产河豚毒素菌株的分离鉴定

从厦门同安海域的黄鳍东方豚(Fugu xanthopterus)卵巢组织中分离纯化出59株菌株,通过小鼠试验筛选出11株产毒菌株,并对其中产毒能力较强的EL10菌株的发酵产物进行高效液相色谱分析,初步认定发酵产物中含有河豚毒素。同时对EL10菌株进行16S rDNA鉴定,结果表明该菌属于希瓦氏菌属(Shewanella)。     

红鳍东方鲀病原鱼肠道弧菌的生物学特性研究

对引起红鳍东方鲀发病死亡的病原细菌进行了分离和主要生物学特性研究,包括病原性、形态特征、理化特性、16S rRNA基因序列及其系统发育学分析、胞外酶及溶血素活性、K抗原及耐药性等.结果表明,引起红鳍东方纯发病死亡的病原细菌为弧菌属(-Vibrio Pacini 1854)的鱼肠道弧菌(V.ichthyoenteri Ishimaru,et al.1996),2株代表菌株16S rRNA基凶序列(GenBank登录号分别为:EF611424和EF635304)与GenBank数据库中鱼肠道弧菌的同源性在98%-100%,且在构建的MP系统发生树中与鱼肠道弧菌聚为一个分支.分离菌不具有淀粉酶、蛋白酶、脂肪酶、DNA酶、脲酶、明胶酶和卵磷脂酶活性,且在含7%家兔脱纤血液营养琼脂培养基上不溶血.不具有K抗原.人工感染试验中分离菌对红鳍东方纯表现出明显的致病性.药敏试验结果显示,4株分离菌对供试37种抗菌药物中的苯唑青霉素和杆菌肽2种耐药.     

一株红景天内生细菌的筛选及初步研究

【目的】从红景天根部筛选并鉴定一株产酪醇的细菌,初步研究其产酪醇特性,为寻找红景天替代资源提供新途径。【方法】用NA培养基从大花红景天根部中分离内生细菌,通过薄层层析(TLC)、高效液相色谱(HPLC)、气相色谱-质谱联用(GC-MS)筛选出产量最大的菌株,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析其分类学地位。单因素实验确定初始pH、培养温度、发酵时间及接种量对菌株产酪醇活力的影响。【结果】从大花红景天根部分离出14株内生细菌,其中8株能产酪醇,筛选出酪醇产量最大的菌株B3,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析初步鉴定为水生拉恩氏菌(Rahnella aquatilis)。研究其发酵条件,其最适pH为6.0,最适温度为32 °C,最佳发酵时间为42 h,最佳接种量为15%。在最适发酵条件下,用改良NA培养基发酵,B3菌株酪醇的产量为15.68 mg/L。【结论】B3菌株是一株具有产酪醇能力的细菌,在最适发酵条件下酪醇产量达到15.68 mg/L,具有潜在的开发价值。     

超广谱β内酰胺酶肠杆菌拮抗菌的鉴定及抑菌产物分析

本研究对从土壤中筛选出的超广谱β内酰胺酶肠杆菌的拮抗菌进行鉴定,并对其发酵产物抑菌效果进行研究。以分离到的污水中的15株产超广谱β内酰胺酶肠杆菌为测试菌株,土壤中分离到的1株拮抗菌为试验菌株,通过点接种法、平板对峙法和发酵产物抑菌试验测试试验菌株对测试菌住的拮抗作用;通过16S r DNA PCR产物测序及生理生化反应鉴定试验菌株种属;通过发酵产物氯仿粗提物薄层色谱分析试验,结合细菌次级代谢产物基因簇预测在线分析,初步研究试验菌株的抑菌成分。显示试验菌株鉴定为铜绿假单胞菌,对15株产超广谱β内酰胺酶肠杆菌均有拮抗作用,其发酵产物氯仿粗提物中组分之一具有明显抑菌作用,次级代谢产物基因簇在线软件分析推测该产物可能为Nrps、Bacteriocin、Phenazine、Siderophore、Hserlactone、Arylpolyene中的一种或几种。本研究提示铜绿假单胞菌含有多种已知或未知次级代谢产物,为开发利用铜绿假单胞菌资源提供研究资料。     

