CN114517162A - 一种涎沫假丝酵母及其发酵产物和应用 - Google Patents
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Abstract
本发明公开了一种涎沫假丝酵母及其发酵产物和应用。所述涎沫假丝酵母为涎沫假丝酵母(Candida zeylanoides)JBB‑MBY‑GW103,保藏编号为CCTCC NO:M20191122。所述涎沫假丝酵母的发酵产物能够清除DPPH自由基,同时对皮肤细胞表皮Hacat细胞和成纤维细胞都有一定的增殖促进作用,并且能够抑制B16细胞的酪氨酸酶活性和黑素生成。此外,该发酵液能抑制金黄色葡萄球菌的菌株活力,同时具有促进表皮葡萄球菌的菌株活力的作用。而这些生物活性使其在化妆品的抗衰,美白,以及调节皮肤微生态菌群上拥有巨大的应用潜力。
Description
技术领域
本发明涉及微生物分离及发酵领域,具体涉及一种涎沫假丝酵母及其发酵产物和应用。
背景技术
在南北极、深海、荒漠、冰川等环境条件下存在着多种嗜极微生物,它们能够成功克服适应极端环境。包括真菌、酵母菌、细菌等有微生物在这种冷环境中具有普遍生物和生态多样性。酵母菌是作为一类多用途的真核微生物,具有代谢途径多样,抗逆性佳的特点,并且能在如冰川这种极端环境中生存。酵母菌常用在发酵技术和生物技术应用中,而在低温环境中的一些酵母菌在其长期的进化过程中,其分子结构及功能发生改变,而使其产生特有的功能,如代谢产低温酶以及多不饱和脂肪酸等大分子物质。而这一特性在生物医药、食品加工、化妆品等行业有极好的应用前景。
发明内容
现有酵母在制备用于清除DPPH自由基、促进皮肤细胞表皮Hacat细胞和成纤维细胞增殖、抑制B16细胞的酪氨酸酶活性和黑素生成和抑制金黄色葡萄球菌的菌株活力等的发酵产物方面存在产物效果不佳的缺陷,本发明的发明人在四零冰川固体冰川中意外分离到一株假丝酵母,实验结果表明其发酵产物能够很好地解决上述现有技术中的问题,可用于制备具备上述能力的高效发酵产物。
为解决上述技术问题,本发明提供的技术方案之一为:一种涎沫假丝酵母,所述涎沫假丝酵母为涎沫假丝酵母(Candida zeylanoides)JBB-MBY-GW103,保藏编号为CCTCCNO:M20191122。
为解决上述技术问题,本发明提供的技术方案之二为:一种涎沫假丝酵母发酵物的制备方法,所述制备方法包括在培养基中加入如技术方案之一所述的涎沫假丝酵母并培养。
较佳地,所述培养基为YPD液体培养基。
较佳地,所述制备方法还包括在培养所述涎沫假丝酵母后,离心取上清液。
较佳地,所述培养的温度为20-40℃,所述培养时进行了振荡培养,所述振荡培养的速度为150-250rpm,所述培养的时间为1-10天。
较佳地,所述温度为28℃,所述振荡的速度为190rpm,所述时间为4 天。
较佳地,在所述培养基中加入的所述涎沫假丝酵母为1%的种子液。
为解决上述技术问题,本发明提供的技术方案之三为:一种通过如技术方案之二所述的制备方法制备的涎沫假丝酵母发酵物。
为解决上述技术问题,本发明提供的技术方案之四为:一种如技术方案之三所述的涎沫假丝酵母发酵物在制备抗衰老、抑菌和/或美白的产品中的应用;优选地,所述产品为护肤品、化妆品或清洁剂。
为解决上述技术问题,本发明提供的技术方案之五为:一种皮肤外用剂,所述皮肤外用剂包含如技术方案之三所述的涎沫假丝酵母发酵物。优选地,所述的皮肤外用剂为化妆水、乳液、精华液或乳霜。更优选地,所述涎沫假丝酵母发酵物的含量为0.01~0.5%,所述百分比为质量百分比。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明得到的涎沫假丝酵母(CCTCC NO:M20191122)发酵得到的发酵产物,具有清除DPPH自由基的能力,同时对皮肤细胞表皮Hacat细胞和成纤维细胞都有一定的增殖促进作用,并且能够抑制B16细胞的酪氨酸酶活性和黑素生成。此外,该发酵液能抑制金黄色葡萄球菌的菌株活力,同时具有促进表皮葡萄球菌的菌株活力的作用。而这些生物活性使其在化妆品的抗衰,美白,以及调节皮肤微生态菌群上拥有巨大的应用潜力。
