Preparation And Bioactivity Evaluation Of Antioxidant Peptides And Lecithin From Tuna Eggs

In this study,the nutritional composition of tuna eggs was be determined,and the results showed that the contents of moisture,crude protein,crude fat and crude ash were 68.76±0.35%,21.00±0.06%,7.61±0.12%and 1.20±0.08%,respectively.The content of mineral elements in tuna eggs was determined by inductively coupled plasma mass spectrometry(ICP-MS)and the results indicated that tuna eggs contained a large amount of K,Na,Mg,Ca,Fe and other metallic elements.Seventeen amino acids were founded in the eggs of tuna,and Glutamic acid(Glu)was measured as the major amino acid,followed by aspartic acid(Asp).Tuna eggs contained 7 kinds of essential amino acids and 8 kinds of non-essential amino acids,accounted for 40.17%and 49.92%of total amino acid content,respectively.The essential amino acids composition was basically met the requirements of the FAO/WHO criteria.In addition,tuna eggs contained 10 kinds of unsaturated fatty acids and 8 kinds of saturated fatty acids,accounting for 44.19%and 55.81%of the total fatty acid content,respectively.Palmitic acid,stearic acid and DHA were three main fatty acids,which accounted for 71.37%of the total fatty acids content.Antioxidant peptides from tuna eggs were prepared through homogenate,skim,enzymatic hydrolysis,and separation process,and their activities were evaluated in chemical and cellular levels.The main research is as follows:Using free radical scavenging capacity of enzymatic hydrolysis products as an evaluation index,the protease and their optimum conditions of enzymatic hydrolysis were optimized.Flavourase was chosen as the optimum hydrolase and its optimal conditions for enzymolysis were determined as:pH7.6,solid-liquid ratio of 1:9.6,enzyme temperature of 50?,enzyme amount of 2%,and enzymolysis time of 7 h.The hydrolysate of protein components was fractionated using ultrafiltration,and four fractions including Z1,Z2,Z3 and Z4 were obtained.Z1(MW<1kDa)with the best antioxidant activity among four fractions was divided into ten peaks(SPH-1,SPH-2,SPH-3,SPH-4,SPH-5,SPH-6,SPH-7,SPH-8,SPH-9 and SPH-10)by anion exchange chromatography and Sephadex G-25 gel chromatography.SPH-6 and SPH-9 has strong scavenging activities on free radical scavenging.Finally,seeventeen peptides were isolated form SPH-6 and SPH-9 by RP-HPLC and their amino acid sequence were indentified as:Asp-Val-Gly-Arg-Gly(L1),Leu-Glu-Pro-His(L2),Ile-Asp-Asp-Lys-Ala(L3),Glu-Asp-Asp-Lys-Ala(L4),Ala-Asp-Met(L5),Ser-Gly-Glu(L6),Val-Asp-Thr-Arg(L7),Ala-Glu-Met(L8),Gln-Asp-His-Lys-Ala(L9),Thr-Val-Me t(L10),Gln-Glu-Ala-Glu(L11),Tyr-Glu-Ala(L12),Val-Glu-Pro—(L13),Ala-Glu-His-Asn-His(L14),Gln-Glu-Pro(L15),Gln-Ala-Glu-Pro(L16),Tyr-Val-Met(L17).Using EC50(Half clearance rate)as an evaluation index,DPPH/ABTS/superoxide anion radical scavenging assays were applied to evaluate the activities of seventeen isolated peptides.Result showed that:L6,L7,L8,L9,L17 exhibited strong antioxidant activities.The H2O2-induced Chang live cell model was built at the H2O2 concentration of 400 ?mol/L for 2h treatment.The results showed that L6(500?mol/L),L7(50?mol/L),L9(50?mol/L),and L17(400?mol/L)had a protective effect on H2O2-induced Change liver cell injury and could significantly(P<0.05)improve the cell survival rate.In addition,L6,L7,L9 and L17 significantly(P<0.05)increased the levels of SOD and GSH-PX in the supernatant of cell culture medium,and decreased the content of MDA.Therefore,L6,L7,L9 and L17 have strong antioxidant capacity.The extraction process of lecithin from tuna eggs was optimized by single factor,orthogonal experiment and response surface optimization experimen,and the lipid-lowering activity of lecithin was determined.Optimization of extraction process.On the experiment results,microwave assisted enzymatic hydrolysis was chosen as the extraction methods.Firstly,the tuna eggs were hydrolyzed by trypsin and the optimal parameters of trypsin were:enzyme amount of 3%,solid-liquid ratio of 1:10,enzymolysis temperature of 37?,pH value of 7.5 and time of 2 h.The trypsin hydrolysate was centrifuged and the resulted precipitated was used for microwave-assisted extraction.The optimum technology was:radiation power 446.64 w,radiation time 101.4 s,and solid-liquid ratio:1:12.4.The yield of lecithin is up to 69.16%.The OA-induced Chang liver cell lipid accumulation model was used to evaluate the hypolipidemic effect of lecithin.The results showed that lecithin could reduced the fat accumulation of hepatocytes at 2.5-15 ?mol/L in a concentration dependent way.In addition,lecithin could significantly reduce the liver TG content.The results indicated that lecition from tuna eggs had a good lipid-lowering activity.In summary,L6,L7,L8,L9,and L17 could protect Chang liver cells against H2O2-induced oxidative damage and significantly improve the cell survival rate.Lecithin could reduced the fat accumulation of hepatocytes induced by OA.Therefore,tuna eggs can serve as raw materials for the development of antioxidant and anti-hyperlipidemia food and drugs.