培养基中铁离子对枯草芽孢杆菌CICC 23659发酵产脂肽的影响研究

高性能的生物表面活性剂脂肽在医药、食品、化妆品及微生物采油等领域具有广泛的应用价值.利用高效液相色谱法和电喷雾质谱法分析了Bacillus subtilis CICC 23659所产脂肽的组成,结果表明其所产脂肽是由surfactin的同系物C13、C14和C15组成,其分子量分别为1008、1022和1036.以生物量、脂肽产量、脂肽组成、乳化能力和临界胶束浓度等指标考察在培养基中投加不同浓度的Fe2+对B.subtilis CICC 23659的影响,结果表明添加Fe2+不仅能够提高B.subtilis CICC 23659的生物量,还能大幅提高脂肽产量.当Fe2+添加浓度为5 mmol/L时,生物量达到最大3.69g/L,提高了6倍,脂肽产量也达到最大234.08 mg/L,提高了9.5倍.Fe2+作用下脂肽同系物中C13、C14、C15在脂肽产物中的相对含量发生了变化,脂肽同系物C13和C15的相对含量降低,脂肽同系物C14的相对含量上升.脂肽同系物中C13的相对含量越高,CMC值越大,脂肽同系物中C14和C15的相对含量越高,CMC值则越小.     

枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定

对一株枯草芽孢杆菌HS-A38产生的脂肽类物质进行分离鉴定及抑菌活性研究。通过酸沉淀分离和有机溶剂抽提的方法,从枯草芽孢杆菌HS-A38发酵液中得到脂肽粗提物LP,产率为1.956 g/L。利用薄层色谱和茚三酮染色法确定该脂肽粗提物中存在四个组分,分别为LP1、LP2、LP3和LP4;抑菌活性检测显示,组分LP3对两株海洋致病菌副溶血性弧菌和铜绿假单胞杆菌的活性较高。组分LP3经硅胶柱层析纯化分离后,应用反相高效液相色谱(RP-HPLC)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对该组分做进一步纯化和鉴定。分析表明,LP3样品在保留时间20 min~28 min产生单峰团LP3-1,其纯度为85.24%;经MALDI-TOF-MS分析和数据比对,组分LP3-1中的主要成分为杆菌霉素Bacillomycin D。     

一种快速分离检测产脂肽类生物表面活性剂枯草芽孢杆菌的方法

脂肽(Lipopeptide)是由枯草芽孢杆菌(Bacillus subtilis)等微生物产生的一类具有较强表面活性的生物表面活性剂.枯革杆菌磷酸泛酰巯基转移酶基因(afp)是枯草芽孢杆菌中参与脂肽代谢的功能性基因.采用sfp基因PCR对从环境中得到的一组产生表面活性剂的微生物进行筛选,结合Tricine-SDS-PAGE电泳对PCR结果呈阳性的菌蛛的代谢粗初提物进行检测,初步鉴定得到两株枯草芽孢杆菌.进一步利用16S rDNA序列的系统发育学分析确定这两种菌株为枯草芽孢杆菌,并利用TLC、HPLC鉴定其产物为脂肽类表面活性剂,从而建立了一套快速分离检测产生脂肽类生物表面活性剂的枯草芽孢杆菌方法.     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性。将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤, 分离得到脂肽类抗生素的纯品。经IC50实验和抗菌谱测定, 考察了脂肽类抗生素对多种植物病原菌的作用, 确定了脂肽类抗生素的抗菌谱。深入研究表明, 枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质, 能够抑制灰霉病菌孢子的萌发和菌丝的生长。脂肽类抗生素和挥发性抑菌物质的协同作用, 有助于提高枯草芽孢杆菌的生物防治效果。     

枯草芽孢杆菌JA产生的脂肽类抗生素－iturin A的纯化 及电喷雾质谱鉴定

枯草芽孢杆菌JA产生的抗生素对植物病原真菌具有广谱抗性,明确抗生素的种类是进一步研究的基础.用6mol/L盐酸沉淀JA菌株的去菌体培养基,再用甲醇抽提获得抗生素的粗提物.利用反相HPLC系统,将粗提物过Diamonsil C18柱,收集有抗小麦赤霉病等病原真菌活性的化合物1、2.运用电喷雾质谱法(ESI/MS)测得其分子量分别为1042.4D和1056.5D.再利用碰撞诱导解离(CID)技术获得化合物的典型结构特征离子碎片,结果表明分子量为1042.4D的化合物一级结构为Pro-Asn-Tyr-βAA-Asn-Tyr-Asn-Gln(βAA为14个碳原子的氨基脂肪酸),属于脂iturin A.化合物1、2为相差一个亚甲基(-CH2)的iturin A同系物.研究结果提供了一种从枯草芽孢杆菌发酵液中快速分离纯化和鉴定脂肽类抗生素iturin A的新方法.     

