Organic Acids in Varietal Red Wines: Influence of Grape Cultivar, Geographical Origin, and Aging

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I have reviewed the manuscript beverages-3647505 by Jesús Heras-Roger and co-authors, “Organic Acids in varietal Red Wines: Influence of Grape Cultivar, Geographical Origin and Ageing.” The goal of this study was to comprehensively analyze the organic acid profiles (both major and minor phenolic acids) of a large set of monovarietal red wines produced from predominantly native grape cultivars of the Canary Islands, using HPLC and enzymatic methods. The authors aim to assess how the organic acid profile differs based on grape cultivar, island of origin, denomination of Origin of the wine and ageing of the wine and to identify relationships between these factors (genetic and environmental) and the organic acid composition. For this purpose, they utilize correlation studies and multivariate analysis to extract maximum information from the data and differentiate wine samples based on the considered factors.

 

Introduction

The authors should expand on the literature review with regard to the use of organic acid profiles for authentication purposes. Moreover, I suggest expanding on the connection of minor acids, to variety and region, in order to be used as markers of geographical and varietal origin of the wines.

 

Originality/novelty

The analytical techniques and classifications (e.g., PCA, LDA for varietal/geographic separation) used in this study have been previously applied in wine studies. However, the specific focus of the study is on native grape cultivars of the Canary Islands, which is a key aspect of its originality. Moreover, the combination of major and minor organic acids is not frequently encountered.

 

Abstract

I suggest to incorporating some numerical data in the abstract in order for it to become more informative.

 

Methodology

• Line 154: using specific enzymatic kits, please specify manufacturer of kits
• It is unclear how many replicates per wine were analyzed. Please provide the number of replications for each analysis. Also add it in Tables 1 and 2.

 

Statistical analysis

Given the size of the dataset, the authors should consider using a validation subset to strengthen the robustness of the LDA model.

 

Discussion

The authors should discuss correlations and explain the biological or chemical mechanisms involved. In the points it isn’t done already, the authors should try interpreting the correlations in enological terms.  

Lines 757-768. Some references should be added here.

 

Conclusions

The authors should expand on the practical value of their study for the wine industry.

Author Response

Thank you very much for your kind comments, corrections and suggestions, which have allowed us to improve our manuscript

• The authors should expand on the literature review with regard to the use of organic acid profiles for authentication purposes. Moreover, I suggest expanding on the connection of minor acids, to variety and region, in order to be used as markers of geographical and varietal origin of the wines.

We appreciate this valuable suggestion. We have expanded the Introduction to include additional literature discussing the use of organic acid profiles for wine authentication and typicity. We have also introduced a brief overview of how minor phenolic acids, particularly hydroxybenzoic and hydroxycinnamic acids, have been used as potential chemical markers of grape variety and terroir in previous works. 

• The analytical techniques and classifications (e.g., PCA, LDA for varietal/geographic separation) used in this study have been previously applied in wine studies. However, the specific focus of the study is on native grape cultivars of the Canary Islands, which is a key aspect of its originality. Moreover, the combination of major and minor organic acids is not frequently encountered.

We thank the reviewer for acknowledging the originality of our study in focusing on native Canary Island grape cultivars and in the combined analysis of both major and phenolic organic acids. We have slightly reworded the closing lines of the Introduction and Abstract to emphasize this novel aspect of our research.

• I suggest to incorporating some numerical data in the abstract in order for it to become more informative.

Thank you for this helpful suggestion. We have revised the Abstract to include representative numerical results to support the relevance of the study’s findings.

• Methodology: Line 154: using specific enzymatic kits, please specify manufacturer of kits. It is unclear how many replicates per wine were analyzed. Please provide the number of replications for each analysis. Also add it in Tables 1 and 2.

1. We have added the manufacturer information for the enzymatic kits used, which are Biosystems (Spain).
2.  Each wine sample was analyzed in triplicate (n = 3). This information has been added to the Methods sections for clarity.

• Statistical analysis. Given the size of the dataset, the authors should consider using a validation subset to strengthen the robustness of the LDA model.

While our dataset comprises a representative and diverse collection of samples, we acknowledge that larger sample sets would enhance the robustness of statistical modeling. To address this limitation, we applied cross-validation to all LDA models and reported both original and post-validation classification accuracies. This approach allows for an assessment of internal reliability and the generalizability of the models within the dataset.

However, we recognize that external validation would provide a more realistic simulation of predictive performance in real-world scenarios. Therefore, we consider the inclusion of a validation subset a promising avenue for future research, especially in longitudinal studies that incorporate new vintages, wineries, or cultivars over time.

