Molecular Identification and VOMs Characterization of Saccharomyces cerevisiae Strains Isolated from Madeira Region Winery Environments
Round 1
Reviewer 1 Report
This article reports the identification and characterizations of some wine yeast strains. The later is well established but the identification is poorly supported by the presented results. Fig3. is not informative at all. I do not see the reason using HinfI enzyme. This one does not differentiate Saccharomyces spp. The HaeIII seems a bit more useful, but many other Sacch. species yield the same pattern as S. cerevisiae. Eight different mtDNA-RFLP patterns were identified, but only 2 of them are shown. The sequence of the amplified ITS fragments would give more and reliable identification, and at least the 8 different representative strains should be sequenced.
My specific comment are as below:
Line 17. change „specie” to „species”
Line 88: change “For the species yeast identification” to For the yeast species identification
Line 119: delete „condition”
Line 121: min..
Line 187: Table 1 shows the number of colonies, not the isolates. Columns “Dilutions and
Nº col.” unnecessary, CFU/ml informative enough.
Lines 84 and 197 are contradictory. „Isolates selected randomly” – assayed only S. cerevisiae like colonies
Line 205: again, change „specie.” to „species.”
Line 206: change „patter with fragment” - to „pattern with fragments”
Line 217: presents – presence?
Line 224 representative – dominant?
Line 226: grapes - grape
Line 304: change „specie.” to „species.”
Line 310: Intraespecific - Intraspecific
Line 438: predominate - predominant
Author Response
This article reports the identification and characterizations of some wine yeast strains. The later is well established but the identification is poorly supported by the presented results. Fig3. is not informative at all. I do not see the reason using HinfI enzyme. This one does not differentiate Saccharomyces spp. The HaeIII seems a bit more useful, but many other Sacch. species yield the same pattern as S. cerevisiae. Eight different mtDNA-RFLP patterns were identified, but only 2 of them are shown. The sequence of the amplified ITS fragments would give more and reliable identification, and at least the 8 different representative strains should be sequenced.
AUTHORS ANSWER:
The published literature reports an amplicon size that varies between 840 [22], 850 [24, 26] and 880 bp [13,25], which makes it possible to identify Saccharomyces of other species. In the present study, a fragment between 850-880 bp (Figure 3) was obtained in all the analyzed isolates, corresponding to the characteristic fragment of Saccharomyces sensu stricto that includes the species S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus. Then, four bands after digestion with the endonuclease HaeIII corresponding to the species S. cerevisiae and S. paradoxus [13,24,26]. Finally, two fragments after digestion with HinfI endonuclease (365 and 155 bp) corresponding to the species S. cerevisiae [13] (lines 207 and 208).
Regarding mtDNA-RFLP patterns, in the literature, only the dominant strains were showed, for example, a large scale survey performed in Vinho Verde Region [9], obtained 267 mtDNA-RFLP patterns, however, only the most 9 dominant mtDNA-RFLP patterns were presented.
We agree with the suggestion to sequence the ITS fragment of the different mtDNA-RFLP patterns.
My specific comment are as below:
Line 17. change „specie” to „species”
Line 88: change “For the species yeast identification” to For the yeast species identification
Line 119: delete „condition”
Line 121: min..
Line 187: Table 1 shows the number of colonies, not the isolates. Columns “Dilutions and
Nº col.” unnecessary, CFU/ml informative enough.
Lines 84 and 197 are contradictory. „Isolates selected randomly” – assayed only S. cerevisiae like colonies
Line 205: again, change „specie.” to „species.”
Line 206: change „patter with fragment” - to „pattern with fragments”
Line 217: presents – presence?
Line 224 representative – dominant?
Line 226: grapes - grape
Line 304: change „specie.” to „species.”
Line 310: Intraespecific – Intraspecific
Line 438: predominate – predominant
AUTHORS ANSWER:
According to the reviewer suggestion all the corrections were done in the revised version of the manuscript
Reviewer 2 Report
I think the author needs to have a native English speaker review the paper first. While I understood the points the authors made, it was difficult to pull out.
