致病性大肠杆菌烈性噬菌体的筛选及其生物学效应研究
发布时间：2019-04-08 08:53
【摘要】： 细菌性食物中毒和感染性腹泻是世界范围内严重的公共卫生问题。其中在鱼、肉、蛋以及各类食品的存放、运输和加工过程中,致病性大肠杆菌是引起污染的重要原因之一,不仅给公众健康造成了严重危害,也使国民经济遭到巨大损失。食品原料和食品的防腐抗菌技术一直以来是控制食物中毒的关键技术,急待需要建立和发展新的方法。 鉴于噬菌体应用的专一宿主性、环境亲和性、经济方便和持续性特点,研究人员继噬菌体技术在临床治疗的探索研究后,已将噬菌体抗菌技术扩展到环境净化和食品抗菌的领域,其结果令人鼓舞。欧美国家已经开始推广并应用此项技术在食品抗菌领域。因此,筛选得到有价值的烈性噬菌体已成为开发应用的有力资源和储备,也是相关技术深入研究的基础。 本课题从环境样本中分离致病性的大肠杆菌噬菌体,观察分析噬菌体生物学特征,并通过观察噬菌体在营养液中抑制宿主菌的规律,为营养液储存的抑菌技术开发奠定实验基础,也为其他食品加工、储存的抑菌技术探索方法。 本课题研究分为两部分: (1)致病性大肠杆菌噬菌体的筛选及分子生物学特征; (2)肠侵袭性大肠杆菌噬菌体LSB-1的应用探索。相关研究内容和结果如下: 1.通过双层培养法从污水样本中分离到4株致病性大肠杆菌的烈性噬菌体。两株为“多价”噬菌体,另两株为“单价”噬菌体。其中LSB-1对大肠杆菌EIEC8401有专一的高溶解作用。 2.通过噬菌体培养、透射电镜、核酸分离电泳、随机序列分析等技术,观察噬菌斑、超微结构、核酸性质及核酸序列。结果显示,噬菌体LSB-1的噬菌斑直径约3～4mm,透明,边缘清楚,无晕环;其结构是由两个大小不同的圆形衣壳体单位组成,似葫芦状(约70nm),其中大衣壳体单位直径为46nm左右,小衣壳体单位直径为24nm左右;核酸大约为23000bp,双链DNA,似覆盖噬菌体科;根据基因比对结果,未发现同源噬菌体株。 3.通过活菌计数试验,观察噬菌体LSB-1对牛奶中宿主菌的抑制作用,试验显示,在非营养环境中,高比配(1:10000)的噬菌体投入在室温下有一定的抑菌趋势,但抑菌效率很低;在营养条件下和在4℃的作用温度下,1:1比配的抑菌效应稳定,宿主菌滴度降低维持在2个对数级左右;同样条件在22℃～37℃作用时,数小时内宿主菌滴度可达到约4个对数级,但抑菌的稳定性较差,随着时间的延长,抑菌作用明显减弱;继续增加噬菌体投入剂量也将明显降低抑菌效果。后两种现象均显示宿主菌有“免疫性”产生。 4.考虑到噬菌体LSB-1的宿主谱相对较窄,而且有宿主菌获得“免疫性”现象,我们提出引入电磁波协同处理和开发噬菌体溶解酶的途径以扩大噬菌体LSB-1在食品及其他领域的抗菌应用效率的设想。
[Abstract]:Bacterial food poisoning and infectious diarrhoea are serious public health problems worldwide. In the process of storage, transportation and processing of fish, meat, eggs and all kinds of food, pathogenic Escherichia coli is one of the important causes of pollution, which not only causes serious harm to public health, but also causes great losses to the national economy. The antiseptic and antibacterial technology of food raw materials and food has been the key technology to control food poisoning. It is urgent to establish and develop new methods. In view of the unique host, environmental affinity, economic convenience and sustainability of bacteriophage applications, researchers have followed the exploration and research of phage technology in clinical therapy. Bacteriophage antibacterial technology has been extended to the field of environmental purification and food antibacterial, and the results are encouraging. European and American countries have begun to promote and apply this technology in the field of food antibacterial. Therefore, screening and obtaining valuable potent bacteriophage has become a powerful resource and reserve for development and application, and it is also the basis of in-depth research on related technologies. In this paper, the bacteriophage of pathogenic Escherichia coli was isolated from environmental samples, the biological characteristics of bacteriophage were observed and analyzed, and the regularity of bacteriophage inhibiting host bacteria in nutrient solution was observed. It lays an experimental foundation for the development of bacteriostasis technology of nutrient solution storage, and also explores the methods of bacteriostasis technology for other food processing and storage. This study is divided into two parts: (1) screening of pathogenic Escherichia coli bacteriophage and its molecular biological characteristics; (2) application of enteroinvasive Escherichia coli bacteriophage LSB-1. Related research contents and results are as follows: 1. Four virulent bacteriophages of pathogenic Escherichia coli were isolated from sewage samples by double-layer culture. Two were "polyvalent" bacteriophages and the other two "monovalent" bacteriophages. Among them, LSB-1 has a specific hyperlytic effect on Escherichia coli EIEC8401. 2. Phage culture, transmission electron microscopy, nucleic acid separation and electrophoresis, random sequence analysis were used to observe phage plaque, ultrastructure, nucleic acid properties and nucleic acid sequence. The results showed that the plaque diameter of bacteriophage LSB-1 was about 3? 4 mm, transparent, with clear edges and no halo ring. Its structure is composed of two round garment shell units of different sizes, similar to cucurbita (about 70nm), in which the unit diameter of coat shell is about 46nm and the unit diameter of small garment shell is about 24nm; The nucleic acid was about 23000bp, and the double-stranded DNA, appeared to cover the bacteriophage family, but no homologous phage strain was found according to the results of gene alignment. 3. The inhibitory effect of bacteriophage LSB-1 on host bacteria in milk was observed by living bacteria counting test. The results showed that in non-nutritious environment, the bacteriophage input with high specific ratio (1 脳 10 000) had a certain inhibitory trend at room temperature. But the bacteriostatic efficiency was very low. Under nutritional conditions and at 4 鈩，