In our study, the sampling aimed to reflect the actual production patterns on the island, resulting in a greater representation of younger wines and certain cultivars or islands where red wines are less common. This distribution may influence model performance and should be taken into account when interpreting results. A note to this effect has been added to the Discussion section, under the statistical limitations of the model.

• Discussion. The authors should discuss correlations and explain the biological or chemical mechanisms involved. In the points it isn’t done already, the authors should try interpreting the correlations in enological terms.  

We thank the reviewer for this recommendation. We have revisited the correlation section in the Discussion and added further interpretation of key significant correlations in enological terms

• Lines 757-768. Some references should be added here.

 Thank you for the suggestion. We have  added supporting references in this section, which help reinforce the relevance for our study.

• Conclusions. The authors should expand on the practical value of their study for the wine industry.

We appreciate this comment. The Conclusions section has been revised to include a short paragraph discussing the practical implications of our findings for the wine sector. Specifically, we highlight how the use of organic acid profiling—particularly the identification of varietal or regional markers—may support efforts in authenticity certification or fraud prevention, winemaking strategies, and the valorization of indigenous cultivars within local and export markets.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Organic acids significantly influence wine quality, particularly in varietal compositions. This comprehensive study investigates the variability of major acids (tartaric, malic, lactic, citric, acetic, and gluconic) and minor phenolic acids (gallic, protocatechuic, syringic, caftaric, caffeic, cutaric, coumaric, and 2-S-glutathionylcaftaric) in red wines produced from autochthonous grape cultivars of the Canary Islands. Analysis of 205 monovarietal red wine samples reveals distinctive organic acid profiles related to grape cultivar, island of origin, denomination of origin, and aging processes, highlighting the interplay between genetic and environmental factors. The research employs high-performance liquid chromatography for minor acids and enzymatic methods for major organic acids. Significant variations observed across cultivars, geographical origins, and aging conditions demonstrate the complex nature of organic acid composition in red wines and its relationship with viticultural factors. Through correlation studies and multivariate analysis, the research identifies key relationships among variables and differentiates samples based on critical factors. This investigation enhances understanding of acid composition and equilibria in red wines, potentially informing future research on quality parameters and regional typicity that characterize wines from the Canary Islands.

Please answer the following questions.

 

L22: Is the degradation of tartaric acid by Acetobacter tropicalis a phenomenon that occurs in normal wine vinification? Please confirm, including other literature.

L125: Why did this study not examine the organic acid composition of the grapes used as raw materials, rather than just the wine? Please discuss whether examining the organic acid composition of the grapes could reflect the geographic distribution of each grape variety.

 

Please add a period at the end of the caption for the following figure and table.

L360: Figure 1. Organic acids concentration with significant differences according to wine ageing

L373: Figure 2. Phenolic acids groups with significant differences according to wine ageing

L412: Table 3. Correlations among the acids considered in the red wines

Table S1. Red wine samples distribution with abbreviations employed

Table S4. Acid profile according to the DO from Tenerife Island

 

Please reconsider the caption for the following figure.

L400: Figure 3. Relationship between Coum and Caft

L408: Figure 4. Relationship between 2SGl and TE.HC.

Author Response

• L22: Is the degradation of tartaric acid by Acetobacter tropicalis a phenomenon that occurs in normal wine vinification? Please confirm, including other literature.

Thank you for this valuable comment. We confirm that tartaric acid degradation by Acetobacter tropicalis is not a typical phenomenon observed during standard wine vinification. Under normal conditions, tartaric acid is highly stable and not significantly metabolized by the microbial populations commonly involved in alcoholic fermentation. The dominant microbial contributors to organic acid transformation during vinification are lactic acid bacteria, primarily through malolactic fermentation, which primarily targets malic acid and might latter metabolize tartaric acid.

While Acetobacter species have been shown under laboratory conditions to degrade tartaric acid when it is the sole carbon source, such activity is unlikely to occur during controlled vinification due to the inhibitory wine matrix, including low pH and high ethanol levels. Typically, lactic acid bacteria, rather than Acetobacter, are responsible for tartaric acid degradation, and this occurs mainly under specific conditions where malolactic fermentation is not well controlled, leading to undesirable “wine illness.”

We have revised the manuscript to reflect this clarification and included references to support the rarity of tartaric acid degradation under typical winemaking conditions

• L125: Why did this study not examine the organic acid composition of the grapes used as raw materials, rather than just the wine? Please discuss whether examining the organic acid composition of the grapes could reflect the geographic distribution of each grape variety.