When describing a conclusion please do not use probably, since this is the major outcome of the manuscript.
I also think there needs to be replicates performed for figure 2.
the gels should be put into contrast and could be cleaner with out any background. (figure 3 and figure 4)
Author Response
I think the author needs to have a native English speaker review the paper first. While I understood the points the authors made, it was difficult to pull out.
AUTHORS ANSWER:
According to the reviewer suggestion the English was corrected by an expert.
When describing a conclusion please do not use probably, since this is the major outcome of the manuscript.
I also think there needs to be replicates performed for figure 2.
the gels should be put into contrast and could be cleaner with out any background. (figure 3 and figure 4)
AUTHORS ANSWER:
We agree with your observation, however, to affirm, DNA sequence is necessary.
During the optimization of the method, microfermentations were performed in triplicate. We found that there are no significant differences in the fermentative profile between replicates.
According to the reviewer suggestion, the corrections were done, (figure 3 and figure 4).
Reviewer 3 Report
Title: Molecular identification and VOMs characterization of Saccharomyces cerevisiae strains isolated from Madeira Region Winery Environments
Authors: Castillo et al.
Summary: The authors conducted an extensive characterization of VOMS and several strains of S. cerevisiae involved in wine production in the Madeira region. The authors identified the molecular characteristics of S. cerevisiae from DRM and characterized the volatile organic metabolites associated with the identified strains of S. cerevisiae. This work is interesting because of the established relationship between the resident S. cerevisiae strains and the quality of wine produced in the region. The authors identified 2 major strains of S. cerevisiae in the sampled areas of DRM producing the highest amounts of alcohol, esters and fatty acids in varying proportions which in turn determine the corresponding aroma of different wines.
Specific comments:
- There are numerous spelling and grammatical errors, use of abbreviations without their full meaning and incorrect representation of metric units abundant in the article make it an unpleasant read.
- Some protocols in the methodology are missing or omitted without any reference such as the composition of the “pure culture” (line 84) and the growth conditions; the incubation conditions (line 100) and the solvent used in washing the cells (line 101) for DNA isolation.
- Other information that should have been included in the methods to make it meaningful were rather found in the result section such as the purity and conc. of DNA used in the analysis.
- The positive (C) and negative controls (MC) used were not clearly explained (line 248). What is meant by rectified clarified or is it another grammatical error? The controls should be clearly stated to be able to justify the results (phenotypes of the isolates).
- The axis of the chart in Figure 5 should be properly labelled. The authors may use fold change to describe the increase in the volatile fraction instead of the ambiguous percentages.
- It is imperative to emphasize the relevance of the study in the concluding section. Is there any relationship between the commercial wine obtained from the region and the fermentation products of the identified strains?
Author Response
cerevisiae involved in wine production in the Madeira region. The authors identified the molecular characteristics of S. cerevisiae from DRM and characterized the volatile organic metabolites associated with the identified strains of S. cerevisiae. This work is interesting because of the established relationship between the resident S. cerevisiae strains and the quality of wine produced in the region. The authors identified 2 major strains of S. cerevisiae in the sampled areas of DRM producing the highest amounts of alcohol, esters and fatty acids in varying proportions which in turn determine the corresponding aroma of different wines.
The authors acknowledge the positive reviewer opinion about the manuscript.
Specific comments:
- There are numerous spelling and grammatical errors, use of abbreviations without their full meaning and incorrect representation of metric units abundant in the article make it an unpleasant read.
AUTHORS ANSWER:
According to the reviewer suggestion the English was revised by an expert.
- Some protocols in the methodology are missing or omitted without any reference such as the composition of the “pure culture” (line 84) and the growth conditions; the incubation conditions (line 100) and the solvent used in washing the cells (line 101) for DNA isolation.
