Evaluation of Biofilm Inhibitory Activity of Probiotics and Postbiotics Using In Vitro Biofilm Model of Canine Periodontal Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors Generally the manuscript is well written: the logic is clear, the experiments are reasonable tests of the hypotheses, the references are sufficient, and the conclusions drawn are appropriate to the presented results.   My major request is more discussion of what I find to be very interesting and somewhat surprising results found in Table 5 describing the inhibition of various combinations of postbiotic derivatives and probiotic bacteria.   Specifically:   Line 282: Can you add a little bit about why YCW-2 was progressed? In light of the next table, this makes sense, but based on the results reported prior to this point, particularly Figure 1, YCW-2 doesn't seem to be a natural choice. Maybe just make the reasoning a little more clear.   Line 351: New paragraph: The results of the combination treatments are very interesting and somewhat unexpected. Could you expand a little more on some possible explanations for the results reported in Table 5?   Additionally I think it would be very compelling for many readers to see some representative images from the FISH evaluation of the biofilm.   There are a number of very minor modifications (language usage and formatting) below that I think would improve the overall manuscript for many readers. Otherwise the manuscript is very well written. The methods, in particular, are very detailed and clear.   Minor requests:   Lines 40-43: Two which clauses can be a little hard to read: "These bacteria can infiltrate the subgingival space and produce several metabolites such as ammonia and volatile sulfur compounds, which lead to halitosis, as well as bacterial endotoxins and proteolytic enzymes, which promote periodontal inflammation and stimulation of the animal’s immune system [3]."   maybe something like:   "These bacteria can infiltrate the subgingival space and produce several metabolites including ammonia and volatile sulfur compounds, which lead to halitosis, as well as bacterial endotoxins and proteolytic enzymes: promoting periodontal inflammation and stimulating the animal’s immune system [3]."   Lines 48-50: This sentence is a little awkward, just punctuate differently: "Although the chemicals currently used, such as chlorhexidine gluconate, offer immediate benefit regarding PD progression, they may also present some adverse effects, such as gastric disorders, dysgeusia, mucosal irritation, and tooth discoloration, which can impair continued use [7]."   maybe:   "Although the chemicals currently used, such as chlorhexidine gluconate, offer immediate benefit regarding PD progression, they may also present adverse effects such as: gastric disorders, dysgeusia, mucosal irritation, and tooth discoloration; which can impair continued use [7]."   Lines 51-52: "Considering that," is probably not necessary here: "Considering that, new preventive and therapeutic approaches are required to control PD in companion animals."   maybe just:   "New preventive and therapeutic approaches are required to control PD in companion animals"   Line 58: "Bacterial collection" maybe just to be absolutely explicit "Biofilm bacterial collection" to distinguish from the probiotic bacteria.   Line 231: Either the word "Data" or the word "statistical" is sufficient. Both are a little unnatural in ordinary scientific English usage.   "Data statistical analysis" Either: "Data analysis" or preferably "Statistical analysis"   Table 4: I am having a little trouble interpreting the sub-titles. If:   "A total of 8 postbiotic yeast derivatives components (YE-2, YE-3, YE-4, YE-5, YE-6, 77 YE-7, YCW-1 and YCW-2) and 3 probiotic bacterial strains (BC-23, BC-05, BC-37) (Lesaffre, France), were evaluated in this study." Lines 77-79   then I think: "Concentrations (%) of postbiotics and probiotic components" should be the title for the whole table i.e. including both yeast derivatives and bacterial strains. And a sub-title: "Concentrations (%) of postbiotic component" should be add for the YE-x yeast derivatives at the top. Then you would have: Title "Concentrations (%) of postbiotics and probiotic components" Sub-title: "Concentrations (%) of postbiotic component" for the YE-x rows Sub-title: "Concentrations (%) of postbiotic component" for YCW-1 rows Sub-title: "Concentrations (%) of postbiotic component" for YCW-2 rows (different concentration columns) Sub-title: "Concentration (CFU/mL) of probiotic bacterial strain" for the BC-x rows.   Line 269: Maybe add a sentence noting that BC-23 and BC-37 were comparatively ineffective with the exception of P. canis which was reduced at least somewhat in all probiotic treatments and all postbiotic treatments except YE-4.   Line 272: "postbiotic" should be "postbiotic or probiotic" (to include the case of the bacterial probiotic)   Line 280: "Based on the previous results obtained" is a little confusing, somehwhat ambiguous between previous section of this manuscript versus previous manuscripts. Maybe just: "Based on the results obtained"  

