Antidiabetic and Hypolipidemic Properties of Newly Isolated Wild Lacticaseibacillus paracasei Strains in Mature Adipocytes

Round 1

Reviewer 1 Report

Grigorova et al tried to investigate the impact of Lacticaseibacillus paracasei supernatants, derived from newly identified wild strains, native ecological niches in Bulgaria, on adipogenesis, glucose uptake, and some genes involved in the insulin signaling pathway. They simulated the condition as adipogenic differentiation successfully mimics obesity-related systematic flux of fatty acids, similar to the insulin resistance condition. The topic is considered original as the identification and characterization of any new strain of probiotics is valuable. However, the authors did not compare their results with similar investigations on probiotics. Therefore, the filled gap in the area is not clarified completely. This point should be further discussed in the discussion and conclusion sections with related references. Depending on the response to the above (general) comment and the following specific comments, the manuscript can be further reconsidered.

 

Other comments

 

1-     Since the authors have not added the strain as a food additive, a shorter title is recommended. For example: “Antidiabetic and Hypolipidemic Properties of Newly Isolated Wild Lacticaseibacillus paracasei Strains in Mature Adipocytes”

 

2-     In the background, the available data regarding the same genus should be provided such as “Health-Promoting Properties of Lacticaseibacillus paracasei:…..” 

 

3-     Authors should define in the materials and methods on page 3 if the designed primers are based on rRNA sequences or any other gene in the bacteria

 

4-     Results, section 3.1, The following sentence should be revised as the use of  “any” is not correct in this context: “The MTT assay evaluated any cytotoxicity effect after 24 or 48 hours of exposure…” 

 

5-     The authors should compare their results with other data having a similar strategy conducted by adipocyte-loaded probiotics and discuss similar or controversial observations 

 

 

Extensive English editing especially in the discussion section is required.

Author Response

• Since the authors have not added the strain as a food additive, a shorter title is recommended. For example: “Antidiabetic and Hypolipidemic Properties of Newly Isolated Wild Lacticaseibacillus paracasei Strains in Mature Adipocytes”

         Answer: The title has been changed according to the reviewer’s suggestion.

• In the background, the available data regarding the same genus should be provided such as “Health-Promoting Properties of Lacticaseibacillus paracasei:…..”Answer: The Introduction section has been rewritten following the suggestions of all reviewers. Health-Promoting Properties of Lacticaseibacillus paracasei have been also included in the paragraph from Line 53 (90).
• Authors should define in the materials and methods on page 3 if the designed primers are based on rRNA sequences or any other gene in the bacteria

        Answer: 

• Primers used were derived from 16S rRNA V1 region of Lactobacillus paracasei, and 16S rRNA conserved sequence of Lactobacillus (Ward, L. J. H., and Timmins, M. J. 1999. Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction. Lett. Appl. Microbiol. 29: 90-92.)
• The following sentences have been added to the text: “Primers used were derived from 16S rRNA V1 region of L. paracasei, and 16S rRNA conserved sequence of Lactobacillus [40]. The procedures have been described in detail in our previous studies [32,37]” (Line 106 (164).

 

• Results, section 3.1, The following sentence should be revised as the use of “any” is not correct in this context: “The MTT assay evaluated any cytotoxicity effect after 24 or 48 hours of exposure…”

        Answer: The sentence has been changed to “The MTT assay evaluated the                             cytotoxicity after 24 or 48 hours of exposure…”

• The authors should compare their results with other data having a similar strategy conducted by adipocyte-loaded probiotics and discuss similar or controversial observations

         Answer: In the Discussion section, a new paragraph has been added                                 (Line 377 (463) to Line 395 (485)), including information about                               recent research similar to our experiment strategies and                                           methodology.

• Extensive English editing especially in the discussion section is required.

        Answer: Proofreading of the manuscript and English editing has been made                      by a Sworn translator and interpreter. English proofreading certificate                      is applied.

Reviewer 2 Report

Grigorova et al have presented a well-structured research article entitled “Exploring the Antidiabetic and Hypolipidemic Properties of Newly Isolated Wild Lacticaseibacillus paracasei Strains in Mature Adipocytes as Perspective Food Additives”. The article is original, well structured; easy to read with main emphasis on the comparative study of various strains of selected microorganism as food additives for antidiabetic activities. In my opinion, the manuscript can be published in this journal, after the authors have addressed the following minor issues:

·       Authors are suggested to improve the introduction section by adding the significance of this research.

·       Authors are suggested to improve the quality of figures and graphs.

·       Mention about the procedure of identification of new strains of microorganism besides species specific PCR and ADRA techniques. Also represent the results of this segment. Can we confirm the newly identified strain by these two techniques?

·       At few places, spelling correction and grammatical proofreading is required.

·       Authors are suggested to improve the conclusion by adding future prospective as per the standard format.

·       The authors are suggested to dually check the citations throughout the manuscript.

Few grammatical and typological errors needs to be rectified.

Author Response

• Authors are suggested to improve the introduction section by adding the significance of this research.

         Answer: The Introduction section has been rewritten following the                                        suggestions of all reviewers. We have tried to put more focus on                              the significance of the current research.

