Research On Antioxidant Activity And Purification Of Hydrolysates Obtained From Yellow-margined Box Turtle (Cistoclemmys Flavomarginata) Muscle

Many diseases and human aging phenomenon are related to the imbalance offree radicals in human body. External antioxidants can protect the body againstdamage by free radicals, and maintain the balance of free radicals in the body. It ispossible to develop health foods and effective antioxidant drugs on the basic of theresearch of natural foods with antioxidant activity. Yellow-margined box turtle（Cistoclemmys flavomarginata） has special nutritional invigoration and medicinalvalue, and it is also a good source of protein. Therefore, if C. flavomarginata wasfully exploited and utilized, it could have important economic and social benefits.The muscle of C. flavomarginata was subjected to chemical compositionanalysis and nutritional evaluation. The examination results indicated that thecontents of moisture, crude protein, crude fat and ash in fresh turtle muscle were79.99%,18.12%,0.58%and0.70%, respectively. The crude protein in the samplecontained18kinds of amino acids with higher levels of essential amino acids,comprising of42.85%of total amino acids, and the constitutional rate of theessential amino acids satisfied FAO/WHO reference standards. The ratio of totaldelicious amino acids to total amino acids was36.53%. The essential amino acidindex was82.68. The ratio of branched chain to aromatic amino acids was2.54,which was close to the normal levels of human body. The fatty acid profile wasdominated by unsaturated fatty acids, accounting for52.84%of total fatty acids,especially DHA and EPA were rich in samples. Meanwhile, C. flavomarginata wasa good minerals source, particularly zinc, selenium and iron. In conclusion, C.flavomarginata was found to be a nutritive and delicious food.The muscle of C. flavomarginata was hydrolyzed by six types of proteases,including pepsin, trypsin, alcalase, neutrase, papain and flavourzyme, and alcalasewas chosen as the optimum enzyme to hydrolyze the muscle for antioxidantpeptides. By using a L9（34） orthogonal array design, the optimum conditions wereas follows: pH8.0, temperature55℃, material-to-liquid ratio1.5:20, enzyme-to-substrate ratio3.0%and reaction time3h. Under the above conditions, thehydroxyl radical scavenging rate of the hydrolysates was82.08%.The antioxidant properties of C. flavomarginata hydrolysates were determinedin vitro. The results showed that the enzymatic hydrolysates had good scavenging abilities on hydroxyl radical （·OH）, superoxide anion radical （O2-·）, α,α-diphenyl-β-picrylhydrazyl radical （DPPH·） and hydrogen peroxide （H2O2）, and they also hadcertain reducing power, ferrous ion chelating capacity and the inhibition of linoleicacid autoxidation. In general, C. flavomarginata hydrolysates showed certainantioxidant activities in different systems in vitro, and they would have a greatprospect for deep development and comprehensive utilization.The C. flavomarginata hydrolysates were separated with ultrafiltration tothree parts （a large molecular weight fraction, a middle molecular weight fractionand a low molecular weight fraction） based on molecular weight cut-offs （MWCOs）of1000Da and3000Da. It was proved that hydrolysates with the molecularweight below1000Da had the highest hydroxyl radical scavenging activity, andthey were orally administered to D-galactose-induced aging mice in order toinvestigate its antioxidant activities in vivo. The results revealed that C.flavomarginata peptides could elevate the activities of T-AOC, SOD and GSH-Px,and reduce the level of MDA in the serum, brain and liver, meanwhile antioxidanteffect was positively correlated with dosage.Antioxidant peptides were purified as hydroxyl radical scavenging activity fortarget. The fraction of molecular weight less than1000Da was separated into twofractions using Sephadex G-25gel filtration chromatography. Fraction F₁exhibitedhigher hydroxyl radical scavenging capability, and its IC₅₀value was6.85mg/mL,so fraction F₁was further purified using anion exchange chromatography. FractionF₁was divided into three parts, including F₁₁, F₁₂and F₁₃, among which fractionF₁₃had the strongest hydroxyl radical scavenging activity, and its IC₅₀value was5.63mg/mL. Fraction F₁₃was identified by RP-HPLC to be a single peak （fractionR）, so peptides that were separated and purified from C. flavomarginatahydrolysates were relatively pure. Fraction R was determined by massspectrometry to get a main peak, and its molecular weight was693.00Da. Thus, itcould be inferred that fraction R was mainly composed of an oligopeptide whichmight contain6-7amino acid residues.