生物材料保藏信息
本发明的涎沫假丝酵母JBB-MBY-GW103,已于2019年12月27日保藏在中国典型培养物保藏中心(CCTCC),保藏地址:湖北省武汉市武昌区八一路299号,邮编:430072,保藏编号为:CCTCC NO:M20191122,培养物名称是JBB-MBY-GW103,分类命名是Candidazeylanoides。
附图说明
图1为分离得到的涎沫假丝酵母的菌落形态。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1菌株的分离、筛选及鉴定
使用灭菌工具采集四零冰川固体冰块(北纬28°28′52″,东经 90°12′45″),并迅速将样品装入灭菌容器中。样品运回实验室后,立即在无菌操作台上进行处理。采用梯度稀释平板涂布法分离并冰川样品中的微生物。取1mL样品加入9mL生理盐水稀释10倍,再从这里面取1mL与 9mL生理盐水混和,依次类推稀释浓度分别为10-2、10-3、10-4。取100μL 不同稀释浓度样品溶液,分别涂布于YPD培养基上。28℃培养48h,4℃保藏备用。定期检查平板上菌落的生长状况。将平板上的菌落都转接到 YPD培养基上。根据菌落表型差异进行分离纯化后进行菌株鉴定及保藏。
YPD培养基配方为:10g酵母膏,20g蛋白胨,20g葡萄糖,蒸馏水1000mL,121℃灭菌20min。
经分离得到一株酵母菌株命名为JBB-MBY-GW103。菌落形态小,呈圆形且边缘整齐。菌落为乳白色,不透明。长势缓慢。同时该菌的ITS序列比对结果鉴定确定其为一株涎沫假丝酵母(Candida zeylanoides),见图1。该菌株已保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO: M20191122。
实施例2菌株发酵产物制备
挑取平板上单一菌落接种于5mLYPD液体培养基中,28℃,190r培养 24h后,制备得到种子液。以1%的接种量接种到50mLYPD培养基中, 28℃,190r培养4d后获得菌株发酵产物。4000rpm离心取上清液冻干,称重保存。
实施例3菌株发酵产物成分测定
3.1多酚含量的测定
标准品没食子酸和待测样品用水溶解。取0.1mL没食子酸标准液和待测样品溶液,加入0.5mL 10%Folin-Ciocalteu试剂,混合,室温放置5min 后加入0.4mL 7.5%Na2CO3溶液,混合后室温放置60min,测定765nm的 OD值。由标准液各管OD765对没食子酸含量作标准曲线,根据标准曲线计算样品中多酚含量。结果见表1。
3.2总糖含量的测定
标准品葡萄糖和待测样品用水溶解。取葡萄糖标准液或者待测样品溶液0.2mL,加5%苯酚0.4mL,振荡后加浓硫酸2.0mL,振荡,在室温放置30min,以空白管为对照测定490nm处的OD值。由标准液各浓度 OD490对葡萄糖含量作标准曲线,根据标准曲线计算样品中总糖含量。所述标准曲线的制备方法同本领域常规。
对菌株JBB-MBY-GW103(CCTCC NO:M20191122,后称T-103)进行发酵产物制备及其成分分析,其结果如下表1。该菌株其发酵产物无难闻的发酵味道产生,并在其发酵产物种多糖含量达到517mg每克。结果见表1。
表1发酵产物基本成分分析
实施例4菌株发酵产物清除自由基能力测试
1,1-二苯基-2-三硝基苯肼(DPPH)是一种稳定的氮中心有机自由基。 DPPH法于1958年被提出,广泛用于地量测定生物试样、分类物质和食品的抗衰老能力。此法是根据DPPH自由基有单电子,在517nm处有一强吸收,其醇溶液呈紫色的特性。当有自由基清除剂存在时,由于与其单电子配对而使其吸收逐渐消失,其褪色程度与其接受的电子数量成定量关系,因而可用分光光度计进行快速的定量分析,来检测自由基清除情况,从而评价样品的抗衰老能力。
