【作者】 刘姝；

【导师】 陆兆新；

【作者基本信息】 南京农业大学 ， 食品科学， 2007， 博士

【摘要】 海洋中蕴藏着大量的微生物资源，在它们中广泛地分布着抗菌活性菌株，是抗菌物质的重要潜在来源。海洋微生物资源还是一个开发不久的新领域，加上海洋环境的独特性和极端性特点，它们所产生的抗菌物质往往是新颖的，可能具有很好的应用前景。因此，从海洋中筛选产高效、新颖、低毒的抗菌活性物质潜力巨大。本研究的目的在于利用我国连云港丰富的海洋微生物资源，从海洋样品中筛选出能产生抗菌活性高、抗菌谱广、安全高效的抗菌物质的菌株，并对抗菌物质的理化特性、组成及其对蜡样芽孢杆菌和大肠杆菌的抑制效果进行研究，旨在获得广谱高效、安全无毒的将来能作为食品防腐剂和饲料抗生素的新型抗菌物质。研究结果分述如下：1．从采自连云港海域的海洋样品中分离到667株菌，其中246株有抗菌活性，包括74株放线菌、156株细菌和16株真菌，拮抗菌株获得比例为36.88％，以放线菌拮抗株得率为最高；选取抗菌活性强的11株菌进行复筛后，发现11株菌株对啤酒酵母和扩展青霉均表现较明显的抑制作用；除1株菌对革兰氏阴性菌没有抑制作用外，其它菌株对革兰氏阳性菌和革兰氏阴性菌均有不同程度的抑制活性。这些菌株大部分有较广泛的抗菌谱，显示了它们较好的应用潜力。其中放线菌GB-2的抑菌谱广，且抑菌活性强，不仅能较好地抑制革兰氏阳性菌和革兰氏阴性菌而且对霉菌和酵母菌也有显著的抑菌作用。因此，确定菌株GB-2为下一步试验菌株。2．对放线菌GB-2进行了分类鉴定。通过形态特征、培养特征、生理生化特性和细胞壁化学组分分析，初步判定其属于链霉菌属。在分子生物学鉴定中，通过总DNA提取、PCR扩增，得到1395bp的16SrRNA序列，与GenBank中相关序列进行同源性比对后，应用Bioedit7.0和Treedrawing软件绘制系统发育树。结果显示，GB-2的16SrRNA序列和数据库中的弗氏链霉菌(Streptomyces fradiae AB184776)的序列同源性达到98％以上。经过与已报道的弗氏链霉菌进行比较分析，确定菌株GB-2为弗氏链霉菌变种。3．通过研究菌株GB-2的生物学活性及其抗菌物质的理化特性发现发酵液色泽的深浅与培养基盐度密切相关，且用人工海水配制培养基可显著提高菌株GB-2的抗菌活性。GB-2菌株的抑菌谱较广，不仅对金黄色葡萄球菌及其耐药性菌株、蜡样芽孢杆菌、大肠杆菌等革兰氏阳性和革兰氏阴性食品致病菌和腐败细菌都有较强的抑制作用，而且可抑制扩展青霉、黄曲霉、点青霉等食品中常见的腐败真菌及番茄灰霉、小麦赤霉病菌、棉花枯萎病菌、稻瘟病菌等植物病原真菌。发酵液稳定性研究表明GB-2菌株所产抗菌物质在121℃pH1和pH12条件下抑菌活性均不变，紫外线照射对其抗菌活性也没有影响，表现出良好的耐酸碱热特性及对紫外线的稳定性，在生防、食品防腐剂及医药方面具有较好的应用潜力。4．菌株GB-2产生的抗菌物质溶于水，微溶于甲醇、乙醇，不溶于其它有机溶剂中。GB-2的发酵液经过超滤后发现其抗菌活性由抗细菌和抗真菌两大组分组成，其中抗细菌组分能通过截留分子量≤1KDa的超滤膜。对含抗细菌物质的超滤液进行纸层析，发现该物质是一种水溶性的中性物质。经Sephadex LH-20和Sephadex G-15色谱柱分离后，过柱液经抗菌活性检测出现3个活性峰，其中a峰的对蜡样芽孢杆菌和大肠杆菌的抑菌活性最强。将a峰的收集液进一步用HPLC纯化，在图谱上出现两个峰，第2个峰具有抑菌活性。根据其特性推测其可能是一种氨基糖苷类抗生素。5．研究了抗细菌物质抑制蜡样芽孢杆菌和大肠杆菌的敏感性及作用效果。结果表明，蜡样芽孢杆菌和大肠杆菌对其均具有较高的敏感性，最小抑菌浓度分别0.663μg/mL和0.904μg/mL，能够较强地抑制这2种菌的生长；菌体生长曲线、膜通透性研究结果显示加了抗细菌物质的培养液菌浓下降、紫外吸收值上升，表明对这2种菌具有杀灭溶解作用；扫描电镜和透射电镜观察发现该抗细菌物质可以导致菌体细胞膜完整性被破坏，细胞内大分子泄漏，进一步导致菌体细胞破裂；但只对生长的菌体细胞起到这一效果，对非繁殖的菌体及芽孢均无明显的抑制效果。更多还原

