本文選題：黃鯽魚 + 美拉德產(chǎn)物　； 參考：《浙江海洋大學》2017年碩士論文

【摘要】：黃鯽魚(Setipinna taty)營養(yǎng)豐富,味美價廉。但由于黃鯽魚體積小,不論是食用還是加工,利用率比較低。在前期研究中,以蛋白酶水解法制備的黃鯽蛋白抗菌肽具有很強的抑菌作用,但是魚腥味較重。而后通過美拉德反應提高了黃鯽蛋白抗菌肽的風味性,同時發(fā)現(xiàn)黃鯽蛋白抗菌肽美拉德產(chǎn)物(HAHp-葡萄糖美拉德產(chǎn)物)具有較強的抑菌活性。因此,本論文在前期研究基礎上,以HAHp-葡萄糖美拉德產(chǎn)物為研究對象,探究其抑菌機制和有效抑菌組分結構,并對其在免疫提高方面的應用進行探討。主要研究包括:(1)HAHp-葡萄糖美拉德產(chǎn)物的抑菌機理;(21)HAHp-葡萄糖美拉德產(chǎn)物中抗菌肽組分的分離與鑒定;(3)HAHp-葡萄糖美拉德產(chǎn)物對小鼠免疫功能的影響。HAHp-葡萄糖美拉德產(chǎn)物與處于對數(shù)生長期的大腸桿菌菌懸液作用,通過掃描電鏡-觀察菌體超微結構變化,結合外膜和內(nèi)膜檢測,分析胞內(nèi)各種酶的釋放情況,同時采用馬爾文電位儀測定人工脂質體與HAHp-葡萄糖美拉德產(chǎn)物作用后粒徑變化。結果表明:HAHp-葡萄糖美拉德產(chǎn)物與大腸桿菌作用1 h即可造成細胞膜損傷;與大腸桿菌作用1 h,外膜和內(nèi)膜的滲透性增強;作用5 h胞內(nèi)乳酸脫氫酶釋放量增加,大腸桿菌的ATP含量也顯著降低。人工脂膜與黃鯽蛋白肽美拉德產(chǎn)物作用后,平均粒徑從170 nm增加到230 nm,說明HAHp-葡萄糖美拉德產(chǎn)物對細胞膜具有聚集作用。由上述實驗結果推斷HAHp-葡萄糖美拉德產(chǎn)物可通過吸附作用達到菌體表面,引起菌體聚集,而后通過膜損傷方式增加外膜和內(nèi)膜滲透性,造成胞內(nèi)物質外泄,從而達到抑菌作用。采用離子交換層析、分子篩分離和反相高效液相色譜分離技術,通過抑菌活性跟蹤檢測法從HAHp-葡萄糖美拉德產(chǎn)物中分離得到一抑菌組分。然后,采用LC-ESI-MS聯(lián)用分析法分離鑒定出7條抗菌肽,分別是RVAPEEHPTL、WLPVVR、FFTQATDLLSR、VLLLWR、VLLVLLR、VLLALWR和LLSWYDNEFGYSNR�；瘜W合成其中5條肽(肽1 RVAPEEHPTL,肽2WLPVVR,肽3 VLLLWR,肽4 VLLVLLR和肽5 VLLALWR),抑菌結果顯示除了肽4外,其余4條合成肽對大腸桿菌均有抑菌活性。進一步選擇肽2(WLPVVR)為出發(fā)肽,合成3條類似肽2-1(WLPVVH)、肽2-2(WLPVVE)和肽2-3(LPVVR),抑菌結果表明合成的類似肽雖然保留了抑菌作用,但抑菌活性發(fā)生不同程度改變,說明肽序列組成對其抑菌性有影響。以昆明種小鼠為對象,通過小鼠動物實驗,測定小鼠胸腺指數(shù)和脾臟指數(shù),同時測定小鼠血清和肝勻漿谷胱甘肽過氧化物酶、總超氧化物歧化酶和過氧化氫酶活力,以及測定脂質過氧化物含量和還原型谷胱甘肽含量。結果表明:HAHp-葡萄糖美拉德產(chǎn)物對胸腺指數(shù)以及脾臟指數(shù)的影響較小,與對照組相比,差異不顯著。高劑量的HAHp-葡萄糖美拉德產(chǎn)物對小鼠肝臟與血清中谷胱甘肽過氧化物酶活力值增加有一定的幫助。高劑量組小鼠肝臟中總超氧化物酶活力也有所提高。HAHp-葡萄糖美拉德產(chǎn)物對于降低小鼠體內(nèi)脂質過氧化物含量與提高還原型谷胱甘肽含量效果極為明顯。從各個指標測定結果看,HAHp-葡萄糖美拉德產(chǎn)物可以提高小鼠體內(nèi)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.

【學位授予單位】：浙江海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TS254.1

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