微生物转化发酵法生产天然香料β-苯乙醇的研究

生物工程论文编号:SW150  论文字数:11914,页数:29

目  录
中文摘要 I
英文摘要 III
目录 v
1. 绪论 1
 1.1 产品概述 2
  1.2国内外技术发展概况和发展趋势 3
 1.3本课题的研究意义 6
 1.4本课题的研究任务 7  

2．实验部分 8
2.1 实验材料 8
2.2 实验方法 9
 2.3结果与讨论 10
    2.4 原生质体制备 16
 3 结果与讨论 17
 3.1 影响原生质体形成因素 17
3.2 紫外诱变剂量选择 18
4 总结与展望 19
4.1 总结 19
4.2 展望 20
致谢 21
参考文献 22

摘 要：本论文以克鲁维氏酵母细胞生物转化生成的天然香料β-苯乙醇为研究对象，主要研内容包括：转化 L-苯丙氨酸生成 β-苯乙醇酵母菌株的确定；发酵液中底物和产物的定量检测方法；摇瓶培养，包括培养基组成的优化及培养工艺
优化；原生质体的制备以及底物诱变，抗反馈抑制优化；β-苯乙醇的结构鉴定。
 从实验室保藏的菌种中筛选出一株克鲁维氏酵母：Kluyveromyces marxious   ，能够将L-苯丙氨酸转化为β-苯乙醇。根据 L-丙氨酸和 β-苯乙醇的化学结构特征，建立了测定发酵液中底物和产物的方法：高效液相色谱法测定β-苯乙醇；高效液相色谱法测定底物 L-苯丙氨酸；该法处理简单、快速、定量准确、回收率高、重复性好，可有效的用于β-苯乙醇产生菌的菌种选育和发酵
工艺研究中。
 为了提高产物浓度，对培养基配方和发酵条件进行优化，确定了优化了的培
 养条件：  菌种培养基（YPD）:酵母浸膏 10g/L, 蛋白胨20g、L, 葡萄糖20g/L    发酵培养基：蔗糖 35g/L、L-苯丙氨酸 6g/L,磷酸二氢钾 0.4g/L，YNB1.8g/L；
种子培养基培养 24h，接种量 6%，优化培养基初始 pH=5.5，装液量 50mL/250mL，温 28℃，转速 220r/min，在此条件下发酵 48h 。

关键词：克鲁维酵母，微生物转化，L-苯丙氨酸，β-苯乙醇
 
Abstract: β-phenylethanol(β-PE) is a higher aromatic alcohol with a rose-like odour. The fragrance of the rose is highly popular and desired, making β-PE the most-used fragrance chemical in perfume and cosmetics. It is also used in flavor compositions for food. Most of the world's annual production of several thousand tons is synthesized by chemical means but, due to increasing demand for natural flavors,  alternative production methods are being sought. Harnessing the Ehrlich pathway of yeasts by bioconversion of L-phenylalanine to β-PE could be an good option.
 In this work, systematic studies were carried out on the screening of yeast strains which can transform L-phenylalanine to β-PE; determination, isolation, purification,  identification, quantification method of β-PE Kluyveromyces marxious was screened from the microorganism strains preserved by our laboratory. According to the chemical structures of L-phenylalanine and β  -phenylethanol, we established some methods for the separation and determination of them: RP-HPLC method for the separation and determination of L-phenylalanine and β-phenylethanol, this method was accurate, sensitive and reliable.
 The conditions of liquid fermentation of β-PE were studied. It showed that the effect of single factors, such as carbon source, nitrogen source, growth factors and inorganic salts on the production of β-PE. Through orthogonal test, the optimum medium composition had been obtained. It was 35g/L sucrose, 6g/L L-phenylalanine, 0.4g/L KH2PO4 and 1.8g/L YNB. The optimum shaking culture condition was as follows: 24 hours strain age, 6% inoculation quantity, initial medium pH=5.48, 50 mL medium in 250mL shaking flask, 28℃, 220r/min shaking speed, 48 hours incubation.        
Keywords: Kluyveromyces marxious ,bio-transform, L-phenylalanine, β-PE