Evaluation of the Bio-Protective Effect of Native Candida Yeasts on Sauvignon Blanc Wines
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors investigated bio-control potential of yeasts [Candida] oleophila and [Candida] boidinii against lactic acid bacteria (LAB), acetic acid bacteria (AAB), as well as Brettanomyces bruxellensis (BB), all 3 considered spoilage microorganism in production of Sauvignon Blanc wine. Few studies exist on at least one of the studied organisms, and no publications appear to be available in context of winemaking, i.e. production of Sauvignon Blanc, in particular.
Experimental data was generated in lab-scale fermentations; there was no report [!] on how replication was implemented. Chemical analysis of volatiles appear to be towards the lower end of state-of-the art, i.e. volatiles lack quantification, or even quantification in as equivalents of some internal standard; only a sub-sample of volatiles were included in multivariate analysis without clear explanation as to the reason; also: Outliers were removed without much detail as to how exactly and why.
Results from statistical analysis (ANOVA and post-hoc procedures) were not reported at all.
Presentation of raw data and of results from statistical analysis, fail to meet acceptable standards. I cannot confirm validity of the reported results and their interpretation from what the manuscript offers. Furthermore, there are many (many!) formal mistakes which require a thorough re-work.
detailed notes
I could not find any indication about any number of replicates. From Figure 5 I could gather that number was 3; it remains unclear whether, for each inoculation modality, 3 measurements were taken or 3 separate fermentations were run. This has to be stated in perfect clarity in the material and methods section.
Using the biocontrol effect on LAB as an example: I could not find, i.e. the manuscript did not contain, any statistical analysis LAB CFU ~ inoculation modality. There was also no reporting of raw CFU counts and no clear explanation of serial dilution scheme, or reporting of counted CFUs: Given the vertical axis in figure 3 was log scaled it appears within group variation may have been large: This depends, however on whether error bars represent standard error of the mean (depends on sample size), range or sample mean, which is not specified. Not observing any significant differences in neither malic acid, nor lactic acid content among the inoculation modalities (table 1) raises the question, if the alleged biocontrol effect had any impact on the wine, since no detrimental impact of LAB in the control was observed. This requires clarification (how confident are you about your CFU counts) and extensive discussion (not observing spoilage effects). Note that in the context of this research, reporting effect sizes, e.g. LAB population size was reduced by at least 10 x, is imperative.
Concerning its form, the manuscript (ms) does not meet basic formal requirements imposed on a contribution to a scientific [any] journal. Specifically, 1) binominal nomenclature is not applied correctly, most frequent: Spelling the epithet with a capital letter and italicizing abbreviations sp., spp. etc.; 2) the scientific names of the study organism(s) were frequently misspelled! 3) Misuse or inconsistent use of specific terminology, for example, the term pathogen is used throughout, albeit not consistently, when – likely – referring to spoilage or deterioration organisms 4) systematic classification of study organisms was not up-to-date: Study organisms [Candida] oleophila and [Candida] boidinii were treated as members of the genus Candida, which neither is (see my notes); among other things, this makes for some unfortunate/incorrect inferences about application of members of the genus Candida in bioprotection; 5) failure to explain acronyms/ use of out-of-the-ordinary acronyms, e.g. LVNS, ufc. 6) There were no reports of the results of statistical analyses, whatsoever: ANOVA and a Tukey Post-Hoc variant were conducted, but never test statistic, p-values and or confidence intervals were reported; 6) sloppy and inconsistent formatting of units-of-measurement, e.g. 72h instead of 72 h, cell/mL instead of cells/mL, etc.; base of log are not specified (CFU) and not converted to widely used numerical formats, e.g. 3.12 × 104.
Second, presentation of the results was, however unfortunate, not adequate and make in nearly impossible to find results mentioned in the text quickly. 1) Important results, e.g. LAB, AAB, BB abundances are given at very hard to grasp text paragraphs instead of a comprehensive table 2) figures are barely accessible due to unnecessary use of abbreviations in color coding; 3) The figures show raw data (e.g. timelines) instead of side-by-side plots of the time-points actually being discussed in the results/discussion section, e.g. comparison of LAB/AAB/BB abundances at 72 h or end point of all species, which is what is discussed, e.g. one study MO was more apt at suppressing one spoilage MO than another: The reads has to find this information by going between multiple panels, all the while deciphering the legend abbreviations. 4) It is not indicated in the figures if error bars are: min-max, i.e. range, standard error of the mean or standard deviation. Note that in normally distributed data, the interval +/- 1 unit of standard deviation contains only approx. 68 % of the population.
Here some general recommendation of mending the most pressing issues
1) Check all instances of scientific binomen: Verify spelling and italicizing
2) Consider use of pathogen/pathogenic and replace with spoilage or deterioration (micro-) organism; whichever you prefer. In case, I misunderstood and the authors had pathogenic microorganisms in mind; please provide both, names of organisms and references backing up claims about their abundance in wine-making and documented consumer health hazards; there is a section on that in the discussion.
