## 4 Discussion  
The current competitive wine market demands increased typicality and differentiation of wines, and as a result, some wine-making methods allow the use of a variety of ancillary products or methods, such as the addition of enzymes to enhance wine aroma [35]. Most flavor precursors are not released during the wine-making process [36; 37]. The addition of exogenous glycosylases to promote the hydrolysis of aroma glycosides is of interest to many winemakers. In particular, the enzyme from non-_Saccharomyces_ yeasts has positive properties such as good tolerance to pH, temperature, or ethanol [38]. In this work, we aimed to characterize newly purified \(\beta\)-glucosidase of the native strains _M. guilliermondii_ NM218 and _H. uvarum_ BF345 isolated from grapes by focusing on the role and possible contribution of these enzymes in the improvement of young Chardonnay wine aroma. The \(\beta\)-glucosidase was purified stepwise by ammonium sulfate precipitation, DEAE-Sepharose-FF column, and Sephacryl1 S-200 column. Both Mg-$ggl and Hu-$ggl were purified 12-fold (Table 1 and Figure 1), and both enzymes showed high levels of specific activity (1.95 U/rng and 2.11 U/mg, respectively). Purified Mg-$ggl and Hu-$ggl exhibited high activity at pH 4.0 and were able to maintain more than 75% activity at pH 2.0, indicating that they are acid tolerant. This optimal pH is significantly different from the reported optimal pH of 5.0-6.0 for the \(\beta\)-glucosidase of _Pichia membranificaciens_ Pm7, _Hansenispora vineae_ Hv3, _H. uvarum_ Hu8, and _Wickerhamomyces anomalus_ Wa1, where only _H. uvarum_ Hu8 maintained 50% activity at pH 3.0, and the other \(\beta\)-glucosidase lost most of their activity at pH 3.0 [13]. The optimal temperature recorded for both Mg-$ggl and Hu-$ggl was 40 \({}^{\circ}\)C, which was lower than the previously reported optimal temperature for \(\beta\)-glucosidase from _Pichia guilliermondii_ G1.2 (55 \({}^{\circ}\)C) [39] and _Talaromyces thermophilus_ (65 \({}^{\circ}\)C) [22]. The use of \(\beta\)-glucosidase, which can maintain activity at low temperatures and low pH, in industrial winemaking increases the solubility of reactants and products and contributes to the hydrolysis of biomass enzymatically [22]. The interaction between metal ions and enzymes can be found by identifying alterations in the secondary and/or tertiary structure \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Triangle Test** & **Number of Trials** & \
\begin{tabular}{c} **Number of Correct** \\ **Answers** \\ \end{tabular} & **Significance Level** & **Odor Descriptors** \\ \hline Mg-$ggl vs. CK & 16 & 14 & \(p\leq 0.001\) &
\begin{tabular}{c} Sweet, floral, fruity, \\ banana, medicinal \\ Sweet, floral, honey, \\ pomelo, banana \\ Sweet, floral, fruity \\ Sweet, floral, fruity \\ Sweet, floral, fruity, \\ banana \\ Floral, fruity, toasty \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Sensory analysis of Chardonnay wine treated with purified enzymes (Mg-$ggl and Hu-$ggl) and commercial enzyme (An-$ggl). of the protein that leads to reduced activity [40]. In this study, Zn\({}^{2+}\), Mn\({}^{2+}\), Fe\({}^{2+}\), and Fe\({}^{3+}\) had a significant activating effect, while the remaining metal ions had a certain inhibitory effect on the enzyme activity of Mg-\(\beta\)gl and Hu-\(\beta\)gl. This result suggests that these four ions can be further investigated and developed as activators of Mg-\(\beta\)gl and Hu-\(\beta\)gl. The influence of enzyme inhibitors (EDTA, SDS, \(\beta\)-ME, and DMSO) on the enzymes' activity was also evaluated. They had an insignificant inhibitory effect on the enzyme activity of Mg-\(\beta\)gl and Hu-\(\beta\)gl, indicating that the enzymes still had catalytic ability amid these inhibitors. Interestingly, non-ionic detergents have been reported to improve the enzyme activity of some \(\beta\)-glucosidase, such as the \(\beta\)-glucosidase of _Feridobacterium islandicum_[41].