Review Reports - The Effect of Yeast, Sugar and Sulfur Dioxide on the Volatile Compounds in Wine

Round 1

Reviewer 1 Report

The article entitled “Influence of the sugar content and sulphur dioxide in the volatile composition of wines fermented with three yeast strains”, submitted to the journal presents a study aimed to comparison the fermentative efficiency of three yeast strains, one commercial and two selected, to assess their tolerance against high sugar concentrations and the presence of sulphur in red must. The study of the reaction of yeast strains to stress factors resulting in changes in the volatile profile of the product is still in a spotlight of research and production practice.

The manuscript is clearly structured and written in a language understandable to potential readers. The range of analyses used is suitable for the declared aim of the study. The results were statistically processed and are mostly satisfactorily discussed.

I have the following comments on the article:

- Line 118,121: What does "Three biological replicates were used to undertake the analysis" mean? On line 108, it says that "Two flasks were inoculated for each strain and each experimental condition".

- Evaluation of aromatic profiles of wines using Odorant activity values (OAVs) should be mentioned in the "Materials and methods" chapter.

- The conclusion is somewhat long and partly repeating the findings described in the discussion. The conclusion should answer the aim of the study and it should capture what new insights have been found and how these insights advance our knowledge in the field.

On my opinion, the work brought a number of findings for further research and practice and manuscript could be accepted for the publication after minor revision.

Author Response

I have the following comments on the article:

The conditions were as follows: two flask for each biological replicate were inoculated with the different yeast strains. On of the flask was used to follow the yeast population. The other flask was used to undertake the oenological and volatile aroma analyses.

For a better understanding the text will be changed.

- Evaluation of aromatic profiles of wines using Odorant activity values (OAVs) should be mentioned in the "Materials and methods" chapter.

A new subsection has now been included in material and methods section.

The conclusion section has been rewritten according with your suggestions.

Reviewer 2 Report

TITLE:Influence of the sugar content and sulphur dioxide in the volatile composition of wines fermented with three yeast strains

Comments to authors:

This paper investigates the effects of yeast, sugar and SO₂ on the physicochemical parameters and aromatic substances of wine. The results showed that each yeast contributes to a different aromatic profile, which can be used by winemakers to highlight specific aromas. The work has some merits and within the scope of Foods, and valuable to the journal’s readership. The work is well-organized and easy to follow. However, the manuscript needs major revision before publication. The followings are some comments and suggestions for authors to consider and improve the manuscript.

1. Lack of summary of the effects of yeast, sugar and SO2 on physicochemical indicators in the conclusion section.

2. Change the title to "The effect of yeast, sugar and SO2 on the volatile compounds in wine"?

3. In the Results and Discussion section, it is recommended that the effect of SC 1 and SC 5 on volatile compounds be properly highlighted. Highlighting the significance of "LC" in the control group.

4. The Results and Discussion section lacks the necessary discussion of the results.

5. Significant analysis for the Tables should be more clearly.

6. The method for identification of the volatiles should be more detail.

7. The conclusion section also requires careful deliberation to highlight the main purpose and significance of this study.

Moderate editing of English language is necessary.

Author Response

Lack of summary of the effects of yeast, sugar and SO2 on physicochemical indicators in the conclusion section.

Abstract has been rewritten according with your suggestions.

Change the title to "The effect of yeast, sugar and SO2 on the volatile compounds in wine"?

Title has been changed.

In the Results and Discussion section, it is recommended that the effect of SC 1 and SC 5 on volatile compounds be properly highlighted. Highlighting the significance of "LC" in the control group.

Result and discussion section has been rewritten according with your suggestion.

The Results and Discussion section lacks the necessary discussion of the results.

The results has now been discussed in extension

Significant analysis for the Tables should be more clearly.

Tables have now been changed for a better understanding.

The method for identification of the volatiles should be more detail.

Minor changes have been included in relation with the identification of volatiles. There are two references in the material and methods section that described in extent the method used. Also, in supplementary material there is additional information about this point.

The conclusion section also requires careful deliberation to highlight the main purpose and significance of this study.

