【作者】 刘海燕；

【导师】 王洪新；

【作者基本信息】 江南大学 ， 食品营养与安全， 2011， 博士

【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。更多还原

【Abstract】 Application of microorganisms in food industry can be traced back thousands of years ago. Now, microorganisms play more and more important role in food-related industry. With the application of transgenic technique new varieties of genetically modified microorganisms （GMM） came into being. Different GMM can be optimized to enhance the existing good qualities, to meet the special needs. It will undoubtedly expand the scope of application of microorganisms, lower production costs. However, as artificial new products, there are some concerns on food safety of GMM existing from the date of the birth of GM products. Currently, there is no systematical, complete and practical food safety evaluation system on GMM and the marketization of GMM is limited. Therefore, developing food safety study on GMM and establishment of evaluation system is the urgent task. It will promote the regulatory progress of GMM, further expand the application.In this study non-GM probiotic Lactobacillus plantarum （Lp） as control, major factors of GM Lactobacillus plantarum 590 （Lp590） introduced with nisI gene influencing on the body health such as in vitro tolerance, sub-chronic toxicity feeding, intestinal immune and gene horizontal transfer were studied. The study according to microorganic characteristics, combined with the existing evaluation model for other genetically modified products, but not confined by other safety evaluation models. The current study was aimed at exploring the systematic, perfect and practical food safety evaluation model of GMM, laying the foundation of development and utilization of GMM.In vitro tolerance of GM Lp590 to multistresses was studied. Compared with Lp, the result of the tolerance to nisin verified that Lp590 can indeed confer resistance to higher concentration nisin, and Lp590 had the similar growth curve as Lp. The ability of Lp590 to tolerate both environmental stresses （like temperatures ranging from 52°C to 4°C, or exposure to ethanol, oxygen, and osmotic stresses） and gastrointestinal transit was assessed. Lp590 showed a tolerance to 4°C and ethanol that was similar to Lp. Notably, Lp590 can tolerate higher temperatures （52°C） and higher levels of H₂O₂ and NaCl than Lp. While, Lp590 has the same gastrointestinal transit tolerance as Lp. These above results indicated that GM Lp590 has many desirable biological characteristics and has good prospects for industrial application, transgenic technique did not lead to negative effect. These results provide the possibility that food safety of GM Lp590 was assessed through animal feeding trials.The sub-chronic toxicity feeding was performed. The results showed that compared with Lp, after feeding with Lp590, no adverse oral administration-related differences in body weight, food consumpation, behavior and condition gross and histopathological examination were observed. Compared with Lp, Lp590 had the similar effect on improving jejunal muscosal structure. These results showed that exogenous gene is not the disadvantage for body health, and GM Lp590 and non-GM probiotic Lp had the same impact on SD rats.Faece as an important metabolic indicator was comprehensively studied. Fecal major flora were studied by both plate count and quantitative PCR methods, the result demonstrated that rats fed with GM Lp590 and non-GM Lp can both increase useful bacteria, decrease harmful bacteria and promote intestinal health. Further, no differences in fecal enzyme activity, SCFA, fecal pH value and fecal water content were observed between rats being administrated Lp590 compared with rats being administrated Lp. These results demonstrated that Lp590 is as safe as Lp, the transgenic composition did not change probiotic function.By several methods such as immunohistochemistry, quantitative PCR and ELISA, intestinal permeability, intestinal mucosal immunity, antioxidant capacity of the body were studied. The results of lowered intestinal permeability, improved tight-junction integrity and mucosal immunity, enhanced antioxidant capacity made cleared that Lp590 and Lp are beneficial to the health of SD rats, there was no side effects on body from exogenous gene.With specific primers for blood, muscle, liver, spleen, kidney, results of PCR showed that transgenic composition was not found in the relevant tissues and organs.According to study, the results of present study have revealed GM Lp590 has the same improvement on body health as Lp, exogenous gene has no any bad effect on SD rats.The first systematic food safety evaluation ensures commercialization and marketization of GM Lp590.The standpoint was first proposed in this study that gut health is the key mark reflecting body health. In the evaluation process, catching microorganic characteristics the study was proceeded form in vitro tolerance to gut health. Around the key point the study performed multi-angle analysis. It made the subject clear, interlocking, highlighted on the important point of food safety evaluation of GMM, but also comprehensive reflection the effect of GMM on the body. This study established a systematic, complete and practical food safety evaluation model of GMM. It can provide scientific standard methods for the regulation and commercial production of GMM. It must be significantly meaningful to the development of GMM.更多还原