【作者】 任龙；

【导师】 陈福生； 陈涛；

【作者基本信息】 华中农业大学 ， 微生物学， 2007， 硕士

【摘要】 番茄红素属于不具有环状结构的一种类胡萝卜素，具有很强的抗氧化活性和多种生理活性，是一种新型功能性天然色素，在食品、药品及医疗保健等方面有良好的应用前景。采用微生物发酵法生产番茄红素，其产品质量和生理活性与天然提取产品完全相同，且采用发酵法能降低成本，污染相对较小。因此，保证菌株高产、优化发酵条件和得到较高的提取率，是实现工业化生产较理想的途径。本论文主要从以下四个方面进行研究：1．从保存的192支沙土管和6支冻干管三孢布拉氏霉菌（Blakeslea trispora，B．t．，下同）菌种中，经划线分离、纯化筛选，通过形态观察，显微结构观察，获得了6⁺、80⁺、83⁺、75^-、94^-、G₃⁺和F₂^-共7株菌。经发酵培养，干菌体含油率为45.1％～62.5％，发酵液分别含453.4～821.2 mg／Lβ-胡萝卜素（β-carotene）和29.2～65.4 mg／L番茄红素（Lycopene）。根据菌株生长情况以及发酵实验结果，从中选出83⁺、94^-、G₃⁺和F₂^-共4株菌作为诱变育种的出发株。2．对分离筛选到的B．t．菌株进行两次⁶⁰Co-γ辐射处理。经过菌体形态、平皿生长、发酵培养和色素实验等研究，选育出能稳定遗传的MG₃⁺和MF₂^-两株产番茄红素的突变株作为发酵生产实验用菌株，其番茄红素的产量达661.8mg／L。3．设定发酵培养基中的玉米淀粉、黄豆饼粉、植物油和葡萄糖作为实验的4个因素，分别取3个水平，以发酵菌体菌丝量和番茄红素产量为指标，进行正交实验。根据正交表L₉（3⁴）设计正交实验，以筛选出最优培养基条件。实验结果用SPSS软件分析，根据直观及方差分析，最后得出最优发酵培养基中的配比为A₃B₁C₂D₂。番茄红素产量（与最佳方案最接近的处理组）比优化前提高了30％以上，达到968.2 mg／L。4．在完成了B．t．诱变选育及其摇瓶发酵实验的基础上，进行了B．t．突变株30 L罐的发酵实验，以便为该菌株的大规模发酵探明工艺流程和技术条件。经过连续5批发酵，番茄红素产量最高达到1295.6 mg／L。更多还原

【Abstract】 Lycopene is a unique caroteniod with non-ring structure. It demonstrates strong antioxdative reactivity and multi-physiological effects. It has great potential in food industry, drug production and medical care. Producing lycopene with fermentation method of microbion, the quality and physiological activityof products are identical with the natural products. It would be to reduce the cost and pollution by applying fermentation method. Accordingly the rather ideal way of implementing industrialized production is to ensure highly producing strain, optimize fermentation condition and get higher extraction ratio.In this paper, the production of lycopene by Blakeslea trispora was investigated in four parts.1. Using 192 sand soil test tubes and 6 freeze dried test tubes, Blakeslea trispora strains 6⁺, 80⁺, 83⁺, 75^-, 94^-, G₃⁺and F₂^- were obtained by separating, purifying and screening. After fermentation, the content of lipid in dry mycelia was 45.1～62.5%,β-carotene and lycopene were 453.4～821.2 mg/L and 29.2～65.4 mg/L respectively. According to growth state and experimental result of fermentation, 83⁺、94^-、G₃⁺and F₂^-strains were chosen as original strains for mutagenesis breeding.2. Blakeslea trispora that had been isolated was treated twice with ⁶⁰Co-γirradiation. According to the research of thalli figure、living in culture utensil、fermentation culture and pigment test, we bred two strains with MG₃⁺ and MF₂^- for fermentability production. Its genetic characteristic was stable and its yield of lycopene reached 661.8mg/L.3. In order to raise the mutant strain production, we designed an orthogonal experiment according to L₉（3⁴） table for screening the optimization medium. The experiment took the following four factors for each at three different levels. And the four factors were corn flour, bean cake flour, vegetable oil and glucose of main medium. The targets of experiment was mycel weight and lycopene yield. Result was analyzed by SPSS. According to the analysis of direct-viewing and variance, we concluded that in the optimization medium the content of four factors was A3B1C2D2. The lycopene yield raised more than 30%, was 968.2 mg/L.4. At the base of mutagenesis selection and shake flask fermentation experiment of Blakeslea trispora, in order to try to find out technology flow-sheet and specification, we completed the experiment by 30 L fermentor. The highest lycopene yield is 1295.6 mg/L through continuously fermenting 5 times.更多还原