The Study On Immunopeptides From Alcalase-Hydrolyzed Wheat Proteins

Immunopeptides are very important and have many immune functions such as stimulate lymphocyte proliferative, enhance the phagocytic activity of macrophages, keep organism from infection of pathogeny, reduce the incidence of disease, anti-tumour and so on. In present study, the immunostimulating activity hydrolysates were got from the wheat protein hydrolyzed by Alcalase. By a series of separating way two peptides were drawn from the hydrolysates. The followings were the results:There were more than 140 immunopeptides collected. It looks like that the molecular weight of these immunopeptides were very small, and it also showed that the hydrophobe amino acid on the bottom and alkaline amino acid were important structurals of the immunopeptides. Then the preparation techniques of wheat peptides through hydrolyzing wheat isolated proteins by Alcalase were studied. The optimum hydrolysis conditions were determined: dosages of enzyme 13×103u/g, pH 9.5, 60℃, substratum concentration 4%, hydrolysis time 100min and the hydrolyzing degree 18.63%. Molecular weight of the hydrolydates were distributed between 147Da~1548Da, in which the molecular weight less than 600Da were 56.12% and 2000Da were 74.18%. Small peptides were the main component.Before the immunostimulating activity of the hydrolysates was assayed, salt was removed by DA201-C macroporous adsorption resins. The result showed that 50mg/mL wheat protein hydrolysates was carried out to remove salt using a DA201-C column in 0.5BV/h flow rate, then the column was washed using water and wheat protein hydrolysates desorbed by 75% ethanol in 1BV/h flow rate. Desalt ratio was reached 91.26%. In vitro study of MTT colorimetry, results showed that wheat protein hydrolysates can enhance lymphocyte proliferative responses especially with 150μg and 200μg (P<0.05).Due to the high frequent appearance of alkaline amino acids in immunopeptides, the hydrolysates were separated by ion exchange chromatography firstly according to the character of charge. The result of MTT colorimetry showed that two fractions got at pH8 and pH10 (fraction B and fraction C) can enhance lymphocyte proliferative responses. These two fractions were separated by gel-chromatography and fraction C3 showed the highest activity. 12 fractions were got by further purification of C3 by RP-HPLC. C3-11 can highly enhance lymphocyte proliferation. The identification of fraction C3-11 by LC/MS showed that there were two components within. The analysis by MassLynx software showed that the molecular weight of the main component was 617.2 and its amino acid sequence was His-Cys-Pro-Val-Tyr. Another component was a peptide with...