CN112843103A - 白蜡多年卧孔菌培养、菌丝体黄酮提取方法及其黄酮复方制品 - Google Patents
白蜡多年卧孔菌培养、菌丝体黄酮提取方法及其黄酮复方制品 Download PDFInfo
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- CN112843103A CN112843103A CN202110245356.1A CN202110245356A CN112843103A CN 112843103 A CN112843103 A CN 112843103A CN 202110245356 A CN202110245356 A CN 202110245356A CN 112843103 A CN112843103 A CN 112843103A
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- Prior art keywords
- flavone
- mycelium
- parts
- extract
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Abstract
本发明涉及了白蜡多年卧孔菌培养、菌丝体黄酮提取方法及其黄酮复方制品。一种白蜡多年卧孔菌菌丝体黄酮提取方法，称取一定质量的白蜡多年卧孔菌菌丝体粉末放入锥形瓶中，加入体积比为85%的乙醇后进行超声提取，收集上层提取液；白蜡多年卧孔菌菌丝体粉末与85%的乙醇的料液比例为1:30～40；将提取液放入离心机内，离心分离，收集上清液；将上清液减压浓缩回收乙醇，待溶液浓缩至原来体积2～5％；将所得膏状物冷冻干燥得到干燥固体粉末，即为白蜡多年卧孔菌菌丝体黄酮物质提取物。一种白蜡多年卧孔菌菌丝体黄酮复方制品，以质量份计，由白蜡多年卧孔菌菌丝体黄酮物质提取物、铁皮石斛提取物、红景天各1～2.5份添加辅料组成。
Description
技术领域
本发明涉及健康产品加工领域，尤其是涉及一种白蜡多年卧孔菌菌丝体高密度发酵获得，菌丝体黄酮复方制品的制备方法。
背景技术
白蜡多年卧孔菌是一种多功能的药用真菌，成分复杂，药用广泛。白蜡多年卧孔菌中含有较强的抗肿瘤、抗心血管病的功效。白蜡多年卧孔菌中还含有大量酮类，具有极强的抗氧化性及抑菌活性，能有效清除机体内部自由基，缓解机体的衰老并对慢性肾炎有很强的治疗效果。白蜡多年卧孔菌黄酮具有免疫调节、抗氧化、消炎杀菌、保护心脑血管系统，缓解疲劳等保健作用。白蜡多年卧孔菌菌丝体黄酮物质的提取及其应用已成为近年新研究方向。
白蜡多年卧孔菌在国内温带和亚热带均有分布，且其菌株经组织分离获得，经过分子生物学手段鉴定（鉴定ITS-DNA条形码序列如下，其基因序列已上传NCBI的genebank，其GenBank号: KU341718.1）。菌种保存于鲁东大学菌种保存中心，菌种保藏编号HMLD090929，河南城建学院生命科学与工程学院，菌种保藏编号HNCJ2019-WKJ。
