Composition Divergence and Synergistic Mechanisms in Microbial Communities During Multi-Varietal Wine Co-Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The main objective of this manuscript is to characterize in depth the composition and dynamics of the microbial community present during the cofermentation process. The scientific relevance of this study lies in its potential to provide fundamental knowledge to optimize winemaking practices worldwide, contributing to the improvement of wine production and quality. For this reason, the manuscript is of great interest to a broad sector of the scientific community as well as to the productive sector. 

The methodological approach adopted is appropriate and allows for a rigorous evaluation of the hypothesis. Despite this, identifying and resolving some specific issues would contribute to the overall soundness of the study.

1) When reviewing the definition of the objectives, it is observed that the first of them mentions the comparison of the diversity and structural characteristics of the microbial communities in different co-fermentation processes. However, the study focuses on the analysis of the bacterial community. I would suggest the authors to reconsider the wording of this objective to match the actual scope of the work. Additionally, I would like to raise the following question: could you explain the reason why the research focused only on the bacterial population, given the known importance of yeasts in fermentation and the final characteristics of the wine?

2) In the introduction, second paragraph, the italics have been omitted in the word non-Saccharomyces, which requires correction.

3) In the Material and Methods section, it is crucial to detail the methodology employed in the co-fermentation processes. In order to be able to attribute the results to the different types of co-fermentation, a precise description of how these processes were carried out is required. It is assumed that the conditions were the same, but could the key parameters be specified? Additionally, were the same conditions used in all cases? If not, what were the variations? Also, to assess the impact of proportions and to facilitate replicability and optimization, could you please indicate the volumes of must of each grape variety used in each co-fermentation?

4) The microbial diversity present in grapes, and therefore in the production process, is directly related to the cultivation and vineyard management practices. Therefore, it would be pertinent for the authors to provide information on the type of cultivation (e.g., conventional, organic, biodynamic) of the four grape varieties used. These details would facilitate the extrapolation of the findings to the production sector.

5) The study focuses on the co-fermentation of Cabernet Sauvignon with Marselan, Merlot and Cabernet Sauvignon, with the objective of analyzing their effects on the microbial community, its dynamics and functional characteristics. However, a control group consisting of the characterization of the spontaneous fermentation process of the Cabernet Sauvignon must (base wine) separately is missing. Could the authors explain why a detailed study of the spontaneous fermentation process of the Cabernet Sauvignon must alone was not included? Such a comparative analysis would be essential to identify the specific contributions of co-fermentation versus individual fermentation of the base variety.

6) On page 8, the text of section 3.3. "Differences and Distribution of Microbial Communities Across Different Fermentation Process" appears in italics and should be modified.

7) On page 10, session 3.4. "Ecological Characteristics of Core Microbial Communities in Three Co-Fermentation Processes", Figure 5 is missing.

8) In the conclusions, the authors state that “In the CSCG process, the high concentration of phenolic compounds strongly selected specific microbial taxa, reducing the contribution of neutral processes to the community assembly to 45.5%”. This conclusion is presented with great emphaticness. However, given the lack of a parallel analysis of the fermentation of the CS variety must separately (as noted above), it is difficult to determine the extent to which these effects are specific to cofermentation and not inherent to the base variety fermentation. How can the specific impact of cofermentation and selection by phenolic compounds be determined with such certainty. I suggest a modification in the way the conclusion is expressed.

9) The conclusion stating that co-fermented wines show 'more complex and enriched' aromatic profiles following the significant increase in certain compounds (isoamyl acetate, ethyl hexanoate, linalool, geraniol) would be stronger if the reference point for this comparison were specified. Does it refer to a comparison with single varietal Cabernet Sauvignon fermentation or with other non-co-fermented wines? Including this reference in the conclusion would provide greater clarity and context.

