CN114107082A - 一种复合菌剂及其应用 - Google Patents
一种复合菌剂及其应用 Download PDFInfo
- Publication number
- CN114107082A CN114107082A CN202010883662.3A CN202010883662A CN114107082A CN 114107082 A CN114107082 A CN 114107082A CN 202010883662 A CN202010883662 A CN 202010883662A CN 114107082 A CN114107082 A CN 114107082A
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- extract
- microbial inoculum
- plant extract
- yeast
- composite
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Abstract
本发明提供一种复合菌剂及其应用。本发明提供一种复合菌剂，所述复合菌剂中包含乳酸菌和复合酵母，所述复合酵母包含克鲁维酵母、产酯香酵母和谷胱甘肽酵母。本发明提供的复合菌剂按一定比例投入到植物提取物中，赋予植物提取物特有的酯香气息，并具有美白，抗氧化，保湿的护肤功能。
Description
技术领域
本发明属于微生物领域，具体涉及一种复合菌剂及其应用。
背景技术
植物提取物(Plant Extract，PE)指以天然的整个植物或植物的一部分为原料，经过物理或化学提取分离过程，定向获取和浓集植物中的某一种或多种有效成分，而不改变其有效成分结构所形成的新型高科技产品。
植物提取物作为活性成份应用到化妆品中，通常具有保湿、护肤、防晒、杀菌、抑制黑色素生成、清除自由基、祛痘，防腐，低毒性及环境友好等特性。与传统化妆品相比，具有很多优点：克服了传统化妆品依赖化学合成，及对皮肤的损伤和刺激性缺点，使产品的安全性能更高；其次含有的天然组分经过某些定向技术手段，如经过益生菌发酵，酶解，水解处理后，更容易被皮肤吸收，也使得产品的作用效果更加显著，功能性更丰富。
发明内容
单一的植物原料中活性成分不足以满足皮肤对各种营养成分的需求，通过益生菌发酵植物提取物，可以改善植物提取物的生物特性，更具体地说，益生菌发酵植物提取物作用更温和，能够最大程度保留提取液中的天然活性成分，降低毒性，产生新的有效活性成分，微生物在代谢过程中会产生多种胞外酶，如纤维素酶、蛋白酶、果胶酶等能够将植物提取物中的多糖分解，生物活性成分进一步释放，能够将大分子物质转化为皮肤较易吸收的氨基酸等小分子活性物质，且微生物发酵产品中富含多种活性酶，有更好的抗氧化及美白能力，而植物提取物除含有功能性活性成分外，也通常含有丰富的供益生菌群可利用的糖类，氨基酸，可利用小分子肽，及生长促进因子，因此选用特定功能的益生菌发酵植物提取物，一方面植物提取物中营养组份能促进益生菌的增殖，另一方面经过二者协同作用，同时还能产生新的活性成分和代谢物，具有新的功能效用。
针对现有技术中存在的问题，本发明提供一种复合菌剂。
本发明还提供一种植物提取物发酵液。
第一方面，本发明提供一种复合菌剂，所述复合菌剂中包含乳酸菌和复合酵母，所述复合酵母包含克鲁维酵母、产酯香酵母和谷胱甘肽酵母。
优选地，按重量份计，所述复合菌剂中包含乳酸菌5-15份和复合酵母56-85份，所述复合酵母包含克鲁维酵母50-70份、产酯香酵母1-5份和谷胱甘肽酵母5-10份。
更优选地，按重量份计，所述复合菌剂中包含乳酸菌7-12份和复合酵母64-78份，所述复合酵母包含克鲁维酵母55-65份、产酯香酵母2-4份和谷胱甘肽酵母7-9份。
本发明中所述的克鲁维酵母为现有技术中任一种或两种以上克鲁维酵母。
优选地，所述克鲁维酵母为马克斯克鲁维酵母和乳酸克鲁维酵母中的任一种或两种以上的组合。
优选地，所述复合菌剂中，所述克鲁维酵母的活菌总数为60-100亿CFU/g；
本发明中所述的乳酸菌为现有技术中任一种或两种以上乳酸菌。
