霍氏肠杆菌B4表面活性剂纯化鉴定及其应用

Purification and Identification of the Biosurfactant Produced by Enterobacter Hormaechei B4 and Its Application

【目的】优化霍氏肠杆菌B4产表面活性剂的液体发酵条件,结合有机试剂萃取、高效液相色谱技术分离纯化所产表面活性剂,并利用质谱分析技术鉴定其结构,进一步研究其促进黄瓜吸收叶面肥的效果,为新型肥料的研制提供理论依据。【方法】利用正交试验优化霍氏肠杆菌B4产表面活性剂的发酵条件,主要包括碳源、氮源、初始pH、发酵温度、接种量、转数和发酵时间,不同处理的评价指标为发酵液的表面张力值,具有最低表面张力的处理为最佳的发酵条件;利用有机试剂萃取后旋转蒸发获得表面活性剂的粗提物;利用高效液相色谱技术,在柱温50℃、进样量为20μL、流速0.5 m L·min^(-1)、检测波长为210 nm条件下分离纯化表面活性剂,并结合傅里叶红外光谱技术分析其所含的不同官能团;在流速10μL·min^(-1)、毛细管电压3.88 k V、锥孔电压为53 V、离子源温度100℃、脱溶温度150℃条件下,利用质心模式进行全扫描,根据已有的质谱数据鉴定所产表面活性剂的结构;通过水培和盆栽试验,验证该表面活性剂提高黄瓜吸收叶面肥的效率。【结果】正交试验结果表明,在添加4%(v/v)甘油、3 g·L^(-1)硝酸钠、初始pH 6.0、6%接种量、35℃、200 r/min、发酵96 h的条件下,发酵液的表面张力下降到44.10 m N·m^(-1),为最优的发酵优化条件。在该优化条件下,生物表面活性剂的粗产物产量高达12.14 g·L^(-1),粗产物可使纯水的表面张力值最低降到34.14 m N·m^(-1)。液相分离结果表明,经有机试剂萃取粗提的生物表面活性剂在1.62—2.33 min处有典型的特征峰,说明该组分为生物表面活性剂粗提物中的主要成分。傅里叶红外光谱分析结果表明,纯化的生物表面活性剂中含有-CH_2、-CO和C-O等官能团,判断其为含碳链的生物表面活性剂。液质联用分析结果表明,m/z 701.54处为[M+Na]+,m/z 723.74处为[M-H+2Na]^+,将该生物表面活性剂鉴定为鼠李糖脂,其结构式为Rha-Rha-C_(10)-C_(12)。黄瓜水培结果表明,与只喷清水(CK)和只喷氨基酸(AA)的处理相比,添加生物表面活性剂的氨基酸叶面肥处理(AAB)的株高分别增加了79.59%和32.90%,鲜重分别增加了43.03%和23.98%。盆栽试验结果表明,与CK和AA处理相比,AAB处理的植株叶绿素含量分别提高了11.72%和10.69%。【结论】利用正交试验获得了霍氏肠杆菌B4产生物表面活性剂最佳液体发酵条件,在该条件下生物表面活性剂产量达到2.07g·L^(-1),并将其主要成分鉴定为鼠李糖脂(Rha-Rha-C_(10)-C_(12)),该物质可显著提高黄瓜对叶面肥的吸收效率,具有很好的应用前景。

【Objective】The objective of this study is to optimize the biosurfacants produced by Enterobacter hormaechei B4, to purify the biosurfacants through organic reagent extraction and high performance liquid chromatography, to identify the biosurfacants by HPLC-MS, and to evaluate the efficiency of improving foliar fertilizer absorption, which will provide theoretical basis for the development of new fertilizers. 【Method】The optimization of biosurfacants production by Enterobacter hormaechei B4 was carried out by orthogonal experiment, and the major parameters were used in this study, including the carbon resources, nitrogen resources, initial pH, temperature, inoculum amount, revolutions, and incubation time. The evaluation index of different treatments was the surface tension value of the fermented liquid, and the treatment with the lowest surface tension was the optimum experimental group. The crude biosurfacants were extracted by extracted by organic regents and then concentrated by using vacuum rotary evaporation apparatus with water bath at 60 centigrade. Different functional groups contained in the purified biosurfacants were analyzed by Fourier infrared spectrum, which could provide favorable information for analyzing the structure. The purification of the biosurfacants was performed by liquid chromatography methods, and the HPLC conditions are： column temperature of 110 ℃, sample size with 20 μL, flow rate of 0.5 m L·min-1. The identification of the purified biosurfacants was carried out by HPLC-MS, and the condition was as following： flow rate of 10 μL·min-1, the velocity of capillary voltage of 3.88 k V, cone voltage of 53 V, ionization temperature of 100 ℃, dissolution temperature of 150 ℃, under the condition of using full scan mode in the center of mass, and the full scan under centroid model was used to collect the mass spectrometry data, and the biosurfactant structure was identified based on existing mass spectrometric data. In the end, the purified biosurfactant was used to evaluate the efficiency of improving foliar fertilizer absorption by cucumber through hydroponic culture and pot experiments.【Result】The results indicated that the optimum condition for biosurfactant production is listed as follow： 4%（v/v） glycerol, 3 g·L-1 sodium nitrate, initial pH 6.0, inoculum of 6%, 35 ℃, 200 rpm and 96 h, under which the surface tension value of the fermented liquid decreases to 44.10 m N·m-1 and the production of the crude biosurfacants was 12.14 g·L-1. Meanwhile, the crude biosurfacants extracts could decrease the surface tension value of pure water to 44.10 m N·m-1, and this condition is considered as the optimum fermentation condition. The liquid phase separation results indicate that the crude biosurfacants extracted by organic reagent have typical characteristic peaks at 1.62-2.33 min, which also showed that it is the main component in the crude extract of the biosurfactant, and it also could decrease the surface tension value to 47.00 m N·m-1 at the concentration of 0.10 g·L-1. The FRIR analysis results indicate that the purified biosurfacants contain various functional groups including-CH_2、-CO and C-O, and it is considered as biosurfacants with carbon chains. The HPLC-MS analysis results show that m/z 701.54 is [M＋Na]~＋, and m/z 723.74 was [M-H＋2 Na]＋, and the biosurfactant is identified as rhamnolipid, and its structure is Rha-Rha-C_（10）-C_（12）. By comparing to the CK（Water） and AA（Amino acids） treatments, the plant height in AAB（Amino acids and biosurfacants） increased by 79.59% and 32.9% and fresh weight increased by 43.03% and 23.98%, respectively. The chlorophyll contents in AAB increase by 11.72% and 10.69%, respectively, by comparing to the CK and AA, in the pot experiment.【Conclusion】In brief, the condition for the biosurfacants production secreted by Enterobacter hormaeche is optimized, under which the production of the crude biosurfacants is 12.14 g·L-1, and the main component of the biosurfacants produced by Enterobacter hormaeche is identified as rhamnolipid（Rha-Rha-C_（10）-C_（12）, which can enhance foliar penetration and will have a good application prospect.