Metschnikowia pulcherrima as a Tool for Sulphite Reduction and Enhanced Volatile Retention in Noble Rot Wine Fermentation

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The article has not been sufficiently revised for acceptance. Some changes have been made in response to the reviewers' comments, but substantial improvements are still pending. 

It has been repeatedly commented that the species Botrytis cinerea, Vitis vinifera, Metchnikowia pulcherima etc. should be in italics and there are still many errors in the text that do not include this basic correction. 

There are certain errors, for example, volatile phenols (line 94) can be produced by yeasts (main subject of this study) and also the descriptors 4-ethylphenol or 4-ethylguaiacol are not positive as described. 

The volatile esters, then analyzed, do not come only from the variety (line 85) and present enough weight in the analysis to describe it. 

The first time a species name is described, it should be complete (line 108). 

Figure 2 is not entirely clear (different scales, it is difficult to make a comparison) and furthermore an increase within the same population range 105 does not indicate an imposition with respect to the other yeasts. 

Table 2 Tertier? Clarify in the text. 

Point 4.1 of the discussion, some behaviors are not attributed to the culture conditions tested. Revise the names of all species described. Data should be provided as to affirm such conclusions, moreover so general. 

Line 924 what does blossom-line aromas mean? Indicated in a conclusion and not commented on before, at least attribute to some earlier reference in the text. 

It should be commented as one of the conclusions whether the treatments carried out have a beneficial effect on the sensory profile of the wines analyzed with or without noble rot. 

 

Author Response

Dear Reviewer,

 

 

We thank the Reviewer for the valuable comments and constructive suggestions!

Comments1. The article has not been sufficiently revised for acceptance. Some changes have been made in response to the reviewers' comments, but substantial improvements are still pending. 

Response1.We thank the Reviewer for their continued engagement with our manuscript. We appreciate the acknowledgment of the revisions made so far and recognize that further improvements were needed. In this revised version, we have carefully addressed all outstanding issues raised in the previous review round. Substantial changes have been made to the Abstract, Introduction, Discussion, Conclusion sections, figures, and data interpretation to enhance clarity, accuracy, and consistency throughout the manuscript. We trust that the current version now meets the expectations for scientific rigor and clarity, and we are grateful for the feedback that guided these improvements.

 

Comments2. It has been repeatedly commented that the species Botrytis cinerea, Vitis vinifera, Metchnikowia pulcherima etc. should be in italics and there are still many errors in the text that do not include this basic correction. 

Response2. We thank the reviewer for their diligence in pointing this out. We apologize for this oversight. We have meticulously reviewed the entire manuscript and have corrected all instances of species names (e.g., Metschnikowia pulcherrima, Botrytis cinerea, Vitis vinifera) to ensure they are correctly italicized as per scientific convention.

 

Comments3. There are certain errors, for example, volatile phenols (line 94) can be produced by yeasts (main subject of this study) and also the descriptors 4-ethylphenol or 4-ethylguaiacol are not positive as described. 

Response3. We appreciate the reviewer's valuable comment regarding the nuanced role of volatile phenols. We have revised the sentence in the Introduction to provide a more balanced description, clarifying that while these compounds can contribute certain aromatic notes, they are often associated with spoilage microorganisms (such as Brettanomyces) and can be considered defects, particularly at higher concentrations. This provides a more accurate context for the reader.

 

Comments4.The volatile esters, then analyzed, do not come only from the variety (line 85) and present enough weight in the analysis to describe it. 

Response4. We thank the Reviewer for this important clarification. We acknowledge that volatile esters are largely fermentation-derived and not solely linked to varietal origin. In response, we have revised the wording in line 85 to avoid this oversimplification and to more accurately reflect the contribution of yeast metabolism to ester formation. Additionally, we have expanded the discussion to better describe the role of esters in the sensory expression of the wines, in proportion to their significance in the analytical results.

 

Comments5. The first time a species name is described, it should be complete (line 108). 

Response5. Thank you for this helpful observation. We have revised the manuscript accordingly and now use the full species name at first mentions in the text.

 

Comments6. Figure 2 is not entirely clear (different scales, it is difficult to make a comparison) and furthermore an increase within the same population range 105 does not indicate an imposition with respect to the other yeasts. 

Response6. We appreciate the Reviewer’s observation regarding the clarity and interpretability of Figure 2. In response, we have revised the figure to use uniform scales across all panels to facilitate more direct comparison between yeast populations.

Furthermore, we acknowledge that the observed increase in M. pulcherrima population to above 10⁵ CFU/ml does not, on its own, demonstrate ecological dominance over other yeasts. We have therefore removed or rephrased any language in the text that may have overstated this point. The revised discussion now more accurately reflects the observed implantation and early proliferation of M. pulcherrima without implying competitive exclusion. These changes improve both the accuracy and transparency of the interpretation.

 

Comments7. Table 2 Tertier? Clarify in the text. 

Response7. We apologize for this lack of clarity. The term 'Tertier' was a typographical error for 'Tertiary,' referring to aromas developed during aging or maturation. We have corrected this term to 'Tertiary / Aging-related' in Tables 1 and 2 and throughout the manuscript text, and have added a brief definition in the 3.2 section for clarity that this category includes compounds associated with wine maturation.

 

Comments8. Point 4.1 of the discussion, some behaviors are not attributed to the culture conditions tested. Revise the names of all species described. Data should be provided as to affirm such conclusions, moreover so general. 

Response8. We thank the Reviewer for this important observation. In response, we have revised section 4.1 to ensure that all discussed microbial behaviors are clearly and directly linked to the conditions tested in our study. Statements that were speculative or derived from literature without direct data support (e.g., the effect of S. cerevisiae on M. pulcherrima decline) have been reworded or removed for clarity. Furthermore, all species names mentioned in this section have been reviewed and adjusted to reflect only those organisms that were either inoculated or relevant to our experimental system. We have avoided generalizations and now refer exclusively to observed data on M. pulcherrima implantation and decline, as measured in our own microbiological analysis. We believe these revisions align the discussion more closely with the data and improve its clarity.

