黄鲫蛋白肽美拉德产物的抑菌机理及对小鼠免疫功能的影响

发布时间：2018-05-04 04:40

本文选题：黄鲫鱼 + 美拉德产物　；参考：《浙江海洋大学》2017年硕士论文

【摘要】：黄鲫鱼(Setipinna taty)营养丰富,味美价廉。但由于黄鲫鱼体积小,不论是食用还是加工,利用率比较低。在前期研究中,以蛋白酶水解法制备的黄鲫蛋白抗菌肽具有很强的抑菌作用,但是鱼腥味较重。而后通过美拉德反应提高了黄鲫蛋白抗菌肽的风味性,同时发现黄鲫蛋白抗菌肽美拉德产物(HAHp-葡萄糖美拉德产物)具有较强的抑菌活性。因此,本论文在前期研究基础上,以HAHp-葡萄糖美拉德产物为研究对象,探究其抑菌机制和有效抑菌组分结构,并对其在免疫提高方面的应用进行探讨。主要研究包括:(1)HAHp-葡萄糖美拉德产物的抑菌机理;(21)HAHp-葡萄糖美拉德产物中抗菌肽组分的分离与鉴定;(3)HAHp-葡萄糖美拉德产物对小鼠免疫功能的影响。HAHp-葡萄糖美拉德产物与处于对数生长期的大肠杆菌菌悬液作用,通过扫描电镜-观察菌体超微结构变化,结合外膜和内膜检测,分析胞内各种酶的释放情况,同时采用马尔文电位仪测定人工脂质体与HAHp-葡萄糖美拉德产物作用后粒径变化。结果表明:HAHp-葡萄糖美拉德产物与大肠杆菌作用1 h即可造成细胞膜损伤;与大肠杆菌作用1 h,外膜和内膜的渗透性增强;作用5 h胞内乳酸脱氢酶释放量增加,大肠杆菌的ATP含量也显著降低。人工脂膜与黄鲫蛋白肽美拉德产物作用后,平均粒径从170 nm增加到230 nm,说明HAHp-葡萄糖美拉德产物对细胞膜具有聚集作用。由上述实验结果推断HAHp-葡萄糖美拉德产物可通过吸附作用达到菌体表面,引起菌体聚集,而后通过膜损伤方式增加外膜和内膜渗透性,造成胞内物质外泄,从而达到抑菌作用。采用离子交换层析、分子筛分离和反相高效液相色谱分离技术,通过抑菌活性跟踪检测法从HAHp-葡萄糖美拉德产物中分离得到一抑菌组分。然后,采用LC-ESI-MS联用分析法分离鉴定出7条抗菌肽,分别是RVAPEEHPTL、WLPVVR、FFTQATDLLSR、VLLLWR、VLLVLLR、VLLALWR和LLSWYDNEFGYSNR。化学合成其中5条肽(肽1 RVAPEEHPTL,肽2WLPVVR,肽3 VLLLWR,肽4 VLLVLLR和肽5 VLLALWR),抑菌结果显示除了肽4外,其余4条合成肽对大肠杆菌均有抑菌活性。进一步选择肽2(WLPVVR)为出发肽,合成3条类似肽2-1(WLPVVH)、肽2-2(WLPVVE)和肽2-3(LPVVR),抑菌结果表明合成的类似肽虽然保留了抑菌作用,但抑菌活性发生不同程度改变,说明肽序列组成对其抑菌性有影响。以昆明种小鼠为对象,通过小鼠动物实验,测定小鼠胸腺指数和脾脏指数,同时测定小鼠血清和肝匀浆谷胱甘肽过氧化物酶、总超氧化物歧化酶和过氧化氢酶活力,以及测定脂质过氧化物含量和还原型谷胱甘肽含量。结果表明:HAHp-葡萄糖美拉德产物对胸腺指数以及脾脏指数的影响较小,与对照组相比,差异不显著。高剂量的HAHp-葡萄糖美拉德产物对小鼠肝脏与血清中谷胱甘肽过氧化物酶活力值增加有一定的帮助。高剂量组小鼠肝脏中总超氧化物酶活力也有所提高。HAHp-葡萄糖美拉德产物对于降低小鼠体内脂质过氧化物含量与提高还原型谷胱甘肽含量效果极为明显。从各个指标测定结果看,HAHp-葡萄糖美拉德产物可以提高小鼠体内抗氧化能力。
[Abstract]:Yellow crucian carp (Setipinna taty) is rich in nutrition and low in taste. But because of the small size of Carassius auratus, the utilization rate of the Carassius auratus is relatively low. In the earlier study, the antibacterial peptide of the protein of the crucian carp, which was prepared by protease hydrolysis, has a strong inhibitory effect, but the fishy smell is heavier. Then the protein resistance of the Yellow crucian carp is improved by the Maillard reaction. At the same time, it was found that the antibacterial peptide Millard product of the Carassius auratus protein (HAHp- glucose mallard product) had strong antibacterial activity. Therefore, on the basis of the previous study, the antibacterial mechanism and the effective antibacterial component structure of HAHp- glucose mallard were studied. The main research includes: (1) the bacteriostasis mechanism of HAHp- glucose mallard products; (21) isolation and identification of antimicrobial peptide components in HAHp- glucose mallard products; (3) the effect of HAHp- glucose meillard products on the immune function of mice,.HAHp- glucose beauty Ladd products and Escherichia coli suspension at logarithmic growth period Using the scanning electron microscope to observe the ultrastructural changes of the mycelium and the detection of the outer membrane and endometrium, the release of various enzymes in the cell was analyzed, and the particle size changes after the action of the artificial liposomes and HAHp- glucose Millard products were measured by Malvin potentiometer. The results showed that the effect of HAHp- glucose mallard products and Escherichia coli