be specified, for instance line124.

9/Line 227-231, should be deleted.

10/For the part “3. results”, authors should draw upon data alteration to support the trend of experiment results. For example, see line 243-247, 258-260, 275-278, 286-287,333-338.

11.For the part “4. Discussion”, Line 391-398 should be deleted because they are independent of this manuscript. For Line 399-411 and Line 423-431, authors should discuss their own data rather than other author results. For Line 414-416, these sentences are correct？

12/The part Conclusion should be redrafted and simplified.

13/For the part References, journal name should be unified for their formats. And the Latin name for species should be written in italic.

Author Response

This manuscript titled “Impact of formulation and freeze drying on the properties and performance of freeze-dried Limosilactobacillus reuteri R2LC” (No. applmicrobiol-2707371) determined the biological and physicochemical properties of freeze-dried Limosilactobacillus reuteri R2LC under different cytoprotectants (trehalose and sucrose), bacteria concentration and freeze-drying parameters. The experiments designed for this study were appropriately performed with some data. However, there several drawbacks in this manuscript should be pointed out.

Firstly, the title of this manuscript covers impact of formulation of freeze-dried Limosilactobacillus reuteri R2LC, but it is hard to find how the cytoprotectants trehalose and sucrose protect strain R2LC cells from injury. Authors used SEM to observe the structure of the freeze-dried bacterial cells embedded by trehalose and sucrose, but they did not support any evidence that illuminated the mixture’s structure.

Thank you for the comment. The matrix structure was studied by SEM and porosity was measured by image analysis. We realize that those results have not been sufficiently described and we therefore have added information about the matrix structure in different bacterial concentrations along with results obtained from porosity analysis and updated the images shown in Figure 5 and results about porosity in Supplementary Table 6.

Secondly, Figure 1 specifies how to design these experiments, but the manuscript’ structure was not well-organized because authors did not provide a clear route to make this manuscript more coherent.

We have elaborated Figure 1 which now describes both the design of experiment and characterization of the freeze-dried products, to provide the reader with a clear route for the structure of the manuscript. We have also reorganized parts of the manuscript to make it more coherent.

Thirdly, several sections, including the parts Results and Conclusion, were generally organized to be more like a textbook rather than a research article. Overall, authors have to make a major revision to their manuscript before resubmitting to the Journal Applied Microbiology.

Thank you for the comment. We have revised the manuscript with the aim to improve the structure and highlighted the changes in yellow.

Specific points are issued as follows.

1/Line 12--Line18, it is too much words for introducing the probiotics and freeze-drying topics, please simply it.

We have modified those lines and the changes are highlighted in yellow.

2/For the part Abstract, it should highlight some symbol data and measuring methods rather than display lots of conclusion, and thus needs to be redrafted for directly indicating the main purpose of this study.

We have revised the abstract accordingly and the changes are highlighted in yellow.

3/The keyword “Live Biotherapeutics” is important for this manuscript? And the key word “Limosilactobacillus reuteri R2LC” should be written in italic.

Formulation and the freeze-drying process are of great importance for both live biotherapeutics and probiotics, and therefore we have included both terms. Limosilactobacillus reuteri R2LC is now changed to italics in the keywords.

4/Line 38-60, too much words with no sense description because of irrelevant information (for example live biotherapeutic products), and should be redrafted to directly indicate the importance of protectants and the effect of freeze-drying on lactic acid bacteria.

We have revised this section and the changes are highlighted in yellow.

5/Line 88, what means “interesting”? please more detail.

Thanks for the comment. We have described some examples of beneficial effects of Limosilactobacillus reuteri R2LC in pre-clinical studies.

6/For the sections 2.6 and 2.7, References should be added.

Reference for subsection 2.7 have been added. The method described in section 2.6 have been optimised and developed by us and no reference has been added.

7/Line 201, the survival of bacteria should be displayed using formula.

Formula for survival of bacteria has been added.

8/The manufacturer and model for the instruments need to be specified, for instance line 124.

Detailed information about the manufacturer and models has been added to the manuscript.

9/Line 227-231, should be deleted.

We agree. The lines 227-231 have been deleted.

10/For the part “3. results”, authors should draw upon data alteration to support the trend of experiment results. For example, see line 243-247, 258-260, 275-278, 286-287,333-338.

Some of the results are of a complex nature, but we have added some text to support the trends of the results.

11.For the part “4. Discussion”, Line 391-398 should be deleted because they are independent of this manuscript. For Line 399-411 and Line 423-431, authors should discuss their own data rather than other author results. For Line 414-416, these sentences are correct？

We have now revised the ‘Discussion’ as commented by the reviewer.

12/The part Conclusion should be redrafted and simplified.

We have excluded the Conclusion section and instead provided a redrafted sense of it towards the end of the Discussion section.

13/For the part References, journal name should be unified for their formats. And the Latin name for species should be written in italic.

We have now rectified the format of ‘References and Journal name’ and also corrected the writing of Latin names to italic.

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript is well written, the experimentation has been correctly designed and teh discussion is scientifically sound. There are some small corections.

In section 2. Material and methods, subsection 2.2, 2.3, 2.4, 2.5, 2.6, 2.7 is without any reference. Please, provide adequate reference on mentioned methods.

Author Response

Manuscript is well written, the experimentation has been correctly designed and the discussion is scientifically sound. There are some small corrections.

 

In section 2. Material and methods, subsection 2.2, 2.3, 2.4, 2.5, 2.6, 2.7 is without any reference. Please, provide adequate reference on mentioned methods.

Thank you for your comments. Reference for subsection 2.4, 2.5 and 2.7 have been added. While method subsections 2.2, 2.3 and 2.6 have been optimised and developed by us and there are no references.