本文编号：2454452
[Abstract]:Bacterial food poisoning and infectious diarrhoea are serious public health problems worldwide. In the process of storage, transportation and processing of fish, meat, eggs and all kinds of food, pathogenic Escherichia coli is one of the important causes of pollution, which not only causes serious harm to public health, but also causes great losses to the national economy. The antiseptic and antibacterial technology of food raw materials and food has been the key technology to control food poisoning. It is urgent to establish and develop new methods. In view of the unique host, environmental affinity, economic convenience and sustainability of bacteriophage applications, researchers have followed the exploration and research of phage technology in clinical therapy. Bacteriophage antibacterial technology has been extended to the field of environmental purification and food antibacterial, and the results are encouraging. European and American countries have begun to promote and apply this technology in the field of food antibacterial. Therefore, screening and obtaining valuable potent bacteriophage has become a powerful resource and reserve for development and application, and it is also the basis of in-depth research on related technologies. In this paper, the bacteriophage of pathogenic Escherichia coli was isolated from environmental samples, the biological characteristics of bacteriophage were observed and analyzed, and the regularity of bacteriophage inhibiting host bacteria in nutrient solution was observed. It lays an experimental foundation for the development of bacteriostasis technology of nutrient solution storage, and also explores the methods of bacteriostasis technology for other food processing and storage. This study is divided into two parts: (1) screening of pathogenic Escherichia coli bacteriophage and its molecular biological characteristics; (2) application of enteroinvasive Escherichia coli bacteriophage LSB-1. Related research contents and results are as follows: 1. Four virulent bacteriophages of pathogenic Escherichia coli were isolated from sewage samples by double-layer culture. Two were "polyvalent" bacteriophages and the other two "monovalent" bacteriophages. Among them, LSB-1 has a specific hyperlytic effect on Escherichia coli EIEC8401. 2. Phage culture, transmission electron microscopy, nucleic acid separation and electrophoresis, random sequence analysis were used to observe phage plaque, ultrastructure, nucleic acid properties and nucleic acid sequence. The results showed that the plaque diameter of bacteriophage LSB-1 was about 3? 4 mm, transparent, with clear edges and no halo ring. Its structure is composed of two round garment shell units of different sizes, similar to cucurbita (about 70nm), in which the unit diameter of coat shell is about 46nm and the unit diameter of small garment shell is about 24nm; The nucleic acid was about 23000bp, and the double-stranded DNA, appeared to cover the bacteriophage family, but no homologous phage strain was found according to the results of gene alignment. 3. The inhibitory effect of bacteriophage LSB-1 on host bacteria in milk was observed by living bacteria counting test. The results showed that in non-nutritious environment, the bacteriophage input with high specific ratio (1 脳 10 000) had a certain inhibitory trend at room temperature. But the bacteriostatic efficiency was very low. Under nutritional conditions and at 4 鈩，
本文编号：2454452
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