## 3 Results and Discussion
### Stuck Fermentation Altered the Profile of Volatile Metabolites in Summer Black Tea  
The aroma of tea is a crucial component of its quality and is formed by a number of volatile metabolites. As a result, following stuck fermentation, we also examined the alterations in the volatile metabolites of summer black tea. GC-MS/MS was used to analyze six biological replicates of YH and FHYH, and the quality control evaluation revealed that the instrument and signals were dependable and stable (Figure S4A,B). In addition, more than 75% of the substances in the QC samples had CVs less than 0.3 (Figure S4C), indicating that the experimental data was stable. We detected 664 volatile metabolites, including 14 acids, 56 alcohols, 41 aldehydes, 12 amines, 41 aromatics, 117 esters, 1 ether, 4 halogenated hydrocarbons, 114 Heterocyclic compounds, 59 hydrocarbons, 51 ketones, 11 nitrogen compounds, 14 phenols, 4 sulfur compounds, 122 terpen, 3 other compounds (Figure 4A and Table S4). PCA showed a clear separation between YH and FHYH based on these metabolites (Figure 4B), and HCA showed that the two samples initially separated into two clusters, while the individual replicates were grouped into one cluster (Figure 4C). In the supervised OP-LSDA model as well (Figure 4D), YH and FHYH were clearly distinct and the model was not overfitted (Figure S5). These results are indicative of significant changes in volatile metabolites after microbial stuck fermentation of summer black tea. Figure 4: GC-MS/MS analysis of YH and FHYH groups. (**A**) The categories of 664 volatile metabolites. (**B**) Principal component analysis (PCA) of YH and FHYH. PC1 is the first principal component. PC2 is the second principal component. (**C**) Hierarchical clustering analysis (HCA) of 664 volatile metabolites. (**D**) Orthonormal partial least squares discriminant analysis (OPLS-DA) of YH and FHYH. (**E**) Heat map of differential metabolites. (**F**) KEGG pathway enrichment analysis of significantly different non-volatile metabolites. A total of 345 volatile metabolites were significantly different between the YH and FHYH samples (VIP \(\geq\) 1, \(p\)\(\leq\) 0.05, \(\mid\) Log\({}_{2}\)FC\(\mid\)\(\geq\) 1), of which 324 were down-regulated and only 21 were up-regulated. (Figure 4E, Table S5). KEGG enrichment analysis did not reveal enrichment of any of the pathways (Figure 4F, \(p\)\(\leq\) 0.05). Based on the FlavorDB, Flavor Library, and Flavorne databases, the odor characteristics of 171 volatile metabolites were identified. Forty metabolites with fruity or sweet aromas, including 4-hexen-3-one (CAS:2497-21-4), linaly acetate (CAS.115-95-7), hexanoic acid, 3-hexenyl ester and (Z)- (CAS:31501-11-8), were down-regulated. In addition, 21 metabolites with floral aroma, such as 1-nonanol (CAS:143-08-8), benzoic acid, 2-propenyl ester (CAS:583-04-0), 5, 9-undecadien-2-one, 6,10-dimethyl- and (Z)- (CAS:3879-26-3), also showed a decreasing trend. The down-regulation of these metabolites explains the diminished floral and sweet aromas of FHYH. Furthermore, 15 metabolites associated with a green aroma, such as heptanal (CAS:111-71-7) and (6Z)-nonen-1-ol (CAS:35854-86-5), decreased after fermentation. [PERSON] et al. showed that metabolites with green aroma are associated with alcohols and aldehydes [35]. However, sensory evaluation indicated similar green notes in YH and FHYH tea samples, which can be attributed to the low aroma threshold metabolites 4-methylthiazol (CAS:693-95-8, aroma threshold: 0.055 mg/m\({}^{3}\)) and 2-isobutylthiazol (CAS:18640-74-9, aroma threshold: 0.0035 mg/m\({}^{3}\)). Consistent with the results reported by [PERSON] et al. [11], the content of acetophenone (CAS:98-86-2) increased after stuck fermentation. Acetophenone has a floral aroma and is a key determinant of the aroma characteristics of Fu brick tea. The increase in acetophenone during fermentation may be related to the moist heat effect and microbial metabolism [35].