The Antioxidant, Anti-Diabetic, and Anti-Adipogenesis Potential and Probiotic Properties of Lactic Acid Bacteria Isolated from Human and Fermented Foods

Round 1

Reviewer 1 Report

The manuscript proposed by the authors is very interesting and specially is on line with the topic to looking for bioactive strains with a biocomplex of activities like the probiotic one, antioxidant, and others.

The article is also well written, well organized, and in my opinion could be accepted with some just minor revision.

-The abstract of the manuscript has to be improved, adding more details related to the test carried out and also introducing more results obtained.

Author Response

Response to Reviewer 1 Comments

July 2nd, 2021

Dear reviewer in Fermentation

We are sincerely grateful for your thorough consideration and scrutiny of our manuscript, “Probiotic properties and the anti-oxidant, anti-diabetic, and anti-adipogenesis potential of lactic acid bacteria isolated from human and foods”, manuscript number Fermentation-1264116. Through the accurate comments made by the reviewers, we better understand the critical issues in this paper. We have revised the manuscript according to the reviewer’s suggestions. We appreciate that the scientific and clinical quality of our manuscript was improved by the scrutinizing efforts of the reviewers and editors. We hope that our revised manuscript will be considered and accepted for publication in the Fermentation.

The changes within the revised manuscript were highlighted (underlined and in red). Point-by-point responses to the reviewers’ comments are provided below.

<GENERAL COMMENTS>

The manuscript proposed by the authors is very interesting and specially is on line with the topic to looking for bioactive strains with a biocomplex of activities like the probiotic one, antioxidant, and others.

The article is also well written, well organized, and in my opinion could be accepted with some just minor revision.

Major points:

Point 1: The abstract of the manuscript has to be improved, adding more details related to the test carried out and also introducing more results obtained.

Response 1: Thank you for your comments. Your comments were greatly improved our data. We summarized results as representing each value in the abstract.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this research article, the authors investigated the probiotic potential of LAB strains isolated from fermented foods, human feces, and breast milk. More specifically, the authors tested the strains for safety features, such as hemolytic activity and antimicrobial resistance, and for functional properties, namely antioxidant and anti-adipogenic potential.  The authors concluded that four out of 15 LAB strains presented significant antioxidant, α-glucosidase inhibitory, and anti-adipogenesis activity.

There are several points in the manuscript that require attention:

Introduction:

Lines 29-31: the authors should add a reference to this claim.

Lines 34-35: in these lines, the authors refer to conditions that are associated with gut dysbiosis, however, they refer to oxidative stress as well, which is not a condition. Maybe the authors could add oxidative-stress-related diseases.

A general comment on the introduction is that the authors could refer to the cellular mechanisms involved in obesity and T2D to better introduce the reader to the rationale of the assays the authors performed to test their hypothesis.

Materials & Methods:

Section 2.2: the authors could add a table presenting the strains tested and their origin.

Section 2.4: did the authors use any positive control to evaluate the antioxidant potential of the CFEs? Also, what was the control sample of the ABTS assay consisted of?

Line 108: the authors should add the abbreviation for PNPC.

Lines 115-116: the way the sample types are described is confusing. Do the authors mean that B is the absorbance with neither α-glucosidase nor the sample? Also, how do these samples correlate to the control sample in Figure 2?

Results & Discussion:

Section 3.1: the authors could present their results as %inhibition, measuring the decrease in radical content compared to the negative control. The authors included the negative control sample in the graphs however that could be mistaken for a positive control sample. Additionally, the authors could add a positive control sample to describe how the antioxidant ability of the CFEs compared to that of compounds with known antioxidant activity, such as ascorbic acid.

Figure 1, legend, line 207-208: did the authors use whole viable bacteria or CFEs?

Lines 232-233: What do the authors mean by “L. rhamnosus GG (LGG), best known as LABs”? Do they mean that LGG is one of the most well studied LAB strains?

Lines 233-237: the authors could include some of these strains in their experimental procedures and directly compare their activity to the studied strains.

