Characterization of Bacterial Exopolysaccharides Produced from Different Fruit-Based Solid Media

Round 1

Reviewer 1 Report

The authors performed an interesting work, but they must correct many issues. The biggest problem of the work is the lack of a result relating to the objective. The text is not convincing that the authors created a new method to recover EPS from fermented fruit or vegetables. The text is also not convincing that the authors developed a new methodology to reliably determine EPS from fermentation media without contamination from plant DF. The text convinces the readers that different strains under several media and plant supplementation produce different types of EPS. These results are important, and the authors must highlight them.

The authors should rewrite the document, highlighting their successful procedure to recover EPS from the media supplemented with fruits. I hope my other comments will help the authors to improve their document.

 

 

**Title:

-The current title is “A new method for recovery allows characterization of bacterial exopolysaccharides from fermented fruit or vegetables.” This title gives me the impression that the authors will compare methods or present analytical parameters for their new method, such as sensitivity, specificity, linearity, limits of detection and quantification, and others. The work shows how different strains produced different types of exopolysaccharides in several media under different fermentation conditions. The title does not represent the performed work and must be changed to better express the research. A possible title could be “Effects of different fruits on the fermentation of bacterial exopolysaccharides on solid media.”

**Abstract

-The “EPS ramified structure differed according to the strain,” but it also differed according to the fruit added to the media and the fermentation conditions, such as adding sucrose or peptone and adjusting the pH. The authors must add these details in the abstract.

**Keywords:

-Why did the authors add “pineapple” as a keyword but not “papaya” and “mango”? The authors must add these other keywords because they used them in the experiment, or they can replace the name of the fruits with “fruit-based media.”

1. Introduction

-The current flow is:

Chemical characteristics of exopolysaccharides (EPS) > Benefits of EPS in humans and factors that impact the EPS production > Recovery of EPS from fermentation medium and problems to identify it without interference from plant dietary fiber (DF) and chemical characteristics of DF > Methods used to quantify EPS > Problems related to the isolation of EPS without contaminating DF and objectives of this work.

The flow is not good. The authors split the same idea into different paragraphs, and they keep moving forth and back in their reasoning. I recommend the following flow:

Chemical characteristics of exopolysaccharides (EPS) > Benefits of EPS in humans > Production of EPS by fermentation > Factors that impact the EPS production > Recovery of EPS from fermentation medium > Methods used to quantify EPS and their limitations > Problems related to the isolation of EPS without contaminating DF > Objectives of this work.

-The authors do not explain the importance of the bacterial strains used in this study or the chosen fruit preparation. It is clear the importance of EPS and the need to quantify them without the influence of DFs, but the authors must make clear why they chose Leuconostoc pseudomesenteroides 56, Weissella cibaria 21, and W. cibaria 64 as target bacteria and why they selected pineapple, mango, and papaya.

2. Materials and Methods

2.1. Bacterial strains and cultivation conditions

-Please explain if the bacteria were freeze-dried and reactivated or if the bacteria came from a refrigerator-kept stock or another type of storage.

2.2. Fruit preparation:

-Please provide a picture of the used fruits. Were they unripe, ripe, rotting, or rotted?

2.3. Screening for EPS production

-Please explain the concentration of bacterial cells as colony-forming units (CFU) in the 100 microliters dropped on the solid media plates.

-This is not a problem, but I would like to suggest to the authors to use in further experiments and this experiment, as possible, to use integers (0, 1, 2, 3, 4) instead of symbols (-, +/-, +, ++, +++) for how mucoid the colonies are.

2.4. Recovery of EPS:

-Please explain a bit more about the manual agitation. Did the authors move the plate with water in an “8” motion for 10 seconds, in a left-right motion for 30 seconds, or another procedure?

-Did the authors perform the washing collection procedure for control plates (plates without bacterial colonies)? Based on subsection 2.6., the authors washed control plates. If the authors did it, they must clarify it in subsection 2.4.

2.5. Phenol-sulfuric colorimetric determination: OK

2.6. Structural characterization by NMR spectroscopy: OK

2.7. Statistical analysis: OK

3. Results

3.1. Determination of EPS content on MRS sucrose

 -The authors cannot claim that “a correlation was observed between quantitative EPS determination and visual score (Figure 1)” because there cannot be a linear regression between qualitative classes of the mucous score and quantitative value of EPS. Regressions are performed between two interval or ratio levels of measurement, not ordinal and ratio levels. Figure 1 shows that the variance of EPS increases for classes with higher mucous amounts. The authors should show the average or median values for the different classes. Please consider presenting the data in Figure 1 as boxplots.

