Assessing the Oenological Potential of Nakazawaea ishiwadae, Candida railenensis and Debaryomyces hansenii Strains in Mixed-Culture Grape Must Fermentation with Saccharomyces cerevisiae

Round 1

Reviewer 1 Report

This study focuses on identifying non-Saccharomyces yeast (NSY) that can be co-cultured with Saccharomyces cerevisiae for improving secondary characteristics of wine. As the Authors aptly point out there are several NSY that are currently in use and this study is an extension of these studies and is focused on identifying additional and unique NSY strain(s) from wine samples. This is a well-structured presentation of their results although the presentation can be improved by careful editing. While novelty is lacking, the study identifies a NSY that increases the portfolio of NSY for co-culture with S. cerevisiae for fermentation leading to predictable wine quality.

Specific comments

1. Line 45: NSY is used as an abbreviation of non-Saccharomyces yeast. Due to this, use of “NSY yeast” here and in other places introduces yeast as a redundant term.
2. Line 69: “…plated out on agar plates…” should read as, “… plated on solid medium …”. The samples were plated on solid growth medium and not on plates!
3. Line 92: Should HLPC read as HPLC?
4. Lines 125-128: The selection used here identified two Debaryomyces hansenii. Only one of the two isolates was used in further studies. Please include pertinent information on the similarity of the two isolates. Since the two D. hansenii were isolated from the same wine sample, is it possible that the two are the same?
5. Lines 139-140: Please italicize the organism names.
6. Abstract, line 16: please remove the second “culture” in “…mixed culture culture…”

Author Response

Line 45: NSY is used as an abbreviation of non-Saccharomyces yeast. Due to this, use of “NSY yeast” here and in other places introduces yeast as a redundant term.

This has been corrected. Line 46

Line 69: “…plated out on agar plates…” should read as, “… plated on solid medium …”. The samples were plated on solid growth medium and not on plates!

This was corrected. Line 70

Line 92: Should HLPC read as HPLC?

This was corrected line 93

Lines 125-128: The selection used here identified two Debaryomyces hansenii. Only one of the two isolates was used in further studies. Please include pertinent information on the similarity of the two isolates. Since the two D. hansenii were isolated from the same wine sample, is it possible that the two are the same?

We added a sentence to address this issue (Line 133-135). Their ITS sequence was identical so we assumed the isolates might perform similar and opted to use just one for further experimentation.

Lines 139-140: Please italicize the organism names.

They are now all in italics

Abstract, line 16: please remove the second “culture” in “…mixed culture culture…”

This has been corrected line 16

Reviewer 2 Report

The manuscript entitled “Assessing the oenological potential of Nakazawaea ishiwadae, Candida railenensis and Debaryomyces hansenii strains in a mixed-culture grape must fermentation with Saccharomyces cerevisiae” aimed to investigate non-Saccharomyces strains to be co-cultured with S. ceevisiae in fermentation of grape must. The manuscript is well-written, and the results support the conclusion.

Minor

• The aroma notes to contribute wine flavors could be described, especially on esters and terpenes.
• Ref 13 should be changed to a MEGA paper from Prof Kumar’s group?
• Line 133, punctuation for the coefficient.
• Line 186, did you possibly observe higher concentrations of the corresponding alcohols in Table 2.

Author Response

The aroma notes to contribute wine flavors could be described, especially on esters and terpenes.

We have added this to tables 2 and 3

Ref 13 should be changed to a MEGA paper from Prof Kumar’s group?

This was added line 20

Line 133, punctuation for the coefficient.

This is the from the MEGA software line 138

Line 186, did you possibly observe higher concentrations of the corresponding alcohols in Table 2.

Apart from 2-Methylbutanol in the C. railenensis, no meaningful increase was observed. The higher alcohols are in the mg/L range whereas many of the esters are only in the ug/L range thus a slight breakdown of a specific ester would not contribute significantly to the overall pool of its respective higher alcohol.

Reviewer 3 Report

1. Would help if there is more information presented on any existing applications of N. ishiwadae, e.g. safely used in food related products.
2. Line 139-141: Use italic for the names of the yeasts.

Author Response

Would help if there is more information presented on any existing applications of N. ishiwadae, e.g. safely used in food related products.