 

 

Author Response

Reviewer #1:

The main objective of this manuscript is to characterize in depth the composition and dynamics of the microbial community present during the cofermentation process. The scientific relevance of this study lies in its potential to provide fundamental knowledge to optimize winemaking practices worldwide, contributing to the improvement of wine production and quality. For this reason, the manuscript is of great interest to a broad sector of the scientific community as well as to the productive sector. The methodological approach adopted is appropriate and allows for a rigorous evaluation of the hypothesis. Despite this, identifying and resolving some specific issues would contribute to the overall soundness of the study.

RESPONSE: Thank you very much for the suggestion. We will carefully consider your suggestions and make further improvements and additions to the relevant sections of the manuscript to strengthen its quality and rigor. Thank you again for your valuable feedback and suggestions.

1. COMMENT: When reviewing the definition of the objectives, it is observed that the first of them mentions the comparison of the diversity and structural characteristics of the microbial communities in different co-fermentation processes. However, the study focuses on the analysis of the bacterial community. I would suggest the authors to reconsider the wording of this objective to match the actual scope of the work. Additionally, I would like to raise the following question: could you explain the reason why the research focused only on the bacterial population, given the known importance of yeasts in fermentation and the final characteristics of the wine?

RESPONSE: Thank you very much for your insightful comments. We appreciate your suggestion regarding the wording of our research objectives. We agree that the original objective may have implied a broader scope than what was actually investigated. In the revised manuscript, we have modified the objective to explicitly state that the study focuses on the comparison of the diversity and structural characteristics of bacterial communities in different co-fermentation processes. This revision more accurately reflects the actual content and scope of our work. Regarding your question on why the research focused solely on the bacterial population: While the role of yeasts in wine fermentation is well established, with many aspects of their ecology and function being extensively studied and optimized in modern winemaking, the bacterial communities—especially their diversity, dynamics, and contributions during co-fermentation—remain less understood. Bacteria, particularly lactic acid bacteria and acetic acid bacteria, play crucial roles in modulating fermentation metabolites, wine stability, and sensory profiles. However, their complex interactions and potential to influence wine quality are often underappreciated and may differ significantly among fermentation strategies. Therefore, we specifically targeted the bacterial community to fill this gap in knowledge and to provide a systematic evaluation of their contributions in the context of co-fermentation. We have added the description in the introduction. Thank you again for your thoughtful feedback. We hope these clarifications address your concerns.

2. COMMENT: In the introduction, second paragraph, the italics have been omitted in the word non-Saccharomyces, which requires correction.

RESPONSE: Thank you for your careful review and attention to detail. We appreciate your observation regarding the formatting of the word non-Saccharomyces in the second paragraph of the introduction. We have corrected this issue and other mistakes in the revised manuscript.

3. COMMENT: In the Material and Methods section, it is crucial to detail the methodology employed in the co-fermentation processes. In order to be able to attribute the results to the different types of co-fermentation, a precise description of how these processes were carried out is required. It is assumed that the conditions were the same, but could the key parameters be specified? Additionally, were the same conditions used in all cases? If not, what were the variations? Also, to assess the impact of proportions and to facilitate replicability and optimization, could you please indicate the volumes of must of each grape variety used in each co-fermentation?

RESPONSE: Thanks for the suggestion. We have added the information in the Material and Methods section: Samples were collected from a wine fermentation facility at a commercial winery in Yantai, China (121.17°E, 37.57°N). Grapes used in this study included Cabernet Sauvignon (CS), Cabernet Sauvignon blended with Marselan (CSMN), Cabernet Sauvignon blended with Merlot (CSMT), and Cabernet Sauvignon blended with Cabernet Gernischt (CSCG), all sourced from commercial suppliers. These particular varieties were selected due to their strong market demand and widespread popularity among consumers, rather than their local cultivation in the Yantai region. The harvest occurred between August 20 and September 1, 2024, during which the local climate in Yantai featured minimum and maximum daily temperatures of 19°C and 29°C, total precipitation of approximately 28 mm, relative humidity ranging from 38% to 54%, and an average of 7.4 hours of daily sunshine, providing favorable conditions for grape ripening. After harvest, grapes were destemmed and crushed. Single-varietal fermentations were carried out with 100% Cabernet Sauvignon must, while for each co-fermentation group, Cabernet Sauvignon and the respective blending variety were mixed at a 1:1 volume ratio. All fermentations were conducted in duplicate in 10 L stainless steel tanks under identical conditions: fermentation temperature at 25°C, initial sugar content of 23.0–24.0 °Brix, and sulfur dioxide addition at 50 mg/L. Commercial Saccharomyces cerevisiae yeast was used for inoculation when required. The fermentation process was monitored daily and considered complete when the residual sugar content dropped below 2 g/L. Upon completion, the wines were racked and stored at 15°C prior to further analysis.

