CN108795775A - 一株中国弯颈霉及其在生产虫草菌丝中的应用 - Google Patents
一株中国弯颈霉及其在生产虫草菌丝中的应用 Download PDFInfo
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- CN108795775A CN108795775A CN201710302225.6A CN201710302225A CN108795775A CN 108795775 A CN108795775 A CN 108795775A CN 201710302225 A CN201710302225 A CN 201710302225A CN 108795775 A CN108795775 A CN 108795775A
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Abstract
本发明公开了一株从天然冬虫夏草分离的中国弯颈霉新种(Tolypocladium sinense C.L.Li sp.nov.)8560菌株,有较多方面的特性与天然冬虫夏草相同,分子生物学rDNA ITS的基因测序序列与有些冬虫夏草相同,中国弯颈霉产生菌丝多糖的化学结构与冬虫夏草子实体多糖的化学结构完全相同,产生多种有效成分与生理功效的特性与冬虫夏草相一致。中国弯颈霉菌丝生长速度快,菌丝产量高,分生孢子数量多,培养周期短,通过发酵培养以求获得在虫草菌丝生产中的应用。
Description
技术领域
本发明涉及一株从天然冬虫夏草分离的中国弯颈霉新种8560及其在生产虫草菌丝和/或与冬虫夏草配伍制备冬虫夏草口服液中的应用。
背景技术
冬虫夏草(简称虫草)是我国特有的传统滋补良药,属于蛇头虫草科Ophiocordycipitaceae,是冬虫夏草菌Cordyceps sinensis寄生在虫草蝙蝠蛾幼虫上继而形成菌核和子座组织体。天然虫草具有抗衰老、抗病菌、抗恶性肿瘤、扩张血管、改善动脉硬化、调节内分泌等多种功效。虫草一般生长在海拔3500~5000m的高原寒冷草甸地区,其冬虫夏草的自然资源较少,近年来的过度采集已使其受到严重破坏,天然虫草已被国家列为濒危保护菌种。人工培育虫草已成为合理保护、利用这一稀有珍贵药用资源的重要技术。人工培养的虫草菌能发挥与天然虫草相同或相近的功效。
冬虫夏草是一种子囊菌,在其生活史中具有分生孢子阶段(无性型)和子囊孢子阶段(有性型)。在人工培养、液体发酵等实际生产中使用的冬虫夏草菌种均为无性阶段,其菌种的正确鉴定至关重要,故而其无性型问题特别引人关注。已报道的冬虫夏草的无性型菌种有22个学名,涉及13个属,但其中部分无性型名称与冬虫夏草的关系有待进一步验证,目前已有证据表明中国被毛孢(Hirsutella sinensis)与冬虫夏草无性型的关系。
中国弯颈霉(Tolypocladium sinense)是从天然虫草中分离出到的无性型新种,有关该新种的研究和应用,国外尚未有报道,国内有少数有关该菌株的报道主要集中在该菌培养条件的探讨、化学成分的初步分析,其发酵培养物的菌丝体具有与冬虫夏草基本相同的化学成分和药理作用。
国外资深菌物学家Solomon P.Wasser等(2011)研究报道,2006年6月从西藏高原采集冬虫夏草标本,共分离30只菌株,经培养和鉴定,其中有27株为中国弯颈霉(Tolypocladium sinense),有2株为中国被毛孢(Hisutella sinensis),研究结果表明冬虫夏草的无性型是中国弯颈霉和中国被毛孢。显然该研究认为中国弯颈霉是冬虫夏草的第二个无性型。由于冬虫夏草生活史的多型现象,寄主昆虫种类的多样性,不同产地生态环境的差异性,土壤微生物种群的多样性,所以中国被毛孢不可能是冬虫夏草的唯一无性型。中国弯颈霉从分子生物和生化方面与天然冬虫夏草具有多方面的相似性。
发明内容