We mention the recent paper by Ruiz et al (2019) as that was the first report to our knowledge on N. ishiwadae in food production

Line 139-141: Use italic for the names of the yeasts.

Was corrected

Reviewer 4 Report

This is a well prepared and enjoyable manuscript to read.  The subject matter of including non-Saccharomyces yeast in wine fermentations to develop more unique taste profiles is timely.  I have 2 necessary concerns and several minor suggestions.  The impact of this study is the potential of N. ishiwadae within a co-culture to improve wine flavor.  The major limitations are that is a relatively small study limited to single grape must, three yeast isolates, and 3 co-cultures performed in triplicate.  However, I think it plays well as a brief study with a tight experimental plan and appropriate conclusions.

Mandatory

1. Discussion for paper #21.  Please check on lines 182 and 141 if the citation should be 21 instead of 22.  More to the point, 21 is an important paper and some increases in esters were observed; please broaden the discussion of this paper here. 
2. Impact: please include a small paragraph towards the end of the discussion giving the reader insight into the importance of the shifts in esters in altering the aroma and taste of the wine.  Also, were there other changes that would fundamentally change the taste of the wine?
3. Repeatability: I have found that phenotypes of yeast isolated can vary considerably within a given species.  It might be an interesting follow up to screen further from this species and even possibly expand it within the clade.  However, using a single isolate is enough for this work and I am NOT suggesting additional experiments.  My concern is rather that the yeast might become lost.  Anyway, to provide assurance that the yeast is properly stored away and has been given a proper catalog number in case someone requests it in the future?

Optional  (line number:  comment/corrrection)

1. 16 please check if it should be micro-vinifications  (usually use dash for fermentations)
2. 20: remarkably is probably not the appropriate word choice here.  Perhaps, use “much” instead or else better yet report the % it is decreased.
3. 22: replace fairly unknown with under appreciated or rarely reported [If it is unknown, it is simply unknown]
4. 23: providing increased -> increasing
5. 31: sluggish fermentations.    Add a citation here, even if it is a general review.
6. 35: needs to be more precise.  How about:  to expand on the breath of possible flavor profiles for wines prepared using defined cultures.
7. 67: where were they isolated (grape surface) and when (e.g. month)
8. 71: not necessary to comment further, however, was the panel expert?  Were they looking for just pleasant aromas or aromas associated with esters?  
9. 81: autoclaved Muscaris:  for how long?  I am a bit concerned as autoclaving can cause many chemical changes.  In the field is it pasteurized?   Is filter sterilization an option?  [this relates to future studies]
10. 82: how were cells counted? 
11. 85: Anchor Yeast – was this also grown in liquid culture or added as a dry formulation?
12. 87: no more weight loss was recovered over a 24 hr period.
13. 88: I assume the samples were not stored for very long at 4C.  Would think that -20 might be more suitable.
14. Figure 1: any what to bold the “strain from this study” to make it more evident for the reader.  Otherwise, a very nice tree.
15. 139-143: yeast needs to be italicized
16. 168: shown to enhance both ways of which the yeast can impact aroma.  Please add a citation here.
17. 172-173: delete “including the main organic acids, residual sugar content (only fructose), glycerol, and ethanol.
18. 176 Upon assessing the …
19. 179-182: as discussed above broaden the discussion around citation (I assume?)  21
20. 190-197: line spacing changed.

217:  avoid remarkable, perhaps, try notable or dramatic

Author Response

Mandatory

Discussion for paper #21.  Please check on lines 182 and 141 if the citation should be 21 instead of 22.  More to the point, 21 is an important paper and some increases in esters were observed; please broaden the discussion of this paper here.

There was a mix-up with the referencing, thanks for pointing it out. It has been fixed. We now mention of the previous paper’s report on an increase of ester content. Line 180-181.

Impact: please include a small paragraph towards the end of the discussion giving the reader insight into the importance of the shifts in esters in altering the aroma and taste of the wine.  Also, were there other changes that would fundamentally change the taste of the wine?