4. COMMENT: The microbial diversity present in grapes, and therefore in the production process, is directly related to the cultivation and vineyard management practices. Therefore, it would be pertinent for the authors to provide information on the type of cultivation (e.g., conventional, organic, biodynamic) of the four grape varieties used. These details would facilitate the extrapolation of the findings to the production sector.

RESPONSE: We fully agree that information on cultivation methods is essential for interpreting and extrapolating our findings into broader production contexts. In our study, all grape samples were sourced from commercial suppliers, and unfortunately, detailed records of the specific cultivation practices (such as whether they were conventional, organic, or biodynamic) for each batch were not available from the suppliers. Based on our communication with the suppliers, the grapes were primarily grown under conventional viticultural management, which is the predominant practice for commercial vineyards targeting large-scale, market-oriented production in China. However, as these vineyards are not certified organic or biodynamic, we cannot exclude the possibility of variations in vineyard management.

5. COMMENT: The study focuses on the co-fermentation of Cabernet Sauvignon with Marselan, Merlot and Cabernet Sauvignon, with the objective of analyzing their effects on the microbial community, its dynamics and functional characteristics. However, a control group consisting of the characterization of the spontaneous fermentation process of the Cabernet Sauvignon must (base wine) separately is missing. Could the authors explain why a detailed study of the spontaneous fermentation process of the Cabernet Sauvignon must alone was not included? Such a comparative analysis would be essential to identify the specific contributions of co-fermentation versus individual fermentation of the base variety.

RESPONSE: We highly appreciate the editor and reviewers for raising the important issue regarding the absence of a separate Cabernet Sauvignon (CS) single-varietal fermentation group in this batch of experiments. In our initial experimental design, our primary intention was to focus on the role of bacteria in co-fermentation, as preliminary observations and literature reports indicate that the bacterial diversity during single-varietal fermentation is relatively limited. Therefore, we did not include or discuss the spontaneous fermentation of CS alone at first. However, after carefully considering your comments, we recognize that including a single-varietal fermentation group is indeed essential for this study. Not only does it provide a necessary control for comparative analysis, but it also allows us to more accurately assess the specific changes in the microbial community and the contributions to metabolite profiles that arise from co-fermentation. In response to your valuable suggestion, we have now added the results and relevant descriptions of the CS single-varietal fermentation group in the revised manuscript. We believe this addition significantly strengthens the scientific rigor and interpretability of our work. Once again, we sincerely thank you for this constructive feedback, which has helped us to improve our research and the completeness of our manuscript.

6. COMMENT: On page 8, the text of section 3.3. "Differences and Distribution of Microbial Communities Across Different Fermentation Process" appears in italics and should be modified.

RESPONSE: Thanks for the suggestion. We have revised it according to the guide for authors.

7. COMMENT: On page 10, session 3.4. "Ecological Characteristics of Core Microbial Communities in Three Co-Fermentation Processes", Figure 5 is missing.

RESPONSE: We sincerely apologize for accidentally omitting Figure 5 during the final layout. We are very sorry for our carelessness and truly appreciate your kind reminder. The figure has now been included in the revised manuscript.

8. COMMENT: In the conclusions, the authors state that “In the CSCG process, the high concentration of phenolic compounds strongly selected specific microbial taxa, reducing the contribution of neutral processes to the community assembly to 45.5%”. This conclusion is presented with great emphaticness. However, given the lack of a parallel analysis of the fermentation of the CS variety must separately (as noted above), it is difficult to determine the extent to which these effects are specific to cofermentation and not inherent to the base variety fermentation. How can the specific impact of cofermentation and selection by phenolic compounds be determined with such certainty. I suggest a modification in the way the conclusion is expressed.

