Effects of Fermented Goat Milk on Adiposity and Gut Microbiota in a Diet-Induced Obesity Murine Model

Round 1

Reviewer 1 Report

The article entitled "Effects of fermented goat milk on adiposity and gut microbiota in a diet-induced obesity murine model" is a good piece of research. It is an interesting addition in the current literature. I have few suggestions to improve this piece of study that should be addressed before being accepted for publication.

Line 35-36:

Sentence does not make sense. Correct it by writing 'Although obesity results from a chronic imbalance between energy intake and expenditure, however there are genetic, behavioral, metabolic, and hormonal factors which can influences on body 36 weight.

Line 49:

Take out "when" before citation.

Line 56-57:

Some authors describe the benefits obtained from the consumption of fermented goat milk (FGM)- you should qoute at least one study here. 

Also the introduction is missing one sentence on why you are choosing this particular strain (Lactobacillus delbrueckii subsp. indicus CRL1447) for fermentation of goat milk.

Section 2.1 misses a citation. Please insert the below citation here (http://dx.doi.org/10.3390/fermentation8090469)

Fig 3a (principal component analysis) is not clear. Increase the font size to make it more visible

Line 364: write "induced" instead of induce

Line 373: Remove double spacing before FER

Line 376: add "the" before increased feeding efficiency

There are some grammatical errors that can be eliminated.

Author Response

Thank you for your helpful and constructive comments. As suggested, all the issues have been carefully addressed. We sincerely hope that the revised manuscript is now satisfactory and acceptable. Revisions were marked in red in the manuscript.

REVIEWER 1:

1) Line 35-36: Sentence does not make sense. Correct it by writing 'Although obesity results from a chronic imbalance between energy intake and expenditure, however there are genetic, behavioral, metabolic, and hormonal factors which can influences on body 36 weight.

RESPONSE: Page 1, lines 38-39: The paragraph in the Introduction section was changed according to the Reviewer's suggestion.

2) Line 49:Take out "when" before citation.

RESPONSE: Page 2, line 51: The word "when" was removed of the sentence according to the Reviewer's suggestion.

3) Line 56-57:Some authors describe the benefits obtained from the consumption of fermented goat milk (FGM)- you should qoute at least one study here. 

RESPONSE: Page 2, lines 59-62: The following paragraph and cite in the Introduction section was added:

" In addition, compared to fermented cow milk, FGM maintains a better nutritional pro-file with higher protein, minerals, and vitamin values and presents a fatty acid profile that is considered more health-promoting [10]".

4) Also the introduction is missing one sentence on why you are choosing this particular strain (Lactobacillus delbrueckii subsp. indicus CRL1447) for fermentation of goat milk.

RESPONSE: Page 2, lines  66-70: The following paragraph and cite in the Introduction section was added:

“Marquez et al., in an intensive study of strain characterization in terms of their probiotic and technological properties, selected Lactobacillus delbrueckii subsp. indicus CRL1447 for developing fermented goat milk [11]. The CRL1447 strain stands out for its high acidification rate in milk, a property of technological interest for developing fermented products”.

5) Section 2.1 misses a citation. Please insert the below citation here (http://dx.doi.org/10.3390/fermentation8090469)

RESPONSE: Page 2, in Material and Methods, section 2.1. 81-83: The following sentence and cite were added:

“These strains, previously characterized for their in vitro probiotic and technology properties, were selected to be assayed on diet-induced obese mice [11]”.

Marquez, A.; Andrada, E.; Russo, M.; Bolondi, M.L.; Fabersani, E.; Medina, R.; Gauffin-Cano, P. Characterization of Autochthonous Lactobacilli from Goat Dairy Products with Probiotic Potential for Metabolic Diseases. Heliyon 2022, 8, e10462, doi:10.1016/j.heliyon.2022.e10462.

The citation suggested by the Reviewer does not correspond to the strain characterization study.

6) Fig 3a (principal component analysis) is not clear. Increase the font size to make it more visible

RESPONSE: Page 9: The figure 3a (currently 5a) was changed

7) Line 364: write "induced" instead of induce

RESPONSE: Page 14, line 404:

The word "induce" was change by "induced" according to the Reviewer's suggestion.