Author Response

Author's Reply to the Review Report (Reviewer 1)
Dear Reviewer
We would like to express our gratitude for the comments and the discussion points presented. We believe they will improve the quality and clarity of the manuscript. Each one of the points described were addressed as you may see in the revised version of the manuscript in track changes. Please see below the response to each question.
Comments and Suggestions for Authors
Generally the manuscript is well written: the logic is clear, the experiments are reasonable tests of the hypotheses, the references are sufficient, and the conclusions drawn are appropriate to the presented results.
My major request is more discussion of what I find to be very interesting and somewhat surprising results found in Table 5 describing the inhibition of various combinations of postbiotic derivatives and probiotic bacteria. Specifically: Line 282: Can you add a little bit about why YCW-2 was progressed? In light of the next table, this makes sense, but based on the results reported prior to this point, particularly Figure 1, YCW-2 doesn't seem to be a natural choice. Maybe just make the reasoning a little more clear.
Response: We understand your question. The selection of the yeast postbiotic YCW-2 was based on its preliminary inhibitory potential, as YCW-2 showed clear inhibition halos against both C. canis and P. canis. Subsequently, in the polymicrobial biofilm assay, we observed a greater reduction of P. canis compared with YCW-1. This led us to include both yeast extracts in the following experiments. Since P. canis is associated with the progression of periodontal disease in dogs, particularly in severe cases, an ingredient with strong activity against this bacterium is especially valuable. Additional information has been added in lines 420-423.
Line 351: New paragraph: The results of the combination treatments are very interesting and somewhat unexpected. Could you expand a little more on some possible explanations for the results reported in Table 5?
Response: We thank you for your question. We observed very interesting results, with distinct activities depending on the combinations tested. This highlights not only the differences between
the components, but also their potential variations in antimicrobial mechanisms. Notably, YE-7 and BC-05 exhibited antimicrobial activity in almost all combinations, with only one combination for each ingredient (YE-7 + YCM-1 and BC-05 + YE-3) showing no inhibitory potential. Furthermore, the inhibitory effect was generally stronger in dual combinations compared with individual evaluations, supporting a possible synergistic interaction between the ingredients. This information has been added to the Discussion section (see lines 427-433).
Additionally I think it would be very compelling for many readers to see some representative images from the FISH evaluation of the biofilm.
Response: We thank you for your suggestion. In response, we have included a new Figure - Figure 2, which presents five FISH images, each corresponding to one of the bacterial species analyzed.
There are a number of very minor modifications (language usage and formatting) below that I think would improve the overall manuscript for many readers. Otherwise the manuscript is very well written. The methods, in particular, are very detailed and clear.
Response: we thank you for your comments. All suggestions were included in the final manuscript as you may see below.
Minor requests:
Lines 40-43: Two which clauses can be a little hard to read: "These bacteria can infiltrate the subgingival space and produce several metabolites such as ammonia and volatile sulfur compounds, which lead to halitosis, as well as bacterial endotoxins and proteolytic enzymes, which promote periodontal inflammation and stimulation of the animal’s immune system [3]." maybe something like: "These bacteria can infiltrate the subgingival space and produce several metabolites including ammonia and volatile sulfur compounds, which lead to halitosis, as well as bacterial endotoxins and proteolytic enzymes: promoting periodontal inflammation and stimulating the animal’s immune system [3]."