本发明提供了一株中国弯颈霉新种8560(Tolypocladium sinense C.L.Lisp.nov.),分类命名:中国弯颈霉(Tolypocladium sinense),保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址:北京市朝阳区北辰西路1号院3号),保藏号CGMCC No.13374,保藏日期为2016年12月21日。其是从云南省迪庆藏族自治州产的冬虫夏草菌核子座及子囊孢子分离所获得的一株纯菌种。将分离出来的中国弯颈霉进行分子生物学鉴定,分析其与冬虫夏草之间的关系,提取基因组DNA,通过PCR扩增其18S- 28S rDNA之间的内转录间隔区及高度保守的5.8S rDNA序列进行测序,将所得的序列与天然冬虫夏草的相应区域的DNA进行比对,结果表明:中国弯颈霉新种8560的 ITS序列与相关产地的冬虫夏草序列的同源性达99%,其rDNA ITS基因序列如SEQ NO 1所示。
一.菌株8560的固体培养及形态特征
1.麦芽汁琼脂培养基上,菌落白色、绒状、圆形、致密,24℃培养7天菌落直径30-36mm。无渗出液,接种3-4天后菌落中部稍凸起,边缘整齐(如图1所示),培养基反面淡黄色,具鲜黄色色素,色素扩散,具放射状沟纹(如图2所示)。菌丝分枝繁茂,细胞壁薄而光滑,直径1-3μm,分生孢子梗直立、光滑,从菌丝上生出,分枝1-2次。细胞7.3-29×1.82-3.27μm,产孢细胞瓶梗型,典型瓶状,在小枝上轮生、单生或顶生,长7.6-19.4μm,基部膨大4.0-12.1×2.9- 3.6μm,颈部细长,常弯曲,3.6-7.3×0.7-1.5μm。分生孢子为瓶梗孢子,单生于产孢细胞上,光滑球形,直径1.45-3.63μm(如图3所示)。
2.菌落在察氏琼脂培养上生长迅速,2-3天菌落边缘出现鲜黄色色素,24℃培养7天,菌落直径23-27mm,圆形、纯白色、绒毛状、表面突起、致密、边缘整齐。反面中央黄棕色,外围淡黄色,色泽扩散,无渗出液,其它同麦芽汁琼脂培养基。
3.菌落在马铃薯葡萄糖琼脂(PDA)培养基上,相似于麦芽汁琼脂上的特征, 24℃,培养7天,菌落直径20-24mm,圆形、白色、绒毛状、致密、表面隆起、边缘整齐,反面中央鲜黄色,外围黄白色。分生孢子梗、瓶梗及瓶梗孢子的形态特征和麦芽汁琼脂相同。
二.菌株8560在液体培养基上的形态特征
中国弯颈霉新种8560在葡萄糖蛋白胨液体培养基进行摇瓶振荡培养,24℃, pH5.2,培养4天,形成菌球,直径5-50mm,胶质、黄色。菌丝分枝繁茂、有隔、透明,直径1.8-2.9μm,分生孢子梗在形态上无明显分化,瓶梗不呈典型瓶状,散生,常偏生于枝的一侧,长7.3-39.9μm,基部宽1.5-4.0μm,最窄处宽为1.1-1.8μm。瓶梗孢子形状多种,卵形、椭圆形、长圆形、柱形等,表面光滑, 2.9-10.3×2.9-4.4μm。接种后第3天瓶梗孢子开始萌发,第5天大量萌发,多从一端长出芽管,也有少数两端同时长出芽管(如图4所示)。
三.菌株8560的深层发酵培养及其有效成分
深层发酵培养液为葡萄糖蛋白胨培养基,温度26℃,pH 5.5,转速为120r/min,发酵终点72-96h。产生大量黄色粘稠的发酵菌液和均匀透明的小菌球,显微检验菌丝分枝繁茂,分生孢子密集,数量极多。每升发酵液含菌丝体干重为18.0— 20.0g,每升发酵液产胞外粗多糖干品3.0-4.0g,粗多糖纯度为20-30%,每100 g菌丝体产胞内粗多糖5.6g。发酵菌液制成的虫草菌粉其中腺苷含量为0.063%,甘露醇为16.2%,虫草素为108.8μg/g。测出24种游离氨基酸,其中有6种新游离氨基酸,并且含有重要功效的牛磺酸。24种微量元素中不含砷、铅、汞等有毒元素。因此,本发明提供的中国弯颈霉新种可以应用于发酵工艺和虫草菌丝的生产中。
四.菌株8560的分子生物学特征
采用分子生物学PCR扩增技术,18S rDNA序列进行测序,结果显示序列长度为544bp,其rDNA ITS基因序列如SEQ NO 1所示。采用Blast将目的DNA与天然冬虫夏草相应序列(AF 291749.1;AY899261.1;AY245638.1;AB067720.1) 进行比对,相似率为99%,中国弯颈霉新种8560很可能是冬虫夏草的另一无性型。
五.菌株8560产虫草多糖的化学特性
