CN115197856B - 一种蛹虫草菌株及其应用 - Google Patents
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Abstract
本发明公开了一种蛹虫草菌株及其应用,涉及微生物技术领域。本发明公开了一种蛹虫草菌株,该菌株为蛹虫草菌株SEUNEU‑113,已于2022年5月23日保藏在中国典型培养物保藏中心,其保藏编号为CCTCC NO:M 2022689。实验表明,SEUNEU‑113具有维持及修护皮肤屏障、保湿、抗衰老、抗自由基的功能,可用于制备药品、化妆品等。
Description
技术领域
本发明涉及微生物技术领域,特别涉及一种蛹虫草菌株及其应用。
背景技术
皮肤是人体内最大的器官,总重量大约占个体体重的16%,一方面维持机体稳定,另一方面也是抵御外界不良因素侵扰的第一道防线。有研究表明,如果外界环境导致皮肤屏障中的相关基因异常,就会诱发皮肤疾病。
皮肤屏障是由角质层的表皮形成细胞和角质间的脂质形成的结构性的屏障。皮肤屏障防止人体过多水分释放,并防止如化学物质或微生物的有害物质进入我们的身体。组成死亡的角质细胞的表面的角质细胞外皮在细胞间脂质的稳定性中起重要作用。皮肤屏障受损将引起皮肤干燥, 皮肤老化、色素沉着异位性皮炎、湿疹、银屑病、鱼鳞病、日光性皮炎等皮肤敏感、刺激性皮炎、激素依赖性皮炎等皮肤油腻, 皮脂溢出性疾病, 如痤疮、酒糟鼻、脂溢性皮炎。
角质间结构性脂质神经酰胺,在基底层向角质分化过程中含量逐渐增加,到达角质层排至细胞间隙,构成防止水分丢失的屏障。角质细胞内含水量高,随着细胞向上代谢分化,角质细胞形状会逐渐变成扁平状,且细胞核及胞器开始退化萎缩,并在角质层形成不具细胞核与胞器的死细胞。角质层本身的亲水、屏障功能,以及角质层中所含有的天然保湿因子即氨基酸类,乳酸盐及糖类等作用,使得角质层通常含有10~30%的水分,这种环境成为了皮肤自身微生物菌落生长的摇篮。但随着年龄的增长,角质层的含水量会逐渐减少,当水分含量低于10%的时候,就会引起皮肤的各种问题。
皮肤衰老,包括由空气污染、吸烟、营养不良和紫外线(UV)等环境因素引起的外在衰老和由时间变化引起的内在衰老。其典型特征是皮肤变薄、产生细纹,这可能是由于细胞增殖减少以及随着年龄增长引起的真皮成分发生显着变化导致的。细胞外基质成分(胶原蛋白、弹性蛋白、糖胺聚糖等)随着皮肤衰老而显着减少。此外,伴随增龄,多种因素如线粒体损伤、炎症反应等产生的活性氧增加,同时,与年龄相关的细胞修复能力下降,使得氧化应激增加,老化受损细胞无法及时清除,从而导致了皮肤衰老。
虫草用在化妆品上,可显着修复皮肤屏障,同时上调保湿基因的表达,有效清除自由基,抵御皮肤衰老,有效增加肌肤对营养物质的吸收,增强免疫力。
发明内容
有鉴于此,本发明提供了一种蛹虫草菌株及其应用。
本发明提供了蛹虫草菌株(Cordyceps militaris),该菌株为蛹虫草菌株SEUNEU-113,已于2022年5月23日保藏在中国典型培养物保藏中心(简称CCTCC,地址为:武汉市武昌区八一路299号,武汉大学,邮编430072),其保藏编号为CCTCC NO: M 2022689的蛹虫草菌株);
所述蛹虫草菌株采用如下分离方法制得:
采样于云南新鲜蛹虫草子实体,将该样品用蒸馏水冲洗后,在超净工作台中用0.1%升汞浸泡1~2min,利用无菌水冲洗掉多余的升汞,滤纸吸干多余水分,用灭菌剪剪成小块,接种于PDA固体培养基,25℃恒温培养,菌落形成后,挑取萌发的菌丝体,在PDA固体培养基上进行划线培养,反复接种筛选,直至分离至菌落性状单一的单菌落,命名为SEUNEU-113。
进一步的,所述蛹虫草菌株SEUNEU-113菌丝体为致密的白色绒毛状,显微镜下观察有隔膜,可形成分生孢子,无锁状联合,生长5~7天转色后,呈橘黄色褶皱。
本发明的另一目的在于提供了上述蛹虫草菌株在制备改善肌肤状况的产品中的应用。
进一步的,所述改善肌肤状况包括修护皮肤屏障、保湿、抗衰老、抗自由基中的至少一种。
在一些实施方式中,所述修复皮肤屏障包括修复皮肤细胞和/或上调屏障修护相关基因的表达;所述屏障修护相关基因包括FLG、IVL和/或OVOL1。
在一些实施方式中,所述保湿为上调保湿相关基因AQP3的表达。
在一些实施方式中,所述抗衰老为上调细胞外基质相关基因的表达;所述细胞外基质相关基因包括MKX、SPTSSA、COL-Ⅰ、COL-1A1中的至少一种。