 

Comments9. Line 924 what does blossom-line aromas mean? Indicated in a conclusion and not commented on before, at least attribute to some earlier reference in the text. 

Response9. We thank the Reviewer for highlighting this ambiguity. The expression “blossom-like aromas” was intended to refer to floral sensory notes such as those associated with nerol, linalool, and phenylethyl acetate, which are described earlier in the Results and Discussion sections. To improve clarity, we have revised the wording in the Conclusion to refer more specifically to “floral aromas”.

 

Comments10. It should be commented as one of the conclusions whether the treatments carried out have a beneficial effect on the sensory profile of the wines analyzed with or without noble rot. 

Response10. Thank you for this valuable comment. We fully agree that the effect of the treatments should be evaluated separately for wines made from healthy and botrytized grapes. Accordingly, we have added a clarifying statement to the end of the conclusion section, explicitly addressing the sensory impact of the treatments in both grape conditions.

 

We once again thank the Reviewer for the thoughtful and helpful comments that have greatly contributed to improving the quality and clarity of our manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The manuscript discusses the effects of a commercial strain of Metschnikowia pulcherrima, which is utilized as a biocontrol agent to reduce sulfur dioxide levels in Tokaj wines made from both healthy and botrytized grapes. Additionally, it highlights the impact of this M. pulcherrima strain on the volatile profile of the produced wines. While the authors have revised the manuscript, the introduction remains excessively lengthy and does not conform to the standard guidelines for scientific papers. The primary weakness lies in the microbiological data, which are crucial for evaluating the effectiveness of the bioprotective action of the M. pulcherrima strain. Indeed, microbial counts that are now included in the revised manuscript were neither properly carried out nor correctly shown in Figure 2. The main issues with these data are reported below: 

• Microbiological data on "L-Late harvest fermented grapes" are lacking
• When performing plate counts in microbiology, the unit of measurement is CFU/mL (Colony Forming Units per millilitre), not just CFU.
• All must samples were filtered, and the membrane was placed onto the WL agar; therefore, it was not possible to assess the colour change around the colonies as described in the "Materials and Methods" section.
• To accurately quantify the non-Saccharomyces yeast populations, a representative number of colonies developed onto WL agar should have been picked up and identified by molecular methods.
• Not all M. pulcherrima colonies are pink or red; hence, the reported counts can be affected by errors.
• Since alcoholic fermentations were conducted using a commercial starter culture containing Torulaspora delbrueckii and Saccharomyces cerevisiae, these populations should also have been detected in all wine samples.

In conclusion, the bioprotective action by the M. pulcherrima strain of healthy and botrytized Tokai grapes cannot be evinced by the results reported in the manuscript.

It has not been clarified why the effect of Starmerella bacillaris on aroma compounds is evaluated in Table 1, given that the presence of this yeast species is not reported.

The "Discussion" should be shortened by discussing only the results achieved in the study in comparison to the existing literature.

The name of microorganisms must be written in Italics.

Author Response

Dear Reviewer,

 

 

We thank the Reviewer for the valuable comments and constructive suggestions!

Comments1. The manuscript discusses the effects of a commercial strain of Metschnikowia pulcherrima, which is utilized as a biocontrol agent to reduce sulfur dioxide levels in Tokaj wines made from both healthy and botrytized grapes. Additionally, it highlights the impact of this M. pulcherrima strain on the volatile profile of the produced wines. While the authors have revised the manuscript, the introduction remains excessively lengthy and does not conform to the standard guidelines for scientific papers.

Response1. We thank the reviewer for this valuable comment. In response, we have substantially revised the Introduction section to improve its conciseness and ensure better alignment with standard scientific structure. Background details have been streamlined, and redundant or tangential information has been removed. The revised version now focuses more clearly on the research context, objectives, and rationale. We appreciate the reviewer’s guidance in improving the clarity and readability of the manuscript.

 

Comments2. The primary weakness lies in the microbiological data, which are crucial for evaluating the effectiveness of the bioprotective action of the M. pulcherrima strain. Indeed, microbial counts that are now included in the revised manuscript were neither properly carried out nor correctly shown in Figure 2. The main issues with these data are reported below: 

Microbiological data on "L-Late harvest fermented grapes" are lacking

Response2. We thank the reviewer for this important observation. We acknowledge that the microbiological data for the "L–Late harvest" fermented grape treatments were missing from the original submission due to an oversight during figure preparation. These data were collected during the experiment but were inadvertently excluded from Figure 2 and the related discussion. In response, we have now included the complete microbiological data for the late-harvest samples in the revised Figure 2, ensuring consistency across all treatment groups. The corresponding text in the Results and Materials and Methods sections has also been updated accordingly. We are grateful for the reviewer’s comment, which allowed us to correct this omission and strengthen the manuscript.

 

Comments3. When performing plate counts in microbiology, the unit of measurement is CFU/mL (Colony Forming Units per millilitre), not just CFU.

Response3. Thank you for this helpful and well-founded observation. We fully agree with the reviewer’s point and have revised the manuscript accordingly.

 

Comments4. All must samples were filtered, and the membrane was placed onto the WL agar; therefore, it was not possible to assess the colour change around the colonies as described in the "Materials and Methods" section.

Response4. We thank the Reviewer for highlighting this methodological inconsistency. We have updated the “Materials and Methods” section to clarify that, due to the use of membrane filtration, the typical pH-based halo effect around colonies on WL agar could be observed with big uncertainty. This limitation has now been stated clearly, along with a note on the broader limitations of morphology-based identification and the potential benefit of molecular confirmation in future work.

 

Comments5. To accurately quantify the non-Saccharomyces yeast populations, a representative number of colonies developed onto WL agar should have been picked up and identified by molecular methods.

