拮抗菌产生的抑菌物质合成银纳米粒子的初步研究及五株新菌的鉴定

发布时间：2018-06-15 00:01

本文选题：银纳米粒子 + 生物合成　；参考：《山东大学》2017年硕士论文

【摘要】：银纳米粒子(silver nanoparticles,AgNPs)具有许多独特的物理化学特性,使其在光学、电子、催化以及医药领域等有着非常广泛的应用。过去的数十年,许多制备技术已经能够控制合成的银纳米粒子的粒径、形状和表面形貌。与传统的物理化学方法相比,生物合成法利用细菌、真菌和植物等合成银纳米粒子,避免有毒化学试剂的使用,具有对环境无污染、成本低等优点,吸引了众多研究者的关注。本文以海洋沉积物样品作为实验对象,通过传统的富集方法结合点种法分离拮抗菌,并筛选出一株拮抗菌,利用其产生的抑菌物质合成银纳米粒子。利用紫外-可见分光光度法(UV-Visible spectrometry,UV-Vis)、扫描电子显微镜(Scanning electron microscopy,SEM)和X射线衍射分析(X-ray diffiraction,XRD)的方法对银纳米粒子进行表征;以大肠杆菌和金黄色葡萄球菌作为抑菌对象,对银纳米粒子的抑菌性能进行检测。本实验以威海小石岛自然海域(37° 31'2"N 122° 1'19" E)采集的底泥样品作为实验对象,通过富集、稀释涂布平板后,以大肠杆菌(Escherichia coli ATCC 25922)、金黄色葡萄球菌(Staphylococcus aureus ATCC 29213)、鲍曼不动杆菌(Acinetobacter baumannii ATCC 19606)、副溶血弧菌(Vibrio parahaemolyticus ATCC 17802T)四株病原菌作为指示菌株,以点种法筛选出242株细菌,其中有177株细菌具有抑菌性。基于16SrDNA基因测序对拮抗菌进行鉴定,发现绝大多数菌株属于厚壁菌门。从分离到的拮抗菌中,挑选出一株拮抗性强的细菌DXFD1,经克隆测序得到其完整的16SrDNA序列(1474bp),经比对分析,发现菌株DXFD1与枯草芽孢杆菌枯草亚种(sucillus subtilis subsp.subtilis NCIB 3610T)的 16SrDNA相似性最高(99.59%)。利用菌株DXFD1产生的抗菌物质溶液与AgNO3溶液混合,置于磁力搅拌器上合成银纳米粒子,并对合成的银纳米粒子进行UV-Vis光谱、SEM和XRD表征。结果显示:银纳米粒子多为球形或近球形,粒径分布在30-70 nm,含银纳米粒子的混合液的UV-Vis光谱显示在480 nm处有吸收峰。以大肠杆菌和金黄色葡萄球菌为检测菌株,对银纳米粒子的抑菌性能进行检测。结果表明:银纳米粒子在实验中表现出明显的抑菌效果,对大肠杆菌和金黄色葡萄球菌的最小抑菌浓度均为5 μg/L,最小杀菌浓度为10 μg/mL。检测去除表面附着物质的银纳米粒子的抑菌活性,发现抑菌效果降低,结果表明抑菌物质在合成银纳米粒子的过程中结合到粒子表面,提高银纳米粒子的抑菌性能。本文对从威海附近海域、文登盐场、肥城精致盐场样品中分离到的编号为P131T、N62T、X7T、WDS2C4T与JZ3A21五株新菌进行了多相分类学鉴定。菌株P131T分离自威海近海沉积物,为革兰氏阴性菌、好氧、无滑动性、细胞呈杆状。菌株P131T的最适生长温度为28-30℃,最适pH为7.0-7.5,可耐受的盐度最为2.0-3.0%(w/v)NaCl。基于16S rDNA序列的分析表明,菌株P131T属于比齐奥氏菌属,与属内物种的16SrDNA相似性范围为94.6-97.0%。菌株P131T 主要脂肪酸组分为 iso-C15:0、iso-C15:0G、iso-C17:03-OH 和 iso-C171 ω9c。主要的极性脂成分包括磷脂酰乙醇胺、两种氨脂质、氨磷脂、磷脂和其他的脂质。主要呼吸醌类型为MK-6。基因组DNA的G+C含量是36.7mol%。根据遗传学和表型特征证据,菌株P131T(=KCTC42587T=MCCC1H00124T)代表比齐奥氏菌属的一个新物种,命名为沉积物比齐奥氏菌(Bizionia sediminis sp.nov.)。菌株N62T分离自自威海近海沉积物,为革兰氏阴性、兼性厌氧、无运动性和细胞短杆状。2216E固体培养基28℃培养2天,形成圆形、光滑、橘色和直径约为0.6-1.5mm的单菌落。菌株N62T最适生长温度为28-30℃,最适pH为7.0-7.5和最适盐度2.0-3.0%(w/v)NaCl。菌株N62T主要的脂肪酸组分是iso-C15:0、iso-C15:0G、iso-C17:03-OH 和 C17:13-OH。主要的呼吸醌类型是 MK-6。菌株N62T基因组DNA的G+C含量是35.3 mol%。主要的极性脂是磷脂酰乙醇胺、两种氨脂质、糖脂和三种其他的脂质。基于16S rDNA序列的分析表明,菌株N62T属于藏红花黄色线菌科(Crocinitomicaceae),与寒冷微菌属的物种的16SrDNA序列相似性范围为95.8-97.2%。根据遗传学和表型特征证据,菌株N62T(=KCTC 42589T=MCCC1H00117T),代表寒冷微菌属的一个新物种,命名为橙色寒冷微菌(Brumimicrobium aurantiacum sp.nov.)。菌株X7T分离自自威海近海沉积物,细胞革兰氏阴性、无滑动性、兼性厌氧和短杆状。最适生长温度为28-30℃,最适pH为7.0和最适盐度2.0-3.0%(w/v)NaCl。基于16S rDNA序列的分析表明,菌株X7T属于盐坑微菌属,与Salinimicrobium gaetbulicola有着最高的 16SrDNA 序列相似性(96.3%)。主要脂肪酸为 anteiso-Ci5:0、iso-C15:0、anteiso-C17:1cω9c、iso-C17:1ω9c、C17;02-OH 和iso-C17;03-OH。主要极性脂是一种磷脂酰乙醇胺、一种磷脂、两种氨脂质和五种其他未确定的脂质。主要呼吸醌类型是MK-6。基因组DNA的G+C含量是46.7 mol%。遗传学、表型特征以及化学组分结果表明,菌株X7T(=KCTC 42585T=MCCC1H00115T)代表盐坑微菌属的一个新物种,命名为黄色盐坑微菌(Salinimicrobium flavum sp.nov.)。菌株WDS2C4T和JZ3A21分别从文登盐场、肥城精致盐场的样品分离到,细胞为革兰氏阴性、长杆状和无滑动性。生长范围:可耐受盐度1.0-20.0%(w/v)NaCl(最适 6.0-10.0%),温度 20-50℃(最适 37-40℃)和 pH6.5-9.5(最适 7.0-8.0)。菌株WDS2C4T和菌株JZ3A21主要脂肪酸组分均是cyclo-C19;0ω8c、summed features 8(C181 ω7cand/or C18:1 ω6c)、C18:0、C17:0、C16:0 和 11-methyl C18:1 ω7c。