产脂肽生物表面活性剂细菌的筛选及培养基优化

利用变色圈法和噬油斑法从土壤样品中分离筛选出一株产生物表面活性剂的细菌菌株LB345,通过薄层层析(TLC)确定其所产生物表面活性剂为脂肽,并通过单因素实验及L9(34)正交实验对菌株的培养基配方进行了优化,确定优化培养基的组成(g/L):葡萄糖30,NaNO32,KH2PO410,Na2HPO410,FeSO42.5×10-4,MgSO4.7H2O 0.03,NaC l 5,CaC l20.4,酵母粉0.3,初始pH值5,培养温度37℃,发酵时间7 d。在上述条件下,菌株LB345的脂肽产量可达到0.7 g/L。     

脂肽生物表面活性剂摇瓶发酵条件的研究

目的:对选育出的脂肽生物表面活性剂高产菌株SD59的摇瓶发酵条件进行了研究。方法:通过正交实验和单因素实验等方法研究了各种营养和环境因素对菌株SD59发酵生产脂肽生物表面活性剂的影响。结果:优化的培养基组成(g/L):可溶性淀粉20.0,硫酸铵6.0,硫酸镁0.3,氯化钙0.08,酵母粉0.6,磷酸二氢钾2.0,磷酸氢二钠8.0,初始pH7.2;最佳培养条件为500mL三角瓶的装液量为200mL,接种量5.0%,培养温度36～38℃。结论:在此优化发酵条件下,脂肽表面活性剂的产量可达到1.2g/L。     

Exploration of Staphylococcus nepalensis (KY024500) Biosurfactant towards Microbial Enhanced Oil Recovery

Oleochemicals have long been used as biolubricants, biopolymers, and biosurfactants; an effective alternative to petroleum-based products. The present study explores the biosurfactant potential of a novel strain, isolated from rocks of earthquake-prone area. On the basis of morphological, biochemical and 16S rRNA sequencing analysis, the isolate was identified as Staphylococcus nepalensis (KY024500). A biosurfactant yield 2.39, 1.39, and 0.9 g L⁻¹ was obtained using glycerol, waste orange peel, and diesel as a sole carbon source, respectively. Based on oil recovery experimental findings through sand pack column, the obtained biosurfactant from waste orange peels as a sole carbon source was carried forward for further analysis. Thus, obtained biosurfactant from waste orange peels were subjected to solvent extraction and purified by column chromatography. The purified biosurfactant thus obtained was characterized with the help of fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), gas chromatography-mass spectroscopy (GC–MS), and MALDI TOF MS/mass spectroscopy (MS) analysis. FTIR spectroscopic analysis revealed the presence of a carbonyl, amine, hydroxyl, and methyl as functional groups. The GC–MS analysis showed the presence of benzene dicarboxylic acid diethyl ester and pthalic acid as fatty acids while MALDI TOF MS/MS analysis shows lysin-glycin as a hydrophilic dipeptide moiety. This study also demonstrates Microbial Enhanced Oil Recovery (MEOR) potential of the biosurfactant as more efficient than commercial ones. The biosurfactant obtained from waste orange peel as carbon source was able to facilitate a 20% higher recovery of diesel from sand pack recovery column.     

Production and Characterization of Surface-Active Lipopeptides by Haloalkaliphilic Bacteria Salibacterium sp. 4CTb

Surface-Active Biomolecules (SAB) obtained from microbial sources are safe alternatives to chemically synthesized counterparts for many environmental and industrial applications. These applications frequently involve the exposure of the SAB to extreme factors making necessary to seek for molecules that are able to function under such conditions. In this work, the polyextremophilic bacterium Salibacterium sp. 4CTb is reported as a SAB producer. Its emulsifier activity increased almost twofold when the C/N ratio and culture conditions were modified. The partially purified molecule was able to form stable emulsions under extreme conditions (70 °C, pH 12, and 4 M NaCl) better than other commercial molecules, and reduced the surface tension of the water to 49.78 mN m⁻¹ with critical micelle concentration (CMC) of 15.1 mg L⁻¹. Carbohydrate, lipid, and protein assays, accompanied by the Fourier Transform Infra-Red (FTIR) and electrospray ionization mass spectroscopy (ESI-MS) results, indicate the lipopeptide nature of the compound with masses around 645 and 746 Da. The SAB obtained from Salibacterium sp. 4CTb may be suitable for electrical, food, pharmaceutical and cosmetic industries, agriculture, and environmental applications, among others.     