8) Line 373: Remove double spacing before FER

RESPONSE: Page 14, line 413:

The space was removed according to the Reviewer's suggestion.

9) Line 376: add "the" before increased feeding efficiency

RESPONSE: Page 14, line 416:

The word "the" was added according to the Reviewer's suggestion.

REVIEWER 2:

1) More detailed results about the gut microbiota and metabolic and immune profiles should be added in the abstract.

RESPONSE: Page 1, lines 21-24: The paragraph in the Abstract section related to microbiota results was added according to the Reviewer's suggestion.

2) Generally, the liver is the most important organ for the obesity mice, and the changes in liver function should be evaluated.

RESPONSE: Our objective was focused on the study of the adipose tissue (AT), the central organ of fat accumulation. Nevertheless, the AT remodeling in obesity not only affects this tissue structure, cellular composition, and secretory profile of adipocytokines but can also promote ectopic fat deposition in peripheral organs such as the heart, skeletal muscle, and liver. Obesity and insulin resistance play the main pathophysiological roles in the development of nonalcoholic fatty liver disease (NAFLD). The pathogenesis begins with lipid accumulation in hepatocytes with alterations in lipid homeostasis associated with obesity, insulin resistance, and abnormalities in adipokine synthesis. However, when animal models are used with an HFD diet, more time than ten weeks is usually required to reach this state (mostly more than fourteen weeks). Also, liver steatosis must be confirmed with liver histology. Furthermore, the addition of fructose to the diet to reach this state is recommended.

In this study, we aimed to analyze the mixes of strains (previously characterized with an effect on the decrease of Tg in a C. elegans model) in the lipid status of an HFD diet obesity model. Considering this, we focused our analysis on the effects on AT status, lipid and glycemic parameters, leptin, and, of course, GM status.

As a projection of this research, to gain insight into the effects of these strains on NAFLD, we are indeed currently evaluating the influence of these mixes on the liver status in a new HFD model of NAFLD.

3) In Table 1, Table 2, and Table 3, how many animals were used in each group ?

RESPONSE: In Tables 1, table 2 (currently figure 3) and table 3 (currently figure 4) the number of animals used were added (n=6) in the figure legend.

4) Figures 4 and 5, the results of three fecal samples from each group should be provided.

RESPONSE:In page 10-11,Figures 4 and 5 (currently figures 6 and 7) with the results of the average abundance of each group  were changed for figures representing relative abundance of each studied mouse (n=3) according to the Reviewer's suggestion.

5) What about the changes in metabolites of each group, such as short chain fatty acids?

RESPONSE: In this model we could not carried out the suggested determination. The determination of short-chain fatty acids will be carried out in the new HFD model of NAFLD previously  mentioned.

6) The correlation between microbiota and metabolic and immune profiles should be discussed in depth.
RESPONSE: Correlations between gut microbiota taxonomic groups and biochemical parameters were performed using Spearmans rank correlation coefficients using  Graphpad prism software. The figure 8 were added in page 13 according to the Reviewer's suggestion

Modifications in Material and Methods, Results and Discussion were made:

Material and Methods, in 2.9 Statistical analysis section, page 4 lines 174-177

Results: Page 13, lines 374-387

Discussion: Page 17, lines 557-579

REVIEWER 3

1) In the manuscript, what is the "Ob" represents, the author should clarify that, and include the full spelling of this word.

RESPONSE: Page 3, line 105: The "Ob" means "Obese". The complete word "obese" was added to explain this abbreviation.

2) The font size of all the notes below the "table" needs to be adjusted, which should not be the same with the font size of the manuscripts. For example, Line 269-272, the font size of the notes for "table 3" should be adjusted to 10pt or 10.5 pt.

RESPONSE: The notes in the Tables were adjusted to the font size of 9 pt according to the template format of Fermentation Journal.

3) The formatting of the main text is a bit disorder and needs to be rearranged, such as Line 239-241, line 268-269, etc.

RESPONSE: The formatting of the main text was improved according to the Reviewer's suggestion.

