Qian Zhu

Lentinus edodes stipe is an under-utilised processing by-product that holds potential as a natural umami source. This study systematically compared nine enzymatic hydrolysis combinations, including single proteases, cellulase, and double enzyme systems comprising both protease–protease and protease–cellulase mixtures, to optimise the release of taste-active and volatile flavor compounds from the stipes. The Flavourzyme-only hydrolysate achieved the highest degree of hydrolysis (50.38%), the richest 5′-nucleotides (5.46 mg/g), free amino acids (59.06 mg/g) and volatile compounds (150.23 μg/kg), and an equivalent umami concentration of 288 g monosodium glutamate equivalent per 100 g matter. It also delivered the strongest umami taste and the lowest bitterness in sensory evaluation. The double-enzyme systems consistently underperformed due to competitive substrate binding, suboptimal pH for companion enzymes, and potential mutual protease digestion. Among the volatile compounds, 1,2,4-trithiolane and cedrol were identified as key aroma differentiators by multivariate analysis. These findings demonstrate that Flavourzyme alone constitutes the optimal enzymatic system for producing clean-tasting, umami-rich condiments from L. edodes stipes and caution against indiscriminate enzyme blending without prior compatibility assessment. The optimized hydrolysis process provides a scalable route for the industrial valorisation of mushroom processing by-products.