摘要本文利用酶解法制备大豆降血糖肽，以酶解产物对 α-葡萄糖苷酶抑制率为 标准，筛选试验用酶；利用超声波预处理大豆蛋白提高产物产量，对超声预处理 过程及酶解过程进行工艺优化，研究超声辅助酶解大豆蛋白的动力学模型，筛选 最佳酶解工艺；通过光谱法研究超声预处理对大豆蛋白结构的影响及酶解过程蛋 白二级结构变化，探讨超声波预处理后大豆蛋白结构与酶解产物的 α-葡萄糖苷 酶抑制活性之间的关系；通过超滤、离子交换树脂、凝胶层析分离纯化大豆降血 糖肽，并对其结构进行表征。 80725

结果表明，在木瓜蛋白酶、菠萝蛋白酶、碱性蛋白酶和胰蛋白酶四种蛋白酶 中胰蛋白酶最适宜酶解；经过单因素试验和响应面试验，得到最佳工艺参数：超 声时间 20。92min、超声功率 245。94W、超声底物浓度 6。27%、酶解时间 22。02min、 加酶量 6416。61U/g、酶解底物浓度 2。10% 

为了探索酶解过程中蛋白质结构的变化，使用荧光光谱、拉曼光谱和傅立叶 红外变换光谱对超声预处理和酶解过程进行研究。结果显示，超声过程中大豆蛋 白没有发生断裂，但二级结构时刻发生变化，并且在超声预处理构成中会出现适 宜酶解的时间点，对应出现特征性二级结构含量变化。酶解过程中，大豆蛋白肽 键断裂，二级结构剧烈变化，但在对应的最佳酶解时间点会出现特征性二级结构 含量变化。 

对超声波酶解制备的多肽进行分离纯化，使用超滤、732 型阳离子离子交换 和 SephadexG-25 葡聚糖凝胶层析等手段分离纯化得出产物 C-Ⅲ-2 的活性最好， 并使用液质联用技术对大豆降血糖肽中抑制活性最高的多肽序列进行结构表征。 结果表明多肽肽序列可能是 Ala-Ile、Ala-Leu、Met-Glu、Phe-Gly-Gly、Ser-His。

分子量小于 5000Da 的多肽对四氧嘧啶高血糖小鼠的试验结果表明，多肽 228mg/kg 具有明显降血糖效果，大豆降血糖肽能够显著降低餐后血糖，并能显 著减轻“三多一少”症状。

毕业论文关键词：大豆降血糖肽；超声预处理；工艺优化；光谱分析；分离纯化 

Abstract This study aimed to establish an optimized method using ultrasonic-assisted hydrolysis for the preparation of soybean-derived α-glycosidase inhibitory peptides。 Based on the α-glycosidase enzyme inhibition rate, the enzymes were screened for the test。 Then selected optimum technology of ultrasonic pretreatment and enzymatic hydrolysis。 Using spectroscopy to study the change of secondary structure of soybean protein  during the ultrasonic  pretreatment  and  enzymatic hydrolysis  processes。   To

purify the soybean-derived hypoglycemic peptides, ultrafiltration，732 cation exchange

chromatography，SephadexG-25 gel chromatography and LC-MS were used。

The results shown that the trypsin was the best one for enzymolysis。 The optimized processing parameters were determined after the single factor test and response surface experiments: enzymolysis for 22。02 min, enzyme dosage 6416。61U/g， enzymolysis

bottom material liquid ratio 2。10%, ultrasonic treatment for 20。92 min at 245。94 W and ultrasonic bottom material liquid ratio 6。27%。

In the process of ultrasonic pretreatment and enzymatic hydrolysis，Fluorescence

spectroscopy，Raman spectroscopy and Fourier Transform Infrared spectra were used to survey the change of secondary structure of soybean-derived protein。 The results shown that the peptide bonds of protein were not broken during the ultrasonic pretreatment process, but the secondary structure was changing all the time。 One of the best time of enzymatic hydrolysis appeared during the ultrasonic pretreatment process。 The Peptide bonds of protein were broken during enzyme hydrolysis process ， secondary structure changed violently。 Similarly, one of the best time to stop the enzyme hydrolysis appeared with an illustrative change of secondary structure。