副溶血性弧菌拮抗菌的分离及其抑菌机理研究

发布时间：2018-05-24 16:38

本文选题：副溶血性弧菌 + 莫海威芽孢杆菌J7　；参考：《沈阳农业大学》2016年博士论文

【摘要】：副溶血性弧菌(Vibrio parahaemolyticus)是我国上报的食源性疾病暴发事件的首要致病因素。同时该菌还是水产动物弧菌病暴发流行的病原菌,给水产养殖业造成巨大的经济损失。而抗生素在水产养殖业的滥用不仅造成多重耐药菌的产生,也会对人体健康造成严重威胁。因此,急需寻找能够防治副溶血性弧菌的抗生素替代品。其中,利用益生菌的生物防治方法具有环境污染少、能从源头控制病原菌基数等突出特点,使其有望成为未来水产养殖业弧菌病害防治的主要发展方向之一。目前,从分子水平上揭示益生菌抑菌机制的尚不多见。因此,本研究从水生环境中探寻对副溶血性弧菌有拮抗作用的益生菌,再应用于水生环境,以减少因益生菌生境改变而造成的效率减退或消失问题。通过对获得的细菌进行分离鉴定,筛选益生菌,确定其分类地位；优化益生菌发酵培养基以获得最大量的抗菌物质；利用色谱技术分离纯化抗菌物质；将纯化的抗菌物质作用于副溶血性弧菌,研究其对菌体的细胞损伤效应；采用蛋白质组学技术筛选抗菌物作用于副溶血性弧菌的反应蛋白,对拮抗作用的机理进行深入研究。主要研究结果如下：1.自海泥和4种水产品中共分离出249株细菌,全部进行点种法初筛,得到24株副溶血性弧菌拮抗菌。其中16株遗传稳定,采用牛津杯法复筛,获得2株具有胞外抗菌活性的副溶血性弧菌潜在益生菌B16和J7。通过形态、生理生化和16S rDNA的分子生物学鉴定,菌株B16和J7分别被鉴定为短小芽孢杆菌(Bacillus pumilus)和莫海威芽孢杆菌(Bacillus mojavensis)。B16和J7都能够分泌胞外酶,对多种指示菌具有胞外抗菌活性,其中,莫海威芽孢杆菌J7产生的胞外酶种类更多、抗菌谱更广确定为后续实验菌株。2.以莫海威芽孢杆菌J7抗菌物质产生的最佳发酵培养基Landy为基础,对该培养基的碳源、氮源、发酵温度、发酵时间、培养基初始pH值和接种量分别进行单因素筛选。结果表明,蔗糖和酵母浸粉为莫海威芽孢杆菌J7的最佳碳源和氮源,30℃、36h培养、培养基初始pH7.0、接种量6%为最佳发酵条件。采用Plackett-Burman实验设计从上述6个因素中筛选出对莫海威芽孢杆菌J7抗菌物质产生的3个主要因素分别为：蔗糖添加量、酵母浸粉添加量和发酵温度。根据这3个主要影响因素的效应大小设计最陡爬坡实验,确定响应面实验的中心点。采用Box-Behnken实验设计建立莫海威芽孢杆菌J7产生抗菌物质的二次多项式数学模型,并利用Design Expert 8.0软件对获得的二次多项式数学模型进行显著性检验。改良后的培养基配方(/L)为：蔗糖16.9g,酵母浸粉6.6 g, KCl0.5g, MgSO4 0.5g, KH2PO4 1.0g, MnSO4 5.0mg, CuSO4 0.16mg, FeSO4 0.15mg。优化后的培养条件为：发酵温度29.7℃,发酵时间36h,培养基初始pH7.0。采用优化的培养基及培养条件进行发酵,结果表明优化后等量培养基内的抑菌物相对产量提高了38.89%。3.采用酸沉淀法加甲醇萃取提取莫海威芽孢杆菌J7发酵上清液中的抗菌物质,得到粗提物后采用Sephadex LH-20凝胶柱层析进行初步分离、分析型HPLC两次纯化,最后得到4种活性稳定的抗菌物质。采用MALDI-TOF-TOF/MS方法对获得的4种活性抗菌物质进行分析鉴定,最终确定活性抗菌物质均为肽类,并获得了其主要的氨基酸序列。研究了抗菌肽组分P3的热稳定性、pH稳定性、紫外稳定性和最小抑菌浓度。结果显示,获得的抗菌肽在100℃ 60min、121℃20min处理,pH2-12处理,紫外灯下照射180min,其抗菌活性基本保持不变。该抗菌肽的MIC为1 mg/mL。4.采用1MIC以上浓度的抗菌肽处理副溶血性弧菌,可以明显抑制该菌的增殖,扫描电镜观察到细胞形态结构受到损伤,细胞膜通透性增加,细胞内容物渗漏,代谢活力降低。采用0.5MIC抗菌肽处理的副溶血性弧菌与对照组相比,SDS-PAGE电泳条带未发现明显缺失,但有少数条带的深浅发生了变化,说明蛋白表达量有所改变。5.采用2-DE技术,以未处理的副溶血性弧菌菌体总蛋白图谱为对照,经0.5MIC抗菌肽处理后获得丰度比大于2或小于0.5,且有显著性差异(p0.05)的蛋白点56个,其中表达上调的蛋白点36个,表达下调的蛋白点20个。将这56个差异表达蛋白全部进行质谱鉴定和生理功能分析,发现这些差异蛋白参与的代谢过程多样,包括碳水化合物代谢、辅因子和维生素代谢、能量代谢、氨基酸代谢、核苷酸代谢、脂代谢、遗传信息加工处理、环境信息处理、维持胞内氧化还原平衡、物质的运输或绑定以及免疫应激等。差异蛋白鉴定结果显示,周质蛋白丰度升高、外膜蛋白W丰度降低,参与遗传信息处理的蛋白丰度普遍降低,推测抗菌肽对副溶血性弧菌的抑制作用可能是造成菌体外膜的损伤、阻断遗传信息处理过程。
[Abstract]:Vibrio parahaemolyticus (Vibrio parahaemolyticus) is the primary pathogenic factor in the outbreak of food borne diseases reported in China. At the same time, it is also the pathogen of the outbreak of Vibrio disease in aquatic animals, causing huge economic losses to the aquaculture industry. The abuse of antibiotics in aquaculture industry not only causes the production of multidrug-resistant bacteria, but also caused by the abuse of antibiotics in the aquaculture industry. It is a serious threat to human health. Therefore, it is urgent to find an antibiotic substitute for the prevention and control of Vibrio parahaemolyticus. Among them, the biocontrol method of probiotics has less environmental pollution and can control the number of pathogenic bacteria from the source, so that it is expected to be the main development side for the prevention and control of Vibrio diseases in the aquaculture industry in the future. At present, it is not very common to reveal