3.2. Visual assessment of EPS content on fruit-based media

-Please clarify if Table 1 and Figure 1 use the same grades. For example, is a ++ grade for W. cibaria 21 on pineapple media, pH 6, sucrose 20%, the same grade as ++ in Figure 1, or is it equivalent to +++? Does it mean that no media presented "very strongly mucoid colonies?"

3.3. Determination of EPS content on fruit purée agar

-Figure 4: Why did the authors not test the W.cibaria 64 in the pineapple juice media? Please, explain the reasoning.

3.6. Structure determination of EPS by NMR spectroscopy

-Where are sections 3.4. and 3.5.? Please, add the missing sections or reorder the number of sections.

-The authors should present data for W. cibaria 21, or at least an explanation for not presenting these data.

4. Discussion

-Weirdly, the authors are critiquing a classic method to determine carbohydrates, suggesting other methods, but without testing them. I strongly recommend removing this discussion because it is out of the scope of the document.

-If the authors want to discuss molecular weight, they must perform an SEC (Size exclusion chromatography) and determine the polymer molecular weight, not use literature information. Please, perform an SEC for EPS from the three strains and add the information to the document.

-It is good to discuss further research, but the authors forgot to discuss the results of this research. Please, explore the obtained data. For example, W. cibaria 64 did not present statistical differences for any treatment, indicating that this strain is somewhat unpredictable. L. pseudomes 56 always had the lowest yield, indicating that they may not be suitable for this type of fermentation. W. cibaria 21 presented predictable yield based on the media, and so on.

**Conclusions

 -The objective was a “simple method for recovery of bacterial EPS from fruit or vegetable foods that can be used prior to their quantification,” and the conclusion was “The developed method, based on a cultivation step on a plate followed by the recovery of EPS from the colony, allowed the quantitative determination of EPS production from fruit juice or puré.” The conclusion is not aligned with the objective. Please, fix it.

-The conclusion “This method resulted in consistent EPS levels and was able to detect differences according to the used strain or medium composition” is not right because W. cibaria 64 did not produce consistent EPS levels in the mango purée and pineapple juice media, with no yield trend based on the sucrose or peptone levels or pH values. The authors need to reformulate their conclusion.

-The authors did not perform any sensorial analysis to determine if the EPS from the fermentation media are pleasant, so they cannot claim any conclusion about it.

**Authors contribution: OK

**Funding: OK

**Data Availability Statement: OK

**Conflicts of Interest: OK

**References:

 

-Please do not capitalize all the nouns of an article, even if it was published this way. For example, please write “Food and health potentials of exopolysaccharides derived from Lactobacilli” instead of “Food and Health Potentials of Exopolysaccharides Derived from Lactobacilli.”

Author Response

Review reports

Reviewer 1

The authors performed an interesting work, but they must correct many issues. The biggest problem of the work is the lack of a result relating to the objective. The text is not convincing that the authors created a new method to recover EPS from fermented fruit or vegetables. The text is also not convincing that the authors developed a new methodology to reliably determine EPS from fermentation media without contamination from plant DF. The text convinces the readers that different strains under several media and plant supplementation produce different types of EPS. These results are important, and the authors must highlight them.

The authors should rewrite the document, highlighting their successful procedure to recover EPS from the media supplemented with fruits. I hope my other comments will help the authors to improve their document.

We are very thankful to the reviewer for critical reading and proposals for improvement of the manuscript. Most of suggested changes were applied and we are convinced thei contribute significantly to improve the manuscript. The absence of contamination of recovered bacterial EPS is showed from NMR analysis but as underlined by the reviewer we missed to highlight this result. Indeed, NMR analysis showed the absence of DF from fruit, since other residues (rhamnose for instance) or β-bonds would have been detected. This was added in section 3.4.

You will find below our other answers and modifications are highlighted in yellow in the MS.

**Title:

-The current title is “A new method for recovery allows characterization of bacterial exopolysaccharides from fermented fruit or vegetables.” This title gives me the impression that the authors will compare methods or present analytical parameters for their new method, such as sensitivity, specificity, linearity, limits of detection and quantification, and others. The work shows how different strains produced different types of exopolysaccharides in several media under different fermentation conditions. The title does not represent the performed work and must be changed to better express the research. A possible title could be “Effects of different fruits on the fermentation of bacterial exopolysaccharides on solid media.”

The reviewer is right and we modified the title. We propose: “Characterization of bacterial exopolysaccharides produced from different fruit-based solid media”.

