【作者】 张俊；

【导师】 易醒； 章辉；

【作者基本信息】 南昌大学 ， 食品工程（专业学位）， 2022， 硕士

【摘要】 磁性纳米粒子(Magnetic nanoparticles,MNPs)是近年来的研究热点之一,利用磁性纳米粒子固定化酶技术可以提高酶的稳定性和重复利用率,并且还可用于传统天然产物活性组分的靶向筛选。茶皂素单体众多,并且结构相似,使其活性组分的筛选变得棘手。乙醇脱氢酶(Alcohol dehydrogenase,ADH)是人体酒精代谢的关键酶,有研究报道了茶皂素具有抑制乙醇脱氢酶的作用。利用固定化酶技术从粗茶皂素溶液中筛选出ADH活性组分不失为一种高效简便的方法,因此,本研究在接续实验室前期提取方法基础上,主要对提取到的ADH活性组分进行理化特性和解酒机理的探究,具体内容和结论如下:(1)研究ADH活性组分(TSI)的理化特性。通过界面张力仪的测定,得到TSI的临界胶束浓度为0.3%,显示出其能显著降低水的表面张力,接触角<90°,且随着浓度减小而增大,表现出良好润湿性能。TSI的起泡性和泡沫稳定性随浓度上升而增强,在50℃时起泡性能最好,具备一定的抗硬水和酸碱的能力。TSI乳化性能良好,对苯的增溶能力为7 m L/g,单独使用时除污能力不强,与其他表面活性剂复配后效果明显,具有润湿、发泡、稳泡、乳化、增溶、去污等性质,是一种优秀的表面活性剂。(2)探讨TSI的解酒机制和对肝胃的影响。通过给小鼠灌胃56°红星二锅头白酒确立了小鼠最适醉酒量为0.14 m L/10g.bw后,发现TSI高剂量组能显著延长小鼠醉酒时间,缩短小鼠的醒酒时间。TSI对醉酒小鼠肝、胃影响的研究发现,TSI中、高剂量组能显著降低小鼠的肝指数和血液中转氨酶含量,提高SOD和GSH-PX活性,降低MDA含量,增强小鼠体内抗氧化水平,减少氧自由基对小鼠的损伤;此外,TSI中、高剂量组还能提高肝细胞和胃细胞的ADH和ALDH活性,促进乙醇代谢。采用荧光定量PCR分析了酒精代谢过程的基因表达情况,发现TSI高剂量组能显著下调促炎因子TNF-α和IL-6的表达量,提高乙醇代谢酶ADH1和ALDH2基因的表达量,这有助于解释TSI对乙醇诱导的胃细胞和肝细胞损伤均有调控保护的作用。(3)制备大蒜油纳米乳(GOE)并初步阐释其抑菌机理。采用超声乳化法制备GOE,发现GOE的最佳制备条件为茶皂素终质量分数为1.5%,大蒜油终质量分数为8%,超声功率为225 W,超声时间为10 min。GOE对金葡菌和单增李斯特菌抑菌作用明显,对大肠杆菌作用不明显。通过抑菌曲线可知GOE浓度为2MIC、MIC时,对细菌的抑制效果较强,GOE浓度为1/2MIC时,对细菌抑制较弱,但仍然有一定抑制作用。通过扫描电镜、AKP酶活力测定、电导率测定及核酸、蛋白质泄漏测定可知,GOE作用于细菌后,先破坏其细胞壁,使得细菌发生扭曲变形,导致细胞壁与细胞膜之间的AKP酶渗出,然后出现细胞膜受损,胞内离子及电解质等物质渗出,电导率升高,DNA及蛋白质泄漏,影响细胞的生理活性,结果引起细菌生长受到抑制或死亡。更多还原

【Abstract】 Magnetic nanoparticles(MNPs)is one of the research hotspots in recent years.Using Magnetic nanoparticles to immobilize enzymes can improve the stability and reusability of enzymes,and can also be used for screening active components of traditional natural products.Tea saponin has many monomers and similar structures,so it’s difficult to screen active components of tea saponin.Alcohol dehydrogenase(ADH)is a key enzyme in human Alcohol metabolism.It has been reported that tea saponin can inhibit the activity of Alcohol dehydrogenase.It is an efficient and simple method to screen ADH-related active components from crude tea saponin solution by immobilized enzyme technology.Therefore,based on the previous extraction method of our laboratory,this study mainly investigated the physical and chemical properties and antialcoholic effect of the ADH-related active components.The specific contents and conclusions are as follows:The physical and chemical properties of ADH-related active components(TSI)has been studied.The Critical Micelle Concentration of TSI is 0.3%,which shows that TSI can significantly reduce the surface tension of water,the Contact Angle is less than90° and increases with decreasing of concentration of TSI,showing good wettability.The foamability and foam stability of TSI increase with the increase of its concentration,and the best temperature of foamability is 50℃,it also has a good ability to resist hard water and acid and alkali.TSI has good emulsifying performance,and the solubilizing ability of benzene is 7 m L/g.When used alone,the decontamination ability of TSI is not strong,but when it was combined with other surfactants,the effect is obvious.With wetting,foaming,foam stability,emulsifying,solubilizing,decontamination and other properties,TSI is an excellent surfactant.The antialcoholic effect of TSI and its effect on liver and stomach was explored.By intragastric administration of 56° Red Star Erguotou to mice,the optimal level of intoxication was 0.14 m L/10 g.bw,and TSI group with high dose could significantly prolong ebriety time and shorten sober time.Studies on the effects of TSI on the liver and stomach of intoxicated mice showed that the medium and high dose groups of TSI could significantly reduce the liver index and blood transaminase content of mice,improve the activities of SOD and GSH-Px,reduce the content of MDA,enhance the antioxidant level in mice,and reduce the damage of oxygen free radicals to mice.In addition,the activities of ADH and ALDH in liver cells and stomach cells were increased of TSI medium and high dose groups,and ethanol metabolism was promoted.Fluorescence quantitative PCR was used to analyze the gene expression during alcohol metabolism.It was found that the expression levels of pro-inflammatory factors of TNF-α and IL-6 were significantly down-regulated in TSI high-dose group,and the expression levels of ethanol metabolism enzymes ADH1 and ALDH2 genes were significantly increased,which helped to explain the regulatory and protective effects of TSI on ethanol-induced gastric cell and liver cell injury.Garlic oil nanoemulsion(GOE)was prepared and its antibacterial mechanism was preliminarily explained.GOE was prepared by ultrasonic emulsification method.It was found that the optimal preparation conditions of GOE were as follows: the content of tea saponin is 1.5%,the content of garlic oil is 8%,ultrasonic power is 225 W and ultrasonic time is 10 min.GOE has obvious inhibitory effect on Staphylococcus aureus and Listeria monocytogenes,but not on Escherichia coli.According to the bacteriostatic curve,when GOE concentration is 2MIC and MIC,the bacteriostatic effect is strong;when GOE concentration is 1/2MIC,the bacteriostatic effect is weak,but it still has a certain inhibitory effect.By scanning electron microscopy(SEM),AKP enzyme activity assay,conductivity measurement and leakage of nucleic acids and proteins,the bacteriostatic effect of GOE is due to destroy the cell wall of bacteria,leading to make the bacteria distort and out of shape,cause AKP enzyme which between the cell wall and cell membrane leak,and then damage the cell membrane,leading to ion and electrolyte materials of cell leak and the conductivity of cell increase.Leakage of DNA and proteins also affects the physiological activity of the cell,resulting in inhibition of bacterial growth or death.更多还原