Inoculation, Growth and Bactericidal Effects of Three Kombucha Cultures

Round 1

Reviewer 1 Report

The manuscript gives some insights on the description of kombucha although it is not particularly innovative. The Innovation aspects should be better outlined by the Authors. Moreover, the description of the 3 kombucha matrixes used as inoculum should be improved, the list of references should be enlarged, the choice of the pathogens better explained and the inhibition data commented in the discussion and compared with literature data, the conclusion re-written.

ABSTRACT:

Line 19: … and had high contamination rates. Please explain better this concept by adding a few words.

Line 22: 10^-3 ampicillin … or 10^-4 penicillin. This does not mean anything for a reader of the sole abstract, please substitute with the respective concentration.

INTRODUCTION:

Line 44. The beneficial effects of Kombucha have to be better explained, in particular the anti-oxidant activity. Please read and quote in the manuscript:

• Gaggìa, F., Baffoni, L., Galiano, M., et al. (2019). Kombucha beverage from green, black and rooibos teas: a comparative study looking at microbiology, chemistry and antioxidant activity. Nutrients, 11(1), 1.
• Villarreal‐Soto, et al. (2018). Understanding kombucha tea fermentation: a review. Journal of food science, 83(3), 580-588.

In general, the list of references is scarce and should be improved.

MATERIALS AND METHODS:

Line 73-78: the main characteristics of the 3 inocula used should be mentioned.

Line 97: what is the purpose of adding different carbon source? What do the authors expected to show? Please comment this point also in the Discussion (lines 210-217)

Line 104: Where do molds come from? Why do you have contamination if the broth is sterile?

Line 126: Why did you choose S. epidermidis among the large array of possible pathogens?

RESULTS:

You cannot refer generally to S. epidermis and E. coli, the inhibition is related to the strain used, so please refer to the strains that you used and not to the

species. e.g. E. coli DH5α.

Line 149: The weight of the original inoculum was not as important as the method for inoculation. This is not clear, please explain better.

Table 2: please add the unit of measure of weight in the Table. Strain is a correct heading for the column describing the different matrixes used. Substitute with a proper word (e.g. inoculum).  

Line 195: Please remove the dot between genus and species

Table 3: please add the concentration of usnic acid and not the dilution factor.

 

DISCUSSION:

Line 226: please refer to the above mentioned paper (Gaggia et al. 2019) for the use of additional herbal infusions

- Please give a possible explanation of why the wet weight of Olinka kombucha with galactose deceased with time, whereas in the other inocula increased.

- Data on the antimicrobial activity presented in the Results are not commented in the Discussion. They need to be compared with what is present in the literature. The few lines presented (263-265) are not sufficient for a paper that claims to study the bactericidal effect.

 

CONCLUSION:

The conclusion is not properly scientific; line 267-275 are not a proper conclusion and it is not clear what the paper wants to reach.

Line 275: Kombucha does not appear to have unique antibacterial properties. This is not clear, please explain.

Author Response

See attached

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript presents basic research on different inoculation formula of Kombucha and some characteristics of the formed pellicle and antimicrobial activity.

General observation: the article refers to relatively old information in the topic; it is recommended to insert in the Introduction and Discussion sections updated and more actual information from the past 5 years in Kombucha topic.

Line 61: see comments on manuscript (Pedioccocus is also a Lab strain isolated in Kombucha)

Line 93: make it clear what is “high”

Line 120: “strain” is used only for pure culture of one microbial strain; in Kombucha there is a consortia of microorganisms, this is why is recommended to use instead “strain”, “consortia” or “starter culture”

Line 162: why  Table S2? Delete S

Line 167: How did you calculated the "amount of contamination". Is it the amount (inside the sample, or the % among all samples?)

Table 2: Replace “strain” with consortia or starter culture

Line 197: why table S3? Delete S

Table 3:  write the microorganisms names in Italic; also, indicate the size range (mm) of the diameters for +, ++ and +++ in the legend

Line 215: Please provide some references

Line 218-224: please link/compare this information to your results, otherwise, there is no need to insert it.

Line 219: Acetobacter xylinum is only one specie; recent publications refers also to other species like Gluconacetobacter or Komagataeibacter (https://doi.org/10.3390/app11041595)

 

Line 262: generally, try to refer to more recent publications in the topic. For the antimicrobial activity many other recent articles are available on the subject.

https://doi.org/10.1016/j.foodres.2019.108782

https://doi.org/10.15586/qas.v13i3.920

 

NOTE: please, also see the manuscript to cross-check the comments

Comments for author File: Comments.pdf

Author Response

See attached

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

• You still haven’t answer to the observation regarding the info in Line 61 (first manuscript): “Pedioccocus is also a Lab strain isolated in Kombucha”. Here is, at least, one reference: (https://doi.org/10.3390/foods9121780)
• Line 111: please, explain in the text what is high (which is the parameter that is high?)
• You haven’t provide an answer for Line 167 (first manuscript): How did you calculated the "amount of contamination". Is it the amount (inside the sample, or the % among all samples?)

Author Response

Dear Editor:  Thank you for your continued careful editing of our manuscript, leading to yet more improvements in the quality and accuracy of our paper.  

We are attaching what we hope is the final, edited version here.  As you can see, we have added mention that Pediococcus species are sometimes recovered as the bacterial component of Kombucha teas, and added a citation to the manuscript by Diguță et al (2020) in References.

In Methods and Materials, we have changed the description of the blending process to read that the macerate pellets were prepared “in a sterile Waring blender on its highest setting for 1 minute.” 

We apologize for missing the comment made by Reviewer 2 in our first revision. (Line 167: How did you calculated the "amount of contamination". Is it the amount (inside the sample, or the % among all samples?)  We simply overlooked this comment, perhaps because our method of calculating contamination was so low tech. We simply looked at the pellicles with our eyes. Those that had visible mold growth were scored as contaminated.  The heading reads “Number of contaminated samples.”    We have added a footnote to the heading that says:  ^aKombucha pellicles with macroscopically visible mold growth on the surface were scored as contaminated.”

The attached copy of the manuscript reflects these changes.   We trust that you will now find our manuscript suitable for publication.  Do not hesitate to contact us should you have any further questions, and we will do our best to respond in a timely manner.