Round 1

Reviewer 1 Report

The study investigated three S. cerevisiae strains under fermentative conditions. They were found to be characterized by different depletion rates of nitrogen (NH4+ and N α-amino acid) in Assyrtiko must fermentation resulting in different fermentation kinetics, production of secondary compounds and eventually in different sensory profiles. The study is important to wine makers as it will enable them to make informed strain selection during wine making.

However, the following issues must be addressed before the manuscript can be accepted.

Methodology

The authors must explain why these two strains (S. cerevisiae Sa and Sb) were investigated. The rationale behind selecting them.

The authors must explain why this commercial strain was selected. What are the fermentation characteristics of this commercial strain? Why was it chosen in the first place? Any particular reason why it was compared with the isolates?

Fermentations: The authors must explain how anaerobic fermentations were maintained when samples were occasionally collected.

The authors must motivate why Yeast Extract (150 glL) and DAP (diammonium phosphate) 250 g/L) were added to the must. Did the authors use response surface methodology to determine this? If not, how were these concentrations chosen?

Minor

Line 88: Include the accession number for S. cerevisiae Sa a

Author Response

RESPONSE TO REVIEWERS

 

On behalf of all the authors, we would like to thank the reviewers for improving our initially submitted manuscript.

 

Reviewer: 1

1. The authors must explain why these two strains (S. cerevisiae Sa and Sb) were investigated. The rationale behind selecting them.

These two S. cerevisiae strains have been previously isolated and belong to the strain collection of the University but have never been tested for their fermentative ability. This is the first work that we have performed fermentation trials to test their potential ability to be used as wine starters (lines 85-91).

1. The authors must explain why this commercial strain was selected. What are the fermentation characteristics of this commercial strain? Why was it chosen in the first place? Any particular reason why it was compared with the isolates?

The commercial S. cerevisiae strain, iYeast® Passion Fruit was chosen as a control inoculation condition as this yeast strain is suitable for the production of white wines with strong aromatic intensity as recommended by the supplier and widely preferred by the wineries. We have compared this commercial strain with the two isolates in order to have a valid control starter both for the fermentation capacity as well as for the aroma production (lines 91-94).

1. Fermentations: The authors must explain how anaerobic fermentations were maintained when samples were occasionally collected.

Samples were collected in a sterile environment (laminar) with the use of an open flame while a special sterile needle was introduced each time for the selection of the samples

1. The authors must motivate why Yeast Extract (150 mglL) and DAP (diammonium phosphate) 250 mg/L) were added to the must. Did the authors use response surface methodology to determine this? If not, how were these concentrations chosen?

The concentrations of YAN were selected on the basis of the minimum (150 mg/L) and the most commonly used (250 mg/L) at industrial scale fermentations. It is necessary to clarify that the authors added the amount required (equal contents of Yeast extract and DAP) to reach a final content of 150 and 250 mg/L respectively (taking into account that the initial YAN content of the must was 80 mg/L). (Lines 111-113).

Minor

1. Line 88: Include the accession number for S. cerevisiae Sa a

This collection is a private one of the Agricultural University of Athens. The stains have not been studied before, and this is the first time that their fermentation activity is tested. It is in our future plans to ask for accession numbers.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper of Stefania Christofi et al. is interesting but requires some revision with a correction of the text used and a more detailed analysis of the results.

The authors compared the characteristics of two strains of Saccharomyces cerevisiae yeast isolated during spontaneous fermentation and a commercial strain and showed the great potential of local yeast strains as a fermentation starter for wine production. For the successful application of the tested strains, it is necessary to clarify the mechanisms that lead to differences between these strains, especially in nitrogen consumption.

In my opinion, the discussion part should be improved due to more discussions. For example, the authors can discuss NCR (nitrogen catabolite repression), which is accompanied by a change in the expression level of many genes and affects the activity of many enzymes. When simultaneously provided with both a good and poor nitrogen source, Saccharomyces cerevisiae cells will exhaust the good source from the medium before using the poor one. The good nitrogen sources are DAP and some amino acids (for example, glutamine). And, perhaps, to offer a more detailed explanation of the differences in nitrogen consumption by strains of Sa and Sb.