Response5. We thank the Reviewer for this insightful observation. We acknowledge that the identification of non-Saccharomyces yeasts based solely on colony morphology on WL agar has its limitations. While our approach provided a general overview of population trends, we agree that molecular identification would have allowed for greater taxonomic resolution. We have now added a sentence in the Methods  section to clarify this limitation and suggest that future studies incorporate molecular techniques to complement culture-based analysis.

 

Comments6. Not all M. pulcherrima colonies are pink or red; hence, the reported counts can be affected by errors.

Response6. We thank the Reviewer for pointing out this important limitation. We agree that colony color alone is not a fully reliable criterion for identifying M. pulcherrima, as pigmentation can vary across strains and cultivation conditions. We have revised the “Materials and Methods” section to clarify that the counts are based on visual identification and may be subject to under- or overestimation. We also acknowledge this limitation in the Discussion and suggest that future work include molecular confirmation to ensure accurate quantification.

 

Comments7. Since alcoholic fermentations were conducted using a commercial starter culture containing Torulaspora delbrueckii and Saccharomyces cerevisiae, these populations should also have been detected in all wine samples.

Response7. We thank the Reviewer for this important observation. Indeed, alcoholic fermentation was carried out using a commercial co-inoculation of T. delbrueckii and S. cerevisiae. However, our microbiological analysis focused primarily on the early phase of fermentation (up to day 14), and our culture-based enumeration was optimized for the detection of M. pulcherrima and general non-Saccharomyces dynamics. As a result, we did not specifically isolate or quantify T. delbrueckii and S. cerevisiae colonies. We have now clarified this point in the Methods sections and acknowledged it as a limitation of our current microbiological dataset. In future work, we plan to include strain-specific molecular quantification (e.g., qPCR) to track individual starter yeast populations.

 

Comments8. In conclusion, the bioprotective action by the M. pulcherrima strain of healthy and botrytized Tokai grapes cannot be evinced by the results reported in the manuscript.

Response8. We thank the Reviewer for this insightful and well-founded observation. We fully acknowledge that the current microbiological dataset does not allow for a conclusive demonstration of the bioprotective mechanism of Metschnikowia pulcherrima, particularly in terms of competitive exclusion on both healthy and botrytized grapes.

In response to this comment, we have substantially revised the manuscript to de-emphasize the mechanistic interpretation of M. pulcherrima's mode of action. Instead, we have refocused the narrative on the chemical and sensory outcomes of applying a bioprotection-based protocol compared to the traditional SO₂-based approach. These aspects are well supported by comprehensive GC-MS analysis and sensory panel data.

We believe this reframing strengthens the manuscript and aligns it more closely with the data presented.

 

Comments9. It has not been clarified why the effect of Starmerella bacillaris on aroma compounds is evaluated in Table 1, given that the presence of this yeast species is not reported.

Response9. The reviewer has identified a significant error in our analysis. The inclusion of Starmerella bacillaris as a factor in Table 1 was an oversight and a remnant of a previous analytical framework. As we did not quantify this species in our samples, this part of the analysis was inappropriate. We have removed the 'Starmerella' row from Table 1 and Table 2 and edited Section 1.2.3 of the Introduction to remove the detailed focus on S. bacillaris, retaining only a general mention.

Comments10. The "Discussion" should be shortened by discussing only the results achieved in the study in comparison to the existing literature.

Response10. We thank the Reviewer for this clear and constructive recommendation. In response, we have substantially revised the Discussion section to focus exclusively on the results obtained in the present study and their comparison to relevant findings in the existing literature. Background explanations and general descriptions of biochemical mechanisms not directly examined in our work have been removed or significantly reduced. The revised text places greater emphasis on the interpretation of our chemical and sensory data in light of published studies and avoids speculative or overly detailed mechanistic discussion. We believe this streamlining improves the clarity, relevance, and focus of the manuscript in accordance with the Reviewer’s valuable feedback.

 

Comments11. The name of microorganisms must be written in Italics.

Response11. We thank the Reviewer for pointing this out. We have carefully reviewed the manuscript and corrected the formatting of all microorganism names to ensure that they appear in italics, in accordance with scientific conventions.

 

We once again thank the Reviewer for the thoughtful and helpful comments that have greatly contributed to improving the quality and clarity of our manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Several non-Saccharomyces yeasts such as Torulaspora delbrueckii, Lactobacillus thermotolerans, and Metschnikowia pulcherrima offer bioprotective benefits, can minimized SO₂additions.  this paper work on two oenological protocols and comparsion,  early harvest and   late harvest grapes were used as fermentation material,  conventional sulphur dioxide-based protocol and an alternative bioprotection-oriented approach that minimized SO₂ additions. Bioprotection was carried out using Metschnikowia pulcherrima, followed by sequential inoculation with Torulaspora delbrueckii and Saccharomyces cerevisiae. Grape-derived tannins (from skin and seed) were also added to inhibit oxidative enzymes such as laccase. Fermentation was monitored using standard analytical techniques, with volatile aroma profiles characterized by HS-SPME-GC-MS. Results showed that harvest timing and botrytization strongly influenced the chemical composition of the wines. Moreover, the treatment protocol had a marked effect on the final sensory profile. Wines produced with the bioprotection-oriented protocol displayed enhanced aromatic complexity, particularly through higher concentrations of esters and higher alcohols. Overall, the alternative protocol involving M. pulcherrima-based bioprotection resulted in wines with more pronounced floral and fruity notes, the use of M. pulcherrima in conjunction with oenological  tannins and reduced SO₂represents a promising tool for shaping wine style while aligning.

this  work is valuable for production Sulphite reduction Wine, I think this paper can be published after the authors make minor revise. My revision opinions are as follows:

1. Figure 2. In all treatments, the Metschnikowia population declined sharply after day 4, becoming undetectable by day 8 or 14.  can author give some explanations?
2. Figure 6. The figure presents a 3D scatterplot visualizing，it is nor clear to show the total polyphenol, calcium, and potassium  contents  of each samples, it is better to use the table to show the date.
3. It is better if the author can detect the bacterial number of conventional sulphur dioxide-based protocol and an alternative bioprotection-oriented approach.

