重组溶菌酶特性及其对小鼠肠道菌群的影响研究

发布时间：2018-09-18 07:00

【摘要】：饲料、食品由于其加工工艺、独特风味、防腐保鲜等方面的要求，必须使用相应的添加剂，各种添加剂由于其操作简单、效果显著且成本低廉，已经得到广泛运用，但随着相关研究的深入，传统添加剂可能对人体造成的潜在危害逐渐受到重视，寻找一种稳定、高效、安全性良好的新型添加剂显得十分重要。人源溶菌酶是溶菌酶类物质的一种，广泛存在于人体体液和组织中。通过基因工程技术将编码人源溶菌酶相关基因导入工程细菌通过微生物发酵法得到的溶菌酶称为重组溶菌酶。重组溶菌酶具有广谱抗菌、保鲜防腐、消炎抗病毒等溶菌酶共有特点，整体效果优于蛋清溶菌酶，可替代传统防腐剂，也可作为功能因子添加于饲料、食品中，应用潜质广阔；此外，重组溶菌酶是人体非特异性免疫因子，具有巨大科研价值是研究分子进化、分子免疫关系的良好模型。本课题旨在通过对重组溶菌酶酶学性质，实际防腐保鲜效果及小鼠肠道菌群及血清生化指标调节作用的研究，探寻其作为饲料、食品添加剂使用的实际效果及对机体产生的影响。首先，本文以经典比浊法为基础，，在不同温度、pH、人工胃肠液及金属离子条件下，以微球菌为底物，测定蛋清、重组溶菌酶酶活变化状况；然后，用不同浓度的重组溶菌酶溶液浸泡斑点叉尾洶鱼肉，分别测定不同时期鱼肉感官指标、菌落总数、pH值、TVB-n值、TBA值、酸度；最后，通过小鼠饮水口服溶菌酶(蛋清溶菌酶和重组溶菌酶酶活分别为2500、5000U/ml)后无菌取肠道内容物，通过微生物培养测定肠道菌群的变化；并测定不同试验时间的血清总蛋白、尿素氮、甘油三酯、胆固醇的含量。酶学性质试验结果表明，蛋清溶菌酶最适温度为40℃，最适pH6.0；重组溶菌酶最适温度45℃，最适pH6.0；两种溶菌酶在温度超过50℃后活性迅速下降，但重组溶菌酶酶活下降较快；室温条件下在pH值6.0-9.0溶液中预处理60min，两种溶菌酶活力均保持80%以上；在人工胃液中，两种溶菌酶活性迅速下降；在人工肠液中，重组溶菌酶酶活下降明显较慢；Fe~(3+)、Ca~(2+)、K~+、Mn~(2+)、Zn~(2+)、Mg~(2+)、Al~(3+)、Cu~(2+)均会不同程度的抑制两种溶菌酶活性。保鲜试验结构表明，不同浓度重组溶菌酶溶液浸泡后鱼肉感官指标下降速度，挥发性盐基氮、TBA值上升速度，菌落总数和酸度不同程度降低，表明重组溶菌酶能够减缓鱼肉腐败进程，达到延长保藏期、最大限度发挥鱼肉食用价值和商品价值的作用。实际应用中，0.25%重组溶菌酶即可得到较好保鲜效果，将斑点叉尾洶保藏期由4天延长至12天。动物试验结果表明，两种溶菌酶均能不同程度降低小鼠肠道金黄色葡萄球菌、粪肠球菌数量，促进双歧杆菌、乳酸杆菌生长，但重组溶菌酶对大肠埃希菌群抑制作用明显(P0.05)，蛋清溶菌酶不明显(P0.05)；不同浓度两种溶菌酶均能一定程度提高血清总蛋白含量，降低尿素氮、胆固醇含量，其中重组溶菌酶(5000U/ml)饲喂21天后血清总蛋白、尿素氮、胆固醇同对照组差异显著(P0.05)。本课题显示，蛋清、重组溶菌酶均具有调节机体肠道菌群平衡，血清指标的作用，重组溶菌酶的效果更为明显，在实际应用中需要注意温度、pH、胃液肠液、金属离子对酶活的影响，此外重组溶菌酶能够显著延长水产品保藏期，非常适合作为饲料、食品添加剂使用。
[Abstract]:Because of its processing technology, unique flavor, preservation and other requirements, it is necessary to use the corresponding additives. Various additives have been widely used because of their simple operation, significant effect and low cost. However, with the deepening of relevant research, the potential harm of traditional additives to human body has gradually been serious. Human lysozyme is a kind of lysozyme and widely exists in human body fluids and tissues. The lysozyme produced by microbial fermentation of engineered bacteria is called weight. Recombinant lysozyme has the common characteristics of broad-spectrum antibacterial, preservative, anti-inflammatory and anti-viral lysozyme, the overall effect is better than egg white lysozyme, can replace traditional preservatives, can also be added as functional factors in feed, food, application potential is broad; in addition, recombinant lysozyme is a human non-specific immune factor, has a huge. Scientific research value is a good model for studying molecular evolution and molecular immunity relationship.The purpose of this study is to explore the practical effect of recombinant lysozyme as feed and food additive and its influence on the organism by studying the properties of recombinant lysozyme, the effect of preservation and preservation, the regulation of intestinal flora and serum biochemical indexes in mice.
Firstly, on the basis of the classical turbidimetric method, the changes of egg white and recombinant lysozyme activity were determined with Micrococcus as substrate under different temperature, pH, artificial gastrointestinal juice and metal ions. Then, the sensory indexes and total bacterial colonies were determined with different concentrations of recombinant lysozyme solution soaked in the pork of T. maculatus. Number, pH value, TVB-n value, TBA value, acidity; finally, after oral administration of lysozyme (egg white lysozyme and recombinant lysozyme activity were 2500,5000U/ml, respectively), the intestinal contents were obtained aseptically and the changes of intestinal flora were determined by microbial culture; and serum total protein, urea nitrogen, triglyceride, cholesterol were determined at different test time. Content.
The results of enzymatic properties test showed that the optimum temperature of lysozyme was 40 C and pH 6.0; the optimum temperature of recombinant lysozyme was 45 C and pH 6.0; the activity of two lysozymes decreased rapidly after the temperature exceeded 50 C, but the activity of recombinant lysozyme decreased rapidly; the activity of two lysozymes was pretreated in pH 6.0-9.0 solution for 60 min at room temperature. In artificial gastric juice, the activity of two kinds of lysozyme decreased rapidly; in artificial intestinal juice, the activity of recombinant lysozyme decreased slowly; Fe ~ (3 +), Ca ~ (2 +), K ~ +, Mn ~ (2 +), Zn ~ (2 +), Mg ~ (2 +), Al ~ (3 +), Cu ~ (2 +) inhibited the activity of two kinds of lysozyme in different degrees. The results showed that recombinant lysozyme could slow down the decay process of fish meat, prolong the shelf life and maximize the edible and commercial value of fish meat. The results of animal experiment showed that the two lysozymes could reduce the number of Staphylococcus aureus and Enterococcus faecalis, promote the growth of bifidobacteria and lactobacillus, but the recombinant lysozyme had obvious inhibition on E. coli (P 0.05). Lysozyme in egg white was not significant (P The effect of recombinant lysozyme on intestinal flora balance, serum index and temperature, pH, gastric juice, intestinal juice and metal ions should be paid attention to. In addition, recombinant lysozyme can significantly prolong the shelf life of aquatic products, which is very suitable for feed and food additives.
【学位授予单位】：中国计量学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R151

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