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1、集美大学 硕士学位论文 柚苷酶发酵放大技术及酶制剂的研究 姓名：丁涛 申请学位级别：硕士 专业：微生物学 指导教师：蔡慧农;肖安风 2011-06-14 I 柚苷酶发酵放大技术及酶制剂的研究柚苷酶发酵放大技术及酶制剂的研究 摘摘 要要 柚苷酶是一种由 -L-鼠李糖苷酶(EC 3.2.1.40)和 -D-葡萄糖苷酶(EC 3.2.1.21)组成的， 以水解柚皮苷至柚皮素为特征的糖苷水解复合酶，它在柑橘类果汁的脱苦、饮料增香、 黄酮类物质和鼠李糖的工业制备等方面具有重要的应用价值。然而由于柚苷酶发酵生产技 术的不成熟和制剂化工艺的缺乏，导致成品柚苷酶酶制剂价格高昂，这严重限制了柚苷酶 应用技术的开
2、发，因此建立成熟的柚苷酶规模化发酵技术和合理的制剂化工艺具有重要意 义。 本论文以黑曲霉 DB056 为柚苷酶产生菌， 在研究了柚苷酶发酵放大之后， 进行了柚苷 酶制剂化工艺的探索和优化，最后对得到的柚苷酶酶制剂进行了食品安全性检测及储藏稳 定性的探讨，主要实验结果如下： 柚苷酶 7 L、20 L 和 200 L 规模的逐级发酵放大研究表明，随着发酵规模的扩大，柚 苷酶的两种组分：-L-鼠李糖苷酶与 -D-葡萄糖苷酶的酶活力水平会有一定的波动，但酶 活力变化趋势基本一致；在 200 L 规模的发酵中，-L-鼠李糖苷酶与 -D-葡萄糖苷酶的最 高酶活力分别为 1069 U/mL 和 727 U/
3、mL，超过了 7 L 和 20 L 规模发酵过程中柚苷酶的活 力水平。实验成功实现了黑曲霉 DB056 产柚苷酶 200 L 规模的发酵放大，显示了该菌株良 好的发酵性能和工业应用价值。 柚苷酶发酵液经冷冻离心、除菌过滤、超滤浓缩及喷雾干燥工艺之后便可得到柚苷酶 固体酶制剂。柚苷酶浓缩发酵液喷雾干燥中选择麦芽糊精为助干剂，固形物含量为 27%， 以 -L-鼠李糖苷酶回收率为标准，在单因素实验的基础上，采用响应面分析法对喷雾干燥 参数进行优化，得到最佳喷雾干燥条件为：进风温度 111.3，进料速度 161.5 mL/h，热风 流量 5.1 m3/min，出风温度 60，雾化器压力 0.4 MPa
4、，在此条件下，-L-鼠李糖苷酶回收 率为 79.7%。采用确定的工艺和参数条件进行柚苷酶酶制剂制备，得到的酶制剂中 -L-鼠 李糖苷酶活力可达 19000 U/g，总回收率为 78.1%；-D-葡萄糖苷酶的活力达到 4700 U/g， 总回收率为 28.3%；酶制剂含水量 7.5%。 通过检测，本研究中制备得到的柚苷酶固体酶制剂的菌落总数、大肠菌群、沙门氏菌 以及重金属含量的检测结果完全符合食品工业用酶制剂的安全标准。 温度、空气和湿度对柚苷酶酶制剂的储藏稳定性影响较大，光照对其影响不明显。柚 苷酶固体酶制剂在-20、真空、避光条件下保存 70 d，-L-鼠李糖苷酶和 -D-葡萄糖苷酶 活力的
5、保留率分别为 97.6%和 86.0%。 本研究实现了柚苷酶 200 L 规模的发酵放大，建立了柚苷酶制剂化工艺流程并优化了 过程参数条件，检测了柚苷酶固体酶制剂的食品安全性，探讨了柚苷酶酶制剂储藏稳定性 的影响因素，为规模化工业生产柚苷酶酶制剂及柚苷酶应用技术的开发奠定了基础。 II 关键词关键词 黑曲霉，柚苷酶，发酵放大，-L-鼠李糖苷酶，-D-葡萄糖苷酶，喷雾干燥， 酶制剂 III Studies on Scale-up Technology of Naringinase Fermentation and its Enzyme Preparation Abstract Naringina
6、se is a kind of glycosidase complex, which composed of -L-rhamnosidase (EC 3.2.1.40) and -D-glucosidase (EC 3.2.1.21). Naringinase can specifically hydrolyze naringin into naringenin and it has many important application value, such as debittering of citrus fruit juice, increasing beverage aroma, in
7、dustrial preparation of rhamnosidase and flavonoids. However, owing to the immaturity of the technology of naringinase fermentation and preparation craft lacking, causes the price of naringinase enzyme preparation is too expensive, which limited the development of the naringinase applied technology.
8、 Therefore, establishes the mature technology of naringinase formalization fermentation and the reasonable preparation craft has the important significance. This paper, Aspergillus niger DB056 was the naringinase-producing strain, after the study on scale-up technology of naringinase fermentation, t
9、he naringinase preparation craft and its optimization were investigated into, followed by the food safety testing and the storage stability discussion. The main results were as follows: 7 L, 20 L and 200 L Naringinases fermentation scale-up research indicated, two components of naringinase, -L-rhamn
10、osidase and -D-glucosidase, of which enzyme activites had a certain range of fluctuation along with the process of gradually scale-up fermentation, but the change tendency of enzyme activities was consistent; The maximum enzyme activities of -L-rhamnosidase and -D-glucosidase were 1069 U/mL and 727
11、U/mL respectively in the 200 L scale fermentation, exceeding that in 7 L and 20 L scale fermentation. In this study, the 200 L scale fermentation of naringinase produced by Aspergillus niger DB056 was achieved successfully, suggesting that the strain owned the favourable fermentation performance and
12、 application value on the industry. The naringinase solid enzyme preparation were obtained by the process of freezing centrifugation, aseptic filtration, ultrafiltration enrichment and spray drying, during which the drying aid was maltodextrin and the content of the solid shape was 27%. Took the rec
13、overy of -L-rhamnosidase as a stand and on the basis of the single factor experimental result, the spray drying parameters were optimized by means of response surface analysis. The best spray drying condition was as follow: inlet air temperature 111.3, feeding speed 161.5 mL/h, hot air flow rate 5.1
14、 m3/min, outlet air temperature 60, atomizer pressure 0.4 MPa. Under this condition, the recovery of -L-rhamnosidase was 79.7%. Using the established craft steps and parameters, IV solid enzyme preparation of naringinase with 7.5% water content was made, in which, the activity and total recovery of
15、-L-rhamnosidase reached 19000 U/g and 78.1%, respectively, and that of -D-glucosidase were 4700 U/g and 28.3%, respectively. As to the results of food safety detection, the aspects of naringinase solid preparation which has been made, including the total number of bacterial colonies, coliform bacter
16、ium, Salmonella and heavy metal content, met completely the requirement for the safety standard of food industry. During the preserve process, the facts of temperature, air and humidity had great influence for the storage stability of naringinase solid enzyme preparations but the light is not obvious. The retention rates of -L-rhamnosidase and -D-glucosidase of the naringinase solid enzyme preparation preserved in the condition that is -20, cacuum and evades the