【Abstract】 Marine microorganisms have a wide range of antimicrobial strains, which are the important potential sources of antimicrobial substances. As a new field of the research of marine microbial resources and because of the uniqueness and extremeness of the marine environment, many biologically active substances the marine microorganisms excreted are relatively newly to us. Therefore, it is a huge potential to screen novel, highly active and low poisonous antimicrobial substances. In order to find an highly active and safe antimicrobial substance using as food preservtives and feed antibiotics in the future, these works were done:(1) to isolate, screen and identify marine microorganisms with novel and highly active antimicrobial substnaces from the marine microbial resources of Lianyungang;(2) to study antimicrobial activity, physics and chemistry nature, composition and the antibacterial mechanism of antimicrobial substances from strain GB-2. The results were described as follows:The 667 marine microorganisms were isolated from the marine samples collected from the sea area of Lianyungang. 246 strains of them(about 36.88% of the total isolates)had antimicrobial activity, consisting of 74 actinomycetes, 156 bacteria and 16 fungi and the isolation rate of antimicrobial active strains from actinomycetes was highest. After duplicate sieves, 11 strains were observed inhibiting Penicillium expansion and Saccharomyces cerevisia obviously, majority of these 11 strains exhibited different extend antagonism against Gram-positive bacteria and Gram-negative bacteria. The antimicrobial activity of these 11 stains demonstrated the potential application in future. Finally, the strain GB-2 of actinomycete from marine sediment collected in the inteltidal zone was determined for the next step of experimental strain because of its strongest and broad-spectrum antibacterial and antifungal activities.Then, the strain GB-2 was identified with classical and molecular methods. According to the morphological and cultural characteristics, the physiological and biochemical properties and chemical components of cell wall, it was indicated that strain GB-2 was belong to Streptomyces. In the 16SrRNA sepuence analysis, the sequence fragment of 1395bp 16S ribosomal RNA was amplified from total DNA of strain GB-2 by PCR, then the sequence analysis was down by the application of BLAST on the websites of NCBI and the phylogenetic tree was drawn by Bioedit 7.0~T and Treedrawing~T. The results showed that GB-2 shared 98% 16SrRNA homology with Streptomyces fradiae(AB184776). After compared with the characteristics of Streptomyces fradiae reported, strain GB-2 was identified as a varation species of Streptomyces fradiae.Influence of different water of medium on the production of antimicrobial substances was examined. And the results showed the high production of antimicrobial substances was significantly associated with aritificial seawater and the color of the fermentation broth was closely related to the salt concentration of the medium. Strain GB-2 displayed significant inhibitory effects not only on Gram-positive and Gram-negative food-borne pathogens and spoilage bacteria but also on the drug-resistant bacteria and the spoilage fungi in diets and plant pathogenic fungi. The results of stability studies of the fermentation broth of Streptomyces GB-2 showed that it was very stable at pH1 and pH12 under 121℃and had little change after ultraviolet treatment. The potential use of substance produced by strain GB-2 in bio-control, food and medical application was predicted.The purification of the antimicrobial substances produced strain GB-2 was studied. The antimicrobial substances were diffluent in the water, dissoluble in the methanol and alcohol, and insoluble in other organic solvents. The results of ultrafiltration of fermented liquid showed that, there were two components of antibacterial activity and antifugal activity respectively, and the molecular weight of antibacterial substance was≤1KDa. The antibacterial substance was proved to be a neutral and water-soluble according to paper chromatogram analysis. After purification on the Sephadex LH-20 and Sephadex G-15, the ultrafiltration fraction with molecular weight≤1KDa exhibited three series of antibacterial activity, among which peak a displayed the strongest activity to inhibit Bacillus cereus and E.coli. The HPLC analysis of peak a showed two series of peaks and the second peak was the one who had the antibacterial activity and might be an aminoglycoside antibiotic according to its property.Sensibility and inhibition effect of antibacterial substance produced by strain GB-2 to Bacillus cereus and E.coli was studied. The results showed that Bacillus cereus and E.coli were very sensitive to antibacterial substance and their MIC were 0.663μg/mL and 0.904μg/ml respectively. Antibacterial substance could strongly inhibit their growth-breeding. Studies of growth curve of thalline, membrane permeability showed that antibacterial substance added into the broth could result in the decrease of bacterial concentration and the increase of ultraviolet absorption, which indicated antibacterial substance could kill or lysis Bacillus cereus and E.coli. SEM and TEM results demonstrated that antibacterial substance could destroy integrity of cell membrance, which led to increasing of membrane permeability and leaking out of macromolecule in the cell, and then thallus cells break. This phenomenon only could be seen in thallus cells which were in the stage of growing. And there was little change of the resting cells and spores after antibacterial substance added.更多还原