在一些实施方式中,所述抗衰老为上调细胞抗氧化相关基因NRF2的表达。
在一些实施方式中,所述抗衰老为上调抑制细胞凋亡相关基因BCL-2的表达。
在一些实施方式中,所述抗衰老为下调降解细胞外基质相关基因的表达;所述降解细胞外基质相关基因包括P38MAPK、MMP家族中的至少一种。
在一些实施方式中,所述抗衰老为上调免疫调节相关基因MOR的表达。
在一些实施方式中,所述抗衰老为下调炎症相关基因TNF-α的表达。
在一些实施方式中,所述抗自由基为:
对羟自由基和/或ABTS自由基的清除作用。
在一些实施方式中,其特征在于,所述产品为药品或化妆品。
本发明公开的SEUNEU-113,其保藏编号为 CCTCC NO: M 2022689。实验表明,SEUNEU-113具有维持及修护皮肤屏障、保湿、抗衰老、抗自由基的功能,可用于制备药品、化妆品等。
生物保藏说明
蛹虫草菌株(Cordyceps militaris)SEUNEU-113,于2022年5月23日保藏在中国典型培养物保藏中心(简称CCTCC,地址为:武汉市武昌区八一路299号,武汉大学,邮编430072),其保藏编号为CCTCC NO: M 2022689。
具体实施方式
本发明提供了蛹虫草菌株及其应用。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明的蛹虫草菌株SEUNEU-113,来源于云南新鲜蛹虫草子实体,通过分离选育,经ITS序列分析为蛹虫草(Cordyceps militaris)。该菌株PDA培养菌丝体为白色绒毛散射状,有隔膜和分生孢子,待见光转色后呈橘黄色。
蛹虫草菌株(Cordyceps militaris)SEUNEU-113,保藏单位:中国典型培养物保藏中心(简称CCTCC,地址为:武汉市武昌区八一路299号,武汉大学,邮编430072),保藏编号为CCTCC NO: M 2022689。
进一步,本发明提供的SEUNEU-113在本发明所述的应用或产品中, 存在形式是活的或死的或经间歇灭菌的,或为真菌产物的形式或为上清液形式或衍生物形式,所述衍生物形式优选地选自:代谢产物、代谢生物产物、益生素、细胞壁及其成分、真菌多糖,和含有免疫原性成分的化合物,优选地选自:上清液、灭活菌体。
体外细胞实验表明,本发明的SEUNEU-113具有上调皮肤屏障修护相关因子丝聚合蛋白(filaggrin) FLG、外皮蛋白(Involucrin)IVL、Ovo样转录因子1(Ovo LikeTranscriptional Repressor 1)OVOL1表达的作用,基因表达量上调1.12~3.24倍。
体外细胞实验表明,本发明的SEUNEU-113具有上调保湿相关的水通道蛋白3基因AQP3表达的作用,基因表达量上调1.79~2.14倍。
体外细胞实验表明,本发明的SEUNEU-113具有上调HaCaT角质细胞抗氧化相关基因核因子E2相关因子2 NRF2基因表达的作用,基因相对表达量上调1.64~1.69倍;具有下调降解细胞外基质相关的丝氨酸/苏氨酸蛋白激酶基因P38MAPK,基因相对表达量下调0.69~0.76倍;具有下调炎症因子肿瘤坏死因子-α基因TNF-α,基因相对表达量下调0.35~0.60倍。
体外细胞实验表明,本发明的SEUNEU-113具有上调HFF人成纤维细胞外基质相关的丝氨酸棕榈酰转移酶基因SPTSSA、Ⅰ型胶原膜蛋白基因COL-I、Ⅰ型胶原膜蛋白α链基因COL-1A1和莫霍克蛋白基因MKX,抑制细胞凋亡相关的B淋巴细胞瘤-2基因BCL-2,以及免疫调节因子β-内啡肽受体基因MOR,基因相对表达量上调1.10~4.97倍;具有下调降解细胞外基质相关的P38MAPK基因及基质金属蛋白酶家族基因MMP2、MMP10,基因相对表达量下调0.53~0.89倍。体外细胞实验表明,本发明的SEUNEU-113具有清除羟自由基和ABTS自由基的功能,自由基清除率22.35%~24.72%。本发明采用的试材皆为普通市售品,均可市售购买得到,下面结合实施例,进一步阐述本发明:
实施例1 SEUNEU-113的分离
于云南新鲜蛹虫草子实体取样。将样品用蒸馏水冲洗后,在超净工作台中用0.1%升汞浸泡1~2min,再用无菌水冲洗掉多余的升汞,滤纸吸干多余水分,用灭菌剪剪成小块,接种于PDA固体培养基,25℃恒温培养,菌落形成后,挑取萌发的菌丝体,在PDA固体培养基上进行划线培养,反复接种筛选,直至分离至菌落性状单一的单菌落,命名为SEUNEU-113。