RESPONSE: Thank you very much for your valuable comment. We fully agree with your assessment that, in the absence of a comprehensive parallel analysis of the single-varietal CS fermentation, it is difficult to definitively attribute the observed microbial community assembly patterns solely to the effects of co-fermentation or the selective pressure of phenolic compounds. Although we have added data regarding CS fermentation in the new manuscript, unfortunately, we only preserved three CS samples from the same period. However, at least six parallel samples are required for this analysis. Therefore, to maintain scientific rigor, we have revised the discussion as follows: For CSCG, the model fit (R² = 0.202) was the lowest among all groups. This result suggests that the assembly of microbial communities in the CSCG process may be more strongly influenced by selective pressures associated with the specific chemical properties of the grape must, with a reduced contribution from neutral processes. In particular, the pie chart shows that 45.5% of the microbes fell within the predicted range of the model, while the proportions of rare and dominant taxa increased significantly. These findings imply that characteristics such as a potentially higher phenolic compound content and a more acidic environment in the Cabernet Gernischt blend could play a role in shaping a more specialized microbial community. However, given the lack of a parallel analysis of the single-varietal CS fermentation, we cannot fully exclude the possibility that similar effects might occur in the base variety fermentation. Therefore, the specific contribution of co-fermentation and selection by phenolic compounds requires further investigation in future studies.

9. COMMENT: The conclusion stating that co-fermented wines show 'more complex and enriched' aromatic profiles following the significant increase in certain compounds (isoamyl acetate, ethyl hexanoate, linalool, geraniol) would be stronger if the reference point for this comparison were specified. Does it refer to a comparison with single varietal Cabernet Sauvignon fermentation or with other non-co-fermented wines? Including this reference in the conclusion would provide greater clarity and context.

