Improving Aroma Complexity with Hanseniaspora spp.: Terpenes, Acetate Esters, and Safranal

Round 1

Reviewer 1 Report

Dear authors, 

thank you for submitting the valuable manuscript with some new insights into the field. I wrote the comments and corrections directly into the text attached during the reading. Most of them consider language and grammar.

The other questions are related to wine production- there are some missing information about the cultivar (please write the name of it correctly in the text), like origin and main properties.

In the part of M&M, put the vintages clearly, together with the information about the place of harvest and production.

It is not clear the dosage of yeast applied or the time of sequential inoculation (why 6^th day ?). What were the parameters in the must when you inoculated the Sc yeast? Did you count the viable Hv or Ho cells? 

Can you explain the separating of Terpenes and the term "Analysis of freshness aroma compounds"- it is not defined what is the criteria for fresh aroma ? The mentioned compounds did not exceed the perception threshold. Did you calculate the Odor threshold values for the compounds? The presentation of Limonene concentration in Fig. 3 is confusing regarding the text (rows 347-350).

The whole text needs language editing and standardization of signs and units. Please include details regarding the statistical analysis in the tables and figures description.

Comments for author File: Comments.pdf

Author Response

Dear authors,

thank you for submitting the valuable manuscript with some new insights into the field. I wrote the comments and corrections directly into the text attached during the reading. Most of them consider language and grammar.

Thank you very much for your excellent review of the manuscript. I have corrected all the errors that you pointed out in the attached text. All corrections suggested by Reviewer 1 have been marked in yellow.

Reviewer 1 suggested the translation of the authors' affiliations into English. However, these affiliations have not been changed, as it is important to maintain the original name for the quality indices of our institutions.

The other questions are related to wine production- there are some missing information about the cultivar (please write the name of it correctly in the text), like origin and main properties.

In the part of M&M, put the vintages clearly, together with the information about the place of harvest and production.

Further information regarding the origin of the grapes has been added in Section 2.2 as follows: The white must used in this study was made from Vitis vinifera Var. Albillo Mayor grapes. This grape was cultivated in the Bodegas Comenge  vineyards located in the D.O. Ribera del Duero in the area of Curiel de Duero, Valladolid, Spain. All fermentations were made with grapes from the 2021 vintage.

It is not clear the dosage of yeast applied or the time of sequential inoculation (why 6th day ?). What were the parameters in the must when you inoculated the Sc yeast? Did you count the viable Hv or Ho cells?

We agree with Reviewer 1; more information needs to be included regarding inoculations and fermentations. The decision for sequential inoculation on day 6 is determined by previous trials with these yeast strains at the laboratory scale. Although optical microscopic observations were made to identify yeasts by their morphology during fermentation, no viable cell count was performed.  During this trial, we tried to apply a simple methodology that would be realistic on an industrial scale. For this reason, the fermentation control was not as exhaustive as in previous laboratory-scale trials. Section 2.2 has been modified as follows:

Fermentations were performed in triplicate in 150 L stainless steel barrels. The Hanseniaspora genus fermentations (Ho and Hv) were inoculated with 5 L (≃3%) of liquid inoculum prepared in YPD media at a population of 7-log CFU/mL. The musts were fortified with 100 mg/L nutrient. After 6 days, these fermentations were sequentially inoculated with Fermivin 3C (S. cerevisiae) in a 20 g/hL dosage as active dried yeast recommended by the manufacturer (Oenobrands SAS, Montpellier, France). The control fermentations (Sc) were inoculated only with Fermivin 3C (20 g/hL). Fermentations proceeded until dryness, and the evolution was monitored daily by measuring density and temperature. Temperature button sensors. Fermentations proceeded until the residual sugars were lower than 2 g/L.

Can you explain the separating of Terpenes and the term "Analysis of freshness aroma compounds"- it is not defined what is the criteria for fresh aroma? The mentioned compounds did not exceed the perception threshold. Did you calculate the Odor threshold values for the compounds? The presentation of Limonene concentration in Fig. 3 is confusing regarding the text (rows 347-350).