the bacteriostasis mechanism of probiotics from the molecular level. Therefore, this study seeks to explore the probiotics that have antagonistic effects on Vibrio parahaemolyticus from the aquatic environment, and then apply it to the aquatic environment in order to reduce the reduction or disappearance of the efficiency caused by the change of probiotic habitats. Identification, screening probiotics, determine their classification status, optimize the fermentation medium of probiotics to obtain the most large amount of antibacterial substances; use chromatography technology to separate and purify the antibacterial substances; use the purified antibacterial substances to act on Vibrio parahaemolyticus, study the cell damage response to the bacteria, and use proteomics technology to screen the antibacterial substances. The reaction protein of Vibrio parahaemolyticus was used to study the mechanism of antagonism. The main results were as follows: 1. a total of 249 strains of bacteria were isolated from sea mud and 4 kinds of aquatic products. All of them were screened and 24 strains of Vibrio parahaemolyticus were isolated. 16 of them were stable, and 2 strains were obtained by Oxford cup method. The potential probiotics B16 and J7. of Vibrio parahaemolyticus, with extracellular antibacterial activity, were identified by molecular biology, physiological and biochemical and 16S rDNA. The strain B16 and J7 were identified as Bacillus subtilis (Bacillus pumilus) and Bacillus subtilis (Bacillus mojavensis).B16 and J7 all secreted extracellular enzymes and were used for a variety of indicative bacteria. There were more extracellular antibacterial activities, among which, the species of extracellular enzymes produced by Bacillus mulHaiwei J7 were more species, and the antimicrobial spectrum was more widely identified as the best fermentation medium of.2., a subsequent experimental strain, based on the best fermentation medium of Bacillus Haiwei Bacillus J7. The carbon source, nitrogen source, fermentation temperature, fermentation time, initial pH value and inoculation amount of the medium were obtained. The results showed that sucrose and yeast were the best carbon source and nitrogen source of Bacillus subtilis J7 J7, 30 C, 36h culture, initial pH7.0 of culture medium and 6% inoculation amount as the best fermentation conditions. 3 factors produced by the above 6 factors were selected from the above 6 factors by Plackett-Burman experimental design. The main factors are the dosage of sucrose, the amount of yeast dipping and the fermentation temperature. According to the effect size of the 3 main factors, the steepest climbing experiment was designed to determine the center of the response surface experiment. The two polynomial mathematical model of the anti bacteria substances produced by Bacillus subtilis J7 was established by Box-Behnken experimental design, and D was used. The esign Expert 8 software tested the obtained two polynomial mathematical models. The improved culture medium formula (/L) was sucrose 16.9g, yeast dipping 6.6 g, KCl0.5g, MgSO4 0.5g, KH2PO4 