灰树花液态发酵多糖富集研究

【作者】 卞冬萍；

【导师】 方维明；

【作者基本信息】 扬州大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 灰树花(Grifola Frondosa)是一种珍稀食药两用真菌,其在工厂快速液态培养过程中可生物合成具有生物活性且对人体无毒副作用的多糖。灰树花液态发酵合成真菌多糖为保健食品的生产提供了新的途径,并具有显著的经济意义。本研究探索了灰树花的液态发酵培养基组成及发酵条件,应用正交法对灰树花多糖提取和纯化条件进行了优化,为进一步开发灰树花保健品提供了理论依据,并为液态发酵生产灰树花菌丝多糖和综合开发利用食用菌多糖资源奠定了基础。为测定灰树花菌丝体内多糖的含量,研究了蒽酮比色法,利用蒽酮遇硫酸脱水生成的糠醛或其衍生物,可与蒽酮试剂发生缩合反应,生成蓝绿色化合物在610nm处有最大吸收,吸收值与糖含量呈线性关系,建立了标准曲线,其回归方程为:y = 0.112x + 0.0154,R2 = 0.9982(x—610nm吸光度,y—葡萄糖浓度μg/mL)。本研究对灰树花液态培养的培养基进行了单因素试验和正交试验。选择培养基中碳源和氮源因素,研究它们在各种水平下对灰树花液态培养(或称发酵)富集多糖的影响。根据单因素试验结果,对碳源(葡萄糖+马铃薯)、氮源(麸皮)的浓度、无机盐以及生长促进剂(酵母膏)这四个因素分别选取四水平进行L16(4~5)正交实验,试验指标为菌丝干重和多糖量,得到优化的培养基组成为:在1L培养基中含马铃薯300g ,葡萄糖30g,麸皮35g,磷酸二氢钾4g,硫酸镁1g,酵母膏1.5g,VB1 1mg/1000mL。试验同时表明,碳源对菌丝干重的影响比较显著,而无机盐的含量及其比例对多糖含量有显著的影响。对灰树花液态培养的工艺条件进行了单因素试验和正交试验,以得到的较优培养基为基础,研究灰树花液态培养和产生多糖的外界条件。对pH值、温度、接种量、摇床转速和摇瓶中装液量,进行了单因素试验和正交试验。由于温度和装液量可变化程度很小,因而温度确定为25℃和装液量确定为10%摇瓶容积,故只选取pH值、接种量和摇床转速三因素,各因素取三水平,进行L9(34)正交实验,实验指标为菌丝干重和多糖量。综合考虑各指标,得到较优的工艺条件是温度25更多还原

【Abstract】 Grifola frondosa, is a rare edible and medicinal fungi which can biosynthesize polysaccharide of bioactivity and less poisonous side-effect on our health during its liquid culture. The liquid culture of Grifola frondosa is a new approach to develop new health food for epiphyte polysaccharide and has prominent economic value. This study explored the composing of culture medium and the condition of liquid fermentation on biosynthesizing polysaccharide of Grifola frondosa. And studied the condition of extracting polysaccharide and purification by orthogonal experiments. These offered theories to develop new health food of Grifola frondosa. Also established bases for making good use of polysaccharide resources from Grifola frondosa mycelium and from edible fungus in biology reactor.In order to determine the content of polysaccharide in Grifola frondosa mycelium, the author studied the anthraceneketone colorimetry. As anthraceneketone reagent can react with furfural and its ramification which was created from the reaction of polysaccharide and vitriol . The reaction can create cyan compound which had the most absorption on 610nm, and the absorption cost had linearity relationship with the content of polysaccharide.The standard curve was set up, and the regressed equation was: y = 0.112x + 0.0154,R² = 0.9982（x- absorbency on 610nm, y-the consistence of glucose （μg/mL ））.By the single factor experiments and orthogonal experiments, the optimum culture medium was established. The influence of carbon resource and nitrogen resource in culture medium on polysaccharide-accumulating fermentation were studied respectively. On the basis of the single factor experiments, orthogonal experiments were performed on carbon resource （potato/glucose）、nitrogen resource （bran）、abio-salt （KH₂PO₄ and MgSO₄） and growth-accelerant （yeast cream） with dry weight of mycelium and the content of polysaccharide as criterion. Considering every criterion synthetically, the更多还原