Response: We thank you for your comment. Changes were made in accordance, please see lines 49-54.
Lines 48-50: This sentence is a little awkward, just punctuate differently: "Although the chemicals currently used, such as chlorhexidine gluconate, offer immediate benefit regarding PD progression, they may also present some adverse effects, such as gastric disorders, dysgeusia, mucosal irritation, and tooth discoloration, which can impair continued use [7]." maybe: "Although the chemicals currently used, such as chlorhexidine gluconate, offer immediate benefit regarding PD progression, they may also present adverse effects such as: gastric disorders, dysgeusia, mucosal irritation, and tooth discoloration; which can impair continued use [7]."
Response: We thank you for your comment. Changes were made in accordance, please see lines 61-62.
Lines 51-52: "Considering that," is probably not necessary here: "Considering that, new preventive and therapeutic approaches are required to control PD in companion animals." maybe just: "New preventive and therapeutic approaches are required to control PD in companion animals" Line 58: "Bacterial collection" maybe just to be absolutely explicit "Biofilm bacterial collection" to distinguish from the probiotic bacteria.
Response: We thank you for your comment. Changes were made in accordance, please see lines 62-63 and 77.
Line 231: Either the word "Data" or the word "statistical" is sufficient. Both are a little unnatural in ordinary scientific English usage. "Data statistical analysis" Either: "Data analysis" or preferably "Statistical analysis"
Response: We thank you for your comment. Changes were made in accordance, please see lines 276.
Table 4: I am having a little trouble interpreting the sub-titles. If: "A total of 8 postbiotic yeast derivatives components (YE-2, YE-3, YE-4, YE-5, YE-6, 77 YE-7, YCW-1 and YCW-2) and 3 probiotic bacterial strains (BC-23, BC-05, BC-37) (Lesaffre, France), were evaluated in this study."Lines 77-79 then I think: "Concentrations (%) of postbiotics and probiotic components" should be the title for the whole table i.e. including both yeast derivatives and bacterial strains. And a sub-title: "Concentrations (%) of postbiotic component" should be
add for the YE-x yeast derivatives at the top. Then you would have: Title "Concentrations (%) of postbiotics and probiotic components" Sub-title: "Concentrations (%) of postbiotic component" for the YE-x rows Sub-title: "Concentrations (%) of postbiotic component" for YCW-1 rows Sub-title: "Concentrations (%) of postbiotic component" for YCW-2 rows (different concentration columns) Sub-title: "Concentration (CFU/mL) of probiotic bacterial strain" for the BC-x rows.
Response: We agree with your comment. Changes were made in the Table to clarify this point (please see table 5).
Line 269: Maybe add a sentence noting that BC-23 and BC-37 were comparatively ineffective with the exception of P. canis which was reduced at least somewhat in all probiotic treatments and all postbiotic treatments except YE-4.
Response: We thank you for your comment. Changes were made as suggested, please see lines 317-318.
Line 272: "postbiotic" should be "postbiotic or probiotic" (to include the case of the bacterial probiotic) Line 280: "Based on the previous results obtained" is a little confusing, somehwhat ambiguous between previous section of this manuscript versus previous manuscripts. Maybe just: "Based on the results obtained"
Response: We thank you for your comment. Changes were made in accordance with the suggestion, please see lines 323 and 338.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments

