Fermentation and Functional Properties of Plant-Derived Limosilactobacillus fermentum for Dairy Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Fermentation and functional properties of plant-derived Limosi-lactobacillus fermentum for dairy applications

Comments article

1.- What is the main question addressed by the research?

The manuscript deals with the probiotic potential of an isolated probiotic bacteria from Prunus padus as an alternative to produce functional foods. Also explains about the characterization of the strain to be considered with probiotic and postbiotic potential.
Limosilactobacillus fermentum showed a good response exhibited strong acid resistance, non-hemolytic safety, and antioxidant activity by DPPH. Also, showed antibacterial activity against 25 five Escherichia coli strains, which remained stable after heat treatment. Is an interesting contribution because these microorganism present postbiotic potential for the formulation of functional products such as yogurt. Nowadays the consumption of prebiotics, probiotics or postbiotics has shown great interest for the preventing disease, due to the increase in metabolic syndrome diseases today.

2.- Do you consider the topic original or relevant to the field? Does it address a specific gap in the field? Please also explain why this is/ is not the case.

Is a topic original because the manuscript is focused on the isolation of probiotic bacteria from vegetable material (Prunus padus) and their characterization in vitro to be used as an alternative in food formulation. Is an important research because the probiotics and postbiotic modulating the immune system and preventing disease related to metabolic syndrome, which today are on the increase.

3.- What does it add to the subject area compared with other published material?

Prunus padus is an underutilized vegetal, the authors mentioned that has been used with both culinary and medicinal, however today, no studies to date have reported the isolation or characterization of acid lactic microorganisms associated with this vegetal. In addition, Limosilactobacillus fermentum showed good results for use as a probiotic, because the microorganism present acid resistance under gastric conditions tested in addition the non-hemolytic nature of the strain supports its safety and suitability for use in foods.

4.- What specific improvements should the authors consider regarding the
methodology?

Indicate date of plant collection line 83.
Indicate that it is CHL medium in line 92.
It is suggested that in point 2.3, separate the information on pathogenic bacteria with respect to lactic acid bacteria. Change it to point 2.8 (Antibacterial activity of CFS derived from the isolated LAB).
Indicate which controls were used in this assay (2.5 Hemolytic activity).
In assay 2.7 antioxidant activity by DPPH, place the bibliographic citation.
Indicate which controls were used in this assay (2.8 Antibacterial activity).
Line 156. 2.8.2.96. well plate assay starts with a capital letter (typing error).
The probiotic strains S. thermophilus KCCM 40430 and L.bulgaricus KCCM 35463 were Line 175 The probiotic strains S. thermophilus KCCM 40430 and L.bulgaricus KCCM 35463 were obtained from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea) is repeated in point 2.3.
Line 188: titratable acidit (error in writing) is titratable acidity.

5.- Are the conclusions consistent with the evidence and arguments presented
and do they address the main question posed? Please also explain why this
is/is not the case.

The conclusions are clear and in line with the study conducted.

6.- Are the references appropriate?
The references are appropriate for this study.

7.- Any additional comments on the tables and figures.
Improve the quality of the figures 6, 7, 8 and 9. Not legible.

In results and discussion, line 218-220 is presented as part of the methodology. Change.

In general, this study is interesting because L. fermentum strain exhibits notable intrinsic resistance to low pH, does not exert cytotoxic effects on red blood cells and present advantages with respect to commercial strains tested, in addition the Prunus padus is a vegetable material underutilized for the isolate and characterization from probiotic microorganisms. The authors do discuss their results with respect to other works. The conclusions are clear and precise.
In my opinion could be accepted with minor changes. 

Author Response

Response to reviewer

Manuscript Title: Fermentation and functional properties of plant-derived Limosilactobacillus fermentum for dairy applications

Manucript ID: fermentation-3621588

 

Reviewer,

We would like to express our sincere gratitude for your time and effort in handling the review of our manuscript. We greatly appreciate the insightful and constructive comments provided by the reviewers. In response, we have carefully addressed all the comments and revised the manuscript accordingly. Our point-by-point responses to each of the reviewers’ comments are provided below, and all changes made in the manuscript have been clearly marked. We are confident that these revisions have significantly improved the quality and clarity of our work.

Thank you again for your kind consideration.

1.- What is the main question addressed by the research?

