【作者】 李干红；

【导师】 丁晓雯；

【作者基本信息】 西南农业大学 ， 农产品加工与贮藏工程， 2004， 硕士

【摘要】 本研究以芝麻蛋白为原料，采用现代生物技术——酶工程技术对芝麻蛋白进行酶解。具体对芝麻蛋白的提取、酶解蛋白酶的筛选、酶解工艺参数的确定、水解液的风味改善、产物抗氧化性及对阻断亚硝胺类物质的合成等进行了研究，现将该实验研究情况报告如下： 1．以蛋白质浸出率为指标，采用单因子实验和正交实验研究芝麻蛋白适宜的提取条件，实验得出最适条件为料液比1：20，pH为12.50，温度为80℃，在此工艺条件下蛋白质浸出率为79.0％。在pH3.03～4.51范围内筛选蛋白质的最适沉淀pH为3.42～3.97，在此范围内，芝麻蛋白的得率为86％～97％。 2．以水解度为指标，对胃蛋白酶、胰蛋白酶、AS1.398中性蛋白酶、胰酶和木瓜蛋白酶五种酶酶解芝麻蛋白的能力进行检测，实验结果表明这五种酶对芝麻蛋白的酶解能力顺序为：AS1.398中性蛋白酶＞胰蛋白酶＞胰酶＞胃蛋白酶＞木瓜蛋白酶。AS1.398中性蛋白酶表现出了最好的酶解能力，它是从微生物中提取出来的酶，酶切位点多，价廉易得，可以作为酶解芝麻蛋白的最适酶。 3．以水解度为指标，采用正交旋转组合实验（四因子两水平（1／2）实施）对AS1.398中性蛋白酶酶解芝麻蛋白的工艺进行研究，试验结果表明水解度（DH，Y）与水解温度（X₁），芝麻蛋白浓度（X₂），酶-底物浓度比（X₃）以及酶解时间（X₄）显著相关，并建立起相关回归模型为： Y=13.18-0.90X₁-1.69X₂+1.87X₃+1.07X₄-0.15X₁X₂+0.92X₁X₃-0.47X₁X₄-0.47X₂X₃+0.92X₂X₄X+0.15X₃X₄-0.12X₁²-1.94X₂²-0.65X₃²-1.05X₄² 从回归模型计算机寻优得出AS1.398中性蛋白酶酶解芝麻蛋白的最适工艺为：在X₁=0，X₂=0，X₃=1.682，X₄=1水平，即酶解温度为45℃，蛋白质底物浓度为5％，酶-底物浓度比为9600u／g，酶解时间为122min，此工艺下水解度为16.74％，酸溶性肽转换率为79.12％，所得肽链的平均长度为6。 4．运用β-环糊精和柠檬酸等对酶解液的风味进行改善，结果表明β-环糊精具有明显的苦味掩蔽作用，当β-环糊精用量为1.5～2.5（w／v）可以很好的掩盖芝麻蛋白酶解液的苦昧。 5．酶解蛋白和未酶解蛋白都对O₂^（?）有一定的清除作用，但弱于阳性对照维生素C(IC₅₀=1.44mg／mL)对O₂^（?）的清除作用。酶解液对O₂^（?）的清除作用随浓度增大，清除效果增强。在酶解液中，酶解80min（DH15.23％）所得的酶解液对O₂^（?）清除作用最强，当酶解液浓度达到8mg／mL时对O₂^（?）的清除作用达85％左右。 6．酶解芝麻蛋白和未酶解蛋白对·OH有着极强的清除作用，并酶解蛋白优于未酶解蛋白对的清除作用，且都优于阳性对照物甘露醇对·OH的清除作用(IC₅₀西南农业大学硕士学位论文摘要里里口口口理口目口里口里里里口口二1.71mg/mL)。当芝麻蛋白酶解液浓度在低于0.6mg/mL时，不同水解度的酶解液对·OH的清除作用基本无差异，但当酶解液浓度大于0.6mg/mL时，高DH（ DH>1 5 .23%）的酶解液对.OH的清除作用优于低DH酶解液的清除作用。 7.酶解液具有抑制脂质过氧化作用，并随浓度的增大抑制作用增强，当酶解液浓度达到3mg/mL时，其不同DH酶解液对不饱和脂肪酸脂质过氧化抑制率都达到60%左右。 8.酶解液和未酶解芝麻蛋白对亚硝胺的合成有一定的阻断作用，酶解液对亚硝胺的阻断作用优于未酶解芝麻蛋白对亚硝胺的阻断作用，且随酶解液浓度的增大阻断作用增强，并随水解度的增大酶解液对亚硝胺的阻断作用增强。在加入酶解122:in（DH=16.74%）酶解液浓度达10mg/mL时对亚硝胺的阻断率达93.74%。但对N仇一抑制作用酶解液却随DH的增大抑制作用下降，在酶解10min（DH 9.73%）时的酶解液对NO厂的抑制最强，当浓度达IOmg/mL时对NO厂抑制率可达90%左右。更多还原

