Preparation Of Antioxidant Peptides From Tuna Head By Enzymatic Hydrolysis

Tuna is a kind of valuable fish in ocean, which were processed mainly for canning or sashimi. About 50% to 70% waste of the total weight including head, viscera bone and skin is generated during processing .Tuna head is rich in protein with high nutritional value. It was considered as good raw materials of functional food.In this paper, tuna head was hydrolysated to prepare antioxidant peptides. The optimum conditions for antioxidant peptide from tuna head protiein by enzymatic hydrolysis were studied. The antioxidant activities in vitro and vivo of the enzymatic hydrolysis were determined. The tuna head protiein hydrolysate (THPH) was purified by ultrafiltration and gel chromatography exchange chromatography. Molecular weight distribution of the hydrolysate was evaluated using HPLC. Main results are as follow:1 The composition of tuna head shows it has high content of protein (63.33% in dry basic), high content of fat(16.72% in dry basic) and reasonable amino acid composition, which makes it a good substrate for hydrolysis. The hydrolysate of alcalase showed the highest radical scavenging activity of 59.44% at 6h compared with the scavenging effect of the hydrolysate of pepsin,neutral protease, trypsin and papain under each optimal condition. The optimum conditions of alcalase were obtained by central composite design of which temperature, enzyme-to-substrate ratio, and time of hydrolysis were chosen for independent variables according to the results of preliminary experiments. The results showed that the optimum hydrolytic conditions was temperature 54℃, 340min, enzyme 0.38% (w/w),with high radical scavenging activity of the hydrolysate to 63.67%.2 The antioxidant activity of hydrolysate obtained by the optimized hydrolysis conditions was evaluated in different systems. The reducing power of THPH was dose dependent and increased steadily with an increasing concentration of the sample, and it is shown that a linear relationship exists between the reducing power and concentration (r2=0.9997). All different dose levels of hydrolysate tested in this experiment showed considerable scavenging abilities over superoxide anion. The superoxide anion scavenging of THPH reached 84.72% at 5mg/mL, the IC50 value is 1.20 mg/mL. DPPH scavenging of THPH increased obviously with its concentration increasing. The DPPH scavenging of THPH was 57.80% at 2mg/mL and the IC50 value is 1.34 mg/mL. In addition, the THPH showed effective inhibition against lipid peroxidation in yolk lecithin system induced by metal ions. The lipid peroxidation inhibition was 51.21% at 5mg/mL and IC50 value is 4.98mg/mL. In soybean oil oxidation system, different concentrations of THPH showed significant difference inhibition ability (p<0.05). There was no significant difference between the inhibition ability of 0.1% BHT and 0.01% THPH. Determined by HPLC, the result showed that the molecular weight of THPH was mainly below 3KDa at a ratio of 59.66 % and almost 40% were the oligopeptides.3 THPH was separated to five fractions through ultrafiltration membrane with a range of molecular weight cutoffs of 8KDa,5KDa,3KDa and 1KDa, respectively. And result showed that fractions with low molecular weight were better anoxidants and the scavenging activity for fractionⅤ(<1K) was highest and the IC50 value was 1.38mg/mL. The reducing power of fractionⅤincreased with the addition amount and was slight lower than that of THPH. FractionⅤshowed a better scavenging activity on superoxide anion and DPPH radical, and the IC50 value were 0.73mg/mL and 0.93mg/mL, respectively. Moreover, fractionⅤdemonstrated good inhibition against lipid peroxidation in yolk ecithin and soybean oil. The inhibition of 0.1% fractionⅤwas almost the same as that of 0.01% BHT.4 FractionⅤwas divided into four peaks by the separation of SephadexG-25 chromatography. The molecular weight distribution of peakⅠwas 1611Da~711Da, peakⅡwas 267Da ~ 82Da. PeakⅡwas a mixture of small peptides and amino acids while peakⅢwas composed of free amino acid and peakⅣmay be some absorption of impurities. Compared with THPH and fractionⅤ, peakⅠand peakⅡshowed better scavenging activities against ? OH and IC50 values were 0.929 mg/mL and 0.527 mg /mL, respectively. IC50 value of peakⅢwas 1.65mg/mL. The analysis of amino acid composition revealed that, 87.12% of the amino acids in peakⅠwas in the form of peptide with rich in Glu, Gly, Ala, Leu and Lys content. Tyr, Phe, Leu and Met were at a high level in peakⅡ, suggesting that may be related to antioxidant activity.5 The Antioxidant activity in vivo of FractionⅤwas tested by using a model of D-galactose induced senile mice .The result showed that fractionⅤcan improve the mental state of injuried mice induced by D-galactose and had no significant effect on body weight in mice. Low dose of fractionⅤcan improve the thymus index (p <0.05) and significantly increased the SOD activity in liver of D-galactose induced mice (p <0.05).High dose and low dose of fractionⅤcould significantly increase GSH-Px activities in serum and liver of D-galactose induced mice (p <0.05). The content of MDA in groups of high dose and low dose significantly decreased comparing with D-galactose induced mice.