Stress Resistance and Adhesive Properties of Commercial Flor and Wine Strains, and Environmental Isolates of Saccharomyces cerevisiae
, Nikolai V. Ravin 1, Svetlana A. Kishkovskaya 2 and Andrey V. Mardanov 1,*Round 1
Reviewer 1 Report
The authors want to apply some simple selection criteria such as the adhesion capacity, the biofilm formation, the growth capacity under stress conditions etc in order to find the best flor yeast strain candidates suitable for commercial winemaking. Their selection would be also based on genetic and phenotypic correlation. I think the authors should make more clear the terms they use about yeast isolates during winemaking. What is the difference between environmental, wine and commercial yeast isolates? The flor yeast contain only strains from the species of S. cerevisiae? Why you write about table wine? It could be nice to add more bibliography about the yeast strains ecology during ex Sherry wines. Spanish and French research teams work a lot on that subject. Additionally, it is really important to mention that there is NO statistical analysis in the Material and Methods section. When we treat physiological characteristics the analysis behind is essential for the comprehension of the results. I think the authors have make a great try but they should spend more time for the manuscript preparation.
Line 2
What do you mean by environmental isolates? wine yeasts are not environmental isolates?
Line 12
Intercellular adhesion?
Line 45
Wine variety or wine style?
Line 44
Biochemical and sensory properties. Can you pls explain more and add more references
Line 53
Flor yeast contain only strains of S. cerevisiae?
Line 57
What about the species?
Line 71-79
Are you sure there is no bibliography on this subject? They are many published papers on Sherry wine production and yeast ecology.
Line 99
Wine strains? You mean commercial strains?
Line 121
Why you have estimated the population level?
Line 178
Why just for 1 hour? Any reference you have been based on. Any replicate?
Table 1
Is better to use the OIV units for the oenological parameters (not dm3 but L)
What is “sherry” tones in aroma and taste? You haven’t talk about them before in the text!!
Table wine? Pay attention to your terms. Pls explain better or correct
Discussion
Do you think the number of used strains in the present study is sufficient to make correlations between genotyping and phenotyping?
Please explain more about the biofilm formation capacity. Any implicated molecules possible excreted?
Line 341
It is normal that the strains isolated from the grapes are less tolerant to the presence of ethanol than the strains isolated from wine. You can not compare them!
Author Response
The authors want to apply some simple selection criteria such as the adhesion capacity, the biofilm formation, the growth capacity under stress conditions etc in order to find the best flor yeast strain candidates suitable for commercial winemaking. Their selection would be also based on genetic and phenotypic correlation.
I think the authors should make more clear the terms they use about yeast isolates during winemaking. What is the difference between environmental, wine and commercial yeast isolates?
RE: We analyzed three groups of S. cerevisiae strains, - flor strains (used for making sherry-like wines), wine strains (used in “usual” winemaking for fermentation of must) and strains isolated from grape samples. The latter group was designated as “environmental isolates”. We added this explanation in Introduction (lines 90-91) and Materials and Methods (lines 117-124).
The flor yeast contain only strains from the species of S. cerevisiae?
RE: yes, all strains in this work were from S. cerevisiae, information added in Materials and Methods section (lines 117 and 121).
Why you write about table wine?
RE: We meant that these strains (“wine strains”) are used in common winemaking but not for biological wine aging. In the revised version this group is defined as “wine strains”
It could be nice to add more bibliography about the yeast strains ecology during ex Sherry wines. Spanish and French research teams work a lot on that subject.
RE: We added several relevant references, mostly to the Introduction.
Additionally, it is really important to mention that there is NO statistical analysis in the Material and Methods section. When we treat physiological characteristics the analysis behind is essential for the comprehension of the results.
RE: Statistically significant differences (p < 0.05) in adhesive properties evaluated in isooctane test and in polystyrene adhesion test were observed between two groups: [flor strains + flor-forming environmental isolates] and [wine strains + non flor-forming environmental isolates]. This is the most important result of this study. We added this information to Results (lines 311-316) and described statistical methods in Material and Methods (lines 211-214).
