Protective Role of Limosilactobacillus fermentum Lf2 and Its Exopolysaccharides (EPS) in a TNBS-Induced Chronic Colitis Mouse Model

Round 1

Reviewer 1 Report

The article examines the effect of a probiotic strain Limosilactobacillus fermentum Lf2 and its exopolysaccharides on a murine model of colitis. In this study, for the first time, a strain and the EPS produced by it are examined simultaneously. The results obtained are interesting and significant. I have some notes that can improve the manuscript.

1. Abstract: Although clear designation with letters, it is better that the groups of mice are given their full names. For example, "treatment with the strain" is better than "L".

2. The legend of the figures diverges in order from the shown in the figures. For example, in most figures, L precedes E in the figure, but follows it in the legend.

3. In tables the phyla should be in italics.

4. Metagenomic studies should be deposited in a database, eg, NCBI, with biosample, bioproject, SRA (rough data), etc.

In my opinion, Cyanobacteria can also be removed from the study. How do you explain the fact that after the treatment with the probiotic strain, the "Lactobacillus" proportion does not increase to the levels of treatment with EPS?

Author Response

The article examines the effect of a probiotic strain Limosilactobacillus fermentum Lf2 and its exopolysaccharides on a murine model of colitis. In this study, for the first time, a strain and the EPS produced by it are examined simultaneously. The results obtained are interesting and significant. I have some notes that can improve the manuscript.

We would like to thank the reviewer for all the valuable suggestions and for taking the time to revise the manuscript. All the feedback was appreciated and carefully considered throughout the revision process.

1. Abstract: Although clear designation with letters, it is better that the groups of mice are given their full names. For example, "treatment with the strain" is better than "L".

The amendment was made as suggested (lines 26 to 37).

2. The legend of the figures diverges in order from the shown in the figures. For example, in most figures, L precedes E in the figure, but follows it in the legend.

The legends were modified following the correct order (lines 242-243, 339-341, 390-392, 427-429, 446-448).

3. In tables the phyla should be in italics.

The amendment was made in Table 1.

4. Metagenomic studies should be deposited in a database, eg, NCBI, with biosample, bioproject, SRA (rough data), etc. 

All raw data was deposited in the Sequence Read Archive, as part of the bioproject PRJNA1063264 (lines 497-498).

In my opinion, Cyanobacteria can also be removed from the study.

This phylum was removed from Table 1 and the discussion (lines 256-259).

How do you explain the fact that after the treatment with the probiotic strain, the "Lactobacillus" proportion does not increase to the levels of treatment with EPS?

In 2020, a taxonomic reorganization of the lactic acid bacteria reclassified over 300 species in 7 genera and 2 families into one family, the Lactobacillaceae, with 31 genera including Lactobacillus, Paralactobacillus, Pediococcus, Weissella, Fructobacillus, Convivina, Oenococcus, Leuconostoc, and 23 new genera that comprise organisms formerly classified as Lactobacillus species (Qiao et al., 2022, https://doi.org/10.3168/jdsc.2021-0183; Zheng et al., 2020, https://doi.org/10.1099/ijsem.0.004107). For this reason, the global impact of the particular strain used in this study is difficult to assess by the levels observed in this family, as many of the bacterial genera included in Lactobacillaceae could have been regulated and modified simultaneously.

Moreover, the intestinal microbiota is quite a complex system in which different factors are involved in the overall response to a certain treatment. In this direction, in a previous study of our group (Ale et al., 2019, https://doi.org/10.1016/j.idairyj.2019.04.014), it was shown that the EPS from Lf2 was able to regulate the intestinal microbiota, effect reflected in an increase in faecal SCFA levels, which could be explained by an increase in the levels of the bacterial groups that produce these acids. In this context, the EPS could have promoted the increase of Lactobacillaceae by different metabolic pathways that did not necessarily take place during the administration of the strain itself.

This response was briefly added to the discussion (lines 280-289).

Reviewer 2 Report

The study entitled "Protective role of Limosilactobacillus fermentum Lf2 and its exopolysaccharides (EPS) in a mouse model of TNBS-induced chronic colitis" is an original work that investigated the effects of strain and metabolites on TNBS-induced chronic colitis.

Comments:

Rewrite the highlights where you will state the achievements of this study.

Rewrite the abstract where you will point out the achievements of this study. The results obtained after the PCA analysis are important, but you did not point them out in this section.

It is necessary to rewrite the Introduction because there is not enough information in this section regarding the current achievements in this field. Additionally, more than 50% of the references in the Introduction are older than five years, so there is not enough state-of-the-art on this topic.

The concentration of the strain Lf2 in the treated group L was selected based on which parameters? In group E, the EPS concentration of 0.6 mg was chosen based on which parameters?

