Comparative study of different commercial enzymes on release of glycosylated volatile compounds in white grapes using SPE/GC–MS
Food Chemistry, Volume 464, Part 2, 2025, 141742: Fig. 1. Principal Component Analysis (PCA) applied on chemical groups of volatile of grape must treated with 12 commercial enzymes.
The goal of this study was to evaluate the effectiveness of twelve commercial enzyme preparations in releasing glycosylated volatile aroma compounds from white grape must. The enzymes were tested under laboratory conditions using SPE and GC–MS techniques.
Although the total volatile compound concentration remained statistically unchanged, significant changes were observed in specific aroma groups—especially acids, alcohols, terpenes, and C13-norisoprenoids. Two enzymes, Rapidase Revelation Aroma and Enozym Extra Aroma, were particularly effective at enhancing the release of key varietal aroma compounds, notably terpenes and norisoprenoids.
The original article
Comparative study of different commercial enzymes on release of glycosylated volatile compounds in white grapes using SPE/GC–MS
Liliana Martínez, Bianca S. da Costa, Mar Vilanova
Food Chemistry, Volume 464, Part 2, 1 February 2025, 141742
https://doi.org/10.1016/j.foodchem.2024.141742
licensed under CC-BY 4.0
Selected sections from the article follow. Formats and hyperlinks were adapted from the original.
Glycoside compounds in grape wines are typically bound to β-d-glucopyranose. In the case of diglycosides, glucose can be bound to other molecules like malonic acid, arabinose, apiofuranose, or rhamnose (Ferreira & Lopez, 2019; Liu et al., 2017; Sarry & Günata, 2004). These odorless compounds, which make up around 90 % of the total precursor concentration, contain aroma and flavor aglycones (Claus & Mojsov, 2018; Dziadas & Jeleń, 2016) and play a crucial role in defining the sensory and varietal attributes of wine (Hjelmeland & Ebeler, 2015) upon the release of free aglycones from glycosides through hydrolysis (Arévalo Villena et al., 2006). The liberation can occur via acid-catalyzed hydrolysis, resulting from the acidic nature of grape must, occurs throughout the winemaking process and participates in the release of bound-aromas (Liu et al., 2017; López et al., 2004) or the activity of endogenous β-glycosidase enzymes (Botelho et al., 2007), that release the associated aromatic compounds in their free form.
Several works report analytical methods for the determination of glycosylated fraction of aroma in grapes. In most of these works, the enzyme Rapidase Revelation Aroma (AR2000) is used to release glycosidically-bound volatiles (Fundira, 2002; Vilanova & Sieiro, 2006; Kang et al., 2012; Ghaste et al., 2015; Wang et al., 1015; Tavernini et al., 2020; Oller-Ruiz et al., 2022). Some comparative studies showed the effect of different commercial enzymes to release glycosylated aroma forms in different cultivars (Fundira, 2002; Armada et al., 2010; Rodríguez-Nogales et al., 2024; Rio Segade et al. 2024) showing different behaviors in basis to the cultivar used.
Despite these findings, there is still limited knowledge regarding the impact of new commercial enzymes on the volatile composition derived from glycosylated compounds in white grape varieties. Understanding and effectively managing the extraction and release of these precursors are essential to know the cultivar aromatic potential.
In this context, the current research explored the effect of twelve different oenological commercial enzymes on the effectiveness of aroma glycoside release by SPE/GC–MS on blended must derived from four aromatic white cultivars (Albariño, Loureira, Treixadura, and Godello) grown in Galicia (NW Spain). In this work a blended of white Galician cultivars were used because they are very aromatic cultivars due to terpenes and C13-norisoprenoids which are responsible for floral and fruity aromas (Falqué et al., 2001; Genisheva & Oliveira, 2009; Losada et al., 2011; Oliveira et al., 2008; Vilanova et al., 2013; Vilanova et al., 2017; Vilanova et al., 2019). Moreover, the blended wines made from white cultivars from Galicia, especially those with Loureira, showed an increase in terpenes and C13-norisoprenoids, improvement the complexity of the wine aroma (Vilanova et al., 2017).
