【作者】 李卓；

【导师】 王莘；

【作者基本信息】 吉林农业大学 ， 生物物理学， 2008， 硕士

【摘要】 紫丁香蘑（Lepista nuda）为伞菌目,白蘑科,香蘑属的一种药食皆用的野生真菌。目前,对紫丁香蘑的林地栽培获得子实体的试验进行了研究,而对紫丁香蘑的液体发酵及有效成分的提取研究的比较少。食药用菌中占70%以上的游离糖和游离糖醇是由一二种糖和糖醇组成的,紫丁香蘑中主要以海藻糖为主,含量较高。海藻糖可应用于食品、医药、化妆品等多方面的研究。我国食用菌资源丰富,借助食用菌开发海藻糖具有独特的优越性。本文对液体发酵紫丁香蘑海藻糖的提取、纯化、鉴定以及应用进行研究。本研究主要试验结果如下:1.以菌丝体干重和海藻糖的含量为测定指标,确定紫丁香蘑液体发酵培养基的组成。试验得出,最适氮源为酵母膏,碳源为葡萄糖,最初pH值为7.0。2.紫丁香蘑菌丝体最佳提取工艺参数研究。通过对紫丁香蘑菌丝体进行单因素和3因素3水平的正交试验,研究提取时间、温度、乙醇浓度对紫丁香蘑菌丝体海藻糖提取的影响。影响提取的各因素顺序:温度>乙醇浓度>提取时间。最佳提取参数最优组合为A₂B₁C₂,即提取时间为1h,温度为70℃,乙醇浓度30%。得到的海藻糖的含量为12.92%,蛋白含量0.62%。3.海藻糖的纯化。采用Sevag法除蛋白。Sevag法中海藻糖溶液与氯仿-正丁醇溶液的比例不同时,不但对蛋白去除率有影响,而且对海藻糖的损失程度也不同,综合考虑,最后确定除蛋白的最佳海藻糖溶液与氯仿及正丁醇的体积比为5:1:0.25。通过测定蛋白含量,从而选择了除蛋白的次数为二次,经二次除蛋白后的海藻糖溶液中的蛋白含量为0.3%。通过单因素和4因素3水平试验,对除蛋白后的海藻糖溶液进行活性炭脱色,活性炭脱色的最佳工艺为:调节pH值为4,加入3%的活性炭,60℃下搅拌10min,脱色率达到72.04%,海藻糖含量为8.06%。脱盐的最佳工艺条件为:1mL/min,pH值6,此时脱色率在90%以上,脱盐率在97%以上。4.薄层层析鉴定海藻糖。通过在不同的展开剂中Rf值的测定,确定展开剂为正丁醇:吡啶:水=15:3:2,点样量1.0ul,硫酸和甲醇配制成1:4的溶液,105℃的干燥箱中加热使之显色。结晶海藻糖得率为2.24%,海藻糖含量92.56%。5.海藻糖应用的研究表明,海藻糖具有一定的抗冷冻和抗疲劳作用。在抗冷冻方面,通过观察不同时间鱼糜色泽、气味、水质三个因素的变化。在抗疲劳方面,海藻糖对小鼠游泳试验、爬绳试验、肝糖原、血清尿素氮都有显著影响。更多还原

【Abstract】 Lepista nuda was belong to Agaricales of Tricholomtaceae.Lepista nuda not only has edible value,but also has officinal value.The research of experiment on collecting fruit body by woodland culture of lepista nuda has been test.For the liquid fermnentation of Lepista nuda and it’s extraction of active component, there are few reaserch about this topic.In domestic fungus,more than 70%of lib-sug and lib-sugar alcohol are made up of one or two kinds of sugar.The main component in Lepista nuda is Trehalose,and it’s content is rather great.The Trehalose can be used to study food,cosmetic and medicine.In our country,the domestic fungus are very plenteous,so developing the Trehalose have great advantage by domestic fungus. In this study,considering the Lepista nuda-hyphal as my studying object,the extraction,purification, assessment and application for the Trehalose of the Lepista nuda liquid fermentation hyphostroma were studied in this exprement.The result that was investigated are following:1.According to the dry weight of hyphostroma and cont of trehalose confirm the medium’s composition in the lepista nuda liquid fermentation.The experiment shows that the most compatible nitrogen source is yeast extract,and the carbon source is glucose,and the inital pH is 7.2.The study of best extract craft parameter for the Lepista nuda hyphostroma.Through single-factor and the orthogonal design,which is to three factors and three levels,I study the extracting time and temperature,and study the effect,which alcohol saturation impact the extraction of Trehalose from Lepista nuda.The best extracting parameter is A₂B₁C₂ The data I have studied as follows:time is 1h;the temperature is70℃;concentration of ethanol is 30%.The cont of Trehalose I gained is 12.92%,and protein level is 0.62%.3.Purification of Trehalose.And I adopted the Sevag to deproteinize.In this experiment,I found as follows:in Sevag,when the ratio of sample volume between Trehalose and chloroform n-butanol is different,it does’t only effect rate of deproteinization,but also effect the lossing degree of Trehalose.I sumed up the conclusion from the materials above as follows:the best volume ratio between the Trehalose, chloroform and n-butanol is 5:1:0.25.According determining protein level,I choose the time of deproteinization is twice.After twice deproteinization,the protein level in the Trehalose solution is 0.3%. Using four factors and three levels for activated carbon decolorization,after the addition of the Trehalose solution,the optimal craft of decaloration by activated carbon was:adjusting pH to 4,put in 3%activated carbon,agitating 10min in 60℃.The bleaching rate is 72.04%;cont of Trehalose is 8.06%.The best craft condition is 1mL/min;pH is 6.In this time,the bleaching rate is more than 90%,and desalting rate is more than 97%.4.Using TLC to accredit Trehalose.spreading agent:n-butanol:Pyr:water=15:3:2;Sample is 1.0ul.The liquor rate:vitriol:Meoh =1:4.heat it under 105℃in baking oven in order to colorate.The ratio of crystallization is 2.24%,and the content of Trehalose is 92.56%.5.The study shows that the Trehalose has the function of anti- freezing and anti-fatigue.In the part of anti-freezing,I observe the color,and smell and water quality of Fish wasteful in different time.In the part of anti-fatigue,the Trehalose can have the influence on the Comparison of time to grasp the rope of mice, Comparison of time to swim of mice,serum urea nitrogen and hepatic glycogen.更多还原