优选地，所述乳酸菌选自植物乳杆菌、干酪乳杆菌和保加利亚乳杆菌中的一种或两种以上的组合。
或者，优选地，所述复合菌剂中，所述乳酸菌的活菌总数为800-1000亿CFU/g。
本发明中所述的产酯香酵母为现有技术中任一种或两种以上产酯香酵母。
优选地，所述产酯香酵母为汉逊酵母、球拟酵母和假丝酵母中任一种或两种以上的组合。
或者，优选地，所述复合菌剂中，所述产酯香酵母活菌总数为500-700亿CFU/g。
本发明中所述的谷胱甘肽酵母为现有技术中任一种或两种以上谷胱甘肽酵母。
优选地，所述谷胱甘肽为面包酵母菌、酿酒酵母菌、假丝酵母和裂殖酵母中的一种或两种以上的组合。
或者，优选地，所述复合菌剂中，所述谷胱甘肽酵母活菌总数为200-300亿CFU/g。
第二方面，本发明提供一种植物提取物发酵液，所述植物提取物发酵液含有本发明提供的复合菌剂、植物提取物和水。
优选地，所述植物提取物为桦树提取物、葡萄籽提取物、金盏花提取物、番石榴提取物、小麦胚芽提取物、迷迭香草提取物、橄榄果提取物、红石榴提取物、雪莲培养物、银耳多糖、香瓜提取物、大米提取物或红葡萄提取物中的一种或者两种以上的组合。
优选地，将下述a和b中的一种或两种与植物提取物的水溶液混合，发酵后得到所述植物提取物发酵液：
a：复合菌剂；
b：复合菌剂活化培养后得到复合菌剂培养物；
优选地，所述植物提取物与水的质量比为1∶5-10；
或者，优选地，所述植物提取物与复合菌剂的质量比为10-100∶1；
或者，优选地，所述植物提取物与复合菌剂的质量比为50-70∶1。
第三方面，本发明提供所述植物提取物发酵液的制备方法，所述制备方法步骤包括将下述a和b中的一种或两种与植物提取物的水溶液混合，发酵后得到所述植物提取物发酵液：
a：复合菌剂；
b：复合菌剂活化培养后得到复合菌剂培养物。
优选地，所述植物提取物与水的质量比为1∶5-10。
或者，优选地，所述植物提取物与复合菌剂的质量比为10-100∶1。
或者，优选地，所述植物提取物与复合菌剂的质量比为50-70∶1。
或者，优选地，所述复合菌剂活化培养20-30h。
或者，优选地，所述活化培养温度为30-35℃。
或者，优选地，所述活化培养基为葡萄糖3-7％，酵母浸粉1-5％，磷酸二氢钾0.1-0.5％，磷酸氢二钾0.1-0.5％，硫酸镁0.01-0.1％，硫酸锰0.01-0.05％，调节培养基的pH为5.5-6.0。
或者，优选地，所述发酵温度为25-35℃。
或者，优选地，发酵时间为20-60h，更优选地，发酵时间为24-48h。
或者，优选地，所述发酵后还包括将发酵产物进行离心过滤，除去菌体，采用滤膜二次除杂过滤。
第四方面，本发明还提供所述复合菌剂或所述植物提取物发酵液或所述的制备方法制备得到的植物提取物发酵液在保健品领域或化妆品领域的应用。
本发明产品经过实验验证，按一定比例投入到植物提取物中，赋予植物提取物特有的酯香气息，并具有美白，抗氧化，保湿的护肤功能，相比仅以植物提取物为主要原料的产品，进一步丰富了植物提取物的营养特性，同时通过益生菌柔性转化了生物活性分子，使得护肤功能成分更丰富及完善，便于化妆品原料开发商生产稳定性，从而保证化妆品原料品质一致性。
本发明已经过实验、模拟和中式试制，本发明的益生菌菌剂产品具有普适性及可行性，证明了植物提取物经过本发明的复合菌剂发酵后，按一定比例复配到化妆品原料中，经过体外验证，其抗氧化，和提高防晒，美白，保湿的功能显著。
菌种保藏信息
本发明所用的汉逊酵母PA-9(Hansenula polymorpha PA-9)于2020年06月02日保藏在中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2020174，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话：(027)-68754052。
本发明所用的植物乳杆菌LP-23(Lactobacillus plantarum LP-23)于2020年06月02日保藏在中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2020175，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话：(027)-68754052。
