## 3 Results
### Identification of Volatile Organic Compounds by HS-SPME-GC-Ms
#### 3.4.2 Multivariate Statistical Analysis  
To gain deeper insights into the differences in VOCs among the GAB fermentation groups, PLS-DA analysis was applied to the GC-MS results. PLS-DA is a supervised discriminant analysis method that allows for visualization of similarities and differences between samples [11]. The greater the difference between the two samples, the further apart they appear on the score plot [35]. The overall discriminant effect of the PLS-DA model is shown in Figure 5A. In the 3D score plot, Component 1 (38.1%), Component 2 (30.2%), and Component 3 (22.1%) together explained 90.4% of the total variance, indicating that the score plot effectively captured the similarities or differences between samples. The statistical validation parameters of model (R\({}^{2}\) = 0.99, Q\({}^{2}\) = 0.97) indicated high accuracy and reliability. And the results of 1000 permutation tests confirmed there was no model overfitting (Figure 5B). The PLS-DA model showed clear separation among the four fermentation groups, indicating significant differences in VOC profiles among the GAB fermented by the four _S. cerevisiae_ strains, consistent with the GC-E-Nose and GC-IMS results.  
Figure 5: (**A**) Partial least squares–discriminant analysis (PLS-DA) of GC-MS data; (**B**) permutation test statistics of PLS-DA; (**C**) variable importance in projection (VIP) scores of each variable.  
Variable importance in projection (VIP) scores were used to assess the influence and explanatory power of each variable for classifying and discriminating between groups. Higher VIP values indicated greater differences in aroma compounds between groups. Using VIP analysis, the key VOCs distinguishing the four fermentation groups were identified. By setting VIP > 1.5 as the selection criterion and combining it with one-way ANOVA (\(p\) < 0.05), nine volatile markers were identified (Figure 5C). These markers included ethyl myristate (VIP = 1.61), methyl laurate (VIP = 1.59), ethyl octanoate (VIP = 1.59), 1-propanol (VIP = 1.59), ethyl linoleate (VIP = 1.58), ethyl laurate (VIP = 1.57), ethyl valerate (VIP = 1.52), phenethyl acetate (VIP = 1.52), and ethyl nonanoate (VIP = 1.51). The heatmap of the VIP score plot indicated the different concentrations of these compounds across the fermentation groups. Among VOCs with VIP > 1.5, ethyl esters were dominant, underscoring their critical role in differentiating the GAB fermented by various _S. cerevisiae_ strains. The total content of ethyl esters with VIP > 1.5 was 1040.48 ug.L-1, 824.58 ug.L-1, 731.44 ug.L-1, and 619.33 ug.L-1 for the SY, ET008-c54, EC-1118, and NM-8 fermentation groups, respectively. Ethyl myristate (pleasant aroma), ethyl octanoate (fresh, banana aroma), ethyl linoleate (fruity aroma), and ethyl laurate (green apple aroma) showed decreasing concentrations in the SY, ET008-c54, EC-1118, and NM-8 groups, respectively. Additionally, phenethyl acetate (rose aroma) had the highest concentration in NM-8, while ethyl valerate (apple, sweet aroma) and ethyl nonanoate (banana, grape aroma) were detected only in the ET008-c54 and EC-1118 fermentation groups.