Biofilm Formation of Probiotic Saccharomyces cerevisiae var. boulardii on Glass Surface during Beer Bottle Ageing

Round 1

Reviewer 1 Report

The authors aimed to reveal biofilm formation on brewing of beers. The authors claimed that results revealed that ageing the beer and maturing from a sugar-rich to a sugar-limited beer led to an increase in biofilm mass. The object is interesting. However, in manuscript, the main finding is difficultly found. Therefore, I strongly suggested that the author should show relationship between sugar-concentration (or such related information) with biofilm formation to make figure or table.

 Minor comments.

1.       The author should check style of references.

2.       Quality of figure 1, figure 2 and figure 3 are poor. The author should remake these figures better.

3.       Scale bars in figure 4 are unclear. The authors should show more clearly.

Author Response

Reviewer 1

Comment

The authors aimed to reveal biofilm formation on brewing of beers. The authors claimed that results revealed that ageing the beer and maturing from a sugar-rich to a sugar-limited beer led to an increase in biofilm mass. The object is interesting. However, in manuscript, the main finding is difficultly found. Therefore, I strongly suggested that the author should show relationship between sugar-concentration (or such related information) with biofilm formation to make figure or table.

Response

We appreciate your thorough review and salient observations. Table 2, showing the sugar content, was linked with Figure 3 A&C illustrating biofilm mass and extracellular matrix, respectively.

Minor comments.

1. The author should check style of references.

Response

The style of references was checked and corrected.

2. Quality of figure 1, figure 2 and figure 3 are poor. The author should remake these figures better.

 Response

The quality of the above-mentioned figures was improved

3. Scale bars in figure 4 are unclear. The authors should show more clearly.

Response

Thanks for your attention. The image size was adjusted to show a clear scale bar.

Reviewer 2 Report

The manuscript “Biofilm formation of probiotic Saccharomyces cerevisiae var. boulardii on glass surface during beer bottle ageing” by Mohammadi and Saris” report the observation of biofilm formation by a probiotic S. cerevisiae var. boulardii strain used to brew beer. The study is interesting, but several procedural details are missing and some parts of the text should be clarified.

The abstract should be revised to clearly report the relevant findings and their impact on probiotic beer production. It is odd to see the statistic reported in the abstract (p. value), especially considering that the authors indicate that the biofilm mass increased over the beer aging and maturation, but not significantly (p>0.05). This seems in contrast with the title of the manuscript.

Then, they report that “however”, the metabolic activity decreased. I believe the authors wanted to indicate that, despite the yeast biofilm formed over the secondary fermentation, it was (potentially) metabolically active only for a certain amount of time, hence suggesting that the presence of the biofilm is not a risk for the end consumer. However, the authors indicate that the biofilm is active, as they write “This study, therefore, provides evidence that Saccharomyces cerevisiae var. boulardii makes biofilm on glass surfaces during beer storage” but, according to the reported results, the biofilm seems to establish before the beer storage.

In addition, the last sentence “Especially, as S. boulardii biofilm probably precedes cases of fungemia in patients having a central venous catheter.” seems to be missing some parts, and it is very speculative.

In table 2, the timepoint of the measurement of metabolites in wort should be indicated. I guess the wort was analyzed at the beginning of the fermentation, as fermentative metabolites are not reported.

I am very surprised by the number of viable cells detected in the probiotic beer, especially after the secondary fermentation. However, it is unclear how the concentration was calculated. I am not sure about the method used to calculate cell viability. In the material and methods section, the authors report “The viable count of yeast was performed on Sabouraud Dextrose agar (HiMedia, PA, USA) using a previously described method”, but I guess this refers to the inoculum preparation and standardization (as per the title of the corresponding section, 2.1). Another method that could be used to infer cell viability is the one based on crystal violet (section 2.4.1), but only for mammalian cells or cells whose viability corresponds with the fact that they are attached to a surface. Hence, crystal violet, as correctly reported by the authors, could be useful to discriminate the yeast cells present in the biofilm, but not to assess their viability. Overall, the authors should provide further details on the methodology used to infer cell viability and contextualize such a high concentration of viable cells at the end of alcoholic fermentation. Would the 10^7 cell/ml concentration result in a very “yeasty” beer, and the beer per se would look very opaque and dense?

Yeast cultures in the lag, exponential and stationary phases should be used as the references for the metabolic analysis of biofilms. The authors report that “a low metabolic activity was recorded despite the presence of a thick biofilm (high biomass and extracellular matrix) at day 60”. However, the metabolic activity, reported as OD492, was apparently higher on day 60 than on day 3. I believe the metabolic activity has not been scaled according to the number of viable cells, as the raw data are reported in the figure. Reporting the same quantification for the reference cultures as indicated above would help the reader understand the raw data.

Authors should clarify which intervals they considered as exponential and stationary growth phases of biofilm formation they referred to in the results section dedicated to FLO11 expression quantification.

Minor comments:

In paragraph 3.1, the authors report “Afterwards, the glucose level remained below 0.2 %, the limit that triggers aggregation and biofilm formation in Saccharomyces”. I would rather say the glucose level remained around 0.2%, considering the values reported in table 2 (glucose contents since D15 do not significantly differ from the values at D3, which was above 0.2%).