2. Materials and methods
2.3. Analysis of volatile composition by gas chromatography coupled to mass spectrometry (GC–MS)
2.3.2. Chromatograph analysis
Volatile composition analysis was performed using an Agilent 7890 gas chromatograph (GC) coupled to an Agilent 7000C triple quadrupole mass spectrometer. Samples were injected in split less mode using a volume of 1.5 μL. Chromatographic separation was performed on a DB-WAX ultra-inert (30 m 0.25 mm i.d., 0.25 μm film thickness, Agilent). The injector temperature was 250 °C. The oven temperature was maintained at 60 °C for 2 min, then programmed to rise from 60 °C to 234 °C with a gradient of 3 °C/min and then with a gradient of 5 °C/min to 250 °C and finally programmed 10 min at 250 °C. The carrier gas is helium N60 (Air Liquide), flow at 1 mL/min. The detector is set to electronic impact mode (70 eV), with an acquisition range from 29 m/z to 360 m/z, and an acquisition rate of 610 ms. Identification was carried out with Mass Hunter Qualitative Analysis software (Agilent) using the NIST library and by comparison with the mass spectra and retention index of chromatographic standards, and data found in the literature. Quantification of volatiles compounds in terms of 4-nonanol was performed with Mass Hunter Quantitative Analysis software (Agilent). The compounds were quantified in terms of 4-nonanol equivalents by comparing the GC retention times Standard compounds 97 % were purchased from Sigma-Aldrich.
3. Results and discussion
3.2. Effect of commercial enzymes on glycosylated groups release in grape must
The aroma of white wines plays an important role in defining their overall quality and expressing their unique varietal character (Pérez et al., 2022). Among the wide range of aroma compounds found in white wines, terpenes and C13-norisoprenoids stand out as the key contributors to their characteristic aroma (Vilanova & Sieiro, 2006). Despite being present in lower proportions compared to other compounds, terpenes have a profound impact on the sensory experience of white wines because their low threshold.
Principal Component analysis (PCA) was applied to understand the effect of different commercial enzymes activity on release of glycosylated chemical groups responsible of white grape must aroma (Fig. 1). This multivariate analysis helped in interpreting the data and identifying the families of volatile compounds that best discriminate among the enzymes used in the study. The first two principal components (PC1 and PC2) accounted for 75.61 % of the total variance (49.54 % and 26.07 %, respectively). PC1 was mostly correlated with esters, acids, alcohols, terpenes and C13-norisoprenoids, while PC2 was mostly correlated with aldehydes and volatile phenols. The enzymes distribution according the two components show the increase of terpenes and C13-norisoprenoide when Rapidase Revelation of Aroma was applied. Acids and esters increased their concentration when Enozym Extra Aroma was used, however Endozym β-split and Lallyzyme Cuvee Blanc showed a hug influence on Aldehydes release. Finally, Trenolin Bouquet and Lallzyme Beta was correlated with C6-compounds release.
Food Chemistry, Volume 464, Part 2, 2025, 141742: Fig. 1. Principal Component Analysis (PCA) applied on chemical groups of volatile of grape must treated with 12 commercial enzymes.
In the context of this study, the conditions employed led to a significant increase in free-volatile compounds, particularly terpenes and C13-norisoprenoids, in the white grape must treated with Rapidase Revelation Aroma. The rise in terpenes and C13-norisoprenoids concentrations contributes to the aromatic complexity and varietal character of the wine. By utilizing Rapidase Revelation Aroma, it can effectively release and amplify these aroma compounds, resulting in white wines with more pronounced floral and fruity notes, thereby suggesting a good candidate to be used for improving white wines quality.
Indeed, it is evident that Enozym Extra Aroma emerged as the enzyme that released the most substantial amounts of volatile compounds compared to the other enzymes used in the study. Notably, Enozym Extra Aroma led to a significant increase in the concentration of acid and ester compounds. Esters are very important in contributing to the fruity and floral aromas in wine, imparting notes of tropical fruits, citrus, pears, apples, and flowers (Iobbi et al., 2023). This enhancement of aromatic complexity and intensity contributes to the overall appeal of the wine. In addition to esters, acids are another important component that significantly influences the sensory experience of white wines. The presence of acids imparts a sensation of freshness, creating a harmonious balance that helps counterbalance any natural sweetness from the fruit or residual sugar in the wine (Chidi et al., 2018).
3.3. Effect of enzyme treatment on individual glycosylated compounds release in grape must
Esters and Terpenes play an important role in the development of fruity and floral aromas. Additionally, C13-norisoprenoids, which are also characterized by floral aromas, are important volatile compounds contributing to the wine aroma due to their low olfactory thresholds (Mateo & Jiménez, 2000). In this sense, Rapidase Revelation Aroma and Enozym Extra Aroma emerge as the top candidates for Galicia winemaking processes, as supported by utilizing these enzymes, winemakers have the opportunity to elevate the terpene content in the must, ultimately resulting in high-quality white wines with enhanced aromatic complexity and pronounced varietal characteristics.
