论文总字数：18373字

摘 要

海藻糖又名蕈糖，海藻糖是一种结构上易于衍生化的含有羟基的二糖化合物，在酵母菌等真菌中存在广泛。海藻糖能增强细胞的抗胁迫能力，提高在恶劣环境下的耐受力。在食品、医疗、化妆品和分子生物学等方面用途广泛。

本次实验以酶-DNS法测定酵母内海藻糖含量，相对于纸层析法、TLC法、HPLC法等方式，酶-DNS法测定结果稳定不易产生波动，结果较为精确，不受实验设备的限制，特异性高。测定市场销售安琪高活性干酵母中的海藻糖含量使用微波-浸提法提取，以酶-DNS法定量测定酵母胞内的海藻糖含量，即通过对比消解破碎酵母膜结构方式，发现微波法很有优势，所需设备易得，操作简单，花费时间少，再通过比较各溶剂的优劣，最终经综合考虑选择了三氯乙酸，并以单因素法与正交法的方式选择最优条件。在以上结果基础上得到最佳提取工艺，对实验室已有的10株酿酒酵母菌株进行筛选，YPD摇瓶培养后，菌乳用上述工艺处理，得到海藻糖含量，通过比较选取出海藻糖含量最高的菌株。

以上实验结果表明提取工艺的最佳条件是微波功率为119W、微波消解处理时间为30s、提取剂三氯乙酸浓度为0.5mol/L、三氯乙酸体积为60mL、浸提温度为25℃。高含海藻糖菌株为标号为7号的菌株。

关键词：活性干酵母 海藻糖 提取 测定

Study on Extraction Technology and Highly Containing Strains of Trehalose from Yeast

Abstract

Trehalose also known as mushroom sugar, is formed by the combination of two glucose molecules through the hemiacetal hydroxyl a non-reducing disaccharide stable, widely exists in yeast and fungi. Trehalose can enhance the ability of cells to resist stress and improve tolerance in harsh environments. In the food, medical, cosmetics, agriculture and molecular biology, etc,have this wide application.

In this study, the intracellular trehalose content of yeast was quantitatively determined by enzyme-DNS method. Compared with paper chromatography, TLC, HPLC and anthrone colorimetric methods, the stability of the enzyme-DNS method was not easy to fluctuate, the results are more accurate, not limited by the experimental equipment, high specificity. The trehalose content in Angelica high activity dry yeast was determined by microwave-extraction method. The trehalose content in yeast cells was quantitatively determined by enzyme-DNS method, that is, the cell membrane was broken by microwave digestion and then treated with trichloroacetic acid the extraction of trehalose from cells was carried out by means of single factor method and orthogonal design. The extraction process of yeast was optimized. The optimum extraction conditions were obtained on the basis of the above experiment. After 10 foliage yeast strains were incubated with YPD medium, the trehalose content in the yeast was quantitatively determined. The highest trehalose content was selected by experiment strain for preservation.

The experimental results show that the optimum extraction process is microwave power 119W, microwave treatment time is 30s, the concentration of trichloroacetic acid is 0.5mol / L, the volume of trichloroacetic acid is 60mL, and the extraction temperature is 25 ℃. High containing trehalose strain is a strain labeled No. 7.

Key Words: Active dry yeast; Trehalose; Extraction; Determination.

目 录

摘要 I

Abstract i

第一章 文献综述 1

1.1 海藻糖概述 1

1.1.1 海藻糖的应用 1

1.2 酵母菌概述 2

1.3 酵母提取海藻糖工艺 2

1.3.1海藻糖的提取方法 2

1.3.2海藻糖的测定方法 3

1.3.3高产海藻糖酵母选育 4

第二章 酵母提取海藻糖工艺条件的优化 6

2.1 材料与方法 6

2.1.1 实验菌种 6

2.1.2 实验仪器与试剂 6

2.1.3 实验主要培养基和溶液配制 7

2.1.4 仪器与设备 7

2.1.5 实验与分析方法 7

2.2 结果与讨论 11

2.2.1葡萄糖标准曲线的建立 11

2.2.2 活性干酵母胞内海藻糖提取工艺优化 12

第三章 高含海藻糖菌株的筛选 18

3.1 材料与方法 18

3.1.1 实验菌种 18

3.1.2 实验仪器与试剂 18

3.1.3 实验主要培养基和溶液配制 18

3.1.4 仪器与设备 19

3.1.5实验操作 19

3.2 结果与分析 20

第四章 结论与展望 21

4.1 主要结论 21

4.2 展望 21

参考文献 22

致谢 25

第一章 文献综述

1.1 海藻糖概述

海藻糖是一种结构上易于衍生化的含有羟基的二糖化合物，在酵母菌等真菌中存在广泛，含量占干重比例大^[1]。大量研究表明^[2],某些酵母菌在特殊外界条件（比如高温）下显现出的抗逆性和海藻糖含量有紧密联系，并且少量外源海藻糖就可以明显增强细胞抵抗不适环境的能力^[3]。海藻糖它能在一些不适状态下保护生物体细胞及蛋白质、核酸等组分，保护功能巨大，应用前景广阔^[4]。

1.1.1 海藻糖的应用^{[5- 11]}

海藻糖的用途十分多样，在多个领域和行业发挥了重要作用。海藻糖最主要的用途是用来保存生物制品，比如酶类（主要是用于生物工程方面的酶类）、生物组织、生物体内的活性物质等。海藻糖也可应用于食品加工,化工等方面。

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