 

Author Response

Dear Reviewer,

 

 

We thank the Reviewer for the valuable comments and constructive suggestions!

Comments1. Figure 2. In all treatments, the Metschnikowia population declined sharply after day 4, becoming undetectable by day 8 or 14.  can author give some explanations?

Response1. We appreciate the reviewer’s comment regarding the sharp decline of Metschnikowia populations. In response, we have incorporated an explanation into the Results section, addressing the possible contributing factors, including ethanol accumulation, killer toxin activity from co-inoculated Saccharomyces strains, and nutrient depletion, as well as differences in fermentation kinetics between Panels A and B. This addition provides a clearer interpretation of the observed population dynamics.

 

Comments2. Figure 6. The figure presents a 3D scatterplot visualizing，it is nor clear to show the total polyphenol, calcium, and potassium  contents  of each samples, it is better to use the table to show the date.

Response2. We appreciate the reviewer’s suggestion regarding the presentation of total polyphenol, calcium, and potassium contents. In response, we have prepared an additional table (now included as Table 3.) that lists these values for each sample. At the same time, we retained the 3D scatterplot in Figure 6, as it provides a useful visual overview of the data distribution.

 

Comments3. It is better if the author can detect the bacterial number of conventional sulphur dioxide-based protocol and an alternative bioprotection-oriented approach.

Response3. We thank the reviewer for this insightful suggestion. While quantifying the initial bacterial load under both protocols is indeed a relevant point, we deliberately omitted this measurement due to significant methodological challenges that could compromise the scientific validity of the results. Given these limitations, we concluded that a simple bacterial count at this early stage would not provide a reliable measure of each protocol's effectiveness. Instead, we focused our analysis on downstream parameters, such as fermentation kinetics, final wine chemistry, and the development of specific spoilage markers, which we believe are more direct and meaningful indicators of the success of the bioprotective strategy versus the conventional SO₂ treatment. We agree that exploring these microbial dynamics is a valuable endeavor. Future studies could employ culture-independent methods, such as quantitative PCR (qPCR) or 16S rRNA gene sequencing, to overcome the limitations of plating and provide a more accurate picture of the microbial succession.

 

We are grateful to the Reviewer for the constructive and insightful feedback, which has significantly helped us enhance both the clarity and overall quality of the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The article has been moderately modified but not in all the points commented by the reviewer. 

The names of yeast or bacterial species (Torulaspora, Metchnikowia, Botrytis etc) included in the text have not been revised (described in italics) and this is a very basic aspect. 

The study has been performed implementing a yeast (Lachancea thermotolerans) and it is described in the text wrongly (Lactobacillus thermotolerans).

Author Response

Dear Reviewer,

 

 

We thank the Reviewer for the valuable comments and constructive suggestions!

Comments1. The names of yeast or bacterial species (Torulaspora, Metchnikowia, Botrytis etc) included in the text have not been revised (described in italics) and this is a very basic aspect. 

Response1. Thank you very much for your valuable feedback.

Regarding the formatting issue with the species names (e.g., Torulaspora, Metchnikowia, Botrytis, etc.), I would like to kindly ask you to refer to the PDF version of the manuscript rather than the Word document. I have carefully revised the text and ensured that all species names are correctly italicized throughout the manuscript.

It appears that during the conversion or rendering of the Word file, the italic formatting may not be displaying properly. I have already notified the editor about this issue.

Please accept my apologies for any inconvenience caused — unfortunately, this formatting inconsistency is beyond my control.

 

Comments2. The study has been performed implementing a yeast (Lachancea thermotolerans) and it is described in the text wrongly (Lactobacillus thermotolerans).

Response2. We thank the reviewer for drawing our attention to this typographical error. In the original text, Lachancea thermotolerans was mistakenly written as Lactobacillus thermotolerans due to an oversight. This has been corrected in the manuscript. Lachancea thermotolerans is a non-Saccharomyces yeast species belonging to the family Saccharomycetaceae, whereas Lactobacillus refers to a genus of lactic acid bacteria; therefore, the incorrect term could indeed cause confusion. We appreciate the reviewer’s attention to detail, thank you again!

 

We are grateful to the Reviewer for the constructive and insightful feedback, which has significantly helped us enhance both the clarity and overall quality of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The manuscript aimed to evaluate the bioprotection activity of a commercial strain of Metschnikowia pulcherrima in Furmint wines, which were fermented using a starter culture that included strains of Torulaspora delbrueckii and Saccharomyces cerevisiae. Unfortunately, the objective of the study was not achieved due to improper microbiological analyses. While the authors have provided a discussion addressing various points that highlight the weaknesses of the results and recommend that proper microbiological analyses be conducted in future studies, the data presented are insufficient to demonstrate a bioprotective effect. 

On the contrary, some effects on the aroma of wines based on the presence of M. pulcherrima during the fermentation were demonstrated, especially in the "Early harvest" samples.

I recommend removing all sections related to the concept and results of bioprotection, keeping only the chemical and sensory data, and altering the title. The introduction should be changed accordingly, avoiding the use of paragraphs and subparagraphs.

Additionally, consistent coding for wine samples is essential throughout the manuscript (see Figures 1, 3, and 4, for example, as different codes can be quite confusing.

Finally, the name of microbial species must be written in italics.

In Tables 1 and 2, what does "Saccharomyces spp." refer to?

Author Response

Dear Reviewer,

 

 

We thank the Reviewer for the valuable comments and constructive suggestions!