大莲湖池杉林土壤细菌的季节性变化

应用稀释平板法对大莲湖池杉林湿地土壤细菌进行分离,采用16S rDNA序列分析法对所分离细菌进行鉴定。结果表明,池杉林不同季节土壤细菌种类和数量有差异。其中夏季土壤细菌数量和种类最多,春、秋季次之,冬季最少。四个季度共分离得到60株菌株,分属15个细菌种属,分别为芽胞杆菌(Bacillus),假单胞菌(Pseudomonas),黄杆菌(Flavobacterium),红球菌(Rhodococcus),北里孢菌(Kitasatosporia),金黄杆菌(Chryseobacterium),不动杆菌(Acinetobacter),鞘氨醇杆菌(Sphingobacterium),丛毛单胞菌(Comamonas),伯克霍尔德氏菌(Burkholderia),链霉菌(Streptomyces),沙雷氏菌(Serratia),肠杆菌(Enterobacter),窄食单胞菌(Stenotrophomonas)和节杆菌(Arthrobacter)。     

渤海沉积物产脂肪酶细菌的筛选及其多样性分析

【目的】从渤海沉积物中分离筛选产脂肪酶细菌,分析其物种多样性,增加人们对渤海生态系统中产脂肪酶菌多样性的认识,获取高效产脂肪酶菌株,为海洋产脂肪酶微生物的挖掘提供菌群资源。【方法】分别将8个渤海沉积物样品梯度稀释涂布至吐温-80筛选平板和三丁酸甘油酯筛选平板,选择性分离产脂肪酶细菌;分析基于16SrRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;利用对硝基苯酚法测定胞外脂肪酶活性,筛选出高效产脂肪酶菌株。【结果】从8个渤海沉积物样品中分离获得51株产脂肪酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria和Firmicutes三个门的8个属,其中Pseudoalteromonas(35.2%)、Marinobacter(23.5%)和Sulfitobacter(17.6%)是优势菌群;脂肪酶酶活性实验表明所有测定菌株都能够分泌脂肪酶,菌株70623分泌的脂肪酶酶活最高,为42.4 U/m L。【结论】渤海沉积物中可培养产脂肪酶细菌类群较为丰富,Pseudoalteromonas、Marinobacter和Sulfitobacter菌株是优势菌群,测定菌株所产胞外脂肪酶能力不同,获得了一株高效产脂肪酶菌株Marinobacter sp.70623。     

文冠果一、二年生植株根系内生菌的分离、鉴定和固氮活性

该文对文冠果(Xanthoceras sorbifolia)根系内生菌的种类和固氮活性进行了首次报道。试验以田间栽培的文冠果一、二年生植株为材料, 以Ashby和YMA为培养基, 对根系内生菌进行了分离, 通过对菌落形态的观察, 划线挑取单菌落培养, 并对7个代表性的单菌落进行扩大培养, 提取其DNA, 采用细菌16S rDNA通用引物序列进行PCR扩增, 胶回收、测序后, 进行Blast比对分析。试验结果表明: YMA培养基上的菌落数量和种类均明显多于Ashby培养基上的菌落。田间一年生文冠果与田间二年生文冠果的根系内生菌情况存在差异。一年生文冠果的根系内生菌数量与种类略多于二年生文冠果。一年生文冠果根系中分离得到的主要内生菌为成团泛菌(Pantoea agglomerans)、根癌土壤杆菌(Agrobacterium tumefaciens)和产酸克雷伯菌(Klebsiella oxytoca)等; 田间二年生文冠果根系中分离得到的主要内生菌为假单胞菌属(Pseudomonas)细菌。固氮活性测定结果表明, 在所分离的7个菌株中, 6个测到了固氮酶活性, 其中鉴定为产酸克雷伯菌的菌株固氮酶活性显著高于其他菌株, 达到9.688 nmol·mg^-1·h^-1, 为文冠果固氮菌肥的菌种的筛选奠定了基础。     

