Preparation Of Peptide And Polysaccharide And Activity Research Of Apostichopus Japonicus Spawn Peptide

In this dissertation, gonads which were byproducts of processed Apostichopusjaponicus were selected as research materials. Spawn and sperm were differentiatedthrough a microscope, whose nutrient compositions were analyzed. The enzymatichydrolysis technologies of spawn were optimized. During preparing step,polysaccharides and cascade peptides were firstly ultrafiltered. Then chemicalantioxidative activities and cell protective activity of cascade peptides were studied.Finally, yield and antioxidative activity were selected as directions for peptideseparation and purification.1. Nutrients detection of the Apostichopus japonicus gonadsNutrients of Apostichopus japonicus spawn were balanced, in which crudeprotein was50.72g/100g and crude polysaccharide was up to27.25g/100g.Phospholipids were the main parts of crude fat. By high performance liquidchromatography (HPLC) analysis, the total amounts of amino acids and essentialamino acids in Apostichopus japonicus spawn were347.19mg/g and176.27mg/grespectively. The contents of Glu、Asp、Leu and Lys were higher than other aminoacids.The content of crude protein in Apostichopus japonicus sperm was up to72.76g/100g, in which the total amounts of amino acids and essential amino acids were536.95mg/g and250.74mg/g respectively. The contents of Glu、Lys、Ala、Asp andArg amino acids were higher than others.2. Optimization of Enzymatic Hydrolysis Technology of Apostichopus japonicusSpawnDifferent enzymes and hydrolysis modes were selected to get the optimumenzymatic hydrolysis technology. After that, we determined the best technology:mixed enzyme (papain:protamex=1:1, enzyme mass ratio) with the enzyme dosage2%, temperature75℃, hydrolysis time4h, boiling to inactivate enzyme, adding flavourzyme with the enzyme dosage1.5%, hydrolyzing1h under temperature45℃.Under such conditions, the hydrolysis degree was up to77.11%.In this experiment, coupling with autolytic enzymes in Apostichopus japonicusspawn, three exogenous enzymes were adopted to establish the mixed enzymehydrolysis system. The optimization of enzymatic hydrolysis technology wassuccessful to save costs and improve economic efficiency, and the hydrolysate wasrich in peptides and amino acids. The processed hydrolysate was fresh, delicious,homogeneous and clear, which was good material to manufacture unique flavorfunctional food.3. Purification of polysaccharides of Apostichopus japonicus SpawnIn this research, a gradient dilution method was used in ultrafiltration separationof polysaccharides and preparation of cascade peptide. After ultrafiltration,polysaccharide without protein was precipitated by trichloroacetic acid. Throughultraviolet spectral scanning, precipitated polysaccharide exhibited a strongabsorption under the wavelength from190nm to200nm, and no absorption underother wavelengths. An elution peak without trailing was obtained by using SephadexG-100gel. Only one peak was existed in sephadex G-200gel online detection.Contrasting to the standard glucose, purified polysaccharide was supposed to havemolecular weight greater than200kDa.4. Cascade peptides Preparation and antioxidative activities testsThree cascade peptides with different molecular weights range were obtained byultrafiltration separation, that is, F1with molecular weight1～10KDa, F2withmolecular weight650Da～1KDa and F3with molecular weight <650Da. Theyields of cascade peptides F1, F2and F3were0.737%,0.295%and0.515%(freshweight) respectively.Chemical antioxidative activities and cell protective activity of spawn cascadepeptides were studied. Components of F1, F2and F3revealed strong abilities toscavenge DPPH, hydroxyl and superoxide anion free radicals, and appeared adose-dependent scavenging effect. Compared to component F2and F3, F1ownedhigher scavenging capacity of DPPH and O2-. For the ability to scavenge OH, threecomponents were substantially equal. Results of cell protective activities indicatedthat cascade peptides played a role in macrophages proliferation. Three cascadepeptides with final concentration100,200,400μg/mL had good cell protectiveabilities which were significantly different (P<0.01) from the injury group. Component F1had slightly better abilities than the other two peptides in cellprotection.5. Peptide purification and physicochemical analysisBy Tricine-SDS-PAGE electrophoresis separation, component F1displayedmultiple bands with concentrated molecular distribution in the range of6.5~35KDa.By using high-speed countercurrent chromatography, component F1was separatedinto four parts with independent spectrum peaks.The four parts of F1all showedhigher radical component F1scavenging ability than former three cascade peptides,where component F1-1showed the best scavenge OH ability than the other threecomponents. Under concentration5mg/mL, component F1-1showed scavenging OH ability of82.95U/mL.Component F1-1was separated into three components by Sephadex gel.Component F1-1-3which was separated with a narrow peak had the maximumcontent in three components. We found that component F1-1-1and F1-1-3had astrong ability to scavenge OH, and they were both higher than component F1-1.Under concentration5mg/mL, component F1-1-3revealed scavenging OH ability of89.82U/mL.By Tricine SDS-PAGE electrophoresis-separation detection, component F1-1-3emerged only one clear band that meant electric charge of the peptide is homogenous.Compared with standard molecular weight protein makers, molecular weight ofcomponent F1-1-3was ascertained about30KDa. Results of UV wavelengthscanning revealed that component F1-1-3had two strong absorption peaks whichwere the characteristic absorption peaks of peptide bond and phenylalanine. Results ofreversed-phase high performance liquid chromatography (HPLC) analysis suggestedthat component F1-1-3was a kind of fairly pure substance.