AUTHORS ANSWER: The yeasts isolation in pure culture (line 84) was performed according to Schuller et. al. [9]. DNA extraction for strain identification was performed at 30 ºC (information added in line 100 – at 30 ºC), we used the same growth temperature in all carried out assays. The information of solvent used in washing cells, mQ, was included in the manuscript (line 102) .
- Other information that should have been included in the methods to make it meaningful were rather found in the result section such as the purity and conc. of DNA used in the analysis.
AUTHORS ANSWER: The information was added in line 97 – DNA concentration was determined using the NanoDrop 2000 Spectrophotometer (Thermo ScientificTM).
- The positive (C) and negative controls (MC) used were not clearly explained (line 248). What is meant by rectified clarified or is it another grammatical error? The controls should be clearly stated to be able to justify the results (phenotypes of the isolates).
AUTHORS ANSWER: Indeed, there is a mistake, the correct name is “rectified concentrated grape must” which, in the present study, has been clarified and diluted. Rectified concentrated grape must is obtained from grape must, dehydrated without using heat. Generally used in the oenological industry for the enrichment of grape musts, especially in years with adverse climatic conditions and/or poor harvests, to increase the potential alcohol of the must. Additionally, the organoleptic properties of final product is not influenced, for this reason, we used it as substrate/negative control. The legislation that regulates the wine sector in Portugal, allows a concentration of rectified concentrated grape must up to 1 %, v/v of potential alcohol. The term was corrected in lines138, 253 and 254.
The positive control used for VOMs characterization was commercial S. cerevisiae strain QA23 (Lavin) widely used in the oenological industry in the production of white table wines and whose technological and organoleptic properties are very well known.
- The axis of the chart in Figure 5 should be properly labelled. The authors may use fold change to describe the increase in the volatile fraction instead of the ambiguous percentages.
AUTHORS ANSWER: Information in Y axis of the Figure 5 and line 249 was updated.
A crucial objective of the present study was to establish the capacity of each strain to biosynthesize a differentiated volatile profile. In this sense, we consider that, in order to establish these differences/similarities between strains, the comparison of proportions in percentage would be more elucidative.
- It is imperative to emphasize the relevance of the study in the concluding section. Is there any relationship between the commercial wine obtained from the region and the fermentation products of the identified strains?
AUTHORS ANSWER:
‘To date, to our knowledge no scientific study has been reported on this subject’, although the DRM is one of the first Demarcated Regions in Portugal.
On the other hand, in this first approach, our results were compared with some studies carried out, referring to the wines of the region and the persistence of VOMs is verified, even in the oldest wines, however, we have to consider that Madeira wines are fortified wines, and further studies are necessary in this sense.
Reviewer 4 Report
The authors present an interesting work. The experimental part is well designed and the results are presented and justified in detail. The Introduction, however, has to be improved. The state-of-the art is not presented. Consider adding more references, especially review arivles to help the readers follow the context.
There are many syntax errors in the manuscript, please revise.
Author Response
The reviewer suggestions were done.
The English was revised by an expert.
Round 2
Reviewer 2 Report
The author has addressed the major grammatical issues.
I would like to see standard deviations on Figure 2.
Figure 4 could be cleaner, there is a lot of back ground.
Author Response
Thanks a lot for your kind suggestions.
On Figure 2 we adeed the following information: "(average standard deviation for each point is lower than 12%)". The inclusion of error bars for each point would make a very complex and difficult-to-interpret figure.
Regarding Figure 4, unfortunately this is the best figure that we achieve. Indeed there is a lot of back ground that makes the figure with low resolution. However, it was the best that we obtained.
Reviewer 3 Report
Authors have made far-reaching efforts to address previous comments and concerns. The current version of the manuscript is greatly improved. Nonetheless, there remain minor grammatical errors that need to be addressed.
Author Response
The manuscript was revised by an English expert, so we hope that this final version be better in terms of grammatical errors.
