Abstract: To investigate the differences in volatile components during the processing of Fuling Zhacai, or pickled mustard tuber, with three stacked layers of mustard tuber and salt, gas chromatography-tandem mass spectrometry (GC-MS/MS)-based non-targeted metabolomics combined with multivariate statistical analysis was used to analyze the changes in volatile metabolites and significantly differential metabolic pathways during the spontaneous fermentation of Zhacai. The results showed that a total of 627 volatile compounds were identified from Zhacai samples at different pickling stages, including terpenoids, esters, heterocyclic compounds, terpenoids, aldehydes, ketones, alcohols, phenols, amines, sulfur compounds, acids, halogenated hydrocarbons, and nitrogen compounds. A significant difference was observed in volatile components among pickling stages. A total 157 unique differential metabolites in the 1st pickling stage vs. the control group, 28 in the 2nd vs. the 1st pickling stage, and one in the 3rd vs. 2nd pickling stage were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the differential metabolites were significantly enriched in metabolic pathways including secondary metabolite biosynthesis, diterpenoid biosynthesis, monoterpenoid biosynthesis, caffeine metabolism, and fatty acid biosynthesis. This study provides a theoretical basis for the standardized production of Fuling Zhacai.

Key words: Fuling Zhacai; gas chromatography-tandem mass spectrometry; non-targeted metabolomics; volatile components