本研究規劃為三年計劃，第一年計劃目的為快速篩選適當的蛋白質原料及蛋白?，並應用小型酵素膜反應系統，連續地生產與區分具有促免疫調節作用的胜?混合液；首先，建立促免疫調節?的篩選技術平台，快速進行蛋白?的篩選，將6種蛋白質分別利用5種蛋白?水解後，測定其蛋白質水解物對促進淋巴細胞增殖與巨噬細胞吞噬作用的影響，並分別以促進指數(stimulating index; SI)與吸光值OD540表示之，SI與OD540值愈高其促免疫調節活性愈高，以此篩選出適當的蛋白質與具有最高促免疫調節作用的生產酵素。其次，採用上述最佳的酵素水解條件，生產免疫調節作用的蛋白水解物，並利用酵素膜反應系統進行區分，探討不同膜分子量限值 (molecular weight cut-off; 簡稱MWCO) 對免疫調節作用之影響，區分所用的膜MWCO從300到100,000 daltons，所得區分物均需測定其總氮含量、分子量分佈情形與SI值等。第二年為上年度的延續性計劃，其目的為建立中間工廠型酵素膜反應系統，量產具有促免疫調節作用的胜?混合液，探討促免疫調節?的?化條件並以液相層析質譜儀(LC-MS-MS)完成其鑑定。第三年探討促免疫調節?於細胞中的作用機制及建立免疫調節的動物功能驗證技術，以開發經動物餵食試驗所驗證的促免疫調節?產品。 This is a three-year project. The purpose of the first year project is to produce and fractionate immunomodulation-stimulating peptides from enzymatic hydrolysis of target protein by using enzymatic membrane reactor system. The activity is evaluated by measuring both lymphocytes proliferation and macrophage phagocytosis in terms of stimulating index (SI) and absorbance OD540, respectively. The higher the SI and OD540 represent the higher the activity. Five proteolytic enzymes will be employed to hydrolyze six protein materials to produce the hydrolysate, respectively. The selected enzyme should be able to produce the maximum immunomodulation-stimulating activity after hydrolysis of target protein. To obtain the better activity, optimum hydrolysis conditions, such as the ratio of substrate to enzyme concentration, hydrolysis time, hydrolysis temperature and pH value, will also be evaluated for the selected enzyme. Moreover, several membranes with molecular weight cut-off (MWCO) of 300-100,000 daltons, individually, will be used to fractionate the hydrolysate produced by the hydrolysis of target protein under the optimum hydrolysis conditions with the enzyme. The fraction obtained from the treatment of the hydrolysate by selected membrane should be able to enhance the activity. Finally, target protein, the enzyme and selected membrane are employed to study the optimum operation conditions for lab scale membrane reactor system. The second year continuous project is to establish a pilot scale membrane reactor system for mass production of peptides mix with immunomodulation-stimulating activity. Optimum operation conditions for pilot scale membrane reactor system will be evaluated. Methods for purification of the peptides will be also studied. Furthermore, identification of the peptides will be completed by LC-MS-MS. The third year will study the mechanism of immunomodulation- stimulating in lymphocytes as well as establish animal feeding test to assess the functionality of the peptides in vivo. Then we will develop a product with immunomodulation-stimulating activity.
