In Vitro Simulated Digestion Unveils the Multifunctional Benefits of Freeze-Dried Cowpea Fiber: Prebiotic,...

Cowpea (Vigna unguiculata) is an underutilized legume rich in dietary fiber (DF) and bioactive compounds. However, the functional potential of its insoluble DF (IDF), particularly that derived from immature or downgraded pods, has not been comprehensively investigated. This study aimed to investigate the physicochemical characteristics, functional properties, antioxidant activity, and biological effects of freeze-dried cowpea powder (CP) and its IDF-rich fraction (CP-IDF). CP was analyzed for proximate composition and techno-functional properties, including water retention capacity (WRC), oil retention capacity (ORC), and glucose-binding capacity. CP-IDF was prepared via sequential enzymatic hydrolysis and characterized for chemical composition, microstructure (scanning electron microscopy), crystallinity (X-ray diffraction), and functional groups (Fourier transform infrared). Simulated in vitro gastrointestinal digestion was performed to obtain digested CP-IDF, and the antioxidant activity of digesta supernatants was evaluated using DPPH and ABTS assays. Prebiotic potential was assessed using Bifidobacterium longum and Lacticaseibacillus casei, while anti-inflammatory activity was determined in LPS-stimulated RAW264.7 macrophages via nitric oxide (NO) inhibition. Results showed that CP possessed high carbohydrate and fiber contents, with WRC (4.27 ± 0.31 g/g) and ORC (3.90 ± 0.19 g/g), indicating strong hydration and lipid-binding capacities. CP-IDF was rich in polysaccharides and uronic acids, consistent with pectic components. Structural analyses revealed a partial disruption of cellulose crystallinity after digestion. Antioxidant activity increased after simulated gastric and intestinal digestion, corresponding to enhanced release of phenolic compounds. Although CP-IDF was not directly fermented under the tested conditions, its fermentation-derived products (cell-free supernatants) exhibited biological activity. Specifically, these products demonstrated anti-inflammatory effects by modulating NO production in macrophages, suggesting a potential postbiotic mechanism. These findings highlight the value-added potential of cowpea-derived IDF as a functional ingredient with antioxidant and postbiotic-associated bioactivities.