【摘要】： 細(xì)菌性食物中毒和感染性腹瀉是世界范圍內(nèi)嚴(yán)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(yùn)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(yán)重危害,也使國(guó)民經(jīng)濟(jì)遭到巨大損失。食品原料和食品的防腐抗菌技術(shù)一直以來是控制食物中毒的關(guān)鍵技術(shù),急待需要建立和發(fā)展新的方法。 鑒于噬菌體應(yīng)用的專一宿主性、環(huán)境親和性、經(jīng)濟(jì)方便和持續(xù)性特點(diǎn),研究人員繼噬菌體技術(shù)在臨床治療的探索研究后,已將噬菌體抗菌技術(shù)擴(kuò)展到環(huán)境凈化和食品抗菌的領(lǐng)域,其結(jié)果令人鼓舞。歐美國(guó)家已經(jīng)開始推廣并應(yīng)用此項(xiàng)技術(shù)在食品抗菌領(lǐng)域。因此,篩選得到有價(jià)值的烈性噬菌體已成為開發(fā)應(yīng)用的有力資源和儲(chǔ)備,也是相關(guān)技術(shù)深入研究的基礎(chǔ)。 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(xué)特征,并通過觀察噬菌體在營(yíng)養(yǎng)液中抑制宿主菌的規(guī)律,為營(yíng)養(yǎng)液儲(chǔ)存的抑菌技術(shù)開發(fā)奠定實(shí)驗(yàn)基礎(chǔ),也為其他食品加工、儲(chǔ)存的抑菌技術(shù)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(xué)特征; (2)腸侵襲性大腸桿菌噬菌體LSB-1的應(yīng)用探索。相關(guān)研究?jī)?nèi)容和結(jié)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(jià)”噬菌體,另兩株為“單價(jià)”噬菌體。其中LSB-1對(duì)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(jī)序列分析等技術(shù),觀察噬菌斑、超微結(jié)構(gòu)、核酸性質(zhì)及核酸序列。結(jié)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(jié)構(gòu)是由兩個(gè)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(jù)基因比對(duì)結(jié)果,未發(fā)現(xiàn)同源噬菌體株。 3.通過活菌計(jì)數(shù)試驗(yàn),觀察噬菌體LSB-1對(duì)牛奶中宿主菌的抑制作用,試驗(yàn)顯示,在非營(yíng)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(shì),但抑菌效率很低;在營(yíng)養(yǎng)條件下和在4℃的作用溫度下,1:1比配的抑菌效應(yīng)穩(wěn)定,宿主菌滴度降低維持在2個(gè)對(duì)數(shù)級(jí)左右;同樣條件在22℃～37℃作用時(shí),數(shù)小時(shí)內(nèi)宿主菌滴度可達(dá)到約4個(gè)對(duì)數(shù)級(jí),但抑菌的穩(wěn)定性較差,隨著時(shí)間的延長(zhǎng),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(xiàn)象均顯示宿主菌有“免疫性”產(chǎn)生。 4.考慮到噬菌體LSB-1的宿主譜相對(duì)較窄,而且有宿主菌獲得“免疫性”現(xiàn)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(kuò)大噬菌體LSB-1在食品及其他領(lǐng)域的抗菌應(yīng)用效率的設(shè)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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