Mezcal as a Novel Source of Mixed Yeasts Inocula for Wine Fermentation

Round 1

Reviewer 1 Report

minor issues:

1, the authors should carefully revise the manuscript. Some of the sentences are too long and are not correct. It would be much better if the authors could cut them into short sentences. Moderate English changes are required. 

2, the statistics of the results should be clearly presented in the figures and tables. 

3, I suggest to include another table to summarize the sources and characteristics of the strains used in the study. This would help the readers to understand the study. 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Major comments:

The proposed manuscript represents novel and relevant results that, in my opinion, will be very useful to readers working on this field. However, two major concerns have to be surpassed before acceptance:

• A deep and extensive revision of english writing has to be done before acceptance.
• A detailed comparison with literature needs to be acomplished. Below, in my minor comments, I propose some examples.

Minnor comments:

Abstract

Line 19-21: please write genera names fully the first time they appear

Introduction

Lines 36 to 44: some important references were missed that need to be cited in this part of the introduction, for the mentioned topic:

• Franco-Duarte R, Umek L, Mendes I, Castro CC, Fonseca N, Martins R, et al. New integrative computational approaches unveil the Saccharomyces cerevisiae pheno-metabolomic fermentative profile and allow strain selection for winemaking. Food Chem. 2016;211: 509–520. doi:10.1016/j.foodchem.2016.05.080
• Romano P, Paraggio M, Turbanti L. Stability in by-product formation as a strain selection tool of Saccharomyces cerevisiae wine yeasts. J Appl Microbiol. 1998;84: 336–341. doi:10.1046/j.1365-2672.1998.00345.x

Methods

Line 68: italics were missed.

Lines 73-81: I suggest to include this information in a table format to clarify.

Lines 126-134: more details about HPLC should be presented. I suggest to include a reference using the same procedure, so readers can replicate the experiments.

Results

Tables 1 and 2: the number of decimal places, or at least the number of significant algharisms, should be equal throughout the table

Figure 1: legends for two of the bars are missing

Discussion

Line 296: replace “S. cerevisiae species….” By “S. cerevisiae strains…”

Lines 296-311: Discussion of S. cerevisiae was made by comparing results with only the ones of Camarasa et al. Several important citations were missed that would improve enourmeously the mentioned analysis. Some suggestions that I leave to authors´ consideration:

• Csoma H, Zakany N, Capece A, Romano P, Sipiczki M. Biological diversity of Saccharomyces yeasts of spontaneously fermenting wines in four wine regions: Comparative genotypic and phenotypic analysis. Int J Food Microbiol. 2010;140: 239–248. doi:10.1016/j.ijfoodmicro.2010.03.024
• Mendes I, Sanchez I, Franco-Duarte R, Camarasa C, Schuller D, Dequin S, et al. Integrating transcriptomics and metabolomics for the analysis of the aroma profiles of Saccharomyces cerevisiae strains from diverse origins. BMC Genomics. 2017;18: 1–13. doi:10.1186/s12864-017-3816-1
• Agnolucci M, Scarano S, Santoro S, Sassano C, Toffanin A, Nuti M. Genetic and phenotypic diversity of autochthonous Saccharomyces spp. strains associated to natural fermentation of “Malvasia delle Lipari.” Lett Appl Microbiol. 2007;45: 657–662. doi:10.1111/j.1472-765X.2007.02244.x
• Mendes I, Franco-Duarte R, Umek L, Fonseca E, Drumonde-Neves J, Dequin S, et al. Computational Models for Prediction of Yeast Strain Potential for Winemaking from Phenotypic Profiles. PLoS One. 2013;8. doi:10.1371/journal.pone.0066523

Lines 371-379: I would like to analyse authors opinion on the comparison of this manuscipt´s results with the ones obtained by Kosel et al. (Kosel J, Cadež N, Schuller D, Carreto L, Franco-Duarte R, Raspor P. The influence of Dekkera bruxellensis on the transcriptome of Saccharomyces cerevisiae and on the aromatic profile of synthetic wine must. FEMS Yeast Res. 2017;17: 1–11. doi:10.1093/femsyr/fox018)

Author Response

Plese see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript by De la Torre-Gonzalez et al. reports a comparison of fermentive properties of 12 S. cerevisiae and 13 non-Saccharomyces yeasts. In particular, the authors tested the effect of pairwise combinations of a S. cerevisiae and one non-Saccharomyces strain.

A few comments that my be considered for improving the manuscript:

The manuscript needs extensive revision with respect to language. In particular, many sentences are extremely long and confusing. A highlight may be the sentence on lines 153-158 - over six rows! There are many others like this.

