论文总字数：22856字

摘 要

花生四烯酸（arachidonicacid；ARA）属于ω-6系列多不饱和脂肪酸（PUFA），系统命名为全顺Δ-5,8,11,14-二十碳四烯酸。ARA是组成人体内生物膜非常重要的部分，并且是如前列腺素、前列环素、血栓烷、白三烯等大量的二十碳酸合成的前体物质。ARA对预防心血管疾病、糖尿病和肿瘤等具有重要功效，还具有酯化胆固醇、增加血管弹性、降低血液粘度、调节血细胞功能等一系列生理活性。因为ARA所具有的这些功能，使得富含ARA的油脂在食品添加剂，化妆品，保健品领域等领域的应用越来越广泛。

传统的ARA油脂来源于蛋黄以及动物内脏，因生产过程复杂价格昂贵已经被微生物发酵法取代。而高山被孢霉（Mortierellaalpina）——ARA油脂的潜在生产菌株也已经被广泛应用于ARA油脂的发酵生产。

利用高山被孢霉发酵生产富含ARA油脂的过程中，仍面临着生物量低，油脂含量低，以及ARA含量低等诸多问题亟待解决。研究发现，菌丝体老化技术可以有效的提高ARA的含量。本论文以高山被孢霉发酵产ARA菌丝体老化过程作为研究对象，分析菌丝体老化过程的调控，研究高山被孢霉发酵产花生四烯酸油脂菌丝老化过程中外源添加对产量的影响。本论文以硝酸钾和硝酸钠为例，研究在老化过程中添加不同浓度的硝酸钾和硝酸钠对高山被孢霉ARA产量的影响。实验结果证明在老化阶段添加硝酸钾对ARA产量会有一定影响，但是结果不是特别明显，添加1g/L的硝酸钾在192h时ARA的含量达到最高；而在老化过程中向培养基中添加2g/L的硝酸钠，在204h，即老化过程的达到60h时，可以达到最高的产率。

菌丝体老化技术和多阶段培养策略都是基于高山被孢霉发酵过程中存在的实际问题提出的行之有效的解决方法，可以有效的提高ARA油脂的产量和ARA的品质，并且操作简便，可用ARA油脂的工业化生产。并且可以为其他类似的PUFA的生产提供借鉴和指导。

关键词：高山被孢霉，油脂组分，花生四烯酸，多阶段发酵调控，老化

ABSTRACT

Arachidonic acid (ARA), 5, 8, 11, 14-cis-eicosatetraenoic acid, is one of the ω-6 polyunsaturated fatty acids. It has various physiological functions, as it can act as a precursor of prostaglandins, thromboxane, prostacyclin, and leukotrienes，some of which play an important role in combating or preventing a number of human diseases, such as diabetes and cardiovascular diseases. Therefore, it has broad applications in medicine, pharmacology, cosmetics, food industry, agriculture, and other fields.

ARA is generally existed in the oil form in the natural world. The traditional sources of ARA-rich oil are egg yolks and some kinds of animal tissues. However, isolating oils from these materials is costly and insufficient to meet the market demand, because these materials only contains mall amounts of ARA. Luckily, microbial oils can be used as an alternative source of ARA. The filamentous fungus, Mortierella alpina, is thought to be the most prominent ARA-rich oil producer.

Until now, various efforts have been made to improve the ARA accumulation in Mortierella alpina, however, the relative low ARA content in the final oil product still limited the product quality and thus restricted its industrial mass scale production. In our previous work, it has been demonstrated that aging technology, which means providing the cell into another culture environment without the carbon sources when normal fermentation ceased (also named as the period after fermentation), could increase the ARA percentage over the total fatty acids (TFA) efficiently. In this paper, we will talk about the aging process and the influence of the exogenous addition on ARA content in the final oil product. Potassium nitrate and sodium nitrate were used as an example to study that different influence of different concentrations of potassium nitrate and sodium nitrate to ARA content in the final oil product. The best concentrations of potassium nitrate is 1g/L in 192h and the best concentrations of sodium nitrate is 2g/L in 204h.

The aging technology and multi-stage fermentation strategy was also designed to resolve the question which hinder ARA-rich oil industrial mass scale production. These strategies proved to be convenient and economical for commercial process. Furthermore, these strategies which was developed to obtain high quality and high yield ARA-rich oil production in M. alpine could be

further improved and applied in other similar polyunsaturated fatty acid-rich oil production by the oleaginous Zygomycete fungi.

KEYWORDS: Mortierella alpina ;lipid composition; arachidonic acid; multi-stage fermentation strategy; aging technology

目录

摘要 II

ABSTRACT III

目录 V

第一章 文献综述 1

1.1 花生四烯酸的性质、来源和生理功能 1

1.1.1 花生四烯酸的理化性质 1

1.1.2 花生四烯酸的用途及生理活性。 1

1.1.3 花生四烯酸的主要来源 2

1.2 微生物发酵产花生四烯酸的研究进展 2

1.2.1 产花生四烯酸菌种的发现和选育 2

1.2.2 微生物发酵产花生四烯酸过程的调控 3

1.2.3 微生物发酵产花生四烯酸的老化过程以及调控 3

1.3本课题的研究内容及意义 4

1.3.1 研究意义 4

1.3.2 研究内容 4

第二章 高山被孢霉发酵产花生四烯酸油脂菌丝体老化过程中外源添加对产量的影响 5

2.1引言 5

2.2 实验材料与方法 5

2.2.1 菌种 5

2.2.2 实验试剂 6

2.2.3 实验仪器 6

2.2.4 培养基 7

2.2.5 培养方法 7

2.2.6 发酵表观数据分析 8

2.3 结果与讨论 9

2.3.1 不同浓度的硝酸钾对高山被孢霉老化过程的影响 9

2.3.2不同浓度的硝酸钾对高山被孢霉老化过程的影响 12

2.3.3 本次实验的不足以及改进方向 14

2.4 本章小结 14

第三章 结论与展望 16

3.1结论 16

3.2 展望 16

参考文献 18

致谢 21

文献综述

1.1 花生四烯酸的性质、来源和生理功能

1.1.1 花生四烯酸的理化性质

花生四烯酸（Arachidonic Acid,简称AA）是一种长链的不饱和脂肪酸，含有二十个碳原子和四个双键，化学名称为5,8,11,14-二十碳四烯酸，分子式为C₂₀H₃₂O₂，结构如图1.1所示，相对分子质量为304.46，熔点为-49.5℃。由于甲基端第六至第七个碳原子之间为第一个双键，故属于n-6系列多不饱和脂肪酸，简记为C20:4(n-6)。

图1.1

1.1.2 花生四烯酸的用途及生理活性。

花生四烯酸为必需脂肪酸（essential fatty acid）（必须脂肪酸还包括亚油酸，亚麻酸），其广泛存在于某些苔藓、海藻和其他植物中, 在牛、猪的肾上腺、肝脏及肺中也有分布^[1]。

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