【作者】 张丽杰；

【导师】 邸进申；

【作者基本信息】 河北工业大学 ， 化学工艺， 2004， 硕士

【摘要】 海藻糖是一种非还原性双糖，其独特的保护功能能使生物大分子(如蛋白质、核酸等)在外界高温、脱水等恶劣环境下免受伤害，近几年成为中外科学家研究的热点。 本文以安琪活性干酵母复壮后的鲜酵母为出发菌株，在确定测定方法和摇瓶培养观察的前提下，分别对其进行紫外、化学和微波诱变。在紫外诱变过程中，对出发菌株共进行了三轮紫外诱变，照射时间是40秒～60秒，每轮照射后，从15株菌中筛选正突变高的菌株进行下一轮的诱变。在进行的第三轮诱变时筛选到一株高产菌株，其海藻糖的产量为19.52g／100g干酵母。化学诱变联合使用了两种低毒性诱变剂，两种诱变剂的最佳配比是：5—溴尿嘧啶300μ mol／L和盐酸平阳霉素5μ mol／L，诱变菌株中最高海藻糖产量为15.98g／100g干酵母。经微波诱变后获得的高产菌株海藻糖的产量为17.01g／100g干酵母。 本文在诱变的同时对酵母菌的操作条件和培养基也进行了优化，在找到海藻糖发酵体系操作最佳条件即最适摇床转速为240r／min、最适温度为35℃的前提下，对培养基中的碳源浓度、氮源浓度、氮碳比、无机盐和微量元素的组成和含量也进行了探讨，从而找出最优培养基配方。 对从活性干酵母中提取海藻糖的工艺进行了研究。作者用蒸馏水从活性干酵母中提取海藻糖的最佳条件是：温度80℃、料液比1：20(干酵母的质量和提取剂的体积比，g／ml)，提取1个小时，海藻糖的产量是16.32g海藻糖／100g干酵母，pH值对提取率没有影响。并比较了不同提取剂对海藻糖提取的影响，其中用1％的氧化钙溶液提取，产量可达21.54g海藻糖／100g干酵母。 在研究从啤酒废酵母中提取海藻糖的工艺时，对啤酒废酵母进行了特殊的处理，即把废酵母在烘箱中烘干一定时间后，用粉碎机制成废酵母粉。并比较了传统提取方法(用冷凝回流装置提取)和索式提取法的产量。实验表明，用索式提取海藻糖的产量约是传统提取产量的3倍，提取工艺为：料液比3：50(废酵母粉的质量和提取剂的体积比，g／ml)，恒温水浴温度为80℃，真空度0.09Mpa下，提取一小时，产量为6.505g海藻糖／kg干废酵母，而所测啤酒废酵母中海藻糖的总含量是7.98g海藻糖／kg干废酵母。 研究了提取液的纯化、精制及结晶工艺。用碱金属除蛋白、活性炭脱色、离子交换树脂除杂离子和进一步脱色。离子交换树脂采用001×7型阳离子交换树脂和201×7型阴离子交换树脂组成的混合床，阴阳离子交换树脂的体积比为2：1。精制后的提取液浓缩到质量百分数为15％左右，用4倍体积的无水乙醇结晶一周，可得柱状晶体，结晶得率为80％。更多还原

【Abstract】 Trehalose is a nonreducing disaccharide of glucose.It has an excellent ability to protect biological cells(such as protein,nucleic acid,etc)from damage caused by drying and dehydration,so it has been the studing focus for scientists in the recent years.In this paper taking AnQi active fresh yeast after rejuvenation as starting strains,mutation was obtained by UV, chemical reagents and microwave heating on the base of making sure the measuring method and observation of fermentation culture.During UV mutation process,there were three turns of UV-mutation on the starting strains in all and the mutation time was 40-60 seconds.After one turn of mutation , strains with high mutation ratio were selected from fifteen strains to receive the next turn for mutation.In the three turns of mutation a high yielding trehalose strain was screened and its trehalose yeilds was 19.52g /100g dry yeast.During chemical mutation two chemical reagents with low toxicity were used together and the best proportion of the two reagents were described as follow: 5-Bu was 300 μ mol/L and Pingyangmycin hydrochloride was 5 μ mol/L. Of the chemical mutation the highest yielding of strain was 15.98g / 100g dry yeast.Through microwave mutation the highest yielding of strain was 17.01g / 100g dry yeast.At the same time the operation conditions and cultrue medium of the yeast strain were also optimized.The best operation conditions for trehalose fermentation system was described as follows: the best ration speed was 240r/min and temperature was 35℃.On this base,the carbon source concentration nitrogen source concentration , proportion of carbon and nitrogen source concentration, constitute and content of inorganic salt and microelement also were investigated to find the best culture medium in which the yeast strain synthesize trehalose furthest.Technology of extraction trehalose from active dry yeast was studied.Taking distilled water as extraction solvent,the maximum extraction ratio of trehalose from active yeast that was 16.32gtrehalose / 100g dry yesat was obtained when the extraction temperature was 80℃, ratio of dry yeast weight and distilled water volum was 1:20 and the extraction time was one hour and pH had no effect on extraction ratio of trehalose.By comparing the effects of different extraction solvent on the extraction ratio of trehalose, high yield of trehalose ,21.54g trehaolse /100g dry yeast, could be obtained when 1 % GaO solution as extraction solvent.To study the technology of extraction trehalose from waste beer yeast,the material was dealed with in a peculiar way in which waste yeast was heated in oven for a period of time then made into powder and compared traditional extraction method with Suoshi extractionmethod.The experimental results in this paper showed that the extraction ratio of trehalose by Suoshi method was almost three times as much as by traditional method.The extraction technology was described as follow: ratio of waste yeast weight and distilled water volum was 3:50,temperature was 80℃, degree of vacuum was 0.09Mpa, the time of extraction was one hour and the yield of trehalose was 6.505gtrehalose / kg dry waste yeast,however the all content of trehalose in waste beer yeast was 7.98g trehalose/kg dry waste yeast.Purification , refining and crystallization of prosess were also studied.Alkali metals were used to remove protein, active carbon was used to remove pigments and ion exchange resin to remove motley ions and pigments further. 001×7 cation exchange resin and 201×7 anion exchange resin composed the mixed ion exchange column and the volume proportion of 201×7 anion exchange resin and 001×7 cation exchange resin was 2:1.When extraction solvent containing trehalose after refining was condensed to almost mass proporation 15 % and crystalled in ethanol of four times as much as extraction solvent volum for one week ,crystal of trehalose with prismatic shape could be obtained and the crystal ratio was 80%.更多还原