一种新型脂肽的分离纯化及其生物学活性

目的从表皮葡萄球菌发酵液中分离纯化脂肽粗提物,并分析其生物学活性。方法通过酸沉淀分离和有机溶剂抽提的方法,从表皮葡萄球菌1457发酵液中得到脂肽粗提物。利用排油圈试验和薄层色谱(TLC)分析脂肽粗提物的性质,HPLC分析其纯度。小鼠皮下注射脂肽粗提物后,再经皮肤感染金黄葡萄球菌,检测其对小鼠体内白细胞的影响及抑菌作用;实时定量PCR检测其对小鼠体内β防御素14(mBD14)表达的影响。结果所得脂肽粗提物F2的排油活性大于F1;TLC分析显示,F2中存在性质相近的脂肽类同系物,纯度>60%;脂肽粗提物能减缓小鼠因感染金黄葡萄球菌引起的急性炎症,降低由感染引起的白细胞增多,并通过上调mBD14的表达抑制细菌在皮肤内的增殖。结论已从表皮葡萄球菌发酵液中成功获得了具有生物学活性的新型脂肽粗提物。     

Biosurfactant from <Emphasis Type="Italic">Lysinibacillus chungkukjangi</Emphasis> from Rice Bran Oil Sludge and Potential Applications

A newly discovered bacterium, Lysinibacillus chungkukjangi, was isolated from the sludge of rice bran oil processing. When the bacterium was grown on rice bran, it produced biosurfactant which reduced the surface tension of the media to 27.9 from 72 mN/m. The biosurfactant was recovered by a solvent extraction method and characterized with the help of various structure elucidation techniques viz. FTIR, ¹H- and ¹³C-NMR spectroscopy and LC–MS analysis. The combined results of FTIR and NMR revealed the presence of carbonyl, olefinic and aliphatic groups, with the typical spectra of lipids. Moreover, LC–MS analysis also supported the same information. The biosurfactant was also studied for its anti-oxidant and microbial enhanced oil recovery (MEOR) potential. The anti-oxidant activity was observed by the DPPH free radical scavenging method using ascorbic acid as the standard. The IC₅₀ (the half maximal inhibitory concentration) was calculated and for standard, it was 0.056 mg/mL and for the biosurfactant it turned out to be 1.3 mg/mL which shows its good anti-oxidant potential. The sandpack test was performed to check its MEOR potential and kerosene was recovered up to 90 %, which shows its excellent applicability in the MEOR processes.     

一种脂肽类生物表面活性剂的理化性质及其对原油的作用

从大港炼油厂污水中筛选到一株地衣芽孢杆菌NK X3,在含糖培养基中培养可产生一种脂肽类生物表面活性剂 ,该生物表面活性剂在 pH 4— 1 2的范围内和 40 0 0mg/L的高钙离子浓度及 1 5%的高盐浓度下仍维持原有表面活性。更为显著的特点是在 1 2 0℃的高温下不失活。该产物可将水的表面张力由 76.6降至 35.5mN/m。其乳化活性值为 1 .50 ,临界胶束浓度 (CMC值 )为 30 .0mg/L。对高含胶质沥青质油的降粘率高达 50 %以上 ,增溶与脱附作用显著 ,可使油水互溶而形成水包油型乳化小滴。可使高含蜡油有效地乳化分散。这些特点有利于原油的增采和输送。     

油菜内生菌BY-2的脂肽类抑菌物质的分子结构鉴定

对油菜内生枯草芽孢杆菌BY-2（Bacillus subtilis）的细胞外分泌型抗菌性纯化物进行双缩脲反应、茚三酮反应、高效液相色谱仪（HPLC）分离、质谱分析和异硫氰酸苯酯（PITC）柱前衍生HPLC分析。结果显示：BY-2的胞外分泌型抑菌物质含肽键,具有环状分子结构。在甲醇/水的流动相条件下,1.969min出现1个抑菌成分峰值;在三氟乙酸/甲醇流动相条件下,抑菌成分形成了6个峰值。组成分析结果表明：该抑菌物质由分子量分别为995Da、1009Da、1023Da、1037Da、1051Da和1087Da六种成分构成,其4种氨基酸比例为：天冬氨酸∶谷氨酸∶缬氨酸∶亮氨酸=1∶1∶1∶1.5。该抑菌物质可归类为环脂肽类抗生素（Cycle lipopeptide）中的枯草菌表面活性素（Surfactin）,其中的1087Da大分子同系物尚未见报道。