Abstract: To investigate the differences in metabolites in fermented grains of Nongxiangxing Baijiu at different fermentation times, volatile and non-volatile substances in fermented grains were analyzed after 30, 75, 172, 236, and 466 days of fermentation. A total of 96 volatile compounds were identified by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). The contents of volatile compounds in were significantly higher in fermented grains at prolonged fermentation periods (236 and 466 days) than at other fermentation periods. Fold change (FC) analysis showed that volatile metabolite levels markedly increased and then tended to be stable with prolonged fermentation. By ultra-high performance liquid chromatography-quadrupole-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap MS) we identified 483 metabolites, mainly including organic acids and derivatives, lipids and lipid-like molecules, and organic heterocyclic compounds. The differential metabolites among fermented grains at different fermentation stages were mainly organic acids, esters, phenols, indoles, flavonoids, and amino acids. Quantitative analysis of 41 organic acids showed that the contents of lactic acid and acetic acid were the highest, and the total content of organic acids in fermented grains at different fermentation stages ranged from 17 725.65 to 29 275.22 μg/g. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differential metabolites revealed that tryptophan metabolism, alanine, aspartate, and glutamate metabolism were involved in all fermentation stages. Glutathione metabolism, glyoxylate and dicarboxylate metabolism, and arginine and proline metabolism pathways were significantly active in the early fermentation stages, while arginine, proline metabolism, butyrate metabolism, and histidine metabolism pathways were notably active in the late stages. This study provides reference data for optimizing the production process of Baijiu.

Key words: Nongxiangxing Baijiu; fermented grains; metabolomics technology; differential metabolites; metabolic pathway