4) Line 13, in the "abstract" section, little of the experimental results about the gut microbiota composition measurement were described, the author should briefly summarize the effect of the dietary intervention of FGM on gut bacteria.

RESPONSE: Page 1, lines 21-24: The paragraph in the Abstract section related to microbiota results was added according to the Reviewer's suggestion.

5) Line 225 and line 245, result 3.3 and 3.4, these data should be presented in a more visual histogram, instead of "tables". The author should modify these two parts, and the "tables", "table 2" and "table 3" are better to be arranged in the "supplementary material" section.

RESPONSE: Tables 2 and 3 were replaced by figures 3 and 4, respectively. Tables 2 and 3 (currently tables S1 and S2) were added to the supplementary material, according to the Reviewer's suggestions

6) Line 237, table 2 presented the data of cytokines "MCP-1", however, in the "materials and methods" section, line 121, which described the measurement methods for "IFN-γ", but there was no data for "IFN-γ" in the "results" section. Thus, the author should verify that and make necessary corrections.

RESPONSE: In Materials and Methods, section 2.6: Page 3, line 132: The sentence was corrected as follows: “Plasma levels of cytokines (TNF-α, MCP-1, IL-6 and IL-10) were determined by flow cytometry.”

ALSO, THE AUTHORS MADE THE FOLLOWING MODIFICATIONS:

-In page 1, line 4: the symbol “ † “ were added to Antonela Marquez and Matias Russo

-In page 1, line 12: the sentence “These authors contributed equally to this work” were added
-In page 1, line 13: the sentence  “*Correspondence”  was change for “*Author to whom -correspondence should be addressed”

-In Abstract, page 1, line 16: the complete name of strain“ Lactiplantibacillus paraplantarum” were added before CRL1472

- In Introduction, page 1-line 31, the word “and”after obesity and overweight were removed.

-In Introduction, page 1-line 43; page 2-line 51 the style cite in text was changed

-In Material and methods, section 2.2: Page 2, line 95-96: The sentence was changed

-In Discussion, page 14-line 435; page 15-line 483 the style cite was changed

-In Discussion page 15 - line 449 and line 479 the corresponding cite were added

- In Discussion page 15, line 500, the word “as” were removed in the sentence “as well as as an antioxidant

-Page 17, line 593, the paragraph “ Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Inflammatory status in mice fed with a high-fat diet (HFD) and a standard diet (SD) with and without supplementation of FGM and mixes; Table S2: Biochemical parameters of mice fed with a high-fat diet (HFD) or a standard diet (SD) with and without supplementation of FGM and mixes” were added after Conclusions.

-In Page 18, line 617: the accesiion number PRJNA1078585 of high throughput sequencing data were added

María Paola Gauffin-Cano

Corresponding author

[email protected]

Author Response File: Author Response.pdf

Reviewer 2 Report

The present study aimed to reveal the effects of fermented goat milk on adiposity and gut microbiota in a diet-induced obesity murine model. Results suggest that the administration of goat milk fermented with Limosilactobacillus fermentum CRL1446 + Lactiplantibacillus paraplantarum CRL1472 improves metabolic and immune profiles in obese mice while positively modulating the gut microbiota, therefore attenuating the risk factors associated with obesity. The topic of this study fully fills in the scope of Fermentation. This study was designed logically and all of the results were good, but there were several suggestions for further improving the quality of the manuscript.

1.     More detailed results about the gut microbiota and metabolic and immune profiles should be added in the abstract.

2.     Generally, the liver is the most important organ for the obesity mice, and the changes in liver function should be evaluated.

3.     In Table 1, Table 2, and Table 3, how many animals were used in each group ?

4.     Figures 4 and 5, the results of three fecal samples from each group should be provided.

5.     What about the changes in metabolites of each group, such as short chain fatty acids?

6.     The correlation between microbiota and metabolic and immune profiles should be discussed in depth.

Author Response

Thank you for your helpful and constructive comments. As suggested, all the issues have been carefully addressed. We sincerely hope that the revised manuscript is now satisfactory and acceptable. Revisions were marked in red in the manuscript.