白蜡多年卧孔菌（Perenniporia fraxinea）ITS-DNA条形码序列：GCGGAAGGATCATTATCGAGTTTTGAAAGGGGTTGTAGCTGGCCTTCCGAGGCATGTGCACGCCCCGCTCAATCCACTCTACACCTGTGCACTTACTGTGGGTTTCGGAGGTGAAGCGTGCTTTCGCTCGCGGATCTAACGGGCCCGCGTTTTACTACAAACACTTTAAAGTAAACGAACGTGTATCGCGATGTAACGCATCTATATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATGAAATTCTCAACCTACCGGTCTTTGCGGATCGGTAAGGCTTGGACTTGGAGGCTTGTCGGCCCGTGTGGTCGACTCCTCTCAAATGCATTAGCCTGGTTCCTTGCGGATCGGCTCTCGGTGTGATAATTGTCTACGCCGCGACCGTGAAGCGTTTGGCTGGCTTCTAACCGTCTCGATGGAGACAACT
但目前白蜡多年卧孔菌通常作为药用真菌被对待，尚无应用于保健品领域。天然采集获取的白蜡多年卧孔菌子实体不能满足资源的集约化利用。为高效利用白蜡多年卧孔菌资源，目前急需一种能够将白蜡多年卧孔菌黄酮物质充分利用的技术方式。
而且，之前公开的白蜡多年卧孔菌菌丝体培养方法，获得的最大白蜡多年卧孔菌菌丝体干重不够理想。
发明内容
本发明针对现有技术的空白，提出一种白蜡多年卧孔菌菌丝体黄酮物质提取方法，并充分利用白蜡多年卧孔菌菌丝体黄酮和红景天、铁皮石斛提取物，提出一种白蜡多年卧孔菌菌丝体黄酮保健品以及该复方制品的制备方法，为高效利用白蜡多年卧孔菌资源提供了一种新的途径。本发明同时针对现有技术白蜡多年卧孔菌培养方法的不足，并对其进行改进，培养获得最大白蜡多年卧孔菌菌丝体干重可达到25.2 g/L。
本发明采用的技术方案：
一种白蜡多年卧孔菌菌丝体黄酮提取方法，包括步骤如下：
1）称取一定质量的白蜡多年卧孔菌粉末放入锥形瓶中，加入体积比为85% 的乙醇后进行超声提取，收集上层提取液；白蜡多年卧孔菌粉末与85% 的乙醇的料液比例为1:30～40；超声提取过程中，提取温度50～65 ℃，超声功率设置为95～100W，提取时间120～160min，其中白蜡多年卧孔菌菌丝体黄酮得率达到1.5～1.8 %.
可选择的，再向残渣中加入体积比为 85%的乙醇进行再次超声提取，合并提取液；
2）将提取液放入离心机内，控制离心机转速在3600-4400 r/min，离心时间为15～20min，离心分离，收集上清液；
3）将上清液减压浓缩回收乙醇，待溶液浓缩至原来体积 2～5％；将所得膏状物冷冻干燥得到干燥固体粉末，使用冷冻干燥机，其参数指标设置真空度40-50 pa，冷阱温度为-45 ℃，过夜24 h。即为白蜡多年卧孔菌菌丝体黄酮物质提取物。
一种白蜡多年卧孔菌菌丝体黄酮复方制品，以质量份计，由白蜡多年卧孔菌菌丝体黄酮物质提取物、铁皮石斛提取物、红景天各1～2.5份，添加辅料玉米微孔淀粉、β-环糊精、海藻酸钠组成，其添加量为：玉米微孔淀粉2～5份、β-环糊精1.5～4份、海藻酸钠1～2.5份。
铁皮石斛包含的主要物质有多糖、石斛碱、氨基酸以及微量元素，因其中的多糖含量最高，包括多糖、氨基酸和微量元素、菲类以及少量生物碱等化学成分，可增强机体免疫力、降低血糖、抵抗疲劳、解热等，抗氧化、降抵血糖、抑菌等功效。红景天有补气、清肺、益智养心、止血、消肿、散瘀以及抗氧化清除自由基的功效。
本发明充分利用了白蜡多年卧孔菌黄酮和红景天、铁皮石斛提取物，具有免疫调节、抗氧化、消炎杀菌、保护心脑血管系统，缓解疲劳等作用。
其中铁皮石斛提取物：将铁皮石斛鲜品，去除叶及茎上的杂物，清水冲洗干净，然后切成1.5厘米左右的小段，放置于60℃烘箱中，干燥至恒重。将干燥的铁皮石斛用粉碎机粉碎，过60目筛，使用超声波提取法进行提取，溶剂采用100%甲醇，时间为2h，提取时功率为160W。无水甲醇混合、揺匀，放进超声波提取装置中进行提取。提取一段时间后，取出冷却至室温减压抽滤，收集滤液。滤液用旋转蒸发仪蒸干。