Comments1. The objective of the study was not achieved due to improper microbiological analyses. While the authors have provided a discussion addressing various points that highlight the weaknesses of the results and recommend that proper microbiological analyses be conducted in future studies, the data presented are insufficient to demonstrate a bioprotective effect. 

Response1. We thank the reviewer for their careful evaluation of our work. We acknowledge the limitations of our microbiological analysis, which did not include species-specific quantification of all yeast strains throughout fermentation. As noted in the manuscript, this is an important area for future research, ideally using more robust molecular tools such as qPCR or metabarcoding. In the revised discussion, we have emphasized this limitation more clearly to ensure it is transparent to readers.

While we recognize that our microbiological dataset does not conclusively demonstrate competitive microbial exclusion in a strict mechanistic sense, we respectfully maintain that the functional impact of early M. pulcherrima inoculation is evident. Specifically, clear and consistent differences in volatile composition and sensory attributes—particularly when compared to non-inoculated controls under identical fermentation protocols—support the practical relevance of this approach.

In contemporary enology, “bioprotection” encompasses not only direct microbial suppression but also the modulation of early fermentation dynamics, including oxygen consumption, redox balance, and metabolic interactions that influence aroma development. The elevated concentrations of key aroma compounds (esters, higher alcohols, terpenes) observed in the bioprotected wines are consistent with prior literature on the early metabolic contributions of non-Saccharomyces yeasts.

Therefore, while we agree that more comprehensive microbiological monitoring would strengthen the mechanistic interpretation, we believe the current study still offers valuable insight into the sensory and chemical outcomes of an M. pulcherrima-based bioprotection strategy under real-world winemaking conditions. We have ensured that all claims are appropriately qualified to align with the strengths and limitations of our dataset, and we respectfully request to retain the conceptual framework of bioprotection as central to the manuscript.

 

Comments2. The introduction should be changed accordingly, avoiding the use of paragraphs and subparagraphs.

Response2. Thank you for your observation. We have revised the introduction accordingly, removing paragraphs and subparagraphs to align with the required structure.

 

Comments3.Additionally, consistent coding for wine samples is essential throughout the manuscript (see Figures 1, 3, and 4, for example, as different codes can be quite confusing.

Response3. Thank you for your helpful comment. We have implemented a consistent coding system for the wine samples throughout the manuscript, including in Figures 1, 3, and 4, to avoid any confusion.

 

Comments4. Finally, the name of microbial species must be written in italics.

Response4. Thank you very much for your valuable feedback.

Regarding the formatting issue with the species names (e.g., Torulaspora, Metchnikowia, Botrytis, etc.), I would like to kindly ask you to refer to the PDF version of the manuscript rather than the Word document. I have carefully revised the text and ensured that all species names are correctly italicized throughout the manuscript.

It appears that during the conversion or rendering of the Word file, the italic formatting may not be displaying properly. I have already notified the editor about this issue.

Please accept my apologies for any inconvenience caused — unfortunately, this formatting inconsistency is beyond my control.

 

Comments5. In Tables 1 and 2, what does "Saccharomyces spp." refer to?

Response5. Thank you for this question. In Tables 1 and 2, the term Saccharomyces spp. refers to yeast isolates that were identified at the genus level as belonging to the Saccharomyces genus, but were not further resolved to species level due to the limitations of our microbiological analysis. Therefore, while S. cerevisiae is the most likely dominant species under the given fermentation conditions, we used the broader term Saccharomyces spp. to reflect that species-level identification was not confirmed.

 

We are grateful to the Reviewer for the constructive and insightful feedback, which has significantly helped us enhance both the clarity and overall quality of the manuscript.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper addresses an issue of practical interest to the Tokaj wine production. The aim is to evaluate the bioprotective potential of Metschnikowia pulcherrima in the context of Tokaji winemaking, focusing on its application in the indigenous grape variety Furmint. The addition of condensed oenological tannins to ensure a more effectively protection against oxidation was also studied. The paper contains a great deal and interesting information to justify publication and it is carefully written and well organized. The experimental design looks robust and the conclusions are supported by the data presented.

However, the paper is excessively long, especially the Introduction. Authors should make an effort to shorten the text substantially in all sections, thus making it much more reader friendly.

Maybe the summary does reflect all the work done. More comments on the abstract:

Lines 18-21: rephrase making clear, specially, the role and time of application of Metchnikowia pulcherrima, and the use of oenological tannin

Lines 22-25: improve English grammar and syntax and rephrase making more clear

Author Response

Dear Reviewer,

 

 

We thank the Reviewer for the valuable comments and constructive suggestions!

 

Comments 1. However, the paper is excessively long, especially the Introduction. Authors should make an effort to shorten the text substantially in all sections, thus making it much more reader friendly.

Response1. We appreciate the Reviewer’s remark regarding the length of the manuscript. In response, we have thoroughly revised the text, with particular attention to the Introduction, and have significantly reduced the overall word count. Superfluous details were removed, and the content was streamlined to enhance clarity and readability while retaining all essential information relevant to the study.

 

Comments 2. Lines 18-21: rephrase making clear, specially, the role and time of application of Metchnikowia pulcherrima, and the use of oenological tannin.

Response 2. Thank you for this suggestion. We have revised the Abstract to clarify the role and timing of the application of Metchnikowia pulcherrima, as well as the specific use of oenological tannins in our experimental design. These modifications aim to present a more precise overview of the methodology and its significance.

 

Comments 3. Lines 22-25: improve English grammar and syntax and rephrase making more clear

Response 3. We acknowledge the need for improved clarity and grammar in this section of the Abstract. The sentences in lines 22–25 have been carefully reworded for better grammatical accuracy and to ensure clearer communication of our findings and conclusions.

 

We once again thank the Reviewer for the thoughtful and helpful comments that have greatly contributed to improving the quality and clarity of our manuscript.

