The Study On Lipopeptid Biosurfactant Productong By Breibactreium Halotolerans Y-1-4

The lipopeptide biosurfactants with unique chemical structure are secondary metabolites produced by microorganism. They have excellent surface activity and special physiological characteristics. Because of potential application advantages in many fields, the lipopeptide biosurfactants have enormous ecological, social and economic benefits in petroleum industry, pharmaceuticals, agriculture, environmental management and cosmetics and other fields. Therefore, exploring the resources of lipopeptide biosurfactant-producing microorganisms, improving their production make lipopeptide biosurfactants a wide range of biological and economic significance.This paper mainly includes the following aspects: the preliminary study on the physico-chemical properties and antimicrobial activity of the lipopeptide produced by Brevibacterium halotolerans Y-1-4, the structure analysis and optimization of its fermentation conditions.(1)The experiment analysis revealed that B. halotolerans Y-1-4 produced lipopeptide biosurfactant in the process of metabolism. The surface tension of the cell-free broth was reduced from the 72.29 mN/m(25 ℃)to less than 27 mN/m. Its critical micelle concentration(CMC)was 21.56 mg/L. The lipopeptide biosurfactant product showed high surface activity and a stable emulsifying ability on paraffin, cyclohexane, cottonseed oil and corn oil and was very stable at wide range of pH(2-12), temperatures(20-120 min incubation in boiling water bath) and salinity(2-21 % NaCl).(2)The lipopeptide cause different degrees of growth inhibition on P. vulgaris, E. aerogenes and E. coli ATCC 25922 especially on B. cereus and S. aureus ATCC 25923. The growth of myceliums of P. pirina CFCC 6862, A. flavus CGMCC 3.2890, M. brunne CFCC 86450, A. flavus CGMCC 3.3950 and were also inhibited by lipopeptide.(3)The lipopeptide were isolated and purified from the cell-free broth of B. halotolerans Y-1-4 by acid precipitation, extraction, thin-layer chromatography, column chromatography and Sephadex LH-20. Its structure was characterized by amino acid analysis and LC-MS method. Five Surfactin homologous series was found, which were C13 Surfactin, C14 Surfactin, C15 Surfactin, C16 Surfactin and C17 Surfactin. It was first time reported that surfactin was produced by Brevibacterium halotolerans.(4)Response surface methodology was employed for the optimization of B. halotolerans Y-1-4 fermentation conditions. Based on the single factor experiments,the influence of NaCl, inoculation arnount and initial pH on lipopeptide production were determined by Plackett-Burman design, and the path of steepest ascent was used to approach the optimal region. Through Box-Behnken design, the fermentation conditions and the optimal medium compositions were were determined as: initial pH 6.3, inoculation arnount 5.2 %, seed age 18 h, medium volume(50 mL/250 mL), rotation speed 150 rpm/min, temperature 30 ℃, molasses sugar 4 g/L, peptone 13 g/L, NaCl 4.3 g/L, MgSO4?7H2O 0.2 g/L. By calculating, in optimal conditions, the inhibition zone diameter be conjectured that was 23.24 mm. After optimization of lipopeptide biosurfactat produced by Y-1-4, the inhibition zone diameter was 23.42 mm, which had 99.23 % consistency with the speculation. It improves 26.58 % than optimization before.