In all the Tables: What are the values? Mean of how many measurements? Standard errors or deviations? Only indicate one decimal.

What is the difference between Figures 3 and 4? This is not obvious. Also, it would have been interesting to compare the mixtures with the pure cultures. Why has this not been done?

Table 4: Remove the rows for those strains that have not been used in the combinations. There is no point in including these here.

Mixtures/discussion: There is no indication that the metabolite/volatile profiles are not just the result of an additive effect. This should be addressed experimentally. As it stands, it cannot be concluded that certain strains prevented the production of any metabolite.

It is a pity that only one mixture was actually studied in detail. It is not really clear why only this particular combination was tested. Would it not have been worthwhile looking at all of them in order to identify interesting effects that are not additive? It certainly seems that this should have been possible and could be expected for such a publication.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

 Overall, the mansuscript is much improved. However, there is stil ample room for improving the text. Some suggestions are given below.

The first and second sentence of the abstract should be split in two.

The first sentence of the introduction should be split or simplified.

The sentence  on lines 41ff could be simplified by separating the conclusion into a separate sentence.

Line 48: Maybe research instead of study group and "has".

Line 52: and "mezcal fermentation is carried out ...."

Line 55: Delete "are" and maybe concentration instead of contents; yeast (not yeasts).

Line 56: in wine (delete the); delete "on this way, "

Line 65f: "and their fermentative performance and potential as starters for wine production were evaluated"

Line 69ff: Split commercial strain from rest in separate sentence.

Line 75f: Separeate last part of sentence.

Line 82: Ringer solution should be specified.

Line 91ff: Simplify and split sentence. “contained” 200 g/L sugars, split part about ammonium sulfate.

Line 102-107: Split/simplify.

Line 107: Delete “for”.

Line 112: Start new sentence after 14’000 rpm.

Comment on biomass production: It may have been advisable (and is for future studies) to expose empty tubes to the same procedure as they may loose weight. The biomass could thus be overestimated.

Line 155: in samples.

Line 162ff: noticed, … isolated at the pressing and early fermentation stages.

Line 167ff: ….but by 72 h of fermentation, less fructose was consumed as compared to glucose, as previously reported [8].

Line 176ff: ….at the 96 h sampling time fermentations with the S. cerevisiae 3D series had lower residual sugar concentrations, but at the end of fermentation (360 h, data not shown) the fermentations with the series 3Y (except Sc3Y2, which was a high glycerol producer) had almost completely consumed both sugars.

Line 185ff: Stop sentence after “(…. 2g/L)”. Then: However, it ….

Line 198: … , but his volatile was not detected ….

Lines 205-212: Simplify/split into several sentences!

Line 219: in the medium.

Line 227f: … all the Saccharomyces and non-Saccharomyces strains.

Table 5: Provide only 1 decimal for all errors.

Line 245: in ethanol production.

Line 247: What are acceptable acetic acid concentrations?

Line 249f: Due to its better overall aroma, lower ethanol production, and low acidity and higher fermentation purity, we decided to analyze the non-Saccharomyces strain Td1AN9 in more detail.

Line 254: Past tense!

Line 283: until the end of the culture; whilst the population of the non-Saccharomyces strain …

Line 284: Start new sentence at “being ….-> This strain was most affected by … but ….

Line 287: Shown.

Line 334ff: More recently, and similar to the work presented here for S. cerevisiae mezcal strains, Franco-Duarte et al [12] established that, for 24 S. cerevisiae strains ethanol and organic acid (in particular acetic acid) concentrations, explained most of the metabolic differences among strains. The S. cerevisiae strains studied in more detail here produced comparable amounts of ethanol as the commercial strain Fermichamp. The selected stains also led to high glycerol levels and were able to almost completely consume glucose and fructose during the fermentation. In general, primary metabolites were produced in higher amounts in the grape juice medium than in the semi-synthetic medium M3.

Discussion: The way how work is cited is strange. Reference numbers should not be included in sentences as if they were subjects.

Lines 370ff: Similarly, it was reported that the main inhibitory mechanism towards K. thermotolerans and T. delbrueckii strains was the physical presence of S. cerevisiae [15]. Our results seem to support this explanation, but this effect may not be species-specific, as suggested by Kosel et al [16]. These authors did not find any effect of the cell-to-cell contact of the commercial S. cerevisiae EC1118 over the growth kinetics of the cultures of Dekkera bruxellensis, which is a spoilage yeast of low growth and a low tolerance to high sugar concentration, as well a weak producer of ethanol. All this indicates that more work is needed to clarify the specific properties of S. cerevisiae and T. delbrueckii co-cultures.

Author Response

Please see the attachment

Author Response File: Author Response.pdf