The study ‘Impact of probiotic and postbiotic on an in vitro biofilm model 2 for canine periodontal disease” reported on evaluate the inhibitory potential of yeast derivative postbiotics components and probiotic bacterial strains using a previous developed in vitro biofilm model mimicking canine PD-associated biofilms. In general, this paper has an interesting scientific information that was well presented. The findings demonstrate that the inhibitory 25 ability of individual components towards the polymicrobial PD-biofilm ranged from 0% to 22.55%, 26 while their eradication ability varied between 0% and 17.28%. This study allowed to select four postbiotic yeast derivatives components and one probiotic bacterial strain as promising for further evaluation aiming at in vivo application.  

However, I have some major/minor concerns;

General comments

1. The manuscript needs extensive English editing

 

1. Title

Please can authors modify the title of the manuscript for clarity; It should be changed to Biofilm inhibitory Activity of probiotic and postbiotic using in vitro biofilm model for canine periodontal disease

 

2. Introduction section
3. Introduction is too brief and does not clearly give the relationship of oral microbiome and their association with PD.
4. Include more information about how oral microbiome dysbiosis can induce PD.
5. Can authors cite some identified bacteria group that has been associated with PD progression and how they cause the disease.
6. Include more information about how pre/post biotics improve oral microbiome and health.
7. Please add information on existing literature that link and mechanistic insight of buccal microbiome to gut microbiome as these biotics end up in the gut.

 

3. Materials and methods
4. How were the bacteria strains used for the experiment confirmed?
5. What was the criteria for classifying a dog as healthy?
6. What was the controls for the Biofilm evaluation by Fluorescent In Situ Hybridization (FISH) and biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC)?
7. Line 182-186- Bacteria quality rating on a scale of 1-5, Can authors add a table to explain the criteria as each scalar unit.
8. What accounted for the Dual combinations of postbiotic yeast derivative components and probiotic bacterial strains selection in this studies?
9. Line 197-205- How was the spot on lawn assay quantified? Please add to manuscript.

 

4. Results
5. Authors should consider comparing among treatment to bring out clear statistical differences to validate the study. Mare quantitative comparison does not validate the conclusions.

Comments on the Quality of English Language

The manuscript needs extensive English editing

 

Author Response

Author's Reply to the Review Report (Reviewer 2)

Dear Reviewer
We would like to express our gratitude for the comments and the discussion points presented. We believe they will improve the quality and clarity of the manuscript. Each one of the points described were addressed as you may see in the revised version of the manuscript in track changes. Please see below the response to each question.
Comments
The study ‘Impact of probiotic and postbiotic on an in vitro biofilm model 2 for canine periodontal disease” reported on evaluate the inhibitory potential of yeast derivative postbiotics components and probiotic bacterial strains using a previous developed in vitro biofilm model mimicking canine PD-associated biofilms. In general, this paper has an interesting scientific information that was well presented. The findings demonstrate that the inhibitory ability of individual components towards the polymicrobial PD-biofilm ranged from 0% to 22.55%, while their eradication ability varied between 0% and 17.28%. This study allowed to select four postbiotic yeast derivatives components and one probiotic bacterial strain as promising for further evaluation aiming at in vivo application.
However, I have some major/minor concerns;
General comments
The manuscript needs extensive English editing
Response: We thank you for your comment. An English revision has been carried out throughout the manuscript, as can be seen in the track changes all over the manuscript.
1. Title
Please can authors modify the title of the manuscript for clarity; It should be changed to Biofilm inhibitory Activity of probiotic and postbiotic using in vitro biofilm model for canine periodontal disease
Response: We thank you for your comment. As suggested the title was changed in accordance.
2. Introduction section
Introduction is too brief and does not clearly give the relationship of oral microbiome and their association with PD.
Include more information about how oral microbiome dysbiosis can induce PD.
Can authors cite some identified bacteria group that has been associated with PD progression and how they cause the disease.
