【摘要】： 細菌性食物中毒和感染性腹瀉是世界范圍內嚴重的公共衛(wèi)生問題。其中在魚、肉、蛋以及各類食品的存放、運輸和加工過程中,致病性大腸桿菌是引起污染的重要原因之一,不僅給公眾健康造成了嚴重危害,也使國民經濟遭到巨大損失。食品原料和食品的防腐抗菌技術一直以來是控制食物中毒的關鍵技術,急待需要建立和發(fā)展新的方法。 鑒于噬菌體應用的專一宿主性、環(huán)境親和性、經濟方便和持續(xù)性特點,研究人員繼噬菌體技術在臨床治療的探索研究后,已將噬菌體抗菌技術擴展到環(huán)境凈化和食品抗菌的領域,其結果令人鼓舞。歐美國家已經開始推廣并應用此項技術在食品抗菌領域。因此,篩選得到有價值的烈性噬菌體已成為開發(fā)應用的有力資源和儲備,也是相關技術深入研究的基礎。 本課題從環(huán)境樣本中分離致病性的大腸桿菌噬菌體,觀察分析噬菌體生物學特征,并通過觀察噬菌體在營養(yǎng)液中抑制宿主菌的規(guī)律,為營養(yǎng)液儲存的抑菌技術開發(fā)奠定實驗基礎,也為其他食品加工、儲存的抑菌技術探索方法。 本課題研究分為兩部分: (1)致病性大腸桿菌噬菌體的篩選及分子生物學特征; (2)腸侵襲性大腸桿菌噬菌體LSB-1的應用探索。相關研究內容和結果如下: 1.通過雙層培養(yǎng)法從污水樣本中分離到4株致病性大腸桿菌的烈性噬菌體。兩株為“多價”噬菌體,另兩株為“單價”噬菌體。其中LSB-1對大腸桿菌EIEC8401有專一的高溶解作用。 2.通過噬菌體培養(yǎng)、透射電鏡、核酸分離電泳、隨機序列分析等技術,觀察噬菌斑、超微結構、核酸性質及核酸序列。結果顯示,噬菌體LSB-1的噬菌斑直徑約3～4mm,透明,邊緣清楚,無暈環(huán);其結構是由兩個大小不同的圓形衣殼體單位組成,似葫蘆狀(約70nm),其中大衣殼體單位直徑為46nm左右,小衣殼體單位直徑為24nm左右;核酸大約為23000bp,雙鏈DNA,似覆蓋噬菌體科;根據基因比對結果,未發(fā)現同源噬菌體株。 3.通過活菌計數試驗,觀察噬菌體LSB-1對牛奶中宿主菌的抑制作用,試驗顯示,在非營養(yǎng)環(huán)境中,高比配(1:10000)的噬菌體投入在室溫下有一定的抑菌趨勢,但抑菌效率很低;在營養(yǎng)條件下和在4℃的作用溫度下,1:1比配的抑菌效應穩(wěn)定,宿主菌滴度降低維持在2個對數級左右;同樣條件在22℃～37℃作用時,數小時內宿主菌滴度可達到約4個對數級,但抑菌的穩(wěn)定性較差,隨著時間的延長,抑菌作用明顯減弱;繼續(xù)增加噬菌體投入劑量也將明顯降低抑菌效果。后兩種現象均顯示宿主菌有“免疫性”產生。 4.考慮到噬菌體LSB-1的宿主譜相對較窄,而且有宿主菌獲得“免疫性”現象,我們提出引入電磁波協(xié)同處理和開發(fā)噬菌體溶解酶的途徑以擴大噬菌體LSB-1在食品及其他領域的抗菌應用效率的設想。
[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，

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