其中红景天：将切好的新鲜红景天单层平铺于真空冷冻盘中，真空度为40～50Pa，冷阱温度为-45℃，自动模式解析和干燥，干燥时间35 h，冻干后的红景天放入高速万能粉碎机中粉碎至粒度0.2～0.3 mm。干燥至恒重。将干燥的铁皮石斛用粉碎机粉碎，过100目筛。
一种白蜡多年卧孔菌菌丝体黄酮复方制品制备方法，1）将玉米微孔淀粉与白蜡多年卧孔菌菌丝体黄酮、红景天、铁皮石斛提取物按照3:1的质量比例进行混匀搅拌；2）搅拌均匀后加入质量份数为40%的β-环糊精溶液；3）继续搅拌待表面的乙醇溶液挥发后加入一定份数海藻酸钠即可得到颗粒状的淡黄色白蜡多年卧孔菌菌丝体黄酮复方制品。
一种白蜡多年卧孔菌培养方法，使用葡萄糖10 g/L，糖蜜25.8 g/L，酵母提取物5.7 g/L，黄豆粉3.2 g/L，磷酸二氢钾3.4 g/L作为发酵培养基，培养温度28 ℃，装液量45%，初始pH值调整为6.2，发酵罐转速325rpm，10%接种量培养7 d，发酵培养物过100目筛绢，冷冻过程使用冷冻干燥机，其参数指标设置真空度40-50pa，冷阱温度为-45℃，过夜24 h，即制得所述白蜡多年卧孔菌菌丝体。该方法获得的最大白蜡多年卧孔菌菌丝体干重达到25.2 g/L。获取的白蜡多年卧孔菌菌丝体保存到-20℃冷冻备用。
发明有益效果：
1、本发明采用超声醇提白蜡多年卧孔菌菌丝体黄酮和铁皮石斛提取物，提取效率高。其中对白蜡多年卧孔菌菌丝体黄酮物质的提取采用优化后的超声辅助乙醇提取法，与传统的双水相提取法和微波提取法相比具有提取率高、操作简单、提取成本低等优点，适用于大规模工业生产，是一种高效生产技术。
本发明对白蜡多年卧孔菌的黄酮的工艺优化尚属首次，目前尚未见对白蜡多年卧孔菌菌丝体黄酮类成分提取的报道。采用单因素和响应面优化法考量到提取温度、乙醇体积分数、提取时间、液料比四个变量考察对白蜡多年卧孔菌总黄酮提取的影响，确定最佳的提取方案。通过方程拟合，实际验证，最终确定白蜡多年卧孔菌菌丝体黄酮得率为1.5～1.8%。
2、本发明白蜡多年卧孔菌菌丝体黄酮复方制品，复方制品稳定，为高效利用白蜡多年卧孔菌资源提供了一种新的途径。充分利用了白蜡多年卧孔菌菌丝体黄酮和铁皮石斛提取物，具有抗氧化、消炎杀菌、保护心脑血管系统，缓解疲劳等保健作用，服务于亚健康人群。本发明充分利用了白蜡多年卧孔菌菌丝体黄酮和红景天、铁皮石斛提取物三者的结合起到抗氧化性能协同增效的作用，同时也充分体现了君臣佐使的中药成方配伍原则。
本发明表征了白蜡多年卧孔菌菌丝体黄酮较好的抗氧化效果，尤其是与抗氧化传统型药物铁皮石斛、红景天的配伍使用起到较理想的协同增效作用。
3、通过本发明制备方法制得的白蜡多年卧孔菌菌丝体黄酮复方制品，将白蜡多年卧孔菌与红景天、铁皮石斛组合制备黄酮复合制品，提供了一种黄酮制品的抗氧化性能的优势组合。复方制品稳定，操作简便。本发明制备的白蜡多年卧孔菌菌丝体黄酮可在人体内长时间多次发挥作用，避免了一次摄入过多黄酮物质对机体的损害，适之更适合老年人服用。
4、本发明使用液体发酵法获取白蜡多年卧孔菌菌丝体黄酮，其中前期有文献报道使用配方“葡萄糖37.7 g/L，酵母粉8.3 g/L，磷酸二氢钾6.31 g/L”，10%接种量培养120 h，最大产量达到19.47 g/L.。而本研究使用新配方，10%接种量培养7 天达到25.2 g/L，每升提高5.73g。在食药用菌高密度发酵领域上生物量干重有了显著性的提高，这为进一步的利用其制备菌丝体黄酮提供了较理想的物质基础和应用可能。