Include more information about how pre/post biotics improve oral microbiome and health.
Please add information on existing literature that link and mechanistic insight of buccal microbiome to gut microbiome as these biotics end up in the gut.
Response: We thank you for your question. Considering the comments provided, additional information was added to the Introduction section, as you may see in lines 43-48 and 64-70.
3. Materials and methods
How were the bacteria strains used for the experiment confirmed?
Response: We thank you for the question. The majority of the bacterial strains were obtained from the commercial culture collections CCUG and DSMZ (please see lines 78-84), and their purity was confirmed both macroscopically and microscopically (Gram staining). E. faecalis is a clinical strain that was identified by PCR in a previous study (13-Oliveira et al. 2016), and its purity was also consistently verified both microscopically and macroscopically. Additional information was added to clarify this point in lines 95-96.
What was the criteria for classifying a dog as healthy?
Response: We thank you for your question. The blood and saliva donor dogs were previously evaluated by a veterinarian, and were considered healthy based on the clinical examination if no signs of disease were observed.
What was the controls for the Biofilm evaluation by Fluorescent In Situ Hybridization (FISH) and biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC)?
Response: We thank you for the question. In the FISH technique, we used only medium (without bacterial suspention/biofilm) as the negative control, and as a positive control, we used bacterial suspension, that allowed to form a biofilm, not exposed to any treatment/compound. Each treatment was compared with the positive control, allowing us to understand the inhibitory potential of the tested compounds. This information can be seen in lines 208-209.
Line 182-186- Bacteria quality rating on a scale of 1-5, Can authors add a table to explain the criteria as each scalar unit.
Response. We thank you for your comment. A new table was added to explain the scoring system used. Please see table 3.
What accounted for the Dual combinations of postbiotic yeast derivative components and probiotic bacterial strains selection in this studies?
Response. We thank you for your question. The evaluation of dual combinations was performed based on the most promising compounds identified when evaluated individually. This assessment served as a preliminary investigation of the potential synergistic effects of the combined compounds. Previous studies have demonstrated that combining probiotics and postbiotics can enhance their individual benefits, leading to improved outcomes. For instance, Çolak and Duran (2025) reported that dual and triple postbiotic formulations exhibited potent antimicrobial activity, and Pørksen et al. (2023) observed positive effects of probiotic combinations in the reduction of dental caries (https://doi.org/10.3389/fcimb.2025.1616501; https://doi.org/10.1016/j.jdent.2023.104599). Additional information regarding this point was added in the discussion section (please see lines 427-435)
Line 197-205- How was the spot on lawn assay quantified? Please add to manuscript.
Response. We thank you for your comment. The spot-on-lawn assay was quantified using three categories: complete inhibition, observed as a clear inhibitory halo around the spot; incomplete inhibition, when an inhibitory halo was present but not well defined, with reduction of bacterial growth; and no inhibition, when no halo was detected and a homogeneous bacterial lawn was observed. This information has been added to the Methods section (please see lines 143-147).
4. Results
Authors should consider comparing among treatment to bring out clear statistical differences to validate the study. Mare quantitative comparison does not validate the conclusions.
Response: We thank you for your comment. In our study, we presented a preliminary evaluation of the antimicrobial potential of 11 biotic components. Statistical comparisons across the three independent assays for each individual ingredient showed low statistical power; therefore, we chose to perform a descriptive statistical analysis, as presented using average values and standard deviations. These results allowed us to identify promising compounds for further studies that can be explored regarding their potential to be use in PD control in dogs. Considering that, limitations of our study were included in the discussion section, as you may see in lines 480-486. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors presented a manuscript submitted to Microorganisms as an Article, entitled: "Impact of probiotic and postbiotic on an in vitro biofilm model for canine periodontal disease".