3) Include a note about C. oleophila and C. boidinii, despite their genus name being identical, do not constitute what would be considered a pair of closely related species, i.e. they must not, conceptually, be confounded with that. Adjust corresponding sections, esp. in the discussion. Check NCBIs taxonomy browser to learn about the species’ placement. Note that on NCBI, both genus’ assignment for both species is given in square brackets to emphasize they are not true Candida (these exist; and these contain some proper pathogens). https://www.ncbi.nlm.nih.gov/taxonomy
C. oleophila is likely to be transferred to genus Kurtzmaniella (cf. Lachance et Starmer 2008) while C. boidinii is currently listed as a member of Ogataea/Candida clade. Browsing through some other papers, it appears taxonomic placement of C. boidinii is not straight forward at the moment.
Lachance et Starmer 2008, Int. J. System. Evol. Microbiol. 58(2): 520-524. Kurtzmaniella gen. nov. and description of the heterothallic, haplontic yeast species Kurtzmaniella cleridarum sp. nov., the teleomorph of Candida cleridarum. https://doi.org/10.1099/ijs.0.65460-0
I’m not affiliated with either NCBI of any of the authors references above.
4) Report how samples were replicated
5) How exactly was cell-counting conducted: Most importantly what was the dilution scheme and how many cells were counted; were cell counts replicated?
6) Analysis of volatiles (GC-MS-SPME): What is the unit-of-measurement of the values reported in table 2: “raw” TIC signal or internal standard equivalents? Please give an explanation why you felt you had to remove analytes; analytes in low abundance may still be impactful to aroma.
7) Re-organize the figures and show, for example side-by-side, the contrasts you analyzed and which you discuss, e.g. larger effect of one study species on LAB/AAB or BB.
8) report the results from all your statistical analysis, even if they were “non-significant”. Limit statistical analysis to analytes what help you build your story. You can google, for example, report results ANOVA, etc.
9) Discuss antagonism and competition as likely explanatory mechanism for your data
Good Luck!
Comments for author File: 
Comments.pdf
1) binominal nomenclature is not applied correctly, most frequent: Spelling the epithet with a capital letter and italicizing abbreviations sp., spp. etc.
2) the scientific names of the study organism(s) are frequently misspelled!
3) Misuse or inconsistent use of specific terminology, for example, the term pathogen is used throughout, albeit not consistently, when – likely – referring to spoilage or deterioration organisms
5) failure to explain acronyms/ use of out-of-the-ordinary acronyms, e.g. LVNS, ufc.
by line notes
Author Response
Please see the attachment. Thank you
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsGeneral assessment
The study aims to find out whether certain types of yeast can be used in wine production instead of harmful SO2 in order to suppress the growth of "pathogenic" microorganisms. This scientific approach is understandable in principle, as are the experiments carried out for this purpose. However, the correct enumeration of the "pathogens" is doubtful with the culture media used. Personally, I also have doubts as to whether the negative fermentation properties outweigh the potentially positive effects of the yeasts.
Unfortunately, the manuscript has some substantial shortcomings, both in terms of form and content.
Some specific points
Are they different species? The names Candida boidini, Candida boidinni, Candida Boidinni, C. boidinni are used arbitrarily in the text. It would be nice if the authors could agree on one version. I have taught my young students that genus names are always capitalized and species names lower case. The genus name is written out in full the first time in the text and then abbreviated. For example, Candida oleophila once and then C. oleophila. Unfortunately, upper or lower case does not play a role in the manuscript.
Line 20 and thereafter: what is meant by "pathogenic". Neither lactic acid bacteria nor most yeasts are dangerous and pathogenic to humans. On the contrary. Do the authors perhaps mean harmful, disruptive or undesirable microorganisms in wine? In any case, the term "pathogenic" is completely inappropriate.
Line 27; "pathogenic bacteria including yeasts"? Yeasts are bacteria?
Line 31: "allergic". SO2 does not trigger a classic immune reaction, but pseudo-allergic reactions.
Line 101: "The grape juice obtained". Were the grapes not pressed beforehand?
Line 125: Interesting, I only know "Sabouraud broth". "Sauvoraud" is probably a new product?
Line 130: strange expression: "maintaining sterility with a lighter".
Line 133: how much grape juice was used to fill it?
Line 137 and thereafter: I don't understand the expression "pre-fermentation maceration". Surely this is pressed grape must? What does "maceration" mean in this context?
Lines 146-172. Nowhere is the pH value of the media specified.
The acetic acid bacteria were counted on WLD medium. Is the culture medium selective for these or do other microorganisms also grow on it? I think so. Please comment and/or refer to the literature!
The lactic acid bacteria were counted on MRS agar. Is the culture medium selective for these or do other microorganisms also grow on it? I think so. Please comment on this and/or refer to the literature!
Line 176: To carry out
Line 176: I expect more details on the HPLC procedure!
Line 178: "Biosytem Y15" Specify manufacturer and location!
Line 216: "helped us to evaluate"
Line 238: "help to analyze"
Line 247 and then: "ufc"? Do you mean colony forming units "cfu"?
Author Response
Please see the attachment. Thank you
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors improved the manuscript in all critical points mentioned by the reviewer. The study can therefore be published in its revised form
