Preparation Of Antioxidant Peptides From Shrimp Byproducts By Enzymolysis

With the rapid development of shrimp processing industry, a large number of byproducts were produced during shrimp processing. In our country, these shrimp byproducts which contain abundant protein are thrown away directly, the potential value of such byproducts for the chemical industry is being ignored. It not only causes environment pollution, but also wastes resources. In this paper, antioxidant peptides were prepared by specific enzyme hydrolysis method, using shrimp byproducts as raw material. The fractions showing high antioxidative activity were isolated from the hydrolysates by dialysis and using consecutive chromatographic methods including Sephadex G-15 gel filtration, DEAE Sephacel ion exchange chromatography, and RP-HPLC.According to the quantitative structure activity relationships of the antioxidant peptide and the cleavage site of common protease, we find that the combination of α-chymotrypsin and thermolysin can get antioxidant peptides. After pre-treatment, shrimp byproduct was smashed into powder. The hydrolysis time was optimized according to the degree of hydrolysis(DH), the results showed that the optimal hydrolysis time for α-chymotrypsin was 4 hours(DH was 16.43 %) and thermolysin was 6 hours(DH was 5.50 %).The separation and purification of antioxidant peptides was optimized according to the degree of hydrolysis. The antioxidant activity was assayed based on 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical scavenging ability and ferric-reducing antioxidant power(FRAP). Then the hydrolysates was separated into two fractions(0~500 Da and 500~1000 Da) by molecular weight cut-off(MWCO) membrane of 500 Da and 1000 Da. The DPPH radical scavenging activity of 0~500 Da fraction was 1.99 ± 0.01 μmol AAE/g and FRAP of 500~1000 Da fraction was 9.46 ± 0.37 μmol AAE/g. The fraction of 500-1000 Da shows higher antioxidant activities. Its DPPH radical scavenging activity was 3.87 ± 0.03 μmol AAE/g and FRAP was 14.65 ± 0.08 μmol AAE/g. Fraction of 500-1000 Da were further separated by gel filtration chromatography and divided into three peaks(G1 to G3). Peak G1 showed highest antioxidant activities, the DPPH radical scavenging activity was 7.96 ± 0.06 μmol AAE/g and FRAP was 53.11 ± 0.21 μmol AAE/g; the DPPH radical scavenging activity of peak G2 was 7.96 ± 0.06 μmol AAE/g and FRAP of peak G2 was 53.11 ± 0.21 μmol AAE/g; peak G3 showed the lowest antioxidant activities. Then peak G1 was concentrated for further separation by ion exchange chromatography and divided into four peaks(G1-H1 to G1-H4). Peak G1-H2 showed highest antioxidant activities, the DPPH radical scavenging activity was 10.54 ± 0.03 μmol AAE/g and FRAP was 89.29 ± 0.01 μmol AAE/g; the DPPH radical scavenging activity of peak G1-H1 was 8.22 ± 0.01 μmol AAE/g and FRAP of peak G1-H1 was 54.79 ± 0.06 μmol AAE/g; peak G1-H3 and peak G1-H4 showed low antioxidant activities. Peak G1-H2 was chosen to be separated by RP-HPLC and got two peaks(G1-H2-F1 to G1-H2-F2). Peak G1-H2-F1 showed higher antioxidant activities. Its DPPH radical scavenging activity was 14.91 ± 0.09 μmol AAE/g and FRAP was 112.53 ± 0.54 μmol AAE/g.Three peptides were found in peak G1-H2-F1 by time-of-flight mass spectrometry analysis: pentadecapeptide YSLKMGGVSVVVIA, tridecapeptide DISHNQRGAILVR, decapeptide GCKVALIVVG. The decapeptide has the highest concentration and antioxidant activity. Its DPPH radical scavenging activity was 16.10 ± 0.02 μmol AAE/g and FRAP was 245.37 ± 0.03 μmol AAE/g. The peptides which showed high antioxidant activity have been obtained from shrimp byproducts, the results provide theoretical basis for the production and application of the byproduct of shrimp.