【Abstract】 This article studied on the enzymatic hydrolysis of sesame protein which extracted from defatted sesame cake .The reseach mainly studied on the sesame protein extracting from the cake .selecting the hydrolysis protease and hydrolysis technological condition parameters.inproving the hydrolysates flavour ,and the hydrolysates antioxidative activity and blocking in N-nitiso and introsamine formation system . The experiment results as following:1 Taking the ratio of protein soluble from sesame as the criterion ,the sesame protein extrating optimum technology was :sesame flour/water ratio 1:20,pH 12.50 temperature 80C.Under this technology the protein solube ratio reach 79%.;and the protein deposition at the pH 3.42 to 3.97was optimum , the protein deposition ratio at 86%~97%.2 Taking degree of hydrolysis(DH) as criterion ,using pepsin typsin ,AS1.398,pancreatin and papain to select the optimum protease .The results indicated the hydrolysis ability was AS1.398> typsin > pancreatin > papain> pepsin, the AS1.398 had the good ability to hydrolyze the protein.The AS1.398 comes from microorgannism , and its is easy to get and it has a advantage of safety, unexpensive ,so it can be as an ideal protease to hydrolyzing sesame protein .3 Taking degree of hydrolysis(DH) as criterion .applying the four factors quadratic regression orthoganal rotation grouping equation experiment(l/2implement) to study the AS 1.398 hydrolyzed sesame protein.The results showed that the degress of hodrolysis (DH).significantly related with temperature (X1),substrate concentration (X2),enzyme/substracte (X3),hydrolyzing time (X4), which affected the DH when hydrolyzing the sesame cake protein.From the result we mormulated the regression equation as:Y=13.18-0.90X1-1.69X2+1.87X3+1.07X4-0.15X1X2+0.92X1X3-0.47X1X4-0.47X2X3+0.92X2X4X+0.15X3X4-0.12X12-1.94X22-0.65X32-1.05X42 .From the regress equation ,The optimum condition draw by computer as: X1=0, X2=0, X3=1.682, X4=1,this is to say ,the temperature at 45℃, protein substrate concentration at 5%, enzyme/substration at 9600u/g,hydrolyzing time at 122min. Under this technology ,the DH could reach 16.74%, acid soluble peptide 79.12%,average peptide chain length 6 oligo-peptide.4 Using P-CD and some flavor reagent improved the hydrolyzates .The experiment showed P-CD masked the bitter of hydrolyzates significantly and superior to the flavor reagent.When added the 1.5 - 2.5(w/v) P-CD,it was good at masking the bitter .5 The hydrolyzates had evident antioxidative effect for scavenging O2- ,the hydrolyzats which hydrolyzed 80min (DH15.23%) had the best scavenging ratio than others hydrolyzats.When the hydrolyzates concentration adding to 8mg/mL ,the scavenging ratio reached 85% or so ,and the scavenging ratio was strengthened as the dosage concentation increasing ,but it was weaker than vitamin C(IC5o is 1.44mg/mL) in scavenging O2-.6 The hydrolyzates had extremely effect on scavenging .OH ,and the ratio of scavenging could be strengthened as the dosage concentration increasing .At the low dose concentration ,the effect of scavenging .OH by different DH hydrolysates were no significant different ,but when the dose concentation incresaing at 0.6mg/mL,high DH hydrolysates scavening .OH better than the low DH hydrolysate .7 The hydrolyzates had the effect of inhibitting LPO,and the inhibitting effect was strengthened as the dosage concentration increasing .when the hydrolyzates concentration adding to 3 mg/mL .different DH hydralyzates inhibitting of PUFA LPO ratio reached 60% or so.8 The hydrolyzates had the effect of inhibitting the N-nitiso and blocking the nitrosamine formation ,and the inhibitting or bloching effect was strengthened as the dosage concentration increasing.When blocking in nitrosamine formation ,the hydrolyzates blocking in nitrosamine formation was better than the sesame protein ,and the hydrolyzates effect were strengthened as the DH更多还原