Reviewer 3 Report

Comments and Suggestions for Authors

For the convenience of the authors, the reviewer's comments and suggestions are noted in the manuscript.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Just a few suggestions - please, see the manuscript (attached).

Author Response

1. Line 14. While "product-specific considerations" are spelled out, "opportunities" are not clear. Delete?

           Thanks for the comment. We have deleted ‘opportunities.

 

2. Line 17. Freeze-drying may be "typical" in food applications when probiotic cultures are used as an additive. However, in many cases they are also involved in fermentation process. Perhaps, "often", instead of "typically"?

We have replaced ‘typically’ with ‘often’.

 

3. Line 22. I am not sure if this term can/should be applied to bacteria. A quick search on PubMed revealed that the term is used to refer to bacteria, but mostly by authors from non-English-speaking countries. Usually, instead of vitality authors mention metabolic activity. However, this is your choice, I guess.

We understand the reviewer’s concern and have changed vitality to metabolic activity throughout the manuscript.

 

4. Line 26. What kind of correlation? Liner, non-linear, etc.?

Pearson’s correlations (i.e. linear correlations; see table 6).

 

5. Lines 27-28, 49. Personally, I would prefer just seeing "metabolic activity", especially since "vitality" was not evaluated, just one parameter was studied, right?

See comment 3 above.

 

6. Line 50. There are food products with probiotics present as additives: snacks, drinks, dried fruits, tea sachet, etc. Perhaps, this should be corrected.

We have changed ‘fermented products’ to ‘food products and probiotic based food products’.

 

7. Line 88. Please, mention at least a few. Were there any clinical studies with this strain? Please, mention that as well (I am unaware of any).

To the best of our knowledge there is no clinical study conducted with this strain but there are several published pre-clinical studies. We have added a brief description of some of these studies to the introduction part of the manuscript.

 

8. Line 115. Here and elsewhere, please, be consistent when referring to the vendor/manufacturer. First time mentioning should contain: name of the company, city, state (for the US), and country. Every next time, just the name of the company is sufficient.

We have added this information.

 

9. Line 120. Here and elsewhere: please, be consistent and follow the journal's instructions. I am guessing, it should be: mL, L.

We have changed to mL.

 

10. Lines 204-210. Why not say that metabolic activity was assessed by the ability of cells isolated from a lyophilized sample to acidify the growth substrate, namely MRS broth. As I read this fascinating technical report, I am increasingly convinced that this is not “vitality”, but the ability to acidify as a reflection of metabolic activity - for instance, see here: doi: 10.3389/fbioe.2021.612285. As for "vitality" and "metabolic activities" covering more than just acidification, of course, more comprehensive analysis will be required (for instance, doi: 10.22207/JPAM.14.1.68; doi: 10.1016/j.ijfoodmicro.2010.06.003, etc.

We understand the reviewer’s concern and have changed vitality to metabolic activity.

 

11. To conclude, I feel the authors should be more specific and just say that the metabolic activity was assessed as ability of the microorganisms to acidify their growth substrate.

We agree, see above.

 

12. Line 206. The suggested reference is irrelevant a) The paper by Wendel is a review and contains no technical details related to the methods used by the authors of this manuscript; b) The paper by Wendel does not contain the word "vitality". Please, consider another, appropriate, reference with technical details.

Thank you for your comment. We have now changed the reference to Ribeiro et al. (2021).

 

13. Line 207. What kind of atmosphere was used? reuteri is a microaerophilic organism.  L. reuteri grow well in MRS broth under aerobic conditions so the tubes were incubated at 37°C in normal atmosphere.
14. Lines 270-272. What exactly the read should see in this microphotographs, what is the take-home message? Please, consider spelling it in the figure legend and pointing with an arrow if appropriate.

The message from SEM images was a) to highlight the matrix structure in general and b) to demonstrate how well the bacterial cells are embedded or protected by the formulation in the matrix. This has now been described in the text and highlighted in figure 5.

 

15. Line 403. Here and elsewhere: first time mentioned microorganism should be spelled in full. The authors may consider saying "Lactiplantibacillus (formerly Lactobacillus) plantarum... "

Thanks for the comment. We have now changed L. plantarum to Lactiplantibacillus plantarum throughout the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript titled “Impact of formulation and freeze drying on the properties and performance of freeze-dried Limosilactobacillus reuteri R2LC” (No. applmicrobiol-2707371) investigated physicochemical and biological properties of freeze-dried Limosilactobacillus reuteri R2LC. It verified that the lyoprotectants sucrose was better than trehalose in terms of survival rate, metabolic activity and storage stability of the strain R2LC. These data could provide a new insight for probiotic starters formula. Additionally, authors have largely revised their manuscript according to reviewers’ comments. However, minor points have to be addressed for authors’ consideration before acceptance.

1/For the part “2. Materials and methods”,“Statistical analysis” about the data should be added.

2/Line 72-75, what is logic connection between authors’ results and previous researches? Hard to understand.

3/Line 383, what means of “quality”? Hard to understand.

Author Response

1/For the part “2. Materials and methods”, “Statistical analysis” about the data should be added.

Thank you for your comment. We have now added subsection 2.13 ‘Statistical analysis’ and highlighted it in blue color.

 

2/Line 72-75, what is logic connection between authors’ results and previous researches? Hard to understand.

The logic behind providing details about previous researches is to offer a concise background of the strain Limosilactobacillus reuteri R2LC.

 

3/Line 383, what means of “quality”? Hard to understand.

Thank you for your comment. We have now replaced ‘quality’ with ‘biological properties’ and highlighted in blue color.