We thank the reviewer for this insightful suggestion. This study was specifically designed to evaluate the organic acid composition of finished red wines from the Canary Islands to explore how these acids vary according to different factors, with the aim of identifying potential markers of authenticity and typicity.

We fully agree that analyzing the initial acid composition of the grapes would provide valuable information and might indeed enhance understanding of how varietal and geographical differences are preserved or transformed during winemaking. However, we consider this to be a distinct objective more suited for a separate investigation focused on grape physiology and pre-fermentative stages. As such, we view this as a promising direction for future research, and we are grateful for the suggestion, which complements our future projects.

• Please add a period at the end of the caption for several figures and tables.

The missing periods have been added at the end of all the mentioned figure and table captions in both the main text and the supplementary materials.

• Please reconsider the caption for the following figures:.

-L400: Figure 3. Relationship between Coum and Caft

-L408: Figure 4. Relationship between 2SGl and TE.HC.

Thank you for your suggestion. We have revised the captions for Figures 3 and 4 to improve clarity and ensure that they accurately reflect the content of the figures. 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The paper, which discusses the characterization and classification of wines from spanish regions is in some places difficult to follow because of the abbreviations used and the fact that their definitions are presented at different points including in the supplementary materials. The statistical analysis presented is sound but needs to discussed further. For PCA I would recommend the use of all three principal components to effect good separation. The concentrations of acids is given in grams, and I would suggest that using moles would be better.

Author Response

Thank you very much for your kind comments, corrections and suggestions, which have allowed us to improve our manuscript.

• The paper, which discusses the characterization and classification of wines from spanish regions is in some places difficult to follow because of the abbreviations used and the fact that their definitions are presented at different points including in the supplementary materials.

We understand that the manuscript may be difficult to follow at times due to the number of abbreviations used throughout the text. To improve clarity and readability, we have now compiled all relevant abbreviations at the end of Section 2.2 (Analytical Methods)

• The statistical analysis presented is sound but needs to discussed further.

Thank you for your valuable suggestion. We have expanded the discussion related to the statistical analyses, particularly the interpretation of the multivariate methods (PCA and LDA), to better highlight their significance and implications. 

• For PCA I would recommend the use of all three principal components to effect good separation.

We sincerely appreciate your recommendation. We have tested the visualization using all three principal components. However, we found that the inclusion of the third component did not improve the visual separation of samples and, in fact, made interpretation more difficult due to overlapping projections. For this reason, we have opted to retain the biplot based on the first two components,

 

• The concentrations of acids is given in grams, and I would suggest that using moles would be better.

Thank you for this thoughtful suggestion. While we agree that expressing acid concentrations in moles would be more chemically rigorous, in the context of winemaking and enology, concentrations are most commonly reported in grams per liter in both industry practice and the literature cited. To maintain consistency and comparability with prior studies, we have chosen to retain the current units.

Reviewer 4 Report

Comments and Suggestions for Authors

A very interesting manuscript on organic acid content of varietal red wines from Canary Islands. The manuscript is very well prepared and loaded with interesting and useful information. The materials and methods and suitable for such a study, the results are clearly presented and thoroughly analyzed and discussed. Only a couple of minor improvements are necessary:

l.  75, 78, 79 please write genera names in italics

l. 75 please specify if 'Lactobacillus' refers to the Lactobacillus genus after the nomenclature change or the Lactobacillus sensu lato genus, i.e. before the nomenclature change

Table 2. please check the TE.HC value of LN 

Author Response

Thank you very much for your kind comments, corrections and suggestions, which have allowed us to improve our manuscript.

• Comment l.  75, 78, 79 please write genera names in italics

Thank you for pointing this out. We have revised the manuscript accordingly and ensured that all bacterial genera (i.e., Acetobacter, Lactobacillus, and Penicillium) are now written in italics to comply with taxonomic conventions.

 

• 75 please specify if 'Lactobacillus' refers to the Lactobacillus genus after the nomenclature change or the Lactobacillus sensu lato genus, i.e. before the nomenclature change

We appreciate this observation. In this context, 'Lactobacillus' refers to the Lactobacillus sensu lato group (before the recent taxonomic reclassification). We have added this clarification in the text to ensure precision and avoid ambiguity.

 

• Table 2. please check the TE.HC value of LN 

Thank you for drawing our attention to this. We have re-checked the data and found a typographical error in the TE.HC value for the LN cultivar in Table 2. The previously reported value of 84.7 has been corrected to 84.72, consistent with the underlying dataset

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed all my comments and the manuscript has been improved.