论文总字数：19660字

摘 要

银耳是具有药用价值的真菌之一，其主要活性成分是银耳多糖。因为其功效丰富，备受关注。大多数情况下，真菌产品大多来自田间栽培的子实体，用热水浸提等方法直接提取多糖，这样很难控制产品的最终质量。并且由于银耳的伴生菌——香灰菌的菌种活力退化，会使其多糖产量降低。因此，运用现代发酵技术液体培养银耳菌是一种有效生产次级代谢物的替代方法，既可以缩短生产周期，节省经济成本，也可以控制多糖产品质量，稳定生产。但目前，对于银耳菌液体发酵的研究较少，所以需要对发酵参数进行确定和优化，通过添加氨基酸作为氮源以提高银耳菌丝体和银耳多糖产量，并研究UDP-葡萄糖焦磷酸化酶这一糖代谢中的关键酶，以期找到UGPase酶活与银耳多糖合成的关系。

（1）确定发酵周期为5 d，碳源为葡萄糖，初始pH为7.0，接种菌龄3 d，接种量为10%，培养条件为28.5℃，180 r/min，3 d。使银耳菌菌丝体和银耳多糖产量达到最大的氨基酸分别是添加浓度为0.1 g/L的天冬氨酸和酪氨酸，菌丝体产量达5.97±0.10 g/L，银耳多糖产量达5.19±0.15 g/L。

（2）将制备的多糖样品经HPLC-RID分析，从多糖样品中分离出相对分子质量为1832 kDa、252 kDa的两个组分。再经PMP柱前衍生化方法测定多糖样品的单糖组成，发现其由甘露糖、葡萄糖、半乳糖组成，摩尔比为19.7:80.1:10.4。

（3）在筛选氨基酸及确定最适添加浓度的基础上，筛选出天冬氨酸、酪氨酸和谷氨酰胺这三种氨基酸，测定了添加氨基酸的发酵液中的UGPase酶活，酪氨酸和天冬氨酸这两个产银耳多糖量较高的氨基酸，其UGPase酶活性分别达到121.1971 nmol (min·mL^-1)和131.0908 nmol (min·mL^-1)，分别是空白对照组酶活的1.96倍和2.12倍。其结果揭示，UGPase酶活性与多糖产量呈现正相关，酶活越高，多糖产量越高。

关键词：银耳菌 发酵法制备 银耳多糖 UGPase 氨基酸

Effect of amino acids on the synthesis of extracellular polysaccharides of Tremella fuciformis

Abstract

Tremella fuciformis was regarded as one of the most valuable medicinal fungi. The main bioactive component of T. fuciformis is polysaccharides. At present, it has attracted much attention because of its rich efficacy . In most cases, most of the fungal products come from the fruiting bodies cultivated in the field, and the polysaccharides are directly extracted by hot water extraction. That will lead to poor quality of the product. Because the companion bacteria of the white fungus whose activity may degrade. Meanwhile, the effect of echinomycin produced by its own metabolism will also reduce its polysaccharide yield. Therefore, the use of modern fermentation technology to culture the Tremella fuciformis is an alternative method for the efficient production of secondary metabolites, which can shorten the production cycle, save economic costs, control the quality of polysaccharide products, and stabilize production. However, there are few studies on the liquid fermentation of Tremella fuciformis. Therefore, it is necessary to determine and optimize the fermentation parameters, then increase the yield of Tremella fuciformis and Tremella polysaccharides by adding amino acids as a nitrogen source and study UDP-glucose pyrophosphorylase which is a key enzyme in the synthesis of glycogen, in order to find the relationship between UGPase activity and the synthesis of Tremella polysaccharides.

（1）The fermentation cycle was determined to be 5 d, the carbon source was glucose, the initial pH was 7.0, the inoculum was 3 d, the inoculum was 10%, and the culture conditions were 28.5°C, 180r/min, 3d. The amino acids that maximize the yield of Tremella mycelium and Tremella polysaccharides were aspartic acid and tyrosine at a concentration of 1 g/L, the mycelium yield was 5.97±0.10 g/L, and the yield of Tremella polysaccharides was 5.19±0.15 g/L.

（2）The polysaccharide sample obtained by liquid fermentation is subjected to suction filtration, rotary evaporation, freeze-drying and the like to obtain a polysaccharide sample. By HPLC-RID analysis, two components were separated from the polysaccharide sample, and the relative molecular mass was 1832 kDa，252 kDa respectively.The monosaccharide composition of the obtained component was determined by a PMP pre-column derivatization method, and it was found that the polysaccharide sample consisted of mannose, glucose, and galactose.

（3）On the basis of screening amino acids and determining the optimum concentration, the three amino acids of aspartic acid, tyrosine and glutamine were screened, and the UGPase activity in the fermentation broth with amino acid added was determined.Tyrosine acid and aspartic acid, two amino acids with higher polysaccharides, have UGPase activity of 121.1971 nmol (min·mL^-1) and 131.0908 nmol (min·mL^-1), respectively, which are 1.96 times and 2.12 times of the blank control enzyme activity. UGPase activity was positively correlated with polysaccharide yield.

Key words: Tremella fuciformis；Fermentation preparation；Tremella polysaccharide；UGP enzyme；Amino acids

目 录

摘要 I

Abstract II

第一章 文献综述 1

1.1 银耳多糖研究进展 1

1.1.1 银耳概述 1

1.1.2 银耳多糖研究进展 1

1.2 银耳菌液态发酵现状 1

1.2.1 基础培养条件优化 2

1.2.2 氨基酸添加对银耳菌生物量和银耳多糖产量的影响 2

1.3 UDP-葡萄糖焦磷酸化酶与真菌胞外多糖合成的关系 3

1.4 课题意义 4

1.5 研究目的 4

第二章 材料与方法 5

2.1 实验材料 5

2.1.1 实验菌 5

2.1.2 实验主要药品和试剂 5

2.1.3 实验主要仪器 5

2.2 银耳菌丝体液体发酵培养条件优化 6

2.2.1 菌种活化 6

2.2.2 发酵参数测定 6

2.2.3配制液体种子培养基 7

2.2.4 制备多糖样品 7

2.2.5确定银耳菌液体培养的发酵参数 7

2.3 银耳菌丝体胞外多糖（EPS）单糖组成和相对分子质量的测定 8

2.3.1 测定EPS的单糖组成 8

2.3.2 测定EPS的相对分子质量 9

2.4 测定UDP-葡萄糖焦磷酸化酶活 9

第三章 结果与分析 11

3.1 银耳菌丝体液体发酵培养条件优化 11

3.1.1 银耳菌最佳发酵周期确定 11

3.1.2 碳源对银耳菌生物量及胞外多糖产量的影响 12

3.1.3 氨基酸对银耳菌生物量及胞外多糖产量的影响 13

3.1.4 氨基酸浓度对银耳菌生物量及胞外多糖产量的影响 13

3.2 菌丝体胞外多糖EPS各组分部分理化性质 15

3.2.1 EPS的单糖组成 15

3.2.2 EPS各组分相对分子质量 16

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