一株高产洛伐他汀红曲霉的筛选与液态发酵条件优化

【背景】洛伐他汀(lovastatin)是红曲霉的次生代谢产物,是重要的临床用降血脂药物。在液态发酵条件下,红曲霉的洛伐他汀产量较低,难以满足工业化生产的要求。【目的】筛选获得一株高产洛伐他汀的红曲霉株,并通过优化液态发酵条件提高洛伐他汀的产量。【方法】从红曲米中筛选获得一株高产洛伐他汀的红曲霉株,依据形态学特征、生理生化特性及18S rRNA基因序列分析对分离菌株进行鉴定;通过响应面法对其产洛伐他汀的液态发酵条件进行优化。【结果】获得一株产洛伐他汀的紫红曲霉(Monascus purpureus M4),该菌在甘油57.80g/L、酵母浸粉5.52 g/L、接种量为6.90%条件下,洛伐他汀产量(173.60 mg/L)较优化前提高了4.8倍。【结论】菌株M4产洛伐他汀最优液态发酵条件的建立,为洛伐他汀的大规模生产及该菌株的工业化应用提供了技术支撑。     

一株高产Monacolin K紫红曲霉菌株筛选、鉴定及发酵条件优化

为从天然发酵红曲米中分离的30株红曲霉菌株中筛选高产MonacolinK的菌株,并对其产MonacolinK的发酵条件进行优化。实验采用高效液相色谱法(HPLC)筛选到9株具有产MonacolinK能力的红曲霉菌株,其中以编号ZX26的菌株产MonacolinK能力最高,发酵液中Monacolin K产量达到107.6mg/L,并且产MonacolinK能力具有良好的稳定性。微生物形态学结合ITS基因同源性分析结果表明,编号ZX26菌株为紫红曲霉。进一步采用单因素试验和正交试验法优化紫红曲霉ZX26产MonacolinK的发酵条件,结果表明在培养基组分为葡萄糖70g/L,牛肉膏15g/L,NaNO32g/L,MgSO4·7H2O0.5g/L,KH2PO41.5g/L时,其最优发酵条件为:发酵初始pH4.0,接种量为7%,培养温度30℃,发酵10天,在此条件下,紫红曲霉ZX26发酵液中MonacolinK产量达到271.36mg/L,相对于培养条件优化前MonacolinK产量提高152.19%,经验证此培养条件下MonacolinK产量最佳。     

枯草芽孢杆菌B47菌株高产抗菌物质的培养基及发酵条件优化

枯草芽孢杆菌Bacillus subtilis B47菌株为番茄内生细菌, 也是玉米小斑病拮抗菌, 能产生对玉米小斑病菌有强烈抑制作用的抗菌物质。以B47菌株发酵液的无菌滤液对玉米小斑病菌的抗菌活性为检测指标, 测定B47菌株产抗菌物质培养所需的最佳碳、氮源和无机盐, 并通过正交试验法对该菌株产抗菌物质的培养基配方和摇瓶发酵条件进行优化。研究结果表明, B47菌株产抗菌物质最佳碳、氮源和无机盐分别为蔗糖、酵母浸膏和MgSO4·7H2O, 最优培养基是YSB (Yeast extract-sucrose-beef extract)培养基, 其配方为: 蔗糖2%, 酵母浸膏2%, 牛肉浸膏1.5%, MgSO4?7H2O 0.06%, FeSO4·7H2O 0.000 9%, 最优发酵条件组合为: 30 °C, pH 7.0, 170 r/min摇床培养6 d, 接种量为1%, 装液量为40 mL/200 mL。     