Does the concentration of 0.6 mg of purified EPS correspond to 3x10⁸ CFU? If the statement is correct, provide proof of it, if it isn't correct, why these ratios of 0.6 mg and 3x10⁸ CFU were used, they can't be compared with each other (it can be in the effect of the Lf2 strain on chronic colitis, but it doesn't have to mean that it's due to EPS)?

Provide s-IgA determination protocol (briefly explain how intestinal fluid was provided).

Rewrite a methodology for measuring oxidative stress parameters in the liver (written like this, it is hard to see how it was done).

Why translocation of bacteria in the liver was done using the classic microbiological method, and not by sequencing? By sequencing it was possible to determine much more precisely which bacteria were translocated to the liver and thus you would add more importance to this study.

In the results, section 3.2, the presentation of translocation results was not presented, but only written "values < 2 log (CFU/g), suggesting that Lf2 and its EPS might be safe to be used as food ingredients" and additionally stated that "further analyzes are needed to confirm their safety". What safety assessment analyses are we talking about here? Present the results in the form of a figure and discuss the obtained result.

Make the figures (2, 3, and 4) so that they are more transparent and that the significance can be more easily observed. Instead of letters (a, b, and c) use asterisks for significance and connect the groups that are being compared.

The discussion is poorly written, and a lot of data is not discussed (the whole section "3.5. Multivariate Analyses" is not discussed and not given importance even though it is perhaps one of the most important analyses of this study.).

Studies examining the effects of bacteria (especially LAB and probiotics) and their metabolites on colitis have already been done, and it is necessary to do something in such studies to make this study recognizable. I think that this study has the potential to be recognizable to other researchers after review and rewriting. Additionally, no mechanism has been demonstrated or even suggested as to how this strain or its metabolite affects colitis.

Specific comments:

Do not start a sentence with abbreviations (Lines 55, 96, 99, 100, …)

Line 97: instead of “plus” write “with”; Name the glycerol manufacturer;

Line 108: Specify the freeze-dried protocol;

Line 110: Briefly describe the purification protocol;

Line 251: introduce an abbreviation MLN 

Author Response

The study entitled "Protective role of Limosilactobacillus fermentum Lf2 and its exopolysaccharides (EPS) in a mouse model of TNBS-induced chronic colitis" is an original work that investigated the effects of strain and metabolites on TNBS-induced chronic colitis.

We would like to thank the reviewer for all the valuable suggestions and for taking the time to revise the manuscript. All the feedback was appreciated and carefully considered throughout the revision process.

Comments:

Rewrite the highlights where you will state the achievements of this study.

The highlights were rewritten (lines 12-18).

Rewrite the abstract where you will point out the achievements of this study. The results obtained after the PCA analysis are important, but you did not point them out in this section.

The abstract was modified and the PCA analysis was added as suggested (lines 26-37).

It is necessary to rewrite the Introduction because there is not enough information in this section regarding the current achievements in this field.

A paragraph about recent advances in this field was added (lines 73-86).

Additionally, more than 50% of the references in the Introduction are older than five years, so there is not enough state-of-the-art on this topic.

Several references were replaced with more recent ones (highlighted in yellow in the list). Those references related to the definition of probiotics, prebiotics and synbiotics are not very recent but we would like to keep them as they are the consensus definitions provided by the ISAPP (International Scientific Association for Probiotics and Prebiotics). Besides, references regarding previous studies of this particular strain (L. fermentum Lf2) were kept as well, as we think they are meaningful to the background of this work.

The concentration of the strain Lf2 in the treated group L was selected based on which parameters? In group E, the EPS concentration of 0.6 mg was chosen based on which parameters?

In a previous study of our group (Ale et al. 2019, https://doi.org/10.1016/j.idairyj.2019.04.014), a daily dose of 9 mg/kg/d EPS from Lf2 showed health-promoting properties in naive mice. In this work, an even higher dose (30 mg/kg/d EPS) was chosen, as we used a mouse model challenged with intrarectal TNBS, seeking a positive effect. The concentration of bacteria was similar to the one used by Burns et al. 2017 (doi: 10.1038/srep43211), 2x10⁸ CFU/day, since we followed the same protocol for the colitis mouse model.

Does the concentration of 0.6 mg of purified EPS correspond to 3x10⁸ CFU? If the statement is correct, provide proof of it, if it isn't correct, why these ratios of 0.6 mg and 3x10⁸ CFU were used, they can't be compared with each other (it can be in the effect of the Lf2 strain on chronic colitis, but it doesn't have to mean that it's due to EPS)?