In relation to freshness compounds, we agree with Reviewer 1 that more explanation is needed to understand the compounds analysed. The perception of freshness is related to some aromas such as mint and liquorice in red wines and some white wines with aromas of peppermint and fresh hay. The criteria for the reporting of the results was that the compounds had this type of aroma and were analysed by the method described in Section 2.7. Therefore, the results presented as "freshness aroma compounds" are not compounds of the same chemical nature. To provide further clarification of the compounds analysed, Section 2.7 has been supplemented as follows:

The freshness aroma compounds analysed were: limonene, menthone, menthol, pulegone, carvone, mintlactone, piperitone, eucalyptol, 4-heptenol, methyl salicylate, ethyl salicylate, ethyl benzoate, safranal, cis-hexenol, trans-hexenol, and n-hexenol.

The thresholds of perception of freshness aroma compounds have not been calculated by us. All of them have been consulted in the literature and are cited in Section 3.5. The mistake referred to by Reviewer 1 in lines 347-350 has been corrected as follows: Wines fermented with Ho showed limonene concentrations of around 1.08 µg/L; these values are statistically higher than the other fermentations.

The whole text needs language editing and standardization of signs and units. Please include details regarding the statistical analysis in the tables and figures description.

All units in the text have been standardised according to the reviewer's suggestion. The language has been revised and edited by a specialised company (marked in pink). The tables and figures have been supplemented with details about the statistical analysis.

Reviewer 2 Report

The manuscript entitled “Improving aroma complexity with Hanseniaspora spp: terpenes, acetate esters and safranal” assessed some oenological parameters, fermentative volatile compounds, and a non-targeted NMR-based metabolomics of wines from the same variety and geographical origin, but different inoculation schemes and vintages. Results showed significant differences due to the presence of the Hanseniaspora species and a good resolution of the methods used with the aim to differentiate the wines. Nevertheless, an extensive revision of the English language is necessary, preferentially by a native speaker, as many sentences are a bit confusing due to several grammatical mistakes. Furthermore, some specific points to be addressed are suggested below:

1.     Lines 22-23: Interestingly, it should be noted that the compound safranal was identified only in the H. vineae wines.

2.     Line 45: “aromatic varieties” is normally used rather than “terpene varieties”, or possibly “high-terpene varieties”

3.     Lines 77-79: it is not clear how this sentence connects with the rest, is it about wine color or body?

4.     Line 87: please specify which strain of Hv was used

5.     Lines 17, 26, 82, 94, 104: in the abstract you cite co-inoculation, but in the aim and in the methods it is specified that sequential fermentations were performed

6.     Lines 98-101: are you describing the grape must from the vintage 2019 or 2021? Was the initial sugar, pH, nutrients similar in both years? Did you use exactly the same fermentation protocol?

7.     Line 104: brand of nutrients is missing

8.     Lines 104-105: in the fermentation SC, did you inoculate S. cerevisiae at time 0 or after 6 days? In the curves of Figure A1, it is clear that a higher sugar consumption was present in SC in the first 6 days, which would be caused by the native yeasts, in case S. cerevisiae was not inoculated at the beginning. Did you check for the presence of native yeasts in the grape must? Did you consider the possibility of a cross contamination with Hanseniaspora between tanks?

9.     Line 158, 345: please define and list the “freshness aroma compounds”

10.  Lines 179-180: why did you use the wines from two vintages only for NMR analysis, and not the other parameters? The must characteristics and fermentation conditions of both vintages should be clearer explained in the Material and Methods section.

11.  Line 231 and elsewhere: please use capital L when referring to liters

12.  Lines 231-237, 447-448: Saccharomyces was inoculated in all fermentations. Thus, if polysaccharides were higher in SC, not only Hanseniaspora species released less polysaccharides than S. cerevisiae, but also caused a reduction on the polysaccharide release by S. cerevisiae. Please comment on and complement the text accordingly

13.  Lines 282-284: diacetyl was significantly higher in SC. Aren’t these compounds more associated with citrate metabolism during malolactic fermentation?