**Abstract

-The “EPS ramified structure differed according to the strain,” but it also differed according to the fruit added to the media and the fermentation conditions, such as adding sucrose or peptone and adjusting the pH. The authors must add these details in the abstract.

The structure analysis has been performed with the two strains on the two fruit-based media and MRS, but not with sucrose or peptone content variations. Apart from this, the abstract was modified.

**Keywords:

-Why did the authors add “pineapple” as a keyword but not “papaya” and “mango”? The authors must add these other keywords because they used them in the experiment, or they can replace the name of the fruits with “fruit-based media.”

Mango and papaya were added to the list.

1. Introduction

-The current flow is:

Chemical characteristics of exopolysaccharides (EPS) > Benefits of EPS in humans and factors that impact the EPS production > Recovery of EPS from fermentation medium and problems to identify it without interference from plant dietary fiber (DF) and chemical characteristics of DF > Methods used to quantify EPS > Problems related to the isolation of EPS without contaminating DF and objectives of this work.

The flow is not good. The authors split the same idea into different paragraphs, and they keep moving forth and back in their reasoning. I recommend the following flow:

Chemical characteristics of exopolysaccharides (EPS) > Benefits of EPS in humans > Production of EPS by fermentation > Factors that impact the EPS production > Recovery of EPS from fermentation medium > Methods used to quantify EPS and their limitations > Problems related to the isolation of EPS without contaminating DF > Objectives of this work.

We thank the reviewer for this useful comment, which makes the introduction straightforward.

-The authors do not explain the importance of the bacterial strains used in this study or the chosen fruit preparation. It is clear the importance of EPS and the need to quantify them without the influence of DFs, but the authors must make clear why they chose Leuconostoc pseudomesenteroides 56, Weissella cibaria 21, and W. cibaria 64 as target bacteria and why they selected pineapple, mango, and papaya.

The reviewer is right and we added these explanations in the introduction.

2. Materials and Methods

2.1. Bacterial strains and cultivation conditions

-Please explain if the bacteria were freeze-dried and reactivated or if the bacteria came from a refrigerator-kept stock or another type of storage.

This was added.

2.2. Fruit preparation:

-Please provide a picture of the used fruits. Were they unripe, ripe, rotting, or rotted?

The text was modified accordingly.

2.3. Screening for EPS production

-Please explain the concentration of bacterial cells as colony-forming units (CFU) in the 100 microliters dropped on the solid media plates.

The text was modified accordingly.

-This is not a problem, but I would like to suggest to the authors to use in further experiments and this experiment, as possible, to use integers (0, 1, 2, 3, 4) instead of symbols (-, +/-, +, ++, +++) for how mucoid the colonies are.

Right, a score is logically a numeric value. This was modified in the text, figure 1 and table 1.

2.4. Recovery of EPS:

-Please explain a bit more about the manual agitation. Did the authors move the plate with water in an “8” motion for 10 seconds, in a left-right motion for 30 seconds, or another procedure?

This was specified.

-Did the authors perform the washing collection procedure for control plates (plates without bacterial colonies)? Based on subsection 2.6., the authors washed control plates. If the authors did it, they must clarify it in subsection 2.4.

This was added in section 2.4.

2.5. Phenol-sulfuric colorimetric determination: OK

2.6. Structural characterization by NMR spectroscopy: OK

2.7. Statistical analysis: OK

3. Results

3.1. Determination of EPS content on MRS sucrose

 -The authors cannot claim that “a correlation was observed between quantitative EPS determination and visual score (Figure 1)” because there cannot be a linear regression between qualitative classes of the mucous score and quantitative value of EPS. Regressions are performed between two interval or ratio levels of measurement, not ordinal and ratio levels. Figure 1 shows that the variance of EPS increases for classes with higher mucous amounts. The authors should show the average or median values for the different classes. Please consider presenting the data in Figure 1 as boxplots.

We would like to acknowledge the reviewer for this very useful comment. By using a numerical score and boxplot, we noticed that our previous classification failed to discriminate scores (++) (now 3) and (+++) (now 4). Hence, we merged those and the visualization of the results gives a much better representation of data in figure 1.

3.2. Visual assessment of EPS content on fruit-based media

-Please clarify if Table 1 and Figure 1 use the same grades. For example, is a ++ grade for W. cibaria 21 on pineapple media, pH 6, sucrose 20%, the same grade as ++ in Figure 1, or is it equivalent to +++? Does it mean that no media presented "very strongly mucoid colonies?"

The score +++ (now 4) was not observed. However, as explained before, this score is eventually not relevant.

3.3. Determination of EPS content on fruit purée agar

-Figure 4: Why did the authors not test the W.cibaria 64 in the pineapple juice media? Please, explain the reasoning.