菌株SEUNEU-113菌丝体为致密的白色绒毛状,显微镜下观察有隔膜,可形成分生孢子,无锁状联合,生长5~7天转色后,呈橘黄色褶皱。经ITS序列分析为蛹虫草(Cordycepsmilitaris)。
实施例2 SEUNEU-113的核酸鉴定
1、ITS基因序列分析:
取培养20d的种子液,离心获得菌体,通过液氮研磨后,用试剂盒提取总DNA。PCR选用真菌鉴定通用引物ITS1和ITS4,对菌株的ITS序列进行PCR扩增。PCR扩增反应体系(50μL)为10×Extaq buffer 5μL,dNTP(800μmol/L)4μL,PrimerITS1(10ng/μL)2μL,PrimerITS4(10ng/μL)2μL,DNA模板(50ng/mL)5μL,Extaq酶(5U/μL)0.5μL,ddH2O31.5μL。PCR反应程序为94℃预变性5min;94℃变性1min,56℃复性1min,72℃延伸2min,共进行30个循环;72℃延伸5min。PCR扩增产物纯化后测序。
2、结果
测序结果在GenBank数据库中进行BLAST同源性比对,选取同源性较高的模式菌株的ITS序列,采用MEGA 6.0中的邻接(neighbor joining,NJ)法构建系统发育树确认SEUNEU-113菌株为蛹虫草(Cordyceps militaris)。
实施例3 SEUNEU-113促进HaCaT屏障修护相关基因表达实验
1、SEUNEU-113上清液制备:
挑取SEUNEU-113菌丝体于PDA液体培养基中,25℃150r/min条件下发酵培养24~48h,取发酵液检测葡萄糖含量至3mmol/L,终止培养,121℃,30min高压灭活,12000转离心2min,经0.22μm滤膜过滤为上清液。
2、促进HaCaT屏障修护相关基因表达实验
接种人永生化角质形成细胞HaCaT(2ml/孔,内含5×105细胞)至6孔板,5%二氧化碳培养箱37℃过夜培养至细胞贴壁。加入上清液5%(对照组以等体积PDA替代上清液),培养24h后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度后反转录为cDNA,以GAPDH为内参基因,采用实时qPCR检测FLG、IVL 、OVOL1基因的表达。以等体积PDA处理组为对照(基因相对表达倍数F=1),利用2-ΔΔCT法计算各样品F值。
公式:F=2-ΔΔCT,其中:
△CT实验=CT实验-CT内参(实验);
△CT对照=CT对照-CT内参(对照);
△△CT=△CT实验-△CT对照。
结果见下表:
结果显示SEUNEU-113上清液具有促进皮肤屏障修护的作用。
实施例4 SEUNEU-113上调HaCaT保湿相关基因表达实验
1、SEUNEU-113上清液制备:
制备方法参照实施例3。
2、上调HaCaT保湿相关基因表达实验
接种人永生化角质形成细胞HaCaT(2ml/孔,内含5×105细胞)至6孔板,5%二氧化碳培养箱37℃过夜培养至细胞贴壁。加入上清液5%(对照组以等体积PDA替代),培养24h后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度后反转录为cDNA,以GAPDH为内参基因,采用实时qPCR检测AQP3基因的表达。以等体积PDA处理组为对照(基因相对表达倍数F=1),利用2-ΔΔCT法计算各样品F值。
结果见下表:
结果显示加入SEUNEU-113具有促进皮肤保湿的作用。
实施例5:SEUNEU-113调节光老化HaCaT角质细胞外基质/细胞自噬/抗氧化相关基因表达实验
1、SEUNEU-113上清液制备:
制备方法参照实施例3。
2、HaCaT细胞制备及紫外线损伤
将HaCaT细胞消化后以0.5ml/孔(内含2×105细胞)接种至24孔板,5%二氧化碳培养箱37℃培养过夜。对孔内细胞进行总剂量为2J/cm2的紫外线UVB照射损伤。
3、SEUNEU-113添加
将上清液5%(V/V)加入刺激过的HaCaT细胞(对照组以等体积PDA替代上清液)。每组3平行,37℃培养过夜。
4、qPCR法检测细胞外基质/抗炎/抗氧化相关基因相对表达倍数
将上述细胞弃去培养基后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度后反转录为cDNA,以GAPDH为内参基因,采用实时qPCR检测细胞外基质相关基因P38MAPK,炎症因子相关基因TNF-α,以及抗氧化相关基因NRF2的表达。以对照组基因相对表达倍数F=1,利用2-ΔΔCT法计算各样品F值。