Section 3.5: the fact that the strains can auto-aggregate does not necessarily mean that they will have good adherence capacity on intestinal cells. For that claim to be made, the authors should perform appropriate experiments. Furthermore, in Lines 302-303 the authors compare the auto-aggregation activity of the tested strains with that of LGG that was not included in this study. It would be optimal if the authors included LGG as a reference strain in these experiments.

Table 4, legend, line 349: maybe the terms reaction/no reaction would be more appropriate.

A general comment on the Results & Discussion section is that the authors could describe in greater detail why the observed phenotypes can be beneficial.

Lines 211-216: the authors could refer in greater detail to the molecular pathways involved in the antioxidant effect of LABs and to the bioactive compounds that mediate this effect.

Lines 301-302: this sentence is quite vague, and it seems that it takes away the focus from the objectives of this paper. The authors could discuss how auto-aggregation and attachment may mediate effects relevant to those observed in this work.

Lines 331-346: what does the metabolic profile of each strain tell us about their attributes and potential beneficial effects on the host? The authors could add a comment on this to contextualize their findings.

Author Response

Response to Reviewer 2 Comments

July 5th, 2021

Dear reviewer in Fermentation

We are sincerely grateful for your thorough consideration and scrutiny of our manuscript, “The antioxidant, anti-diabetic, and anti-adipogenesis potential and probiotic properties of lactic acid bacteria isolated from human and fermented foods”, manuscript number Fermentation-1264116. Through the accurate comments made by the reviewers, we better understand the critical issues in this paper. We have revised the manuscript according to the reviewer’s suggestions. We appreciate that the scientific and clinical quality of our manuscript was improved by the scrutinizing efforts of the reviewers and editors. We hope that our revised manuscript will be considered and accepted for publication in the Fermentation.

The changes within the revised manuscript were highlighted (underlined and in red). Point-by-point responses to the reviewers’ comments are provided below.

<GENERAL COMMENTS>

In this research article, the authors investigated the probiotic potential of LAB strains isolated from fermented foods, human feces, and breast milk. More specifically, the authors tested the strains for safety features, such as hemolytic activity and antimicrobial resistance, and for functional properties, namely antioxidant and anti-adipogenic potential.  The authors concluded that four out of 15 LAB strains presented significant antioxidant, α-glucosidase inhibitory, and anti-adipogenesis activity.

There are several points in the manuscript that require attention.

Major points:

Point 1: Introduction: Lines 29-31: the authors should add a reference to this claim.

Response 1: Thank you for your comment, your comments were greatly improved our data. According to Derosa et al. (2012), cardiovascular outcomes trial study in 2010 showed a 16% rise in the risk of non-fatal myocardial infarction or stroke in people taking sibutramine. Thus, we added the reference (Derosa et al., 2012) to support the sentence in line 32, and Reference part.

Point 2: Introduction: Lines 34-35: in these lines, the authors refer to conditions that are associated with gut dysbiosis, however, they refer to oxidative stress as well, which is not a condition. Maybe the authors could add oxidative-stress-related diseases.

A general comment on the introduction is that the authors could refer to the cellular mechanisms involved in obesity and T2D to better introduce the reader to the rationale of the assays the authors performed to test their hypothesis.

Response 2: Thank you for your comment. Your comments helped make this study more convincing. We revised and clarified the sentence as your comments in line 36-37.  

Point 3: Materials & Methods: Section 2.2: the authors could add a table presenting the strains tested and their origin.

Response 3: Thank you for your comment, your comments helped make this study more convincing. We represented the information in Supplementary table 1 and briefly described in line 72-73. This data would be better to show in supplemental data rather than the main text.

Point 4: Materials & Methods: Section 2.4: did the authors use any positive control to evaluate the antioxidant potential of the CFEs? Also, what was the control sample of the ABTS assay consisted of?

Response 4: Thank you for your comment. Your comments were greatly improved our data. We found that we missed the positive control (10 μg/ml of Ascorbic acid) and negative control (PBS) in Figure 1.  In this regard, the main text (line 98-100 and line 109) and legend of Figure 1 (line 250) were corrected to clarify.

Point 5: Materials & Methods: Line 108: the authors should add the abbreviation for PNPC.