菌株8560深层发酵培养,发酵终点72h,获得黄色粘稠的发酵菌液,经离心分离,获得发酵液与菌丝体,发酵液浓缩,95%乙醇沉淀,其沉淀物为胞外粗多糖。菌丝体热水提取,提取液浓缩,95%乙醇沉淀,其沉淀物为胞内粗多糖。胞外和胞内粗多糖经葡聚糖凝胶Sephadex G-200柱层析,纯化,分部收集,获胞外和胞内纯多糖。
经分析虫草胞外多糖和胞内多糖的单糖组成相同,由甘露糖、半乳糖和少量葡萄糖组成,三种单糖的摩尔比为5:3:1,核磁共振氢谱(1H-NMR)分析糖苷键为α-型。高碘酸氧化、Smith降解、甲基化分析提示虫草多糖是一高度分枝的多糖,主链由甘露糖组成支链由半乳糖和少量葡萄糖组成,主链的甘露糖之间的连接方式为1→2、1→4和1→6连接,支链的单糖之间的连接方式是1→3、1→5、1 →6连接,非还原末端是半乳糖和甘露糖。因此,本发明提供的中国弯颈霉新种可以应用于虫草多糖的生产。
六.菌株8560的游离氨基酸的测定
菌株8560经深层发酵培养产生大量菌丝体,将菌丝体用80%乙醇进行超声提取,提取液用HPLC进行游离氨基酸分析,共测出24种游离氨基酸,比常见的18 种游离氨基酸的分析多6种,即:牛磺酸、γ-氨基丁酸、谷氨酰胺、天冬酰胺、瓜氨酸、鸟氨酸,它们都具有重要生理功效。
七.菌株8560的抗菌活性测定
菌株8560的发酵液用乙酸乙酯进行提取,提取液中含有抗菌活性的次生代谢的产物,对真菌和细菌具有抑制作用,采用滤纸片扩散法测定抑菌的活性。测定结果表明对丝状真菌、酵母菌、细菌等都显示了明显的抑菌作用,如黑曲霉 (Aspergillus niger)、新月弯孢霉(Curvularia lunata)、粗糙脉孢菌(Neurospora crassa)、弯长蠕孢(Helminthosporium curvatum)、白地霉(Geotrichum candidum)、白色念珠菌(Candidaalbicans)、深红酵母(Saccharomyces ruber)、热带假丝酵母(Candida tropicalis)、枯草芽孢杆菌(Bacillis subtilis)、大肠杆菌(Escherichia coli)。经测定抑菌圈的抑菌直径在20-35mm之间(如图5所示)。
八.菌株8560的药理活性
对小鼠的游泳、常压、耐缺氧试验和肌力紧张测定结果证明菌株8560具有明显的抗疲劳作用。
采用荧光法计数血小板及放射免疫法测定CAMP,证明菌株8560和天然冬虫夏草一样,无论肌注或灌胃,均能显著增加小鼠的血小板数(P<0.01),小鼠的血浆CAMP也升高(P<0.001),为治疗血小板减少等疾病提供了实验依据。
菌株8560具有增强和调节小鼠的免疫功能的作用,采用多种免疫学检测指标,证明菌株8560虫草菌丝和天然冬虫夏草一样,均有明显的保护T淋巴细胞、激活单核巨噬细胞系统的吞噬功能,在腹腔巨噬细胞吞噬鸡红血球的吞噬百分率和吞噬指数中,给药组明显高于对照组(P<0.001),巨噬细胞内酸性磷酸酶增加,脾中的巨噬细胞处于激活状态,实验结果表明虫草菌丝8560与天然虫草具有相同的免疫功能。
九.菌株8560的安全性质量检测
菌株8560虫草菌丝浓缩液经江苏省卫生防疫站进行了安全性质量检测,其检测结论如下:
1.急性经口毒性实验:
雌雄小鼠急性经口LD50均大于21.5g/Kg,该检品属无毒级。
2.小鼠骨髓嗜多染红细胞微核试验:
剂量达10.0g/Kg体重,雌雄小鼠均未检出对骨髓嗜多染红细胞的诱变活性。
3.Ames试验
对标准测试菌株TA97a、TA98、TA100、TA102,平板掺入法,检品剂量达5 mg/皿,无论加或不加S-9,均未发现诱变活性。
综上所述,本发明提供的中国弯颈霉新种8560与冬虫夏草序列的同源性达99%,很可能是冬虫夏草的另一无性型,其深层发酵可产生虫草菌丝、虫草素、虫草多糖和24种游离氨基酸,此外本菌种还具有抗菌抑菌作用和显著的抗疲劳作用,可提高机体的免疫功能,因此可用于发酵工艺中和虫草多糖、虫草素、虫草菌丝的生产中,以及冬虫夏草口服液等保健品的制备中。
进一步,本发明提供了中国弯颈霉新种8560深层发酵及生产虫草菌丝的方法,包括步骤:
1)将中国弯颈霉新种8560接种入灭菌后的液体培养基中;
2)摇瓶发酵培养;
3)种子罐发酵培养;
4)生产罐发酵培养;
5)发酵终点:72h,放罐;
6)收集发酵菌液并分离菌丝体与发酵液。
优选地,步骤1)中所述液体培养基为葡萄糖蛋白胨液体培养基。