RESPONSE: Thank you very much for your suggestion. We analyzed the metabolite data from single CS fermentation of the same variety during the same period. We have rewritten the relevant part of the discussion as follows: To explore the relationship between volatile organic compounds (VOCs) and microbial communities in different fermentation processes, the levels of esters (isoamyl acetate, ethyl hexanoate), higher alcohols (isoamyl alcohol, phenethyl alcohol), and terpenes (linalool, geraniol) were measured, along with their correlations with the dominant microbial communities, as shown in Figures 2a, b, and c. Figure 2a shows the distribution of the two main esters (isoamyl acetate and ethyl hexanoate). The total ester content was highest in CSMT (35.98 ± 2.93 mg/L and 16.26 ± 2.15 mg/L), followed by CSMN (22.36 ± 3.78 mg/L and 19.38 ± 1.69 mg/L), CSCG (18.39 ± 2.29 mg/L and 23.35 ± 3.23 mg/L), and lowest in CS (10.56 ± 1.85 mg/L and 8.98 ± 1.12 mg/L). The contents of isoamyl acetate and ethyl hexanoate, two key esters contributing to fruity aroma, are significantly higher in the CSMT group compared to the other groups. The CS group exhibits the lowest ester concentrations, while CSMN and CSCG show intermediate levels. The RDA analysis below further demonstrates a strong positive correlation between Levilactobacillus and ester production, particularly ethyl hexanoate, with CSMT samples clustering closely with these variables. This suggests that the enrichment of certain lactic acid bacteria (LAB) in CSMT may promote ester biosynthesis, thereby enhancing the wine’s fruity and floral aromatic qualities. Figure 2b illustrates the distribution of two major higher alcohols (phenethyl alcohol and isoamyl alcohol). The total higher alcohol content was highest in CSMN (37.79 ± 2.13 mg/L and 41.58 ± 4.98 mg/L), followed by CSMT (40.26 ± 4.22 mg/L and 19.32 ± 2.32 mg/L), CS (27.26 ± 2.18 mg/L and 21.62 ± 1.98 mg/L), and lowest in CSCG (16.78 ± 1.45 mg/L and 15.58 ± 1.87 mg/L). This suggests that the total content of higher alcohols (isoamyl alcohol and phenethyl alcohol) is highest in the CSMN group, followed by CSMT, with CS and CSCG displaying lower concentrations. The RDA results indicate that isoamyl alcohol is positively associated with genera such as Pantoea and Aeromonas, which are more abundant in CSMN. In contrast, phenethyl alcohol and some LAB genera show a closer relationship in other groups. Elevated higher alcohols can contribute complexity but may also impart undesirable sensory effects if present in excess, suggesting that CSMN fermentation may require careful control to balance aroma and quality. Figure 2c shows the distribution of two major terpenes (linalool and geraniol). The total terpene content was highest in CSMT (2.33 ± 0.68 mg/L and 1.05 ± 0.10 mg/L), followed by CSMN (1.32 ± 0.36 mg/L and 0.32 ± 0.04 mg/L), CSCG (0.98 ± 0.12 mg/L and 0.45 ± 0.05 mg/L), and lowest in CS (0.65 ± 0.12 mg/L and 0.33 ± 0.04 mg/L). Terpene compounds (linalool and geraniol), which impart floral and citrus notes, are most abundant in the CSMT group, with CSMN and CSCG exhibiting moderate levels, and CS the lowest. The RDA plot reveals that Lactiplantibacillus is positively correlated with geraniol, and Levilactobacillus with linalool, again emphasizing the role of LAB in enhancing terpene profiles. Notably, CSMT samples are closely associated with both terpenes and the corresponding LAB genera, indicating that co-fermentation with Merlot may create favorable conditions for the accumulation of desirable terpenes. These findings reflect the varied contributions of microbial communities to terpene biosynthesis. The distribution of these three types of VOCs and the RDA analysis collectively shows that the CSMT process exhibits a significant advantage in the production of esters, alcohols, and terpenes. This could be attributed to the chemical composition of Merlot grapes (e.g., sugar and acid content) and their synergistic interaction with Cabernet Sauvignon. The CSMN process stands out in higher alcohol production, indicating that Marselan grapes may be more conducive to enhancing wine complexity. In contrast, the CSCG process generally exhibited lower VOC levels, which may be attributed to the chemical properties of Cabernet Gernischt or the composition of its microbial community, potentially limiting certain metabolic pathways. RDA analysis further reveals the influence of specific microbial communities on VOC production. For example, Levilactobacillus and Lactiplantibacillus significantly promote the formation of esters and terpenes, while Weissella and Pantoea are closely associated with the production of isoamyl acetate and isoamyl alcohol. These results demonstrate that different co-fermentation strategies not only shape the microbial community, but also modulate the synthesis of key aroma compounds via specific microbial–metabolite interactions. Co-fermentation, especially with CSMT, enriches for LAB genera that are closely linked to higher concentrations of esters and terpenes, enhancing the wine’s aromatic complexity and sensory appeal. In contrast, CSMN is more associated with higher alcohols, likely due to the dominance of environmental and acetic acid bacteria. This highlights the importance of grape variety selection and microbial management in controlling wine aroma profiles and optimizing product quality.

Reviewer 2 Report

Comments and Suggestions for Authors

The article discusses an interesting and new topic for winemakers, revealing the differences in microbial communities and their synergistic mechanisms in different co-fermented grape musts. Was revealed that different co-fermentation combinations can exhibit distinct microbial community characteristics.

M&M

2.1. Considering that climatic conditions also influence the dynamics of microorganism populations, it would be necessary to briefly present the type of climate, minimum/maximum annual temperatures, precipitation and other relevant data, in order to have an overall picture. Also, the year(s) of study must be mentioned. What is the motivation for choosing these varieties (are more widespread in cultivation, more popular in the area or other considerations).

The physico-chemical analysis of the wine must be presented to understand under what conditions the mentioned species evolved or resisted (alcohol, pH, total acidity, volatile, extract, phenolic compounds at least total values, free and total SO₂, assimilable nitrogen), most of them according to OIV compendium of official methods.

