Preparation And Purification Of Tilapia Skin Antioxidant Peptides

In this paper, the gelatin was extracted from the raw materials of tilapia skin with hot water method. Two different enzymes were selected and the optimum hydrolysis condition of each enzyme was determined using the orthogonal experiment. Then the best hydrolysis style was settled based on the results of single and double-enzymatic hydrolysates. Meanwhile, the antioxidant properties in vitro of tilapia skin gelatin hydrolysates were investigated. The antioxidant peptides among the tilapia skin gelatin enzymatic hydrolysates were isolated and purified finally.(1)To obtain the gelatin, tilapia skin was dealed with acid solution and alkali solution, then rinsed with distilled water at45℃for12h, and the extraction rate reached to68.10%. Alcalase, trypsin and neutral proteinase were used to hydrolyzed the tilapia skin gelatin. The results showed that the hydrolysates by neutral and Alcalase proteinase exhibited higher degree of hydrolysis (DH) than the others. So the optimal conditions of neutral and Alcalase proteinase by the orthogonal test were D3C2B3A3(4.5h, pH9.0, E/S5%and55℃) and C3D3A3B1(pH8.0,4.5h, E/S5%and35℃). Based on the optimal conditions of two single enzymes, the double-enzyme hydrolysis was further applied. And the optimal compound hydrolytic technology was first hydrolyzed by neutral proteinase then by Alcalase proteinase. The DH and hydroxyl radical scavenging activity of the enzymic hydrolysates by optimal compound hydrolytic technology was highest, which was22.11%and99.24%, respectively.(2)The tilapia skin gelatin hydrolysate (TSGH) was obtained by optimal compound hydrolytic technology, and its antioxidant properties were determined in vitro. The results showed that the scavenging ability of TSGH on hydroxyl radical, DPPH, ABTS·+and superoxide radical were all increased with increasing concentration used, and their IC50values were2.21、21.99、26.55and0.94mg/mL, respectively. At the same time, its reductive ability exibited positive correlation with the concentration used, and the reductive ability of TSGH at7.31mg/mL (FRAP values965.00μMFeSO4) was equal to that of Vc at62.52μg/mL. Besides, Cu2+chelating activity of TSGH had a bell-shaped curves with contentration, arriving to the peak (the highest cheating activity,42.72%) at the concentration of5.15mg/mL While the Fe2+cheating activity of TSGH was better than its’ Cu2+, the Fe2+cheating activity could reach to76.16%at the concentration of0.2mg/mL, and the cheating avtivity of TSGH at0.3mg/mL(82.65%) was equal to that of EDTA at20μg/mL(88.82%). Generally speaking, the tilapia skin gelatin hydrolysate showed certain antioxidatative activities in the different systems in vitro and it promised a good prospect of isolation and purified.(3)The peptides with hydroxyl radical scavenging activity were isolated and purified from tilapia skin gelatin hydrolysates. Five different peaks, named A-E throughed the Sephadex G-25gel filtration column. The third peak C showed the highest hydroxyl radical scavenging ability (IC50value was380.13μg/mL)among these peaks. Then the peak C was load onto SP Sephadex C-25ion exchange column for further purification and three peaks was eluted. The third peak C3exhibited higher hydroxyl radical scavenging ability (IC50value was135.95μg/mL). To remove the salts, C3peak was then purified by Sephadex G-15gel filtration column, and four peaks were got. Among which, the third peak C3c had the highest hydroxyl radical scavenging ability (IC50value was110.80μg/mL). then the peak C3c was further purified by reversed-phase high performance liquid chromatography on an semi-preparative C18and analysis column until a single peak was obtained. We obtained two single peaks at last, namely C3c1-pand C3c14-p, The IC50value of them was4.61and6.45μg/mL, respectively. The molecular mass and amino acid sequences of C3c1-p and C3c14-p were determined using nano-LC-ESI mass spectrometry. The molecular weights of the two peptides were317.33Da and645.21Da, and they were composed of three and five amino acids with the sequences Glu-Gly-Leu and Tyr-Gly-Asp-Glu-Tyr, respectively.