Aroma Profiles of Vitis vinifera L. cv. Gewürztraminer Must Fermented with Co-Cultures of Saccharomyces cerevisiae and Seven Hanseniaspora spp.

Round 1

Reviewer 1 Report

This study reports the oenological potential of seven NSY Hanseniaspora strains co-inoculated with SY strain, in regard to fermention curves and aroma compounds production. All the methods are well described and seem to be strong enough to produce significant results. HOwever, litterature and results are not always well commented and documented and must be revised:

line 362- I could not find this publication. Is it already published? Please give more references about the subject in wine.

line 136- please modify Polydimethylsiloxane and Divinylbenzene fiber

line 223- Can the authors explain why they did not observe a decrease in ethanol contents while it is a consequence of co-inoculation particularly highlighted in the literature.

line 282- Authors explain that they give strong evidence about decrease of citronellol due to its esterification to citronellol acetate. However, the increase of citronellol acetate's peak intensity is very small, when compared to the decrease of citronellol’s peak intensity (divided by 4). It seems to me that esterification is not the only reason explaining the decrease of citronellol. In addition, authors report a decrease of concentration in the co-innoculated fermention with H. guilliermondii., but when they tested this strain alone with citronellol, they observe an increase in its concentration. This result shows that this strain can produce citronellol but this production is affected when co-innoculated with S.C. Can authors comment and explain this phenomenon. Results and discussions about citronellol must be clarified.

line 285- The PCA description and discussion must be improved in order to give significant additional information to this study.

Author Response

Fermentation-2120691

Response to the Reviewer’s comments

We would like to thank the reviewers for their insight comments and suggestions. We have addressed all the comments below (in red). We have altered the main text in red.

Reviewer 1

This study reports the oenological potential of seven NSY Hanseniaspora strains co-inoculated with SY strain, in regard to fermentation curves and aroma compounds production. All the methods are well described and seem to be strong enough to produce significant results. However, literature and results are not always well commented and documented and must be revised:

Comment 1

Line 362- I could not find this publication. Is it already published? Please give more references about the subject in wine.

The manuscript was recently accepted and has a DOI, but the journal has not yet published the final version online. We have added “in press”. We have also added two more relevant references, but if the reviewer has any suggestions, he/she is welcome to suggest appropriate references.  (line 31 and line 367)

Comment 2

Line 136- please modify Polydimethylsiloxane and Divinylbenzene fiber

This has been corrected (lines 136-137)

Comment 3

line 223- Can the authors explain why they did not observe a decrease in ethanol contents while it is a consequence of co-inoculation particularly highlighted in the literature.

There are many conflicting findings on this topic of alcohol reduction by using NSYs. In theory, the NSY should consume enough sugars and divert carbon away from ethanol production (being non-Crabtree yeasts) which would leave a reduced amount of sugars for Saccharomyces yeast to ferment to ethanol. The reality is often different, and a lot of anecdotal data supported by literature show no meaningful reduction in alcohol levels which is our finding as well. We have reported on this finding previously with H. occidentalis (10.3390/app12146919). In a simultaneous inoculation, this observation of no reduction in is even more expected as the Hanseniaspora cultures (or most other NSYs) are not capable of competing very well with the S. cerevisiae in oenological settings (low pH, low O₂, high sugar, SO₂). We did, however, measure a lot of significant production of many aroma components meaning that the Hanseniaspora are indeed active and had to utilize “some” of the sugars yet “not enough” to have an impact in reducing the alcohol levels.

Comment 4

line 282- Authors explain that they give strong evidence about decrease of citronellol due to its esterification to citronellol acetate. However, the increase of citronellol acetate's peak intensity is very small, when compared to the decrease of citronellol’s peak intensity (divided by 4). It seems to me that esterification is not the only reason explaining the decrease of citronellol. In addition, authors report a decrease of concentration in the co-inoculated fermentation with H. guilliermondii., but when they tested this strain alone with citronellol, they observe an increase in its concentration. This result shows that this strain can produce citronellol but this production is affected when co-inoculated with S.C. Can authors comment and explain this phenomenon. Results and discussions about citronellol must be clarified.

The “fermentation” experiment where we supplemented the must with higher levels of citronellol was only conducted over one day. We wanted to see the “fate” of citronellol i.e., if any citronellol-derived compounds can be observed on the chromatogram after one day (when fewer other aroma compounds are formed which would make the chromatogram visually more complex). We were able to see a peak corresponding to its acetate. This by no means a quantitative approach, but the fact that we were able to see esterification of citronellol just after one day let us to believe this is a valid explanation why citronellol’s level drop compared to the pure S. cerevisiae fermentation. An explanation for the visually bigger peak in the H. guilliermondii fermentation could be due to pipetting errors as small amounts lead to dramatic increase in peak size. We were really focused on the presence of other citronellol derived peaks rather than the exact quantity.

Comment 5

line 285- The PCA description and discussion must be improved in order to give significant additional information to this study.