A sentence was added.

3.6. Structure determination of EPS by NMR spectroscopy

-Where are sections 3.4. and 3.5.? Please, add the missing sections or reorder the number of sections.

Section numbering presented an error. This was modified.

-The authors should present data for W. cibaria 21, or at least an explanation for not presenting these data.

Right. This was modified.

4. Discussion

-Weirdly, the authors are critiquing a classic method to determine carbohydrates, suggesting other methods, but without testing them. I strongly recommend removing this discussion because it is out of the scope of the document.

-If the authors want to discuss molecular weight, they must perform an SEC (Size exclusion chromatography) and determine the polymer molecular weight, not use literature information. Please, perform an SEC for EPS from the three strains and add the information to the document.

-It is good to discuss further research, but the authors forgot to discuss the results of this research. Please, explore the obtained data. For example, W. cibaria 64 did not present statistical differences for any treatment, indicating that this strain is somewhat unpredictable. L. pseudomes 56 always had the lowest yield, indicating that they may not be suitable for this type of fermentation. W. cibaria 21 presented predictable yield based on the media, and so on.

The discussion was entirely re-written to follow the order: method (analysis of EPS without interference with fruit DF, limitations of visual scoring, variability of results and further improvement) > diversity of production levels according to the strain and the conditions and selection of strain > adjustments of conditions to favor production of EPS during fruit fermentation.

Hence, some parts were removed, such as other methods to determine carbohydrates and discussion on EPS length. Discussion on the results, regarding diversity of production levels according to the strain, was added. To help the reader, numerical data were added in the result section for the maximal EPS production levels.

**Conclusions

 -The objective was a “simple method for recovery of bacterial EPS from fruit or vegetable foods that can be used prior to their quantification,” and the conclusion was “The developed method, based on a cultivation step on a plate followed by the recovery of EPS from the colony, allowed the quantitative determination of EPS production from fruit juice or puré.” The conclusion is not aligned with the objective. Please, fix it.

-The conclusion “This method resulted in consistent EPS levels and was able to detect differences according to the used strain or medium composition” is not right because W. cibaria 64 did not produce consistent EPS levels in the mango purée and pineapple juice media, with no yield trend based on the sucrose or peptone levels or pH values. The authors need to reformulate their conclusion.

-The authors did not perform any sensorial analysis to determine if the EPS from the fermentation media are pleasant, so they cannot claim any conclusion about it.

We thank the reviewer for these suggestions. The conclusion was rewritten to better fit the objective and use right conclusions.

**Authors contribution: OK

**Funding: OK

**Data Availability Statement: OK

**Conflicts of Interest: OK

 

**References:

-Please do not capitalize all the nouns of an article, even if it was published this way. For example, please write “Food and health potentials of exopolysaccharides derived from Lactobacilli” instead of “Food and Health Potentials of Exopolysaccharides Derived from Lactobacilli.”

We used the available formatted style for the Fermentation journal, which uses capitalization of each nouns. However, we applied the proposed modification.

 

Reviewer 2

The authors of the Manuscript fermentation-2473950, Guérin et al., describe the characterization of the exopolysaccharides produced on media based on fruit puree, solidified by agar addition. The manuscript needs improvement before publication.

The title needs to be changed to reflect better the article content. The method is not new – a media containing carrot puree, solidified by agar addition, was used to recover exopolysaccharides produced by lactic acid bacteria and further characterized it - Juvonen, R.; Honkapää, K.; Maina, N.H.; Shi, Q.; Viljanen, K.; Maaheimo, H.; Virkki, L.; Tenkanen, M.; Lantto, R. The Impact  of Fermentation with Exopolysaccharide Producing Lactic Acid Bacteria on Rheological, Chemical and Sensory Properties of Pureed  Carrots (Daucus Carota L.). International journal of food microbiology 2015, 207, 109–118, doi:10.1016/j.ijfoodmicro.2015.04.031. 508. This method is quite similar to the “new” method described by the authors – the authors cited this method several times. The recovery was not done “from fermented fruit or vegetables”, but from media solidified with agar that contain pineapple juice or fruit puree from mango or papaya (and not puree from vegetables) and, in some cases, sucrose and peptone.

We thank the reviewer for its critical reading. Reviewer 2 comments were perfectly in line with those of Reviewer 1. We took into account both and modified deeply the manuscript.

The title was changed to “Characterization of bacterial exopolysaccharides produced from different fruit-based solid media”.

More keywords must be added – for example, mango and papaya, (template allows up “to ten pertinent keywords specific to the article”).

