| In order to make better use of tuna milt,its nutrient composition was firstly analyzed.Secondly,protamine and antioxidant peptides were prepared from tuna milt,and their activity was also evaluated using in vitro chemical experiments and cell level experiments.Part ?:Nutrient composition analysis of the tuna milt.(1)Basic component analysis indicated that contents of moisture,nucleic acid,and crude proteins were(76.36±1.14)%,(27.59±0.43)%(dry weight),and(57.71±0.42)%(dry weight),respectively.Therefore,the tuna milt is a low-fat and high-protein food resource.(2)Amino acid analysis suggested that seven essential amino acids were found in the tuna milt,and arginine(Arg)was the main amino acid with the content of(9.29±0.09)%.The lysine score was the highest among all amino acids on the CS score(chemical score)and AAS score(amino acid score).In addition,the EAAI(essential amino acid index)of the tuna milt was 120.96.(3)Elemental analysis showed that the content of potassium in the tuna milt was the higher than those of other elements.Thirteen different types of fatty acids were identified from tuna milt by the national standard method.Furthermore,docosahexaenoic acid(DHA)was abundant in tuna milt.Part ?:Preparation and activity evaluation of tuna protamine.(1)The preparation process of tuna protamine was optimized by single factor and response surface methodology.The optimum conditions were temperature of 360C,H2SO4 concentration of 0.4 M,liquid-to-solid ratio of 6:1,and extraction time of 65 min.Under the optimum conditions,the yield of protamine is(11.25± 1.2)%.(2)Tricine-SDS-PAGE electrophoresis showed that the protein molecular weight of crude protamine was below 31 kDa.In addition,protamine with high arginine content was composed of a and ? chains and have the maximum absorption wavelength of around 220 nm.(3)The crude tuna protamine was further purified by CM Sephaorse CL-6B and tested by Tricine-SDS-PAGE electrophoresis and Sakaguchi reaction.The results suggested that there were 3 components(?,? and ?)in protein pattern.The component ? was the target protein with a molecular weight of 6.5-21.1 kDa.The total and basic amino acid contents of protamine were(76.90±0.37)%and(38.99±0.27)%,respectively.The amino acid of tuna protamine is similar with related literatures.(4)The results of Heparin titration method showed that the potency of the component III was about 2/3 of the protamine standard,and the potency of the components I and II was 0.In a summary,the component III was confirmed as the tuna protamine.Part ?:Antioxidant peptides from protein hydrolysate of tuna milt(1)Using DPPH free radical scavenging rate as the indicator,the preparation process of protein hydrolysate of tuna milt(TMH)with microwave-assisted extraction were optimized by single factor and response surface methodology,and the optimum conditions were microwave time of 147 s,material-liquid ratio of 1:10,microwave power of 420 W,dosage of enzyme of 3%,and pH value of 7.0.At these optimum conditions,DPPH free radical scavenging rate of resulted hydrolysate was 11.25±1.2%.(2)Using ultrafiltration membrane,TMH-?(MW<3.5 kDa),TMH-?(3.5<MW<5 kDa),TMH-?(5<MW<10 kDa)and TMH-?(MW>10 kDa)were retained from TMH.TMH-?(MW<3.5 kDa)has the highest activity including DPPH,ABTS radical scavenging activity and Ferric-reducing power among four fractions.subsequently,nine components(A1,A2,B1,B2,B3,C1,C2,D1 and E1)were isolated from TMH-? by Q Sepharose Fast Flow and Sephadex G-25 chromatography.The results on radical scavenging activity and Ferric-reducing power showed that E1 component has the highest among all fractions.Finally,thirteen pentapeptides were isolated from E1 component by UHPLC-Q-TOF-MS and identified as GHHAA(TMH1),PHPR(TMH2),AKHQ(TMH3),GRVPR(TMH4),ADMYW(TMH5),VDDDD(TMH6),SVTEV(TMH7),VKIYI(TMH8,Da),VRDQY(TMH9),IRDDY(TMH10),YREY(TMH11),AQRPR(TMH12),SMDV(TMH13)with molecular weights of 562.59 Da,505.58 Da,482.54 Da,654.77 Da,684.77 Da,577.50 Da,533.58 Da,634.82 Da,679.73 Da,680.72 Da,629.67 Da,626.72 Da and 450.51 Da,respectively.The isolated oligopeptides exhibited good activity on DPPH and ABTS radical scavenging activity and Ferric-reducing power apart from TMH4,TMH8 and TMH11(EC50>3 mg/mL).Therefore,these isolated oligopeptides(apart from TMH4,TMH8 and TMH11)were used to the following activity evaluation.(3)To discuss the protective effects of these antioxidant oligopeptides on oxidative damage cells,the oxidative damage model of HUVECs(human umbilical vein endothelial cells)was induced by H2O2.The assay suggested that the cell viability of H2O2-induced HUVECs increased significantly(p<0.05)with the protection of TMH1,TMH2,TMH7,TMH9 and TMH13 at the concentration of 200 ?M because they could significantly(p<0.05)increase the activity levels of SOD and GSH-Px,and decrease the content of MDA in HUVECs. |
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