Impact of salting-in/out assisted extraction on rheological, biological, and digestive, and proteomic properties of Tenebrio molitor larvae protein isolates

In this study, NaCl (salting-in) and (NH4)₂SO₄ (salting-out) treatments were employed in alkaline extraction and acid precipitation procedures, respectively, to enhance the extraction of Tenebrio molitor larvae protein isolates (TPIs). The in vitro digestibility, rheological properties, biological activities, and proteomic analysis of TPIs were investigated. The results showed that salting-in treatment did not result in significant differences (P > 0.05) in antioxidant activities (except for ABTS radical scavenging), but increased tumor necrosis factor β (TNF-β) and the degree of hydrolysis (DH). Salting-out treatment significantly (P ˂ 0.05) enhanced trichloroacetic acid-soluble peptide yield (Tsp) and ACE inhibitory activities but negatively affected antioxidant and antibacterial activities. The combined salting-in-out treatment produced the highest values of DH (45.80 %), Tsp (72.46 %), and TNF-β (0.86 mg/kg). Proteomic analysis using UPLC-MS/MS identified 141 proteins, including metabolic enzymes and actin, in the TPIs. While the salting treatments did not significantly alter the protein compositions, they primarily affected protein content. Overall, salting-in and salting-out treatments can effectively enhance specific biological properties of T. molitor protein isolates, particularly digestibility and ACE inhibitory activities, while salting-out treatments may reduce antioxidant functions. These findings suggest the potential of salting-assisted extractions for optimizing insect protein functionality in food and nutraceutical applications.