Line 2
What do you mean by environmental isolates? wine yeasts are not environmental isolates?
RE: All these strains are natural isolates. The term “environmental isolates” was used for strains isolated from grape samples, to distinguish them from flor and wine strains. We added this explanation in Materials and Methods section (line 122).
Line 12
Intercellular adhesion?
RE: Corrected to “adhesive properties”
Line 45
Wine variety or wine style?
RE: Wine style. The text was modified
Line 44
Biochemical and sensory properties. Can you pls explain more and add more references
RE: we added more references. More detail description of biochemical and sensory properties of sherry-like wines is out of scope of this paper.
Line 53
Flor yeast contain only strains of S. cerevisiae?
RE: At present, only S. cerevisiae strains of flor yeast have been described. Despite the fact that in addition to S. cerevisiae other yeast species (W. anomalus, P. membranaefaciens, P. kudriavzevii and P. manshurica) were found in industrial biofilms in minor amounts their role in the formation of a biofilm has not been studied (included in lines 45-48).
Previously recognized Saccharomyces yeasts S. aceti, S. beticus, S. cheresiensis, S. cordubensis, S. gaditensis, S. hispanica, S. oxidans, and S. prostoserdovii (differed by their ability to ferment various carbon sources) belongs of the biological species S. cerevisiae.
Line 57
What about the species?
RE: S. cerevisiae (see line 35)
Line 71-79
Are you sure there is no bibliography on this subject? They are many published papers on Sherry wine production and yeast ecology.
RE: Certainly there are many papers on the production of sherry-like wines. Currently, studies of flor yeast are carried out on well-known commercial strains, which were obtained in the course of long-term selection. But the question of the evolutionary origin of these strains remains open. That is why the approaches proposed in this work for the search for such yeasts are of interest and will provide new information for the study of the evolution of this group of yeasts. We changed the word “ecology” to “evolution” to be more precise (line 79). We also referenced a recent relevant study (lines 80-82).
Line 99
Wine strains? You mean commercial strains?
RE: commercial strains used for “usual” winemaking.
Line 121
Why you have estimated the population level?
RE: In order to set the same amount of cells in each flask. It is important for comparison of different strains.
Line 178
Why just for 1 hour? Any reference you have been based on. Any replicate?
RE: This time was taken form a protocol described by Hsu et al., 2015 (https://0-doi-org.brum.beds.ac.uk/10.1371/journal.pone.0135174), there is a reference in the text. All experiments were performed in five replicas (line 188).
Table 1
Is better to use the OIV units for the oenological parameters (not dm3 but L)
RE: corrected
What is “sherry” tones in aroma and taste? You haven’t talk about them before in the text!!
RE: It was an expert opinion based on the results of the tasting (information added to table legend).
Table wine? Pay attention to your terms. Pls explain better or correct
RE: modified
Discussion
Do you think the number of used strains in the present study is sufficient to make correlations between genotyping and phenotyping?
RE: This is not exactly what we investigated in this work. We analyzed such characteristics of the strains as resistance to stress (ethanol, acetaldehyde, oxidative stress) and adhesive properties using simple tests. Known flor and wine strains were analyzed, as well as a set of natural isolates with alleles, characteristic for flor strains (Table 1). The aim of the work was to understand whether rapid tests can be used for screening of candidate flor strains among “environmental” yeast isolates. It turned out that the high hydrophobicity determined in the isooctane test and the test with polystyrene clearly correlated with the ability of the strains to form a flor, while in stress resistance tests there was no such correlation. Thus, genotyping at three loci followed by a simple test for hydrophobicity can be used to search for new candidate flor strains.
Please explain more about the biofilm formation capacity. Any implicated molecules possible excreted?
