Alkaline hydrogen peroxide-xylanase sequential modification enhances the prebiotic functionality of citrus peel and pulp fibers.

Citrus pomace represents a sustainable yet underutilized source of dietary fibers. This study investigated whether alkaline hydrogen peroxide-xylanase sequential modification could enhance the prebiotic functionality of citrus peel and pulp pomace fibers. Relative to native fibers, sequential modification significantly increased soluble dietary fiber content (from ∼10% to ∼31%), reduced lignin encapsulation, and created porous, cracked surface morphologies that enhanced enzyme accessibility and binding capacity. Eight fiber substrates (native and modified peel/pulp) were evaluated through simulated gastrointestinal digestion, digestive enzyme inhibition assays, cholesterol/bile salt binding, pure-culture fermentation with Lactobacillus acidophilus and Bifidobacterium animalis, and human colonic fermentation with 16S rRNA sequencing. Alkaline hydrogen peroxide-xylanase sequentially modified citrus peel fiber (AXCPF) and alkaline hydrogen peroxide-xylanase sequentially modified citrus pulp fiber (AXCRF) retained porous surface morphologies post-digestion, exhibiting 95% matrix retention, significant cholesterol binding (18 mg/g), and potent α-amylase inhibition (30%). Structural divergence between peel and pulp fractions underpinned distinct fermentation behaviors: pure-culture screening revealed strain-specific fermentation patterns; L. acidophilus produced elevated lactate (0.94 mg/mL) on AXCPF, whereas B. animalis generated higher acetate (1.38 mg/mL) and propionate (0.39 mg/mL) on AXCRF. In human colonic fermentation, AXCRF yielded 77% more total short-chain fatty acids (SCFAs) than fructo-oligosaccharide (FOS), shifting the molar ratio toward equimolar acetate/propionate/butyrate (1:1.1:1.2) and enriching Prevotella, while AXCPF enriched Bacteroides and sustained butyrate production. These compositional and metabolic divergences demonstrate that sequential modification creates structurally programmable dietary fibers, enabling targeted design for distinct colonic health applications.