發(fā)布時間：2018-05-24 16:38

  本文選題：副溶血性弧菌 + 莫海威芽孢桿菌J7��； 參考：《沈陽農(nóng)業(yè)大學》2016年博士論文

【摘要】：副溶血性弧菌(Vibrio parahaemolyticus)是我國上報的食源性疾病暴發(fā)事件的首要致病因素。同時該菌還是水產(chǎn)動物弧菌病暴發(fā)流行的病原菌,給水產(chǎn)養(yǎng)殖業(yè)造成巨大的經(jīng)濟損失。而抗生素在水產(chǎn)養(yǎng)殖業(yè)的濫用不僅造成多重耐藥菌的產(chǎn)生,也會對人體健康造成嚴重威脅。因此,急需尋找能夠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環(huán)境污染少、能從源頭控制病原菌基數(shù)等突出特點,使其有望成為未來水產(chǎn)養(yǎng)殖業(yè)弧菌病害防治的主要發(fā)展方向之一。目前,從分子水平上揭示益生菌抑菌機制的尚不多見。因此,本研究從水生環(huán)境中探尋對副溶血性弧菌有拮抗作用的益生菌,再應用于水生環(huán)境,以減少因益生菌生境改變而造成的效率減退或消失問題。通過對獲得的細菌進行分離鑒定,篩選益生菌,確定其分類地位；優(yōu)化益生菌發(fā)酵培養(yǎng)基以獲得最大量的抗菌物質(zhì)；利用色譜技術分離純化抗菌物質(zhì)；將純化的抗菌物質(zhì)作用于副溶血性弧菌,研究其對菌體的細胞損傷效應；采用蛋白質(zhì)組學技術篩選抗菌物作用于副溶血性弧菌的反應蛋白,對拮抗作用的機理進行深入研究。主要研究結果如下：1.自海泥和4種水產(chǎn)品中共分離出249株細菌,全部進行點種法初篩,得到24株副溶血性弧菌拮抗菌。其中16株遺傳穩(wěn)定,采用牛津杯法復篩,獲得2株具有胞外抗菌活性的副溶血性弧菌潛在益生菌B16和J7。通過形態(tài)、生理生化和16S rDNA的分子生物學鑒定,菌株B16和J7分別被鑒定為短小芽孢桿菌(Bacillus pumilus)和莫海威芽孢桿菌(Bacillus mojavensis)。B16和J7都能夠分泌胞外酶,對多種指示菌具有胞外抗菌活性,其中,莫海威芽孢桿菌J7產(chǎn)生的胞外酶種類更多、抗菌譜更廣確定為后續(xù)實驗菌株。2.以莫海威芽孢桿菌J7抗菌物質(zhì)產(chǎn)生的最佳發(fā)酵培養(yǎng)基Landy為基礎,對該培養(yǎng)基的碳源、氮源、發(fā)酵溫度、發(fā)酵時間、培養(yǎng)基初始pH值和接種量分別進行單因素篩選。結果表明,蔗糖和酵母浸粉為莫海威芽孢桿菌J7的最佳碳源和氮源,30℃、36h培養(yǎng)、培養(yǎng)基初始pH7.0、接種量6%為最佳發(fā)酵條件。采用Plackett-Burman實驗設計從上述6個因素中篩選出對莫海威芽孢桿菌J7抗菌物質(zhì)產(chǎn)生的3個主要因素分別為：蔗糖添加量、酵母浸粉添加量和發(fā)酵溫度。根據(jù)這3個主要影響因素的效應大小設計最陡爬坡實驗,確定響應面實驗的中心點。采用Box-Behnken實驗設計建立莫海威芽孢桿菌J7產(chǎn)生抗菌物質(zhì)的二次多項式數(shù)學模型,并利用Design Expert 8.0軟件對獲得的二次多項式數(shù)學模型進行顯著性檢驗。改良后的培養(yǎng)基配方(/L)為：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。優(yōu)化后的培養(yǎng)條件為：發(fā)酵溫度29.7℃,發(fā)酵時間36h,培養(yǎng)基初始pH7.0。采用優(yōu)化的培養(yǎng)基及培養(yǎng)條件進行發(fā)酵,結果表明優(yōu)化后等量培養(yǎng)基內(nèi)的抑菌物相對產(chǎn)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桿菌J7發(fā)酵上清液中的抗菌物質(zhì),得到粗提物后采用Sephadex LH-20凝膠柱層析進行初步分離、分析型HPLC兩次純化,最后得到4種活性穩(wěn)定的抗菌物質(zhì)。采用MALDI-TOF-TOF/MS方法對獲得的4種活性抗菌物質(zhì)進行分析鑒定,最終確定活性抗菌物質(zhì)均為肽類,并獲得了其主要的氨基酸序列。研究了抗菌肽組分P3的熱穩(wěn)定性、pH穩(wěn)定性、紫外穩(wěn)定性和最小抑菌濃度。結果顯示,獲得的抗菌肽在100℃ 60min、121℃20min處理,pH2-12處理,紫外燈下照射180min,其抗菌活性基本保持不變。該抗菌肽的MIC為1 mg/mL。4.采用1MIC以上濃度的抗菌肽處理副溶血性弧菌,可以明顯抑制該菌的增殖,掃描電鏡觀察到細胞形態(tài)結構受到損傷,細胞膜通透性增加,細胞內(nèi)容物滲漏,代謝活力降低。采用0.5MIC抗菌肽處理的副溶血性弧菌與對照組相比,SDS-PAGE電泳條帶未發(fā)現(xiàn)明顯缺失,但有少數(shù)條帶的深淺發(fā)生了變化,說明蛋白表達量有所改變。5.采用2-DE技術,以未處理的副溶血性弧菌菌體總蛋白圖譜為對照,經(jīng)0.5MIC抗菌肽處理后獲得豐度比大于2或小于0.5,且有顯著性差異(p0.05)的蛋白點56個,其中表達上調(diào)的蛋白點36個,表達下調(diào)的蛋白點20個。將這56個差異表達蛋白全部進行質(zhì)譜鑒定和生理功能分析,發(fā)現(xiàn)這些差異蛋白參與的代謝過程多樣,包括碳水化合物代謝、輔因子和維生素代謝、能量代謝、氨基酸代謝、核苷酸代謝、脂代謝、遺傳信息加工處理、環(huán)境信息處理、維持胞內(nèi)氧化還原平衡、物質(zhì)的運輸或綁定以及免疫應激等。差異蛋白鑒定結果顯示,周質(zhì)蛋白豐度升高、外膜蛋白W豐度降低,參與遺傳信息處理的蛋白豐度普遍降低,推測抗菌肽對副溶血性弧菌的抑制作用可能是造成菌體外膜的損傷、阻斷遺傳信息處理過程。
[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.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S942.3

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2 隋R，

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