RE: The molecular mechanism of biofilm formation is beyond the scope of this paper. There are several review articles on this topic, in particular, Zara et al. 2010 (doi:10.1128/AEM.00111-10). Formation of the biofilm appears to be an adaptive mechanism because it ensures access to oxygen and therefore permits continued growth on nonfermentable ethanol. In general, non-buoyant cells cease growth at the end of completed wine fermentations not for lack of carbon but for lack of oxygen. Biofilm cells have been found to have an elevated and/or altered lipid content and increased surface hydrophobicity. While both Hsp12, a small heat shock protein, and Flo11, a hydrophobic cell wall mannoprotein, have been shown to be required for the flor biofilm, other genetic or environmental requirements, other than an absence of glucose and the presence of ethanol and oxygen, have not been demonstrated.
Line 341
It is normal that the strains isolated from the grapes are less tolerant to the presence of ethanol than the strains isolated from wine. You can not compare them!
RE: But this turned out to be just not the case. Some strains isolated from grapes (23, 54, 109, 112, 90) were more resistant to ethanol than most of wine strains. Although ethanol resistance is an important factor for wine yeast, the degree of resistance varies greatly between different industrial wine strains. For example, among 14 commercial wine strains some were more sensitive to ethanol even than the laboratory strain of S. cerevisiae (Carrasco et al. Analysis of the stress resistance of commercial wine yeast strains. Arch Microbiol. 2001, 175, 6, 450-7).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The paper have been slightly improved, but has a low scientific soundness and it's not interesting to readers. I have some major concerns after read the new version:
There are too many old references. They should be replaced by references from the last ten years.
Line 57-58: …Osmotic stress is induced by the high content of sugars in the grape must (about 20% w/v) at the beginning of vinification, and the yeasts must resist it in order to start the alcoholic fermentation… It is not fully true, not all Sherry wines have the same “base” wine. While Spanish Sherry Base Wine is a white young wine which is fortified till 15% (v/v) and the DYNAMIC ageing system (“criaderas and soleras”) mixes wines from different years, Vin Jaune (Jura, France) is carried out by with a sugar concentrated base wine in a static biological aging, as there is no mixing between wines of different vintages or aging stages. Please, see the reference Ruiz-Muñoz et al., 2021: https://0-doi-org.brum.beds.ac.uk/10.1016/j.fm.2020.103553
Line 62: (up to 15%) in Spanish Sherry wines up to 15,5%.
Line 66: Microsatellite analysis revealed a high degree of phylogenetic relationship of sherry flor strains…Not always, please see Ruiz-Muñoz et al., 2021: https://0-doi-org.brum.beds.ac.uk/10.1016/j.fm.2020.103553.
Author Response
The paper have been slightly improved, but has a low scientific soundness and it's not interesting to readers. I have some major concerns after read the new version:
There are too many old references. They should be replaced by references from the last ten years.
RE: Of the 49 references, more than a half (30) refer to publications over the last 10 years. Since flor yeast has been studied for quite a long time, we consider it relevant to also mention the relevant older piblications.
Line 57-58: …Osmotic stress is induced by the high content of sugars in the grape must (about 20% w/v) at the beginning of vinification, and the yeasts must resist it in order to start the alcoholic fermentation… It is not fully true, not all Sherry wines have the same “base” wine. While Spanish Sherry Base Wine is a white young wine which is fortified till 15% (v/v) and the DYNAMIC ageing system (“criaderas and soleras”) mixes wines from different years, Vin Jaune (Jura, France) is carried out by with a sugar concentrated base wine in a static biological aging, as there is no mixing between wines of different vintages or aging stages. Please, see the reference Ruiz-Muñoz et al., 2021: https://0-doi-org.brum.beds.ac.uk/10.1016/j.fm.2020.103553
RE: In this part of Introduction we are talking about general aspects of stress tolerance of wine yeasts. Osmotic and ethanol stresses are most important during alcoholic fermentation (line 51). Biological wine aging is a different process in which yeast does not meet with a high concentration of sugars (as noted in line 57-58). Therefore we did not analyzed our strains for tolerance to osmotic stress.
Line 62: (up to 15%) in Spanish Sherry wines up to 15,5%.