They were added.

In the Abstract, the sentence ” This method for recovery of EPS opens new perspectives to pilot EPS production in lactic fermented fruit or vegetable foods” must be nuanced – the described method is helpful to select strains that efficiently produce EPS in industrial relevant conditions.

This was modified.

In the last paragraph of the Introduction Section, the authors must “briefly mention the main aim of the work and highlight the principal conclusions”. The authors describe not only a method to produce exopolysaccharides that does not involve several purification steps. They also made a structural characterization by NMR spectroscopy.

The change was applied and the introduction final paragraph deeply modified.

In subsection 2.1 Bacterial strains and cultivation conditions, authors must describe the exact cultivation condition – anaerobic, microaerophilic, aerated, and the devices used to cultivate the tested bacterial strains.

This was specified.

The Conclusion Section must also include the main conclusions from structural characterization by NMR spectroscopy, e.g., “EPS  ramified structure differed according to the strain.”

This was added.

Reviewer 2 Report

The authors of the Manuscript fermentation-2473950, Guérin et al., describe the characterization of the exopolysaccharides produced on media based on fruit puree, solidified by agar addition. The manuscript needs improvement before publication.

The title needs to be changed to reflect better the article content. The method is not new – a media containing carrot puree, solidified by agar addition, was used to recover exopolysaccharides produced by lactic acid bacteria and further characterized it - Juvonen, R.; Honkapää, K.; Maina, N.H.; Shi, Q.; Viljanen, K.; Maaheimo, H.; Virkki, L.; Tenkanen, M.; Lantto, R. The Impact  of Fermentation with Exopolysaccharide Producing Lactic Acid Bacteria on Rheological, Chemical and Sensory Properties of Pureed  Carrots (Daucus Carota L.). International journal of food microbiology 2015, 207, 109–118, doi:10.1016/j.ijfoodmicro.2015.04.031. 508. This method is quite similar to the “new” method described by the authors – the authors cited this method several times. The recovery was not done “from fermented fruit or vegetables”, but from media solidified with agar that contain pineapple juice or fruit puree from mango or papaya (and not puree from vegetables) and, in some cases, sucrose and peptone.

More keywords must be added – for example, mango and papaya, (template allows up “to ten pertinent keywords specific to the article”).

In the Abstract, the sentence ” This method for recovery of EPS opens new perspectives to pilot EPS production in lactic fermented fruit or vegetable foods” must be nuanced – the described method is helpful to select strains that efficiently produce EPS in industrial relevant conditions.

In the last paragraph of the Introduction Section, the authors must “briefly mention the main aim of the work and highlight the principal conclusions”. The authors describe not only a method to produce exopolysaccharides that does not involve several purification steps. They also made a structural characterization by NMR spectroscopy.

In subsection 2.1 Bacterial strains and cultivation conditions, authors must describe the exact cultivation condition – anaerobic, microaerophilic, aerated, and the devices used to cultivate the tested bacterial strains.

The Conclusion Section must also include the main conclusions from structural characterization by NMR spectroscopy, e.g., “EPS  ramified structure differed according to the strain.”

Author Response

Review reports

Reviewer 1

The authors performed an interesting work, but they must correct many issues. The biggest problem of the work is the lack of a result relating to the objective. The text is not convincing that the authors created a new method to recover EPS from fermented fruit or vegetables. The text is also not convincing that the authors developed a new methodology to reliably determine EPS from fermentation media without contamination from plant DF. The text convinces the readers that different strains under several media and plant supplementation produce different types of EPS. These results are important, and the authors must highlight them.

The authors should rewrite the document, highlighting their successful procedure to recover EPS from the media supplemented with fruits. I hope my other comments will help the authors to improve their document.

We are very thankful to the reviewer for critical reading and proposals for improvement of the manuscript. Most of suggested changes were applied and we are convinced thei contribute significantly to improve the manuscript. The absence of contamination of recovered bacterial EPS is showed from NMR analysis but as underlined by the reviewer we missed to highlight this result. Indeed, NMR analysis showed the absence of DF from fruit, since other residues (rhamnose for instance) or β-bonds would have been detected. This was added in section 3.4.

You will find below our other answers and modifications are highlighted in yellow in the MS.

**Title:

-The current title is “A new method for recovery allows characterization of bacterial exopolysaccharides from fermented fruit or vegetables.” This title gives me the impression that the authors will compare methods or present analytical parameters for their new method, such as sensitivity, specificity, linearity, limits of detection and quantification, and others. The work shows how different strains produced different types of exopolysaccharides in several media under different fermentation conditions. The title does not represent the performed work and must be changed to better express the research. A possible title could be “Effects of different fruits on the fermentation of bacterial exopolysaccharides on solid media.”

