Extraction-driven structural transitions and functional enhancement of mycelial protein concentrates under homogenization pressure and alkaline conditions.

Mycelium-derived proteins from edible fungi have emerged as sustainable, nutritionally balanced, and industrially scalable alternative protein sources. Nevertheless, the limited functional performance of native mycelia restricts their broader incorporation into food formulations. This study investigated the effects of high-pressure homogenization (300-900 bar) and alkaline extraction (pH 9-12) on the structural and functional properties of Pleurotus citrinopileatus mycelial protein concentrates (PCMPCs). Extraction at 600 bar and pH 12 achieved the highest yield of 93.01% and a protein content of 79.53%. Under these conditions, solubility reached 62.27%, representing a 4.3-fold increase over that at pH 9, while foaming capacity increased 1.9-fold to 61.67%. The emulsifying activity and emulsion stability indices reached 27.82 m2/g and 62.08 min, representing improvements of 83.2% and 53.1%, respectively. Structural analyses revealed that alkaline pH played a dominant role in driving protein conformational unfolding and increasing surface charge and hydrophobicity, whereas homogenization pressure exerted an indispensable synergistic role by enhancing protein release and extraction efficiency and fine-tuning the aggregation state. Notably, pH 12 promoted disulfide-mediated covalent cross-linking at the molecular level, which together with enhanced electrostatic repulsion shaped a unique structural state accounting for the marked improvements in solubility and interfacial properties. Compared with untreated mycelial powder, PCMPCs exhibited substantially superior functionality and were competitive with commercial proteins (soy protein isolate, whey protein concentrate, and yeast protein). These findings highlight the potential of combined HPH-alkaline extraction for producing functional mycelial protein ingredients suitable for food formulations.