CN108034590A - 一种猴头菌的培养基、生物转化菌丝体、生物转化菌丝体的提取物及其用途 - Google Patents
一种猴头菌的培养基、生物转化菌丝体、生物转化菌丝体的提取物及其用途 Download PDFInfo
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Abstract
本发明公开了一种猴头菌的培养基、生物转化菌丝体、生物转化菌丝体的提取物及其用途。该猴头菌的培养基包括如下重量份的各组分:绿豆490~350份,麦麸140~100份,谷糠70~50份,水400~600份。该生物转化菌丝体是将猴头菌接入上述培养基经菌丝转化培养所得的产物。该生物转化菌丝体的提取物是将猴头菌丝体经水提后再减压浓缩,最后真空冷冻干燥而制备出来的。该生物转化菌丝体的提取物具有提高免疫力及抗肿瘤作用,可用于制备提高免疫力保健品或抗肿瘤药物。
Description
技术领域
本发明涉及生物制药技术领域,特别涉及一种猴头菌的培养基、生物转化菌丝体、生物转化菌丝体的提取物及其用途。
背景技术
猴头菌是一种猴头菌科蘑菇(真菌),在中药用于治疗胃肠道疾病已经超过2000年的历史了,一系列小分子化合物如erinacines,被认为具有神经再生,并且可以透过血脑屏障修复受损神经组织。猴头菌还有抗溃疡,抗炎,抗微生物,免疫调节,提高肝功能,抗衰老,降低血糖和血脂,提高抗缺氧能力,增加心脏输出、和改善备注循环等作用,包含猴头菌的医用制剂在中国广泛使用。
猴头菌丝体中的活性物质最重要的是多糖及糖蛋白,目前国内外对猴头多糖的研究表明,猴头菌多糖具有多种生物活性和药理作用,能增强巨噬细胞的吞噬功能,促进溶血素的形成,抗白细胞下降,降血糖、抗凝血、抗血栓、抗突变和抗衰老等。因此,猴头菌多糖备受人们关注,成为近年来分子生物学、医药、食品科学等领域研究和开发应用的热点。《中国药典》记载了猴头菌丝体(参见2015年版一部的第1614-1615页),本发明所述的猴头菌丝体就是该标准记载的猴头菌丝体。
当前,从高等真菌及其培养物中进行筛选和发现活性成分,应用于抗肿瘤、抗病毒、和治疗糖尿病等,不仅成为国家重点研究新药的发展方向,也是当今世界各国开发生物医药的热点。
生物转化“菌丝体”是指利用菌丝体(包括真菌)的代谢功能,使有机物分解的生物化学反应过程。以适宜的培养基为营养,通过菌丝体的生长代谢和生命活动产生丰富的次生代谢产物。将绿豆作为培养基组成部分,通过菌丝体转化,次生代谢出生物活性成分,再实行“定向培养、双向转化”生物转化工艺,获得生物活性物质原料,菌类多糖、多肽、牡荆苷等有效成分对提高免疫力或抗肿瘤有良好的作用。
绿豆(Vigna radiata(Linn.)Wilczek.),属于豆科,别名青小豆(因其颜色青绿而得名)、菉豆、植豆等,在中国已有两千余年的栽培史。原产地在印度、缅甸地区。含蛋白质(主要是球蛋白类)、脂肪、碳水化合物、钙、磷、铁、维生素A、B、C等成分。绿豆的药理作用为降血脂、降胆固醇、抗过敏、抗菌、抗肿瘤、增强食欲及保肝护肾。
本发明旨在提供一种新的猴头菌培养基,菌类或菇类培养,实际运用及文献报道的使用棉籽壳、木屑、玉米芯、麦麸、玉米等来培养,很少甚至没有报道使用绿豆来进行菌类培养,本发明使用绿豆作为主要培养基,产生意想不到的效果。
本专利中所述的猴头菌丝体参考专利号为ZL 201410393488.9的《一种猴头菌丝体及其培育方法》,符合《中国药典》2015年版一部的第1614-1615页。
发明内容
本发明的目的在于提供一种新材料(新原料),即一种猴头菌的培养基、生物转化菌丝体、生物转化菌丝体的提取物及其用途,所述生物转化菌丝体的提取物的制备方法简单,成本较低,提取物用于制备提高免疫力保健品或抗肿瘤药物。
基于上述目的,本发明的内容包括一种猴头菌的培养基,包括如下重量份的组分:
绿豆50~70份,麦麸40~20份,谷糠10份,水40~50份。
