Production of surface-active sophorolipid biosurfactant and crude oil degradability by novel Rhodotorula mucilaginosa strain SKF2

Biosurfactants are produced by important types of microorganisms such as bacteria, yeast, and filamentous fungi and have been used in a variety of industries. Among the 15 crude oil-degrading fungi, the two molds and one yeast were identified by 18S rDNA sequences as Mucor circinelloides strain SKMC, Fusarium fujikuroi strain DB2, and Rhodotorula mucilaginosa strain SKF2. These strains were isolated from crude oil–contaminated soil, diesel oil–contaminated soil, and activated sludge in the Oil Refinery Plant in Isfahan, Iran, respectively. The yeast strain was identified as a novel crude oil–degrading and biosurfactant-producing fungi in the presence of (1% v/v) Iranian light crude oil in the minimal salt medium (MSM). The highest amount of the dry weight of produced biosurfactant was measured at 6.2 g L⁻¹. Chemical nature of produced biosurfactant was determined as a surface-active sophorolipid biosurfactant compound by thin-layer chromatography, Fourier transform infra-red spectroscopy, and gas chromatography–mass spectrometry (GC–MS) analysis. The residual hydrocarbons in the MSM were analyzed by GC–MS, and it was shown that octadecane and docosane were eliminated by this novel strain completely.     

7.5%多杀霉素悬浮剂的制备与分析

采用湿法研磨工艺,单因素优选法结合正交试验优化7.5%多杀霉素悬浮剂的制备条件,并采用高效液相色谱-质谱联用(HPLC-MS)法对热贮14 d产品进行质量检测。结果表明:以槐糖脂水溶液(200 mg/L)为分散介质,悬浮剂最优制备条件为Well-301和Well-303(质量比2∶1)用量6.0%,黄原胶用量0.25%,尿素用量4.5%,研磨时间2.0 h。热贮后有效成分组成和质量分数均保持不变,符合悬浮剂的要求。HPLC-MS法适用于多杀霉素类多组分混合物制剂的质量检测。     

Cloning and functional characterization of the UDP-glucosyltransferase UgtB1 involved in sophorolipid production by Candida bombicola and creation of a glucolipid-producing yeast strain

Sophorolipids produced by the non-pathogenic yeast Candida bombicola ATCC 22214 are glycolipid biosurfactants applied commercially as biodegradable and eco-friendly detergents. Their low cell toxicity, excellent wetting capability and antimicrobial activity attract the attention of high-value markets, such as the cosmetic and pharmaceutical industries. Although sophorolipid production yields have been increased by the optimization of fermentation parameters and feed sources, the biosynthetic pathway and genetic mechanism behind sophorolipid production still remains unclear. Here we identify a UDP-glucosyltransferase gene, UGTB1, with a key function in this economically important pathway. The protein shows sequence and structural homology to several bacterial glycosyltransferases involved in macrolide antibiotic synthesis. Deletion of UGTB1 in C. bombicola did not affect cell growth and resulted in a yeast producing glucolipids, thereby opening the route for in vivo production of these glycolipid intermediates. Activity assays on cell lysates confirmed that the identified gene is responsible for the second glucosylation step during sophorolipid production and illustrated that sophorolipid production in C. bombicola involves the stepwise action of two independent glucosyltransferases. The complete UGTB1 sequence data have been submitted to the GenBank database (http://www.ncbi.nlm.nih.gov) under Accession No. HM440974.     

新型柑橘贮藏保鲜剂的研究进展

柑橘类果实在贮藏运输中果实表面易被腐败菌浸染而导致货架期和贮藏时间缩短,柑橘的贮藏保鲜方法一直以来都是业界关注的热点问题。本文介绍几种具有无毒、可生物降解、对环境无污染等特点的新型杀菌保鲜方法,其中包括动植物多糖涂膜保鲜（壳寡糖）、植物源提取物保鲜（精油）、生物保鲜剂（微生物代谢产物槐糖脂和拮抗菌）等处理方法。综合分析不同处理方法对柑橘类果实的贮藏效果及其对腐败菌的抑制作用,并对柑橘类果实采后防腐保鲜技术的发展趋势及其商业化前景进行展望。     

Head group-modified sophorolipids: Synthesis of new cationic, zwitterionic, and anionic surfactants

Amino acid-glycolipid conjugates were prepared using carbodiimide-mediated coupling methods. The amino acid units were multifunctional and possessed a para-aminobenzoic acid linker. The glycolipid used was a stearic sophoroside. The aim of preparing these modified sophorolipids was to increase their water solubility as well as to introduce sites at the polar head groups that permitted further chemical derivatization. After acidolytic or hydrogenolytic deprotection of the amino acid N-terminus or side chain, water-soluble compounds were obtained that displayed good surfactant properties. Critical micelle concentration values were clustered in the range of high 10⁻⁶ to low 10⁻⁵ M, and minimum surface tension values were below 40 mN m⁻¹. Two of the compounds represented more complicated structural classes, namely, gemini and bolaform surfactants.     

