生物转化天然香料2-苯乙醇调控与分离纯化技术研究
发布时间：2018-08-27 19:11
【摘要】：2-苯乙醇(2-Phenylethanol,2-PE)是一种带有玫瑰香气的芳香醇,本研究以L-苯丙氨酸为前体,以活性酿酒酵母(Saccharomyces cerevisiae)作为生产天然香料2-PE的菌种。以葡萄糖和乙醇交替氧化策略作为生物转化生产天然香料2-PE的基本体系,该体系既可以解决2-PE和乙醇对生物转化体系的协同抑制作用,也可以提高碳基质的利用效率。通过Plackett-Burman设计和响应面实验优化酵母细胞内提取辅因子NAD+和NADH的方法,并建立该辅因子NAD+和NADH的含量检测方法。从辅因子调控水平探索NADH/NAD+比率和2-PE生成速率的关系。结果表明：在葡萄糖代谢(阶段Ⅰ)和乙醇氧化降解(阶段Ⅱ)两个阶段中,2-PE的生成速率分别为0.38g/(L·h)和0.14g/(L·h);阶段Ⅰ的NADH/NAD+的比率是阶段Ⅱ的2-7倍,阶段Ⅱ是还原力供应的受限阶段。不论存在产物抑制与否,通过补加葡萄糖的方式保持较高的NADH/NAD+比率时,均有利于产物2-PE的合成。另外,二丁基羟基甲苯和过氧化钠可分别提升阶段Ⅰ和Ⅱ的NADH/NAD+比率,进而提高2-PE的生成速率。生物转化85h时,2-PE浓度积累峰值为15.1g/L。2-PE的分离纯化技术也是限制整个天然香料产业化的主要瓶颈。产品分离成本、品质和纯度是2-PE分离的关键。本文分离纯化研究主要从大孔径吸附树脂筛选、副产物控制和香气品质提升入手,降低成本的同时提升2-PE的香气品质。首先选择了价格低廉的国产树脂,通过饱和吸附、平衡吸附、动态吸附与解吸和树脂磨损实验选择了一种适合该体系的大孔径吸附树脂CZ90,其饱和吸附容量为133mg/g,进行200h以上的磨损实验后,其吸附容量为125mg/g,且该树脂吸附容量大、解吸方便,可用于放大生产中。其次,在生物转化生产香料2-PE香气品质时,发现添加抗坏血酸的加入不仅可以提升反应速率也可以抑制副产物的产生,在葡萄糖代谢阶段加入抗坏血酸,产生的2-PE与标准品相似度达到99.19%。最后,对2-PE样品主要杂质进行分析和含量测定,采用Aspen plus精馏模拟的手段,将96.5%纯度的天然2-PE粗品经双塔精馏优化最终2-PE纯度和香气相似度均达到99.9%以上。
[Abstract]:2-Phenylethanoline (2-Phenylethanolium 2-PE) is a kind of aromatic alcohol with rose aroma. L- phenylalanine was used as precursor and active Saccharomyces cerevisiae (Saccharomyces cerevisiae) was used as the strain to produce natural spice 2-PE. The alternative oxidation strategy of glucose and ethanol was used as the basic system of bioconversion to produce natural spice 2-PE. The system could not only solve the synergistic inhibition of 2-PE and ethanol on the biotransformation system, but also improve the utilization efficiency of carbon matrix. The method of extracting cofactor NAD and NADH from yeast cells was optimized by Plackett-Burman design and response surface experiment, and the detection method of NAD and NADH was established. To explore the relationship between NADH/NAD ratio and 2-PE production rate from the level of cofactor regulation. The results showed that in the two stages of glucose metabolism (stage I) and ethanol oxidative degradation (stage II), the formation rate of 2-PE was 2-7 times that of NADH/NAD in 0.38g/ (L h) and 0.14g/ (L h); 鈪，
本文编号：2208180
[Abstract]:2-Phenylethanoline (2-Phenylethanolium 2-PE) is a kind of aromatic alcohol with rose aroma. L- phenylalanine was used as precursor and active Saccharomyces cerevisiae (Saccharomyces cerevisiae) was used as the strain to produce natural spice 2-PE. The alternative oxidation strategy of glucose and ethanol was used as the basic system of bioconversion to produce natural spice 2-PE. The system could not only solve the synergistic inhibition of 2-PE and ethanol on the biotransformation system, but also improve the utilization efficiency of carbon matrix. The method of extracting cofactor NAD and NADH from yeast cells was optimized by Plackett-Burman design and response surface experiment, and the detection method of NAD and NADH was established. To explore the relationship between NADH/NAD ratio and 2-PE production rate from the level of cofactor regulation. The results showed that in the two stages of glucose metabolism (stage I) and ethanol oxidative degradation (stage II), the formation rate of 2-PE was 2-7 times that of NADH/NAD in 0.38g/ (L h) and 0.14g/ (L h); 鈪，
本文编号：2208180
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