蜜环菌产甘露醇发酵条件与甘露醇测定方法优化研究.pdf

河南大学硕士学位论文蜜环菌产甘露醇发酵条件与甘露醇测定方法优化研究姓名:范书军申请学位级别:硕士专业:微生物学指导教师:朱显峰 2011-05 I 摘要蜜环菌具有重要的药用价值,而甘露醇的含量是蜜环菌系列产品的重要质量指标之一。因此,对蜜环菌菌粉中甘露醇的含量进行有效精确测定研究,以及对蜜环菌产甘露醇的发酵条件进行研究至关重要、意义重大。本研究分两部分进行。第一部分以确立蜜环菌菌粉中甘露醇含量的测定方法为目标,通过对醇提取法、水提取法、超声波破碎法的比较,筛选出最优的蜜环菌菌粉预处理方法;通过样品液与浓盐酸沸水浴共热的方法除去果糖对甘露醇含量测定的干扰;通过对碘量法与分光光度法的深入比较, 选择出最佳的甘露醇含量测定方法。最终结果为: 以水为溶剂, 80 ℃操作温度下对蜜环菌菌粉进行超声波破碎处理,相对醇提取法和水提取法能有效提高甘露醇测定值;浓盐酸与样品液沸水共热可有效去除果糖对甘露醇含量测定的干扰;在存在葡萄糖等单糖对甘露醇干扰的情况下,分光光度法测定值相对碘量法更准确。通过精密度试验、呈色稳定性试验、供试样品液稳定性试验、重复性试验以及回收率实验的验证,最终确立蜜环菌菌粉的预处理方法为超声波破碎法、去除果糖对甘露醇含量测定的干扰的方法为样品液与浓盐酸共热法,甘露醇含量的最佳测定方法为分光光度法。第二部分以提高蜜环菌中甘露醇含量为目标,对蜜环菌发酵培养基及发酵条件进行了系统优化。通过碳源、氮源的单因素试验、碳氮比例的筛选试验、无机盐与 VB 1 以及微量元素混合液的正交试验,最终确立了最优发酵培养基,其配比为:葡萄糖: 2 % 、红薯粉: 2 % 、玉米粉: 30 g 、麸皮: 20 g 、 KH 2 PO 4 : % 、 MgSO 4?7H 2 O : % 、 VB 1 : 5 mg 、微量元素混合液: 6 mL 、水: 1000 mL ;通过对培养基的初始 pH 、接种量、装液量、温度、转速进行筛选,最终确立了最适发酵条件,内容为:培养基初始 pH : 5 ,接种量: 6 % ,装液量: 60 mL ,温度: 25 ℃,转速: 160 r/min 。关键词:蜜环菌,甘露醇,分光光度法,发酵条件 III ABSTRACT The Amillariella mellea has the important value for medicinal purposes. The mannitol content is an important indicators of quality for Amillariella mellea . So it is necessary to study the mannitol content in Amillariella mellea and the fermented conditions of using Amillariella mellea to produce mannitol. This research has two purpose of the first part is to establish a method to quickly determinate the mannitol in Amillariella mellea powder. The experimentation were studied from three aspects: the first was to choose an effective method of extraction, the second was a method to eliminate the interference from frucdose and the third was to choose an accurate mannitol determination method between the iodometric method and the colorimetric method. The result was that The ultrasonic wave breaking method which choose water as the solution and set the operating teperature in 80 °C is more effective than the other methods of water extraction method and ethanol extraction method. When 3 mL concentrated hydrochloric acid a