Review Reports

Round 1

Reviewer 1 Report

The manuscript addresses the study of adhesion of a strain Lactobacillus namurensis - an isolate from salted rice bran - to porous fine ceramics  in order to develop a LAB-containing pickle container for the production of fermented vegetables. To be honest, I do not see a deeper sense of this. The authors focused on the importance of pore size of the material in order to increase the adhesion ability of LAB. Indeed, there are numerous other, more important factors influencing the overall adhesion process. This point is not reflected in the paper, it is extremly focusing on only one parameter - pore size and thus, physico-chemical properties of the material of the conntainer. Limitations and shortcomings of the study are not discussed at all. It should be included in the discussion part, and also introduction should  give more comprehensive information and background and describe more in detailed all the potential factors involved in overall adhesion.  

Comments for authors:

How do you want to ensure bacterial viability and sufficient LAB-counts? What about the survival rate? 

line 48: not all LABs are probiotics

line 63: "fermented container"? or "fermentation container"?

Introduction: Please, include more background related to adherence. There are so many...

GYP agar is used for lactobacilli? or LAb? or which bacterial group? Later used MRS agar in the experiment, why?

You isolated twenta strains (line 181) but you have chosen only the strain GYP-74. Based on what? Some properties of the isolates? 

For future, it is better to test a bigger group of bacteria to have a stronger conclusion.

English grammar/style check would be recommended.

Include limitations of the study. Discussion part: You state that the mechanisms of adherence to porous materials are significantly different than those related to GIT - please, elaborate and discuss more...

Conclusion: avoid to generalize ("LAB adherence"), you tested only one strain of Lbc. 

 

Author Response

Thank you very much for valuable comments. 

1) How do you want to ensure bacterial viability and sufficient LAB-counts? What about the survival rate?

In this study, we focused on number of adherent bacterial cells, and adherent to ceramics was evaluated by colony-counting. We can confirm the “viability” of strain GYP-74 based on colony counting because bacterial colonies cannot be formed without bacterial viability. When we consider both viability and survival rate, if we expose bacterial cells to severe conditions such as low pH, high temperatures, we can evaluate the “survival rate” by colony-counting at the same time. However, in section 2.5. (Adherent test), strain GYP-74 was exposed to mild condition meaning immersion to dilute MRS medium because this assay was for checking how many bacterial cells can attach to each kind of ceramics.

When we think “sufficient LAB-counts”, the roles expected by enriched LAB should be considered. In this study, we isolated LABs from the salted rice bran used for pickling (Nukadoko), and carried out adherent test using one strain GYP-74. In this test, we wanted to confirm which type of ceramics is adequate to enrich the cells of this strain. This is a model case. In future, we’ll try to check the adherent level of various LAB strains, and can expect the role of each LAB such as modification of taste of Nukazuke (fermented vegetables), probiotics which can enhance immunity of human… Based on the used LAB and objective, we can define the sufficient LAB-counts.

 

2) line 48: not all LABs are probiotics

  Yes, that’s right. We also understand that all LABs are not probiotics. In the entire projects, firstly, we’d like to develop the specific vessel to easily enrich LABs by arranging the size distribution of pottery. This study is first step of this project. Secondly, we plan to do screening test to select LABs which have probiotic ability.

To explain more, we added one sentence “Some species of LABs have been expected as probiotics for various host animals including aquacultured species” and references.

 

3) line 63: "fermented container"? or "fermentation container"?

  Here, it should be “fermentation”. I’m sorry, it is confused. We changed this to “fermentation”.

 

4) Introduction: Please, include more background related to adherence. There are so many...

Thank you for suggestions. I added more information about adherence and references.

 

5) GYP agar is used for lactobacilli? or LAb? or which bacterial group? Later used MRS agar in the experiment, why?

GYP agar medium has been used for the isolation of wide varieties of LABs. Some species of LABs can not grow or weakly grow on MRS medium. For isolation, we targeted all LABs, not only lactobacilli. That’s why we used GYP medium in the first half of this study. After purification, we used MRS medium because the strain GYP-74 was identified to genus Lactobacillus which can grow well on this medium

 

5) You isolated twenta strains (line 181) but you have chosen only the strain GYP-74. Based on what? Some properties of the isolates?

　We understood the reviewer’s question well. In this study, we attempted to isolate LABs from pickling (Nukadoko), and all isolates were identified to L. namurensis. We checked the growth rate in MRS medium among isolates, and the growth of strain GTP-74 used in this study was slightly faster than another isolates. We could not find select any other factors to choose strains for this study. Therefore, we chose strain GYP-74.

 

6) For future, it is better to test a bigger group of bacteria to have a stronger conclusion.

Yes, off course. As I answered in question 2, our team plan to check the possibility of this ceramics to enrich LABS by using wide varieties of LABs. Thank you for this comment.

 

7) English grammar/style check would be recommended.

As I noted in the cover letter, English sentences was checked by a native English speaker in KN-international Company before submission. Ok, we’ll check this in entire manuscript again. Thank you very much.

 

8) Include limitations of the study. Discussion part: You state that the mechanisms of adherence to porous materials are significantly different than those related to GIT - please, elaborate and discuss more...

  Thank you for valuable suggestions. Yes, it is better to add this point to enrich the content of this manuscript. We added more information and references in the revised manuscript.

 

9) Conclusion: avoid to generalize ("LAB adherence"), you tested only one strain of Lbc.

  Yes, I agree with the reviewer’s suggestion. I changed this point from LAB to L. namurensis.

