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Study on the Fermentation Process of Antimicrobial Lipopeptide Produced by Bacillus Amyloliquefacien

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Tutor: BieXiaoMei
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: The antimicrobial lipopeptide,Defoamer,Macroreticular resin,Dissolved oxygen
CLC: TQ936.16
Type: Master's thesis
Year:  2011
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Antimicrobial lipopeptides are produced by Bacillus sp.with the function of antibacterial, antifungal, antivirus and antimycoplasmal. Lipopeptides with highly active and low poisonous have an important application value in biomedicine. In addition, lipopeptides as a biosurfactant can be used in the fields of petroleum industry, food, cosmetic and agriculture.In the last few years, a endophyte existing in plants Bacillus amyloliquefaciens ES-2 strain has been screened out in our lab, which could produce antibacterial substances. These substances are stable to heat and some enzymes, and have very high antimicrobial activity and broad antibacterial spectrum. Previous studies have demonstrated that the substances mainly included surfactin and fengycin, both of cyclolipopeptide.To achieve the industrialization of antibacterial lipopeptide, the foaming problem must been solved urgently in the process of antibacterial lipopeptide fermentation produces, and the production process must be simple and feasible. Therefore, this study starts from defoaming agent.The defoaming possibility of defoaming agent, the extraction and purification methods and the influence of dissolved oxygen were researched in this study.The main results are as following.1. Soybean oil, silicone defoamer, and polyether defoamer three defoamers are used to Bacillus amyloliquefaciens ES-2 lipopeptide antibacterial lipopeptides fermentation, the research shows that all three types of defoamers defoaming and inhibiting foam can be achieved, in which soybean oil and silicone defoamers defoaming show better defoaming and foam surpresstion performance. The cell content of the fermentation broth with adding the soybean oil and the polyether defoamers is more than do not add defoamer broth, in which the fermentation liquid adding polyether defoamer with 2.14 times the control for the dry cell weight get the maximum increase. The content of the antimicrobial lipopeptides of the fermentation broth adding the soybean oil and the polyether defoamer compared to broth without defoamer had been greatly improved, which soybean oil brings increasment of the production,especially obviously, lipopeptide production reached 2497.67mg/L.2. The addition of soybean oil is benefit for the extraction of acid precipitation of the antimicrobial lipopeptide. The extraction rate reached 92.29%.The antimicrobial lipopeptides fermented by adding soybean oil as defoamer is more easily extracted by methanol and ethanol.The extraction rate reached high levels. The purity of product is also slightly higher than the purity of the antimicrobial lipopeptide fermented without defoamer. Therefore soybean oil can be used as suitable defoamer for the antimicrobial lipopeptide fermentation.The diatomite filtration was selected for obtaining the acid precipitation, and the ethanol as organic solvent for extraction.Ethanol and fermentation with the total volume ratio was 1:2.5.The extraction rate of antibacterial lipopeptides reached 88.72%.3. The purification of antimicrobial lipopeptide extracts by macroporous resin was researched, the results showed that the macroporous resin X-5 shown excellent adsorption capacity in the static adsorption of the antimicrobial lipopeptide. The adsorption capacity reached 57.73 mg¡€g-1, the adsorption rate reached 85.05%. In the desorption results, the resolution rate reached 65.59%. Integrated the absorption and desorption result, select the macroporous resin X-5 as the purified material. After the dynamic adsorption,the preliminary results showed that of the purity of the antimicrobial lipopeptide by adsorption purification reached 72.86%, the relative purity of the original crude extracts has been greatly improved, so the adsorption purification by using macroporous resin was selected for improving the purity of the antimicrobial lipopeptide.The purification use 100% ethanol for elution.The optimized technological parameters of the purification were as following: loading concentration 14.4mg/mL, flow rate for adsorption lmL/min,desorption flow rate 2 mL/min, and elution volume was 2.5 BV.With above conditions,the recovery ratio and purity of product reached 90.01% and 74.4% reseparately.4. Experimental observations by oxygen electrodes in the dissolved oxygen change in the fermentation process showed that the fermentation dissolved oxygen began to decrease after 6h, and decrease gradually increased to 0.6% in 18h,dissolved oxygen reached a very low level and to maintain the low dissolved oxygen levels to the end of fermentation. To study the impact of oxygen on the fermentation, three different dissolved oxygen control schemes were used in the antimicrobial lipopeptide fermentation. After all detected in the whole process of fermentation to maintain the dissolved oxygen level above 5%, the cell concentration, cell specific growth rate and cell production intensity has been raised, but the antimicrobial lipopeptide production, production specific synthetic rate and production intensity decreased. After cell growth into the stable phase increase the dissolved oxygen level above 5%.The result showed that the cell growth did not change relative to the normal fermentation, and antimicrobial lipopeptide production, production specific synthetic rate and production intensity increased.It can explain that dissolved oxygen is an important factor that adjusts cell growth and product synthesis and metabolism during the antimicrobial lipopeptide fermentation.
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