Response 5: Thank you for your comment. Your comments were greatly improved our data. We mentioned the full name (4-nitrophenyl α-D-glucopyranoside) in 2.1. Materials in M&M part (line 62-63).

Point 6: Materials & Methods: Lines 115-116: the way the sample types are described is confusing. Do the authors mean that B is the absorbance with neither α-glucosidase nor the sample? Also, how do these samples correlate to the control sample in Figure 2?

Response 6: Thank you for your comment. Your comments helped make this study more convincing. We revised and clarified the sentence as representing their description (line 126). In addition, we described the control sample (PBS) in line 123. Your comments helped make this study more convincing.

Point 7: Results & Discussion: Section 3.1: the authors could present their results as %inhibition, measuring the decrease in radical content compared to the negative control. The authors included the negative control sample in the graphs however that could be mistaken for a positive control sample. Additionally, the authors could add a positive control sample to describe how the antioxidant ability of the CFEs compared to that of compounds with known antioxidant activity, such as ascorbic acid.

Response 7: Thank you for your comment. Your comments helped make this study more convincing. We corrected the Figure 1 as showing negative (PBS) and positive controls (10 μg/mL of Ascorbic acid).

Point 8: Results & Discussion: Figure 1, legend, line 207-208: did the authors use whole viable bacteria or CFEs?

Response 8: Thank you for your comment. Your comments were greatly improved our data. For the antioxidant activity, we used cells as whole viable cells. To avoid ambiguity, we clarified the type of the sample in line 96 and line 109.

Point 9: Results & Discussion: Lines 232-233: What do the authors mean by “L. rhamnosus GG (LGG), best known as LABs”? Do they mean that LGG is one of the most well studied LAB strains?

Response 9: Thank you for your comment. Your comments were greatly improved our data.  We corrected the sentence in line 261 as “~, one of the most well studied LAB, ~”.

Point 10: Results & Discussion: Lines 233-237: the authors could include some of these strains in their experimental procedures and directly compare their activity to the studied strains.

Response 10: Thank you for your comment. We agree with your opinion. However, we would like to avoid the legal disputes between company vs. company. Therefore, we compared the results with other previous studies as an alternative.

Point 11: Results & Discussion: Section 3.5: the fact that the strains can auto-aggregate does not necessarily mean that they will have good adherence capacity on intestinal cells. For that claim to be made, the authors should perform appropriate experiments. Furthermore, in Lines 302-303 the authors compare the auto-aggregation activity of the tested strains with that of LGG that was not included in this study. It would be optimal if the authors included LGG as a reference strain in these experiments.

Response 11: Thank you for your comment. Your comments were greatly improved our data. We agreed with your opinion. We performed cell adhesion activity using human colonic cell line HT-29, and replaced the results in section 2.9 and 3.5 with the newly obtained data.

Point 12: Results & Discussion: Table 4, legend, line 349: maybe the terms reaction/no reaction would be more appropriate.

Response 11: Thank you for your comment. Your comments helped make this study more convincing. We changed the terms as your comments in line 394. Thank you.

Point 13: Results & Discussion: Lines 211-216: the authors could refer in greater detail to the molecular pathways involved in the antioxidant effect of LABs and to the bioactive compounds that mediate this effect.

Response 13: Thank you for your comment. Your comments were greatly improved our results. Although the antioxidative activity of LAB have been confirmed in vitro and in vivo, the mechanism has not been fully understood in the present study. However, antioxidant potential of our strains might be the effect of enzymes or metabolites. According to review of Feng et al., (2020), LAB exhibit antioxidant functions through various cellular mechanisms (enzymes, repair of damaged protein and DNA, metabolites). We discussed this in main text in line 229-244 in revised MS.

Point 14: Results & Discussion: Lines 301-302: this sentence is quite vague, and it seems that it takes away the focus from the objectives of this paper. The authors could discuss how auto-aggregation and attachment may mediate effects relevant to those observed in this work.

Response 14: Thank you for your comments. Your comments were greatly improved our data.  We performed cell adhesion activity using human colonic cell line HT-29, and replaced the results in section 2.9 and 3.5 with the newly obtained data.