优选地,步骤2)-4)的培养温度为26±2℃。
优选地,步骤2)中所述摇瓶的装液量为瓶体积的1/4-1/3,摇瓶转速为100- 120r/min。
优选地,步骤3)和4)的发酵罐(种子罐、生产罐)装料系数为0.7,发酵罐搅拌速度为120-160r/min,接种方法优选为差压法,接种量为5%-10%,罐压为0.4-0.5Kg/cm2,通气量为每分钟1:0.5-1:1V/V。
优选地,步骤2)的发酵终点为72小时,步骤3)的发酵终点为48小时,步骤4)的发酵终点为72-96h。
优选地,步骤5)的放罐标准为:菌丝长出繁茂分枝,菌球稍密、发酵液粘稠、清澈菌丝湿重占发酵液体积10-15%,还原糖降至1.0%以下,pH降至5.5以下,氨基氮降至15.0%以下。
优选地,步骤6)所述分离菌丝体与发酵液的方法为离心分离或过滤分离。
本发明的深层发酵及生产虫草菌丝的方法生产出来的菌丝分枝繁茂,每升发酵液含菌丝体干重为18.0-20.0g,进一步可冷冻干燥和超细粉碎,虫草菌粉中腺苷含量为0.063%,甘露醇为16.2%,虫草素为108.8μg/g,另有24种游离氨基酸;每升发酵液产胞外粗多糖干品3.0-4.0g,粗多糖纯度为20-30%,每100g菌丝体产胞内粗多糖5.6g。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是固体培养基上中国弯颈霉新种8560的菌落形态(正面);
图2是培养中国弯颈霉新种8560的固体培养基背面照片;
图3是在固体培养基上中国弯颈霉新种8560菌丝分生孢子梗、瓶梗及分生孢子的显微形态;
图4是在液体培养基中中国弯颈霉新种8560的菌丝、分生孢子梗及分生孢子的显微形态;
图5是中国弯颈霉新种8560的抑菌活性(抑菌圈直径,左图:白地霉;右图:黑曲霉)。
具体实施方式
菌株8560的固体培养基
1.麦芽汁琼脂
12Brix,麦芽汁1.0升,琼脂20.0克。
2.马铃薯葡萄糖琼脂(PDA)
马铃薯提取液1.0升,葡萄糖20.0克,琼脂20.0克。
3.察氏培养琼脂
蔗糖30.0克,NaNo3 3.0克,MgSO4·7H2O 0.5克,FeSO4·7H2O 0.01克, K2HPO4 1.0克,琼脂20.0克,蒸馏水1.0升,pH 6.0-6.5。
菌株8560的液体培养基
葡萄糖蛋白胨培养液:
葡萄糖30.0克,蛋白胨10.0克,酵母膏1.0克,KH2PO4 2.0克,MgSO4·7H2O 0.5克,水1.0升。
实施例1菌株8560的菌种分离
从云南省迪庆藏族自治州的高海拔地区采集天然冬虫夏草标本,用低温处理带回实验室内。冷开水冲洗虫草标本表面的泥土,用75%酒精消毒1分钟和再用 0.1%升汞消毒3分钟,对剥去菌膜的冬虫夏草进行表面消毒。将消毒过的冬虫夏草从子座基部切成子座和菌核两部分,每一部分各切成1-2mm厚的薄片,将所有切成的薄片分别移植到PDA平板和PDA试管斜面上,置于10-15摄氏度的培养箱中培养。
实施例2菌种的保藏
1.斜面低温保藏
菌种在PDA培养基上培养成熟后,选择菌丝生长健壮的试管母钟,菌种口用牛皮纸包好,保藏温度4-6℃,每隔6个月重新移植一次。
2.真空冷冻干燥保藏
试管斜面菌种,用无菌脱脂牛奶制成孢子悬浮液,无菌吸管分别吸取0.2mL 孢子悬浮液,各注入多支无菌安瓿管中,冷冻干燥,抽真空,熔封。低温或室温下保藏,保藏期长达10-20年仍不降其原有性能。定期使用时将安瓿管启封复苏,安瓿管封端加热,无菌冷水滴在受热部位,使其管壁造成裂缝,然后,再轻轻隔断,安瓿管注入无菌生理盐水,摇动即成悬浮液,接入相应的培养基,复苏培养。
实施例3菌株8560作为虫草菌丝的生产工艺流程
天然冬虫夏草的组织分离→纯菌种→PDA试管斜面→菌种保藏→菌种活化→试管斜面母种→摇瓶发酵培养→种子罐发酵培养→生产罐发酵培养→放罐→收集发酵菌液离心过滤→菌丝体和发酵液。
实施例4菌株8560深层发酵培养过程
