CN105420132B - 一种酿酒酵母及其在制备皮肤外用剂中的应用 - Google Patents
一种酿酒酵母及其在制备皮肤外用剂中的应用 Download PDFInfo
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- CN105420132B CN105420132B CN201610050717.6A CN201610050717A CN105420132B CN 105420132 B CN105420132 B CN 105420132B CN 201610050717 A CN201610050717 A CN 201610050717A CN 105420132 B CN105420132 B CN 105420132B
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Abstract
本发明公开了一种酿酒酵母及其在制备皮肤外用剂的应用。所述酿酒酵母保藏在中国典型培养物保藏中心，保藏号为：CCTCC No:M 2015725。酿酒酵母CCTCC No:M 2015725具有抵御紫外线能力强、β‑糖苷酶酶活性高的特性，能够应用于皮肤外用剂的制备中，以达到抗氧化、抗衰老的目的。
Description
技术领域
本发明涉及微生物领域，具体涉及一种酿酒酵母及其在制备皮肤外用剂中的应用。
背景技术
自由基衰老学说(Harman，D.Aging:A theory based on free radical andradiation chemistry.J.Gerontol.1956，11:289-300)，认为过多的自由基是造成生物老化的重要原因。根据这一理论，体内过量的活性氧自由基与不饱和脂肪酸作用产生丙二醛等物质，丙二醛与细胞膜上的蛋白质等作用产生褐色素，沉淀于皮肤成为各种色斑。过量的自由基还能使皮肤内的胶原纤维、弹性纤维发生交联变性、变脆、失去弹性，当皮肤水分不足时，容易使弹性纤维断裂，出现暗纹、细纹、皱纹等皮肤老化现象。此外，环境中的离子辐射以及诸如空气污染与化学物质的环境污染物，也会使生物体内不断地产生自由基。例如紫外线会使皮肤真皮中的纤维母细胞及粒线体受到刺激，进而释放出超氧阴离子，而过量的超氧阴离子则会转换成其它破坏性更强的自由基。
虽然人体内具有能够维持氧化与抗氧化之间平衡状态的抗氧化防御***，用以减缓活性氧及自由基的产生，但若长期过度地曝晒在阳光下，则人体内大量产生的自由基会导致皮肤的抗氧化防御能力降低，造成皮肤的伤害，例如光老化、表皮皱纹、皮肤免疫力失调等。目前已知有维生素E、维生素C等生物体内的清除自由基型抗氧化剂，另外也报道了植物来源的抗氧化剂如莲花、梅果提取物等。
皮肤老化的原因可以分为光老化和内因引起的老化，紫外线(UV)暴露引起的光老化会引起皮肤弹性蛋白酶的活性增加。目前已知有植物来源的抗老化活性物有蓼蓝、香豌豆等。
酿酒酵母(Saccharomyces cerevisiae)是单细胞真菌，形态通常有球形、卵圆形等，一般为1～5微米至5～30微米，有细胞壁、细胞膜、细胞核、细胞质结构。酿酒酵母主要生长在偏酸性的潮湿的含糖环境中，菌体含有丰富的蛋白质、脂肪、糖分和B族维生素等，以及酶、辅酶、核糖核酸、甾醇和一些新陈代谢的中间产物，具有丰富的营养价值。酿酒酵母丰富的代谢产物可利用于药物或化妆品的制备中，如SAM、NAD(H)激酶、Pos5p、胞外多糖、金属硫蛋白等。SAM是半胱氨酸、牛磺酸和谷胱甘肽等重要物质的前体。如谷胱甘肽是美国MerleNorman经典作品，风行欧美50年，堪称世纪美白祛斑经典。NAD(H)激酶的缺失将导致细胞抗氧化性能出现障碍。在细胞面临不同类型的氧化胁迫时，Pos5p都能有效行使其NAD(H)激酶活性，补充NADP(H)的损耗，从而对细胞起到抗氧化保护作用。酿酒酵母多糖作为多糖中的一种，研究发现其具有多种生理活性，能够提高机体免疫力，还具有抗氧化、抗病毒和抑制肿瘤等作用。