菌株WDS2C4T和JZ3A21主要的呼吸醌类型是Q-10。菌株WDS2C4T的基因组DNA的G+C含量为67.7 mol%,菌株JZ3A21的基因组DNA的G+C含量为67.3 mol%。菌株WDS2C4T主要极性脂成分是两种磷脂、氨脂质、磷脂酰甘油、磷脂酰胆碱和两种糖脂。菌株 WDS2C4T与Rhodovulum marinum JCM 13300T 的 16S rDNA 序列相似性最高为 95.11%。与物种 Albidovulum xiamenense、Paracoccus aestuarii、Rhodobacter vinaykumarii 和Roseivivax pacificus 的模式菌株的相似性分别为94.75%、94.00%、93.88%和93.85%。根据16SrDNA序列建立的三个系统发育树(Neighbour-joining,maximum-likelihood 和 maximurn-parsimony)表明,菌株WDS2C4T和JZ3A21位于Rhodobacteraceae科的单独分支上。根据遗传学和表型特征证据,菌株 WDS2C4T(=KCTC 52227T=NBRC 112331T=MCCC 1H00148T)代表红细菌科的一个新属新种,命名为嗜盐白色盐杆菌(Albihalobacter halophilus gen.nov.sp.nov.)。
[Abstract]:Silver nanoparticles (AgNPs) has many unique physical and chemical properties, which makes it very widely used in the field of optics, electron, catalysis and medicine. In the past few decades, many preparation techniques have been able to control the size, shape and surface morphology of the synthesized silver nanoparticles. Compared with the method, biosynthesis method uses bacteria, fungi and plants to synthesize silver nanoparticles, avoids the use of toxic chemical reagents, has the advantages of no pollution to the environment and low cost, and attracts the attention of many researchers. A strain of antagonist was screened and silver nanoparticles were synthesized by the antibacterial substances produced by them. The silver nanoparticles were characterized by UV visible spectrophotometry (UV-Visible spectrometry, UV-Vis), scanning electron microscope (Scanning electron microscopy, SEM) and X ray diffraction analysis (X-ray diffiraction, XRD). The Bacteriostasis of silver nanoparticles was detected by Bacillus and Staphylococcus aureus as a bacteriostasis object. This experiment took the sediment samples collected in the Weihai small Shidao natural sea area (37 31'2 "N 122 1'19" E) as the experimental object, and by the enrichment and dilution coating plate, the Escherichia coli (Escherichia coli ATCC 25922) and golden yellow grapevine ball were used. Bacteria (Staphylococcus aureus ATCC 29213), Acinetobacter Bauman (Acinetobacter baumannii ATCC 19606), Vibrio parahaemolyticus (Vibrio parahaemolyticus ATCC 17802T) four strains of pathogenic bacteria as indicative strains, 242 strains of bacteria were screened by seed seed method, of which 177 bacteria were bacteriostasis. Based on the sequencing of 16SrDNA gene, the antagonistic bacteria were identified. It was found that most of the strains belonged to the phylum asiasis. From the isolated bacteria, a strain of resistant bacteria DXFD1 was selected and the complete 16SrDNA sequence (1474bp) was cloned and sequenced. The strain DXFD1 was similar to the 16SrDNA of Bacillus subtilis subspecies (sucillus subtilis subsp.subtilis NCIB 3610T). The highest (99.59%). The antibacterial substance produced by the strain DXFD1 is mixed with the AgNO3 solution, and the silver nanoparticles are synthesized on the magnetic stirrer, and the silver nanoparticles