| Lipid oxidation, which might results in undesirable off-flavors and potentially toxic reaction products, and some chronic diseases due to unbalance in the body related to free radical damage, could be inhibited or weakened in the addition of natural antioxidant without toxicity and side effects. It is theoretical and practical significances on studying about the preparation and application of squid antioxidant from by-products, which could also enhance the utilization rate of the squid industry.The preparation of squid meat meal and skin gelatin from residuals, were invested at first, the extraction ratio of which were 15.26% and 5.94%, respectively. The hydrolysates from squid meat and gelatin using five proteases (pepsin, trypsin, papain, alcalase and flavourzyme) were detected for the hydrolysate with the maximum antioxidative effect on DPPH and hydroxyl radical scavenging activities. Results showed that, the target hydrolysate was present in the hydrolysis of squid meat with papain in 30 minute, and the highest ratio of scavenging DPPH and hydroxyl radical were respective 59.51% and 57.58%.Based on the results of one-factor-at-a-time-technique, a three-level Box-Behnken factorial design combined with response surface methodology (RSM) was employed to optimize the enzymatic hydrolysis condition of squid meat with papain. It was showed that, the optimum conditions obtained to prepare OSMH (optimal squid meat hydrolysate), were pH of 7.0, substrate concentration of 4%, E/S ratio of 1.74%, temperature of 31℃and time of 46 min. The maximum DPPH radical scavenging activity of 74.25% derived from the optimum conditions, was in accordance with the predicted value (74.40%) within a 99% confidence interval.Besides, antioxidant activities of the antioxidant hydrolysate including free radicals scavenging, reducing power, chelating capability and inhibition of lipid peroxidation were also investigated. Results were as follows:the ratio of scavenging hydroxyl, superoxide anion and hydrogen peroxide radical with the hydrolysate concentration of 1.5 mg/mL were 72.46±1.21%,54.95±1.76% and 48.71±1.16%, respectively. The reducing power of the hydrolysate in the concentration of 10mg/mL was stronger than that of 0.01 mg/mL BHT and 0.1 mg/mL VE. The iron-chelating ability was respective 28.94±0.71% and 30.19±0.63% with the concentration of 8 mg/mL and 10 mg/mL. Moreover, it was found that the optimum hydrolysate concentration of 8 mg/mL exhibited a relative stronger inhibitory effect on lipid peroxidation compared with a-Tocopherol of 0.1 mg/mLIn order to determine structures of the most active peptides fraction, the two pure components were analyzed by Mass Spectrometer (Waters Platform ZMD 4000) and Biolynx Software, after ultrafiltration, ion exchange chromatography, gel chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) utilized one after another to separate and purify the peptides in the rule of high DPPH scavenging ability, with the result that Pro-Glu-Trp (430.18 Da) and Ser-Ser-Leu-Ala-Gly-Pro (530.27 Da) obtained.Finally, the antioxidative effect of OSMH was studied on the model of Chinese sausages. Results showed that, peroxide value (POV) and acid value of sausages in the addition of 0.5%,1.0%,1.5% and 2.0% OSMH, respectively, stored at 37℃,25℃and 4℃, were better than that of blank group. The higher the OSMH concentration, the stronger the antioxidant activity. The antioxidant activity of 2% OSMH was significantly higher than that of synthesized antioxidant, butylated hydroxytoluene (BHT). Furthermore, results of sensory evaluation of experimental Chinese sausages were not affected badly by the addition of OSMH.
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