Point 15: Results & Discussion: Lines 331-346: what does the metabolic profile of each strain tell us about their attributes and potential beneficial effects on the host? The authors could add a comment on this to contextualize their findings.

Response 15: Thank you for your comments. Your comments helped make this study more convincing. We added more elucidative description for the carbohydrate fermentation profiles in line 383-387.

Author Response File: Author Response.pdf

Reviewer 3 Report

Manuscript 1264116

The manuscript deals with the evaluation of probiotic, antioxidant, antidiabetic, and anti-adipogenesis lactic acid bacteria isolated from humans and foods. With this aim diverse tests were performed. Results for several strains were promising. Overall, the manuscript includes valuable information. However, explanations of graphs require clarification. In its current version is not straightforward. Particularly, the way comparisons are achieved is unclear. Another observation; there are numerous comparisons between percentages. However, comparison of percentages would require specific statistical tools, which apparently were not used. Some explanations?

Specific

L13 and through text. “…bacteria (LABs)…”. The “s” is not necessary because bacteria is plural

Table S1. The antioxidant activity is missing

L147. Apparently, the concentration of the washed cells is not in the proper position.

L191. The name of the program is missing.

L198. The sentence is confusing. The range compared to the control?

L202. Idem previous. Please clarify this type of sentence through text.

L203. Please, indicate the range.

L207. The sentence needs clarification.

L232.  “.., best known as LABs,…” . the meaning of this sentence is unclear.

L248. Was considered the influence of taking several differences simultaneously?

L259-265. Please, include the p-values corresponding to these correlations or at least an overall significance.

264. Please, include the full name of all organisms the first time they are mentioned.

Table 2. This table requires a better explanation (e.g. log10CFU/mL, below Initial). Idem below the rest of the headings. Percentages below survival may lead to confusion; use an additional column.

L293-294. The sentence could be removed since it was already mentioned in Material & Methods.

L325. Idem previous.

Figures. The use of black bars is not appealing. Use color or different textures in bars

Author Response

Response to Reviewer 3 Comments

July, 5th, 2021

Dear reviewer in Fermentation

We are sincerely grateful for your thorough consideration and scrutiny of our manuscript, “The antioxidant, anti-diabetic, and anti-adipogenesis potential and probiotic properties of lactic acid bacteria isolated from human and fermented foods”, manuscript number Fermentation-1264116. Through the accurate comments made by the reviewers, we better understand the critical issues in this paper. We have revised the manuscript according to the reviewer’s suggestions. We appreciate that the scientific and clinical quality of our manuscript was improved by the scrutinizing efforts of the reviewers and editors. We hope that our revised manuscript will be considered and accepted for publication in the Fermentation.

The changes within the revised manuscript were highlighted (underlined and in red). Point-by-point responses to the reviewers’ comments are provided below.

<GENERAL COMMENTS>

The manuscript deals with the evaluation of probiotic, antioxidant, antidiabetic, and anti-adipogenesis lactic acid bacteria isolated from humans and foods. With this aim diverse tests were performed. Results for several strains were promising. Overall, the manuscript includes valuable information. However, explanations of graphs require clarification. In its current version is not straightforward. Particularly, the way comparisons are achieved is unclear. Another observation; there are numerous comparisons between percentages. However, comparison of percentages would require specific statistical tools, which apparently were not used. Some explanations?

Major points:

Point 1: L13 and through text. “…bacteria (LABs)…”. The “s” is not necessary because bacteria is plural

Response 1: Thank you for your comment. Your comments were greatly improved our data. We deleted the alphabet ‘s’ from ‘LABs’, and double checked the word in MS, Tables, and Figures.

Point 2: Table S1. The antioxidant activity is missing

Response 2: Thank you for your comment. Your comments were greatly improved our data. We changed the table legend as “Inhibitory effects of lipid accumulation in 3T3-L1 adipoctytes cells of lactic acid bacteria (n = 3).”

Point 3: L147. Apparently, the concentration of the washed cells is not in the proper position.

Response 2: Thank you for your comment. Your comments were greatly improved our data. We revised the sentence to be clearer as representing final cell count inoculum in line 161-163.

Point 4: L191. The name of the program is missing.

