Fruit pretreatment and extraction method govern macadamia protein functionality and ACE2 inhibitory peptides: A flag-ACE2 pull-down study

This study examined the combined effects of fruit pretreatment (fresh, air-dried, and roasted) and extraction method (alkaline versus salt) on the structure, physicochemical properties, and function of macadamia protein. Roasting increased protein recovery but decreased water-holding capacity. In contrast, salt extraction improved water-holding capacity and surface hydrophobicity despite lower recovery. Principal component analysis (PCA) revealed that emulsifying properties were primarily determined by the extraction method rather than by the pretreatment method. Simulated gastrointestinal digestion (SGID) significantly enhanced both DPPH radical-scavenging and ACE2 inhibitory activities. Following SGID, the roasted samples (both alkaline- and salt-extracted) exhibited stronger DPPH scavenging activity. Meanwhile, the roasted alkaline-extracted samples showed higher ACE2 inhibition (84.67%). A novel strategy was then established: Flag-ACE2 affinity pull-down coupled with peptidomics to identify ACE2-inhibitory peptides from the most active gastrointestinal digest. Using this method, a total of 37 novel ACE2-binding peptides were identified across three independent experiments. Among them, three peptides (AAYQVAALPK, GGGGFGGGGFGGGGIGGGGF and YYHKPSVF) with relatively low binding energies to ACE2 were selected for functional validation. The synthetic peptides inhibited ACE2 in a concentration-dependent manner, with respective IC₅₀ values of 40.21, 9.14, and 152.5 μM. The glycine-rich peptide GGGGFGGGGFGGGGIGGGGF exhibited the strongest inhibitory activity. These results illustrate the critical role of the method of fruit pretreatment and extraction in determining the techno-functional and bioactive properties of macadamia protein. Furthermore, the target protein affinity pull-down strategy effectively bypasses conventional fractionation workflows, enabling the direct identification of ACE2-inhibitory peptides in complex food digests. This offers a platform for screening bioactive peptides against other protein targets.