RE: corrected
Line 66: Microsatellite analysis revealed a high degree of phylogenetic relationship of sherry flor strains…Not always, please see Ruiz-Muñoz et al., 2021: https://0-doi-org.brum.beds.ac.uk/10.1016/j.fm.2020.103553.
RE: Here we wanted to say that flor strains formed a distinct phylogenetic cluster and the genetic diversity in this group is lower than of wine strains. We have modified this paragraph as follows.
Comparative genomic studies revealed that flor yeast represents a distinct lineage that separated from the wine clade through a relatively recent bottleneck event (Coi et al., 2017). We added this sentence (line 61-62).
Regarding microsatellite analysis it should be noted that Ruiz-Muñoz et al. concluded that “Biodiversity analysis showed there were significant differences between the three wineries in the three aging scales, although the overall diversity was relatively low”. We modified the sentence in lines 63-64 as follows: “Microsatellite analysis revealed overall low genetic diversity of flor strains, although genetic differences between strains from different wineries were observed”.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
1) Thank you for your answers. I still don't agree with the term enviromental isolates in the title (maybe indigenous?), but it's my personal opinion. I agree for publication.
2) I think the authors should focus more on the interpretation of their results and the conclusion they make. Major revisions to propose. Some commnets:
What about the wine strains in the title?
Keywords: you don’t have any genotyping in this paper
How you study the biofilm formation you are referring in the abstract and discussion section? In the Material & Methods the authors described the MATH hydrophobicity test (section 2.4) and the ability to adhere to polystyrene (section 2.5).
How you compare the three groups of strains by applying two samples t-test?
Table 1 **Expert opinion based on the results of the tasting. What do you mean by expert? How many experts? all the experts gave the same answer in all the tested samples? there is no such section in the Material & Methods
Do you think the number of flor yeast in the study is sufficient for the conclusion you make and that the rapid test for flor yeast selection is valuable? I think is more an indication
Author Response
We thank the reviewer for useful comments and recommendations. All of them were taken into account, the corresponding corrections were made in the text of the article.
What about the wine strains in the title?
RE: we modified the title: “Stress resistance and adhesive properties of commercial flor and wine strains, and environmental isolates of Saccharomyces cerevisiae”
Keywords: you don’t have any genotyping in this paper
RE: yes, we removed genotyping from the keywords
How you study the biofilm formation you are referring in the abstract and discussion section? In the Material & Methods the authors described the MATH hydrophobicity test (section 2.4) and the ability to adhere to polystyrene (section 2.5).
RE: The data on flor growth are shown in Table 1. We added description in Material & Methods and in the legend to Table 1.
How you compare the three groups of strains by applying two samples t-test?
RE: we compared two groups: flor strains and biofilm-forming environmental strains (10 strains, group 1) with the rest of the strains (12 strains, group 2) revealed statistically significant differences between these groups both in the isooctane test (p value 0.016) and in the test with polystyrene (p value 0.0018).
Table 1 **Expert opinion based on the results of the tasting. What do you mean by expert? How many experts? all the experts gave the same answer in all the tested samples? there is no such section in the Material & Methods
RE: We added description to the Material & Methods section. A positive assessment was given in case of consent of at least 4 out of 5 experts.
Do you think the number of flor yeast in the study is sufficient for the conclusion you make and that the rapid test for flor yeast selection is valuable? I think is more an indication
RE: We agree that this is an indication but it has statistical support (10 flor strains and biofilm-forming environmental strains versus 12 other strains). Anyway, we put this sentence in the Discussion and it was formulated as an indication: “Overall, our results indicate that relatively simple tests for cell hydrophobicity, together with the identification of molecular markers characteristic for flor yeasts, can be used for the rapid screening of new candidate flor strains in yeast culture collections and among environmental isolates”.
Author Response File: Author Response.pdf
Reviewer 2 Report
This paper has been improved. However it is not very interesting to readers.
Author Response
We respect your opinion, but we believe that the results of our study indicating that relatively simple tests for cell hydrophobicity can be used for the rapid screening of new candidate flor strains in yeast culture collections and among environmental isolates are of practical importance and general interest. Almost all studies on flor yeast were carried out on industrial strains that have been used and selected for many years, and the question of whether such yeasts could be found in natural sources like grapes has been little studied.