The conclusion section has been rewritten according with your suggestions.

Reviewer 3 Report

In this manuscript, in the process of wine fermentation, brewing characteristic or metabolic compounds of three yeast strains has been compared, especially aromatic compounds. The authors measured contents of the aromatic series, and studied different flavors in wine-making.

Generally, the complete fermentation and performance evaluation has finished for yeasts, however, some works might be deeply explored if conditions allowed.

The problems are stated as follows:

1) Maybe the word “climate change” is not suitable as the one of “Keywords”.

2) “Three S. cerevisiae yeast strains were used in this work, and one is commercial strain”. So the readers will more concern about the different genetic background of three strains, such as sequence variations in genome-scale, or genetic evolution, and even differential expression regulation of crucial genes in carbon metabolism during wine-making.

This will improve the manuscript to reach a certain theoretical depth.

3) “Sterilized musts containing 220 and 250 g/L of glucose and fructose (equimolecular) were used”.

Why the authors use the concentration of 220 and 250 g/L? Pls explain the reason for given concentrations

4) For the tolerance assay of SO₂, how about dotted plate assay in YPD medium with inhibitor’s addition? It is better to intuitively characterize cell tolerance, as well as physiological or biochemical indicators of yeasts with the addition of 1-2 figures.

5) References should be updated in recent 5 years, some cited papers are from the 1980s, and few paper that cited from the year 2019-2021.

6) If, adding one synthetical figure (Data and information presentation of the Fig1./ Fig2./ Fig3. should be integrated) for comprehensive understanding for this part of work are recommended.

Finally, the language of manuscript should be polished.

Author Response

Generally, the complete fermentation and performance evaluation has finished for yeasts, however, some works might be deeply explored if conditions allowed.

The problems are stated as follows:

1) Maybe the word “climate change” is not suitable as the one of “Keywords”.

The keyword has been deleted.

This will improve the manuscript to reach a certain theoretical depth.

The aim of the manuscript was to test the yeast in enological conditions. Nowadays we are analyzed the proteome, but this will be the objective of future papers.

3) “Sterilized musts containing 220 and 250 g/L of glucose and fructose (equimolecular) were used”.

Why the authors use the concentration of 220 and 250 g/L? Pls explain the reason for given concentrations.

In recent years, the increased temperatures have given rise to increasing the sugar contents of the grape. 220 g/L of sugar equals to 12,9-13% (v/v) of ethanol, a normal content for wine under typical conditions of grape maturation. 250 g/L equals to 14,7% of ethanol a content frequently reached in the last years due to the increased temperatures reached during maturation. A new paragraph explaining this fact has been included in the revised version.

4) For the tolerance assay of SO2, how about dotted plate assay in YPD medium with inhibitor’s addition? It is better to intuitively characterize cell tolerance, as well as physiological or biochemical indicators of yeasts with the addition of 1-2 figures.

Yeasts are known to be affected in greater or lesser extent by SO2. The grade of affectation is not the aim of the paper but to test the previously selected yeast in oenological conditions and to compare them with a commercial yeast whose main characteristic is its capability of ferment must with high sugar contents. In future works it could be taken into account.

5) References should be updated in recent 5 years, some cited papers are from the 1980s, and few paper that cited from the year 2019-2021.

We have added updates reference section.

6) If, adding one synthetical figure (Data and information presentation of the Fig1./ Fig2./ Fig3. should be integrated) for comprehensive understanding for this part of work are recommended.

The results and discussion section has been rewritten. We hope that at this point figures are more comprehensive.

