Optimization of Enzymatic Hydrolysis and Leuconostoc mesenteroides MKJW Fermentation of Gryllus bimaculatus Using Response Surface Methodology

Gryllus bimaculatus (GB) is a promising sustainable protein source, and its bioprocessing performance is strongly influenced by processing conditions. Enzymatic hydrolysis and lactic acid bacterial fermentation represent complementary approaches for modifying GB components and developing value-added products. In this study, response surface methodology (RSM) with a Box-Behnken design was used to optimize two GB bioprocesses: Neutrase-mediated enzymatic hydrolysis and fermentation with Leuconostoc mesenteroides MKJW. For enzymatic hydrolysis, GB concentration, Neutrase concentration, and hydrolysis time were evaluated using degree of hydrolysis (DH) as the response. The model-derived condition associated with a high predicted DH within the tested range was 20% (w/v) GB, 136.2 U/mL Neutrase, and 31 h, under which the experimental DH was 78.59% compared with a predicted value of 87.03%. For fermentation, GB concentration, sucrose concentration, and fermentation time were simultaneously evaluated using viable cell count and in vitro α-glucosidase inhibition (AGI) as responses. Multi-response optimization identified 7.1% (w/v) GB, 6.7% (w/v) sucrose, and 27 h as the model-derived condition associated with simultaneously high viable cell count and AGI within the tested range, yielding experimental values of 9.94 log CFU/mL and 92.94% AGI compared with predicted values of 9.98 log CFU/mL and 86.57%, respectively. Overall, RSM enabled the quantitative identification of distinct process drivers for GB hydrolysis and fermentation and the experimental evaluation of model-derived operating conditions, providing a process-level basis for subsequent compositional, mechanistic, and functional characterization.