are characterized by UV-Vis, SEM and XRD. The results show that the silver nanoparticles are mostly spherical or near the spherical shape, the particle size distribution is 30-70 nm, and the mixing of silver nanoparticles is mixed. The UV-Vis spectrum of the liquid showed a peak of absorption at 480 nm. The Bacteriostasis of silver nanoparticles was detected with Escherichia coli and Staphylococcus aureus. The results showed that silver nanoparticles showed obvious bacteriostasis effect in the experiment, and the minimum inhibitory concentration for Escherichia coli and Staphylococcus aureus were 5 g/L. The bacteriostasis activity of the silver nanoparticles which removed the surface attachment material was detected by the small bactericidal concentration of 10 g/mL.. The results showed that the bacteriostatic effect was reduced. The results showed that the bacteriostatic substance was combined with the surface of the particles in the process of synthesizing silver nanoparticles and improved the antibacterial properties of the silver nanoparticles. This paper is a refined salt field from the sea area near Weihai, Wendeng salt field and Feicheng. Five strains of P131T, N62T, X7T, WDS2C4T and JZ3A21 were identified by multiphase taxonomy. The strain P131T was isolated from the coastal sediments of Weihai. It was Gram-negative bacteria, aerobic, non gliding, and rod-shaped. The optimum growth temperature of the strain P131T was 28-30, the optimum pH was 7.0-7.5, and the tolerable salinity was the most 2.0-3.0%. (w/v) NaCl. based on the analysis of 16S rDNA sequence, the strain P131T belongs to the genus of Bacillus, and the 16SrDNA resemblance to the internal species is the 94.6-97.0%. strain P131T main fatty acids in iso-C15:0, iso-C15:0G, iso-C17:03-OH, and iso-C171 Omega respectively, including phosphatidyl ethanolamine, two kinds of ammonia lipid, ammonia and phosphorus. Lipid, phospholipids and other lipids. The G+C content of the main respiratory quinone type is the MK-6. genome DNA, which is 36.7mol%. based on genetic and phenotypic characteristics. The strain P131T (=KCTC42587T=MCCC1H00124T) represents a new species of the genus Qi, which is named as the sediment than the Bizionia sediminis sp.nov.. The strain N62T is isolated from the self power. The marine sediments were gram-negative, facultative, facultative anaerobic, no motion and cell short rod like.2216E solid medium at 28 C for 2 days, forming a single colony of round, smooth, orange and 0.6-1.5MM diameter. The optimum growth temperature of strain N62T was 28-30, and the optimum pH was 7.0-7.5 and 2.0-3.0% (w/v) NaCl. strain N62T main fat. The acid components are iso-C15:0, iso-C15:0G, iso-C17:03-OH and C17:13-OH., the main type of respiratory quinone is the G+C content of the N62T genome DNA of the MK-6. strain is 35.3 mol%., the main polar lipid is phosphatidyl ethanolamine, two kinds of lipid, sugar and three other lipids. Based