REVIEWER 1:

1) Line 35-36: Sentence does not make sense. Correct it by writing 'Although obesity results from a chronic imbalance between energy intake and expenditure, however there are genetic, behavioral, metabolic, and hormonal factors which can influences on body 36 weight.

RESPONSE: Page 1, lines 38-39: The paragraph in the Introduction section was changed according to the Reviewer's suggestion.

2) Line 49:Take out "when" before citation.

RESPONSE: Page 2, line 51: The word "when" was removed of the sentence according to the Reviewer's suggestion.

3) Line 56-57:Some authors describe the benefits obtained from the consumption of fermented goat milk (FGM)- you should qoute at least one study here. 

RESPONSE: Page 2, lines 59-62: The following paragraph and cite in the Introduction section was added:

" In addition, compared to fermented cow milk, FGM maintains a better nutritional pro-file with higher protein, minerals, and vitamin values and presents a fatty acid profile that is considered more health-promoting [10]".

4) Also the introduction is missing one sentence on why you are choosing this particular strain (Lactobacillus delbrueckii subsp. indicus CRL1447) for fermentation of goat milk.

RESPONSE: Page 2, lines  66-70: The following paragraph and cite in the Introduction section was added:

“Marquez et al., in an intensive study of strain characterization in terms of their probiotic and technological properties, selected Lactobacillus delbrueckii subsp. indicus CRL1447 for developing fermented goat milk [11]. The CRL1447 strain stands out for its high acidification rate in milk, a property of technological interest for developing fermented products”.

5) Section 2.1 misses a citation. Please insert the below citation here (http://dx.doi.org/10.3390/fermentation8090469)

RESPONSE: Page 2, in Material and Methods, section 2.1. 81-83: The following sentence and cite were added:

“These strains, previously characterized for their in vitro probiotic and technology properties, were selected to be assayed on diet-induced obese mice [11]”.

Marquez, A.; Andrada, E.; Russo, M.; Bolondi, M.L.; Fabersani, E.; Medina, R.; Gauffin-Cano, P. Characterization of Autochthonous Lactobacilli from Goat Dairy Products with Probiotic Potential for Metabolic Diseases. Heliyon 2022, 8, e10462, doi:10.1016/j.heliyon.2022.e10462.

The citation suggested by the Reviewer does not correspond to the strain characterization study.

6) Fig 3a (principal component analysis) is not clear. Increase the font size to make it more visible

RESPONSE: Page 9: The figure 3a (currently 5a) was changed

7) Line 364: write "induced" instead of induce

RESPONSE: Page 14, line 404:

The word "induce" was change by "induced" according to the Reviewer's suggestion.

8) Line 373: Remove double spacing before FER

RESPONSE: Page 14, line 413:

The space was removed according to the Reviewer's suggestion.

9) Line 376: add "the" before increased feeding efficiency

RESPONSE: Page 14, line 416:

The word "the" was added according to the Reviewer's suggestion.

REVIEWER 2:

1) More detailed results about the gut microbiota and metabolic and immune profiles should be added in the abstract.

RESPONSE: Page 1, lines 21-24: The paragraph in the Abstract section related to microbiota results was added according to the Reviewer's suggestion.

2) Generally, the liver is the most important organ for the obesity mice, and the changes in liver function should be evaluated.

RESPONSE: Our objective was focused on the study of the adipose tissue (AT), the central organ of fat accumulation. Nevertheless, the AT remodeling in obesity not only affects this tissue structure, cellular composition, and secretory profile of adipocytokines but can also promote ectopic fat deposition in peripheral organs such as the heart, skeletal muscle, and liver. Obesity and insulin resistance play the main pathophysiological roles in the development of nonalcoholic fatty liver disease (NAFLD). The pathogenesis begins with lipid accumulation in hepatocytes with alterations in lipid homeostasis associated with obesity, insulin resistance, and abnormalities in adipokine synthesis. However, when animal models are used with an HFD diet, more time than ten weeks is usually required to reach this state (mostly more than fourteen weeks). Also, liver steatosis must be confirmed with liver histology. Furthermore, the addition of fructose to the diet to reach this state is recommended.

