Olive flounder (OF) is a widely aqua-cultivated and recognized socioeconomic resource in Korea. However, more than 50% of by-products are generated when processing one OF, and there is no proper way to utilize them. With rising awareness and interest in eco-friendly bio-materialization recycling, this research investigates the potential of enzymatic hydrolysis of OF by-products (OFB) to produce functional ingredients. Various enzymatic hydrolysates of OFB (OFBEs) were generated using 11 commercial enzymes. Among them, Prozyme 2000P-assisted OFBE (OFBP) exhibited the highest protein content and yield, as well as low molecularization. The muscle regenerative potential of OFBEs was evaluated using C2C12 myoblasts, revealing that OFBP positively regulated myoblast differentiation. In an in vitro Dex-induced myotube atrophy model, OFBP protected against muscle atrophy and restored myotube differentiation and Dex-induced reactive oxygen species (ROS) production. Furthermore, zebrafish treated with OFBEs showed improved locomotor activity and body weight, with OFBP exhibiting outstanding restoration in the Dex-induced muscle atrophy zebrafish in vivo model. In conclusion, OFBEs, particularly OFBP, produce hydrolysates with enhanced physiological usability and muscle regenerative potential. Further research on its industrial application and mechanistic insights is needed to realize its potential as a high-quality protein food ingredient derived from OF processing by-products.
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