Lipopeptide Biosurfactant Process And Its Emulsifying Properties

Lipopeptide biosurfactant is a class of secondary metabolites and extracelluar secreted by Bacillus. They have biocompatible, biodegradable, high surface activity, antimicrobial activity and other characteristics, its super surfactants are widely used in oil exploration, environmental management, bio-medical, agricultural, comestic, food industry and other fields. Currently, Against the surfactant lipopeptide, there are more research on surfactin, but it is difficult to scale the indusrial production due to cost and other reasons and limiting their wider application. Therefore, breeding high-yeild strains, improving fermentation and extraction processes and reduce production costs are urgent problems needed to solve.Based on the earlier study of soybean oil as defoamers significantly improved antimicrobial lipopeptide yield. This study use soybean oil as a defoaming agent, for high-yielding strains of Bacillus amyloliquefaciens fmb50fermentation production, carry on the efficient extraction process of surfactin production for Bacillus. Meanwhile, the purified surfactin emulsification and its applications conducted a preliminary study. Intention for the basis of industrial production and application of surfactin. The results are as follows:1. The extraction of lipopeptide biosurfactant. Using the method of flocculation pretreat for fermentation broth. First, the best flocculant was screening by single factor experiments. Secondly, the use of Plackett-Burman Design for the extraction of the key factors of surfactin filter, and the key factors were optimized with Central Composite Design. Finally, the antimicrobial of the broth was studied with punching method. The results showed that the flocculants CaCl2, AlCl3, CaCl2and Na2HPO4, AlCl3and Na2HPO4, biochemical flocculant, the biochemical flocculant had the best effect, and when the biochemical flocculant was added0.2%, recovery rate of surfactin was88.43%; Screening the key factors of extraction were ethanol addition and initial fermentation pH; determining the optimum amount of flocculating conditions were160%ethanol addition, initial pH was8.0and0.2%of biochemical flocculant addition, surfactin recovery rate was94.54%under this condition, the surfactin recovery rate was93.21%in validation experiments. Identified a wide rang of surfactin with antibacterial spectrum, it had a strong inhibitory effect on Gram-positive bacteria, Gram-negative bacteria and fungi.2. Purification and identification of lipopepetide biosurfactant. Using macroporus adsorption method purified the pretreated fermentation. The results showed that D101macroporous resin had the highest surfactin recovery. Among them, the adsorption amount166mg.g-1, absorption rate89.28%, desorption rate80.64%, the recovery rate of surfactin is72.00%, purity32.90%. Dynamic adsorption conditions on the concentration of the sample8.853g/L, sample volume is5BV, elution flow rate is2mL/min, eluent consumption is13BV, recovery rate of surfactin is82.36%and purity is85.75%. The purified sample was further purified by preparative HPLC,1a, lb,2a,2b,2c,3peaks collected, the LC-MS/ESI-MS analysis showed the results that the m/z of the sample is994.10,994.31,1008.26,1008.27,(1008.34and1022.32),1036.48respectively. All of them are homologues of surfactin with C13～C15. Secondary and tertiary mass spectrometry showed, peak2a,2b and3have leu or Ile in seventh place of the peptide chain. IR spectrum showed: the substance had a peptide chain and aliphatic hydrocarbon chain structure.3. Study on emulsification and application of the surfactin sample which was purified by resin. The surface activity of surfactin was studied by Platinum plate method, emulsifying property was studied by turibidity and centrifugation method, and application of surfactin in peanut beverage was studied. The results show that, the critical micelle concentration of surfactin is9.03×10-5mol/L, the surface of water can be droped down from73.98mN/m to27.48mN/m. When the Sodium chloride solution at a concentration of1×10-3mol/L-1×10-2mol/L, the surface tension of surfactin solution decreased with increasing salt concentration and stabilized at34mN/m eventually. Surfactin with the treatment of121℃20min showed that the surface tension was still remained at37.10mN/m; surface activity of surfactin stay a good stability in the pH range of2-12. Above shows surfactin has a strong tolerance of salt, temperature, pH. Hydrophile-Lipophile Balance (HLB) value measured by turbidity method was14, and has a good emulsifying property of the oil phase which required the HLB value between12～16, even had a good enmulisiying effect on peanut drink and better than sucrose esters.