## 3 Results and Discussion
### Aroma Concentration and Composition Analysis of B. roseflavus Samples  
The majority of precursors for the synthesis of volatile aromatic compounds in mushrooms are derived from various non-volatile substances, such as amino acids, fatty acids, carbohydrates, and terpen [36]. Identifying the volatile aroma components and key flavor substances in mushrooms can provide insights into how metabolism and the growth environment impact the types and quantities of flavor compounds. This process also helps to identify internal factors that influence the production of these compounds [37]. Using the HS-SPME-GC-MS method, volatile compounds and their concentrations in the samples were detected and analyzed. A total of 1056 volatile compounds were identified (Figure 4A), including 17.54% terpen, 18.2% lipids, 12.61% ketones, 10.33% alcohols, 9.38% heterocyclic compounds, 5.97% aldehydes, 5.59% hydrocarbons, 5.31% acids, 4.27% aromatics, 3.89% phenols, 2.46% amines, 2.18% ethers, and 2.27% other compounds. OPLS-DA analysis was conducted on the detected volatile compound content (Figure 4B). The R\({}^{2}\)X value (fitting index for independent variables) was 0.978, the R2Y value (fitting index for dependent variables) was 1, and the Q\({}^{2}\) value (prediction index) was 0.999. R\({}^{2}\) and Q\({}^{2}\) values above 0.5 indicate good sample separation [38]. After 200 permutation tests (Figure 4C), the Q\({}^{2}\) regression line intersected the vertical axis below zero, showing no overfitting and validating the model. This result clearly demonstrates the overall aroma differences between CN and DBG. Analysis of the relative content of the identified volatile compounds (Figure S3A) showed that DBG had a significantly higher relative total content than CN, with all categories except for ethers and other compounds being higher in DBG. To further analyze the characteristic aroma substances of CN and DBG, differential metabolites were selected based on VIP > 1, fold change \(\geq\) 2, and fold change \(\leq\) 0.5. A total of 218 differential metabolites were screened, and the volcano plot (Figure 4D) showed that 154 metabolites were significantly upregulated and 48 metabolites significantly downregulated. The heat map of differential metabolite clustering (Figure 5) revealed that the three largest categories of volatile substances were terpen, aldehydes, and alcohols. In fungi, terpen are synthesized via the mevalonate (MVA) pathway, which uses acetyl-CoA as the starting substrate [36]. Aldehydes are an important class of volatile components in edible fungi, contributing green, fatty, almond, and waxy aromas [39]. Alcohols primarily provide a mushroom-like and green aroma, mainly produced via the fatty acid pathway. For example, octenol is formed during the oxidative decomposition of linoleic acid [40]. Figure 4: Comprehensive analysis of volatile metabolites: (**A**) volatile metabolite species; (**B**) scatter plot of scores from the orthogonal partial least squares discriminant analysis (OPLS-DA) model; (**C**) results of the 200-permutation test; and (**D**) volcano plot of differential metabolites. Figure 5: Heat map of volatile substance differential metabolites. Relative odor activity value (rOAV) is a key tool used to assess the contribution of key aroma components to the overall mushroom aroma from both the concentration and threshold perspectives [41]. By combining rOAV, key aroma substances in DBG and CN were analyzed (Table 3). The results showed that 17 volatile compounds in CN had OAV > 1, including 4 aromatic hydrocarbons, 3 aldehydes, 3 esters, 2 ketones, 2 ethers, 1 phenol, 1 terpene, and 1 heterocyclic compound. In DBG, 10 volatile compounds had OAV > 1, including 2 ketones, 2 aldehydes, 2 esters, 1 aromatic hydrocarbon, 1 ether, 1 terpene, and 1 heterocyclic compound. These compounds play key roles in the aroma formation of _B. roseflavus_ from different regions.