14.  Lines 294-295: this sentence is not in accordance with the cited reference (Swiegers, Bartowsky, Henschke & Pretorius, 2005)

15.  Lines 302-305: please cite the compounds and concentrations in the text. It is not possible to claim that certain wines are fruitier if you did not carry out sensory analysis. Esters could contribute to the fruity character, but there are other factors involved

16.  Did you measure the concentration of acetic acid?

17.  Table 1: please reorganize the compounds in their respective families, like in Table 2

18.  Lines 349-350: looking at Figure 3a, concentration of limonene in HO is between 0.6-0.8 mg/L, statistically lower than HV and similar to SC.

19.  Lines 359-360: in the Figure 3c it is indicated a threshold of 400 instead of 1550 mg/L

20.  Lines 364-365, 456-457: what could be the olfactory impact of safranal in the aroma of Albillo Mayor? Is it associated with freshness? Why would it significantly increase the complexity of wines?

21.  Lines 397-407: the name Candida zemplinina was updated to Starmerella bacillaris, and Saccharomyces bayanus ex uvarum is not legitimate, it should be either S. bayanus or S. uvarum, please refer to the current names

22.  Lines 409-412: it is not clear why the two vintages of wines were only examined in this analysis, and not in the previous ones. It is also the first time that Ribera del Duero is mentioned as the geographical origin. What happened to the fermentation with H. opuntiae in 2019?

23.  Lines 450-455: did you perform a PCA with data from the fermentative volatile compounds?

24.  Figure 1A: Please add the standard deviations to each curve. In the first stage, it looks like SC and HO had similar kinetics

Author Response

The manuscript entitled “Improving aroma complexity with Hanseniaspora spp: terpenes, acetate esters and safranal” assessed some oenological parameters, fermentative volatile compounds, and a non-targeted NMR-based metabolomics of wines from the same variety and geographical origin, but different inoculation schemes and vintages. Results showed significant differences due to the presence of the Hanseniaspora species and a good resolution of the methods used with the aim to differentiate the wines. Nevertheless, an extensive revision of the English language is necessary, preferentially by a native speaker, as many sentences are a bit confusing due to several grammatical mistakes. Furthermore, some specific points to be addressed are suggested below:

Thank you very much for your excellent review of our manuscript. All grammatical errors have been checked and edited by a company specialising in this field. All modifications suggested by Reviewer 2 are marked in the text in blue.

Lines 22-23: Interestingly, it should be noted that the compound safranal was identified only in the H. vineae wines.

Lines 22-23 have been modified according to the reviewer's suggestion.

Line 45: “aromatic varieties” is normally used rather than “terpene varieties”, or possibly “high-terpene varieties”.

Line 45 has been corrected according to the reviewer's suggestion.

Lines 77-79: it is not clear how this sentence connects with the rest, is it about wine color or body?

The text refers to the body and not to the colour. The absorbance of intracellular components at wavelengths in the ultraviolet (UV) can be an indication of a higher release of molecules in hydroalcoholic solutions contributing to changes in the mouth feel or body. In an attempt to clarify lines 77-79, the sentence has been modified as follows:

A higher absorbance has been observed at 260 nm and 280 nm compared with other Saccharomyces and non-Saccharomyces species [14]. Absorbance values at these wavelengths can be used to estimate the nucleic acid and protein content in hydroalcoholic solutions. This might be related to the higher elution of intracellular components [27] compared to Sc. This sensory effect is clear and it has been observed in the evaluation by tasting panels.

Line 87: please specify which strain of Hv was used.

Line 87 has been completed according to Reviewer 2's suggestion.

Lines 17, 26, 82, 94, 104: in the abstract you cite co-inoculation, but in the aim and in the methods it is specified that sequential fermentations were performed

In this study, only sequential fermentations were performed. The cite “co-inoculation” in the abstract is a mistake, and it has been corrected.

Lines 98-101: are you describing the grape must from the vintage 2019 or 2021? Was the initial sugar, pH, nutrients similar in both years? Did you use exactly the same fermentation protocol?

Lines 90-101 only concern fermentations carried out in the 2021 vintage year. The previous analysis of this must was only a basic analysis in the winery, which included density, pH and total acidity. Wines fermented in 2019 were included only in the nuclear magnetic resonance analysis. This has been described in the Materials and Methods Section 2.8. Nuclear magnetic resonance spectroscopy, to clarify that this sole analysis was performed on both vintages. The fermentative protocol is the same for both fermentations and is described in Reference 14.