深层发酵培养基为葡萄糖蛋白胨液体培养基,灭菌温度为121℃,维持20-30 min,培养温度为26±2℃,发酵罐装料系数为0.7,摇瓶装液量为瓶体积的1/3,摇瓶转速120r/min,发酵罐搅拌速度为140r/min,接种方法为差压法,接种量为 5%-10%,罐压为0.4-0.5Kg/cm2,通气量为1:0.5-1:0.8。发酵终点摇瓶72h,种子罐48h,生产罐为72-96h。每天取样测总糖、还原糖、氨基氮、pH、菌丝浓度、菌丝形态。放罐标准:菌丝长出繁茂分枝,菌球稍密、发酵液粘稠、清澈菌丝湿重占发酵液体积10-15%,还原糖降至1.0%以下,pH降至5.5以下,氨基氮降至15.0%以下。收集发酵菌液,管式离心机分离,分开菌丝体与发酵液,菌丝体冷冻干燥,超细粉碎。发酵液浓缩,95%酒精沉淀,其醇析的沉淀物为虫草菌丝胞外多糖。
序列表
<110> 中国人民解放军海军医学研究所
<120> 一株中国弯颈霉及其在生产虫草菌丝中的应用
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 544
<212> DNA
<213> 中国弯颈霉新种8560
<400> 1
tagggtgaac ctgcggaggg atcattaccg agttatcaac tcccaaaccc ctgtgaacat 60
acccaacgtt gcttcggcgg gaccgccccg gcgcctcggc gtcccggaac caggcgcccg 120
ccggaggacc caaactcttg tttaaccata gtggcatatt ctgagtctca caagaaaaat 180
gaatcaaaac tttcaacaac ggatctcttg gctctggcat cgatgaagaa cgcagcgaaa 240
tgcgataagt aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcacattg 300
cgcccgccag tattctggcg ggcatgcctg ttcgagcgtc atttcaaccc tcaagcccca 360
gcggcttggt gttggggacc ggccccggcc gccccccaaa tgcagtggcg acctcgccgc 420
agcctcccct gcgtagtagc acaactcgca ccggagcgcg gagacggtca cgccgtaaaa 480
cgcccaactt ctcagagttg acctcggatc aggtaggaat acccgctgaa cttaagcata 540
tcaa 544
Claims (10)
1.一株中国弯颈霉(Tolypocladium sinense C.L.Li sp.nov.),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号CGMCC No.13374,保藏日期为2016年12月21日。
2.根据权利要求1所述的中国弯颈霉,其rDNA ITS基因序列如SEQ NO 1所示。
3.根据权利要求1所述的中国弯颈霉在发酵工艺中的应用。
4.根据权利要求1所述的中国弯颈霉在虫草多糖和虫草素生产中的应用。
5.根据权利要求1所述的中国弯颈霉在生产虫草菌丝中的应用。
6.根据权利要求1所述的中国弯颈霉在抗菌抑菌产品生产中的应用。
7.根据权利要求1所述的中国弯颈霉在抗疲劳产品制备中的应用。
8.根据权利要求1所述的中国弯颈霉在提高免疫功能产品制备中的应用。
9.根据权利要求1所述的中国弯颈霉在冬虫夏草口服液制备中的应用。
10.根据权利要求1所述的中国弯颈霉生产虫草菌丝的方法,其特征在于所述方法包括步骤:
1)将所述中国弯颈霉接种入灭菌后的液体培养基中;
2)摇瓶发酵培养;
3)种子罐发酵培养;
4)生产罐发酵培养;
5)发酵终点:72—96小时放罐;
6)收集发酵菌液并分离菌丝体与发酵液。
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CN117025407B (zh) * | 2023-06-28 | 2024-06-18 | 中国人民解放军海军特色医学中心 | 一株弯颈霉属真菌及其在制备抗辐损伤药物中的应用 |
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