准确称取15mgDPPH(购置于sigma,货号D9132),95%乙醇溶解后定容于5mL容量瓶中,超声辅助溶解。移取0.25mL原液用95%乙醇溶解定容于25mL容量瓶中。获得DPPH乙醇溶液(30ug/mL)。
取发酵液溶液0.lmL加入试管中,再加入0.1ml DPPH乙醇溶液,混匀,室温下避光在黑暗处反应30min,在525nm处测定OD值,按照下面公式计算清除率:清除率I(%)=[1-(T-T0)/(C-C0)]×100%,式中:T0: 0.1ml样品液加0.1mL 95%乙醇的吸光度;T:0.1ml样品液加0.1ml DPPH溶液的吸光度;C0:0.1ml水加0.1ml 95%乙醇的吸光度;C: 0.1ml水加0.1ml DPPH溶液的吸光度。结果如表2所示。
表2发酵产物DPPH自由基清除率
实施例5菌株发酵产物的抑菌能力测试
将金黄色葡萄球菌S.a(Staphylococcus aureus ATCC 12600)和表皮葡萄球菌S.e(Staphylococcus epidermidis12228)分别接入LB液体培养基,摇床37℃,150rpm培养16小时;4000rpm离心移除培养基,灭菌水稀释重悬菌液获得等OD600=0.5的菌悬液。将S.a和S.e的菌悬液,分别接种于含样品和含对照组的LB培养基中,测试体系总体积4mL。每个样品平行3 管。培养16小时后,采用比浊法通过测定吸光值OD600表示微生物的生物量。取1mL菌液,4000rpm离心移除培养基,加入1mL灭菌水重悬菌液测定吸光值OD600。并以此计算菌株活力。菌株活力=(1-T样品/C对照) *100%。
其中样品浓度分别为:C-空白对照:400μL灭菌水+3600μL液体LB培养基;T-样品:400μL发酵产物+3600μL液体LB培养基。发酵产物样品配置浓度为50mg/mL。
对该酵母菌发酵产物抑菌活性的实验结果显示,该发酵产物在终浓度为5mg/mL时对金黄色葡萄球菌有明显的抑制作用,抑制其24.01%的菌株活力,与此同时对表皮葡萄球菌则表现出促进作用,增加了8.04%的菌株活力。结果见表3。
表3发酵产物的抑菌活性
与Control比,***p<0.001,*p<0.05
实施例6菌株发酵产物促进皮肤细胞增殖能力测试
角质细胞是表皮层中最主要的细胞,参与形成皮肤的物理屏障,防止物理、化学性及微生物等不良因素的侵袭,对皮肤起保护作用。皮肤衰老后的一个主要特征就是表皮变薄,伤口愈合速度变慢,这主要是由于表皮层中角质细胞的再生能力下降,角质细胞体系的增殖能力降低所导致。
一般来说,真皮衰老表现为真皮对外来化学物清除力下降,真皮厚度变薄、胶原蛋白和弹性蛋白合成减少、分解增加,分解酶活性增强。这些现象都与成纤维细胞数减少以及分泌合成功能下降或异常有关。人皮肤成纤维细胞是皮肤真皮网织层中最重要的细胞,是皮肤衰老和细胞受损后的主要修复细胞之一。它不但能够促进表皮细胞的迁移、增殖和分化,还能分泌大量的胶原蛋白、弹性纤维蛋白及多种细胞修复因子,具有强大的自我更新能力,从而修复老化的皮肤。
发酵样品按常规方法冻干,用PBS溶解后过滤除菌,加入到人永生化表皮Hacat细胞和人原代成纤维细胞培养液中,以不含样品的PBS水为空白对照。培养48小时后,用MTT法对细胞染色后,用酶标仪测定550nm处吸光度,空白对照的增殖率为100%,参比空白对照,评估对人角质形成细胞增殖的影响。结果如表4所示。
表4皮肤细胞增殖结果
与Control比,**p<0.01
T-103发酵产物对皮肤细胞表皮Hacat细胞和成纤维细胞都有一定的增殖促进作用。
实施例7菌株发酵产物对小鼠B16黑色素瘤细胞酪氨酸酶抑制作用的测试