附图说明
图1为白蜡多年卧孔菌菌丝体黄酮芦丁样品标准曲线；其中，芦丁标准的回归方程为Y=0.705X+0.0057，R2=0.9976（n=3）；
图2：白蜡多年卧孔菌黄酮与红景天、铁皮石斛提取物配伍后抗氧化试验DPPH指标；
图3：白蜡多年卧孔菌黄酮与红景天、铁皮石斛提取物配伍后抗氧化试验羟基指标。
具体实施方式
下面通过具体实施方式，对本发明技术方案做进一步的详细描述。
实施例1
本发明白蜡多年卧孔菌菌丝体黄酮提取方法，包括步骤如下：
1）称取一定质量的白蜡多年卧孔菌粉末放入锥形瓶中，加入体积比为85% 的乙醇后进行超声提取，收集上层提取液；白蜡多年卧孔菌粉末与85% 的乙醇的料液比例为1:30～40；
可选择的，再向残渣中加入体积比为 85%的乙醇进行再次超声提取，合并提取液；
2）将提取液放入离心机内，离心分离，收集上清液；
3）将上清液减压浓缩回收乙醇，待溶液浓缩至原来体积 2～5％；将所得膏状物冷冻干燥得到干燥固体粉末，即为白蜡多年卧孔菌菌丝体黄酮物质提取物。
实施例2
本发明白蜡多年卧孔菌菌丝体黄酮的取方法，与实施例1不同的是：步骤1)超声提取过程中，提取温度50～65 ℃，超声功率为95～100W，提取时间120～160 min；步骤2）的离心分离过程中，控制离心机转速在3600-4400 r/min，离心时间为15～20min；步骤3）的冷冻过程使用冷冻干燥机，其参数指标设置真空度40-50 pa，冷阱温度为-45 ℃，过夜24 h。
本实施例具体采用的白蜡多年卧孔菌粉末与85% 的乙醇的料液比例为36 mL/g ，超声提取功率为95～100W，提取温度61°C，提取时间151 min n，其中得率为1.64 %，离心时间20min。其中菌丝体黄酮检测使用酶标仪检测，使用的黄酮芦丁样品标准曲线如图1所示。经过测定白蜡多年卧孔菌菌丝体黄酮得率达到1.5～1.8 %。
参见表1、表2、表3。其中：
表1：影响白蜡多年卧孔菌菌丝体黄酮提取的4个主要参数响应面实验设计的因素与水平梯度
表2：影响白蜡多年卧孔菌菌丝体黄酮提取的4个主要参数对提取率影响的Box-Behnken实验设计及实验数据结果
表3：影响白蜡多年卧孔菌菌丝体黄酮提取的4个主要参数回归方程系数显著性检验结果
注：*表示在5 %的水平内呈显著；**表示在1 %的水平内呈显著
根据软件Design Expert 11分析得到的数据结果详见。以回归系数来建立白蜡多年卧孔菌菌丝体中提取黄酮类化合物的得率（Y）与提取时间（A）、乙醇体积分数（B）、提取时间（C）以及液料比（D）的二次回归方程见式（1）：
Y=1.64+0.0308×A+0.0451×B+0.0124×C+0.0293×D+0.015×AB-0.0057×AC+0.0057×AD+0.0520×BC+0.0132×BD-0.016×CD-0.2019×A2-0.2165×B2-0.2353×C2-0.2152×D2 （1）
依照表3回归方程系数显著性检验结果的方差分析即可知：白蜡多年卧孔菌菌丝体中提取黄酮响应面实验的P值小于0.0001，而提取温度的二项式、乙醇体积分数的二项式、提取时间的二项式、液料比的二项式结果均小于0.0001，即非常显著，而失拟项为0.1406大于0.05为不显著，说明本次实验结果能够有效地表明实际情况。同时回归方程的R2=0.9486，即表示该响应面数据有94.86 %是可靠的，能够依照此回归模型的结果来确定白蜡多年卧孔菌菌丝体中提取黄酮类化合物的最佳提取工艺。