on the rDNA sequence of 16S, the strain N62T belongs to the yellow line of saffron. The family of bacteria (Crocinitomicaceae), similar to the 16SrDNA sequence of the species of the cold microbacteria, is 95.8-97.2%. based on the evidence of genetic and phenotypic characteristics, strain N62T (=KCTC 42589T=MCCC1H00117T), representing a new species of cold microbacteria, named orange cold microbacteria (Brumimicrobium aurantiacum sp.nov.). The strain X7T is isolated from self self. The sediments of Weihai offshore were gram-negative, non gliding, facultative and short rod-shaped. The optimum growth temperature was 28-30, the optimum pH was 7 and the optimum salinity 2.0-3.0% (w/v) NaCl. based on 16S rDNA sequence analysis showed that the strain X7T belonged to the salt pit Micrococcus, and the highest 16SrDNA sequence resemblance with Salinimicrobium gaetbulicola (96) .3%). The main fatty acids are anteiso-Ci5:0, iso-C15:0, anteiso-C17:1c Omega 9C, iso-C17:1 Omega 9C, C17, 02-OH and iso-C17; 03-OH. major polar lipid is a phosphatidyl ethanolamine, a kind of phospholipid, two kinds of lipids, and five other undetermined lipids. The results showed that the strain X7T (=KCTC 42585T=MCCC1H00115T) represented a new species of salt pit microbacteria, named as the yellow salt pit microbacteria (Salinimicrobium flavum sp.nov.). The strain WDS2C4T and JZ3A21 were separated from the Wendeng salt field and the Feicheng delicate salt field, and the cells were gram-negative, long rod-shaped and non gliding. Growth range: tolerance to salinity 1.0-20.0% (w/v) NaCl (optimum 6.0-10.0%), temperature 20-50 C (optimum 37-40 C) and pH6.5-9.5 (optimum 7.0-8.0). The main fatty acid components of strain WDS2C4T and strain JZ3A21 are cyclo-C19, 0 Omega 8C, summed features 8 The main respiratory quinone type of S2C4T and JZ3A21 is the G+C content of the genomic DNA of Q-10. strain WDS2C4T is 67.7 mol%, the G+C content of the genomic DNA of the strain JZ3A21 is 67.3 mol%., and the main polar lipids are two kinds of phospholipids, the ammonia lipid, the phosphatidyl glycerol, the phosphatidylcholine and the two kinds of glycolipids. The similarity of the 16S rDNA sequence of 3300T was the highest of 95.11%. and the species Albidovulum xiamenense, Paracoccus aestuarii, Rhodobacter vinaykumarii and Roseivivax pacificus were 94.75%, 94%, 93.88% and three phylogenetic trees established according to the sequence. Elihood and maximurn-parsimony) showed that strain WDS2C4T and JZ3A21 were located in the separate branch of the family Rhodobacteraceae. According to the evidence of genetic and phenotypic characteristics, strain WDS2C4T (=KCTC 52227T=NBRC 112331T=MCCC 1H00148T) represented a new species of a new genus of the family red bacteria family, named halophilic white halophilus (Albihalobacter halophilus). V.sp.nov.).
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.1;Q93

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