In this study, we aimed to analyze the mixes of strains (previously characterized with an effect on the decrease of Tg in a C. elegans model) in the lipid status of an HFD diet obesity model. Considering this, we focused our analysis on the effects on AT status, lipid and glycemic parameters, leptin, and, of course, GM status.

As a projection of this research, to gain insight into the effects of these strains on NAFLD, we are indeed currently evaluating the influence of these mixes on the liver status in a new HFD model of NAFLD.

3) In Table 1, Table 2, and Table 3, how many animals were used in each group ?

RESPONSE: In Tables 1, table 2 (currently figure 3) and table 3 (currently figure 4) the number of animals used were added (n=6) in the figure legend.

4) Figures 4 and 5, the results of three fecal samples from each group should be provided.

RESPONSE:In page 10-11,Figures 4 and 5 (currently figures 6 and 7) with the results of the average abundance of each group  were changed for figures representing relative abundance of each studied mouse (n=3) according to the Reviewer's suggestion.

5) What about the changes in metabolites of each group, such as short chain fatty acids?

RESPONSE: In this model we could not carried out the suggested determination. The determination of short-chain fatty acids will be carried out in the new HFD model of NAFLD previously  mentioned.

6) The correlation between microbiota and metabolic and immune profiles should be discussed in depth.
RESPONSE: Correlations between gut microbiota taxonomic groups and biochemical parameters were performed using Spearmans rank correlation coefficients using  Graphpad prism software. The figure 8 were added in page 13 according to the Reviewer's suggestion

Modifications in Material and Methods, Results and Discussion were made:

Material and Methods, in 2.9 Statistical analysis section, page 4 lines 174-177

Results: Page 13, lines 374-387

Discussion: Page 17, lines 557-579

REVIEWER 3

1) In the manuscript, what is the "Ob" represents, the author should clarify that, and include the full spelling of this word.

RESPONSE: Page 3, line 105: The "Ob" means "Obese". The complete word "obese" was added to explain this abbreviation.

2) The font size of all the notes below the "table" needs to be adjusted, which should not be the same with the font size of the manuscripts. For example, Line 269-272, the font size of the notes for "table 3" should be adjusted to 10pt or 10.5 pt.

RESPONSE: The notes in the Tables were adjusted to the font size of 9 pt according to the template format of Fermentation Journal.

3) The formatting of the main text is a bit disorder and needs to be rearranged, such as Line 239-241, line 268-269, etc.

RESPONSE: The formatting of the main text was improved according to the Reviewer's suggestion.

4) Line 13, in the "abstract" section, little of the experimental results about the gut microbiota composition measurement were described, the author should briefly summarize the effect of the dietary intervention of FGM on gut bacteria.

RESPONSE: Page 1, lines 21-24: The paragraph in the Abstract section related to microbiota results was added according to the Reviewer's suggestion.

5) Line 225 and line 245, result 3.3 and 3.4, these data should be presented in a more visual histogram, instead of "tables". The author should modify these two parts, and the "tables", "table 2" and "table 3" are better to be arranged in the "supplementary material" section.

RESPONSE: Tables 2 and 3 were replaced by figures 3 and 4, respectively. Tables 2 and 3 (currently tables S1 and S2) were added to the supplementary material, according to the Reviewer's suggestions

6) Line 237, table 2 presented the data of cytokines "MCP-1", however, in the "materials and methods" section, line 121, which described the measurement methods for "IFN-γ", but there was no data for "IFN-γ" in the "results" section. Thus, the author should verify that and make necessary corrections.

RESPONSE: In Materials and Methods, section 2.6: Page 3, line 132: The sentence was corrected as follows: “Plasma levels of cytokines (TNF-α, MCP-1, IL-6 and IL-10) were determined by flow cytometry.”

ALSO, THE AUTHORS MADE THE FOLLOWING MODIFICATIONS:

-In page 1, line 4: the symbol “ † “ were added to Antonela Marquez and Matias Russo

-In page 1, line 12: the sentence “These authors contributed equally to this work” were added
-In page 1, line 13: the sentence  “*Correspondence”  was change for “*Author to whom -correspondence should be addressed”

-In Abstract, page 1, line 16: the complete name of strain“ Lactiplantibacillus paraplantarum” were added before CRL1472

- In Introduction, page 1-line 31, the word “and”after obesity and overweight were removed.

