论文总字数：19320字

摘 要

苹果酸是一种常见的天然有机酸，也是三羧酸循环的中间产物，因此在人体、动物、微生物体中广泛存在。所以，从外部摄入的时候也很容易被人体接受，这决定了苹果酸在食品加工和医疗医药行业的使用非常广泛。以为有手性分子的存在，苹果酸以L-苹果酸和D-苹果酸两种信使存在，并且以L-苹果酸的存在居多。从许多水果中都可以提取出苹果酸。目前,L-苹果酸的主要生产方法分为化学合成法和生物合成法两种，生物合成法方法较多，有酶转化法、一步、二步发酵法等。

本课题以酿酒酵母为研究对象，研究酿酒酵母生产苹果酸的发酵条件优化。为了进行优化，以达到提高苹果酸的产量目的，研究采用单因素实验，通过控制单一变量来研究其最佳值。首先将发酵温度变量梯度化，结果发现在发酵温度为30℃时，苹果酸产量获得最大值，故选取温度区间28-33℃作为自变量进行响应面实验。然后设计生物素浓度梯度实验，试验结果表明当培养基生物素浓度取0.05 mg/L时，产量达到峰值，故选取生物素浓度区间0.025-0.15mg/L作为自变量进行响应面实验。最后进行葡萄糖的单因素实验，实验结果表明葡萄糖浓度取100 g/L时，苹果酸产量获得最大值，因此选取葡萄糖浓度80-120g/L作为自变量进行响应面实验。

以发酵温度、生物素浓度和葡萄糖浓度这三个因素为自变量，苹果酸产量为响应值，设计响应面实验，并建立模型的回归方程。为了确定模型的准确性以及各因素对最终产量的影响程度，本研究对模型的回归方程进行了方差分析以及验证实验。最终得出了各影响因素的最终优化准确值：最佳发酵温度32.6℃、生物素浓度0.09 mg/L、葡萄糖浓度90.32 g/L以及苹果酸产量的预测值14.15 g/L。

通过在得到的最优发酵条件下进行验证实验，得到苹果酸产量的平均值15.08 g/L，其与模型回归方程预测值十分接近，因此回归模型准确。

关键词：苹果酸 酿酒酵母 条件优化 响应面

Abstract

Malic acid is a common natural organic acid and an intermediate product of the tricarboxylic acid cycle, and thus is widely present in humans, animals, and microorganisms. It is known from its spatial structure that there is an asymmetric carbon atom in the malic acid molecule, so it has two spatial isomers. In the nature, it is mainly L-malic acid(the left-handed body), which is present in the fruit pulp of fruits such as grapes and apples, and can also be obtained by biofermentation of fumaric acid. It is an important intermediate product of the internal circulation of the human body and is also easily absorbed by the human body. Therefore, it is widely used in food and medicine industries. At present, the main production methods of L-malic acid are extraction method, chemical synthesis method, one-step fermentation method, two-step fermentation method, immobilized enzyme or cell transformation method. The lack of good production strains is also a major problem at present. The cultivation of excellent strains and optimization of extraction techniques are hot topics at home and abroad.

In this paper, Saccharomyces cerevisiae was used as the research object to study the fermentation conditions for the production of malic acid by Saccharomyces cerevisiae. In order to optimize the purpose of increasing the yield of malic acid, the study used a single factor experiment to study the optimal value by controlling a single variable. First, the fermentation temperature variable was graded. It was found that the malic acid yield was the highest when the fermentation temperature was 30 °C. Therefore, the response surface experiment was performed with the temperature range of 28-33 °C as the independent variable. Then the biotin concentration gradient experiment was designed. The results showed that when the concentration of biotin in the medium was 0.05 mg/L, the yield peaked. Therefore, the biotin concentration range of 0.025-0.15 mg/L was used as the independent variable for the response surface experiment. Finally, a single factor experiment of glucose was carried out. The results showed that the yield of malic acid was the highest when the glucose concentration was 100 g/L. Therefore, the response surface experiment was performed with the glucose concentration of 80-120 g/L as the independent variable.