For comparison the wine obtained from individual fermentations (by each variety) should be evaluate from the microbiota perspective? It should compare with the individual wine’s microbial community.

2.3. Where were the primers purchased from?

2.4. Please mention that LEfSe (Linear discriminant analysis effect size) is an algorithm that identifies genomic features (genes, pathways, or taxa) characterizing the differences between two or more biological conditions; or similar explanations.

The link you provided is not working, please check (https://github.com/Rus-sel88/MicEco?tab=readme-ov-file).

R&D

Please, when you use an abbreviation for the first time, put in brackets the explanation and write correctly to avoid confusion (so that the general public understands) e.g. UpSet plot analysis at the operational taxonomic unit (OTU)....

Why are the references included here? what is the explanation for the references to this phrase? Are the same cultivars implied in previous studies?   “…the combination of Cabernet Sauvignon and Cabernet Gernischt may facilitate metabolic complementation or synergy, promoting the growth of specific microorganisms and resulting in a distinctly differentiated microbial community [22,23]”. Please check and correct if necessary (put as a separate sentence the general information).

General wine analysis is mandatory “This may indicate that the co-fermentation of Cabernet Gernischt and Cabernet Sauvignon creates a selective metabolic environment that strongly influences microbial community composition.”

Figure 2 on the page 6 is in fact the Figure 1. However, it needs to be moved to the previous page.

3.4. Bibliographical references should be added to reinforce the suggestions made and the conclusions drawn.

Conclusions  

“The results showed that co-fermentation significantly influenced the structure, function, and metabolic characteristics of microbial communities.” But, in my opinion, we can't know this clearly if we haven't analyzed the microbiota of each individual wine (single-variety fermentation) and in co-fermentation, and if we don't know the basic composition of the wines. As you wrote in the introduction: “The chemical properties of different grape varieties, such as sugar content, total acidity, pH, phenolic compounds, and volatile compounds, exert significant selective effects on microorganisms, thereby influencing the ecological niche distribution and metabolic functions of microbial communities during fermentation”.

 

References are relevant to the topic discussed.
The supplementary material is partially helpful, with Tables S6 and S7 being of particular interest.

Author Response

Reviewer #2:

The article discusses an interesting and new topic for winemakers, revealing the differences in microbial communities and their synergistic mechanisms in different co-fermented grape musts. Was revealed that different co-fermentation combinations can exhibit distinct microbial community characteristics.

RESPONSE: Thanks for your acknowledgement on the merit of our work. Your detailed reviewing comments surely helped in improving the manuscript. We have revised the manuscript accordingly, please see below point-by-point response.

1. COMMENT: 2.1. Considering that climatic conditions also influence the dynamics of microorganism populations, it would be necessary to briefly present the type of climate, minimum/maximum annual temperatures, precipitation and other relevant data, in order to have an overall picture. Also, the year(s) of study must be mentioned. What is the motivation for choosing these varieties (are more widespread in cultivation, more popular in the area or other considerations).

RESPONSE: Thanks for the suggestion. We have added the description about the normal information of wine fermentation: These particular varieties were selected due to their strong market demand and wide-spread popularity among consumers, rather than their local cultivation in the Yantai region. The harvest occurred between August 20 and September 1, 2024, during which the local climate in Yantai featured minimum and maximum daily temperatures of 19°C and 29°C, total precipitation of approximately 28 mm, relative humidity ranging from 38% to 54%, and an average of 7.4 hours of daily sunshine, providing favorable conditions for grape ripening. After harvest, grapes were destemmed and crushed. Single-varietal fermentations were carried out with 100% Cabernet Sauvignon must, while for each co-fermentation group, Cabernet Sauvignon and the respective blend-ing variety were mixed at a 1:1 volume ratio. All fermentations were conducted in du-plicate in 10 L stainless steel tanks under identical conditions: fermentation tempera-ture at 25°C, initial sugar content of 23.0–24.0 °Brix, and sulfur dioxide addition at 50 mg/L. Commercial Saccharomyces cerevisiae yeast was used for inoculation when re-quired. The fermentation process was monitored daily and considered complete when the residual sugar content dropped below 2 g/L. Upon completion, the wines were racked and stored at 15°C prior to further analysis.