The manuscript deals with the probiotic potential of an isolated probiotic bacteria from Prunus padus as an alternative to produce functional foods. Also explains about the characterization of the strain to be considered with probiotic and postbiotic potential. Limosilactobacillus fermentum showed a good response exhibited strong acid resistance, non-hemolytic safety, and antioxidant activity by DPPH. Also, showed antibacterial activity against 25 five Escherichia coli strains, which remained stable after heat treatment. Is an interesting contribution because these microorganism present postbiotic potential for the formulation of functional products such as yogurt. Nowadays the consumption of prebiotics, probiotics or postbiotics has shown great interest for the preventing disease, due to the increase in metabolic syndrome diseases today.

Response: Thank you for your comment.

2.- Do you consider the topic original or relevant to the field? Does it address a specific gap in the field? Please also explain why this is/ is not the case.

Is a topic original because the manuscript is focused on the isolation of probiotic bacteria from vegetable material (Prunus padus) and their characterization in vitro to be used as an alternative in food formulation. Is an important research because the probiotics and postbiotic modulating the immune system and preventing disease related to metabolic syndrome, which today are on the increase.

Response: Thank you for your comment.

3.- What does it add to the subject area compared with other published material?

Prunus padus is an underutilized vegetal, the authors mentioned that has been used with both culinary and medicinal, however today, no studies to date have reported the isolation or characterization of acid lactic microorganisms associated with this vegetal. In addition, Limosilactobacillus fermentum showed good results for use as a probiotic, because the microorganism present acid resistance under gastric conditions tested in addition the non-hemolytic nature of the strain supports its safety and suitability for use in foods.

Response: Thank you for your comment.

4.- What specific improvements should the authors consider regarding the
methodology?

Reviewer’s Comment: Indicate date of plant collection line 83.

Response: The collection date has been indicated in the revised manuscript.

 

Reviewer’s Comment: Indicate that it is CHL medium in line 92.

Response: This has been corrected in the manuscript.

 

Reviewer’s Comment: It is suggested that in point 2.3, separate the information on pathogenic bacteria with respect to lactic acid bacteria. Change it to point 2.8 (Antibacterial activity of CFS derived from the isolated LAB).

Response: We have revised the manuscript accordingly by moving the relevant content to section 2.8.

 

Reviewer’s Comment: indicate which controls were used in this assay (2.5 Hemolytic activity).

Response: The control strain used in this assay has been added to the manuscript.

 

Reviewer’s Comment: In assay 2.7 antioxidant activity by DPPH, place the bibliographic citation.

Response: The appropriate reference has been cited.

 

Reviewer’s Comment: Indicate which controls were used in this assay (2.8 Antibacterial activity).

Response: The control strain has been included in the description of the assay.

 

Reviewer’s Comment: Line 156. 2.8.2.96. well plate assay starts with a capital letter (typing error).

Response: This typographical error has been corrected.

 

Reviewer’s Comment: The probiotic strains S. thermophilus KCCM 40430 and L.bulgaricus KCCM 35463 were Line 175 The probiotic strains S. thermophilus KCCM 40430 and L.bulgaricus KCCM 35463 were obtained from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea) is repeated in point 2.3.

Response: The duplicated sentence has been deleted.

 

Reviewer’s Comment: Line 188: titratable acidit (error in writing) is titratable acidity.

Response: The typo has been corrected.

 

Reviewer’s Comment:

5.- Are the conclusions consistent with the evidence and arguments presented and do they address the main question posed? Please also explain why this is/is not the case.

The conclusions are clear and in line with the study conducted.

6.- Are the references appropriate?

The references are appropriate for this study.

7.- Any additional comments on the tables and figures.

Improve the quality of the figures 6, 7, 8 and 9. Not legible.

In results and discussion, line 218-220 is presented as part of the methodology. Change.

In general, this study is interesting because L. fermentum strain exhibits notable intrinsic resistance to low pH, does not exert cytotoxic effects on red blood cells and present advantages with respect to commercial strains tested, in addition the Prunus padus is a vegetable material underutilized for the isolate and characterization from probiotic microorganisms. The authors do discuss their results with respect to other works. The conclusions are clear and precise.In my opinion could be accepted with minor changes. 

Response: Thank you for your overall positive evaluation. All suggested revisions have been made.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Limosilactobacillus fermentum plays an important role in the production of fermented dairy products due to its robust fermentation capabilities and potential health-promoting properties. Recently, this species has gained growing attention for its probiotic traits, and several new studies published this year have further highlighted its possible impact on human health. The article aligns well with this research trend, contributing to the expanding body of knowledge on the functional and probiotic potential of L. fermentum in the context of modern food biotechnology.