Then, through the three factors of fermentation temperature, biotin concentration and glucose concentration, the malic acid yield was the response value, the response surface experiment was designed, and the regression equation of the model was established. In order to determine the accuracy of the model and the extent to which each factor affects the final yield, this study performed a variance analysis and validation experiment on the regression equation of the model. Finally, the final optimized value of each influencing factor was obtained: the optimal fermentation temperature was 32.6 °C, the biotin concentration was 0.09 mg / L, the glucose concentration was 90.32 g / L and the predicted value of malic acid production was 14.15 g / L. Through the verification experiment under the optimal fermentation conditions obtained, the average value of malic acid production was 15.08 g/L, which is very close to the predicted value of the model regression equation, so the regression model is accurate.

Key words: malic acid, Saccharomyces cerevisiae, condition optimization, response surface

目 录

摘要…………………………………………………………………………………Ⅰ

Abstract……………………………………………………………………………Ⅱ

1. 文献综述 …………………………………………………………………1

1.1引言 ………………………………………………………………………1

1.2 苹果酸的理化性质 ………………………………………………………1

1.3 苹果酸的应用 ……………………………………………………………1

1.4 苹果酸的合成方法 ………………………………………………………1

1.4.1 苹果酸的化学合成 ……………………………………………1

1.4.2 苹果酸的生物合成 ……………………………………………2

1.5 生物法制备苹果酸的研究进展 …………………………………………3

1.5.1 苹果酸生产菌株的研究进展 …………………………………3

1.5.2 苹果酸生产发酵过程研究进展 ………………………………3

1.6本文的研究目的与研究内容 ……………………………………………4

第二章 材料与方法 ………………………………………………………………6

2.1引言 ………………………………………………………………………6

2.2菌株 ………………………………………………………………………6

2.3试剂 ………………………………………………………………………6

2.4仪器 ………………………………………………………………………7

2.5培养基 ……………………………………………………………………8

2.6菌体活化培养方法 ………………………………………………………8

2.7发酵方案 …………………………………………………………………8

2.8分析与检测 ………………………………………………………………9

第三章 结果与分析 ………………………………………………………………10

3.1 单因素实验及讨论 ………………………………………………………10

3.1.1种子培养基选取的影响 …………………………………………10

3.1.2温度的影响 ………………………………………………………10

3.1.3葡萄糖浓度的影响 ………………………………………………11

3.1.4生物素浓度的影响 ………………………………………………12

3.2 响应面实验及讨论 ………………………………………………………13

3.2.1回归模型的建立 …………………………………………………13

3.2.2回归模型方差分析 ………………………………………………14

3.2.3 响应曲面图及其等高线 …………………………………………13

3.2.4回归模型的验证 …………………………………………………16

3.3本章小结 …………………………………………………………………16

第四章 结论与展望 ………………………………………………………………17

4.1结论 ………………………………………………………………………17

4.2展望 ………………………………………………………………………17

参考文献 ……………………………………………………………………………18

致谢 …………………………………………………………………………………20

1. 文献综述
   1. 引言

苹果酸^[1]，也称2－羟基丁二酸，一般状态下以白色结晶体或粉末状晶体呈现，味道以酸味为主，由于对液体有很强的吸湿性，所以易溶于水、乙醇等溶液。在自然世界中以两种异构体的左旋体L-苹果酸为主要形式存在。由于苹果酸参与了三羧酸循环，这证明了苹果酸与人体的亲和性很高。所以目前被广泛运用于食品加工和医疗医药等行业。就目前来看，苹果酸的世界年产量已经到达了一个非常庞大的数字，并且在未来市场需求还有很大的上升空间。若利用好这个庞大的市场，进行降低成本的研究，一定会带来巨大的收益。

• 1. 苹果酸的理化性质

苹果酸^[2]，又名2－羟基丁二酸，分子式为C₄H₆O₅，结构式为HOOC(COH)CCOOH。苹果酸无色无味，有稍许酸味，一般状态下为晶体且溶于水，溶解度为55．59/100mL（20℃），溶于乙醇，不溶于乙醚。有吸湿性，1%(质量)水溶液的pH值2.4。L－苹果酸密度为1.601；熔点131-132℃，分解点150℃。

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