Abstract: 【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 ^oC, sample size with 20 μL, flow rate of 0.5 mL·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 kV, cone voltage of 53V, ionization temperature of 100 ^oC, dissolution temperature of 150 ^oC, 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 ^oC, 200 rpm and 96 h, under which the surface tension value of the fermented liquid decreases to 44.10 mN·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 mN·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 mN·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 -CH₂、-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+2Na]⁺, and the biosurfactant is identified as rhamnolipid, and its structure is Rha-Rha-C₁₀-C₁₂. 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-C₁₀-C₁₂), which can enhance foliar penetration and will have a good application prospect.

Key words: Enterobacter hormaechei, biosurfactants, optimization, foliar fertilizer