-In Introduction, page 1-line 43; page 2-line 51 the style cite in text was changed

-In Material and methods, section 2.2: Page 2, line 95-96: The sentence was changed

-In Discussion, page 14-line 435; page 15-line 483 the style cite was changed

-In Discussion page 15 - line 449 and line 479 the corresponding cite were added

- In Discussion page 15, line 500, the word “as” were removed in the sentence “as well as as an antioxidant

-Page 17, line 593, the paragraph “ Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Inflammatory status in mice fed with a high-fat diet (HFD) and a standard diet (SD) with and without supplementation of FGM and mixes; Table S2: Biochemical parameters of mice fed with a high-fat diet (HFD) or a standard diet (SD) with and without supplementation of FGM and mixes” were added after Conclusions.

-In Page 18, line 617: the accesiion number PRJNA1078585 of high throughput sequencing data were added

María Paola Gauffin-Cano

Corresponding author

[email protected]

Author Response File: Author Response.pdf

Reviewer 3 Report

This manuscript investigated the administration of goat milk fermented with different lactobacilli strains on the metabolism parameters and gut microbiota composition in high fat diet fed mice. The work was well conceived, and the data were carefully analyzed. However, some errors in the manuscript should be modified by the author.  

(1)   In the manuscript, what is the “Ob” represents, the author should clarify that, and include the full spelling of this word.

(2)   The font size of all the notes below the “table” needs to be adjusted, which should not be the same with the font size of the manuscripts. For example, Line 269-272, the font size of the notes for “table 3” should be adjusted to 10pt or 10.5 pt.

(3)   The formatting of the main text is a bit disorder and needs to be rearranged, such as Line 239-241, line 268-269, etc.

(4)   Line 13, in the “abstract” section, little of the experimental results about the gut microbiota composition measurement were described, the author should briefly summarize the effect of the dietary intervention of FGM on gut bacteria.

(5)   Line 225 and line 245, result 3.3 and 3.4, these data should be presented in a more visual histogram, instead of “tables”. The author should modify these two parts, and the “tables”, “table 2” and “table 3” are better to be arranged in the “supplementary material” section.

(6)   Line 237, table 2 presented the data of cytokines “MCP-1”, however, in the “materials and methods” section, line 121, which described the measurement methods for “IFN-γ”, but there was no data for “IFN-γ” in the “results” section. Thus, the author should verify that and make necessary corrections.

Author Response

Thank you for your helpful and constructive comments. As suggested, all the issues have been carefully addressed. We sincerely hope that the revised manuscript is now satisfactory and acceptable. Revisions were marked in red in the manuscript.

REVIEWER 1:

1) Line 35-36: Sentence does not make sense. Correct it by writing 'Although obesity results from a chronic imbalance between energy intake and expenditure, however there are genetic, behavioral, metabolic, and hormonal factors which can influences on body 36 weight.

RESPONSE: Page 1, lines 38-39: The paragraph in the Introduction section was changed according to the Reviewer's suggestion.

2) Line 49:Take out "when" before citation.

RESPONSE: Page 2, line 51: The word "when" was removed of the sentence according to the Reviewer's suggestion.

3) Line 56-57:Some authors describe the benefits obtained from the consumption of fermented goat milk (FGM)- you should qoute at least one study here. 

RESPONSE: Page 2, lines 59-62: The following paragraph and cite in the Introduction section was added:

" In addition, compared to fermented cow milk, FGM maintains a better nutritional pro-file with higher protein, minerals, and vitamin values and presents a fatty acid profile that is considered more health-promoting [10]".

4) Also the introduction is missing one sentence on why you are choosing this particular strain (Lactobacillus delbrueckii subsp. indicus CRL1447) for fermentation of goat milk.