尽管来源于微生物的具有抗氧化清除自由基和抗衰老活性作用的抗老化物质正日渐得到护肤产业的重视，但是目前仍然缺乏具有抵御紫外线能力强、糖苷酶活性强的酿酒酵母。
发明内容
本发明所要解决的技术问题是，针对目前缺乏具有抵御紫外线能力强、糖苷酶活性强的酿酒酵母的不足，提供一种抵御紫外线能力强、糖苷酶活性强的酿酒酵母及其在制备皮肤外用剂中的应用。
本发明的技术方案之一是：一种酿酒酵母(Saccharomyces cerevisiae)，其保藏在中国典型培养物保藏中心(CCTCC)，保藏号为：CCTCC No:M2015725。
将购自中国工业微生物菌种保藏管理中心(China Center of IndustrialCulture Collection，CICC)的酿酒酵母菌株通过神舟十号载人飞船搭载进入太空，历经15天的太空搭载后，经选择获得所述的酿酒酵母CCTCC No:M2015725。所述的酿酒酵母CCTCCNo:M 2015725在PDA液体培养基中生长良好，具有抵御紫外线能力强、糖苷酶活性强的特性。对该菌株进行鉴定，结果为酿酒酵母(Saccharomyces cerevisiae)，命名为JBA-DZ-49-Gly-3-05。该菌株已于2015年12月7日保藏于中国典型培养物保藏中心，并收到保藏编号CCTCC No:M 2015725。
本发明的技术方案之二是，一种培养酿酒酵母CCTCC No:M 2015725的方法，其包括以下的步骤：在培养基中培养所述的酿酒酵母CCTCC No:M 2015725即可。
其中，所述的培养基为本领域常规的培养基，能够生长所述的酿酒酵母CCTCC No:M 2015725即可，较佳地为PDA液体培养基、YPD培养基或GMMY培养基。所述的PDA液体培养基为本领域常规的PDA液体培养基，较佳地，其包括5g/L马铃薯浸粉、15g/L葡萄糖、10g/L蛋白胨和5g/L NaCl。所述培养的温度为本领域常规的温度，能够生长所述的酿酒酵母CCTCCNo:M 2015725即可，较佳地为28～30℃，更佳地为28℃。所述培养的时间为本领域常规的时间，较佳地为2～3天，更佳地为2天。
较佳地，所述的培养前还包括使用种子培养基进行种子培养的步骤。所述的种子培养为本领域常规的种子培养。所述的种子培养基为本领域常规的种子培养基，较佳地为PDA液体培养基。所述种子培养的时间为本领域常规的时间，较佳地为48～60小时，更佳地为48小时。所述种子培养的温度为本领域常规的温度，较佳地为28～30℃。所述种子培养的接种量为本领域常规的接种量，较佳地为5％，所述百分比为体积百分比。
本发明的技术方案之三是：一种酿酒酵母CCTCC No:M 2015725在制备皮肤外用剂中的应用。
本发明所述酿酒酵母CCTCC No:M 2015725具有抵御紫外线能力强、β-糖苷酶酶活性高的特性，能够应用于皮肤外用剂的制备中，以达到抗氧化、抗衰老的目的。
本发明中，所述皮肤外用剂是通常用于皮肤外部的所有成分的统称概念，例如可以是化妆品或药品。所述化妆品中可以是基础化妆品、面部妆容化妆品、头部用护理品、身体用化妆品等，对其剂型无特殊限制，根据不同目的可合理选择。
