Sequential Enzymatic and Ultrasonic Extraction of Lentinula edodes Mushroom Proteins Leading to Enhanced Yield and Significant Immunoactivity

Lentinula edodes is a widely consumed edible fungus and a rich source of proteins with both nutritional and medicinal value. This study discovered an effective process to extract proteins from L. edodes mushroom by comparison of water extraction (WE), enzyme-assisted extraction (EAE), ultrasound-assisted extraction (UAE), and various combinations of EAE with UAE. The two-step and sequential scheme by EAE and then UAE, designated EUE resulted in the highest protein yield compared with EAE after UAE (UEE) and simultaneous EAE and UAE (SEUE). The soluble protein yield by EUE (9.4%) was nearly three times that by UEE (3.6%) and 1.4 times and around two times higher than by UAE (6.9%) and EAE (4.9%), respectively. Compared with those by other extraction methods, the protein fraction by EUE had the highest protein content (56.0%) and β-sheet content (55.8%) and exhibited the strongest in vitro immunostimulatory activity. Through statistically designed experiments and response surface methodology, EUE conditions were optimized as enzyme 0.28% (w/v), ultrasound amplitude 62%, and (NH ₄ ) ₂ SO ₄ saturation 69%, achieving 9.7% protein yield and 58.4% protein content. The distribution of protein molecular weight (MW) was below 10 kDa and between 25-75 kDa. The protein fraction contained nutritional amino acids and significant immunostimulatory activities in vitro . EUE has shown promising potential for efficient extraction of proteins from mushrooms in the food industry.