本研究目的為分離、純化pepsin-大豆分離蛋白水解物(P-ISPH)中免疫調節肽，並鑑定其胜肽序列與探討免疫調節機制。首先探討不同水解時間P-ISPH對免疫調節活性的影響，結果顯示pepsin水解大豆分離蛋白4h所得水解物(P-ISPH4h)能顯著提升細胞吞噬活性且產量最高。其次，探討不同濾膜分子量限值(molecular weight cut-off ; MWCO)區分對P-ISPH4h免疫調節活性之影響，將P-ISPH4h進行30 kDa、10 kDa及1 kDa MWCO濾膜序列區分，其中以1 kDa濾液(1 kDa MWCO permeate ;1P)最能顯著提升細胞吞噬活性且不引起過度發炎反應。為進一步純化並提升免疫調節活性，將1P以逆向高效能液相層析管柱區分，並進行小鼠體內免疫調節活性試驗，結果以管柱區分物fraction 1(F1)對小鼠脾臟中巨噬細胞及中性顆粒球細胞皆能顯著提升其吞噬活性，但其吞噬活性的增加並非來自誘導巨噬細胞向M1或M2極化，且對於LPS誘導之促發炎細胞激素不具有抑制的效果，表示F1不具有抗發炎活性，但能透過提升巨噬細胞及中性顆粒球的吞噬活性達到免疫調節的功能。最後將F1以LC-MS/MS鑑定其中免疫調節胜肽序列，從可信度最高的五個胜肽片段中挑選一段帶正電荷胺基酸最多之胜肽片段進行合成，後續將以此合成肽進行小鼠體內試驗並比對F1之免疫調節活性，以了解免疫調節肽之作用機制。 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.