Among the volatile phenols chemical family, the impact of different enzymes was relatively limited, with only two compounds (eugenol and 3,4,5-trimethoxyphenol) being significantly affected. For Eugenol, the most substantial increase in content was observed when Rapidase Revelation Aroma, Lallzym Cuve Blanc, and Enozym Extra Aroma were used. However, only Enozym Extra Aroma displayed the highest release of 3,4,5-trimethoxyphenol. Interestingly, Enozym FW proved to be less effective in breaking the glycosylated link of Eugenol from its precursor, resulting in lower liberation of this compound, as well as 3,4,5-trimethoxyphenol, showing a contrast performance compared to Rapidase Revelation Aroma and Enozym Extra.
The efficiency of the commercial enzymes varied as differences in their ability to release individual compounds when eight commercial preparations were assessed on Verdejo grapes to evaluate their wine aromatic potential (Rodríguez-Nogales et al., 2024).
These findings demonstrate the unique impact of each commercial enzyme on the individual aroma compounds, underscoring the importance of enzyme selection in winemaking to achieve specific aromatic profiles and sensory characteristics in the final product.
Finally, we generated a heatmap based on the commercial glycolytic enzymes used, focusing on the statistically significant twenty-one volatile compounds identified (Fig. 2). The heatmap visually represented the relationships between the enzymes and the concentration levels of these key volatile compounds. By examining the heatmap, we gained valuable insights into how each glycolytic enzyme influenced the production of specific volatile compounds. The clustering patterns in the heatmap provided a clear visualization of enzyme-chemical interactions, highlighting which enzymes were particularly efficient in releasing certain aroma compounds.
Food Chemistry, Volume 464, Part 2, 2025, 141742: Fig. 2. Heat map representations of the 21 significative volatile compounds according to the commercial enzyme used.
The analysis of different metabolite intensities of color led to a clear separation of the 12 enzymes used, revealing two main clusters denoted as A and B (Fig. 2). Cluster A encompassed Enozym FW, Lafazym Arom, Endozym β-Split, Enovien Varietal, and Trenolin Mash DF, exhibiting, on average, lower concentrations of these volatile compounds. In contrast, cluster B consisted of the remaining 7 enzymes, primarily characterized by the presence of terpenes, C13-norisoprenoids, acids, esters, and alcohols. Notably, the enzymes Rapidase Revelation Aroma and Enozym Extra Aroma displayed particularly high abundances of these volatile compounds, outperforming the others and confirming the results obtained from the Principal Component Analysis (PCA). Furthermore, Rapidase Revelation Aroma, is a unique enzymatic formulation of β-D-glucosidase to maximize aromatic potential that has been used in the protocols proposed by several authors (Oliveira et al., 2008; Vilanova et al., 2010; Vilanova et al., 2021).
4. Conclusion
In this study, we conducted an evaluation of 12 commercial enzymes to assess their effectiveness in releasing aromatic compounds from their conjugated forms in a mixed white variety must under laboratory conditions. The results revealed a significant effect of glycolytic enzymes, which led to a notable increase in the concentration of twenty-one aroma compounds. This finding confirms the importance of utilizing glycolytic enzymes in the production of more aromatic wines. However, it is essential to be careful when assuming that all commercial enzymes would exhibit equal effectiveness in hydrolyzing aroma precursors at the specific conditions employed in this research (40 °C and pH of 3.27). Enzymes can show different glycosidase activities depending on the temperature and pH conditions they are subjected to. Hence, their performance may vary under different experimental setups. Among the commercial enzymes studied in this work, Rapidase Revelation Aroma and Enozym Extra Aroma emerged as the most powerful ones on the must's volatile composition. These enzymes demonstrated an important ability to release higher concentrations of essential varietal aroma compounds, particularly release of terpenes, C13-norisoprenoids, and esters compounds. As a result, they hold significant potential to enhance the aromatic profile of the must, thereby contributing to the overall floral and fruity characteristics of white Galician wines. The findings suggest that these enzymes could play a crucial role in improving the quality of the end product. To improve understanding of these enzymes' mechanisms and optimize aroma extraction during the winemaking process, more in-depth exploration and analysis have to be conducted.