We are mentioning this in the Conclusion section and it will form part of a follow-up study. The reality is that the Corona-epidemic has prevented any “proper” science-based tastings in our institute for the foreseeable future so we can now only show analytical data. Line 217-222

Repeatability: I have found that phenotypes of yeast isolated can vary considerably within a given species.  It might be an interesting follow up to screen further from this species and even possibly expand it within the clade.  However, using a single isolate is enough for this work and I am NOT suggesting additional experiments.  My concern is rather that the yeast might become lost.  Anyway, to provide assurance that the yeast is properly stored away and has been given a proper catalog number in case someone requests it in the future?

This is true and although the positive role of NSY in wine is undisputed, the anecdotal evidence of problems of repeatability is sadly not reflected in literature. The word triplicate for most winemaking experiments (including here) means conducting a fermentation in three vessels at the same time. Although perfectly acceptable, a better pursuit would be to conduct fermentations where one follows the next one – but issues like changes in must compositions over time provides a convenient excuse. Our research here is not helping in this regard.

When you google “Nakazawaea” it asks if you made a spelling mistake which illustrates how new/unknown this species is in literature, but together with the recent publication that was published last year on it, we hope it paves the way to more investigations on this yeast and its “family members”. The yeast including the others mentioned in the paper are all part of the Geisenheim yeast collection so when the corresponding author is contacted, the yeast can be provided.

Optional  (line number:  comment/corrrection)

16 please check if it should be micro-vinifications  (usually use dash for fermentations)

We have changed it to micro-vinifications throughout the manuscript

20: remarkably is probably not the appropriate word choice here.  Perhaps, use “much” instead or else better yet report the % it is decreased.

The word was replaced with “much” line 22

22: replace fairly unknown with under appreciated or rarely reported [If it is unknown, it is simply unknown]

We changed it to “rarely reported” line 22

23: providing increased -> increasing

This was changed line 23

31: sluggish fermentations.    Add a citation here, even if it is a general review.

We have added an appropriate citation. Christ et al (2015) line 238

35: needs to be more precise.  How about:  to expand on the breath of possible flavor profiles for wines prepared using defined cultures.

We have added this line to the text line 35-36

67: where were they isolated (grape surface) and when (e.g. month)

Unfortunately, this was not well-documented and is thus not known exactly where in the vineyard each yeast was isolated so we cannot expand this

71: not necessary to comment further, however, was the panel expert?  Were they looking for just pleasant aromas or aromas associated with esters? 

We now mention in the discussion this will be a future experiment to include sensory data

81: autoclaved Muscaris:  for how long?  I am a bit concerned as autoclaving can cause many chemical changes.  In the field is it pasteurized?   Is filter sterilization an option?  [this relates to future studies]

The must used for preculturing was autoclaved (a standard 15 min 121 degrees centigrade program) just to ensure the precultures (for both the NSY and VIN13) were grown in sterile conditions without the influence other organisms that still might be present in a pasteurized must. The must used for the vinifications were NOT autoclaved (mentioned line 84) and the amount of the autoclaved must that forms part of the inoculum was negligible.

82: how were cells counted?

With a hemocytometer (now mentioned) line 83

85: Anchor Yeast – was this also grown in liquid culture or added as a dry formulation?

The S. cerevisiae yeast were treated similar to the NSY i.e. liquid culture. Line 85

87: no more weight loss was recovered over a 24 hr period.

We have changed it to 24 hr line 88

88: I assume the samples were not stored for very long at 4C.  Would think that -20 might be more suitable.

The samples were all analyzed within a week after the fermentation stopped.

Figure 1: any what to bold the “strain from this study” to make it more evident for the reader.  Otherwise, a very nice tree.

The software does not provide options of making certain entries bold. We placed a red frame around the strains to “highlight” them.

139-143: yeast needs to be italicized

This is now fixed in the manuscript

168: shown to enhance both ways of which the yeast can impact aroma.  Please add a citation here.

A citation is now given, Belda et al (2017). Line 310

172-173: delete “including the main organic acids, residual sugar content (only fructose), glycerol, and ethanol.

This was removed. line 173

176 Upon assessing the …

Line was changed to “Upon…”. Line 177

179-182: as discussed above broaden the discussion around citation (I assume?)  21

We have added another sentence showing that more esters were also measured in the previous Nakazawaea mixed-culture fermentation. Line 181-182

190-197: line spacing changed.

Fixed

217:  avoid remarkable, perhaps, try notable or dramatic

We changed it to notable line 218