The concentration chosen for EPS does not necessarily correspond to the concentration used for Lf2. The reason why these levels were selected is explained above. We fully agree with the reviewer, in order to directly relate the properties of the strain to its EPS, a knock-out mutant unable to produce EPS would have been necessary as a negative control. This latter statement is already mentioned in the conclusion (lines 480-481), as we were aware of this limitation.

Provide s-IgA determination protocol (briefly explain how intestinal fluid was provided).

This information was added as requested (lines 188-191).

Rewrite a methodology for measuring oxidative stress parameters in the liver (written like this, it is hard to see how it was done).

The information was provided (lines 193-205).

Why translocation of bacteria in the liver was done using the classic microbiological method, and not by sequencing? By sequencing it was possible to determine much more precisely which bacteria were translocated to the liver and thus you would add more importance to this study.

We appreciate the suggestion, and we agree with the reviewer on the fact that sequencing samples from the liver would have provided much more information, but, unfortunately, sequencing services are quite expensive in Argentina, and the economic situation of our country is delicate. For this reason, we give priority to sequencing DNA from faecal samples to study the impact of the treatment on gut microbiota, which is our main objective, we hope the reviewer understands. Furthermore, and according to preliminary in vivo studies of the strain and its EPS, we expected to have no translocation to the liver.

In the results, section 3.2, the presentation of translocation results was not presented, but only written "values < 2 log (CFU/g), suggesting that Lf2 and its EPS might be safe to be used as food ingredients" and additionally stated that "further analyzes are needed to confirm their safety". What safety assessment analyses are we talking about here? Present the results in the form of a figure and discuss the obtained result.

Translocation of bacteria to the liver is just a routine assay we perform to corroborate this does not occur during the treatments, so we consider that adding a figure or table might not provide much valuable information. In this case, the majority of mice presented levels <1 log(CFU/mL), which is the detection limit of this technique.

By "further analyses are needed to confirm their safety" we meant assessing translocation to extra-gut parts such as the spleen, kidney and blood, but as lactobacilli are generally regarded as safe (Pradhan et al. 2020, https://doi.org/10.1016/j.foodcont.2019.106872), and no negative side effects were observed during the assay, these determinations were not included. A statement about the safety of the strain was added (lines 325-328).

Make the figures (2, 3, and 4) so that they are more transparent and that the significance can be more easily observed. Instead of letters (a, b, and c) use asterisks for significance and connect the groups that are being compared.

Figures 2, 3 and 4 were modified so that significant differences are shown only. The use of (*) might be confusing as 4 treatments were compared simultaneously by ANOVA. For this reason, we think that letters are clearer to indicate differences among all the treatments.

The discussion is poorly written, and a lot of data is not discussed (the whole section "3.5. Multivariate Analyses" is not discussed and not given importance even though it is perhaps one of the most important analyses of this study.).

This section was discussed as suggested (lines 454-473).

Studies examining the effects of bacteria (especially LAB and probiotics) and their metabolites on colitis have already been done, and it is necessary to do something in such studies to make this study recognizable. I think that this study has the potential to be recognizable to other researchers after review and rewriting. Additionally, no mechanism has been demonstrated or even suggested as to how this strain or its metabolite affects colitis.

The mechanisms were discussed (lines 459-473).

Specific comments:

Do not start a sentence with abbreviations (Lines 55, 96, 99, 100, …)

The amendments were made (lines 66, 122, 126, 127).

Line 97: instead of “plus” write “with”; Name the glycerol manufacturer;

This part was modified as indicated (lines 123-124).

Line 108: Specify the freeze-dried protocol;

The protocol was added (line 136).

Line 110: Briefly describe the purification protocol;

This information was added (lines 139-143).

Line 251: introduce an abbreviation MLN 

The abbreviation was clarified (line 314).

Round 2

Reviewer 2 Report

Dear Authors,

After the review, the general impression of this Manuscript has been significantly improved and I suggest that it be accepted for publication.

I would have a suggestion to bring back data that was deleted after review (I assume due to reviewer suggestions, such as lines 130, 132, 133, 134, 157, 178-181). I think it is necessary to enter data such as centrifugation conditions, preparation of anesthetic cocktail, etc. in the Methodology section.

Best Regards

Author Response

After the review, the general impression of this Manuscript has been significantly improved and I suggest that it be accepted for publication.

We are glad that the manuscript met the reviewer’s expectations, and we are grateful for all the fruitful feedback received, which noticeably improved its quality.

I would have a suggestion to bring back data that was deleted after review (I assume due to reviewer suggestions, such as lines 130, 132, 133, 134, 157, 178-181). I think it is necessary to enter data such as centrifugation conditions, preparation of anesthetic cocktail, etc. in the Methodology section.

The data was kept as suggested (lines 120-123, 162-166), except for the part in line 157 which was formerly mentioned (now line 142) (only the order changed in R1).