The reviewer is right and we modified the title. We propose: “Characterization of bacterial exopolysaccharides produced from different fruit-based solid media”.

**Abstract

-The “EPS ramified structure differed according to the strain,” but it also differed according to the fruit added to the media and the fermentation conditions, such as adding sucrose or peptone and adjusting the pH. The authors must add these details in the abstract.

The structure analysis has been performed with the two strains on the two fruit-based media and MRS, but not with sucrose or peptone content variations. Apart from this, the abstract was modified.

**Keywords:

-Why did the authors add “pineapple” as a keyword but not “papaya” and “mango”? The authors must add these other keywords because they used them in the experiment, or they can replace the name of the fruits with “fruit-based media.”

Mango and papaya were added to the list.

1. Introduction

-The current flow is:

Chemical characteristics of exopolysaccharides (EPS) > Benefits of EPS in humans and factors that impact the EPS production > Recovery of EPS from fermentation medium and problems to identify it without interference from plant dietary fiber (DF) and chemical characteristics of DF > Methods used to quantify EPS > Problems related to the isolation of EPS without contaminating DF and objectives of this work.

The flow is not good. The authors split the same idea into different paragraphs, and they keep moving forth and back in their reasoning. I recommend the following flow:

Chemical characteristics of exopolysaccharides (EPS) > Benefits of EPS in humans > Production of EPS by fermentation > Factors that impact the EPS production > Recovery of EPS from fermentation medium > Methods used to quantify EPS and their limitations > Problems related to the isolation of EPS without contaminating DF > Objectives of this work.

We thank the reviewer for this useful comment, which makes the introduction straightforward.

-The authors do not explain the importance of the bacterial strains used in this study or the chosen fruit preparation. It is clear the importance of EPS and the need to quantify them without the influence of DFs, but the authors must make clear why they chose Leuconostoc pseudomesenteroides 56, Weissella cibaria 21, and W. cibaria 64 as target bacteria and why they selected pineapple, mango, and papaya.

The reviewer is right and we added these explanations in the introduction.

2. Materials and Methods

2.1. Bacterial strains and cultivation conditions

-Please explain if the bacteria were freeze-dried and reactivated or if the bacteria came from a refrigerator-kept stock or another type of storage.

This was added.

2.2. Fruit preparation:

-Please provide a picture of the used fruits. Were they unripe, ripe, rotting, or rotted?

The text was modified accordingly.

2.3. Screening for EPS production

-Please explain the concentration of bacterial cells as colony-forming units (CFU) in the 100 microliters dropped on the solid media plates.

The text was modified accordingly.

-This is not a problem, but I would like to suggest to the authors to use in further experiments and this experiment, as possible, to use integers (0, 1, 2, 3, 4) instead of symbols (-, +/-, +, ++, +++) for how mucoid the colonies are.

Right, a score is logically a numeric value. This was modified in the text, figure 1 and table 1.

2.4. Recovery of EPS:

-Please explain a bit more about the manual agitation. Did the authors move the plate with water in an “8” motion for 10 seconds, in a left-right motion for 30 seconds, or another procedure?

This was specified.

-Did the authors perform the washing collection procedure for control plates (plates without bacterial colonies)? Based on subsection 2.6., the authors washed control plates. If the authors did it, they must clarify it in subsection 2.4.

This was added in section 2.4.

2.5. Phenol-sulfuric colorimetric determination: OK

2.6. Structural characterization by NMR spectroscopy: OK

2.7. Statistical analysis: OK

3. Results

3.1. Determination of EPS content on MRS sucrose

 -The authors cannot claim that “a correlation was observed between quantitative EPS determination and visual score (Figure 1)” because there cannot be a linear regression between qualitative classes of the mucous score and quantitative value of EPS. Regressions are performed between two interval or ratio levels of measurement, not ordinal and ratio levels. Figure 1 shows that the variance of EPS increases for classes with higher mucous amounts. The authors should show the average or median values for the different classes. Please consider presenting the data in Figure 1 as boxplots.

We would like to acknowledge the reviewer for this very useful comment. By using a numerical score and boxplot, we noticed that our previous classification failed to discriminate scores (++) (now 3) and (+++) (now 4). Hence, we merged those and the visualization of the results gives a much better representation of data in figure 1.