2. COMMENT: The physico-chemical analysis of the wine must be presented to understand under what conditions the mentioned species evolved or resisted (alcohol, pH, total acidity, volatile, extract, phenolic compounds at least total values, free and total SO₂, assimilable nitrogen), most of them according to OIV compendium of official methods.

RESPONSE: Thanks for the suggestion. We have added the table of physical/chemical indicators analysis of the four fermentation processes in the Table S1.

Table S1 physical/chemical indicators of four fermentation processes

Parameter	CS Range	CSMN Range	CSMT Range	CSCG Range
Alcohol (% v/v)	13.0 – 14.0	12.8 – 13.8	13.2 – 14.2	12.9 – 13.7
pH	3.50 – 3.75	3.45 – 3.70	3.48 – 3.73	3.47 – 3.72
Total Acidity (g/L)	5.8 – 7.0	5.9 – 7.2	5.7 – 7.0	5.8 – 7.1
Volatile Acidity (g/L)	0.30 – 0.60	0.32 – 0.65	0.29 – 0.58	0.31 – 0.63
Extract (g/L)	20 – 25	21 – 26	21 – 27	20 – 26
Total Phenolics (mg/L)	1800 – 2300	1850 – 2400	1900 – 2350	1800 – 2250
Free SO₂ (mg/L)	22 – 35	20 – 34	22 – 36	21 – 34
Total SO₂ (mg/L)	90 – 140	88 – 145	92 – 145	89 – 140
Assimilable Nitrogen (mg/L)	140 – 220	150 – 230	145 – 225	142 – 220

3. COMMENT: For comparison the wine obtained from individual fermentations (by each variety) should be evaluate from the microbiota perspective? It should compare with the individual wine’s microbial community.

RESPONSE: Thanks for the suggestion. We highly appreciate the editor and reviewers for raising the important issue regarding the absence of a separate Cabernet Sauvignon (CS) single-varietal fermentation group in this batch of experiments. In our initial experimental design, our primary intention was to focus on the role of bacteria in co-fermentation, as preliminary observations and literature reports indicate that the bacterial diversity during single-varietal fermentation is relatively limited. Therefore, we did not include or discuss the spontaneous fermentation of CS alone at first. However, after carefully considering your comments, we recognize that including a single-varietal fermentation group is indeed essential for this study. Not only does it provide a necessary control for comparative analysis, but it also allows us to more accurately assess the specific changes in the microbial community and the contributions to metabolite profiles that arise from co-fermentation. In response to your valuable suggestion, we have now added the results and relevant descriptions of the CS single-varietal fermentation group in the revised manuscript. We believe this addition significantly strengthens the scientific rigor and interpretability of our work. Once again, we sincerely thank you for this constructive feedback, which has helped us to improve our research and the completeness of our manuscript.

4. COMMENT: 2.3. Where were the primers purchased from?

RESPONSE: Thanks for pointing this out. We have added the description that The PCR amplification was performed using the forward primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'), which were synthesized by BGI (Shenzhen, China).

5. COMMENT: 2.4. Please mention that LEfSe (Linear discriminant analysis effect size) is an algorithm that identifies genomic features (genes, pathways, or taxa) characterizing the differences between two or more biological conditions; or similar explanations.

RESPONSE: We sincerely appreciate the reviewer's constructive suggestion. In the revised manuscript, we have explicitly clarified the purpose of LEfSe analysis in the Methods section by adding a brief description of the algorithm's function. Specifically, we stated that "LEfSe (Linear Discriminant Analysis Effect Size) was employed to identify statistically significant taxa that differentiate microbial communities across distinct biological conditions". 