However, I have a few doubts that I have to ask the authors of the manuscript to clarify.

Materials and methods:

lines 86-87: Please provide the reference according to which such a quantity of sodium azide was chosen.

line 141: The optimal wavelength for reading absorbance after the reaction with DPPH is 517 nm. Please explain why 490 nm was used?

Results and discussion:

lines 239-241: Information on distinguishing between the various types of hemolysis mechanisms has already been explained in the methodology. There is no need to repeat it. Please move the citation to the methodology section.

Figures 3, 4, 6, and 7: I suggest marking the significance (level of significance on the charts).

Figures 6, 7, 8, and 9 are not sufficiently legible. Please provide the figures in better resolution, including the significance level of the results on the charts (it can be marked with * or letters).

In Figure 7, I did not notice any information about whether the experiment was performed in 3 repetitions (there is also no such information in the methodology). Please clarify.

In the case of Figure 5, please provide complete photos with visible descriptions of the reference strains used for the test.

General note on the charts:

The standard deviation in the case of each graph seems to be a percentage of the mean; with small values, it is always a small error value, while with larger values, it is greater and almost identical. Isn't this an error in determining the deviation and creating the chart? It seems a bit unnatural. Please explain.

Author Response

Response to reviewer

Manuscript Title: Fermentation and functional properties of plant-derived Limosilactobacillus fermentum for dairy applications

Manucript ID: fermentation-3621588

 

Dear Reviewer,

We would like to express our sincere gratitude for your time and effort in handling the review of our manuscript. We greatly appreciate the insightful and constructive comments provided by the reviewers. In response, we have carefully addressed all the comments and revised the manuscript accordingly. Our point-by-point responses to each of the reviewers’ comments are provided below, and all changes made in the manuscript have been clearly marked. We are confident that these revisions have significantly improved the quality and clarity of our work.

Thank you again for your kind consideration.

Reviewer’s Comment: Limosilactobacillus fermentum plays an important role in the production of fermented dairy products due to its robust fermentation capabilities and potential health-promoting properties. Recently, this species has gained growing attention for its probiotic traits, and several new studies published this year have further highlighted its possible impact on human health. The article aligns well with this research trend, contributing to the expanding body of knowledge on the functional and probiotic potential of L. fermentum in the context of modern food biotechnology.

Reviewer’s Comment: lines 86-87: Please provide the reference according to which such a quantity of sodium azide was chosen.

Response: The appropriate reference has been added.

 

Reviewer’s Comment: line 141: The optimal wavelength for reading absorbance after the reaction with DPPH is 517 nm. Please explain why 490 nm was used?

Response: A supporting reference has been included to justify the use of 490 nm.

 

Reviewer’s Comment: lines 239-241: Information on distinguishing between the various types of hemolysis mechanisms has already been explained in the methodology. There is no need to repeat it. Please move the citation to the methodology section.

Response: As suggested, we have relocated the relevant explanation to the methodology section.

 

Reviewer’s Comment: Figures 3, 4, 6, and 7: I suggest marking the significance (level of significance on the charts).

Response: Significance levels have been marked on the figures as recommended.

 

Reviewer’s Comment: Figures 6, 7, 8, and 9 are not sufficiently legible. Please provide the figures in better resolution, including the significance level of the results on the charts (it can be marked with * or letters).

Response: Figures 6 through 9 have been revised to enhance clarity.

 

Reviewer’s Comment: In Figure 7, I did not notice any information about whether the experiment was performed in 3 repetitions (there is also no such information in the methodology). Please clarify.

Response: We have clarified in both the figure legend and the methodology that all experiments were conducted in triplicate.

 

Reviewer’s Comment: In the case of Figure 5, please provide complete photos with visible descriptions of the reference strains used for the test.

Response: The figure has been updated to include complete images with visible reference strain labels.

 

Reviewer’s Comment: The standard deviation in the case of each graph seems to be a percentage of the mean; with small values, it is always a small error value, while with larger values, it is greater and almost identical. Isn't this an error in determining the deviation and creating the chart? It seems a bit unnatural. Please explain.

Response: We have reviewed the calculations and revised the standard deviation presentation accordingly.