Response 4: Thank you for your comment. Your comments helped make this study more convincing. We found a flaw in the section, and revised the paragraph as representing statistics tools (SPSS and GraphPad Prism). Overall, the data analysis was performed using R statistics or SPSS, and figures were presented by using GraphPad.

Point 5: L198. The sentence is confusing. The range compared to the control?

Response 5: Thank you for your comments. Yes, it is. Each data compared to the negative control (PBS). We added this to be more clarify the section (line 224-225).

Point 6: L202. Idem previous. Please clarify this type of sentence through text.

Response 6: Thank you for your comments. Your comments were greatly improved our data. We deleted the repeated word or phrase through whole text, Table, and Figures.

Point 7: L203. Please, indicate the range.

Response 4:  Thank you for your comments. As your comment, the revised the sentence as showing the range of scavenging activity of the LAB in line 226-228.

Point 8: L207. The sentence needs clarification.

Response 4: Thank you for your comments. Your comments helped make this study more convincing. We corrected the legend of Figure 1 as showing negative (PBS) and positive controls (10 μg/mL of Ascorbic acid). In addition, we revised and clarified the related sentences as representing their description in M&M part (line 98-100).

Point 9: L232.  “.., best known as LABs,…” . the meaning of this sentence is unclear.

Response 9: Thank you for your comments. As your comment, we corrected the sentence in line 261 as “~, one of the most well studied LAB, ~”.

Point 10: L248. Was considered the influence of taking several differences simultaneously?

Response 10: Sorry for that we could not understand your question. If you mean statistical analysis, yes, we conducted a significance test through analysis of variance (ANOVA) analysis and confirmed significant differences with post-hoc Tukey HSD test.

Point 11: L259-265. Please, include the p-values corresponding to these correlations or at least an overall significance.

Response 11: Thank you for your comment, your comments were greatly improved our data. We included the p-values in line 290-291. Your comments were greatly improved our data.

Point 12: L264. Please, include the full name of all organisms the first time they are mentioned.

Response 12: Thank you for your comment, your comments were greatly improved our data. We mentioned the full name of the organisms when it was used first in text. Thank you for your comments.  

Point 13: Table 2. This table requires a better explanation (e.g. log10CFU/mL, below Initial). Idem below the rest of the headings. Percentages below survival may lead to confusion; use an additional column.

Response 13: Thank you for your comments. Your comments were greatly improved our data. We changed Table 2.

Point 14: L293-294. The sentence could be removed since it was already mentioned in Material & Methods.

Response 14: Thank you for your comments. We deleted the sentence and show the other new data (cell adhesion). Also, as the comment, we tried to avoid the sentences mentioned in M&M part.

Point 15: L325. Idem previous.

Response 15: Thank you for your comments. We deleted the sentence as your comments.

Point 16: Figures. The use of black bars is not appealing. Use color or different textures in bars

Response 16: Thank you for your comments. Your comments were greatly improved our data. We changed color and thickness of bars in all figures. The results seem to be more visible.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors successfully addressed my comments.

The revised manuscript is now suitable for publication.

Author Response

Response to Reviewer 2 Comments (round2)

July 14th, 2021

Dear reviewer in Fermentation

We are sincerely grateful for your thorough consideration and scrutiny of our manuscript, “The antioxidant, anti-diabetic, and anti-adipogenesis potential and probiotic properties of lactic acid bacteria isolated from human and fermented foods”, manuscript number Fermentation-1264116. Through the accurate comments made by the reviewers, we better understand the critical issues in this paper. We have revised the manuscript according to the reviewer’s suggestions. We appreciate that the scientific and clinical quality of our manuscript was improved by the scrutinizing efforts of the reviewers and editors. We hope that our revised manuscript will be considered and accepted for publication in the Fermentation.

<GENERAL COMMENTS>

The authors successfully addressed my comments.

The revised manuscript is now suitable for publication.

Major points:

Point 1: English language and style are fine/minor spell check required

Response 1: Thank you for your comments. As the comment, we received English language editing service. We checked the terms and grammar in the manuscript and edited. 

Author Response File: Author Response.pdf