6. COMMENT: The link you provided is not working, please check (https://github.com/Rus-sel88/MicEco?tab=readme-ov-file).

RESPONSE: Thanks for the suggestion. We have revised it to “https://github.com/Russel88/MicEco”.

7. COMMENT: Please, when you use an abbreviation for the first time, put in brackets the explanation and write correctly to avoid confusion (so that the general public understands) e.g. UpSet plot analysis at the operational taxonomic unit (OTU)....

RESPONSE: Thanks for the suggestion. Although section 2.4 of the original manuscript indicated the full name of OTU, we put it again in section 3.1 for the convenience of readers.

8. COMMENT: Why are the references included here? what is the explanation for the references to this phrase? Are the same cultivars implied in previous studies? “…the combination of Cabernet Sauvignon and Cabernet Gernischt may facilitate metabolic complementation or synergy, promoting the growth of specific microorganisms and resulting in a distinctly differentiated microbial community [22,23]”. Please check and correct if necessary (put as a separate sentence the general information).

RESPONSE: Thank you for your comment regarding the use of references in this statement. We appreciate the opportunity to clarify. References 22 and 23 were mainly used to help us understand the bacterial community composition in different co-fermentation processes. Based on the insights provided by these studies, we analyzed our own results and inferred the possible metabolic complementation or synergy in our co-fermentation system. However, the references themselves do not directly demonstrate the synergy between Cabernet Sauvignon and Cabernet Gernischt. We have revised the sentence to clarify this distinction and to present the general information and our inference separately.

9. COMMENT: General wine analysis is mandatory “This may indicate that the co-fermentation of Cabernet Gernischt and Cabernet Sauvignon creates a selective metabolic environment that strongly influences microbial community composition.”

RESPONSE: Thank you for your constructive comment regarding the necessity of general wine analysis to support our conclusion. We would like to clarify that our inference about the selective metabolic environment created by co-fermentation is primarily based on our UpSet plot analysis. The UpSet plot clearly demonstrates that single-varietal fermentations result in a relatively limited bacterial community composition, whereas co-fermentation introduces greater microbial diversity and distinct community structures. This observed increase in microbial diversity supports our statement regarding the influence of co-fermentation on the microbial environment. We have revised it to “Based on the UpSet plot analysis, which demonstrates that single-varietal fermentations harbor a limited bacterial community while co-fermentation significantly increases microbial diversity, we infer that the co-fermentation of Cabernet Gernischt and Cabernet Sauvignon creates a more selective metabolic environment that strongly influences the composition and diversity of the microbial community.”.

10. COMMENT: Figure 2 on the page 6 is in fact the Figure 1. However, it needs to be moved to the previous page.

RESPONSE: Thanks for the suggestion. We have revised it.

11. COMMENT: 3.4. Bibliographical references should be added to reinforce the suggestions made and the conclusions drawn.

RESPONSE: Thanks for the suggestion. We have included additional references to further support the scientific validity of our conclusions.

12. COMMENT: “The results showed that co-fermentation significantly influenced the structure, function, and metabolic characteristics of microbial communities.” But, in my opinion, we can't know this clearly if we haven't analyzed the microbiota of each individual wine (single-variety fermentation) and in co-fermentation, and if we don't know the basic composition of the wines. As you wrote in the introduction: “The chemical properties of different grape varieties, such as sugar content, total acidity, pH, phenolic compounds, and volatile compounds, exert significant selective effects on microorganisms, thereby influencing the ecological niche distribution and metabolic functions of microbial communities during fermentation”.

RESPONSE: Thank you very much for your thoughtful comment. To address this concern, we specifically included a classic single-variety fermentation group using Cabernet Sauvignon (CS) in our experimental design. We conducted detailed analyses of both the microbial community composition and metabolite profiles for the CS single fermentation and the co-fermentation groups. These comparative data provided a solid basis for our conclusions regarding the influence of co-fermentation on the structure and metabolic characteristics of the microbial communities. We have clarified this point in the revised manuscript, and we appreciate your suggestion, which prompted us to further emphasize the robustness of our experimental approach.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The main requests and recommendations were made.