RESPONSE: Page 2, lines  66-70: The following paragraph and cite in the Introduction section was added:

“Marquez et al., in an intensive study of strain characterization in terms of their probiotic and technological properties, selected Lactobacillus delbrueckii subsp. indicus CRL1447 for developing fermented goat milk [11]. The CRL1447 strain stands out for its high acidification rate in milk, a property of technological interest for developing fermented products”.

5) Section 2.1 misses a citation. Please insert the below citation here (http://dx.doi.org/10.3390/fermentation8090469)

RESPONSE: Page 2, in Material and Methods, section 2.1. 81-83: The following sentence and cite were added:

“These strains, previously characterized for their in vitro probiotic and technology properties, were selected to be assayed on diet-induced obese mice [11]”.

Marquez, A.; Andrada, E.; Russo, M.; Bolondi, M.L.; Fabersani, E.; Medina, R.; Gauffin-Cano, P. Characterization of Autochthonous Lactobacilli from Goat Dairy Products with Probiotic Potential for Metabolic Diseases. Heliyon 2022, 8, e10462, doi:10.1016/j.heliyon.2022.e10462.

The citation suggested by the Reviewer does not correspond to the strain characterization study.

6) Fig 3a (principal component analysis) is not clear. Increase the font size to make it more visible

RESPONSE: Page 9: The figure 3a (currently 5a) was changed

7) Line 364: write "induced" instead of induce

RESPONSE: Page 14, line 404:

The word "induce" was change by "induced" according to the Reviewer's suggestion.

8) Line 373: Remove double spacing before FER

RESPONSE: Page 14, line 413:

The space was removed according to the Reviewer's suggestion.

9) Line 376: add "the" before increased feeding efficiency

RESPONSE: Page 14, line 416:

The word "the" was added according to the Reviewer's suggestion.

REVIEWER 2:

1) More detailed results about the gut microbiota and metabolic and immune profiles should be added in the abstract.

RESPONSE: Page 1, lines 21-24: The paragraph in the Abstract section related to microbiota results was added according to the Reviewer's suggestion.

2) Generally, the liver is the most important organ for the obesity mice, and the changes in liver function should be evaluated.

RESPONSE: Our objective was focused on the study of the adipose tissue (AT), the central organ of fat accumulation. Nevertheless, the AT remodeling in obesity not only affects this tissue structure, cellular composition, and secretory profile of adipocytokines but can also promote ectopic fat deposition in peripheral organs such as the heart, skeletal muscle, and liver. Obesity and insulin resistance play the main pathophysiological roles in the development of nonalcoholic fatty liver disease (NAFLD). The pathogenesis begins with lipid accumulation in hepatocytes with alterations in lipid homeostasis associated with obesity, insulin resistance, and abnormalities in adipokine synthesis. However, when animal models are used with an HFD diet, more time than ten weeks is usually required to reach this state (mostly more than fourteen weeks). Also, liver steatosis must be confirmed with liver histology. Furthermore, the addition of fructose to the diet to reach this state is recommended.

In this study, we aimed to analyze the mixes of strains (previously characterized with an effect on the decrease of Tg in a C. elegans model) in the lipid status of an HFD diet obesity model. Considering this, we focused our analysis on the effects on AT status, lipid and glycemic parameters, leptin, and, of course, GM status.

As a projection of this research, to gain insight into the effects of these strains on NAFLD, we are indeed currently evaluating the influence of these mixes on the liver status in a new HFD model of NAFLD.

3) In Table 1, Table 2, and Table 3, how many animals were used in each group ?

RESPONSE: In Tables 1, table 2 (currently figure 3) and table 3 (currently figure 4) the number of animals used were added (n=6) in the figure legend.

4) Figures 4 and 5, the results of three fecal samples from each group should be provided.

RESPONSE:In page 10-11,Figures 4 and 5 (currently figures 6 and 7) with the results of the average abundance of each group  were changed for figures representing relative abundance of each studied mouse (n=3) according to the Reviewer's suggestion.

5) What about the changes in metabolites of each group, such as short chain fatty acids?

RESPONSE: In this model we could not carried out the suggested determination. The determination of short-chain fatty acids will be carried out in the new HFD model of NAFLD previously  mentioned.