本发明所用的克鲁维酵母KW-6(Kluyveromyces marxious KW-6)于2020年06月02日保藏在中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2020173，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话：(027)-68754052。
本发明所用的酿酒酵母GT-58(Saccharomyces cerevisiae GT-58)于2020年06月02日保藏在中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2020172，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话：(027)-68754052。
具体实施方式
本发明提供一种复合菌剂，所述复合菌剂包含乳酸菌和复合酵母，所述复合酵母包含克鲁维酵母、产酯香酵母和谷胱甘肽酵母。
本发明中所述的产酯香酵母是指能够生成含有酯香味物质的酵母菌。
本发明中所述的谷胱甘肽酵母是指能够产生谷胱甘肽的酵母菌。
本领域技术人员依据本发明记载，可利用现有技术中任一种或两种以上乳酸菌、任一种或两种以上克鲁维酵母、任一种或两种以上产酯香酵母、任一种或两种以上谷胱甘肽酵母来实现本发明。
本申请发明人利用现有技术中的乳酸菌、克鲁维酵母、产酯香酵母和谷胱甘肽酵母来实施本发明，并且将所述克鲁维酵母、乳酸菌、产酯香酵母、谷胱甘肽酵母进行保藏，保藏编号分别为CCTCC NO：M 2020173、CCTCC NO：M 2020175、CCTCC NO：M 2020174、CCTCCNO：M 2020172，均保藏于中国典型培养物保藏中心(CCTCC)。
以下以非限制方式更具体的介绍适用于本发明的复合菌剂及其应用。
本发明实施例中总还原力、·OH清除率和DPPH·清除率的检测方法如下：
(1)总还原力测定：取1mL发酵液，加入0.2mol/L的pH值为6.6的磷酸缓冲液2.5mL和2.5mL质量分数为1％的铁氰化钾，混合物在50℃避光保存20min，再加入2.5mL质量分数10％三氯乙酸溶液，混合均匀。在3000r/min下离心10min，取2.5mL上清液，加入2.5mL蒸馏水和0.5mL质量分数0.1％的三氯化铁溶液混合均匀，静置10min，在700nm下测定吸光度，以吸光度值表示还原力大小。
(2)DPPH·清除率：DPPH自由基清除率：取3mL等体积的待测液与2x104mol/L的DPPH溶液混匀(A1管)；取等体积的无水乙醇与2×104mol/L的DPPH溶液混匀(A2管)；取等体积的无水乙醇与待测液混匀(A3管)；30℃避光反应30min后，以蒸馏水为空白组，在517nm下测A1、A2、A3管的吸光度值，分别记作ODA1、ODA2和ODA3。DPPH自由基清除率根据式如下公式计算：
DPPH清除率＝(ODA2+ODA3-ODA1)/ODA2×100％。
(3)·OH清除率测定：吸取1mL样品溶液，加入0.05mol/LpH8.2的Tris-HC1-EDTA缓冲液9mL，混匀，25℃水浴反应15min，反应结束后，取3mL该反应液，加入45mmol/L的邻苯三酚溶液(用0.01molL盐酸配制)0.1mL，混匀，静置反应3min，测其在340nm处的吸光度A1，同时检测未加邻苯三酚的混合物吸光值A2，以蒸馏水代替样品溶液，得到吸光值A3，以蒸馏水作为空白。超氧离子清除率根据如下公式计算：
·OH清除率＝A3-(A1-A2)/A3*100。
下面实施例中所用到的微生物及试剂来源如表1所示。
表1实施例中用到的试剂信息表
| 名称 | 厂家 |
| 桦树提取物 | 西安文竹生物科技公司 |
| 葡萄籽提取物 | 中鑫生物 |
| 番石榴提取物 | 沐凡生物 |
| 小麦胚芽提取物 | 西安首禾生物科技有限公司 |
| 迷迭香草提取物 | 上禾生物科技 |
实施例1-5和对比例1-6