优选地,所述基础培养基包括如下重量份的各组分:绿豆60份,麦麸30份,谷糠10份,水45份。
本发明还提供一种生物转化菌丝体,制备方法为将猴头菌类菌株接种至所述猴头菌的培养基中,在20~27℃下,猴头菌培育30~40天,烘干,得到生物转化菌丝体。
优选地,所述猴头菌为猴头菌科真菌猴头菌Hericium erinaceum(Bull.ex Fr.)Pers.。
优选地,所述生物转化菌丝体是猴头菌科真菌猴头菌(Hericium erinaceum(Bull.ex Fr.) Pers.)的菌丝体与其附生含有绿豆成份的固体培养基的干燥混合物。
本发明还提供一种生物转化菌丝体的提取物,制备方法为:
(1)将所述生物转化菌丝体进行水提,40-60℃下用水浸泡2-3次,每次浸泡2-5小时,水的重量为生物转化菌丝体重量的6~10倍,得到提取液;
(2)将步骤(1)中的提取液50℃减压浓缩,将减压浓缩所得的浓缩液冷冻干燥,即得生物转化菌丝体的提取物。
优选地,所述用水浸泡的温度为50℃。
优选地,步骤(2)所述冷冻干燥的工艺为:-40℃冷冻2-4小时,-10℃一次升华2-5小时,0℃二次升华5-10小时,30℃三次升华24-36小时。
优选地,所述冷冻干燥的工艺为:-40℃冷冻3小时,-10℃一次升华3小时,0℃二次升华6小时,30℃三次升华30小时。
本发明还提供一种所述的生物转化菌丝体的提取物在制备提高免疫力保健品或抗肿瘤药物中的用途。
本发明的有益效果有:
本发明低温水浸泡生物转化菌丝体,在40-60℃下用水浸泡,无高温破坏,既能溶解有效成分且又不破坏有效成分;为了不影响有关活性物质,本发明优选采用真空冷冻干燥方法,不会破坏有效成分,特别是活性酶类物质。
具体实施方式
实施例1
一种生物转化菌丝体的提取物,其制备方法包括如下步骤:
(1)取农副产品绿豆500g、麦麸400g、谷糠100g,混匀,加入400g水拌匀,即得培养基。再装瓶、灭菌,接入猴头菌,在27℃条件下转化培养约40天,取出菌丝体,烘干,即得生物转化菌丝体;
(2)将生物转化菌丝体进行水提,40℃下用水浸泡2次,每次浸泡5小时,水的重量为生物转化菌丝体重量的6倍,得到提取液;
(3)将上述提取液50℃减压浓缩,将减压浓缩所得的浓缩液真空冷冻干燥,-40℃冷冻2小时,-10℃一次升华2小时,0℃二次升华10小时,30℃三次升华24小时,得到干燥的生物转化菌丝体的提取物(代号:HL-5)。
实施例2
一种生物转化菌丝体提取物,其制备方法包括如下步骤:
(1)取农副产品绿豆600g、麦麸300g、谷糠100g,混匀,加入450g水拌匀,即得培养基。再装瓶、灭菌,接入猴头菌,在27℃条件下转化培养约40天,取出菌丝体,烘干,即得生物转化菌丝体;
(2)将生物转化菌丝体进行水提,50℃下用水浸泡2次,每次浸泡4小时,水的重量为生物转化菌丝体重量的8倍,得到提取液;
(3)将上述提取液50℃减压浓缩,将减压浓缩所得的浓缩液真空冷冻干燥,-40℃冷冻 3小时,-10℃一次升华3小时,0℃二次升华6小时,30℃三次升华30小时,得到干燥的生物转化菌丝体的提取物(代号:HL-6)。
实施例3
一种生物转化菌丝体提取物,其制备方法包括如下步骤:
(1)取农副产品绿豆700g、麦麸200g、谷糠100g,混匀,加入500g水拌匀,即得培养基。再装瓶、灭菌,接入猴头菌,在27℃条件下转化培养约40天,取出菌丝体,烘干,即得生物转化菌丝体;
(2)将生物转化菌丝体进行水提,50℃下用水浸泡3次,每次浸泡3小时,水的重量为生物转化菌丝体重量的10倍,得到提取液;
(3)将上述提取液50℃减压浓缩,将减压浓缩所得的浓缩液真空冷冻干燥,-40℃冷冻 4小时,-10℃一次升华5小时,0℃二次升华5小时,30℃三次升华36小时,得到干燥的生物转化菌丝体的提取物(代号:HL-7)。
实施例4绿豆掺入比例对比试验
