Purification and Identification of Immunomodulating Peptide from Pepsin-Soy Protein Hydrolysate

• Main Authors: Ming-Shing Lu, 呂明興
• Other Authors: Wen-Dee Chiang
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/mkjvh3

碩士 === 東海大學 === 食品科學系 === 103 === The purpose of this study was to isolate and purify immunomodulating peptides from pepsin-isolated soy protein hydrolysates (P-ISPH) and identified peptide sequence to investigate its mechanisms of immunomodulation. At first, the effect of hydrolysis time on immunomodulating activity of hydrolysate was investigated. The result showed hydrolysis of isolated soy protein with pepsin for 4 h to obtain hydrolysate (P-ISPH4h) which had the highest phagocytosis activity and the highest yield. Furthermore, the effect of fractionation of P-ISPH4h with different molecular weight cut-off (MWCO) membranes on immunomodulating activity was studied. The P-ISPH4h was sequentially fractionated by 30 kDa, 10 kDa and 1 kDa molecular weight cut-off (MWCO) membrane, in which 1 kDa MWCO permeate (1P) showed the most significant increase of phagocytosis activity as compared with P-ISPH4h without causing excessive inflammation. To further purified and enhanced the immunomodulating activity, 1P was distinct by high performance liquid chromatography equipped with reverse-phase column and in vivo immunomodulating activity of column fractions was test in mice. The result showed column fraction 1 (F1) could significantly enhance phagocytosis activity of mice spleen macrophages and neutrophils. But increase of phagocytosis activity did not result from inducing macrophages M1 or M2 polarization, and did not have a suppression effect of pro-inflammatory cytokines induced from LPS. The result indicated that F1 did not have anti-inflammatory activity, but it had immunomodulating function through stimulating phagocytosis activity of macrophages and neutrophils. Finally, the immunomodulating peptide sequence from F1 was identified by LC-MS/MS. Peptide fragment which have most positively charged amino acids was selected and synthesized from five of the most credible peptide fragments. The synthetic peptides will test in vivo immunomodulating activity in mice and compare with F1 to understand the mechanisms of the immunomodulating peptides.