称取如表2中所示的克鲁维酵母KW-6、植物乳杆菌LP-23、汉逊酵母PA-9和酿酒酵母GT-58，混匀后得到实施例1-5和对比例1-6的复合菌剂。
表2实施例1-5和对比例1-6的复合菌剂的组分含量
实施例6一种桦树提取物发酵液
取12份桦树提取物，每份20g，分别标记为实施例1-5组、对比例1-6组，每份桦树提取物均溶于100g水中，121℃，灭菌20min。
分别取实施例1-5和对比例1-6中复合菌剂各1g分别加入到50ml活化培养基中，在35℃条件下，200rpm活化培养24h，活化培养基中，葡萄糖5wt％，酵母浸粉3wt％，磷酸二氢钾0.2wt％，磷酸氢二钾0.2wt％，硫酸镁0.04wt％，硫酸锰0.025wt％，其余为水，pH为5.5。活化培养后的复合菌剂培养液在无菌环境下分别加入到对应组别的桦树提取物水溶液中，另一份桦树提取物水溶液作为空白对照不加复合菌剂。将12份桦树提取物水溶液在30℃条件下，200rpm发酵48h，5000rpm离心过滤，除去菌体，上清液经过0.22μm滤膜，二次除杂过滤。
测定得到的12份桦树提取物水溶液发酵液的总还原力、·OH清除率和DPPH·清除率，测定结果如表3所示。
表3
实施例7一种葡萄籽提取物发酵液
取12份葡萄籽提取物，每份10g，分别标记为实施例1-5组、对比例1-6组，每份葡萄籽提取物均溶于100g水中，121℃，灭菌20min。分别取实施例1-5和对比例1-6中复合菌剂各1g，在无菌环境下分别加入到对应组别的葡萄籽提取物水溶液中，另一份葡萄籽提取物水溶液作为空白对照不加复合菌剂。将12份葡萄籽提取物水溶液在30℃条件下，200rpm发酵48h，5000rpm离心过滤，除去菌体，上清液经过0.22μm滤膜，二次除杂过滤。
测定得到的12份葡萄籽提取物水溶液发酵液的总还原力、·OH清除率和DPPH·清除率，测定结果如表4所示。
表4
实施例8一种番石榴提取物发酵液
取12份番石榴提取物，每份60g，分别标记为实施例1-5组、对比例1-6组，每份番石榴提取物均溶于300g水中，121℃，灭菌20min。分别取实施例1-5和对比例1-6中复合菌剂各1g，在无菌环境下分别加入到对应组别的番石榴提取物水溶液中，另一份番石榴提取物水溶液作为空白对照不加复合菌剂。将12份番石榴提取物水溶液在30℃条件下，200rpm发酵48h，5000rpm离心过滤，除去菌体，上清液经过0.22μm滤膜，二次除杂过滤。
测定得到的12份番石榴提取物水溶液发酵液的总还原力、·OH清除率和DPPH·清除率，测定结果如表5所示。
表5
实施例9一种小麦胚芽提取物发酵液
取12份小麦胚芽提取物，每份100g，分别标记为实施例1-5组、对比例1-6组，每份小麦胚芽提取物均溶于1000g水中，121℃，灭菌20min。分别取实施例1-5和对比例1-6中复合菌剂各1g，在无菌环境下分别加入到对应组别的小麦胚芽提取物水溶液中，另一份小麦胚芽提取物水溶液作为空白对照不加复合菌剂。将12份小麦胚芽提取物水溶液在30℃条件下，200rpm发酵48h，5000rpm离心过滤，除去菌体，上清液经过0.22μm滤膜，二次除杂过滤。
测定得到的12份小麦胚芽提取物水溶液发酵液的总还原力、·OH清除率和DPPH·清除率，测定结果如表6所示。
表6
实施例10一种迷迭香草提取物发酵液
取12份迷迭香草提取物，每份10g，分别标记为实施例1-5组、对比例1-6组，每份迷迭香草提取物均溶于100g水中，121℃，灭菌20min。分别取实施例1-5和对比例1-6中复合菌剂各1g，在无菌环境下分别加入到对应组别的迷迭香草提取物水溶液中，另一份迷迭香草提取物水溶液作为空白对照不加复合菌剂。将12份迷迭香草提取物水溶液在30℃条件下，200rpm发酵48h，5000rpm离心过滤，除去菌体，上清液经过0.22μm滤膜，二次除杂过滤。
测定得到的12份迷迭香草提取物水溶液发酵液的总还原力、·OH清除率和DPPH·清除率，测定结果如表7所示。
表7
从表3-7中可以看出，植物提取物水溶液经微生物发酵后的总还原力、·OH清除率和DPPH·清除率均提高，但是本发明实施例1-5组的总还原力、·OH清除率和DPPH·清除率比对比例1-6组的高，且实施例2组、实施例4组和实施例5组的总还原力、·OH清除率和DPPH·清除率比实施例1组和实施例3组的高，说明本发明提供的特定配比的复合菌剂能够提高植物提取物水溶液发酵液的总还原力、·OH清除率和DPPH·清除率。