 

Sincerely yours

Reviewer 2 Report

Comments and Suggestions for Authors

The study aims to assess the impact of a commercial Metschnikowia pulcherrima strain on volatile retention in Tokaj wine fermentation conducted with healthy and botrytised grape berries, with the ultimate goal of reducing the use of SO₂. In addition to M. pulcherrima, used as a biocontrol agent, a commercial product containing Torulaspora delbrueckii and Saccharomyces cerevisiae strains was used to induce alcoholic fermentation. 

Overall, the manuscript resembles a treatise on oenology rather than a scientific paper, featuring an excessively lengthy introduction that covers general aspects of viticulture and oenology, which are well known to experts in the field. Therefore, novelties are not highlighted, and the aims appear to be confused.

The primary issue with the manuscript is that all the chemical analyses conducted on the experimental wines assume that the commercial yeasts used have successfully dominated the natural microbiota. However, no microbiological analysis is presented. Since the M. pulcherrima strain was utilised as a biocontrol agent, it was essential to verify its effectiveness against undesirable microorganisms. Instead, the manuscript only discusses its effects on wine aroma. Other general remarks follow:

• Details on the use of the M. pulcherrima strain are lacking (since the manufacturer's instructions indicate a range dosage of 2-7 g/100L)
• The compound category in tables 1 and 2, which is indicated as "Starmerella", what does it mean?  
• Microbial species names (M. pulcherrima, T. delbrueckii, S. cerevisiae) should consistently be italicised throughout the text, including all tables and figure captions.

Author Response

Dear Reviewer,

 

Thank you for your detailed and constructive comments, which will significantly improve our manuscript. We agree with the issues you have raised and will revise the paper accordingly.

We will shorten the extensive introduction to focus on the study's novel aspects: the dual role of Metschnikowia pulcherrima in a sequential fermentation of healthy and botrytised grapes, aimed at reducing SO₂. The research aims will be clarified throughout.

We fully agree that the lack of microbiological analysis is a key limitation. We will clearly state this in the manuscript and revise the discussion to emphasize that our results focus on aroma, while direct confirmation of biocontrol efficacy requires further study.

Furthermore, we will make all other necessary corrections: specifying the M. pulcherrima dosage, clarifying the 'Starmerella' category in the table footnotes, and ensuring consistent italicization of all microbial species names.

We acknowledge that the applied dosage of 10 g/hL exceeds the manufacturer's general recommendation of 2–7 g/hL. However, this higher concentration was intentionally chosen in the context of an experimental setting to ensure a more pronounced and measurable effect under the specific conditions of our study. This decision was also based on preliminary trials and literature reports suggesting that, under certain oenological conditions (e.g., must composition, microbial load), higher doses may be required to achieve the desired technological impact. We have now clarified this reasoning in the Methods section of the manuscript.

 

Thank you again for your valuable feedback. We are confident these revisions will make the manuscript suitable for publication.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Line 11: The term 'specialised yeast strains' is ambiguous and requires clarification. Does it refer to non-Saccharomyces species? 

Line 14: 'Strain-specific commercial yeast remain unavailable' – this is not the objective of the study, and in addition, commercial strains are used. 

Line 19: Please clarify whether this refers to the use of these yeasts in co-inoculation or sequential inoculation. 

Line 24: 'Enhance organoleptic properties” – “compensate negative effects of climate change”. It is necessary to specify which properties or characteristics detected in the study enhance or compensate for the described effects. 

In the introduction, line 26 should provide a description of how bioproduction can lead to a reduction in sulphur use. 

It is imperative that 'Vitis vinifera' is italicised throughout the text. 

Line 70-72: In this instance, the specific information pertaining to the variety utilised in the study has been employed with reference to the corresponding study. It is imperative that this is given due consideration and incorporated into the remainder of the introduction. For instance, in lines 83 and 88, it is imperative to insert the relevant reference that accurately describes these characteristics. 

It is imperative that only 'primary aromas' and 'fermentative aromas' are used on lines 80 and 81. 

Lines 92 to 124: In these paragraphs, there is a necessity to include various bibliographic references concerning the aldehydes or esters formed/present. Furthermore, a more specific approach to the Furmint variety or the type of processing used is required. The introduction is very general and lengthy (78 references out of 128 are used in the introduction). The primary focus of the presentation should be on the conditions or particularities of the study being presented. Moreover, it is necessary to acknowledge that a substantial proportion of this information can be employed in the discourse surrounding the results. 

Ltnes 128-135 must be unified with the description of terpenic compounds, as these compounds have been described in other paragraphs, and the repetition is superfluous. 

Line 170-181: This paragraph discusses the concept of 'bioprotection', a term that is also employed in other paragraphs with the objective of unifying information. 

Line 195-208: It is recommended that the paragraph outlining 'non-Saccharomyces' yeasts be unified with the paragraph below it in Section 1.2.1. 

It is imperative to verify that all yeast species delineated are italicised. 

Following the description of the full species name (Metschnikowia pulcherrima), the abbreviation (M. pulcherrima) should be used throughout the text (error lines 386, 391). It is imperative to meticulously revise the text to ensure its accuracy for all species, as evidenced by the erroneous designation of B. cinerea on line 329 and the inaccuracy on line 639. 

Line 275-284: necessary to ascertain whether there are specific studies that focus on the Furmint variety. 

Line 370: Please provide clarification of the term 'blue varieties'. 

It is recommended that the information contained on lines 377-379 be removed, as it is repetitive. 

In order to be included in the description of all commercial preparations of micro-organisms or tanin, as appropriate, and all information on the preparations, such as the supplier. 

It is recommended that the sentence commencing on line 430 and concluding on line 433 be removed, since the detailed information required is described below. 

Line 436: Subindices and spaces require revision. 

Line 436: It is necessary to provide a detailed account of the number of times or frequency with which sampling was performed. 

In Table 1, the term 'M. pulcherrima' is written with italics. 

Note that Starmerella has not been referenced in preceding sections, and its role requires elucidation. 