1. Lines 56-58: «Grape-must contains several different nitrogen sources including amino acids, ammonium, and small peptides although not all of these forms can be metabolized by yeasts». Which of the listed compounds cannot be metabolized by yeast and why? If peptides are meant, then the authors on page 8 talk about extracellular proteases.
2. It is necessary to correctly present links to articles with multiple authors in the text: the first author (last name only) et al., year. Lines 46-47; 49-50; 52-53; 61-62; 67-68; 74-75; 118-119; 160-161$ 260-261; 265-266; 291; 297-298; 308-309 and so on.
3. Line 254: check the spelling of the abbreviationΥΑΝ
4. Lines 258-259: What hydrolytic enzymes are talking about? Which enzymes polymerize and how does this affect their activity?
5. Lines 271-272: How was cell autolysis tested?
6. Line 306: oxyglutarate dehydrogenase replace with oxoglutarate dehydrogenase

Comments for author File: Comments.pdf

Author Response

RESPONSE TO REVIEWERS

 

On behalf of all the authors, we would like to thank the reviewers for improving our initially submitted manuscript.

 

Reviewer: 2

1. In my opinion, the discussion part should be improved due to more discussions. For example, the authors can discuss NCR (nitrogen catabolite repression), which is accompanied by a change in the expression level of many genes and affects the activity of many enzymes. When simultaneously provided with both a good and poor nitrogen source, Saccharomyces cerevisiae cells will exhaust the good source from the medium before using the poor one. The good nitrogen sources are DAP and some amino acids (for example, glutamine). And, perhaps, to offer a more detailed explanation of the differences in nitrogen consumption by strains of Sa and Sb.

Lines 267-272 and 295-306 have been added to discussion

1. Lines 56-58: «Grape-must contains several different nitrogen sources including amino acids, ammonium, and small peptides although not all of these forms can be metabolized by yeasts». Which of the listed compounds cannot be metabolized by yeast and why? If peptides are meant, then the authors on page 8 talk about extracellular proteases.

Utilization of small peptides in different environments is possible under specific conditions (Ref.5) Lines 57-59 were added

 

1. It is necessary to correctly present links to articles with multiple authors in the text: the first author (last name only) et al., year. Lines 46-47; 49-50; 52-53; 61-62; 67-68; 74-75; 118-119; 160-161$ 260-261; 265-266; 291; 297-298; 308-309 and so on.

References have been numbered and linked to articles (References section)

 

1. Line 254: check the spelling of the abbreviationΥΑΝ

In the specific paragraph (lines 264-306) authors are referred to α-amino acids as FAN (free amino acids) and to yeast assimilable nitrogen as YAN

 

1. Lines 258-259: What hydrolytic enzymes are talking about? Which enzymes polymerize and how does this affect their activity? The authors are referred to metabolic (hydrolytic) enzymes that are responsible for amino acids utilization, which when polymerized alter or acquire secondary functions

Lines 281-283 were modified

 

1. Lines 271-272: How was cell autolysis tested?

Cell autolysis could be tested with further investigation in mannoproteins’ concentration (lines 295-296)

 

1. Line 306: oxyglutarate dehydrogenase replace with oxoglutarate dehydrogenase

Replaced (Line 337)

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript “Effect of yeast assimilable nitrogen content on fermentation kinetics, wine chemical composition and sensory character in the production of Assyrtiko ‘terroir’ wines with autochthonous yeasts” concerns the chemical and sensory characteristics of the wine fermented by isolated S. cerevisiae strains at different YAN levels. The manuscript is interesting, but there are some issues that should be addressed:
-       References are not in line with the journal requirements
-       There are some references in the text that are not included in the references section (e.g. L118)
-       Name of microorganisms should be written in italics – please, check the manuscript carefully
Also, some specific comments:
-       L 116 – “OIV (2020) method” should be included in the references
-       L118 – “while ammonium nitrogen was determined based on a modified method of Berthelot…” – what was the modification of the method?
-       L142 – “In more detail, 2 ml of each wine were pipetted into a 20 ml glass vial containing 7.5 ml of preboiled deionized water, 1 g ammonium sulfite, and 500 μl 1-octanol.” – was 1-octanol an IS? If yes, please add this information
-       L185-186 – “…finally, the ethanol content of the three wines produced was almost similar, without statistically important differences …” if there are no statistically important differences, there is no need to write that something was similar (or almost)
-       L189-192 – “(Fig.2), demonstrating the potential of the employed wt strains for the production of wines containing high concentrations of ethanol, or even their capability to convert sugar-rich agro-industrial residues (i.e. molasses, hydrolyzed starchy materials, etc) into ethanol, with significant final product concentrations and yields” – please explain this sentence, how does Fig. 2 indicate that?
-       L271-273 – “Fermentations carried out with Sa strain, independently from the initial level of YAN, after 25 days of fermentation, contained the highest concentration of total amino acids probably due to extended yeast cells’ autolysis” – not true for YAN=250 mg N/L
-       Tab. 2 – please check the results of the statistical analysis 
-       L329-331 – “In addition, higher increase in VA was observed in ferments with 250 mg/L compared to ferments with 150 mg/L YAN, when curried out with the commercial and Sb strains, unlike to ferments curried out with Sa strain” – according to Tab. 3 there are no differences between different YAN 
-       L345-346 – “However, Sb strain was characterized by the highest glycerol and lower ethanol production” – there are no significant differences in the ethanol content 
-       L362 (Fig 5) – there should be “b” Sa, not “d”
-       L373 – “(Romano et al., 1994)” – why is this reference cited here?
-       L419-420 – “The same ferments were also judged as less reductive then the pivot, probably due to lower production of volatile sulfurous compounds” – were VSC also analyzed? 