本发明中，所述的皮肤外用剂的形式可以为任何合适的产品形式，所述的产品形式包括，但不限于气溶胶型喷雾剂、霜剂、乳液、固体、液体、分散体、泡沫、凝胶、化妆水、摩丝、软膏、粉剂、贴剂、润发油、手按泵型喷雾剂、棒状物、面膜和湿纸巾。众所周知地，本发明的皮肤外用剂可为化妆品、皮肤病学或药物局部施用产品，其制备方法可为本领域常规的制备方法。
所述的皮肤外用剂还可以进一步包含可药用载体。所述的可药用载体是本领域常规可药用载体，较佳地为防腐剂、香精、亲水性活性剂和亲脂性活性剂中的一种或多种。所述的可药用载体的含量为本领域常规的含量。
所述的皮肤外用剂还可以进一步包括一种或多种下述成分：抗过敏剂、抗微生物剂、抗氧化剂、螯合剂、着色剂去色素剂、润肤剂、乳化剂、表皮脱落剂、香料、保湿剂、昆虫驱避剂、润滑剂、药物活性剂、增湿剂、耐光剂、防腐剂、护肤剂、皮肤渗透增强剂、防晒剂、稳定剂、表面活性剂、增稠剂、粘度调节剂、维生素或其任意组合。该些成分的含量可为本领域常规的含量。
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：本发明通过太空搭载诱变及后续的筛选方法，获得了一株抵御紫外线能力强、糖苷酶活性强的酿酒酵母CCTCC No:M 2015725，并且提供了其在制备皮肤外用剂中的应用。酿酒酵母CCTCC No:M 2015725具有抵御紫外线能力强、β-糖苷酶酶活性高的特性，并且可以通过发酵提高植物提取物的生物可利用性和活性，因此该酿酒酵母能够应用于皮肤外用剂的制备中。所制得的皮肤外用剂拥有更强的抗老化和抗衰老能力，该皮肤外用剂能够帮助皮肤清除的自由基，抵御UV辐射，防止光老化。
生物材料保藏信息
本发明的所述的酿酒酵母(Saccharomyces cerevisiae)，已于2015年12月7日保藏在中国典型培养物保藏中心(CCTCC)，保藏地址：中国武汉武汉大学，邮编：430072，保藏编号为CCTCC No:M 2015725，培养物名称为酿酒酵母JBA-DZ-49-Gly-3-05，分类命名为Saccharomyces cerevisiae JBA-DZ-49-Gly-3-05。
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。
实施例中所述室温是本领域常规的室温，较佳地为10～30℃。
实施例中所述培养基的组分如下所示：
YPD培养基：1％(w/w)酵母膏、2％(w/w)蛋白胨、2％(w/w)葡萄糖和水，pH为6.0。
PDA液体培养基：5g/L马铃薯浸粉、15g/L葡萄糖、15g/L蛋白胨和5g/L NaCl。
实施例1本发明新微生物的获得
将购自中国工业微生物菌种保藏管理中心(China Center of IndustrialCulture Collection，CICC)的酵母菌菌株(菌株保藏编号CICC1210)等分为两份，一份通过神舟十号载人飞船搭载进入太空，历经15天的太空搭载后，分离获得太空诱变的酵母菌菌株DZ-39、DZ-49，另外一份为出发菌株CK作为对照，以便进行以下的试验。
1)紫外辐射(UV)环境中存活率测试
紫外辐射(UV)环境中存活率的具体步骤参照文献方法进行(Qadri I,Fatima K,AbdeL-Hafiz H.Hepatitis B virus X protein impedes the DNA repair via itsassociation with transcription factor,TFIIH.BMC Microbiol.2011；11:48.)。
实验结果如表1所示，其中，存活率的含义是UV光照后存活的酵母菌菌落数目占空白对照组未进行UV光照存活的酵母菌菌落数目的百分比；1、2分别表示重复组1、2。表1说明菌株DZ-49表现出更好的抵御UV的能力，30秒时存活率都在60％以上，优于对照CK和DZ-39，CK和DZ-39在30秒时存活率在40％左右。
