The Study On Enzymatic Process Of Pig Bone Immune Peptides And Their Separation

Fresh pig bone were enzymolyzed by trypsin,papain or Alcalase to obtain collagen peptides.The proliferation of spleen lymphocyte in mouse were selected as an main indicator,Referencing the degree of hydrolysis(DH).The best hydrolysis conditions were got by single factor experiment and orthogonal experiment.The immunologically active peptides from pig bone hydrolyzate were separated and purified by ultrafiltration,gel filtration chromatography and ion exchange chromatography.The main findings are as follows:The optimal condition of papain were 4h(t), pH5.5,7000U/g (E/S),55℃(T) and 6%(S).Under these conditions the hydrolysis of degree could reach 18.25%,while proliferation rates on spleen lymphocyte of hydrolysates could reach 79.50%; The optimal condition of trypsin were 5h(t)5 pH7.5.5000U/g (E/S),50℃(T) and 6%(S). Under these conditions the hydrolysis of degree could reach 21.25%,while proliferation rates on spleen lymphocyte of hydrolysates could reach 67.14%;The optimal condition of Alcalase were 4h(t), pH9.5,6000U/g (E/S),45℃(T) and 6%(S).Under these conditions the hydrolysis of degree could reach 16.82%,while proliferation rates on spleen lymphocyte of hydrolysates could reach 98.41%.The hydrolysates hydrolyzed by Alcalase had the strongest proliferation rate on spleen lymphocyte.Using four specifications ultrafiltration centrifugal tubes to separate the higher proliferation rates on spleen lymphocyte component of the hydrolysates hydrolyzed by Alcalase,whose intercept molecular weight was 10Ku,5Ku,3Ku,2Ku.After ultrafiltration, the component of molecular size less than 2Ku had the highest proliferation rates on spleen lymphocyte of 100.89%.The component of molecular size less than 2Ku were load onto Sephadex G-25 gel filtration column and four peaks were eluted.The second peak had the highest proliferation rates on spleen lymphocyte of 109.83% when the peptide concentration was 100μg/mL.Then the second components were separated by SP-Sephadex C-25 ion-exchange chromatography into nine peaks.The forth peak showed the highest highest proliferation rates on spleen lymphocyte of 114.30% when the peptide concentration was 100μg/mL.The component of molecular size less than 2Ku were load onto SP-Sephadex C-25 ion exchange column and eight peaks were eluted.The fifth peak had the highest proliferation rates on spleen lymphocyte of 107.97% when the peptide concentration was 100μg/mL.Then the fifth components were separated by Sephadex G-25 gel filtration column into five peaks. The third peak showed the highest highest proliferation rates on spleen lymphocyte of 110.95%when the peptide concentration was 100μg/mL.Comparison the effect of two methods for separating pig bone protein hydrolysates, determining the best method for the separation were:Using ultrafiltration centrifugal tubes to separate the hydrolysates,then subjected to gel filtration chromatography, ion exchange chromatography final.The proliferation rates on spleen lymphocyte of Peptides by isolated was 114.30%.