Enzymatic Preparation And Immunocom Pepence Research Of Sipunculus Nudus L.

Sipunculus nudus L.is mainly distributed in coasts of Hainan,Guangxi,Guangdong and Fujian,which is an aquatic with high nutrinal value and high protein content.Previous studies have already been carried on various physiological activities of Sipunculus nudus L.,such as anti-oxidation activity,radiation resistant activity,anti-inflammatory activity,immunomodulatory activity and antiviral activity,etc.Its immunomodulatory activity has been focused on polysaccharide and crude extract from Sipunculus nudus L.,however,very few studies have mentioned in its protein and peptide.Therefore,Sipunculus nudus L.was used as raw material in this paper,firstly,the physical and chemical properties of Sipunculus nudus L.protein were studied,secondly,the enzymatic hydrolysis process of isolated proteins that isolated by pH adjustment method was optimized,and then the immunologically activies of ultrafiltration components of enzymolysis products were evaluated by cell experiment in vitro and in vivo experiments,respectively.Finaly,the immunological peptides of ultrafiltration components were separated and purified by column chromatography and then identified by mass spectrometry,on the basis of immunologically active tracking.This study will provid theoretical basis for high value utilization and development of immune products of Sipunculus nudus L.The main research contents and results are as follows:?1?Sipunculus nudus L.is an aquatic product with high protein content and low fat content,those of 80.87%and 2.37%,respectively.Its water-soluble protein,salt-soluble protein and insoluble protein accounted for 19.05%,43.58%and 29.35%of the total protein,respectively.SDS-PAGE electrophoresis shows that water soluble protein strips widely distributed between 6 kDa and 200 kDa.Salt soluble protein strips mostly ranged from 29 kDa to 50 kDa and 66 kDa to 200 kDa.Insoluble protein had obviously distinct bands around 48,102.3 and 200 kDa.It contains variety of amino acid,and the contents of immune-related basic amino acid and hydrophobic amino acid in these three protein components exceed 50%of the total amino acid.DSC result shows that the denaturation temperatures of water-soluble protein,salt soluble protein and insoluble protein were77.9?,69.2?and 54.9?,respectively.?2?Based on the value-added rate of mouse spleen lymphocytes,the complex enzyme consisting of animal and papain?complex ratio 1:1?was determined by enzyme screening experiment.Enzymatic conditions were determined by a single factor and response surface optimization.The optimized enzymatic hydrolysis conditions are as follow:liquid ratio?v/m?is 5:1,hydrolysis time is 4.5 hours,enzyme amount is 2460 U/g,temperature is 56°C.The value of spleen lymphocyte proliferation rate of enzymolysis products hydrolysed under these conditions was 60.22%,which is similar to the predicted value of the model of 60.61%?p>0.05?.?3?The enzymolysis products of isolated protein from Sipunculus nudus L.was subjected to ultrafiltration to separated to two components with molecule weights of<2.5kDa and>2.5 kDa.The result in vitro shows that the higher immunological activities were determined on the concentrations of 0.6mg/mL of<2.5 kDa component and0.9mg/mL of>2.5 kDa component,respectively.And the immunological activity of<2.5kDa component?0.6mg/mL?is higher than component of>2.5 kDa?0.9mg/mL??p<0.05?.The result in vivo shows that the functions of cellular immune,humoral immune,and monocyte macrophage and the activity of NK cell were intensified by three dose groups of the<2.5 kDa component.It means the<2.5 kDa component has function of enhancing immunity,according to the evaluation method of the?Technical Specification for Health Food Testing and Evaluation?.?4?A high immunologically activity fraction named F₄₃₁ was seperated and purified from<2.5kDa component by Sephadex G-25 column chromatography followed with C₁₈HPLC,based on the immunologically activity evaluation assay in vitro.Finally,two peotides?phe-lys,phe-gln?from fraction F₄₃₁31 were identified by mass spectrometry analysis.