以下是本发明白蜡多年卧孔菌菌丝体黄酮复方制品及其制备方法。以质量份计，由白蜡多年卧孔菌菌丝体黄酮物质提取物1～2.5份、铁皮石斛提取物1～2.5份、红景天1～2.5份，添加辅料玉米微孔淀粉、β-环糊精、海藻酸钠组成，其添加量为：玉米微孔淀粉2～5份、β-环糊精1.5～4份、海藻酸钠1～2.5份。
实施例3
本发明白蜡多年卧孔菌菌丝体黄酮复方制品，以质量份计，包括玉米微孔淀粉2份、β-环糊精1.5份、白蜡多年卧孔菌菌丝体黄酮物质提取物1份、海藻酸钠1份、红景天1份、铁皮石斛提取物1份。
实施例4
本实施例的白蜡多年卧孔菌菌丝体黄酮复方制品，含有玉米微孔淀粉3份、β-环糊精1.5份、白蜡多年卧孔菌菌丝体黄酮物质提取物1份、海藻酸钠1份、红景天1份、铁皮石斛提取物1份。
实施例5
本实施例的白蜡多年卧孔菌菌丝体黄酮保健品，含有玉米微孔淀粉4份、β-环糊精2份、白蜡多年卧孔菌菌丝体黄酮物质提取物1.5份、海藻酸钠1.5份,包括红景天1.5份、铁皮石斛提取物1.5份。
实施例6
本实施例的白蜡多年卧孔菌菌丝体黄酮保健品，含有玉米微孔淀粉3份，β-环糊精2份、白蜡多年卧孔菌菌丝体黄酮物质提取物1.5份、海藻酸钠1份、包括红景天1.5份、铁皮石斛提取物1.5份。
实施例7
本实施例的白蜡多年卧孔菌菌丝体黄酮保健品，含有玉米微孔淀粉2份，β-环糊精2份、白蜡多年卧孔菌菌丝体黄酮物质提取物、海藻酸钠、红景天、铁皮石斛提取物各1份。
实施例8
本实施例的白蜡多年卧孔菌菌丝体黄酮保健品，含有玉米微孔淀粉3份，β-环糊精2份、白蜡多年卧孔菌菌丝体黄酮物质提取物1.5份、海藻酸钠1份、红景天1.5份、铁皮石斛提取物1.5份。
实施例9
本实施例为白蜡多年卧孔菌菌丝体黄酮复方制品制备方法，包含步骤如下：
1）将玉米微孔淀粉与白蜡多年卧孔菌菌丝体黄酮、红景天、铁皮石斛提取物按照3:1的质量比例进行混匀搅拌；
2）搅拌均匀后加入质量份数为40%的β-环糊精溶液；
3）继续搅拌待表面的乙醇溶液挥发后加入一定份数海藻酸钠即可得到颗粒状的淡黄色白蜡多年卧孔菌菌丝体黄酮复方制品。
其中，白蜡多年卧孔菌菌丝体黄酮、红景天、铁皮石斛提取物按照配伍范围选取。
实施例10
本实施例所述的白蜡多年卧孔菌菌丝体黄酮复方制品制备方法，与实施例7的区别在于，进一步的：α淀粉酶和糖化酶与玉米微孔淀粉的质量比为1:100，其中α淀粉酶和糖化酶用量比例为1：1；超声波清洗器温度为38～43°C，超声功率为95～100W，超声反应时间为30～40min。
实施例11
本实施例的白蜡多年卧孔菌菌丝体黄酮复方制品制备方法，与实施例7或实施例8不同的是：白蜡多年卧孔菌菌丝体黄酮提取物的提取过程如下：
1）称取一定质量的白蜡多年卧孔菌粉末放入锥形瓶中，加入 85% 的乙醇后进行超声提取，收集上层提取液，再向残渣中加入体积比为 85%的乙醇进行再次超声提取，合并提取液；将提取液放入离心机内，以 4000 r/min的转速离心，时间为15～20min，收集上清液；
2）将上清液减压浓缩回收乙醇，待溶液浓缩至原来体积 2～5％；将所得膏状物冷冻干
燥得到干燥固体粉末，即为白蜡多年卧孔菌菌丝体黄酮物质提取物。
其中，白蜡多年卧孔菌粉末与85% 的乙醇料液比例为1:30，超声提取功率为90～95W，提取温度37～45°C，提取时间25～35min。离心机离心时间为15～20min。
实施例12
