Laboratory-to-pilot-scale purification of gastrointestinal-tolerant superoxide dismutase from peanut kernels: Process optimization and in vitro evaluation for oral delivery

L.) are among the richest plant sources of superoxide dismutase (SOD), with seed activity reaching 1,400-2,200 U/g fresh weight. This study established a systematically optimized, laboratory-to-pilot-scale workflow for preparing high-purity SOD from kilogram-scale peanut kernels. Because SOD is consumed rather than synthesized during germination, overnight-soaked seeds were directly homogenized for extraction. Single-factor optimization identified the key extraction conditions: 2.5 mM sodium phosphate buffer (pH 7.8), a 2:1 (v/w) liquid-to-solid ratio, and 35 °C for three 60-min rounds. Impurity removal combined n-hexane defatting (1:1, v/v, 30 min), heat treatment (55 °C, 40 min), and ammonium sulfate fractionation (50% (w/v) for impurities; 85% for SOD). Sequential Sephadex G-100 size-exclusion and DEAE-52 anion-exchange chromatography yielded a specific activity of approximately 506 U/mg protein with 79.5-fold purification and 26% overall recovery. The freeze-dried SOD powder exhibited robust thermal stability (no significant loss at 55 °C for 60 min), broad pH tolerance (5.0-11.0), and room-temperature stability (48 h in 2.5 mM phosphate buffer, pH 7.8). Notably, the enzyme retained up to 81% activity after 120 min in simulated gastrointestinal fluid, suggesting favorable oral bioavailability. This work provides a comprehensive process engineering foundation for the pilot-scale production of plant-derived SOD for oral applications.