Optimization of Enzymatic Hydrolysis and Fermentation Processes for Daylily Flowers and Analysis of Their Anti-depressive Metabolic Pathways

Objective: To determine the optimal enzymatic hydrolysis and fermentation processes for daylily flowers and to analyze their effects on physicochemical components and anti-depressive related metabolic pathways. Methods: The best process combination was selected using mixed strain fermentation, single-factor experiments, response surface methodology, and orthogonal experiments. Total flavonoids, total phenolic acids, and total polysaccharides were measured using aluminum nitrate method, Folin-Ciocalteu method, and phenol-sulfuric acid method, respectively. Metabolic changes before and after fermentation were determined using UPLC-QQQ-MS/MS. Results: The optimal enzymatic hydrolysis conditions were a material-to-liquid ratio of 1:25, enzyme addition of 0.2%, enzyme ratio of 1:1, at 50 ℃ for 1.5 h. The optimal fermentation conditions were at 37 ℃ for 22 hours with an inoculum size of 3% (bacterial count of 108 CFU/mL). Fermentation significantly (P<0.05) increased the content of total flavonoids and phenolic acids and the number of viable cells, while decreasing the total polysaccharide content. Metabolomics identified 66 differential metabolites, mostly related to anti-depression, predominantly enriched in metabolic pathways such as tryptophan metabolism, glycerophospholipid metabolism, biosynthesis of pantothenic acid and coenzyme A, degradation of valine, leucine, and isoleucine, metabolism of nicotinate and nicotinamide, and metabolism of alanine, aspartate, and glutamate. Conclusion: This study established the optimal enzymatic hydrolysis and fermentation processes for enhancing the effective components of daylily flowers and elucidated the metabolic pathways related to the fermentation-improved anti-depressive effects at the metabolomic level, providing a foundation for the future in-depth development and utilization of daylily resources.