Bioactive and pathway modeling in functional frozen dessert enriched with spinach (Spinacia oleracea L.) extract: An untargeted metabolomics driven study

This study aimed to elucidate the biochemical composition, molecular interactions, and metabolomic pathways of frozen dessert (FD) and spinach extract (SE)-fortified functional frozen dessert (FFD). Incorporation of 2.41 g SE significantly enhanced total phenolic content, flavonoids, and antioxidant activity while maintaining physicochemical and sensory attributes. During 72 days of frozen storage, gradual oxidation and chlorophyll degradation occurred, though bioactivity remained higher than in the control. Fourier-transform infrared spectroscopy (FTIR) confirmed Fe–O coordination and protein–polyphenol complexation, indicating stable structural integration. Proton nuclear magnetic resonance ( 1 H NMR) identified amino acids, organic acids, carbohydrates, and polyols, suggesting improved biochemical diversity and cryostability. High-resolution accurate mass spectrometry (HRAMS) detected metabolites previously reported to possess antioxidant, antimicrobial, and anti-inflammatory pathways. Pathway enrichment analysis indicated enrichment of amino acid metabolism, tricarboxylic acid (TCA), and glutathione metabolism, demonstrating enhanced synergistic metabolic adaptation in FFD. These findings confirm that spinach fortification enhanced the nutritional and metabolomic characteristics of FFD.