Impacts of fixation processing workflows on the volatile and non-volatile metabolomic profiles of Gougunao green tea

Jie Zhou, Wenhua Li, Jinyun Peng, Yifan Cai, Junjie Lu, Yongfa Guo, Shuaiwen Yin, Xiaojin Liang, Huimin Sun, Xinjun Liao

Fixation is a central thermal step in green tea processing, but Gougunao tea uses a distinctive two-stage fixation and rolling workflow that has not been systematically evaluated at the metabolomic level. Here, four fixation processing workflows were compared: fully mechanical fixation (M1), mechanical–manual hybrid fixation (M2), manual–mechanical hybrid fixation (M3), and fully manual fixation (M4). An integrated UHPLC-MS/MS and HS-SPME-GC-MS strategy was used to profile non-volatile and volatile metabolites. In total, 2,105 non-volatile metabolic features and 1,066 volatile metabolites were putatively annotated. PCA showed clear workflow-associated separation in both LC-MS and GC-MS datasets, while OPLS-DA supported pairwise discrimination among most comparisons. Differential screening using FDR < 0.05 and |log₂FC| > 1 identified 16–183 differential non-volatile metabolites and 0–26 differential volatile metabolites across the six pairwise comparisons. Non-volatile differences were mainly distributed among lipids and lipid-like molecules, organoheterocyclic compounds, organic acids and derivatives, benzenoids, and phenylpropanoids and polyketides, indicating broad changes in metabolite pools related to tea taste formation, phenolic transformation, lipid-derived reactions, and secondary metabolism. Representative taste-associated compounds, including amino acids, catechins, methylxanthines, phenolic acids, and theaflavin-related metabolites, showed workflow-dependent abundance patterns. For volatile compounds, FDR-significant markers and relative odor activity value (rOAV) ranking highlighted aldehydes, esters, alcohols, ketones, and sulfur-containing compounds as candidate aroma-related volatiles. These results provide metabolomic evidence that different fixation processing workflows are associated with distinct chemical profiles in Gougunao green tea, while sensory validation remains necessary to confirm their direct quality implications.