Reviewer 4 Report

The manuscript of Martín-García et al. reports the effect three strains of Saccharomyces cerevisiae yeast in final volatile wine composition. For that purpose, 3 pre-selected yeast strains and one commercial strain (control) were pre-inoculated into YPD medium at 28 °C for 48 hours at 165 rpm. Fermentations were conducted with an initial cell concentration of 10⁶ cells/mL at 22 °C, in 150 mL of pasteurized red must, containing 220 or 250 g/L of equimolecular concentrations of glucose and fructose, being the effect of SO2 evaluated only in grape-must containing 250 g/L sugar. The experimental work is quite a simple one: fermentations were conducted in duplicate (220 g/L sugar, 250 g/L sugar, and 250 g/L sugar + 75 mg/L SO2) X 3 strains, resulting in total of 18 flasks. General physic-chemical analysis, including pH, titratable acidity, volatile acidity, ethanol, residual sugar, volatile compounds were performed after alcoholic fermentation. Authors concluded that the strains tested displayed a similar fermentative behaviour, and no significant differences on final aroma composition were identified.

This is a very common type work that has already been carried out in different fermentable raw materials, particularly in grape-juice. Authors would enrich the results if sensory and volatile analysis had been carried out. It would be interesting try to find a correlation between volatile substances (namely those present above the perception threshold) and sensory analysis. The discussion should be enhanced. The conclusions are quite insubstantial. The manuscript would benefit if authors could make it enough shorter to be presented as “short communication”.

In general, the manuscript is easy to follow but some parts are not so well presented or discussed therefore it needs improvement. Aspects that need to be explained/modified:

The abstract must include the background, the results and methodology must be clearly presented, avoiding the abbreviations, Sc1, Sc5 and LC (this limits the interest of the reader, who has to go to the body of the manuscript to understand the experimental work), also the objectives of the work must be clearly presented;

Material and Methods:

Fermentations were conducted in 150 mL of pasteurized red must, specify the volume of juice in flasks;

General physic-chemical analysis, were or not performed prior and after alcoholic fermentation. It is not clear in the text. It is recommendable the presentation of the results of titratable acidity, pH, sugar content or ^oBrix before and after alcoholic fermentation. The initial sugar content may give an idea about the ethanol yield of each strain used in the experiments.

Apparently sensory analyses of final wines were not performed. It would be interesting to carry out a descriptive sensory analysis of the wine samples, making possible to demonstrate organoleptic differences between the different yeasts under study. Thus is not clear how figure 1 was constructed, using descriptors of sensory evaluation;

Table 1 and 3: needs improvement, legend, spacing between columns and rows (too may rows and columns) and font size.

Results and Discussion section must be focused in what really happened, comparing the difference among the experiments but avoiding overlapping and unnecessary repetitions.

Moreover, conclusions must highlight the novelty of the work.

Author Response

The manuscript of Martín-García et al. reports the effect three strains of Saccharomyces cerevisiae yeast in final volatile wine composition. For that purpose, 3 pre-selected yeast strains and one commercial strain (control) were pre-inoculated into YPD medium at 28 °C for 48 hours at 165 rpm. Fermentations were conducted with an initial cell concentration of 106 cells/mL at 22 °C, in 150 mL of pasteurized red must, containing 220 or 250 g/L of equimolecular concentrations of glucose and fructose, being the effect of SO2 evaluated only in grape-must containing 250 g/L sugar. The experimental work is quite a simple one: fermentations were conducted in duplicate (220 g/L sugar, 250 g/L sugar, and 250 g/L sugar + 75 mg/L SO2) X 3 strains, resulting in total of 18 flasks. General physic-chemical analysis, including pH, titratable acidity, volatile acidity, ethanol, residual sugar, volatile compounds were performed after alcoholic fermentation. Authors concluded that the strains tested displayed a similar fermentative behaviour, and no significant differences on final aroma composition were identified.

In general, the manuscript is easy to follow but some parts are not so well presented or discussed therefore it needs improvement. Aspects that need to be explained/modified:

Abstract has been rewritten according to your suggestion-

Material and Methods:

Fermentations were conducted in 150 mL of pasteurized red must, specify the volume of juice in flasks;

“Fermentations were conducted in 250 mL flask containing 150 mL of grape must”. This sentence has now been included in the text.

General physic-chemical analysis, were or not performed prior and after alcoholic fermentation. It is not clear in the text. It is recommendable the presentation of the results of titratable acidity, pH, sugar content or oBrix before and after alcoholic fermentation. The initial sugar content may give an idea about the ethanol yield of each strain used in the experiments.

