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Abstract: To optimize the hydrolysis conditions to prepare hydrolysates of jellyfish umbrella collagen with the highest hydroxyl radical scavenging activity, collagen extracted from jellyfish umbrella was hydrolyzed with trypsin, and response surface methodology (RSM) was applied. The optimum conditions obtained from experiments were pH 7.75, temperature (T) 48.77 °C, and enzyme-to-substrate ratio ([E]/[S]) 3.50%. The analysis of variance in RSM showed that pH and [E]/[S] were important factors that significantly affected the process (P<0.05 and P<0.01, respectively). The hydrolysates of jellyfish umbrella collagen were fractionated by high performance liquid chromatography (HPLC), and three fractions (HF-1>3000 Da, 1000 Da<HF-2<3000 Da, and HF-3<1000 Da) were collected. The HF-2 fraction had the highest hydroxyl radical scavenging activity with the highest yield compared with the other two fractions. Furthermore, HF-2 also showed the strongest Cu²⁺-chelating ability and the best tyrosinase-inhibitory activity.

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