一株枯草芽孢杆菌的分离鉴定、生物学特性及其对水质净化的作用

[背景]随着水产养殖业的发展和养殖集约化程度的提高,养殖水环境日趋恶化,养殖动物病害频发,而水产益生菌因其环境友好、安全而被广泛应用于水产养殖中.[目的]从南美白对虾养殖池底泥分离枯草芽孢杆菌,探究其体外生物学特性及对水质的净化作用,以期扩充微生态制剂的种质资源.[方法]采用稀释涂布平板法分离菌株,通过形态学观察、生理...     

枯草芽孢杆菌BI1产抗真菌脂肽培养条件的优化

采用Plackett—Burman设计对枯草芽孢杆茵BI1产抗菌脂肽培养条件中相关影响因素(即种龄、接种量和pH值)的效应进行评价并筛选出重要的影响因素,然后用Box-Behnken设计及响应面分析确定了重要影响因素的最佳条件,优化后的溶血透明圈直径达到17.5 mm,比优化前的13.75 mm提高了27.27%。     

一种脂肽类生物表面活性剂产生菌的筛选

从油田地层水中筛选分离得到1株能够产生表面活性剂的细菌,经鉴定为枯草芽孢杆菌。分析了该菌株的生理形态和生长特性,以及该菌株代谢产生的生物表面活性剂的性质。薄层色谱与原位水解显色和红外光谱分析表明,培养后菌株代谢产生的生物表面活性为脂肽。它能使水的表面张力降低到26mN/m,其临界胶束浓度为0.025mg/mL。     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性.将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤,分离得到脂肽类抗生素的纯品.经IC50实验和抗菌谱测定,考察了脂肽类抗生素对多种植物病原菌的作用,确定了脂肽类抗生素的抗菌谱.深入研究表明,枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质,能够抑制灰霉病菌孢子的萌发和菌丝的生长.脂肽类抗生素和挥发性抑菌物质的协同作用,有助于提高枯草芽孢杆菌的生物防治效果.     

一株种内拮抗的海洋枯草芽孢杆菌的分离与鉴定

从中国东海海域筛选到好氧耐盐菌株A01,此菌株显示出抗枯草芽孢杆菌和白色念珠菌的活性采取对该菌株形态特征、培养特征、生理生化特征和遗传特性进行研究的方法,结果表明此菌株与枯草芽孢仟菌（Bacillus subitilis）的特征一致;将此菌株的16S rDNA序列在GenBank中进行序列比对,结果亦显示其与Bacillus subitilis的16SrDNA的序列片段的相似性为100％;以相似性为基础构建系统发育树,分析表明该菌株与Bacillus subitilis同源关系最近最终得出结论为菌株A01是一株来源于海洋的枯草芽孢杆菌,且具有种内拮抗的特性。     

产表面活性素和伊枯草菌素A菌株的筛选及其脂肽类产物的特性

采用血琼脂平板法, 从菜园土壤中分离到8株代谢表面活性剂的菌株, 比较各菌株的排油性、抑菌性, 根据合成脂肽类物质表面活性素(Surfactin)和伊枯草菌素A (Iturin A)必需的sfp、ituD和lpa-14基因设计引物, 结合PCR的方法筛选到一株具广谱抗菌性且含有sfp、ituD和lpa-14 3个关键基因的细菌XZ-173。经过生理生化试验测定和16S rDNA序列系统发育学分析, 将其鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。通过红外光谱(FT-IR)分析该菌株代谢产物, 初步鉴定为脂肽类物质, 并对照高效液相色谱(HPLC)与标准品比对结果, 确定含有Surfactin和Iturin A组分。该菌株产生的脂肽粗品能使纯水的表面张力降低至26.6 mN/m, 临界胶束浓度(CMC)为500 mg/L, 具有很好的乳化性能, 对立枯丝核菌和青枯菌表现出很好的拮抗活性。因此, 产脂肽细菌XZ-173是一株应用前景广阔的功能菌。     