The initial sugar contents of the must were indicated in material and methods section (220 and 250 g/L). Tables haves been remake for a better understanding.

The values of the oenological variables prior to fermentation has now been include in the text.

Due to the low volume of wine it was not possible to perform an organoleptic analysis. But we can know some olfactory attributes by grouping the aroma compounds in aromatic series. This procedure is described now in material and methods section. Aromatic series can help us to differentiation wines according to the aroma descriptors of the volatile analyzed and ultimately the contribution of individual yeast to wine aroma. Figure 1 was constructed with the aromatic series obtained as described in material and methods section.

Table 1 and 3: needs improvement, legend, spacing between columns and rows (too may rows and columns) and font size.

Tables 1 to 3 has been changed for a better understanding of them.

Results and Discussion section must be focused in what really happened, comparing the difference among the experiments but avoiding overlapping and unnecessary repetitions.

The results and discussion section has been rewritten for a better understanding.

Moreover, conclusions must highlight the novelty of the work.

Conclusion section also has been rewritten.

Reviewer 5 Report

The manuscript investigates the contribution to the aromatic profile of selected strains to be used for modulating the organoleptic characteristics of wine. Although the evaluation of the volatile compounds and aroma profile is of interest, in this work there are several concerns regarding to microbiological methods and oenological parameters.

The criteria of selection of the strains used and fermentation conditions have been little described:

- Data about selection criteria to choose Sc1 and Sc5 should be reported. Where were this strains selected? Is it from spontaneous fermentation in the winery or in the laboratory under aseptic conditions? Variety of grapes?

- Data about the red must used in the study should be indicated. Is it the same variety from which the yeasts used were isolated? Was it pasteurized or sterilized? It is not the same concept and may affect microbial activity.

- In line 111, “The fermentation was considered finished when the observed mass loss was equal to that of a yeast-free control flask only containing must”. If there is not inoculation of yeasts, no changes in mass should be detected neither in the beginning or in the end of fermentation. The end of fermentation should correspond to no changes in the mass of the individual flasks.

In the results and discussion section:

- Fermentation curves must be represented. The statement “No remarkable differences…” should be justified as Table S2 shows different values. Difference in fermentation kinetics should be expected, as fermentation with isolated and commercial strains was carried out during different periods of time. This statement is again repeated in conclusions (line 441), “…fermentative behaviour similar to….” Please, more precise data should be shown to justify this.

- Commercial yeast does not finished fermentation (RS data) although it was chosen for its good fermentative capacity. How do you explain this? If the control used is not able to finish the fermentative process it is not an appropriate control. Why do you considered fermentation finished before Sc1 and Sc5?

- How can you explain equal ethanol contain for the three yeasts but significant differences in residual sugars? The results should be discussed in depth.

Author Response

The criteria of selection of the strains used and fermentation conditions have been little described:

Sc1 and Sc5 yeast strain were selected by the collaborative institute “Institut Català de la Vinya i el Vi (INCAVI), Barcelona, Spain” as a part of a research project financed by the Spanish government. The yeasts were isolated from Vitis Vinifera sylvestris, according to their ability, among other parameters, to ferment must with high sugar contents. Both yeast strains were supplied by the INCAVI. A paragraph detailing this fact will be included in the text.

Must was pasteurized. Data about the supplier has been included. The aim is only to test the effect of the studied factors son the rest of variables such as must were equal for all the experiments.

Of course, no changes in mass should be detected in must with no inoculation, but in other assays we detected a loss of weight due to the humidity conditions of the chamber where the fermentations were carried out. So as a control we always included a flask with only must. However, to avoid confusion de sentence will be changed.

In the results and discussion section:

Of course, we provide confusing information regarding to the fermentation kinetic. Figure S1 are now included in supplementary material and a better explanation has been included in the text.

We choose Lalvin Clos because is a commercial yeast that is characterized, according to the description given by the supplier, by its capacity of ferment must with high sugar concentration. As it has now been explained in the text the low content in assimilable nitrogen of the must could explain that the commercial yeast was unable to finish fermentation. However, wild yeast finish fermentation. So, this yeast can be an alternative to ferment must with low nitrogen content. On the other hand, as has now been best described in material and methods section “Fermentation was considered finished when no difference of weight for a given flask was observed in 48 hrs.”