槐糖脂发酵液预处理及不同结构槐糖脂的分离纯化

优化确定了槐糖脂（sophorolipids，SLs）的工业化生产过程中发酵液预处理及槐糖脂的分离纯化新工艺。对下层发酵液利用自然沉降法替代传统乙酸乙酯萃取法获得内酯型槐糖脂；对上层发酵液利用工业化生产中常用的板框过滤替代传统实验室离心法去除菌丝体，利用树脂吸附及超滤法除杂替代实验室及生产中常用的有机溶剂萃取法及醇沉法，获得高纯度的酸型槐糖脂产品。通过上述步骤的实施，实现了菌液的有效分离，成功获得了高浓度、高纯度的不同结构的槐糖脂产品。所得产品经槐糖脂含量测定及HPLC组成测定检验发现，产品标准达到市售标准，产品质量优于市售产品。该预处理及分离纯化工艺不仅丰富了槐糖脂类产品的品种，填补了市售产品均为内酯型和酸型槐糖脂混合物的缺陷，还可以减少有机试剂的使用量，提高槐糖脂生产的安全性与可行性，具有重要的生产指导意义，有望产生巨大经济价值。     

Sophorolipid biosynthesis from a biodiesel co-product stream

We applied a biodiesel co-product stream (BCS) as a fermentation feedstock for the microbial synthesis of sophorolipids (SL). The BCS was composed of 40% glycerol, 34% hexane-solubles (made up of 92% FA soaps/FAME and 6% MAG/DAG), and 26% water. Batch culture fermentations of the yeast Candida bombicola on pure glycerol resulted in low-level synthesis of SL (∼ g/L). HPLC associated with atmospheric pressure CI-MS (LC/APCI-MS) revealed that the SL derived from pure glycerol had 99% of the FA side chains linked to the 4″ hydroxyl group of the sophorose sugar, resulting in a lactonic structure. In contrast, the use of the BCS as feedstock increased the SL yield to 60 g/L and the open-chain form to 75% including both oleic acid and linoleic acid (along with their methyl esters) as the dominant species comprising the side chains. By favoring the open-chain structure, the SL molecules (particularly the FA side chain) can be chemically modified without the need to open a lactone ring first. The ability to use the BCS as a feedstock for SL synthesis will provide an outlet for this residual material, thus helping to stimulate growth in the biodiesel market and the use of agricultural fats and oils from which the biodiesel was synthesized.     

槐糖脂为助剂的多杀霉素悬浮剂的润湿分散剂筛选

采用湿法研磨工艺,通过对比实验确定槐糖脂对多杀霉素悬浮剂的助分散性。在此基础上,通过不断提高多杀霉素的质量分数,以粒径和稳定性为考察指标筛选适用于不同质量分数多杀霉素悬浮剂的润湿分散剂。结果表明,槐糖脂具有降低多杀霉素悬浮剂粒径,提高悬浮剂稳定性的作用,适合用作多杀霉素悬浮剂的助分散剂。以槐糖脂为助分散剂,Well-301、Well-303和7227-A分别适合用作多杀霉素质量分数不大于5%、7.5%和10%悬浮剂的单一分散剂。生物表面活性剂有望部分替代化学表面活性剂应用于农药悬浮剂中。     

Characterization and emulsification properties of rhamnolipid and sophorolipid biosurfactants and their applications

Due to their non-toxic nature, biodegradability and production from renewable resources, research has shown an increasing interest in the use of biosurfactants in a wide variety of applications. This paper reviews the characterization of rhamnolipid and sophorolipid biosurfactants based on their hydrophilicity/hydrophobicity and their ability to form microemulsions with a range of oils without additives. The use of the biosurfactants in applications such as detergency and vegetable oil extraction for biodiesel application is also discussed. Rhamnolipid was found to be a hydrophilic surfactant while sophorolipid was found to be very hydrophobic. Therefore, rhamnolipid and sophorolipid biosurfactants in mixtures showed robust performance in these applications.     

槐糖脂对蜜橘果实采后青霉病的控制及其机理

从自然发病的蜜橘果实上分离出一株致病真菌,通过形态学特征、rDNA-内转录间隔区（rDNA-internal transcribed spacer,rDNA-ITS）分析以及致病性检测,发现该真菌为柑橘意大利青霉（Penicillium italicum）。离体实验发现,当槐糖脂质量浓度为0.1?g/L时,青霉的生长被完全抑制;在果实实验中,当槐糖脂质量浓度为35?g/L时,蜜桔果实的病斑直径最小,且发病率最低。采用质量浓度为35?g/L的槐糖脂对蜜桔果实进行接种和浸泡处理,均能有效提高蜜桔果实的抗病性,且蜜桔果皮的过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶的活力得到了提高。该实验为蜜桔果实采后青霉病的防治提供了一定的理论依据。