6) The correlation between microbiota and metabolic and immune profiles should be discussed in depth.
RESPONSE: Correlations between gut microbiota taxonomic groups and biochemical parameters were performed using Spearmans rank correlation coefficients using  Graphpad prism software. The figure 8 were added in page 13 according to the Reviewer's suggestion

Modifications in Material and Methods, Results and Discussion were made:

Material and Methods, in 2.9 Statistical analysis section, page 4 lines 174-177

Results: Page 13, lines 374-387

Discussion: Page 17, lines 557-579

REVIEWER 3

1) In the manuscript, what is the "Ob" represents, the author should clarify that, and include the full spelling of this word.

RESPONSE: Page 3, line 105: The "Ob" means "Obese". The complete word "obese" was added to explain this abbreviation.

2) The font size of all the notes below the "table" needs to be adjusted, which should not be the same with the font size of the manuscripts. For example, Line 269-272, the font size of the notes for "table 3" should be adjusted to 10pt or 10.5 pt.

RESPONSE: The notes in the Tables were adjusted to the font size of 9 pt according to the template format of Fermentation Journal.

3) The formatting of the main text is a bit disorder and needs to be rearranged, such as Line 239-241, line 268-269, etc.

RESPONSE: The formatting of the main text was improved according to the Reviewer's suggestion.

4) Line 13, in the "abstract" section, little of the experimental results about the gut microbiota composition measurement were described, the author should briefly summarize the effect of the dietary intervention of FGM on gut bacteria.

RESPONSE: Page 1, lines 21-24: The paragraph in the Abstract section related to microbiota results was added according to the Reviewer's suggestion.

5) Line 225 and line 245, result 3.3 and 3.4, these data should be presented in a more visual histogram, instead of "tables". The author should modify these two parts, and the "tables", "table 2" and "table 3" are better to be arranged in the "supplementary material" section.

RESPONSE: Tables 2 and 3 were replaced by figures 3 and 4, respectively. Tables 2 and 3 (currently tables S1 and S2) were added to the supplementary material, according to the Reviewer's suggestions

6) Line 237, table 2 presented the data of cytokines "MCP-1", however, in the "materials and methods" section, line 121, which described the measurement methods for "IFN-γ", but there was no data for "IFN-γ" in the "results" section. Thus, the author should verify that and make necessary corrections.

RESPONSE: In Materials and Methods, section 2.6: Page 3, line 132: The sentence was corrected as follows: “Plasma levels of cytokines (TNF-α, MCP-1, IL-6 and IL-10) were determined by flow cytometry.”

ALSO, THE AUTHORS MADE THE FOLLOWING MODIFICATIONS:

-In page 1, line 4: the symbol “ † “ were added to Antonela Marquez and Matias Russo

-In page 1, line 12: the sentence “These authors contributed equally to this work” were added
-In page 1, line 13: the sentence  “*Correspondence”  was change for “*Author to whom -correspondence should be addressed”

-In Abstract, page 1, line 16: the complete name of strain“ Lactiplantibacillus paraplantarum” were added before CRL1472

- In Introduction, page 1-line 31, the word “and”after obesity and overweight were removed.

-In Introduction, page 1-line 43; page 2-line 51 the style cite in text was changed

-In Material and methods, section 2.2: Page 2, line 95-96: The sentence was changed

-In Discussion, page 14-line 435; page 15-line 483 the style cite was changed

-In Discussion page 15 - line 449 and line 479 the corresponding cite were added

- In Discussion page 15, line 500, the word “as” were removed in the sentence “as well as as an antioxidant

-Page 17, line 593, the paragraph “ Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Inflammatory status in mice fed with a high-fat diet (HFD) and a standard diet (SD) with and without supplementation of FGM and mixes; Table S2: Biochemical parameters of mice fed with a high-fat diet (HFD) or a standard diet (SD) with and without supplementation of FGM and mixes” were added after Conclusions.

-In Page 18, line 617: the accesiion number PRJNA1078585 of high throughput sequencing data were added

María Paola Gauffin-Cano

Corresponding author

[email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The revised manuscript could be accepted in the current form.

Reviewer 3 Report

ok