皮肤的颜色来自于角质形成细胞内存储的黑色素。一般来讲,存储黑色素多的人肤色更深,也更受到保护,远离阳光辐射。黑色素数量和质量在皮肤黑色素是决定皮肤颜色的重要因素。酪氨酸酶是结构复杂的含铜氧化还原酶,广泛存在于微生物、动植物及人体中。在人体中酪氨酸酶是皮肤合成黑色素的关键酶。
采用L-Dopa氧化法测定样品对酪氨酸酶的抑制作用。小鼠黑色素瘤B16 细胞以1×105密度培养在96孔板中,经24h后,将提取物的样品粉末用PBS溶解后过滤除菌,加入到细胞中培养48h,去掉培养液,每孔加入含1% TritonX-100的PBS缓冲液100μL,然后加入50μL 0.2mg/mL的L-DOPA, 37℃处理3h后再测定490nm的吸光值。按如下公式计算酶活力:酪氨酸酶抑制率=[1-(实验组OD值/对照组OD值]×100%。同时用MTT法测定样品对 B16细胞的活力影响。
采用NaOH裂解法测定细胞内黑色素的含量。小鼠黑色素瘤B16细胞以 1×105密度培养在12孔板中,经24h后,将提取物的样品粉末用PBS溶解后过滤除菌,加入到细胞中,培养48h弃去上清液,胰酶消化后离心收集细胞到离心管,加入150μL的1mol/L的NaOH溶液(含10%DMSO),在80℃条件下充分裂解细胞1h,测定405nm的吸光值。B16细胞的蛋白浓度以BCA法测定,用于吸光值的校正。数据统计以对照组的百分比表示。
结果如表5所示。
表5小鼠B16黑色素瘤细胞酪氨酸酶抑制作用
与Control比,***p<0.001,**p<0.01,*p<0.05
T-103发酵产物对B16细胞活力没有影响,但是能够抑制B16细胞的酪氨酸酶活性和黑素生成。
Claims (10)
1.一种涎沫假丝酵母,其特征在于,所述涎沫假丝酵母为涎沫假丝酵母(Candidazeylanoides)JBB-MBY-GW103,保藏编号为CCTCC NO:M20191122。
2.一种涎沫假丝酵母发酵物的制备方法,其特征在于,所述制备方法包括在培养基中加入如权利要求1所述的涎沫假丝酵母并培养。
3.如权利要求2所述的制备方法,其特征在于,所述培养基为YPD液体培养基。
4.如权利要求2所述的制备方法,其特征在于,所述制备方法还包括在培养所述涎沫假丝酵母后,离心取上清液。
5.如权利要求2-4任一项所述的制备方法,其特征在于,所述培养的温度为20-40℃,所述培养时进行了振荡培养,所述振荡培养的速度为150-250rpm,所述培养的时间为1-10天。
6.如权利要求5所述的制备方法,其特征在于,所述温度为28℃,所述振荡的速度为190rpm,所述时间为4天。
7.如权利要求2或3所述的制备方法,其特征在于,在所述培养基中加入1%的涎沫假丝酵母种子液。
8.一种通过如权利要求2-7任一项所述制备方法制备的涎沫假丝酵母发酵物。
9.一种如权利要求8所述的涎沫假丝酵母发酵物在制备抗衰老、抑菌和/或美白的产品中的应用;优选地,所述产品为护肤品、化妆品或清洁剂。
10.一种皮肤外用剂,所述皮肤外用剂包含如权利要求8所述的涎沫假丝酵母发酵物;优选地,所述的皮肤外用剂为化妆水、乳液、精华液或乳霜;更优选地,所述涎沫假丝酵母发酵物的含量为0.01~0.5%,所述百分比为质量百分比。
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Address after: 201403 Shanghai City, Fengxian District Lifenglu No. 12 Patentee after: Shanghai Natural Hall Group Co.,Ltd. Country or region after: China Address before: 201403 Shanghai City, Fengxian District Lifenglu No. 12 Patentee before: JALA Group Co. Country or region before: China |