【作者】 刘丽；

【导师】 李俊峰；

【作者基本信息】 青岛科技大学 ， 轻工技术与工程， 2016， 硕士

【摘要】 脂肽类生物表面活性剂是一类由微生物产生的次级代谢物,具有独特的化学结构,表现出优良的表面活性和生理特性,在石油工业、医药、农业生产、环境治理及化妆品等领域具有巨大的生态、社会和经济效益。因此,发掘产脂肽类生物表面活性剂微生物的资源,提高产量,具有广泛的生物学和经济意义。本论文的主要内容包括耐盐短杆菌(Brevibacterium halotolerans)Y-1-4所产脂肽理化性质和抑菌活性的初步研究及其结构分析,并对其发酵条件进行了优化。(1)B.halotolerans Y-1-4的代谢产物为脂肽类生物表面活性剂,能将水的表面张力从72.29 mN/m(25℃)降低到27 mN/m以下,CMC值为21.56 mg/L,对石蜡、环己烷、棉籽油、玉米油都具有稳定的乳化活性,对盐浓度的耐受性为21%,在pH 2~12范围内其表面张力变化不大,热稳定性好,沸水浴120 min表面张力保持稳定。(2)B.halotolerans Y-1-4所产脂肽分别对变形杆菌(P.vulgaris)、产气肠杆菌(E.aerogenes)、大肠埃希氏杆菌(E.coli ATCC 25922)有不同程度的抑制作用,对蜡样芽孢杆菌(B.cereus)和金黄色葡萄球菌(S.aureus ATCC25923)的抑制效果尤其明显;分别对梨叶点霉菌(P.pirina CFCC 6862)、黄曲霉(A.flavus CGMCC 3.2890)、褐色盘二孢(M.brunne CFCC 86450)和黄曲霉(A.flavus CGMCC 3.3950)的菌丝生长有不同程度的抑制效果。(3)对B.halotolerans Y-1-4所产脂肽进行了分离和纯化,采用经酸沉淀、二氯甲烷抽提、薄层层析、硅胶柱层析和Sephadex LH-20凝胶层析等方法,经氨基酸分析和LC-MS质谱等进行结构分析,发现该脂肽由五种Surfactin同系物组成,分别是C13 Surfactin、C14 Surfactin、C15 Surfactin、C16 Surfactin和C17 Surfactin。这也是首次报道从耐盐短杆菌(Brevibacterium halotolerans)发酵液中分离出莎梵婷(Surfactin)。(4)用响应面分析法优化了B.halotolerans Y-1-4产脂肽的发酵培养基,以单因素实验结果为依据进行Plackett-Burman设计,确定了NaCl浓度、接种量和初始pH值为影响脂肽产量的主要因素;设计最陡爬坡实验确定最佳实验中心点;通过Box-Behnken实验,最终确定最佳培养条件为:初始pH值6.3、接种量5.2%、接种种龄18 h、装液量(50 mL/250 m L)、摇床转速150 rpm/min、培养温度30℃、蔗糖糖蜜4 g/L、蛋白胨13 g/L、NaCl 4.3 g/L、MgSO4·7H2O 0.2 g/L。在最优条件下,推测其抑菌圈直径为23.24 mm,经培养发酵验证后,抑菌圈直径达到23.42 mm,与推测值的相符性为99.23%,较原发酵培养基提高了26.58%。更多还原

【Abstract】 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.更多还原