• Authors are suggested to improve the quality of figures and graphs.

         Answer: The quality of all figures and graphs have been improved.

• Mention about the procedure of identification of new strains of microorganism besides species specific PCR and ADRA techniques. Also represent the results of this segment. Can we confirm the newly identified strain by these two techniques?

         Answer: 

• The procedure for the identification of newly isolated stains has been described in our previous study including the results obtained by usage of species-specific PCR and ARDRA techniques (Dinev, T.; Rusenova, N.; Velichkova, K.; Beev, G. Antimicrobial potential of eleven Lacticaseibacillus paracasei strains isolated from mountain anthills.  J. Agric. Sci.2022, 28, 949–955. Beev, G.; Michaylova, M.; Dinev, T.; Naydenova, N.; Tzanova, M.; Urshev, Z. ARDRA Analysis on biodiversity of lactobacilli isolated from Bulgarian raw buffalo milk. Acta Microbiol. Bulg. 2021, 37, 22–26.
•  Primers used for species-specific identification were derived from 16S rRNA V1 region of Lactobacillus paracasei, and 16S rRNA conserved sequence of Lactobacillus and are successfully used by: Ward, L. J. H., and Timmins, M. J. 1999. Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction. Lett. Appl. Microbiol. 29: 90-92; Roy, D.; Ward, P.; Vincent, D.; Mondou, F. Molecular identification of potentially probiotic lactobacilli. Curr. Microbiol. 2000, 40, 40–46; Walter, J.; Tannock, G.W.; Tilsala-Timisjarvi, A.; Rodtong, S.; Loach, D.M.; Munro, K.; Alatossava, T. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 2000, 66, 297–303. And Patent US20100196341A1

 

• At few places, spelling correction and grammatical proofreading is required.

        Answer: Proofreading of the manuscript and English editing has been made                        by a Sworn translator and interpreter. English proofreading                                      certificate is applied.

• Authors are suggested to improve the conclusion by adding future prospective as per the standard format.

        Answer: The following sentences have been included in the Conclusion                              section, Line 521 (617): “The obtained results reveal that P4 and                          M2.1  L. paracasei strains provide a solid reason for further investigation                 and could emerge as a promising method to overcome obesity- related                  metabolic disorders. However, additional investigations and in vivo                          studies are required to add value to the reported effects.”

• The authors are suggested to dually check the citations throughout the manuscript.

         Answer: The citations have been checked and corrected where needed.

Reviewer 3 Report

Dear authors, here you will find some comments on your work, in general it seems very well written.

 

The keywords must be completely different from those used in the title, to give the document greater visibility in search engines.

 

The introduction is well written, however, I recommend defining the general objective of the research and the working hypothesis, so that the conclusions can be contrasted, and the research developed carried out.

 

In the materials and methods separate the °C, h, µm from the number, change the nomenclature of M per mol.

 

Line 234, colors ® as super index.

 

Line 257, because a p<0.01 is reported if the statistical analysis was carried out with a p≤0.05. Regardless of the probability results, significant differences are reported as p≤0.05 and non-significant ones as p>0.05 (when the significance level is 95%).

 

Figure 1 reports a p<0.01, indicate in the methodology if different statistical differentiation tests were performed.

 

The Figures present standard deviations, how many times were the tests performed? It is not mentioned in the methodology.

 

Separate Figures 1 and 2 for greater visibility of the images, there are too many images together.

 

The discussion seems appropriate. Once the working hypothesis has been established, contrast it in this section.

 

The conclusions must respond to the stated objectives.

Author Response

Dear authors, here you will find some comments on your work, in general it seems very well written.

• The keywords must be completely different from those used in the title, to give the document greater visibility in search engines.

         Answer: The keywords have been replaced by: “probiotics, glucose consumption, insulin sensitivity, lipolysis, lipid accumulation, 3T3-L1, cell-free supernatants, food additives.”.

• The introduction is well written, however, I recommend defining the general objective of the research and the working hypothesis, so that the conclusions can be contrasted, and the research developed carried out.

        Answer: The Introduction section has been rewritten following the suggestions of all reviewers. We tried to clarify the overall aim of the current study at the end of the Introduction section as well as in the paragraph added from Line 377 (463) in the Discussion.

•  In the materials and methods separate the °C, h, µm from the number, change the nomenclature of M per mol.

         Answer: The suggested corrections have been made.

•  Line 234, colors ® as super index.

          Answer:The index has been corrected.

•  Line 257, because a p<0.01 is reported if the statistical analysis was carried out with a p≤0.05. Regardless of the probability results, significant differences are reported as p≤0.05 and non-significant ones as p>0.05 (when the significance level is 95%). 

         Answer: Statistical analyses of the obtained results were performed via the nonparametric Mann-Whitney U test, by which the exact magnitude of differences was estimated. We have presented them as p<0.05 (when the significance level is 95%); p<0.01 (when the significance level is 98%); p<0.001 (when the significance level is 99%).

Therefore, we have corrected the explanation included in the Material and Method section from: “P values ≤ 0.05 were assumed as statistically significant.” to “P values less than 0.05 were assumed as statistically significant.”