B．animalis V9对腹泻动物的保护性作用及其机制研究

目的研究益生菌B.animalis V9对腹泻动物的保护作用及其对腹泻致病菌的拮抗效果。方法采用自制的腹泻SPF鼠,喂服B．animalis V9,观察并检测腹泻致病菌,并在体外试验益生菌B．animalisV9对腹泻致病菌的拮抗作用。结果SPF鼠致泻动物模型喂食B．animalisV9,剂量为1．0×10^8CFU/（只·d）,3d后腹泻治愈率为90％,死亡率为9％,对照组自然恢复率为16％,死亡率为41％,其余仍有不同程度的腹泻现象。B．animalis V9在体外对志贺痢疾杆菌、沙门菌、铜绿假单胞菌和大肠埃希菌有不同程度的拮抗作用,共同培养60h后均未检测到上述4种致病菌。结论B．animalisV9可以有效的对腹泻小鼠进行治疗,其机制在于B．animalisV9对致泻菌株具有一定程度的拮抗作用。     

The effect of fumonisin B1 on the growth of bacteria

Fumonisins are mycotoxins produced by several Fusarium species which are common contaminants of maize and maize products. Their toxicity and carcinogenicity for animals ingesting these toxins have been clearly demonstrated, but, there are no studies on the effect of fumonisins on bacteria. In this study various Gram-positive and Gram-negative bacteria including typical representatives of the human intestinal flora were grown in the presence of 50 to 1000 M fumonisin B1 and the fumonisin concentrations in the culture medium were determined after incubation for a certain period. No inhibition of bacterial growth was observed, indicating that this mycotoxin is non-toxic for the bacteria investigated. There was no indication that fumonisin B1 was metabolized by the bacteria as its concentration in the culture medium did not decrease during the incubation period.     

影响多粘菌素B对绿脓杆菌杀菌效力的主要环境因素的研究

本文通过改变温度,水活度,气体条件和营养含量等影响绿脓杆菌生长的主要环境因素,测定多粘菌素B对绿脓杆菌的最小杀菌浓度(MBC)。结果表明环境因素导致或显著影响绿脓杆菌对抗生素的生态耐受性。实验表明多粘菌素B对绿脓杆菌的杀菌效力,除药物对细菌特有的药理学作用外,还取决于细菌的生长环境。结合冷休克率试验表明,环境影响细菌群体处于分裂状态的菌数。若分裂状态菌数下降表明生长速度减慢。提示了多粘菌素B对绿脓杆菌的效力指数,定量分析可以作为其综合效力作用的表现。以同步培养法确定在单个细胞周期中的抗生素敏感阶段。同时以冷休克率试验资料证明细菌处于分裂状态和幼龄期是其敏感阶段。初步阐述了生长速度缓慢与药物的生态耐受性密切相关。     

B. thuringiensis is a Poor Colonist of Leaf Surfaces

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10⁶ cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10⁷ cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.     

B-animalisV9质粒检测及抗生素敏感性试验

本实验对动物双歧杆菌Vg（B．animalisvg）菌株进行质粒检测及抗生素敏感性测试。结果表明,Baninalis V9菌株无质粒检出。该菌株对针对G＋菌的抗生素,如青霉素类（青霉素G、氨苄青霉素）和大环内酯类（氯霉素、红霉素）表现出高度敏感;对抑制细菌蛋白质合成的广谱（G＋、G-）抗生素（四环素、利福平、痢特灵）表现出中度敏感;对G-菌的抗生素,如氨基糖苷类（庆大霉素、链霉素）、喹诺酮类（诺氟沙星）及内酰胺类（卡那霉素、丁胺卡那霉素）表现出耐药性;而对广谱抗生素磺胺甲基异嗯唑也表现为耐药。     

Effect of antiserum against aflatoxin B1-bovine serum albumin complex on aflatoxin B1-induced lysogenesis

Escherichia coli K12 bacteria lysogenic for the lambda phage were used to study the effect of antiserum against aflatoxin B1-induced lysogenesis. The antiserum was obtained from rabbits immunized with water in oil emulsion of aflatoxin B1-bovine serum albumin complex (AFB1-BSA). A marked reduction in the degree of lysogenesis was observed when the antiserum was added to the reaction medium prior to microsomal enzyme activation of aflatoxin B1. There was no detectable effect when the antiserum was added after aflatoxin B1 activation. The result presented suggests that the antibodies in the AFB1-BSA antiserum can interact with aflatoxin B1 prior to its activation. This implies that an immune-protective effect can only be exerted if the antibodies intervene before activation.     