培养基绿豆的比率分别占50%、60%、70%,设计实验方案,总投料5000g,分别按上述实施例1、实施例2、实施例3配制培养基,加水混匀、灭菌、接种、培养,平行对照,培养条件完全一致,培养过程中观察菌丝体生长状况、生长周期、污染情况、生长速度,挖瓶后烘干称重计算收率,检测样品中的纤维素酶、麦角甾醇含量(考察转化是否彻底)、牡荆苷含量,总黄酮含量,干燥后的样品分别标注为HG-5、HG-6、HG-7。
参考申请号为201410393488.9的发明专利《一种猴头菌丝体及其培育方法》,同样灭菌、接种、培养,平行对照,培养条件完全一致,得到猴头菌丝体,将样品标注为HTJ。
试验结果统计如下表1。
表1
试验结果表示:绿豆占70%时污染率高,纤维素酶含量低,生物转化不彻底;而绿豆占 50%时,菌类多糖、菌类多肽、牡荆苷含量低;绿豆占60%比率时最为合适,菌丝体生长情况较好,兼顾各优势,为最佳比率。
此外,与HJT组相比,绿豆占60%比率时的HG-6组的污染率低于HTJ组,且生长周期相对较短,生长速度快,纤维素酶、麦角甾醇、菌类多糖、菌类多肽以及牡荆苷的含量也都高于HTJ组。由于菌类多糖、多肽、牡荆苷对提高免疫力及抗肿瘤有良好的作用,所以本发明在培养基中加入绿豆成分,使所得生物转化菌丝体的提取物可用于制备提高免疫力保健品或抗肿瘤药物。
实施例5抗癌细胞实验
(1)实验细胞株
人肝癌Bel-7402细胞株、人肝癌SMMC-7721细胞株、人结肠癌HT-29细胞株和人结肠癌 HCT-116细胞株,均购于美国模式培养物研究所(American Type CultureCollection,ATCC; Manassas,VA,USA)
(2)体外抗肿瘤活性观察
①细胞培养
Bel-7402、SMMC-7721、HT-29和HCT-116细胞株购进后,用含10%牛血清的RPMI1640(GIBCO)培养基在37℃,5%的CO2条件下培养,传3代后,取对数生长期细胞用于实验。
②药物的配制
药物浓度:电子天平称取少量生物转化菌丝体提取物,用少量DMSO溶解后,再用相应培养基配制成5个浓度梯度的药液,过0.2μm滤膜,浓度分别为100、50、25、12.5、6.25μg/mL, 备用;
顺铂注射液:作为阳性对照药物,用培养基配制成5个浓度梯度的药液,即50、25、12.5、 6.25、3.125μg/mL,备用。药物均在种板24h后,临时配制,再加入96孔板中。
③细胞增殖抑制试验
取各对数生长期细胞,调整细胞浓度为8*104个/mL,接种于96孔板中,每孔100μL,24h 后加入不同浓度含药培养基100μL,对照组每孔加培养基100μL,对照组与药物组均设3个复孔,另设空白对照组(不加细胞只加培养基)。将96孔板置37℃,5%CO2培养箱培养48h,加入5mg/mL MTT 20μL,继续培养4h后,弃上清,加入DMSO 150μL,振荡溶解10min,酶标仪于490nm处测定OD值。
④观察指标
抑制率(%)=*100%
(3)结果
数据见下表2和表3。
表2
表3
本实验结果表明,绿豆生物转化菌丝体提取物具有较好的体外抗肿瘤作用,体外对人肝癌细胞Bel-7402、SMMC-7721有较好的抑制作用,在100μg/mL抑制率可达100%,对人结肠癌HT29、HCT-116亦有较好的抑制作用。
实施例6增强免疫力动物实验
(1)动物分组及给药剂量实验动物
SPF级KM小鼠,随机分为6大组(1、ConA诱导小鼠脾淋巴细胞转化,2、抗体生成细胞检测,3、二硝基氟苯(DNFB)耳肿胀法诱发小鼠迟发型变态反应(DTH),4、小鼠腹腔巨噬细胞吞噬鸡红细胞,5、血清溶血素的测定,6、NK细胞活性测定),每组40只,每大组内分为溶剂对照组、猴头菌丝体对照组、绿豆对照组,以及生物转化菌丝体提取物的高、中、低剂量组。
(2)给药剂量和方法
给药剂量:小鼠给药按人体推荐用量10、20、60倍,分别设0.51、1.02、3.06g/kg·BW三个剂量组,以及溶剂对照组。绿豆提取物和猴头菌丝体提取物对照组浓度同中剂量组。