Line 104: brand of nutrients is missing

The brand name of the nutrient has been added according to the reviewer's suggestion.

Lines 104-105: in the fermentation SC, did you inoculate S. cerevisiae at time 0 or after 6 days? In the curves of Figure A1, it is clear that a higher sugar consumption was present in SC in the first 6 days, which would be caused by the native yeasts, in case S. cerevisiae was not inoculated at the beginning. Did you check for the presence of native yeasts in the grape must? Did you consider the possibility of a cross contamination with Hanseniaspora between tanks?

Sc fermentations were inoculated at time 0 and used as controls in this study. These controls were also inoculated with Sc on day 6 to add the same volume in all triplicates. The presence of indigenous yeasts was not verified, but periodic optical microscopic observations indicated the correct implantation of yeasts of the Hanseniaspora species.

Line 158, 345: please define and list the “freshness aroma compounds”

Two sentences have been added to the manuscript as follow:

The perception of freshness in white wines is related to the aromas of peppermint and fresh hay. In this respect, the freshness aroma compounds were analysed by gas chromatography with thermal desorption coupled to tandem mass spectrometry GC-MS/MS.

The freshness aroma compounds analysed were: limonene, menthone, menthol, pulegone, carvone, mintlactone, piperitone, eucalyptol, 4-heptenol, methyl salicylate, ethyl salicylate, ethyl benzoate, safranal, cis-hexenol, trans-hexenol, and n-hexenol.

Lines 179-180: why did you use the wines from two vintages only for NMR analysis, and not the other parameters? The must characteristics and fermentation conditions of both vintages should be clearer explained in the Material and Methods section.

This work is a study of the fermentations carried out in the 2021vintage, and their characteristics are described in Section 2.2 of the manuscript. However, we considered that including 2019 results from the same Hv and Sc strains with the same grape variety and geographical area in the NMR study better clarifies the observation by clustering the results to differentiate fine features between wine samples. The characteristics of the fermentations for the 2019 vintage have already been described in a previous publication, which is attached below (Reference 14).

[14] Del Fresno, J. M., Escott, C., Loira, I., Herbert-Pucheta, J. E., Schneider, R., Carrau, F., ... & Morata, A. (2020). Impact of Hanseniaspora vineae in alcoholic fermentation and ageing on lees of high-quality white wine. Fermentation, 6(3), 66.

Line 231 and elsewhere: please use capital L when referring to liters

This mistake has been corrected throughout the manuscript.

Lines 231-237, 447-448: Saccharomyces was inoculated in all fermentations. Thus, if polysaccharides were higher in SC, not only Hanseniaspora species released less polysaccharides than S. cerevisiae, but also caused a reduction on the polysaccharide release by S. cerevisiae. Please comment on and complement the text accordingly.

We agree with Reviewer 2. The manuscript has been supplemented in the “Results” and “Conclusions” sections as follows:

These results seem to indicate that Hanseniaspora yeasts have lower polysaccharide release kinetics than Saccharomyces yeasts, with no significant differences between Hv and Ho species. Since on the sixth day, S. cerevisiae was inoculated into the Hv and Ho barrels, it appears that the interaction of yeasts of the genera Hanseniaspora and Saccharomyces results in a lower release of polysaccharides from the latter.

The cell wall polysaccharide release capacity was lower in Hanseniaspora genus yeast than in Sc. In addition, it appears that these species interact with the polysaccharide transfer capacity of S. cerevisiae yeast.

Lines 282-284: diacetyl was significantly higher in SC. Aren’t these compounds more associated with citrate metabolism during malolactic fermentation?

None of the white wines carried out malolactic fermentation, so we assumed that the diacetyl contents were produced during the alcoholic fermentation of the yeasts studied.

Lines 294-295: this sentence is not in accordance with the cited reference (Swiegers, Bartowsky, Henschke & Pretorius, 2005)

The sentence is in accordance with the cited reference; the aromatic descriptor of the compound is listed in Table 1 under the name of 2-Phenylethyl alcohol (Swiegers, Bartowsky, Henschke & Pretorius, 2005).