3.2. Visual assessment of EPS content on fruit-based media

-Please clarify if Table 1 and Figure 1 use the same grades. For example, is a ++ grade for W. cibaria 21 on pineapple media, pH 6, sucrose 20%, the same grade as ++ in Figure 1, or is it equivalent to +++? Does it mean that no media presented "very strongly mucoid colonies?"

The score +++ (now 4) was not observed. However, as explained before, this score is eventually not relevant.

3.3. Determination of EPS content on fruit purée agar

-Figure 4: Why did the authors not test the W.cibaria 64 in the pineapple juice media? Please, explain the reasoning.

A sentence was added.

3.6. Structure determination of EPS by NMR spectroscopy

-Where are sections 3.4. and 3.5.? Please, add the missing sections or reorder the number of sections.

Section numbering presented an error. This was modified.

-The authors should present data for W. cibaria 21, or at least an explanation for not presenting these data.

Right. This was modified.

4. Discussion

-Weirdly, the authors are critiquing a classic method to determine carbohydrates, suggesting other methods, but without testing them. I strongly recommend removing this discussion because it is out of the scope of the document.

-If the authors want to discuss molecular weight, they must perform an SEC (Size exclusion chromatography) and determine the polymer molecular weight, not use literature information. Please, perform an SEC for EPS from the three strains and add the information to the document.

-It is good to discuss further research, but the authors forgot to discuss the results of this research. Please, explore the obtained data. For example, W. cibaria 64 did not present statistical differences for any treatment, indicating that this strain is somewhat unpredictable. L. pseudomes 56 always had the lowest yield, indicating that they may not be suitable for this type of fermentation. W. cibaria 21 presented predictable yield based on the media, and so on.

The discussion was entirely re-written to follow the order: method (analysis of EPS without interference with fruit DF, limitations of visual scoring, variability of results and further improvement) > diversity of production levels according to the strain and the conditions and selection of strain > adjustments of conditions to favor production of EPS during fruit fermentation.

Hence, some parts were removed, such as other methods to determine carbohydrates and discussion on EPS length. Discussion on the results, regarding diversity of production levels according to the strain, was added. To help the reader, numerical data were added in the result section for the maximal EPS production levels.

**Conclusions

 -The objective was a “simple method for recovery of bacterial EPS from fruit or vegetable foods that can be used prior to their quantification,” and the conclusion was “The developed method, based on a cultivation step on a plate followed by the recovery of EPS from the colony, allowed the quantitative determination of EPS production from fruit juice or puré.” The conclusion is not aligned with the objective. Please, fix it.

-The conclusion “This method resulted in consistent EPS levels and was able to detect differences according to the used strain or medium composition” is not right because W. cibaria 64 did not produce consistent EPS levels in the mango purée and pineapple juice media, with no yield trend based on the sucrose or peptone levels or pH values. The authors need to reformulate their conclusion.

-The authors did not perform any sensorial analysis to determine if the EPS from the fermentation media are pleasant, so they cannot claim any conclusion about it.

We thank the reviewer for these suggestions. The conclusion was rewritten to better fit the objective and use right conclusions.

**Authors contribution: OK

**Funding: OK

**Data Availability Statement: OK

**Conflicts of Interest: OK

 

**References:

-Please do not capitalize all the nouns of an article, even if it was published this way. For example, please write “Food and health potentials of exopolysaccharides derived from Lactobacilli” instead of “Food and Health Potentials of Exopolysaccharides Derived from Lactobacilli.”

We used the available formatted style for the Fermentation journal, which uses capitalization of each nouns. However, we applied the proposed modification.

 

Reviewer 2

The authors of the Manuscript fermentation-2473950, Guérin et al., describe the characterization of the exopolysaccharides produced on media based on fruit puree, solidified by agar addition. The manuscript needs improvement before publication.

The title needs to be changed to reflect better the article content. The method is not new – a media containing carrot puree, solidified by agar addition, was used to recover exopolysaccharides produced by lactic acid bacteria and further characterized it - Juvonen, R.; Honkapää, K.; Maina, N.H.; Shi, Q.; Viljanen, K.; Maaheimo, H.; Virkki, L.; Tenkanen, M.; Lantto, R. The Impact  of Fermentation with Exopolysaccharide Producing Lactic Acid Bacteria on Rheological, Chemical and Sensory Properties of Pureed  Carrots (Daucus Carota L.). International journal of food microbiology 2015, 207, 109–118, doi:10.1016/j.ijfoodmicro.2015.04.031. 508. This method is quite similar to the “new” method described by the authors – the authors cited this method several times. The recovery was not done “from fermented fruit or vegetables”, but from media solidified with agar that contain pineapple juice or fruit puree from mango or papaya (and not puree from vegetables) and, in some cases, sucrose and peptone.

