Evaluation of Four Indigenous Non-Saccharomyces Yeasts Isolated from the Shangri-La Wine Region (China) for Their Fermentation Performances and Aroma Compositions in Synthetic Grape Juice Fermentation

Round 1

Reviewer 1 Report

The paper “Evaluation of four indigenous non-Saccharomyces yeasts isolated from the Shangri-La wine region (China) for their fermentation performances and aroma compositions in synthetic grape juice fermentation” deals with the oenological characterization of four indigenous non-Saccharomyces yeasts of different species isolated from the Shangri-La wine region (China). The study was performed in synthetic grape must in comparison to a commercial strain of Saccharomyces cerevisiae.  The work is not particularly novel, but it is interesting for the different species of non-Saccharomyces used. The work is well organized, the methods are well described and reproducible. I have concerns that fermented samples have a significantly high residual sugar content. Consequently, these yeasts failing to complete the fermentation process, if used in grape must, however, would produce unstable wines.The comparison with the fermentation by S. cerevisiae obviously underlines this aspect. I wonder why not to program the fermentations in mixed or sequential cultures of non-Saccharomyces with the S. cerevisiae strain. This would have allowed us to really understand the potential of the 4 non-Saccharomyces strains as industrial cultures.

However, the work is well done and detailed and has produced a lot of data. The concept that these yeasts could be considered useful as co-cultures in fermentations should be deepened in the work.

Minor comments:

1) The first time a species is cited, it must be written in full, i.e genus and species, such as in the line 20 of the  abstract: Saccharomyces cerevisiae

2) Since some genera have the same initial, to avoid confusion I suggest differentiating the abbreviations of the genus, for example: S. for Saccharomyces, Sac. for Saccharomycopsis, Sat. for Saturnispora.

3) lines 66-67: “Despite of diverse non-Saccharomyces yeasts found in vineyard and on the surface of grapes, very few non-Saccharomyces yeasts are commercially available for wine production”. In this last decade, numerous non-Saccharomyces wine yeast strains are available and marketed. Therefore, I suggest to change very few in few.

4) in Table 1 correct Reducing

Author Response

Comments:

“Why not to program the fermentations in mixed or sequential cultures of non-Saccharomyces with the S. cerevisiaes strain”

Response: It is exhaustively described in numerous researches that most non-Saccharomyces yeasts are not able to consume all the sugars during wine fermentation, and therefore they are generally used in multistarter or sequence fermentations with S. cerevisiae.

The reason that we researched on single culture fermentation is to eliminate the interference that may occurred in mixed fermentation and confirm the varieties and contents of aroma compounds produced by these four non-Saccharomyces yeast isolates. Based on the results of the present study, we will definitely plan to carry out mixed fermentation experiments and evaluate the performance of these four non-Saccharomyces yeasts in multistarter fermentation and sequence fermentation. This would provide us more insights about the potential of these non-Saccharomyces yeasts in wine production.

 

Minor comments:

1) The first time a species is cited, it must be written in full, i.e genus and species, such as in the line 20 of the abstract: Saccharomyces cerevisiae

Response 1: Thank you for prompt us, we have corrected “S. cerevisiae” to “Saccharomyces cerevisiae” in the “Abstract”.

 

2) Since some genera have the same initial, to avoid confusion I suggest differentiating the abbreviations of the genus, for example: S. for Saccharomyces, Sac. for Saccharomycopsis, Sat. for Saturnispora.

Response 2: Thank you for your suggestion. To avoid confusion, we have differentiating the abbreviations of the genus throughout the manuscript (including Table 1, 2, 3; Figure 1, 2B, 3).

 

3) lines 66-67: “Despite of diverse non-Saccharomyces yeasts found in vineyard and on the surface of grapes, very few non-Saccharomyces yeasts are commercially available for wine production”. In this last decade, numerous non-Saccharomyces wine yeast strains are available and marketed. Therefore, I suggest to change very few in few.

Response 3: Thank you for your suggestion. Besides change “very few” in “few”, is it more appropriate to change the original statement into the following one?

“Diverse non-Saccharomyces yeasts are widely distribute in the vineyards and on the surface of grapes. Although some of them have been confirmed to exhibit unique performances during fermentation, there are still many undiscovered oenological behaviors of non-Saccharomyces yeasts that deserve to be explored.”

 

4) in Table 1 correct Reducing

Response 4: Thank you for your careful review, we have corrected “educing” to “Reducing” in “Table 1”.

Reviewer 2 Report

The manuscript "Evaluation of four indigenous non-Saccharomyces yeasts isolated from the Shangri-La wine region (China) for their fermentation performances and aroma compositions in synthetic grape juice fermentation" submitted by Zhao et al.  to publication in Journal of Fungi is interesting, but the authors revise it according to the following comments before considering for publication:

- The English of the manuscript is checked with the help of an English language expert.

- Title of the manuscript is too long. It would be concise.  