本实施例公开了一种白蜡多年卧孔菌菌丝体黄酮复方制品实验室制备方法，其原料组分为：玉米微孔淀粉60g、β-环糊精25g、白蜡多年卧孔菌菌丝体黄酮物质提取物20g、海藻酸钠15g、红景天20g、铁皮石斛提取物20g。
制备方法如下：
取上述配方量的玉米微孔淀粉、白蜡多年卧孔菌菌丝体黄酮提取物和β-环糊精，将玉米微孔淀粉、白蜡多年卧孔菌菌丝体黄酮提取物、红景天、铁皮石斛提取物混匀搅拌。搅拌均匀后加入质量分数为40 %的 β-环糊精溶液。继续搅拌待表面的乙醇溶液挥发后即可得到的颗粒状的淡黄色颗粒即为白蜡多年卧孔菌菌丝体黄酮复方制品。
实施例13
一种白蜡多年卧孔菌菌丝体黄酮保健品原料组分为：玉米微孔淀粉40g，、β-环糊精15g、白蜡多年卧孔菌菌丝体黄酮物质提取物14g、海藻酸钠14g、红景天20g、铁皮石斛提取物20g。制备方法操作步骤如实施例12所述。
实施例14
一种白蜡多年卧孔菌菌丝体黄酮保健品原料组分为：其原料组分为：玉米微孔淀粉30g，白蜡多年卧孔菌菌丝体黄酮物质提取物8g、β-环糊精9g、海藻酸钠7g、红景天8g、铁皮石斛提取物8g。操作步骤如实施例12所述。
本实施例白蜡多年卧孔菌菌丝体黄酮复方制品，减少了黄酮提取物的添加含量。白蜡多年卧孔菌复方制品在人体内随着摄入时间的延长可持续发挥作用，可有效避免一次摄入过多黄酮造成对人体的危害。
以上实施例中抗氧化的协同增强在于白蜡多年卧孔菌菌丝体黄酮与红景天、石斛提取物，三者比例为1:1:1的量效比关系。实施例基于此比例原则进行拟定。
图2、图3为白蜡多年卧孔菌菌丝体黄酮物质提取物与红景天、石斛提取物配伍后抗氧化主要指标实验结果DPPH指标和羟基指标。由图2、图3可知，随着溶液浓度的增加各溶液对自由基的清除率呈现正相关，而三者配伍后对DPPH自由基和·OH自由基的清除率明显优于配伍前，提高了白蜡多年卧孔菌黄酮复合制品的抗氧化性。
实施例15
本实施例为白蜡多年卧孔菌培养方法，使用葡萄糖10 g/L，糖蜜25.8 g/L，酵母提取物5.7 g/L，黄豆粉3.2 g/L，磷酸二氢钾3.4 g/L作为发酵培养基，培养温度28 ℃，装液量45 %，初始pH值调整为6.2，发酵罐转速325rpm，10%接种量培养7 d，发酵培养物过100目筛绢，冷冻过程使用冷冻干燥机，其参数指标设置真空度40-50pa，冷阱温度为-45℃，过夜24 h，即制得所述白蜡多年卧孔菌菌丝体。该方法获得的最大白蜡多年卧孔菌菌丝体干重达到25.2 g/L。获取的白蜡多年卧孔菌菌丝体保存到-20℃冷冻备用。用于菌丝体黄酮的提取以及白蜡多年卧孔菌黄酮物质复方制品的制备。
Claims (10)
1.一种白蜡多年卧孔菌菌丝体黄酮提取方法，包括步骤如下：
1）称取一定质量的白蜡多年卧孔菌菌丝体粉末放入锥形瓶中，加入体积比为85% 的乙醇后进行超声提取，收集上层提取液；白蜡多年卧孔菌菌丝体粉末与85% 的乙醇的料液比例为1:30～40；
可选择的，再向残渣中加入体积比为 85%的乙醇进行再次超声提取，合并提取液；
2）将提取液放入离心机内，离心分离，收集上清液；
3）将上清液减压浓缩回收乙醇，待溶液浓缩至原来体积 2～5％；将所得膏状物冷冻干燥得到干燥固体粉末，即为白蜡多年卧孔菌菌丝体黄酮物质提取物。
2.根据权利要求1所述的白蜡多年卧孔菌菌丝体黄酮的提取方法，其特征在于：超声提取过程中，提取温度50～65 ℃，超声功率设置为95～100W，提取时间120～160 min。