Pseudomonas pseudoalcaligenes B50铁载体合成相关基因pyrD的克隆与功能分析

从棉花根际分离的一株细菌B50在低铁条件下可以产生铁载体。通过形态学特征、生理生化特征及其16SrDNA序列分析,将其鉴定为Pseudomonas pseudoalcaligenes。采用三亲本杂交的方法将转座子Tn5-1063a(含luxAB)导入B50中,进行转座子插入诱变,获得突变株。用TAIL-PCR方法得到与铁吸收有关的pyrD基因序列。通过互补实验验证pyrD与铁载体的合成有关。Pseudomonas pseudoalcaligenes能够产生铁载体属于首次报道。     

Formation of Bacteriochlorophyll Form B820 in Light Harvesting 2 Complexes from Purple Sulfur Bacteria Treated with Dioxane

Treatment of some sulfur bacteria (Allochromatium minutissimum, Thiorhodospira sibirica, and Ectothiorhodospira halovacuolata WN22) with dioxane results in formation of the bacteriochlorophyll form B820 in the light harvesting complex LH2. This form characterized by absorption maximum at 820 nm has the same absorption spectrum as B820 subcomplex from LH1 complex. Appearance of the B820 form was accompanied by a sharp decrease in absorption in the carotenoid region. This phenomenon observed in all LH2 complexes investigated may be attributed to formation of colorless carotenoid aggregates. This is very similar to the previously reported dissociation of the LH1 complex with carotenoids into B820 subcomplexes. Although the B820 form corresponded the bacteriochlorophyll dimer, its circular dichroism spectrum showed that pigment molecules in this dimer exhibit different interaction than those in the B820 subcomplex. The dioxane treatment of LH2 complexes isolated from Rhodopseudomonas palustris bacteria grown under normal or low intensity illumination did not result in formation of such dimers. It is suggested that bacteriochlorophyll B820 formation is related to unique structure of LH2 complexes from the sulfur bacteria.     

Characterization and purification of acidocin 1B, a bacteriocin produced by Lactobacillus acidophilus GP1B

In the present study, acidocin 1B, a bacteriocin produced by Lactobacillus acidophilus GP1B, exhibited profound inhibitory activity against a variety of LAB and pathogens, including Gram-negative bacteria, and its mode of action was to destabilize the cell wall, thereby resulting in bactericidal lysis. Acidocin 1B was found to be heat stable, because it lost no activity when it was heated up to 95 degrees C for 60 min. It retained approximately 67% of the initial activity after storage for 30 days at 4 degrees C, and 50% of its initial activity after 30 days at 25 degrees C and 37 degrees C. The molecular mass of acidocin 1B was estimated to be 4214.65 Da by mass spectrometry. Plasmid curing results indicated that a plasmid, designated as pLA1B, seemed to be responsible for both acidocin 1B production and host immunity, and that the pLA1B could be transformed into competent cells of L. acidophilus ATCC 43121 by electroporation. Our findings indicate that the acidocin 1B and its producer strain may have potential value as a biopreservative in food systems.     

内生细菌B10对稻瘟病菌抑制作用的初步研究

目的:研究内生细菌B10对水稻稻瘟病菌的抑制作用,为菌株的应用提供理论依据。方法:采用菌丝生长速率法、孢子萌发法和田间试验测定内生细菌发酵上清液和菌液对稻瘟病菌的抑制作用。结果:内生细菌B10发酵上清液对稻瘟病菌菌丝生长和孢子萌发有较强的抑制作用,100倍稀释液对菌丝生长的抑制率为79.37%,对孢子萌发的抑制率为63.42%,对稻瘟病的田间防治效果达70.2%以上。结论:内生细菌B10对稻瘟病有较强的抑菌作用。