We have added an additional sentence (line 293-295) to show how the PCA also showcase the acid modulation by H. osmophila. Moreover, we have modified figure 2A to show the separation of the data points of the strain belonging to the FEL and the SEL (lines 293-295).

Reviewer 2 Report

see attached file

Comments for author File: Comments.pdf

Author Response

Fermentation-2120691

Response to the Reviewer’s comments

We would like to thank the reviewers for their insight comments and suggestions. We have addressed all the comments below (in red). We have altered the main text in red.

Reviewer 2

A generally well performed study, written by renowned authors, easy to read.

Comment 1

Title: Assessing the aroma profiles of seven Hanseniaspora spp. in a simultaneously inoculated mixed-culture fermentation of Vitis vinifera L. cv. Gewürztraminer must could more concisely read

Aroma profiles of Vitis vinifera L. cv. Gewürztraminer must fermented with co-cultures of Saccharomyces cerevisiae and seven Hanseniaspora spp.

The original title might be misunderstood: Were mixed cultures of two (or more?) Hanseniaspora strains inoculated? Para 2.2 Microvinification: Here would be the place to state, which strains were simultaneously inoculated.

We thank the reviewer for the comment, and we have subsequently changed the title to their suggestion.

Comment 2

P 3 “polydimethylsiloxane and divinylbenzol fibre” Is somewhat misleading; the SPME fibre is coated with polydimethylsiloxane, which is crosslinked with divinylbenzol to increase chemical stability. The authors should be aware that HS-SPME is a non-quantitative “quick and dirty” method. However, you list microgram concentrations with two digits after the point. I think the digits after the points should be omitted: abc ± xy. HS-SPME does not deliver this level of precision. Errors of SPME arise from matrix effects, from the different partial pressures of the aroma compounds in the gas phase, from different adsorption coefficients on the fibre and displacement of minor volatiles by major or more tightly binding ones. The use of internal standards and calibration curves is only a weak remedy. Solvent extraction and concentration of extracts by rectification has been established as the gold standard years ago (Schreier, Tressl, Drawert, Rapp) – but is of course more laborious.

We thank the reviewer for their insight on this method. We have removed the digits after the comma for the compounds measured in the mg/L range in Table 2. We have rewritten the passage in the materials and methods Line 136-137.

Comment 3

Fig. 3 Something must have been confused here. In Table 2, H. guillermondii showed the lowest citronellol concentration (9.5 mg/L), but an overloaded peak is shown in the Figure, whereas H. uvarum showed a somewhat higher concentration (13.9), but only a tiny peak in the displayed chromatogram. Anyway, the chromatograms do not add new information and should be omitted. Data for citronellyl acetate, emphasized in the conclusion, are missing in Table 2.

The chromatograms shown in Fig 3 were from ”fermentations” conducted with must supplemented with higher levels of citronellol. This was shortly explained in Lines 105-107. The fermentations were only conducted over one day followed by a subsequent analysis to minimize the formation of other aroma compounds. We were interested to see if any “citronellol-derived” compounds are formed, and we could detect citronellyl acetate after one day which we believe is at least one explanation for the reduction in citronellol we observe through the entire fermentation with H. uvarum and H. guilliermondii additions. The idea was not to quantify (as we did not have a citronellyl acetate at high level of purity), but just to visually confirm a “fate” of citronellol. In addition, the terpenes are also quantified while being run in SIM-mode as explained in the materials and methods thus we were only looking at “expected” ions and citronellyl acetate is not included in our analysis. Thus, we would like to keep Fig 3 as visual proof of citronellol conversion.

Comment 4

What I missed most is a sensory evaluation. High levels of ethyl acetate, present in most of the experimental wines, impart an undesirable glue-like “organic solvent” odour. Authors should add sensory data, at least in a descriptive way, and comment on possible sensory improvements by the co-inoculates. Was there a benefit?

Sensorial evaluations are arguably the most important aspect of fermentation experiments. With microvinifications of ~150 ml, not enough volume was available to conduct a proper sensorial evaluation and we were focussed on how the different Hanseniaspora yeast (including unexplored Hanseniaspora spp) changed the must-to-wine composition analytically. As mentioned, (Line 256), ethyl acetate levels above 150 mg/L as measured with the FEL-inoculations would most likely result in the perception of these unpleasant solvent-like notes and would definitely not make for an acceptable wine (some even exceeded double that amount). Nevertheless, the yeast belonging to the SEL (H. osmophila and H. occidentalis actually did produce levels that might be more acceptable.

Comment 5

Language: Use past tense in all reporting paragraphs. “Which”, “whereas” etc. should be preceded by commas throughout the text to indicate the subordinate clause.

We have added commas to indicate subordinate clauses throughout the manuscript (Line 19, 183, 207). Some sentences written in the present tense were corrected (line 121, 134, 152-154). Minor grammatical errors were also noted (line 148, line 182).

Round 2

Reviewer 1 Report

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