Lines 302-305: please cite the compounds and concentrations in the text. It is not possible to claim that certain wines are fruitier if you did not carry out sensory analysis. Esters could contribute to the fruity character, but there are other factors involved

We agree with Reviewer 2, that a sensory analysis in combination with the analysis of volatile compounds would have defined more exactly the fruit character of the wines. However, volatile esters are the compounds that mainly define the fruit character, thus the sentence of the text. Line 302 discusses the total content of esters; we have cited them all together with their concentration, as individual concentrations are shown in Table 1, already mentioned at the beginning of Section 3.3.

Did you measure the concentration of acetic acid?

Acetic acid was not measured in this study. However, one commercial yeast and two previously selected yeasts were used in these fermentations, one of the selection criteria being low volatile acidity production.

Table 1: please reorganize the compounds in their respective families, like in Table 2

Table 1 has been modified according to the reviewer’s suggestion.

Lines 349-350: looking at Figure 3a, concentration of limonene in HO is between 0.6-0.8 mg/L, statistically lower than HV and similar to SC.

Sorry, but these results correspond with HV wines. Finally the sentence has been modified as follows: Wines fermented with Ho showed limonene concentrations of around 1.08 µg/L, these values are statistically higher than the other fermentations.

Lines 359-360: in the Figure 3c it is indicated a threshold of 400 instead of 1550 mg/L

We agree with the Reviewer 2 that there is a mistake in the manuscript, whereas the threshold of perception shown in the figure is correct. This has been corrected in the text.

Lines 364-365, 456-457: what could be the olfactory impact of safranal in the aroma of Albillo Mayor? Is it associated with freshness? Why would it significantly increase the complexity of wines?

Safranal is a powerful aromatic compound that can be associated with the freshness descriptors of wines. Considering that it has been identified at values three times higher than its perception threshold, we consider that it can have a strong impact on the global sensory profile of the wine. Especially in the case of Albillo Mayor wines, which is considered a grape that provides few varietal aromas

Lines 397-407: the name Candida zemplinina was updated to Starmerella bacillaris, and Saccharomyces bayanus ex uvarum is not legitimate, it should be either S. bayanus or S. uvarum, please refer to the current names.

The yeast names have been updated according to the reviewer's suggestion.

Lines 409-412: it is not clear why the two vintages of wines were only examined in this analysis, and not in the previous ones. It is also the first time that Ribera del Duero is mentioned as the geographical origin. What happened to the fermentation with H. opuntiae in 2019?

The D.O. Ribera del Duero is the geographical origin of the grapes used in this study. This information has also been added in Section 2.2 of Material and Methods. The study is based on the fermentations of the 2021 vintage. However, we decided to include 2019 fermentations of the same Hv and Sc strains in the NMR study. The reason was to improve this study by contributing to the same grape variety, different yeast species and vintages. We do not have any data on fermentations with H. opuntiae in the 2019, vintage because this yeast was not used.

Lines 450-455: did you perform a PCA with data from the fermentative volatile compounds?

Yes, a principal component analysis was performed for fermentative volatile compounds. However, no results were obtained that allowed a clear grouping of the different, and it was decided not to include this statistical analysis in the manuscript.

Figure 1A: Please add the standard deviations to each curve. In the first stage, it looks like SC and HO had similar kinetics

Standard deviations have been added to Figure 1A. These values were not added because the 57-day measurements made the figure very complicated to read.

Round 2

Reviewer 1 Report

Dear authors,

thank you for adopting the comments and corrections that you made, which enabled the acceptance of the manuscript in the present form.

Author Response

We thank reviewer 1 for his work, which has significantly improved the manuscript.

Reviewer 2 Report

Thanks for the thorough revision of the manuscript following both reviewers’ suggestions, it greatly improved the text. Nevertheless, I have a few further suggestions regarding information that you put on the rebuttal letter, but could also be added to the text:

-       Lines 108-114: Sc fermentations were inoculated at time 0 and used as controls in this study. These controls were also inoculated with Sc on day 6 to add the same volume in all triplicates. The presence of indigenous yeasts was not verified, but periodic optical microscopic observations indicated the correct implantation of yeasts of the Hanseniaspora species.