3.根据权利要求1或2所述的白蜡多年卧孔菌菌丝体黄酮的取方法，其特征在于：步骤2）离心分离过程中，控制离心机转速在3600-4400 r/min，离心时间为15～20min；步骤3）冷冻过程使用冷冻干燥机，其参数指标设置真空度40-50 pa，冷阱温度为-45 ℃，过夜24 h。
4.一种白蜡多年卧孔菌菌丝体黄酮复方制品，其特征在于：以质量份计，由白蜡多年卧孔菌菌丝体黄酮物质提取物1～2.5份、铁皮石斛提取物1～2.5份、红景天1～2.5份，添加辅料玉米微孔淀粉、β-环糊精、海藻酸钠组成，其添加量为：玉米微孔淀粉2～5份、β-环糊精1.5～4份、海藻酸钠1～2.5份。
5.根据权利要求4所述的白蜡多年卧孔菌菌丝体黄酮复方制品，其特征在于：以质量份计，包括玉米微孔淀粉2～4份、β-环糊精1.5～3份、白蜡多年卧孔菌菌丝体黄酮物质提取物1份、海藻酸钠1份、红景天1份、铁皮石斛提取物1份。
6.根据权利要求4所述的白蜡多年卧孔菌菌丝体黄酮复方制品，其特征在于：以质量份计，含有玉米微孔淀粉3-5份、β-环糊精2～4份、白蜡多年卧孔菌菌丝体黄酮物质提取物1.5份、海藻酸钠1.5份,包括红景天1.5份、铁皮石斛提取物1.5份。
7.根据权利要求4所述的白蜡多年卧孔菌菌丝体黄酮复方制品，其特征在于：以质量份计，含有玉米微孔淀粉2-5份，β-环糊精2份、白蜡多年卧孔菌菌丝体黄酮物质提取物2.5份、海藻酸钠2.5份、包括红景天2.5份、铁皮石斛提取物2.5份。
8.一种白蜡多年卧孔菌菌丝体黄酮复方制品制备方法，由白蜡多年卧孔菌菌丝体黄酮物质提取物1～2.5份、铁皮石斛提取物1～2.5份、红景天1～2.5份以及10～25份辅料组成，其中所述辅料由玉米微孔淀粉、β-环糊精、海藻酸钠组成，其特征在于：
1）将玉米微孔淀粉与白蜡多年卧孔菌菌丝体黄酮、红景天、铁皮石斛提取物按照3:1的质量比例进行混匀搅拌；
2）搅拌均匀后加入质量份数为40%的β-环糊精溶液；
3）继续搅拌待表面的乙醇溶液挥发后加入一定份数海藻酸钠即可得到颗粒状的淡黄色白蜡多年卧孔菌菌丝体黄酮复方制品。
9.根据权利要求8所述的白蜡多年卧孔菌菌丝体黄酮复方制品制备方法，其特征在于：铁皮石斛提取物的提取过程包括步骤如下：将铁皮石斛鲜品，去除叶及茎上的杂物，清水冲洗干净，然后切成1.5厘米左右的小段，放置于60℃烘箱中，干燥至恒重；将干燥的铁皮石斛用粉碎机粉碎，过60目筛，使用超声波提取法进行提取，溶剂采用100%甲醇，时间为2h，提取时功率为160W；无水甲醇混合、揺匀，放进超声波提取装置中进行提取；提取一段时间后，取出冷却至室温减压抽滤，收集滤液，滤液用旋转蒸发仪蒸干。
10.一种白蜡多年卧孔菌培养方法，使用葡萄糖10 g/L，糖蜜25.8 g/L，酵母提取物5.7g/L，黄豆粉3.2 g/L，磷酸二氢钾3.4 g/L作为发酵培养基，培养温度28 ℃，装液量45 %，初始pH值调整为6.2，发酵罐转速325rpm，10%接种量培养7 d，发酵培养物过100目筛绢，冷冻过程使用冷冻干燥机，其参数指标设置真空度40-50pa，冷阱温度为-45℃，过夜24 h，即制得所述白蜡多年卧孔菌菌丝体。
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| CN114292343B (zh) * | 2021-12-31 | 2022-11-04 | 华南师范大学 | 一种白蜡多年菌胞外多糖和胞内多糖制备方法及其在调节肠道微生物菌群与降血糖应用 |
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