【作者】 赵丽青；

【导师】 孙志浩；

【作者基本信息】 江南大学 ， 发酵工程， 2006， 博士

【摘要】 香草醛(4-羟基-3-甲氧基苯甲醛)是最重要的香味物质之一,具有香兰荚豆特有的香气,广泛应用于食品、糖果、饮料、香料、化妆品及制药等行业。随着人们越来越关心健康和营养,崇尚“绿色”、“天然”,美国和欧盟等纷纷对化学食品添加剂的使用进行了严格的限制规定,天然香草醛的需求量越来越大。从香荚兰豆荚中提取的天然香草醛价格昂贵,产量低,成本高,原料受多方因素限制,不能满足日益增长的需求。利用生物转化法生产天然香草醛具有周期短、成本价格较低、可获得天然等同产品等优点,是最有发展前途的替代方法之一。本论文对微生物法转化异丁香酚生产香草醛进行了研究,在从土壤中筛选获得具有完全自主知识产权、能高效转化异丁香酚生产香草醛的菌株Bacillus sp. SW-B9的基础上,对该菌株进行了生理生化特性鉴定以及16S rDNA序列分析,命名为纺锤芽孢杆菌(Bacillus fusiformis)CGMCC1347;继而对该菌株的发酵条件进行了研究,首次建立了纺锤芽孢杆菌CGMCC1347转化异丁香酚生成香草醛的树脂分离耦合体系和异丁香酚—水两相体系,同时对此二体系的底物和产物的分离提取进行了研究,成功实现了两相体系中固定化细胞的反复利用。对实验室所保藏的细菌、放线菌、真菌进行初步筛选发现细菌中枯草芽孢杆菌(Bacillus subtilus)7658、粪产碱杆菌(Alcaligenes)AS1.7617、黄色短杆菌(Brevibacterium flavum)1.7a、黄色短杆菌(Brevibacterium flavum)AS1.013能产生少量香草醛,最高浓度为0.31 g/L;放线菌中诺卡氏菌(Nocardia sp.)33、酒石酸诺卡氏菌(Nocardia tartic)58、诺卡氏菌(Nocardia sp.)56、诺卡氏菌(Nocardia sp.)44和13-1.58能生成香草醛,最高浓度为0.63 g/L;真菌中酿酒酵母(Saccharomyces cerevisiae)4.1及酵母2.16-7、2.16-8、2.21f、2.21h、SW-57等菌株能生成部分香草醛。在含有高浓度异丁香酚的筛选平板上获得的14支菌株均有转化异丁香酚生成香草醛的能力,其中SW-B9的转化能力最好,能从2%异丁香酚生成1.10 g/L香草醛,高于文献报道的菌种转化水平,并且其进一步降解产物香草醛的能力较为微弱。通过对实验室保藏菌株和土壤中筛选获得菌株生成香草醛能力的对比,最终选用从土壤中筛选获得的Bacillus sp. SW-B9作为进一步研究的出发菌株。生理生化实验结合16S rDNA序列分析表明,该菌株为纺锤芽孢杆菌,命名为纺锤芽孢杆菌CGMCC1347,保藏于中国北京中关村中国微生物菌种保藏管理委员会普通微生物保藏中心,保存号CGMCC No.1347。纺锤芽孢杆菌CGMCC1347的发酵条件对其转化异丁香酚生成香草醛影响的研究表明,其最佳发酵培养基组成为(g/L):玉米浆55,尿素1,K2HPO4 2,MgSO4?7H2O 1,pH7.5,发酵条件为:37 oC,200 r/min,装液量50 mL/250-mL三角瓶,发酵时间更多还原

【Abstract】 As one of the most important components of natural flavours, vanillin (4-hydroxy-3- methoxybenzaldehyde) is widely used in foods, beverages, perfumes, pharmaceuticals and medical industries. Natural vanillin extracted from vanilla pods features both a high price and a limited supply. The increasing concern for health and nutrition and the limit of chemistry food additive in USA and EU stimulate a worldwide demand for natural vanillin.Biotransformation is an alternative or equivalent technology to get natural vanillin. Natural isoeugenol from essential oil are the available and economical substrate for the vanillin production. Therefore, the bioconversion of isoeugenol to vanillin becomes more and more attractive.This paper focused on the biotransformation of isoeugenol to vanillin by microorganisms. After the isolation of Bacillus sp. SW-B9 from soil, its physiological, biochemical characteristics and the 16S rDNA were analyzed. Condition optimization for its efficient fermentation was also carried out. The reaction and separation system with the addition of resin HD-8 and the isoeugenol/aquoeus biphasic system of the biotransformation of isoeugenol to vanillin by Bacillus fusiformis CGMCC1347 were set up. The isolation and purification of isoeugenol and vanillin in both systems were studied. The reuse of the immobilized cells in biphasic system was successfully applied.The biotransformations of isoeugenol to vanillin with various strains available in our lab were tested. It was found that several baterica including Bacillus subtilus 7658, Alcaligenes AS1.7617, Brevibacterium flavum 1.7a and Brevibacterium flavum AS1.013 could convert isoeugenol to vanillin. The maximum vanillin concentration was 0.31 g/L. Nocardia sp. 33, Nocardia tartic 58, Nocardia sp. 56, Nocardia sp. 44 and Nocardia sp. 13-1.58 could produce 0.12 g/L~0.63 g/L vanillin. Some other fungi yielding vanillin were Saccharomyces cerevisiae 4.1, yeast16-7, 2.16-8, 2.21f, 2.21h and yeast SW-57.14 strains beneficial to transform isoeugenol to vanillin were isolatied from soil based on the screening model using high concentration of isoeugenol. Strain SW-B9 could produce 1.10 g/L vanillin from 2% (v/v) isoeugenol and its degrading capacity of vanillin was weak. Among the strains avaible in lab and isolatied from soil, Bacillus sp. SW-B9 was selected for further study due to its highest vanillin production ability.According to physiological and biochemical characteristics of strain SW-B9 and the 16S rDNA gene sequence analysis, strain SW-B9 was identified as a kind of Bacillus fusiformis. The sequence data has been assigned accession number AY907676 in the GenBank database. Strain SW-B9 was conserved in China General Microbiological Culture Collection Center更多还原