表1紫外辐射(UV)环境中存活率
2)β-糖苷酶酶活性测试
2.1菌株培养
将突变的酿酒酵母菌株和出发菌株CK按25μL/50mL的接种量接种于YPD培养基，30℃、150rpm摇床培养48小时，分别得培养液。用去离子水配制使突变的酿酒酵母菌株与CK的培养液的浓度相同。将相同浓度的上述培养液8000rpm离心5分钟，取上清液。
2.2 β-糖苷酶酶活性测试
取4μL上清液加入80μL 5mmol/L的pNPG溶液(将购自aladdin的对硝基苯-β-D-半乳糖吡喃糖苷，用磷酸-柠檬酸缓冲液调pH至5)滴加于酶标板，并在酶标仪(购自美国分子仪器公司)内50℃温热30分钟进行反应，然后加入160μL 1mol/L的碳酸钠终止反应，立即测量OD405。代入下面公式计算β-糖苷酶酶活性：
其中，V：反应液的总体积(mL)；v：反应体系内酶液量(mL)；T：反应时间(分钟)；OD405：反应体系405nm吸光度值。
结果如表2所示，结果说明菌株DZ-49的β-糖苷酶酶活性高于对照CK和太空诱变的菌株DZ-39。
表2 β-糖苷酶酶性活检测
根据上述试验，选择抵御UV能力强、β-糖苷酶酶活性高的突变的菌株DZ-49。由于研究表明，在UV辐射下，酿酒酵母能产生自我保护物质，这些物质中的抗氧化成分能够帮助皮肤清除自由基，抵御UV辐射，具有防止光老化的活性。β-糖苷酶是生物转化有关的关键酶，酿酒酵母通过β-糖苷酶的作用可以将植物提取物中含有糖苷结构的分子进行生物转化，提高植物提取物的功效活性。由此可见，菌株DZ-49能够应用于皮肤外用剂的制备中。
实施例2菌株DZ-49的形态学和培养学特征
形态学和培养特征：采用PDA液体培养基28℃培养菌株DZ-49。48小时后，观察菌落，菌落质地柔软，干燥，菌落为乳白色，圆形，微凸，边缘光滑，细胞呈圆形至椭圆形，增殖方式为芽殖，有假菌丝形成。
实施例3菌株DZ-49的生理生化特性
参照祖若夫，胡宝龙，周德庆《微生物学实验教程》(复旦大学出版社1993年)对菌株DZ-49进行生理生化特征分析。
结果发现，菌株DZ-49可以利用碳源葡萄糖、蔗糖、麦芽糖、半乳糖、棉子糖；不发酵乳糖、松三糖、D-纤维二糖；不同化木糖、甘油、乙醇葡萄糖、蔗糖、麦芽糖、半乳糖或棉子糖，不能利用碳源乳糖或松三糖。菌株DZ-49不同化木糖、甘油或乙醇。
将菌株DZ-49于2015年12月7日在中国典型培养物保藏中心(CCTCC)保藏，获得保藏编号为：CCTCC No:M 2015725，培养物名称是JBA-DZ-49-Gly-3-05，分类命名是酿酒酵母(Saccharomyces cerevisiae)。
实施例4菌株DZ-49的培养
菌株DZ-49接种至PDA液体培养基中，28℃培养48小时，得种子液；
接种5％(v/v)种子液于至PDA液体培养基中，28℃，200rpm摇床培养2天。
实施例5菌株DZ-49的培养
菌株DZ-49接种至PDA液体培养基中，28℃培养60小时，得种子液；
接种5％(v/v)种子液于至PDA液体培养基中，30℃，200rpm摇床培养2天。
实施例6菌株DZ-49的培养
菌株DZ-49接种至PDA液体培养基中，30℃培养48小时，得种子液；
接种5％(v/v)种子液于至PDA液体培养基中，28℃，200rpm摇床培养3天。
实施例7酵母菌人参花发酵物的制备
(1)、人参花提取物的制备
将200g市售的人参花粉碎，按1:10(g/mL)加入去离子水，于水浴锅中90℃微沸搅拌提取2次，每次60分钟，固液分离后的滤液，将滤液除去水分即为人参花提取物。