上清液上调抗氧化基因NRF2的表达,并下调降解细胞外基质基因P38MAPK、炎症因子TNF-α的表达,结果见下表:
结果显示加入SEUNEU-113具有增加HaCaT角质细胞抗氧化能力,降低细胞外基质降解、及降低炎症的抗衰老作用。
实施例6:SEUNEU-113调节氧化损伤人成纤维细胞外基质/细胞凋亡/抗氧化相关基因表达实验
1、SEUNEU-113上清液制备:
制备方法参照实施例3。
2、HFF人成纤维细胞制备及H2O2诱导氧化损伤
将DMEM培养的HFF细胞消化后以0.5ml/孔(内含2×105细胞)接种至24孔板,5%二氧化碳培养箱37℃培养过夜。每孔加入终浓度为200μM的H2O2进行刺激,37℃静置1h。
3、SEUNEU-113添加
将上清液5%(V/V)加入刺激过的HFF细胞,对照组以等体积PDA替代上清液。每组3平行,37℃培养过夜。
4、qPCR法检测细胞外基质/细胞凋亡/抗氧化/免疫调节相关基因相对表达倍数
将上述细胞弃去培养基后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度后反转录为cDNA,以GAPDH为内参基因,采用实时qPCR检测细胞外基质、抗氧化、细胞外基质降解、细胞凋亡和免疫调节因子相关基因的表达。以对照组基因相对表达倍数F=1,利用2-ΔΔCT法计算各样品F值。
上清液调节HFF人成纤维细胞相关基因结果见下表:
结果显示加入SEUNEU-113具有促进HFF人成纤维细胞外基质合成、减少细胞外基质降解、抑制细胞凋亡、以及促进免疫调节的抗衰老作用。
实施例7: SEUNEU-113抗氧化清除自由基的作用
1、SEUNEU-113上清液制备:
制备方法参照实施例3。
2、SEUNEU-113上清液羟自由基清除能力检测
试剂配制和检测方法按照索莱宝羟自由基清除能力检测试剂盒说明书进行。测定各样品536nm吸亮度,求平均值并计算各样品清除率。
计算公式及结果如下表:
3、SEUNEU-113上清液ABTS自由基清除能力检测
试剂配制和检测方法按照索莱宝ABTS自由基清除能力检测试剂盒说明书进行。测定各样品405nm吸亮度,求平均值并计算各样品清除率。
计算公式及结果如下表:
结果显示SEUNEU-113具有清除羟自由基和ABTS自由基的作用,自由基清除率22.35%~24.72%。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.一种蛹虫草(Cordyceps militaris)菌株,该菌株为蛹虫草菌株SEUNEU-113,已于2022年5月23日保藏在中国典型培养物保藏中心,其保藏编号为CCTCC NO: M 2022689;
所述蛹虫草菌株采用如下分离方法制得:
采样于云南新鲜蛹虫草子实体,将该样品用蒸馏水冲洗后,在超净工作台中用0.1%升汞浸泡1~2min,利用无菌水冲洗掉多余的升汞,滤纸吸干多余水分,用灭菌剪剪成小块,接种于PDA固体培养基,25℃恒温培养,菌落形成后,挑取萌发的菌丝体,在PDA固体培养基上进行划线培养,反复接种筛选,直至分离至菌落性状单一的单菌落,命名为SEUNEU-113。
2.权利要求1所述的蛹虫草菌株在制备改善肌肤状况的产品中的应用,所述产品为药品或化妆品,所述改善肌肤状况为修护皮肤屏障、保湿、抗衰老、抗自由基中的至少一种;
所述修护 皮肤屏障为上调屏障修护相关基因的表达;所述屏障修护相关基因为FLG、 IVL和/或OVOL1;
所述保湿为上调保湿相关基因AQP3的表达;
所述抗衰老为上调细胞外基质相关基因的表达;所述细胞外基质相关基因为MKX、 SPTSSA、COL-Ⅰ、COL-1A1中的至少一种;
所述抗衰老为上调细胞抗氧化相关基因NRF2的表达;
所述抗衰老为上调抑制细胞凋亡相关基因BCL-2的表达;
所述抗衰老为下调降解细胞外基质相关基因的表达;所述降解细胞外基质相关基因为P38MAPK、MMP2和MMP10中的至少一种;
所述抗衰老为上调免疫调节相关基因MOR的表达;
所述抗衰老为下调炎症相关基因TNF-α的表达;
所述抗自由基为:对羟自由基和/或ABTS自由基的清除作用。
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