1.0g, MnSO4 5.0mg, the fermentation temperature was 29.7, fermentation time, culture The initial pH7.0. was fermented by the optimized medium and culture conditions. The results showed that the relative yield of the bacteriostat within the optimized medium was improved by 38.89%.3. using acid precipitation method and methanol extraction to extract the antibacterial substances from the J7 fermentation supernatant of Bacillus Haiwei bacillus, and then the crude extract was obtained by Sephadex LH-20 gel column chromatography Preliminary separation, analytical type HPLC two times purification, and finally obtained 4 kinds of active antibacterial substances. 4 kinds of active antibacterial substances obtained by MALDI-TOF-TOF/MS method were analyzed and identified. Finally, the active antibacterial substances were all peptides, and their main amino acid sequences were obtained. The thermal stability of the antibacterial peptide component P3 was studied, pH Stability, UV stability and minimum bacteriostasis concentration. The results showed that the antibacterial peptides were treated at 100 60min, 121 20min, pH2-12 and 180min under UV light, and the antibacterial activity of the peptide was basically unchanged. The MIC of the antibacterial peptide was 1 mg/mL.4. to treat Vibrio parahaemolyticus with the antimicrobial peptide above 1MIC concentration, which could obviously inhibit the bacteria. The cell morphology and structure were damaged, cell membrane permeability increased, cell contents leaked, and metabolic activity decreased. Compared with the control group, the SDS-PAGE electrophoresis strips had not found obvious loss, but the depth of a few bands changed, indicating the expression of protein. The amount of.5. was changed by 2-DE technology, and the total protein atlas of untreated Vibrio parahaemolyticus was compared. After 0.5MIC antimicrobial peptide treatment, there were 56 protein points with more than 2 or less than 0.5 and significant difference (P0.05), of which 36 were up to up protein points and 20 down-regulated protein points were expressed. The 56 differences of eggs were expressed. A variety of metabolic processes involving carbohydrate metabolism, cofactors and vitamin metabolism, energy metabolism, amino acid metabolism, nucleotides metabolism, lipid metabolism, genetic information processing, environmental information processing, maintenance of intracellular redox balance, and transport of substances are found in the metabolic process of various proteins involved in various metabolic processes. The results of differential protein identification showed that the abundance of periplasmic protein, the abundances of outer membrane protein W and the abundances of proteins involved in the processing of genetic information generally decreased, and the inhibitory effect of antimicrobial peptides on Vibrio parahaemolyticus might be caused by the damage of the outer membrane of the mycelium and blocking the process of genetic information processing.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S942.3

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