The title is quite expressive of the main achievments of the study. The abstract is comprehensive, bring quantitative results and a conclusion based on them.

Keywords are ok. GA was not presented.

Introduction is focused and well-written.

Experimental section could be less detailed with observing the references.

The results section should be shortened removing the information that belongs to the experimental section. It is not enough to describe, as line 246, the "Detailed results are presented in Suppelementary file 1. The results must be better presented and only the tables and graphics presented as SM, but they must be nominated at the main text, as FS1, FS2... and so on.

At the results section, authors must avoid to present discussion or conclusions.

At the Figure 1, the title is also the experimental and the discussion, must be shortened.

Table 5 brings data with SD so high that sometimes CV is 200%, but should be 10%. Please review. Table 6 has some similar data.

The first paragraph of the discussion (310-322) is not the discussion of the results from the present study. So was the paragraph from line 352 to 364. 

The study does not present a conclusion.

References are relevant and updated, but about 20% are self-citations and could be carefully observed as this percentual can be considered high.

Author Response

Author's Reply to the Review Report (Reviewer 3)

Dear Reviewer
We would like to express our gratitude for the comments and the discussion points presented. We believe they will improve the quality and clarity of the manuscript. Each one of the points described were addressed as you may see in the revised version of the manuscript in track changes. Please see below the response to each question.
Authors presented a manuscript submitted to Microorganisms as an Article, entitled: "Impact of probiotic and postbiotic on an in vitro biofilm model for canine periodontal disease".
The title is quite expressive of the main achievements of the study. The abstract is comprehensive, bring quantitative results and a conclusion based on them.
Keywords are ok. GA was not presented.
Introduction is focused and well-written. Experimental section could be less detailed with observing the references.
Response: We appreciate your comment. We chose to provide a detailed explanation of the methodology to allow the fully replication of the study, and facilitate readers to understand all methodologies. The reports from the other reviewers have described that they appreciated the detailed methodology description. However, considering your comment, some reductions were made in the Methods section, as you may see in track changes. If you consider that further reduction is needed, we are willing to implement it.
The results section should be shortened removing the information that belongs to the experimental section. It is not enough to describe, as line 246, the "Detailed results are presented in Supplementary file 1. The results must be better presented and only the tables and graphics presented as SM, but they must be nominated at the main text, as FS1, FS2... and so on.
Response: We understand your question. The option to include the table with the results from the inhibitory ability of the components observed after the spot-on-lawn as supplementary was related to the size of the table. However, we can include it as a table, if the formatting rules of the journal allowed that. Besides that, additional information in the results was added, as you may see in lines 291-292, 317-318 and 330-335.
At the results section, authors must avoid to present discussion or conclusions.
Response: We thank you for your comment. We have revised the manuscript in accordance.
At the Figure 1, the title is also the experimental and the discussion, must be shortened.
Response: We thank you for the question. Figure 1 shows the results of FISH detection of individual bacterial species in the biofilm after exposure to the highest concentration of selected postbiotic and probiotic components, compared to the positive control (untreated polymicrobial biofilm). To facilitate the readers’ understanding, we included a description of the scale used for this characterization in the figure. Considering your comment, the title legend was shortened, removing that description (please see lines 325-328). Considering the discussion section, we have reduced some parts (please see lines 286-288 and 414-416). However, in order to include all comments provided by other readers, some other information was added in the discussion section.
Table 5 brings data with SD so high that sometimes CV is 200%, but should be 10%. Please review. Table 6 has some similar data.
Response: We understand your question. In fact, in Table 5 we observed a high SD, which results from the complexity and variability of biofilm formation. Each biofilm produced in each peg is different, as biofilm structure and formation are inherently complex mechanisms, and variations between replicates and assays are expected. Similar high SD were also reported by Cunha et al. (2020; https://doi.org/10.1186/s12917-020-02646-3) in their study on the effects of antimicrobial peptides against polymicrobial biofilms. In addition, in Table 6, high variability was observed, which may be a result of the different blood samples obtained from different dogs for each assay. Considering that additional information regarding this point was added in the discussion section (please see lines 482-483)
The first paragraph of the discussion (310-322) is not the discussion of the results from the present study. So was the paragraph from line 352 to 364.
Response: We understand your comment. In fact, the first paragraph of the Discussion section provides a contextualization of the study, highlighting the potential of probiotics and postbiotics in relation to periodontal pathogens and periodontal disease. We believe that the Discussion should include information that supports both the study itself and the viability of its underlying concept. For this reason, we included previous reports that describe relevant findings on the use of probiotics and
postbiotics in the context of this study. Considering the second paragraph mentioned, we also included in the Discussion section information from other reports that describe potential molecules capable of conferring antimicrobial activity to postbiotics and probiotics. This information helps to better understand some of the potential compounds that may also be present in our products. However, following your comment, we have reduced the discussion of other reports in the discussion section (please see lines 286-288 and 414-416).
The study does not present a conclusion.
Response: Usually the conclusion in this journal is optional. However, considering your indication we have included a conclusion section in the manuscript, as you may see in lines 491-505.
References are relevant and updated, but about 20% are self-citations and could be carefully observed as this percentual can be considered high.
Response: We understand your comment. In fact, the majority of the self-citations refer to methodologies used during the experiments, which were developed by some of the researchers who participated in this study. However, additional references were included in the manuscript, as you may see in the new reference list.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The peer-reviewed manuscript by Lesaux et al. evaluates the effect of postbiotic yeast derivative components and probiotic bacterial strains on an in vitro biofilm model composed of selected bacterial species that may be associated with canine periodontal disease.