-       Lines 298-300: However, no significant differences were identified for quantitatively important compounds in wines such as acetaldehyde, acetoin, or 2,3-butanediol. (As already pointed out in the first-round revision, you showed in Table 1 that diacetyl was significantly different)

-       Lines 310-312: 2-phenylethanol has a positive sensory impact in wines, which is described as ‘rose’-like and ‘floral’ [36]. (As reported by Swiegers, Bartowsky, Henschke & Pretorius (2005), the ester 2-phenylethyl acetate is associated with honey, fruity, and flowery aromas, and it is not said that 2-phenylethanol is the only higher alcohol with positive sensory impact)

-       Lines 320-321: in Sc wines, suggesting that fermentations with Hv could result in wines with more pronounced fruity aromas.

-       Lines 379-380: This compound has a perception threshold in water of 400 μg/L according to [48]. (The perception threshold was not corrected in the revised manuscript)

-       Lines 423-428: (Please update the yeasts’ names in the whole paragraph)

-       Lines 191-192, 432-433, 451-452: (Please you should add a sentence in the Material & Methods and Results to precisely tell that “We do not have any data on fermentations with H. opuntiae in the 2019, vintage because this yeast was not used”)

 

-       Line 524: In this first stage, Ho and Sc wines showed similar kinetics.

Author Response

Thanks for the thorough revision of the manuscript following both reviewers’ suggestions, it greatly improved the text. Nevertheless, I have a few further suggestions regarding information that you put on the rebuttal letter, but could also be added to the text:

Thank you for the new suggestions, all of which are added to the manuscript and highlighted in green.

Lines 108-114: Sc fermentations were inoculated at time 0 and used as controls in this study. These controls were also inoculated with Sc on day 6 to add the same volume in all triplicates. The presence of indigenous yeasts was not verified, but periodic optical microscopic observations indicated the correct implantation of yeasts of the Hanseniaspora species.

The sentence has been added in accordance with the reviewer's suggestion.

  Lines 298-300: However, no significant differences were identified for quantitatively important compounds in wines such as acetaldehyde, acetoin, or 2,3-butanediol. (As already pointed out in the first-round revision, you showed in Table 1 that diacetyl was significantly different)

The compound diacetyl has been removed from the sentence according to the reviewer's suggestion.

Lines 310-312: 2-phenylethanol has a positive sensory impact in wines, which is described as ‘rose’-like and ‘floral’ [36]. (As reported by Swiegers, Bartowsky, Henschke & Pretorius (2005), the ester 2-phenylethyl acetate is associated with honey, fruity, and flowery aromas, and it is not said that 2-phenylethanol is the only higher alcohol with positive sensory impact)

The sentence has been modified in the manuscript in line with the reference cited.

Lines 320-321: in Sc wines, suggesting that fermentations with Hv could result in wines with more pronounced fruity aromas.

The sentence has been modified according to the reviewer's suggestion.

Lines 379-380: This compound has a perception threshold in water of 400 μg/L according to [48]. (The perception threshold was not corrected in the revised manuscript)

The perception threshold has been corrected. I am sorry that this suggestion was not included in the previous version.

Lines 423-428: (Please update the yeasts’ names in the whole paragraph)

Names have been updated throughout the paragraph.

Lines 191-192, 432-433, 451-452: (Please you should add a sentence in the Material & Methods and Results to precisely tell that “We do not have any data on fermentations with H. opuntiae in the 2019, vintage because this yeast was not used”)

Trying to explain the different yeasts used, three sentences have been included in accordance with the reviewer's suggestion:

Lines 195-197: is noted that we do not have any data on fermentations with Ho in the 2019 vintage because this yeast was not used.

Lines 438-439: It is highlighted that Ho yeast was not used in the 2019 vintage and therefore no data is presented.

Lines 445-446: wine samples from the same variety but different fermentation schemes, Sc, Hv and Ho in 2021 vintage, and Sc and Hv in 2019 vintage.

 Line 524: In this first stage, Ho and Sc wines showed similar kinetics.

The mistake has been corrected.