经过以上实施例说明，植物提取液与经过本益生菌菌剂产品发酵后，在抑制黑色素，防晒美白功效上均得到一定程度提高。
Claims (12)
1.一种复合菌剂，其特征在于，所述复合菌剂中包含乳酸菌和复合酵母，所述复合酵母包含克鲁维酵母、产酯香酵母和谷胱甘肽酵母。
2.根据权利要求1所述复合菌剂，其特征在于，所述乳酸菌选自植物乳杆菌、干酪乳杆菌和保加利亚乳杆菌中的一种或两种以上的组合；
优选地，所述克鲁维酵母为马克斯克鲁维酵母和乳酸克鲁维酵母中的任一种或两种以上的组合；
优选地，所述产酯香酵母为汉逊酵母、球拟酵母和假丝酵母中任一种或两种以上的组合；
优选地，所述谷胱甘肽为面包酵母菌、酿酒酵母菌、假丝酵母和裂殖酵母中的一种或两种以上的组合。
3.根据权利要求1或2所述复合菌剂，其特征在于，按重量份计，所述复合菌剂中包含乳酸菌5-15份和复合酵母56-85份，所述复合酵母包含克鲁维酵母50-70份、产酯香酵母1-5份和谷胱甘肽酵母5-10份；
优选地，按重量份计，所述复合菌剂中包含乳酸菌7-12份和复合酵母64-78份，所述复合酵母包含克鲁维酵母55-65份、产酯香酵母2-4份和谷胱甘肽酵母7-9份。
4.根据权利要求1-3任意一项所述复合菌剂，其特征在于，所述乳酸菌的活菌总数为800-1000亿CFU/g；
或，优选地，所述克鲁维酵母的活菌总数为60-100亿CFU/g；
或，优选地，所述产酯香酵母活菌总数为500-700亿CFU/g；
或，优选地，所述谷胱甘肽酵母活菌总数为200-300亿CFU/g。
5.一种植物提取物发酵液，其特征在于，所述植物提取物发酵液含有权利要求1-4任意一项所述的复合菌剂、植物提取物和水。
6.根据权利要求5所述的植物提取物发酵液，其特征在于，所述植物提取物为桦树提取物、葡萄籽提取物、金盏花提取物、番石榴提取物、小麦胚芽提取物、迷迭香草提取物、橄榄果提取物、红石榴提取物、雪莲培养物、银耳多糖、香瓜提取物、大米提取物或红葡萄提取物中的一种或者两种以上的组合。
7.根据权利要求5或6所述的植物提取物发酵液，其特征在于，将下述a和b中的一种或两种与植物提取物的水溶液混合，发酵后得到所述植物提取物发酵液：
a：复合菌剂；
b：复合菌剂活化培养后得到复合菌剂培养物；
优选地，所述植物提取物与水的质量比为1∶5-10；
或，优选地，所述植物提取物与复合菌剂的质量比为10-100∶1；
或，优选地，所述植物提取物与复合菌剂的质量比为50-70∶1。
8.权利要求5-7任意一项所述的植物提取物发酵液的制备方法，其特征在于，所述制备方法步骤包括将下述a和b中的一种或两种与植物提取物的水溶液混合，发酵后得到所述植物提取物发酵液：
a：复合菌剂；
b：复合菌剂活化培养后得到复合菌剂培养物；
优选地，所述植物提取物与水的质量比为1∶5-10；
或，优选地，所述植物提取物与复合菌剂的质量比为10-100∶1；
或，优选地，所述植物提取物与复合菌剂的质量比为50-70∶1。
9.根据权利要求8所述的制备方法，其特征在于，所述活化培养20-30h，
或，优选地，所述活化培养温度为30-35℃，
或，优选地，所述活化培养基为葡萄糖3-7％，酵母浸粉1-5％，磷酸二氢钾0.1-0.5％，磷酸氢二钾0.1-0.5％，硫酸镁0.01-0.1％，硫酸锰0.01-0.05％。
10.根据权利要求8或9所述的制备方法，其特征在于，所述发酵温度为25-35℃；
或，优选地，发酵时间为20-60h，更优选地，发酵时间为24-48h。
11.根据权利要求8-10任意一项所述的制备方法，其特征在于，所述发酵后还包括将发酵产物进行离心过滤，除去菌体，采用滤膜二次除杂过滤。
12.权利要求1-4任意一项所述的复合菌剂或权利要求5-7任意一项所述植物提取物发酵液或权利要求8-11任意一项所述的制备方法制备得到的植物提取物发酵液在保健品领域或化妆品领域的应用。
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