## 3 Results
### Free Amino Acid Profile of the Mushroom Samples  
The total free amino acid contents of the pretreated mushroom samples are presented in Figure 7. The oyster mushroom samples contained 22 free amino acids, with their total amounts ranging from 25.95 to 55.68 mg/g for the pretreated samples (Appendix C). The total free amino acid content of the fresh oyster mushroom was 34.07 mg/g. The blanching (23.8%) and microwaving (14.9%) pretreatments caused a significant decrease in the free amino content of the mushroom samples, while the HHP (63.4%), oven (61.9%), UV light (60.1%), and steam (8.0%) pretreatments caused an increase (\(p<0.05\)). Among these, the HHP pretreatment increased the free amino acid concentration the most, reaching 55.68 mg/g. In the fermented sample group (Figure 8), the fresh fermented samples exhibited the highest free amino acid content at 53.64 mg/g, while the blanched fermented samples showed the lowest content at 26.23 mg/g. This suggests that fermentation alone significantly increases the total free amino acid content, underscoring the importance of pretreatments before fermentation. The blanching, steaming, and microwaving pretreatments followed by fermentation led to lower free amino acid contents compared to not only the other pretreatments but also the fresh oyster mushrooms themselves (\(p<0.05\)). However, the oven-, HHP-, and UV-pretreated fermented samples retained their high free amino acid contents with a slight reduction compared to their pretreated counterparts (Appendix D). The free amino acid profile of mushroom samples was evaluated using a principal component analysis, as shown in Figure 9. The first two principal components accounted for 66.3% of the variance, representing the majority of the dataset's variation. Figure 7: Total free amino acid content of the pretreated oyster mushroom samples (mean \(\pm\) SE, n = 4). Values with different letters (a–c) show significant differences (\(p<0.05\)). In Figure 9, the upper-right quadrant contains markers corresponding to four samples: oven-, UV-light-, and HHP-pretreated mushrooms, while the lower-right quadrant features markers for these samples' fermented counterparts and the fresh fermented samples. The upper-left quadrant contains four samples: microwaved, steamed, fresh, and blanched  
Figure 8: Total free amino acid content of the pretreated and fermented oyster mushroom samples (mean \(\pm\) SE, n = 4). Values with different letters (a–f) show significant differences (\(p<0.05\)). Figure 9: PCA plot (PC 1 vs. PC 2) of free amino acid profile of mushroom samples. mushrooms, while the lower-left quadrant contains their fermented counterparts. The clear separation of pretreated and fermented samples in principal component 2 highlights the significant impact of fermentation on the free amino acid profiles of the samples. Additionally, principal component 1 distinctly differentiates the various pretreatments. Samples located in the same quadrant (oven/UV/HHP and microwave/blanched/steamed) indicate similarities in their amino acid profiles, with the degree of similarity being reflected by their proximity to one another, forming distinct clusters. The greater distance between fermented samples and their pretreated counterparts in the oven-, UV-light-, and HHP-pretreated groups suggests that these samples underwent more substantial changes in their free amino acid composition compared to the other samples. Notably, the fresh mushroom samples exhibited the most significant change in composition, shifting from the upper-left quadrant to the lower-left quadrant upon fermentation, indicating that the application of fermentation without any pretreatments causes a pronounced alteration in the free amino acid profile. The arrows represent the free amino acids' contribution to the principal components. The fresh, HHP-pretreated, and UV-light-pretreated fermented samples are positioned further along the positive PC1 axis, suggesting high levels of free amino acids such as glycine, valine, leucine, and isoleucine.