was 1 h. Cell membrane damage; the permeability of the outer membrane and the intima increased with the effect of 1 h with the Escherichia coli; the release of lactate dehydrogenase in 5 h increased and the ATP content of Escherichia coli decreased significantly. The average particle size increased from 170 nm to 230 nm after the action of artificial lipid membrane and the protein peptide mallard of Carassius auratus, indicating that the HAHp- glucose Maillard product was fine. It is concluded from the experimental results that the HAHp- glucose mallard products can reach the surface of the bacteria by adsorption, cause the accumulation of the bacteria, and then increase the permeability of the outer membrane and intima through the membrane damage, resulting in the leakage of the intracellular material and thus achieve the bacteriostasis. The ion exchange chromatography, molecular sieve separation and reverse phase are used. A bacteriostasis component was isolated from HAHp- glucose Millard products by high performance liquid chromatography (HPLC). Then, 7 antimicrobial peptides were separated and identified by LC-ESI-MS combined analysis, and 5 peptides were synthesized by chemical synthesis of RVAPEEHPTL, WLPVVR, FFTQATDLLSR, VLLLWR, VLLVLLR, VLLALWR and LLSWYDNEFGYSNR.. Peptide 1 RVAPEEHPTL, peptide 2WLPVVR, peptide 3 VLLLWR, peptide 4 VLLVLLR and peptide 5 VLLALWR). The bacteriostasis results showed that the other 4 synthetic peptides had antibacterial activity to Escherichia coli except peptide 4. The peptide 2 (WLPVVR) was selected as the starting peptide, and 3 similar peptides 2-1 (WLPVVH), peptide 2-2 (WLPVVE) and peptide 2-3 (LPVVR) were synthesized. The antibacterial results showed that similar peptides were synthesized although the peptide was synthesized. The bacteriostasis was retained, but the bacteriostasis activity changed in varying degrees, indicating that the sequence composition of the peptide had an influence on its bacteriostasis. The thymus index and spleen index of mice were measured by the mice in Kunming, and the caspase peroxidase, total superoxide dismutase and peroxy in the mice serum and liver homogenate valley were measured. The activity of hydropase and the content of lipid peroxide and glutathione were measured. The results showed that the effects of HAHp- glucose Maillard products on thymus index and spleen index were small, and the difference was not significant compared with the control group. The high dose of HAHp- glucose Maillard produced glutathione peroxidation in the liver and serum of mice. The activity of enzyme activity increased to some extent. The activity of total superoxide enzyme in the liver of high dose mice was also improved. The effect of.HAHp- glucose Maillard product on reducing the content of lipid peroxide in mice and increasing the reduced glutathione content in mice was very obvious. From the results of each index, the product of HAHp- glucose Maillard It can improve the antioxidant capacity of mice in vivo.

【学位授予单位】：浙江海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS254.1

【参考文献】