Bioactive Substances Characterization in Probiotic-Fermented Guava (Psidium guajava L.) Juice and Systematic Evaluation of its Anti-Hyperglycemic Activity.

The development of natural anti-hyperglycemic agents with minimal side effects is a critical objective in functional food research. This study comprehensively investigated the anti-diabetic potential and bioactive substances in guava juice fermented with Lacticaseibacillus paracasei NCUH012072 (FM). Using an integrated in vitro and metabolomic approach, the anti-hyperglycemic activity was systematically assessed through a series of bioassays (enzyme inhibition, antioxidant capacity, and insulin-resistant cell models), untargeted metabolomics, and bioinformatic and affinity-based validation. The results demonstrated that FM significantly inhibited key carbohydrate-metabolizing enzymes (α-amylase, α-glucosidase) as well as the insulin signaling regulator protein tyrosine phosphatase 1B (PTP1B) (P < 0.05). Additionally, FM exhibited enhanced antioxidant capacity and improved glucose uptake in insulin-resistant HepG2 cells (P < 0.05). Untargeted metabolomics revealed that fermentation modulated 13 pivotal metabolic pathways. Integrated analysis using Spearman correlation and random forest modeling identified key anti-hyperglycemic metabolites, including flavonoids (e.g., liquiritigenin), phenolic acids (e.g., cis-caffeic acid), and redox-active compounds such as (R)-lipoic acid. Affinity ultrafiltration combined with liquid chromatography-mass spectrometry (AF-LC-MS) further characterized quercetin 3-galactoside and resveratrol as potent inhibitors of the target enzymes. This study not only systematically demonstrates the anti-hyperglycemic properties of probiotic-fermented guava juice but also proposes a comprehensive strategy for identifying bioactive compounds in complex food matrices. The findings offer a theoretical basis for developing fermented fruit-based functional foods as dietary strategies in hyperglycemia management.