促脂解活性大豆?最適水解條件探討、純化及定序

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Title:	促脂解活性大豆?最適水解條件探討、純化及定序
Other Titles:	Optimized production, purification and sequencing of soy peptide with lipolysis-stimulating activity
Authors:	高豪駿 Kao,Hao-Chun
Contributors:	江文德 Chiang, Wen-Dee 東海大學食品科學系
Keywords:	脂肪分解;脂肪細胞;反應曲面法;Flavourzyme;大豆? lipolysis;adipocytes;response surface methodology;Flavourzyme;soy peptide
Date:	2010
Issue Date:	2011-10-12T06:44:23Z (UTC)
Abstract:	本研究依據 3T3-L1 脂肪細胞的丙三醇釋放量作為促脂解?篩選的指標，藉由反應曲面法 (RSM)　探討 Flavourzyme 水解分離大豆蛋白 (ISP) 的最適水解條件，以生產水解物可增加脂肪細胞的丙三醇釋放量。水解的變數分別為反應pH值 5.32-8.68、反應溫度 33.2-66.8 ?C 與水解時間 19.2-220.8 min。反應曲面迴歸分析 (RSREG) 顯示， pH 7.12 、 48.8 ?C 、 124.9 min ，其水解物對3T3-L1脂肪細胞丙三醇釋放量最大化可達 359.93 nmol/mg protein ，依照最適化水解條件進行驗證實驗，結果顯示水解物對丙三醇釋放量為 359.92 nmol/mg protein ，與預測值相符。為了進一步?化與定序促脂解活性?，首先利用 1,000-30,000 Da 的分子量限值 (MWCO) 濾膜初步區分水解物，結果指出 F-ISPH 經1 kDa MWCO 濾膜區分所得 1 kDa retentate 促脂解活性最為顯著，400 ppm 的 F-ISPH 1 kDa retentate 可使丙三醇釋放量從318.73 nmol/mg protein 提升至 378.19 nmol/mg protein (p<0.05) 。後續以膠體層析細部分離出分子量介於 189-2,080 Da 胜?片段區分物具有最顯著的促脂解活性，於 4 ppm 添加濃度可顯著提升丙三醇釋放，從318.73 nmol/mg protein 提升至 507.06 nmol/mg protein (p<0.05) 。最後透過逆相層析分離並?化出兩種活性胜? RHF4-2 和 RHF4-3 ，各別添加 4 ppm 對丙三醇釋放量即顯著高於控制組 (p<0.05) ，丙三醇釋放量從 316.18 nmol/mg protein 分別提升至 580.59 nmol/mg protein 與 615.87 nmol/mg protein 。 RHF4-2 經鑑定其序列可能為 Leu-Leu-Leu 、 Ile-Leu-Leu 或 Ile-Ile-Ile ，而 RHF4-3 則為 Val-His-Val-Val 。 The research was based on glycerol release in 3T3-L1 adipocytes as a marker for screening the lipolysis-stimulating peptides. The optimum hydrolysis conditions of isolated soy protein (ISP) with Flavourzyme for increasing glycerol release in 3T3-L1 adipocytes were investigated by response surface methodology (RSM). The independent variables were reaction pH 5.32-8.68, reaction temperature (RT) 33.2-66.8 ?C and hydrolysis time (HT) 19.2-220.8 min. Based on response surface regression (RSREG) procedure, the optimum hydrolysis of ISP with Flavourzyme for maximizing glycerol release (359.93 nmol/mg protein) in the cells were: pH = 7.12, RT = 48.8 ?C and HT = 124.9 min. According to optimum hydrolysis conditions, the verification studies proved that experimental value (359.92 nmol/mg protein) and predicted value were very closely. In order to purify and sequence the lipolysis-stimulating peptides from Flavourzyme-ISP hydrolysates (F-ISPH), the first purification step employed several membranes with molecular weight cut-off (MWCO) of 1,000-30,000 Da to fractionate F-ISPH. The F-ISPH 1 kDa retentate obtained from the treatment of F-ISPH using 1,000 Da MWCO membrane could significantly (p<0.05) increase glycerol release from 318.73 nmol/mg protein to 378.19 nmol/mg protein at 400 ppm level. The F-ISPH 1 kDa retentate was further fractionated by gel chromatography. The fraction with molecular weight between 189 to 2,080 Da could significantly (p<0.05) increase glycerol release from 318.73 nmol/mg protein to 507.06 nmol/mg protein at 4 ppm level. The reverse-phase chromatography was further employed to purify lipolysis-stimulating peptides including RHF4-2 and RHF4-3. Their glycerol release were significantly higher than control (p<0.05). Glycerol release of RHF4-2 and RHF4-3, respectively, increased from 316.18 nmol/mg protein to 580.59 nmol/mg protein and 615.87 nmol/mg protein at 4 ppm level. Amino acid sequence of RHF4-2 may be Leu-Leu-Leu, Ile-Leu-Leu or Ile-Ile-Ile. The sequence of RHF4-3 was Val-His-Val-Val.
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