Author Response

Reviewer 2

The manuscript “Biofilm formation of probiotic Saccharomyces cerevisiae var. boulardii on glass surface during beer bottle ageing” by Mohammadi and Saris” report the observation of biofilm formation by a probiotic S. cerevisiae var. boulardii strain used to brew beer. The study is interesting, but several procedural details are missing and some parts of the text should be clarified.

The abstract should be revised to clearly report the relevant findings and their impact on probiotic beer production. It is odd to see the statistic reported in the abstract (p. value), especially considering that the authors indicate that the biofilm mass increased over the beer aging and maturation, but not significantly (p>0.05). This seems in contrast with the title of the manuscript.

Response

Thank you very much for your comments that helped us to improve this manuscript. The abstract is revised, and the p-value is removed from the abstract.

Then, they report that “however”, the metabolic activity decreased. I believe the authors wanted to indicate that, despite the yeast biofilm formed over the secondary fermentation, it was (potentially) metabolically active only for a certain amount of time, hence suggesting that the presence of the biofilm is not a risk for the end consumer. However, the authors indicate that the biofilm is active, as they write “This study, therefore, provides evidence that Saccharomyces cerevisiae var. boulardii makes biofilm on glass surfaces during beer storage” but, according to the reported results, the biofilm seems to establish before the beer storage.

Response

You are exactly right. In this case, the formed biofilm on the glass surface is not considered a potential risk for the end consumer. After "bottle ageing" the bottles are stored in cold room. So we changed the "storage" to "aging".

In addition, the last sentence “Especially, as S. boulardii biofilm probably precedes cases of fungemia in patients having a central venous catheter.” seems to be missing some parts, and it is very speculative.

Response

The last sentence was removed.

In table 2, the timepoint of the measurement of metabolites in wort should be indicated. I guess the wort was analyzed at the beginning of the fermentation, as fermentative metabolites are not reported.

Response

The time point in table 2 is clarified. As you mentioned, in the produced wort, there were no fermentative metabolites to report.

I am very surprised by the number of viable cells detected in the probiotic beer, especially after the secondary fermentation. However, it is unclear how the concentration was calculated. I am not sure about the method used to calculate cell viability. In the material and methods section, the authors report “The viable count of yeast was performed on Sabouraud Dextrose agar (HiMedia, PA, USA) using a previously described method”, but I guess this refers to the inoculum preparation and standardization (as per the title of the corresponding section, 2.1). Another method that could be used to infer cell viability is the one based on crystal violet (section 2.4.1), but only for mammalian cells or cells whose viability corresponds with the fact that they are attached to a surface. Hence, crystal violet, as correctly reported by the authors, could be useful to discriminate the yeast cells present in the biofilm, but not to assess their viability. Overall, the authors should provide further details on the methodology used to infer cell viability and contextualize such a high concentration of viable cells at the end of alcoholic fermentation. Would the 10^7 cell/ml concentration result in a very “yeasty” beer, and the beer per se would look very opaque and dense?

Response

During beer bottle aging, the viable yeast cells were enumerated on Sabouraud dextrose agar plates. As you have pointed out, crystal violet has been used to discriminate the yeast cells in the biofilm.

At the end of batch fermentation, we did not filter the beer prior to bottling, and that's the reason for the high number of yeast in bottled beer. There are studies confirming the survival of S. boulardii at the end of alcoholic fermentation. (Ref. EMİRLEROĞLU, E., & SARİS, P. The effect of hop concentrations on probiotic viability in beer). You are quite right that the beer is opaque, but from the prespective of potential probiotic effect it is good to have many cells alive. After more than 4 months the cells settle and a clear beer is formed.

Yeast cultures in the lag, exponential and stationary phases should be used as the references for the metabolic analysis of biofilms. The authors report that “a low metabolic activity was recorded despite the presence of a thick biofilm (high biomass and extracellular matrix) at day 60”. However, the metabolic activity, reported as OD492, was apparently higher on day 60 than on day 3. I believe the metabolic activity has not been scaled according to the number of viable cells, as the raw data are reported in the figure. Reporting the same quantification for the reference cultures as indicated above would help the reader understand the raw data.

Response

On day 60, the OD492 was apparently higher than on Day 3; however, compared with days 15, 30 and 45, it was decreasing. In fact, fluctuations of OD492 during the whole beer aging period was not significant therefore we clarified this fact by adding statistical significance (p > 0.05) at the end of this statement "However, a low metabolic activity was recorded despite the presence of a thick biofilm (high biomass and extracellular matrix) at day 60"

Authors should clarify which intervals they considered as exponential and stationary growth phases of biofilm formation they referred to in the results section dedicated to FLO11 expression quantification.

Response

Thank you for pointing this out. The time intervals were clarified.

Minor comments:

In paragraph 3.1, the authors report “Afterwards, the glucose level remained below 0.2 %, the limit that triggers aggregation and biofilm formation in Saccharomyces”. I would rather say the glucose level remained around 0.2%, considering the values reported in table 2 (glucose contents since D15 do not significantly differ from the values at D3, which was above 0.2%).

Response

Many thanks for your attention to the words. Around 0.2% demonstrates glucose fluctuations after D3.

Round 2

Reviewer 1 Report

The authors have addressed my concerns.