•  Figure 1 reports a p<0.01, indicate in the methodology if different statistical differentiation tests were performed.

Answer: We applied the same statistical method to analyze the data achieved in our investigation.

• The Figures present standard deviations, how many times were the tests performed? It is not mentioned in the methodology.

         Answer: The entire experiment was performed once, as each group was presented in six biological replicates. (Line 136 (196))

• Separate Figures 1 and 2 for greater visibility of the images, there are too many images together.

        Answer: We have separated the images included in Figures 1a and 1b and have also increased the amount of text included in the remaining figures.

• The discussion seems appropriate. Once the working hypothesis has been established, contrast it in this section.

        Answer: In the Discussion section, a new paragraph (Line 377 (463) to Line 395 (482)) has been added, including information about recent research involved similar to our experiment strategies and methodology but contradicting the obtained results (Line 396 (483)).

• The conclusions must respond to the stated objectives.

         Answer: In conclusion we have added information concerning the effects of supernatants from C8 and C15 strains (Line 521(612)).

Reviewer 4 Report

The manuscript entitled "Exploring the Antidiabetic and Hypolipidemic Properties of Newly Isolated Wild Lacticaseibacillus paracasei Strains in Mature Adipocytes as Perspective Food Additives" is interesting to know how the bacterial byproducts influence adipocyte functional regulation. However, the author of this manuscript has lots of issues to be clarified before it goes for publication.

1 Introduction. It is too long and hard to follow the story and needs to be rewritten and made precise. 

2.. Methodology: Glucose uptake and glucose utilization? I need clarification on what is the difference between uptake and utilization. The author used uptake and then said utilization in the same sentence. Uptake is different from utilization. If the author wants to use utilization, he needs to measure the product of glucose after utilization of glucose. The uptake protocol and the description should have been provided in that section. 

3. Not only mRNA analysis will confirm the functional relationship with protein. Needing Western Blot for that protein is important to show the effect of cell-free supernatant on those proteins. 

4. In figure bar diagrams. No need to provide the percentage values on top of the bars instead author can mention in the text where it will be appropriate.

 

Hence, authors need provide more data using additional experiments, and rewriting the manuscript will help the manuscript is publishable.

Author Response

• Introduction. It is too long and hard to follow the story and needs to be rewritten and made precise. 

         Answer: The Introduction section has been rewritten following the suggestions of all reviewers.

• Methodology: Glucose uptake and glucose utilization? I need clarification on what is the difference between uptake and utilization. The author used uptake and then said utilization in the same sentence. Uptake is different from utilization. If the author wants to use utilization, he needs to measure the product of glucose after utilization of glucose. The uptake protocol and the description should have been provided in that section. 

        Answer: We have replaced “glucose utilization” with “glucose consumption” in the whole manuscript and have added a brief explanation of the method used in Line 178 (338).

• Not only mRNA analysis will confirm the functional relationship with protein. Needing Western Blot for that protein is important to show the effect of cell-free supernatant on those proteins. 

        Answer: The objective of the present study was to conduct preliminary tests on the potential anti-obesity and hypoglycemic effects of newly isolated L. paracasei strains. Therefore, protein-level investigations were not planned at this stage. We anticipate conducting more extensive in vitro and in vivo studies to further explore the potential health benefits of these microorganisms in the future.

In Line 521 (617) we have included the following paragraph: “The obtained results reveal that P4 and M2.1 L. paracasei strains provide a solid reason for further investigation and could emerge as a promising method to overcome obesity-related metabolic disorders. However, additional investigations and in vivo studies to add value to the reported effects are required.”

• In figure bar diagrams. No need to provide the percentage values on top of the bars instead author can mention in the text where it will be appropriate.

         Answer: In all figures, we have removed the numerical values that were previously displayed above the bars.

 

Round 2

Reviewer 1 Report

The manuscript can be accepted.

Minor to moderate English quality might be required.

Author Response

• Comments on the Quality of English Language: Minor to moderate English quality might be required.

         Answer: Language editing for the manuscript has been reconsidered, and additional minor corrections have been made by the Sworn translator and interpreter.

Reviewer 4 Report

Appreciate the authors' response. However, authors should provide some clarification and information mentioned below.

References: I could not see reference 21 in the reference section but I do see the 21 references were cited in the text. 

I also did not see references 24-27 in the reference sections, however, I do see it was cited in the text.

The author needs to either provide the appropriate references in the reference sections or remove them from the cited text and make sure the information in the text does not lack reference citations.

Author Response

Comments and Suggestions for Authors: Appreciate the authors' response. However, authors should provide some clarification and information mentioned below.

• References: I could not see reference 21 in the reference section but I do see the 21 references were cited in the text. 

         Answer:Thank you very much for your note. It seems that a technical problem occurred during the PDF conversion of the manuscript file and some newly added References in the Word file were hidden in the PDF version of our manuscript.

• I also did not see references 24-27 in the reference sections, however, I do see it was cited in the text.

Answer: Thank you very much for your note. It seems that a technical problem occurred during the PDF conversion of the manuscript file and some newly added References in the Word file were hidden in the PDF version of our manuscript.