【作者】 姚婷婷；

【导师】 赵培城；

【作者基本信息】 浙江工业大学 ， 生物化工， 2006， 硕士

【摘要】 本论文开展了乳蛋白源抗菌肽的生物学研究：采用胰蛋白酶和胃蛋白酶作为工具酶，分别确立了乳蛋白经两种蛋白酶酶解产生抗菌肽的条件；酶解产物通过凝胶层析和离子交换层析进行分离，获得了两个具有抗菌活性组分；并对两个抗菌肽制剂的抑菌作用和热稳定性进行具体的研究。通过正交试验和抗菌试验，综合水解效果和抑菌效果两方面考虑，确立了两种蛋白酶酶解乳蛋白产生抗菌肽的最佳水解条件为：胰蛋白酶水解乳蛋白条件为底物浓度10％，[E]／[S]为2.5％，反应温度50℃，pH值8.0，反应时间3h；胃蛋白酶水解乳蛋白条件为底物浓度10％，[E]／[S]为14％，反应温度37℃，pH值1.5，反应时间3h。选用Sephadex G-75和Sephadex G-15凝胶色谱，SP-Sepharose Fast Flow离子交换色谱来进行分离纯化乳蛋白的两种酶解产物，分别分离得到了较纯的具有抗菌活性的组分。对获得的两个抗菌组分进行微量稀释法抗菌试验，体外抑菌实验显示两个抗菌组分对金黄色葡萄球菌、大肠杆菌、链球菌、枯草杆菌、鸡巴氏杆菌、兔沙门氏菌这些革兰氏阳性菌和革兰氏阴性菌均具有抑菌作用，表明乳蛋白酶解产生的抗菌肽有广谱抑菌活性。纯化后所得的两种抗菌肽组分的热稳定性实验表明，乳蛋白水解所得到的抗菌组分对热有很高的稳定性。更多还原

【Abstract】 In this study, we detect the bioactive effect of antibacterial peptides in milk. We chose trypsin and pepsin, established the method to produce the antibacterial peptides hydrolisated from milk protein. We seperated the hydrolysate by gel filtration chromatography and ion exchange chromatography, acquired two antibacterial peptides, then studied the antibacterial activity and thermal stability of these two antibacterial peptides.The study established the method to produce the antibacterial peptides from milk protein by trypsin and pepsin on the basis of the good hydrolyzing degree and the best effect of antibacterial activity. The optimum conditions of two kinds of enzymes hydrolyze milk protein were determined by orthogonal design test and antibacterial activity experiment. The research showed that the best condition of trypsin hydrolyze milk protein was: concentration of milk protein 10%, [E]/[S] 2.5%, temperature 50°C, pH 8.0 and reaction time 3h. The best condition of pepsin hydrolyze milk protein was: concentration of milk protein 10%, [E]/[S] 14%, temperature 37°C, pH 1.5 and reaction time 3h. The study seperated the hydrolysates by Sephadex G-75 and Sephadex G-15 gel filtrate chromatography first, then chose SP-Sepharose Fast Flow to purify the collected antibacterial activity fractions. In conclusion, two antibacterial activity fractions were achieved.In order to detect the antibacterial activity of two antibacterial fractions, the study mensurate Minimal Inhibitory Concentration of two antibacterial activity fractions against S.aureus (ATCC25923), E.coli (ATCC25922), Streptococcus, Bacilli subtilis, Pasteuralla and Salmonella by Micro-dilution. The results indicated that two antibacterial fractions both have antibacterial activity against these Gram postive and negative bacterias. And the antibacteria peptides from milk protein have broad-spectrum antibacterial activity.The experiment of thermal stability of these two antibacterial peptides indicated that the antibacterial peptides from milk protein were heat-resistant.更多还原