 

Reviewer 2 Report

In general, this paper is interesting and might have potential impact for relevant industries if it can be better presented with in-depth discussions. There were several technical issues to be addressed. Furthermore, the manuscript needs intensive editing.  Please see details below. 

1) The authors should also measure the surface wetting (e.g. contact angle) and surface roughness.

2)  However, there was lack of in-depth discussion of surface properties effect on bacteria attachment on different surfaces.

More detailed discussions about the effect of surface roughness and surface wetting are required. For example, the authors could talk about the XDLVO theories and its extended version by considering surface roughness.

Some useful details were covered in the following papers. 

R. An, Y. Dong, J. Zhu, C. Rao, Colloids Surfaces B: Biointerfaces 159 (2017) 108-117.

Modelling the combined effect of surface roughness and topography on bacterial attachment, Journal of Materials Science and Technology 81 (2021) 151–161

Influence of surface roughness on the initial formation of biofilm, Surface and Coatings Technology 284 (2015) 410-416

Nanostructured titanium surfaces exhibit recalcitrance towards Staphylococcus epidermidis biofilm formation, Sci Rep 8(1) (2018) 1071.

R.J. Crawford, H.K. Webb, V.K. Truong, J. Hasan, E.P. Ivanova, Adv Colloid Interface Sci 179-182 (2012) 142-9.

 

2) The Figure captions were either too wordy or too concise. They need to be rewritten. For example, in Fig.2, what is the surface that bacteria adherent to?

3) Method: Some sections should be tightened. There was some repetition in the description. For example, section 2.4 and 2.5 should be combined and be trimmed.

4) In Section 2.4: Strain GYP-74 was cultured in a GYP liquid medium at 28 °C for 48 h. However, in section 2.5: The test tube was incubated at 27oC for 45 hrs.  Why these two culture temperature and duration were different?

5) The results were not well organized. The authors should present SEM images of different porous surfaces and then add representative images of bacteria on these surfaces.

6) The conclusions were not satisfactory. It should be rewritten.

7) I suggest that the authors ask a native English speaker to do a careful proofreading.

Author Response

Thank you very much for valuable comments for our manuscript.

 

1) The authors should also measure the surface wetting (e.g. contact angle) and surface roughness.

Actually, our research team also has considered this point. However, at present, it is difficult to analyze these subjects in our facilities. Our project will be continued, and this study was first step in entire projects. In future, we have plan to collaborate Miyazaki Prefectural Industrial Research Institute which can analyze these parameters. In next study, we’d like to incorporate these analyses and collect more detailed data. Thank you for valuable suggestions.

 

2)  However, there was lack of in-depth discussion of surface properties effect on bacteria attachment on different surfaces. More detailed discussions about the effect of surface roughness and surface wetting are required. For example, the authors could talk about the XDLVO theories and its extended version by considering surface roughness. Some useful details were covered in the following papers.

  Thank you very much for suggestions. As we answered, at present, it is difficult to determined surface roughness and surface wetting. However, as a reviewer commented, it is better to discuss more using references related to these parameter. I added more discussion with references which a reviewer recommended.

 

2) The Figure captions were either too wordy or too concise. They need to be rewritten. For example, in Fig.2, what is the surface that bacteria adherent to?

  Fig. 2 is to present cell morphology of this strain, meaning that the SEM picture shows only cells cultured in liquid medium. In all Figures, We checked the Figure captions, and rewrote the Figure captions. And also, as we answered to question 3, we combined section 2.4 and 2.5. We rewrote the explanation about SEM pictures due to this.

 

3) Method: Some sections should be tightened. There was some repetition in the description. For example, section 2.4 and 2.5 should be combined and be trimmed.

　Thank you for indicating the construction of this manuscript. We combined section 2.4 and 2.5, and deleted unnecessary sentences according to ta reviewer’s suggestion.

 

4) In Section 2.4: Strain GYP-74 was cultured in a GYP liquid medium at 28 °C for 48 h. However, in section 2.5: The test tube was incubated at 27 ^oC for 45 hrs.  Why these two culture temperature and duration were different?

  Actually, the incubator used in this study has heating function, but not cooling function. Our University was located on south area, Kyushu, Japan, and the temperature increased to more than 30 ^oC in summer. In other seasons except for summer, we can control the temperature at 27 ^oC, but the temperature in summer increase to 28 ^oC. And also, we estimated bacterial density by measuring turbidity with a spectrophotometer, and adjust it every experiments. That’s why temperature and duration were slightly different.

 

5) The results were not well organized. The authors should present SEM images of different porous surfaces and then add representative images of bacteria on these surfaces.

  In this study, the adherent ability to ceramics was evaluated by plate-counting method. And, as you indicated, it is better to evaluate this by SEM pictures. However, it is difficult to express the adherence of total bacteria based on SEM pictures. That’s why we added only one image in this manuscript. However, we agreed with the reviewer’s suggestion.Therefore, we put one more picture of control group in Fig. 4. At least, it is possible to understand the effective of porous on bacterial adherence by comparing between two pictures. Actual adherent cell number can be confirmed by seeing the data in Fig. 3.

 

6) The conclusions were not satisfactory. It should be rewritten.

　We rewrote the conclusions according to the suggestion by a reviewer.

 

7) I suggest that the authors ask a native English speaker to do a careful proofreading.

  As I noted in the cover letter, English sentences was checked by a native English speaker in KN-international Company before submission. Ok, we’ll check this in entire manuscript again. Thank you very much.

Round 2

Reviewer 1 Report

The quality of the manuscript has been improved and the questions have been answered and explained.

Reviewer 2 Report

The authors have reasonably addressed the comments raised.