Round 3
Reviewer 1 Report
I think the paper should be written again with a clear aim and clear conclusion based on the proven results. More bibliography on wine yeast should be conducted as well as on the mechanism of biofilm formation and the meaning in winemaking.
Author Response
We respect your opinion, but we believe that the results of our study indicating that relatively simple tests for cell hydrophobicity can be used for the rapid screening of new candidate flor strains in yeast culture collections and among environmental isolates are of practical importance and general interest. The search for new natural strains of flor yeast has been little studied. We added a section about biofilm formation and adhesive properties of commercial wine strains and wine spoilage yeasts to the Discussion.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The authors want to simplify the selection of candidate strains for obtaining sherry wines, but they did not perform their tests in triplicate and the data were not evaluated with a statistical analysis.
In my opinion, without the support of statistics to evaluate the results it is not possible to select yeast strains.
Reviewer 2 Report
In the present work the authors have used a large collection of S. cerevisiae strains isolated during winemaking in order to make a correlation between their genotype and phenotype. Their aim was to valorise the yeast strains for sherry type wines production. In the beginning the authors should be clearer by using appropriately the term ‘’sherry’’. What do you mean by sherry wine and sherry strains? Maybe you want to say type of sherry wine. The authors don’t mention any replicate of their experiments neither in the materials&methods section neither in their presentation of their results. Especially in the study of adhesion capacity on polystyrene plates the method is characterized by high heterogeneity among the wells. Subsequently there is no statistical analysis in the hole paper. The discussion section is really poor. You should compare deeper your results with other relative works. The authors should consider that studying the hydrophobicity by the isooctane test don’t permit them to generalise about the adhesion properties of the yeast cells especially under winemaking conditions, but of course could give them an indication. The bibliographic references are quite few (only 29), it would be nice to be enriched.
Line 82
‘’substances that damage the cell wall’’, can you pls explain more this phrase?
Line 92
Which are the ‘’sherry strains’’ that you are later referred to?
Linen105
Pls write the exact conditions of must pasteurization
Line 110
Stress may have an impact on cell morphology (especially in the case of Saccharomyces), how you know that measuring cell viability by absorbance is accurate? Why you didn’t test viability with culture plates by counting colonies?
Line 155
Why you standardize your initial population on the plates? pls describe/explain
Line 155
Any biological replicates? Is rare to obtain homogeneity with this method
Line 277
Sherry isolates are not also natural isolates? Pls explain/ describe more
Line 301
How important is the choice of the organic solvent? Do you think that the results will be different by changing the used solvent?
Line 303
Do you think is possible to compare yeast strains coming from the grapes with yeasts isolated from the flor film?
Reviewer 3 Report
This paper entitled "Stress resistance and adhesive properties of collection and natural strains of S. cerevisiae yeast in relation to the possibility of their use for producing sherry wines" shows some experiments to characterize the possible potential of some yeast strains from a collection or from the nature to be used as veil-forming yeasts for Sherry-likes wine.
I think the the use of the word Sherry or naming yeasts as Sherry yeast should be carefully used since this term is linked to the Jerez-Xèrés-Sherry area. Authors could use the words veil-forming yeasts, Sherry-like or Sherry-type yeasts.
This paper has a low scientific soundness and it's not interesting to readers. Authors applied some well stablished experiments done by other authors or from the OIV-OENO 370-2012 resolution. Moreover, results have shown that none of the characterised yeasts strains could be used as veil-forming yeast in Sherry-like wines, none of them showed a hihg resistence to ethanol (about 15,5% v/v).
Title is too long and this title should be shortened. The text requires some revisions. eg: the word microflora should be replaced by microbiota, in line 59 "omix" by "omics" or Sherry biofilm by veil of flor.
I liked the content of the manuscrispt since it could be useful to be applied in High School laboratory practices or at the University level. But not for a publication in the Fermentation journal. This paper is too basic and authors do not conclude anything.
