【摘要】：2-苯乙醇(2-Phenylethanol,2-PE)是一種帶有玫瑰香氣的芳香醇,本研究以L-苯丙氨酸為前體,以活性釀酒酵母(Saccharomyces cerevisiae)作為生產(chǎn)天然香料2-PE的菌種。以葡萄糖和乙醇交替氧化策略作為生物轉(zhuǎn)化生產(chǎn)天然香料2-PE的基本體系,該體系既可以解決2-PE和乙醇對(duì)生物轉(zhuǎn)化體系的協(xié)同抑制作用,也可以提高碳基質(zhì)的利用效率。通過Plackett-Burman設(shè)計(jì)和響應(yīng)面實(shí)驗(yàn)優(yōu)化酵母細(xì)胞內(nèi)提取輔因子NAD+和NADH的方法,并建立該輔因子NAD+和NADH的含量檢測(cè)方法。從輔因子調(diào)控水平探索NADH/NAD+比率和2-PE生成速率的關(guān)系。結(jié)果表明：在葡萄糖代謝(階段Ⅰ)和乙醇氧化降解(階段Ⅱ)兩個(gè)階段中,2-PE的生成速率分別為0.38g/(L·h)和0.14g/(L·h);階段Ⅰ的NADH/NAD+的比率是階段Ⅱ的2-7倍,階段Ⅱ是還原力供應(yīng)的受限階段。不論存在產(chǎn)物抑制與否,通過補(bǔ)加葡萄糖的方式保持較高的NADH/NAD+比率時(shí),均有利于產(chǎn)物2-PE的合成。另外,二丁基羥基甲苯和過氧化鈉可分別提升階段Ⅰ和Ⅱ的NADH/NAD+比率,進(jìn)而提高2-PE的生成速率。生物轉(zhuǎn)化85h時(shí),2-PE濃度積累峰值為15.1g/L。2-PE的分離純化技術(shù)也是限制整個(gè)天然香料產(chǎn)業(yè)化的主要瓶頸。產(chǎn)品分離成本、品質(zhì)和純度是2-PE分離的關(guān)鍵。本文分離純化研究主要從大孔徑吸附樹脂篩選、副產(chǎn)物控制和香氣品質(zhì)提升入手,降低成本的同時(shí)提升2-PE的香氣品質(zhì)。首先選擇了價(jià)格低廉的國產(chǎn)樹脂,通過飽和吸附、平衡吸附、動(dòng)態(tài)吸附與解吸和樹脂磨損實(shí)驗(yàn)選擇了一種適合該體系的大孔徑吸附樹脂CZ90,其飽和吸附容量為133mg/g,進(jìn)行200h以上的磨損實(shí)驗(yàn)后,其吸附容量為125mg/g,且該樹脂吸附容量大、解吸方便,可用于放大生產(chǎn)中。其次,在生物轉(zhuǎn)化生產(chǎn)香料2-PE香氣品質(zhì)時(shí),發(fā)現(xiàn)添加抗壞血酸的加入不僅可以提升反應(yīng)速率也可以抑制副產(chǎn)物的產(chǎn)生,在葡萄糖代謝階段加入抗壞血酸,產(chǎn)生的2-PE與標(biāo)準(zhǔn)品相似度達(dá)到99.19%。最后,對(duì)2-PE樣品主要雜質(zhì)進(jìn)行分析和含量測(cè)定,采用Aspen plus精餾模擬的手段,將96.5%純度的天然2-PE粗品經(jīng)雙塔精餾優(yōu)化最終2-PE純度和香氣相似度均達(dá)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，

本文編號(hào)：2208180