Author Response

RESPONSE TO REVIEWERS

 

On behalf of all the authors, we would like to thank the reviewers for improving our initially submitted manuscript.

 

Reviewer: 3

1. References are not in line with the journal requirements
   -       There are some references in the text that are not included in the references section (e.g. L118)

References were corrected to be in line with journal requirements and the missing ones have been added

 

1. Name of microorganisms should be written in italics – please, check the manuscript carefully

Revised in both the article and the reference section

Also, some specific comments

1. L 116 – “OIV (2020) method” should be included in the references

Corrected

1. L118 – “while ammonium nitrogen was determined based on a modified method of Berthelot…” – what was the modification of the method?

 

Corrected (Line 127)

 

1. L142 – “In more detail, 2 ml of each wine were pipetted into a 20 ml glass vial containing 7.5 ml of preboiled deionized water, 1 g ammonium sulfite, and 500 μl 1-octanol.” – was 1-octanol an IS? If yes, please add this information

 

Information has been added (line158)

 

1. L185-186 – “…finally, the ethanol content of the three wines produced was almost similar, without statistically important differences …” if there are no statistically important differences, there is no need to write that something was similar (or almost)

 

Corrected (line 202)

 

1. L189-192 – “(Fig.2), demonstrating the potential of the employed wt strains for the production of wines containing high concentrations of ethanol, or even their capability to convert sugar-rich agro-industrial residues (i.e. molasses, hydrolyzed starchy materials, etc) into ethanol, with significant final product concentrations and yields” – please explain this sentence, how does Fig. 2 indicate that?

 

Fig.2 provides evidence regarding the conversion yield of ethanol produced per unit of total sugars consumed by each S. cerevisiae strain in fermentations showing that the conversion yield of ethanol could reach in many cases the theoretical yield (Terpou et al., 2019). Nevertheless, the indication that this could be applied in sugar-rich agro-industrial residues has been removed from the manuscript.  The sentence has been modified as follows: “The conversion yield of ethanol produced per unit of total sugars consumed was c. 0.50-0.51 g/g (the absolute value of the slope of the line) for all strains tested, that is the 98-100% w/w (Fig.2) of the maximum theoretical yield (=0.51 g/g), demonstrating the potential of the employed wild type strains for the production of wines containing high concentrations of ethanol.”

 

1. L271-273 – “Fermentations carried out with Sa strain, independently from the initial level of YAN, after 25 days of fermentation, contained the highest concentration of total amino acids probably due to extended yeast cells’ autolysis” – not true for YAN=250 mg N/L

 

It has been revised. Indeed, that was the case for ferments with low initial YAN level (line 293)

 

1. Tab. 2 – please check the results of the statistical analysis 

 

We thank the reviewer for this comment. The results of the statistical analysis of arginine content were corrected.

 

1.  L329-331 – “In addition, higher increase in VA was observed in ferments with 250 mg/L compared to ferments with 150 mg/L YAN, when curried out with the commercial and Sb strains, unlike to ferments curried out with Sa strain” – according to Tab. 3 there are no differences between different YAN

 

Modified (line 360)

 

1. L345-346 – “However, Sb strain was characterized by the highest glycerol and lower ethanol production” – there are no significant differences in the ethanol content 

 

Modified (Lines 376-378)

 

1. L362 (Fig 5) – there should be “b” Sa, not “d”

Revised (line 393)

 

1. L373 – “(Romano et al., 1994)” – why is this reference cited here?

 

We thank the reviewer for his/her comment. It was a mistake. The reference has been deleted.

 

14. 
    L419-420 – “The same ferments were also judged as less reductive than the pivot, probably due to lower production of volatile sulfurous compounds” – were VSC also analyzed? 

 

Unfortunately, we haven’t analyzed the VSC compounds but indeed, further investigation is required (line 453-454)

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed all my concerns. Thanks

Author Response

We would like to thank the reviewer for his effort and for helping us improving our manuscript.