In Figure 4, there is a clear necessity for enhancement, as the descriptor names are not clearly visible, and the figure's caption provides an insufficient level of detail. 

Line 773-785: A detailed description of the figures in which these results are presented is required. 

Line 775: In this study, the term 'broader aromatic variability' is employed to denote the presence of Saccharomyces and non-Saccharomyces, as well as Botrytis, in the examined samples. It is imperative to incorporate references that either substantiate or contradict these findings. 

Line 805-808: describe if a suitable method exists to evaluate the release of aroma was performed. 

Line 811: It is necessary to ascertain whether the events described in references 91 and 92 also occur in this study. 

Line 818: Has this study demonstrated the characteristics or circumstances that can be described as 'synergistic effect', 'wine balance' or 'modulatory role'. Describe it in detail. 

The abbreviations and italics on line 869 be revised. 

Appropriate reference must be included before the bullet point on line 870. 

Which would be the recommendation derived from the results of this study, as outlined in lines 893-896. 

Line 899:  'improved sustainability' is important and must be detailed or addressed in reference to the results. 

Lines 909-912, the final sentence is not comprehensible due to its excessive generalisation. It is imperative that the supporting details are delineated prior to the utilisation of this particular expression. 

Line 922: which aromas improve the expression of the Furmint variety'. 

Line 932: Would be the addition of tannin is essential for botrytised grapes?, and if so, the underlying reasons why. 

In table S1, species should be denoted by italics. 

The extensive introductory section should be refocused and made more concise, and the discussion section should be reinforced with references (perhaps with many of the references in the introduction), since the amount of data generated is important and should be more thoroughly contrasted. 

Author Response

Dear Reviewer,

We thank the Reviewer for the valuable comments and constructive suggestions!

Comments1. Line 11: The term 'specialised yeast strains' is ambiguous and requires clarification. Does it refer to non-Saccharomyces species? 

Response1. We thank the Reviewer for this observation and fully agree that the term ‘specialised yeast strains’ was ambiguous in its original form. We have now clarified this expression in the manuscript to explicitly indicate that it refers to non-Saccharomyces yeast species.

 

Comments 2. Line 14: 'Strain-specific commercial yeast remain unavailable' – this is not the objective of the study, and in addition, commercial strains are used. 

Response 2. We can agree and repaired it.

 

Comments 3. Line 19: Please clarify whether this refers to the use of these yeasts in co-inoculation or sequential inoculation. 

Response 3. This has been corrected in the revised manuscript.

 

Comments 4. Line 24: 'Enhance organoleptic properties” – “compensate negative effects of climate change”. It is necessary to specify which properties or characteristics detected in the study enhance or compensate for the described effects. 

Response 4. We thank the Reviewer for this important comment and fully agree that more specificity was needed and we did it.

 

Comments 5. In the introduction, line 26 should provide a description of how bioproduction can lead to a reduction in sulphur use. 

Response 5. This has been corrected in the revised manuscript.

 

Comments 6. It is imperative that 'Vitis vinifera' is italicised throughout the text. 

Response 6. We thank the Reviewer for pointing this out. All instances of Vitis vinifera have been checked and correctly italicised throughout the revised manuscript.

 

Comments 7. Line 70-72: In this instance, the specific information pertaining to the variety utilised in the study has been employed with reference to the corresponding study. It is imperative that this is given due consideration and incorporated into the remainder of the introduction. For instance, in lines 83 and 88, it is imperative to insert the relevant reference that accurately describes these characteristics. 

Response 7. We thank the Reviewer for this insightful comment. We acknowledge the importance of properly referencing the specific characteristics of the grape variety used in our study. Accordingly, we have now incorporated the relevant citation in the indicated sections (lines 83 and 88) and ensured that the source is consistently acknowledged throughout the Introduction.

 

Comments 8. It is imperative that only 'primary aromas' and 'fermentative aromas' are used on lines 80 and 81. 

Response 8. Thank you and we corrected it.

 

Comments 9. Lines 92 to 124: In these paragraphs, there is a necessity to include various bibliographic references concerning the aldehydes or esters formed/present. Furthermore, a more specific approach to the Furmint variety or the type of processing used is required. The introduction is very general and lengthy (78 references out of 128 are used in the introduction). The primary focus of the presentation should be on the conditions or particularities of the study being presented. Moreover, it is necessary to acknowledge that a substantial proportion of this information can be employed in the discourse surrounding the results. 

Response 9. We are grateful to the Reviewer for this comprehensive and constructive feedback. In response, we have revised the Introduction thoroughly to address these concerns. Additional bibliographic references have been incorporated to support the discussion of aldehydes and esters, particularly with respect to their formation and relevance in wine aroma. We have refined the focus of the Introduction by adding more specific information on the Furmint grape variety and the particular processing techniques employed in this study. To reduce the length and improve the focus, we have removed some general background content and relocated several paragraphs to the Discussion section, where they more appropriately support the interpretation of our findings. As a result, the number of references in the Introduction has been reduced, and the content now more clearly highlights the specific objectives and context of our research.

 

Comments 10. Ltnes 128-135 must be unified with the description of terpenic compounds, as these compounds have been described in other paragraphs, and the repetition is superfluous. 

Response 10. We thank the Reviewer for this helpful observation. We agree that the discussion of terpenic compounds contained unnecessary repetition. To address this, we have now unified the relevant content into a single, coherent paragraph. Redundant statements have been removed, and the discussion has been streamlined to avoid overlap while maintaining the necessary detail.

 

Comments 11. Line 170-181: This paragraph discusses the concept of 'bioprotection', a term that is also employed in other paragraphs with the objective of unifying information. 

Response 11. Thank you for highlighting this point, we corrected it.

 

Comments 12. Line 195-208: It is recommended that the paragraph outlining 'non-Saccharomyces' yeasts be unified with the paragraph below it in Section 1.2.1.

Response 12. Thank you and we corrected it.