分解玉米赤霉烯酮菌株的分离、鉴定及其产酶特征

【目的】从发酵食品材料中筛选出对玉米赤霉烯酮(ZEN)有分解作用的微生物,研究其分解效率及产酶特征并进行菌种鉴定。【方法】利用添加ZEN毒素类似物(PL)的固体培养基对25种发酵食品材料进行初筛,获得毒素类似物耐受菌株,经过用ZEN复筛,得到高效分解ZEN的细菌。用高效液相色谱法(HPLC)分析培养物残留ZEN,评价菌株对ZEN的分解效率。初步分析该菌株产纤维素酶、木聚糖酶及β-葡萄糖苷酶的特性。通过微生物形态学、分子生物学方法进行菌种鉴定,确定该菌的系统分类学地位。【结果】从发酵食品材料中筛选出一株分解ZEN的菌株BF-B-3,经初步鉴定该菌株为枯草芽孢杆菌(Bacillus subtilis)。其ZEN分解率达62.48%,测定该菌产纤维素酶、木聚糖酶及β-葡萄糖苷酶活力分别为160.38、84.51和4.14 U/mL。【结论】枯草芽孢杆菌属于饲用微生物,生物安全性高,所分离到的枯草芽孢杆菌疑似株(Bacillus subtilis)BF-B-3菌株,可作为具有分解ZEN功能的益生菌使用,具有较好的应用前景。     

The effect of Bacillus subtilis producing Surfactin in ROS production and transformation efficiency of tobacco cells

Surfactin secreted by bacilli has biological functions in plant. Surfactin C14 and C15 have the highest effect on inducing hydrogen peroxide species release in the plant. Surfactin production in the two Bacillus strains ACCT21332 and FKR3 were analysed by HPLC and the phytotoxicity of the Bacilli-derived surfactins was determined in Tobacco cell culture. Surfactin C14 and C15 were detected in ACCT21332 but not in FKR3 strain. Extracellular hydrogen peroxide produced by tobacco cell culture cells exposed to ATCC21332 and FKR3 strains increased compared to untreated ones. The Agrobacterium mediated transformation rate of tobacco cells drops from 4% transformed cells to 0.8 and 1.2% when pretreated with ATCC21332 or FKR3 strain, respectively. The strong drop in transformation rate of plant cell culture after FKR3 strain pre-treatment indicates that Surfactin C14 and C15 are not the major or the only cause in protecting plant cells from Agrobacterial infection and transformation.     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997     

Effect of different Bacillus subtilis lipopeptides on surface hydrophobicity and adhesion of Bacillus cereus 98/4 spores to stainless steel and Teflon

Various lipopeptides produced by Bacillus subtilis were examined for their ability to modify the surface hydrophobicity of two substrata, stainless steel (SS) and Teflon. These modifications were evaluated by water contact angle measurements. The effects depended on the lipopeptide, its concentration, and the tested substratum. Treatment of SS with different concentrations of surfactin S1 showed an increase of the hydrophobicity between 1 and 100 mg l^?1. On the same substratum, fengycin increased hydrophobicity up to its critical micelle concentration (6.25 mg l^?1). With higher concentrations of fengycin, hydrophobicity decreased. Surfactin, mycosubtilin, and iturin A decreased hydrophobicity on Teflon. The different effects of these three families of lipopeptides were related to their structural differences. A good correlation was shown between hydrophobicity modifications of surfaces and the attachment of B. cereus 98/4 spores. Enhancement in the hydrophobicity of the surfaces increased the number of adhering spores.     

Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis

Aims:  Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics.
Methods and Results:  The antagonistic activity of JA was tested in vitro ; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l⁻¹ HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C₁₈ column (5  μ m, 250 × 4·6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization–mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments.
Conclusions:  Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens.
Significance and Impact of the Study:  This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis .     

Effects of Bacillus subtilis O9 biosurfactant on the bioremediation of crude oil-polluted soils

The application of a surfactant from Bacillus subtilis O9 (Bs) on the bioremediation of soils polluted with crude oil was assayed in soil microcosms under laboratory conditions. Three concentrations of biosurfactant were assayed (1.9, 19.5, and 39 mg kg(-1) soil). Microcosms without biosurfactant were prepared as controls. During the experiment, the crude oil-degrading bacterial population, the aliphatic and aromatic hydrocarbons were monitored in each microcosm. The results indicated that applying Bs did not negatively affect the hydrocarbon-degrading microbial population Concentrations of 19 and 19.5mg (Bs) per kilogram of soil stimulated the growth of the population involved in the crude oil degradation, and accelerated the biodegradation of the aliphatic hydrocarbons. However, none of the assayed Bs concentrations stimulated aromatic hydrocarbon degradation.