- The materials and methods section would be written carefully and in more detail especially isolation and identification of yeasts.

- Are molecular tests used for the identification of yeasts only? If yes, authors should be done the morphological and biochemical tests to confirm yeast strains.  

- I recommend DNA sequences register in Genbank and draw a phylogenetic tree according to the information.  

- Organic acids and some volatile compounds inhibit alcohol production. Is it relational to the isolated yeast strains with lower fermentative capacity and lower conversion rate of sugar to alcohol used in alcohol fermentation?  

- Discussion is weak and results need to be better discussed! The manuscript needs to be better discussed with related and more references and don’t repeat the content of the results.

Author Response

- The English of the manuscript is checked with the help of an English language expert.

Response: Indeed, before submitted the manuscript to “Journal of Fungi”, one English language expert helped us improved the language quality of this manuscript.

 

- Title of the manuscript is too long. It would be concise.

Response: Thank you for your suggestion. Considering the topic of this manuscript and make the title more concise, is it more appropriate to adjust the title into “Aroma-producing characteristics of four indigenous non-Saccharomyces yeasts isolated from the Shangri-La wine region (China) in synthetic grape juice fermentation”.

 

- The materials and methods section would be written carefully and in more detail especially isolation and identification of yeasts.

Response: We are very sorry for fail to provide the detailed methods about the isolation and identification of these yeasts. Actually, the origin, isolation and identification of these yeasts were explicitly described in our previously research entitled “Biodiversity of non-Saccharomyces yeasts associated with spontaneous fermentation of Cabernet Sauvignon wines from Shangri-La wine region, China”. To make it more obvious, we inserted this previously research as reference in “Microorganisms” section.

 

- Are molecular tests used for the identification of yeasts only? If yes, authors should be done the morphological and biochemical tests to confirm yeast strains.

Response: These yeasts were identified by using morphological and molecular methods. To provide more complete identification information, the colony morphologies of these non-Saccharomyces yeast isolates that grow on YPD and WL agar were attached as “Figure 1S” in “Supplementary data”.

 

- I recommend DNA sequences register in Genbank and draw a phylogenetic tree according to the information.

Response: Thank you for your suggestion, we have submitted the sequences to Genbank, and the accession numbers of these sequences were listed in Table 1S. Additionally, phylogenetic trees of four non-Saccharomyces yeast isolates based on the sequence analysis of the 26S rRNA D1/D2 region and 5.8S rRNA ITS region were attached as “Figure 2S” and “Figure 3S” in “Supplementary data”.

 

- Organic acids and some volatile compounds inhibit alcohol production. Is it relational to the isolated yeast strains with lower fermentative capacity and lower conversion rate of sugar to alcohol used in alcohol fermentation?

Response: This is really a good question. Several studies demonstrated that organic acids (e.g. medium-chain fatty acids) and some volatile compounds (e.g. terpenes) are toxic to yeast cells, the presence of these compounds might affect the growth and metabolism of yeasts. However, it is very likely that metabolic characteristics may vary from the different species of yeasts, and they may also respond differently to various inhibit factors. The lower fermentative capacity and lower conversion rate of sugar to alcohol of these yeast isolates probably related to the inhibiting effect of organic acids or some other volatiles, but further experiments are still needed to confirm it.

Anyway, thank you for providing us with new idea for future research.

 

- Discussion is weak and results need to be better discussed! The manuscript needs to be better discussed with related and more references and don’t repeat the content of the results.

Response: Really, we should admit that the “Discussion” of this manuscript seems not enough when compared with other relevant literatures. This is mainly because little study has been conducted for these four species of non-Saccharomyces yeasts in wine or other alcoholic beverages fermentation. In this case, very little relevant study could be referred to discuss, especially for M. guilliermondii, S. vini and S. diversa. As for W. anomalus, there was a study about one strain (YN6) belonging to this species in cider fermentation, which could be helpful to discuss the different production behavior of ester during synthetic grape juice or cider fermentation.

The following content have been inserted in the manuscript after “Esters” section.

“However, these compounds were not be detected in apple cider fermented by W. anomalus (YN6) [Ye, M.Q.; Yue, T.L.; Yuan, Y.H.; Effects of sequential mixed cultures of Wickerhamomyces anomalus and Saccharomyces cerevisiae on apple cider fermentation. FEMS Yeast Research 2014, 14, 873-882.]. This could be explained as the difference of ester metabolism among different W. anomalus strains, and the difference of components between apple juice and SGJ could be another possibility for the different results. Therefore, the distinctive ester production ability of W. anomalus (DR-110) required further study.”

Round 2

Reviewer 2 Report

- The new title "Aroma-producing characteristics of four indigenous non-Saccharomyces yeasts isolated from the Shangri-La wine region (China) in synthetic grape juice fermentation" set for the revised manuscript.

- "Non-published Material" add to text or supplementary data.