We thank the reviewer for its critical reading. Reviewer 2 comments were perfectly in line with those of Reviewer 1. We took into account both and modified deeply the manuscript.

The title was changed to “Characterization of bacterial exopolysaccharides produced from different fruit-based solid media”.

More keywords must be added – for example, mango and papaya, (template allows up “to ten pertinent keywords specific to the article”).

They were added.

In the Abstract, the sentence ” This method for recovery of EPS opens new perspectives to pilot EPS production in lactic fermented fruit or vegetable foods” must be nuanced – the described method is helpful to select strains that efficiently produce EPS in industrial relevant conditions.

This was modified.

In the last paragraph of the Introduction Section, the authors must “briefly mention the main aim of the work and highlight the principal conclusions”. The authors describe not only a method to produce exopolysaccharides that does not involve several purification steps. They also made a structural characterization by NMR spectroscopy.

The change was applied and the introduction final paragraph deeply modified.

In subsection 2.1 Bacterial strains and cultivation conditions, authors must describe the exact cultivation condition – anaerobic, microaerophilic, aerated, and the devices used to cultivate the tested bacterial strains.

This was specified.

The Conclusion Section must also include the main conclusions from structural characterization by NMR spectroscopy, e.g., “EPS  ramified structure differed according to the strain.”

This was added.

Round 2

Reviewer 1 Report

The authors performed a compelling work and altered the commented points in the previous round of reviews. The overall quality increased a lot. Good job!
There are still minor points to fix in the document:

**Title: OK
**Abstract: OK
**Keywords: OK
1. Introduction
-The current flow is:
Chemical characteristics of exopolysaccharides (EPS) > Benefits of EPS in humans > Production of EPS by fermentation > Methods to quantify EPS > Impact of fruit dietary fibers (DF) on EPS quantification > Objectives of this work.

The current flow is good enough for the document.

-The authors used three strains, but they mentioned five. Please, fix the Introduction to align it with the Materials and Methods.

2. Materials and Methods
2.1. Bacterial strains and cultivation conditions: OK
2.2. Fruit preparation: OK
2.3. Screening for EPS production:
-Please keep a space between units and the numbers, but do not keep a space between units and “°C” and “%.”
2.4. Recovery of EPS: OK
2.5. Phenol-sulfuric colorimetric determination: OK
2.6. Structural characterization by NMR spectroscopy: OK
2.7. Statistical analysis: OK
3. Results
3.1. Determination of EPS content on MRS sucrose: OK
3.2. Visual assessment of EPS content on fruit-based media: OK
3.3. Determination of EPS content on fruit purée agar: OK
3.4. Structure determination of EPS by NMR spectroscopy: OK
4. Discussion: OK
**Conclusions: OK
**Authors contribution: OK
**Funding: OK
**Data Availability Statement: OK
**Conflicts of Interest: OK
**References: OK

Author Response

Review report

Answers and changes in the manuscript are in red.

Reviewer 1

The authors performed a compelling work and altered the commented points in the previous round of reviews. The overall quality increased a lot. Good job!

There are still minor points to fix in the document:

 

**Title: OK

**Abstract: OK

**Keywords: OK

1. Introduction

-The current flow is:

Chemical characteristics of exopolysaccharides (EPS) > Benefits of EPS in humans > Production of EPS by fermentation > Methods to quantify EPS > Impact of fruit dietary fibers (DF) on EPS quantification > Objectives of this work.

 

The current flow is good enough for the document.

 

-The authors used three strains, but they mentioned five. Please, fix the Introduction to align it with the Materials and Methods.

The mention of supplementary strains in the introduction was removed.

 

2. Materials and Methods

2.1. Bacterial strains and cultivation conditions: OK

2.2. Fruit preparation: OK

2.3. Screening for EPS production:

-Please keep a space between units and the numbers, but do not keep a space between units and “°C” and “%.”

Changes were made in this section as well as in the entire document.

2.4. Recovery of EPS: OK

2.5. Phenol-sulfuric colorimetric determination: OK

2.6. Structural characterization by NMR spectroscopy: OK

2.7. Statistical analysis: OK

3. Results

3.1. Determination of EPS content on MRS sucrose: OK

3.2. Visual assessment of EPS content on fruit-based media: OK

3.3. Determination of EPS content on fruit purée agar: OK

3.4. Structure determination of EPS by NMR spectroscopy: OK

4. Discussion: OK

**Conclusions: OK

**Authors contribution: OK

**Funding: OK

**Data Availability Statement: OK

**Conflicts of Interest: OK

**References: OK