(2)、酿酒酵母人参花发酵物的制备
将菌株DZ-49接种至PDA液体培养基中，28℃培养48小时，得种子液；
接种5％(v/v)种子液于至含有20％(w/w)人参花提取物的PDA液体培养基中，28℃，200rpm摇床培养5天，得培养液；
将培养液于8℃，8000rpm离心5分钟，取上清液，冷冻干燥即得酿酒酵母人参花发酵物。
实施例8酿酒酵母人参花发酵物的抗氧化活性的检测
将实施例7制备的酿酒酵母人参花发酵物和人参花提取物，用DPPH法测定其清除自由基的抗氧化活性。
测定步骤为：将上述待测样品用去离子水配制成0.5mg/mL的溶液A，移取2mL溶液A于10mL具塞试管中，加入2mL 2×10-4mol/L的DPPH乙醇溶液(DPPH购自国药集团化学试剂有限公司)，充分混匀，室温静置30min后用分光光度计测定517nm波长的吸光度A517；同时测定2mL溶液A与2mL乙醇混合后的吸光度A0，2mL去离子水与2mL 2×10-4mol/L的DPPH乙醇溶液混合后的吸光度C，以及2mL去离子水与2mL乙醇溶液混合后的吸光度C0。平行测定三次，取平均值，根据以下公式计算自由基清除率，自由基清除率越大，说明酵母人参发酵物的抗氧化能力越强。
自由基清除率(％)＝[1-(A517-A0)/(C-C0)]×100％
表3 DPPH法清除自由基活性的检测结果
| 检测对象 | 浓度(mg/mL) | 自由基清除率(％) |
| 酿酒酵母人参花发酵物 | 0.25 | 95 |
| 人参花提取物 | 0.25 | 41 |
表3的结果说明菌株DZ-49的人参花发酵物能够有效地清除自由基，能够应用于制备皮肤外用剂。由此可见，菌株DZ-49能够发挥在制备皮肤外用剂中的作用。
实施例9含有酵母菌人参花发酵物的化妆水
使用实施例7步骤(2)制得的酿酒酵母人参花发酵物制备含有酵母菌人参花发酵物的化妆水，其具体组成见下表4。其中“-”表示不含有该种组分。
表4化妆水
按照本领域常规的化妆水的制备方法，制得实施例9的化妆水。
以上所述仅为本发明的较佳实施例而已，并不构成对权利要求范围的限制，本领域技术人员可以想到的其他实质上等同的替代，均在本发明保护范围内。
Claims (7)
1.一种酿酒酵母(Saccharomyces cerevisiae)，其保藏在中国典型培养物保藏中心，保藏号为：CCTCC No:M 2015725。
2.一种培养酿酒酵母CCTCC No:M 201572的方法，其特征在于，其包括以下的步骤：在培养基中培养如权利要求1所述的酿酒酵母CCTCC No:M 2015725即可。
3.如权利要求2所述的方法，其特征在于，所述的培养基为PDA液体培养基；所述的培养的温度为28～30℃；所述的培养的时间为2～3天；和/或，所述的酿酒酵母CCTCC NO:M2015725的接种量为5％，所述百分比为体积百分比。
4.如权利要求3所述的方法，其特征在于，所述的PDA液体培养基包括5g/L马铃薯浸粉、15g/L葡萄糖、15g/L蛋白胨和5g/L NaCl；所述的培养的温度为28℃；和/或，所述的培养的时间为2天。
5.如权利要求2所述的方法，其特征在于，所述的培养前还包括使用种子培养基进行种子培养的步骤；所述种子培养的时间为48～60小时；和/或，所述种子培养的温度为28～30℃。
6.如权利要求5所述的方法，其特征在于，所述种子培养的时间为48小时。
7.如权利要求1所述的酿酒酵母CCTCC No:M 2015725在制备皮肤外用剂中的应用。
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