Untargeted Metabolomic Profiling of <em>Astragalus membranaceus</em> Aqueous Extracts Fermented by <em>Pediococcus acidilactici</em> Using UHPLC—Orbitrap MS

Astragalus membranaceus (AM) is a traditional medicinal and edible herb with well-documented immunomodulatory activities; however, its application in functional beverages is limited by the low bioavailability of its bioactive constituents. Probiotic fermentation has emerged as an effective strategy to enhance the nutritional and functional properties of herbal materials, yet the underlying metabolic mechanisms remain insufficiently understood. In this study, untargeted metabolomics based on ultra-high-performance liquid chromatography coupled with Orbitrap mass spectrometry (UHPLC-Orbitrap MS) was employed to comprehensively characterize metabolic alterations in AM aqueous extracts before and after fermentation with Pediococcus acidilactici (P. acidilactici) for 48 h. Multivariate statistical analyses combined with pathway enrichment analysis were used to identify differential metabolites and key metabolic pathways affected by fermentation. A total of 659 significantly altered metabolites were identified, including 350 upregulated and 309 downregulated metabolites after fermentation. These metabolites were mainly associated with organic acids, flavonoids, amino acid derivatives, nucleotides, and phenylpropanoids. Notably, fermentation markedly enhanced metabolites related to arginine biosynthesis, carbon metabolism, and nicotinate and nicotinamide metabolism, accompanied by a substantial accumulation of functional compounds such as lactate, phenyllactic acid, indolelactic acid, and nicotinamide adenine dinucleotide (NAD+). Overall, P. acidilactici fermentation induced extensive metabolic reprogramming of AM aqueous extracts, leading to the enrichment of multiple bioactive metabolites and the activation of key functional processes. These findings provide mechanistic insights into probiotic fermentation of medicinal and edible herbs and offer a scientific basis for the development of value-added fermented AM beverages with improved nutritional and functional properties.