- How can you explain equal ethanol contain for the three yeasts but significant differences in residual sugars? The results should be discussed in depth.

As can be seen in table 1, ethanol content depends on the initial sugar concentration and of the yeast strain. The same is true for residual sugars, although from an oenological point of view only the residual sugar content is relevant when the concentration is above 5 g/L. Below this concentration wines is considered dry and no microbial contamination are expected (wine is considered microbiologically stable). The differences in ethanol, for the same content in sugar, could be that different yeast show a different conversion ratio of sugar to ethanol. This fact has now been explained in the text.

Reviewer 6 Report

The manuscript evaluated the impact of yeast strain and sulphur on the wine characteristics, giving a special focus on volatile compounds. The manuscript brings interesting information for the wine making science, elucidating that volatile compounds depend significantly on the used yeast strain, instead of the presence of sulphur dioxide. However some important point need to be reviewed:

· An English revision is necessary

· Abstract needs substantial improvement. As it is, it is difficult to comprehend. A scientific abstract must provide background, methodology, results and main conclusions, setting clear the novelty of the work.

· Introduction: authors bring a great background about the changes in grapes composition due to the climate change and the importance of studying its impact on wine quality. However, this is not much explored in the manuscript. Sugar content is only varied from 220 to 250 g/L, which cannot be considered an evaluation of sugar concentration impact on the wine. The two isolated saccharomyces yeasts capacity to ferment high sugar loads in the presence of sulphur is much more explored than sugar concentration. In this sense, introduction is not appropriate and must be revised to be in accordance with the manuscript content.

Please perform an english revision

Author Response

An English revision is necessary

English has been revised carefully.

Abstract needs substantial improvement. As it is, it is difficult to comprehend. A scientific abstract must provide background, methodology, results and main conclusions, setting clear the novelty of the work.

Abstract has been rewritten according with your suggestion.

Introduction: authors bring a great background about the changes in grapes composition due to the climate change and the importance of studying its impact on wine quality. However, this is not much explored in the manuscript. Sugar content is only varied from 220 to 250 g/L, which cannot be considered an evaluation of sugar concentration impact on the wine. The two isolated saccharomyces yeasts capacity to ferment high sugar loads in the presence of sulphur is much more explored than sugar concentration. In this sense, introduction is not appropriate and must be revised to be in accordance with the manuscript content.

Although it seen that a variation between 220 and 250 g/L is a small variation, since a oenological and microbiological point of view is quite relevant. 250 g/L is nowadays a usual concentration of sugar in must due to the increase of temperatures during grape maturation. Many yeast strains cannot deplete completely the sugar due to osmotic stress and fermentation process stop. Changes in yeast metabolism due to the increasing sugar concentration will be included in the introduction and result and discussion sections.

Round 2

Reviewer 2 Report

Accept.

Author Response

Thank you for considering our paper as appropriate for publication

Reviewer 3 Report

No.

Author Response

Thank you for your comments.

New paragraph and references have been included in the introduction section regarding to the influence of sugar concentration in wine composition.

Major volatile compounds analysis has been deeper described.

Reviewer 4 Report

It is difficult to follow a fully marked text. However, it appears that the authors have answered most of the questions posed by the reviewers. I still think that, given the limited novelty of this paper, it could be accepted as a short communication.

Author Response

We have included in the introduction section new paragraphs and references to better explain the influence of sugar content in wine composition.

Thank you for your suggestion. We have made the changes below described:

Major volatile aroma compounds determination has been deeper described.

At this moment is difficult to modify the experimental design

References were updated in the previous revision.

We also believe, that the number of analyses conducted, the quantity of determined compounds, and the data treatment performed are more comprehensive than what would be expected for a short communication.

Reviewer 5 Report

The manuscript has been improved.

Author Response

Thank you for considering our paper as appropriate for publication