I only have methodological comments about the manuscript, which should be explained and supplemented.

1. For an objective assessment of the manuscript, the results of preliminary screening of the inhibitory activity of yeast derivative postbiotic components and probiotic bacterial strains should be supported by photo documentation in the form of a supplementary file.
2. To assess the cultivation conditions, the reviewer should be familiar with the species of microorganisms of probiotics or the source of postbiotic components. If the relevant postbiotic and probiotic products are protected by a patent, then this should be stated in the manuscript. The provided Link https://www.mdpi.com/xxx/s1 (l. 404) is unavailable / non-functional.
3. P. canis CCUG 57081 and P. cangingivalis DSMZ VPB 4874 were grown under anaerobic conditions. At the same time (second step), polymicrobial biofilms were incubated in microaerophilic conditions. The original methodology by Sanguansermsri et al. (2018), referred to by Cunha et al. (2020), however, maintains anaerobic conditions in both steps of the preparation of polymicrobial biofilms. Can you explain why you chose microaerophilic conditions in the second step? Could these different conditions have influenced the assessment of the results? 

Author Response

Author's Reply to the Review Report (Reviewer 4)

Dear Reviewer
We would like to express our gratitude for the comments and the discussion points presented. We believe they will improve the quality and clarity of the manuscript. Each one of the points described were addressed as you may see in the revised version of the manuscript in track changes. Please see below the response to each question.
The peer-reviewed manuscript by Lesaux et al. evaluates the effect of postbiotic yeast derivative components and probiotic bacterial strains on an in vitro biofilm model composed of selected bacterial species that may be associated with canine periodontal disease.
1. For an objective assessment of the manuscript, the results of preliminary screening of the inhibitory activity of yeast derivative postbiotic components and probiotic bacterial strains should be supported by photo documentation in the form of a supplementary file.
Response 1: We thank you for your comment. An additional Supplementary file 2 was added, that includes some photographs about the preliminary screening of the inhibitory activity of the components.
2. To assess the cultivation conditions, the reviewer should be familiar with the species of microorganisms of probiotics or the source of postbiotic components. If the relevant postbiotic and probiotic products are protected by a patent, then this should be stated in the manuscript. The provided Link https://www.mdpi.com/xxx/s1 (l. 404) is unavailable / non-functional.
Response 2: We understand your point of view. In fact, the probiotics and postbiotics used in this study are currently under patent consideration, for that reason no additional information was added. Considering the culture conditions of probiotic strains, all were propagated under aerobic conditions using general medium (BHI agar). Additional information was included in lines 107-108.
3. P. canis CCUG 57081 and P. cangingivalis DSMZ VPB 4874 were grown under anaerobic conditions. At the same time (second step), polymicrobial biofilms were incubated in microaerophilic conditions. The original methodology by Sanguansermsri et al. (2018), referred to by Cunha et al. (2020), however, maintains anaerobic conditions in both steps of the preparation of polymicrobial biofilms. Can you explain why you chose microaerophilic
conditions in the second step? Could these different conditions have influenced the assessment of the results?
Response 3: We thank you for your question. The methodology used in this study to establish the polymicrobial biofilm has been previously performed and published (see https://doi.org/10.1186/s12917-020-02646-3). The use of a microaerophilic environment allowed all species to grow within the biofilm, as demonstrated by FISH in the biofilm evaluation. We believe that the combination of reduced oxygen concentration and the complex biofilm composition are primarily responsible for the survival of all species, as demonstrated in the initial study. Indeed, in the subgingival sulcus, where dental plaque is established and contributes to periodontal disease in dogs, a similar environment is thought to exist, facilitating polymicrobial participation. Nevertheless, it is always challenging to fully replicate in vivo conditions, and this in vitro model of dental plaque can only provide an approximate model of the real situation. 

Author Response File: Author Response.pdf