酵母菌以玉米浆为氮源发酵产柠檬酸的研究

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摘要：在含有葡萄糖和酵母提取物的培养基中，解脂耶罗维亚酵母SWJ-1b（Yarrowia lipolytica SWJ-1b）能产生大量的柠檬酸。我们发现玉米浆是一种能够刺激柠檬酸生产的经济氮源，并且能够通过调节柠檬酸合成途径中的相关酶活力促使代谢途径中的碳代谢流向柠檬酸。同时，我们以CaCO3作为发酵培养基的中和剂进行实验。结果发现，添加在柠檬酸发酵培养基中的各成分的最佳含量为玉米浆1.0 g/l，CaCO3 40.0 g/l，葡萄糖60.0 g/l。在此条件发酵下，摇瓶中的柠檬酸产量为49.2 g/l，异柠檬酸产量为5.9 g/l。进一步以3-L发酵体系进行发酵，柠檬酸产量达到52.3 g/l，产率达到0.9 g/g 葡萄糖，在288小时内的柠檬酸生产力为0.18 g/l/h。而异柠檬酸产量仅为5.5 g/l，发酵结束时葡萄糖利用率达到96.7%。65062

关键词：柠檬酸，玉米浆，Yarrowia lipolytica，CaCO3，发酵

Abstract: Yarrowia lipolytica SWJ-1b is able to produce a large amount of citric acid in the medium containing glucose and yeast extract. It was found that corn steep liquor (CSL) could stimulate citric acid production as an economic alternative providing nitrogen, and could improve carbon flux to citric acid by adjusting the activities of enzymes involves in citric acid biosynthesis pathway. At the same time, CaCO3 was added in the medium as a neutralizer. It was found that 1.0 g/l of CSL, 40.0 g/l of CaCO3, 60.0 g/l of glucose were the most suitable for citric acid production. Under the optimal conditions, 49.2 g/l of citric acid and 5.9 g/l of isocitric acid was yield in shake flask cultivations. During 3-L fermentation, 52.3 g/l of citric acid, a yield of 0.9 g/g of glucose, and a productivity of 0.18 g/l/h were reached within 288 h of the fermentation. Only 5.5 g/l of isocitric acid were attained and 96.7% of glucose was used in this process.

Key words: citric acid, corn steep liquor, Yarrowia lipolytica, CaCO3, fermentation

1 前言 3

2 材料和方法 5

2.1 材料 5

2.2 方法 6

2.3 产物产量测定 6

2.4 酶活力测定 7

3 结果 7

3.1 玉米浆对Y. lipolytica柠檬酸发酵的影响 7

3.2 以玉米浆为氮源时Y. lipolytica胞内碳通量变化 8

3.3 发酵最佳CaCO3添加量的确定 8

3.4 发酵最佳碳源初始浓度确定 9

3.5 发酵过程的产物产量及葡萄糖含量变化 10

4 讨论 11

结论 13

参考文献 14

致谢 16

1 前言

柠檬酸（Citric acid）又名枸橼酸, 其学名为3-羟基-3-羧基戊二酸，是三羧酸循环中的关键中间产物，在自然界中分布广泛，主要存在于柠檬、柑橘、梅子等果实中，广泛应用于食品、医药及日化等众多领域，是一种十分重要的有机酸。目前，柠檬酸全世界消耗量已达120万吨，年产能达160万吨，市场贸易量为90多万吨，且每年以7%的增长率增长。柠檬酸的生产方法主要有天然植物提取法及生物发酵法两种，其中，90.0%以上的柠檬酸是通过微生物发酵的方式获取的。我国发酵柠檬酸的主要原料有淀粉、玉米粉、葡萄糖、稻米粉及木薯粉等，而欧美等国家则采用葡萄糖，淀粉等精料进行发酵，因而我国的柠檬酸产品一直因较低的价格在世界柠檬酸市场上占绝对优势[1]。