 

Comments 13. It is imperative to verify that all yeast species delineated are italicised. 

Response 13. We thank the Reviewer for drawing our attention to this detail. We have carefully reviewed the manuscript and tried to use correctly italicised name of yeast strains throughout the text.

 

Comments 14. Following the description of the full species name (Metschnikowia pulcherrima), the abbreviation (M. pulcherrima) should be used throughout the text (error lines 386, 391). It is imperative to meticulously revise the text to ensure its accuracy for all species, as evidenced by the erroneous designation of B. cinerea on line 329 and the inaccuracy on line 639. 

Response 14. Thank you and we corrected the abbreviations in the whole text.

 

Comments 15. Line 275-284: necessary to ascertain whether there are specific studies that focus on the Furmint variety. 

Response 15. We thank the Reviewer for this valuable suggestion. In response, we have reviewed the literature and identified relevant studies that specifically investigate the Furmint grape variety.

 

Comments 16. Line 370: Please provide clarification of the term 'blue varieties'. 

Response 16. We have clarified this terminology in the text.

 

Comments 17. It is recommended that the information contained on lines 377-379 be removed, as it is repetitive. 

In order to be included in the description of all commercial preparations of micro-organisms or tanin, as appropriate, and all information on the preparations, such as the supplier. 

It is recommended that the sentence commencing on line 430 and concluding on line 433 be removed, since the detailed information required is described below. 

Response 17. We removed it.

 

Comments 18. Line 436: Subindices and spaces require revision. 

Response 18. We coorected it.

 

Comments 19. Line 436: It is necessary to provide a detailed account of the number of times or frequency with which sampling was performed. 

Response 19. We have revised the manuscript to include a detailed description of the sampling procedure. Specifically, we now indicate the number of sampling points and the frequency at which samples were collected during the fermentation process.

 

Comments 20. In Table 1, the term 'M. pulcherrima' is written with italics. 

Response 20. We corrected it, thank you.

 

Comments 21. Note that Starmerella has not been referenced in preceding sections, and its role requires elucidation. 

Response 21. Thank you, we agree and clarified the role of Starmerella.

 

Comments 22. In Figure 4, there is a clear necessity for enhancement, as the descriptor names are not clearly visible, and the figure's caption provides an insufficient level of detail. 

Response 21. We corrected the Figure 4.

 

Comments 23. Line 773-785: A detailed description of the figures in which these results are presented is required. 

Response 23. We tried to provide more detailed descriptions and interpretations of the figures in the revised text.

 

Comments 24. Line 775: In this study, the term 'broader aromatic variability' is employed to denote the presence of Saccharomyces and non-Saccharomyces, as well as Botrytis, in the examined samples. It is imperative to incorporate references that either substantiate or contradict these findings. 

Response 24. We agree that the use of the term ‘broader aromatic variability’ in the context of mixed microbial presence requires proper scientific support. Accordingly, we have revised the manuscript to include relevant references that demonstrate how the coexistence of Saccharomyces, non-Saccharomyces yeasts, and Botrytis cinerea can contribute to enhanced aromatic diversity in wine.

 

Comments 25. Line 805-808: describe if a suitable method exists to evaluate the release of aroma was performed. 

Response 25. We precised the original sentence in the text.

 

Comments 26. Line 811: It is necessary to ascertain whether the events described in references 91 and 92 also occur in this study. 

Response 26. We have carefully re-examined our data in light of the findings reported in references 91 and 92. We have now included a discussion of this comparison in the Discussion section to clarify the relationship between our findings and those reported in the cited studies.

 

Comments 27. Line 818: Has this study demonstrated the characteristics or circumstances that can be described as 'synergistic effect', 'wine balance' or 'modulatory role'. Describe it in detail. 

Response 27. We did the description in the text,

 

Comments 28. The abbreviations and italics on line 869 be revised. 

Appropriate reference must be included before the bullet point on line 870. 

Response 28. We did it.

 

Comments 29. Which would be the recommendation derived from the results of this study, as outlined in lines 893-896. 

Response 29. We have revised the conclusion section to more explicitly articulate the main recommendation based on our findings.

 

Comments 30, Line 899:  'improved sustainability' is important and must be detailed or addressed in reference to the results. 

Response 30. We agree that the term “improved sustainability” should be supported by specific results from the study. Accordingly, we have revised the manuscript to clarify how the applied biotechnological approach—namely the use of Metchnikowia pulcherrima and oenological tannins—can contribute to sustainability.

 

Comments 31. Lines 909-912, the final sentence is not comprehensible due to its excessive generalisation. It is imperative that the supporting details are delineated prior to the utilisation of this particular expression. 

Response 31. We agree that the final sentence, as originally written, was overly general and lacked sufficient supporting detail. In response, we have revised this part of the conclusion to first outline the specific results and observations that justify our final statement.

 

Comments 32. Line 922: which aromas improve the expression of the Furmint variety. 

Response 32. We have clarified it in the text.

Comments 33. Line 932: Would be the addition of tannin is essential for botrytised grapes?, and if so, the underlying reasons why. 

Response 33. We clarified it in the text.

 

Comments 34. In table S1, species should be denoted by italics. 

Response 34. We corrected it.

 

Comments 35. The extensive introductory section should be refocused and made more concise, and the discussion section should be reinforced with references (perhaps with many of the references in the introduction), since the amount of data generated is important and should be more thoroughly contrasted. 

Response 35. We thank the Reviewer for this constructive and well-founded suggestion. In response, we have substantially revised the manuscript to improve its structure and focus. Specifically, the Introduction has been shortened and streamlined to highlight only the most relevant background information directly related to the aims of the study. As recommended, several references previously cited in the Introduction have been relocated to the Discussion section to strengthen the interpretation and contextualisation of our results.

We once again thank the Reviewer for the thoughtful and helpful comments that have greatly contributed to improving the quality and clarity of our manuscript.