转nisI基因植物乳杆菌Lactobacillus plantarum 590对SD大鼠肠道健康的影响研究

发布时间：2018-05-28 17:09

本文选题：转基因微生物 + 食用安全　；参考：《江南大学》2011年博士论文

【摘要】：微生物在食品中的应用可以追溯到几千年之前。如今,微生物在食品相关生产加工过程中扮演的角色越来越重要。转基因技术在微生物中的应用使转基因微生物新品种应运而生。不同的转基因微生物可以优化提升微生物原有的优良品质,能够满足人们某一方面的特殊需求,这无疑扩大了微生物的应用范围,降低了生产成本。然而,转基因产品毕竟是一种人工的新产品,人们对转基因产品的食用安全从转基因产品诞生之日起就存在疑虑。目前,针对转基因微生物系统、完善、实用的食用安全评价体系还没有出台,使得转基因微生物的市场化受到限制。因此,开展转基因微生物的食用安全性研究,建立系统完善的评价体系成为当前急需解决的任务。这对于促进转基因微生物规范发展,进一步扩大微生物的实际应用范围具有重要意义。本课题以转nisI基因植物乳杆菌Lactobacillus plantarum 590(Lp590)为研究对象,以非转基因的亲本植物乳杆菌Lp为对照,根据微生物自身特点,结合已有的对转基因植物的评价模型,而又不拘泥于已有的其他转基因产品的安全评价模式,从体外耐受性质、亚慢性毒性喂养、肠道相关免疫、基因水平转移等对机体有重要影响的作用过程及影响因素进行了研究。本论文通过全方位、较系统的研究,旨在探索完善、系统、实用的转基因微生物的食用安全评价模型,为转基因微生物大规模的开发利用奠定基础。对转基因植物乳杆菌Lp590体外耐受性质进行了研究。与Lp菌株相比,对乳酸链球菌素(nisin)的耐受性试验结果表明:Lp590确实能够耐受较高浓度的nisin,并且Lp590与Lp的生长特性相似。对实际生产中可能遇到的极端环境如,低温(4℃)和较高浓度乙醇时Lp590表现出与Lp相似的耐受性质,而且对于高温(52℃)、较高浓度过氧化氢和氯化钠三种环境的耐受性Lp590还要优于Lp。体外模拟胃肠道消化的结果表明,Lp590与Lp相似,都能耐受胃肠道环境。以上结果表明,基因改造没有对Lp590体外性质产生负面影响,Lp590有多种适合工业生产的优良特性。这些结论的获得也为之后通过动物喂养实验评价转基因植物乳杆菌Lp590的食用安全提供了可能。对SD大鼠进行了亚慢性毒性喂养试验。结果表明,与灌喂Lp相比,灌喂Lp590 28天后大鼠生长状态良好;体重、进食正常;血液、尿液各项指标正常;常规病理切片无特异性毒性变化;Lp590与亲本Lp相似,对空肠粘膜结构有显著改善。一系列结果都表明,Lp590与菌Lp对机体有相似的影响,没有发现转基因成分对大鼠机体产生不利于健康的影响。对粪便这一对肠道健康有重要影响的代谢指标进行了较全面的研究。平板计数和定量PCR两种方法对粪便主要菌群的研究表明,灌喂Lp590与Lp相似,都能促进肠道有益菌的增加抑制有害菌的增长,起到整肠作用。灌喂Lp590与Lp后,粪便主要酶活、粪便主要短链脂肪酸(SCFA)、粪便pH值及粪便水分含量的变化相似。从研究结果来看,没有发现转基因成分改变Lp590对机体的益生作用。用免疫组化、定量PCR和ELISA等方法对反映肠道渗透性、肠道粘膜免疫及机体整体免疫和抗氧化能力的相关研究表明,灌喂Lp590和Lp后,肠道渗透性降低;肠道粘膜免疫增加;机体抗氧化能力增强。Lp590表现出与Lp相似的对机体的有益作用,没有发现因外源基因转入而对机体产生副作用。设计特异的引物,对血液、肌肉、肝脏、脾脏及肾脏组织器官进行了PCR检测。结果没有在上述相关组织器官中发现外源基因成分。通过一系列试验,结果没有发现Lp590因为外源基因转入对机体产生不利影响,而且Lp590与Lp都对机体健康有改善作用。本研究首次对转基因微生物进行了针对肠道健康的食用安全评价。本课题首次提出了肠道健康是反映微生物对机体健康影响的主要标志。在具体评价过程中,课题抓住微生物的特点,从体外耐受到对肠道健康的重点评价思路,围绕重点,多角度分析,突出了对转基因微生物食用安全评价重点,建立了关于肠道健康的系统、完善、实用的转基因微生物食用安全评价体系,为转基因微生物的规范提供科学的标准方法。这必将为转基因微生物大规模的科学的开发利用和商业化奠定基础,并对进一步扩大微生物的实际应用范围具有重要指导意义。
[Abstract]:The application of micro - organisms in food can be traced back to thousands of years . Nowadays , the role of microbe in food - related production is more and more important .

In this paper , we studied the effect and influencing factors of transgenic plants in vitro tolerance , subchronic toxicity feeding , intestinal related immunity , gene level transfer and so on .

Lp590 has a similar tolerance to Lp , and Lp590 has a similar tolerance to Lp . The results show that Lp590 has no negative effect on the in vitro properties of Lp590 , and Lp590 has many excellent properties suitable for industrial production .

The results showed that Lp590 had a similar effect on the structure of jejunum . Lp590 had a similar effect on the structure of jejunum mucosa .

The results showed that Lp590 and Lp were similar to Lp , which could promote the growth of intestinal beneficial bacteria and inhibit the growth of harmful bacteria .

By means of immunohistochemistry , quantitative PCR and ELISA , the effects of Lp590 and Lp on intestinal permeability , intestinal mucosal immunity and body immunity and anti - oxidation ability showed that the intestinal permeability decreased after Lp590 and Lp were fed . The immunity of intestinal mucosal immunity increased , and the anti - oxidation ability of organism increased . Lp590 showed a similar beneficial effect on organism and found no side effect on organism due to the transfer of exogenous gene .

Specific primers were designed to detect the organs of blood , muscle , liver , spleen and kidney .

Through a series of experiments , it was found that Lp590 has no adverse effect on organism because of exogenous gene transfer , and Lp590 and Lp have an improved effect on organism health .

This paper first puts forward the main marker of the influence of microorganism on organism ' s health . In the process of specific evaluation , the subject grasps the characteristics of microorganism , from outside tolerance to the key evaluation method for the health of the intestinal tract . The key point and the multi - angle analysis indicate that the safety evaluation system of the transgenic microbe edible safety is emphasized , and the scientific standard method is provided for the regulation of the transgenic microbe . This will surely lay the foundation for the large - scale development and utilization of the transgenic microbe and the commercialization . It will be of great significance to further expand the practical application range of the microorganism .
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：TS201.3;R371

【参考文献】