摘要：本课题通过探究添加海藻酸钠的含量、添加凹凸棒石的含量、固定化温度、氯化钙浓度以及添加吐温含量对固定化酵母产生γ-癸内酯的影响，从而确定最佳固定化工艺。与此同时，还进行了发酵时间对产物含量的影响以及细胞重复性使用测试，旨在验证固定化细胞较游离态细胞的优势。发酵条件: 发酵培养基(体积为30.0mL)在摇床转速为 250r/min，培养温度为30℃的环境下发酵 48h。发酵产生的γ-癸内酯利用气象色谱仪进行分析测定。结果发现当海藻酸钠的含量为2%；固定化温度为30℃；氯化钙浓度为2%；凹凸棒石的加入量为2%；反应培养基中吐温-80的含量为0.2%时；γ-癸内酯的含量为该因素中的最大值。同时，在发酵时间以及重复利用次数的比较中，固定化细胞产物的减少量小于游离态细胞；在二次重复利用中，固定化细胞产物较上次的减少量远远小于游离态细胞，说明固定化细胞较游离态细胞在发酵时间以及重复性利用方面具有一定优势。

关键词：固定化工艺；耶式酵母；γ-癸内酯；海藻酸钠

Effects of Different Immobilized Bacteria Processes on Biotransformation Production of γ-decanolide

Abstract: In this study, the effects of adding sodium alginate, adding attapulgite, immobilization temperature, calcium chloride concentration and adding Tween content on immobilized yeast to produce γ-decalactone were studied to determine the optimal immobilization Process. At the same time, the effect of fermentation time on product content and cell repetitive use tests were also performed to verify the advantages of immobilized cells compared to free cells. Fermentation conditions: Fermentation medium (30.0 mL in volume) was fermented for 48 h at a shaking speed of 250 r/min and a culture temperature of 30°C.The γ-decalactone produced by fermentation was analyzed by gas chromatography. The results showed that when the sodium alginate content was 2%; the immobilization temperature was 30°C; the calcium chloride concentration was 2%; the attapulgite addition amount was 2%; the content of Tween-80 in the reaction medium was 0.2% Gamma-decalactone content is the maximum of this factor. At the same time, in the comparison of fermentation time and number of reuses, the amount of immobilized cellular product was less than that of free cells; in the second repeated use, the amount of immobilized cellular product was much less than that of the free cells, indicating that the immobilized cells were immobilized. Cells have some advantages over free cells in terms of fermentation time and reusability.

Key words：Immobilization process； Yarrowia sp.;   γ-decalactone； Sodium alginate

目  录

1绪论 1

1.1γ-癸内酯的合成及研究现状 1

1.1.1γ-癸内酯的合成 1

1.1.2γ-癸内酯的研究现状 2

1.2固定化工艺 3

1.3海藻酸钠包埋原理 4

1.4本课题的目的及研究意义 4

1.4.1研究目的 4

1.4.2研究意义 5

2材料与方法 5

2.1原料和试剂的准备以及配置方法 5

2.2实验仪器 6

2.3实验方法 6

2.3.1酵母菌悬液的制备 6

2.3.2单因素实验 6

2.3.3发酵时间对产物含量的影响 7

2.3.4细胞重复性使用测试 8

2.3.5样品的处理 8

2.3.6标准曲线的绘制 8

2.3.7待测样品中γ-癸内酯含量的测定 8

3 结果与分析 9

3.1γ-癸内酯标准曲线 9

3.2海藻酸钠浓度的确定 9

3.3固定化温度的确定 10

3.4氯化钙浓度的确定 10

3.5凹凸棒石浓度的确定 11

3.6吐温添加量的确定 12

3.7发酵时间对产物含量的影响 12

3.8细胞重复性使用测试 13

4 结论 14

致谢 15

参考文献 16

1绪论

1.1γ-癸内酯的合成及研究现状

1.1.1γ-癸内酯的合成

γ-癸内酯，别名丙位癸内酯，为γ内酯类的包含十个碳原子的化合物。分子式为C10H18O2，相对分子质量为170.25[19]  （结构式如图1.1）。γ-癸内酯在常温下是一种无色液体，呈愉快的桃子和椰子香气[7]。