给药方法:每一实验组小鼠按体重随机分为6组,每组10只,按高、中、低剂量组灌胃 (BW)给药,0.2mL/10g·BW,对照组给予相同体积的试样,空白对照组给予蒸馏水,连续给药30天。
(3)检验提高免疫力试验
按照《保健食品检验与评价技术规范》对各试验组动物进行如下实验:ConA诱导小鼠脾淋巴细胞转化实验(MTT);抗体生成细胞检测(Jerne改良玻片法);二硝基氟苯(DNFB)耳肿胀法诱发小鼠迟发型变态反应(DTH)试验;小鼠腹腔巨噬细胞吞噬鸡红细胞实验(滴片法);血清溶血素的测定(血凝法);NK细胞活性测定(乳酸脱氢酶LDH测定法)。
(4)增强免疫力的实验结果
①受试药对小鼠脾淋巴细胞转化和抗体生成细胞的影响见下表4。
表4
结果表明3个剂量组均能增强淋巴细胞增值力和增加溶血空斑数,但存在剂量依赖关系。
②受试药对小鼠迟发性变态反应和巨噬细胞吞噬鸡红细胞的影响见下表5。
表5
结果表明高剂量和中剂量组明显增加耳片重量差值(mg-1)、吞噬率(%)和吞噬指数。
③受试药对小鼠血清溶血素和NK细胞活性的影响见下表6。
表6
结果表明,高剂量和中剂量组明显增加半数溶血值(HC50)和NK细胞活性(%)。
综上所述,绿豆生物转化菌丝体提取物能提高小鼠的淋巴细胞增值力、脾脏生成抗体细胞数、半数溶血值及巨噬细胞吞噬能力,从而提高小鼠的免疫力。
所属领域的普通技术人员应当理解:以上任何实施例的讨论仅为示例性的,并非旨在暗示本公开的范围(包括权利要求)被限于这些例子;在本发明的思路下,以上实施例或者不同实施例中的技术特征之间也可以进行组合,并存在如上所述的本发明的不同方面的许多其它变化,为了简明它们没有在细节中提供。因此,凡在本发明的精神和原则之内,所做的任何省略、修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种猴头菌的培养基,其特征在于,包括如下重量份的组分:
绿豆50~70份,麦麸40~20份,谷糠10份,水40~50份。
2.如权利要求1所述的猴头菌的培养基,其特征在于,所述培养基包括如下重量份的组分:
绿豆60份,麦麸30份,谷糠10份,水45份。
3.一种生物转化菌丝体,其特征在于,将猴头菌类菌株接种至如权利要求1或2所述的猴头菌的培养基中,在20~27℃下,猴头菌培育30~40天,烘干,即得生物转化菌丝体。
4.如权利要求3所述的生物转化菌丝体,其特征在于,所述猴头菌类菌株为猴头菌科真菌猴头菌Hericium erinaceum(Bull.ex Fr.)Pers。
5.如权利要求4所述的生物转化菌丝体,其特征在于,所述生物转化菌丝体是猴头菌科真菌猴头菌(Hericium erinaceum(Bull.ex Fr.)Pers.)的菌丝体与其附生含有绿豆成份的固体培养基的干燥混合物。
6.一种生物转化菌丝体的提取物,其特征在于,所述提取物通过以下步骤制备得到:
(1)将如权利要求3-5任意一项所述的生物转化菌丝体进行水提,40-60℃下用水浸泡2-3次,每次浸泡2-5小时,水的重量为生物转化菌丝体重量的6~10倍,得到提取液;
(2)将步骤(1)中的提取液减压浓缩,将减压浓缩所得的浓缩液冷冻干燥,即得生物转化菌丝体的提取物。
7.如权利要求6所述的生物转化菌丝体的提取物,其特征在于,所述用水浸泡的温度为50℃。
8.如权利要求6或7所述的生物转化菌丝体的提取物,其特征在于,所述冷冻干燥的工艺为,-40℃冷冻2-4小时,-10℃一次升华2-5小时,0℃二次升华5-10小时,30℃三次升华24-36小时。
9.如权利要求8所述的生物转化菌丝体的提取物,所述冷冻干燥的工艺为:-40℃冷冻3小时,-10℃一次升华3小时,0℃二次升华6小时,30℃三次升华30小时。
10.一种如权利要求6-9任意一项所述的生物转化菌丝体的提取物在制备提高免疫力保健品或抗肿瘤药物中的用途。
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