CN117017843A - 米曲霉与结合酵母发酵复合液的保湿和/或皮肤屏障修护应用 - Google Patents
米曲霉与结合酵母发酵复合液的保湿和/或皮肤屏障修护应用 Download PDFInfo
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- CN117017843A CN117017843A CN202311161297.5A CN202311161297A CN117017843A CN 117017843 A CN117017843 A CN 117017843A CN 202311161297 A CN202311161297 A CN 202311161297A CN 117017843 A CN117017843 A CN 117017843A
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- aspergillus oryzae
- skin
- fermentation filtrate
- yeast fermentation
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Landscapes
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Abstract
米曲霉与结合酵母发酵复合液在皮肤保湿和/或皮肤屏障修护中的应用,所述米曲霉与结合酵母发酵复合液包含米曲霉发酵滤液和结合酵母发酵滤液,其中,米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:3至3:1。本发明还涉及米曲霉与结合酵母发酵复合液在制备具有皮肤保湿和/或皮肤屏障修护作用的皮肤外用剂、药品及保健食品中的应用。
Description
技术领域
本发明涉及天然药物化学领域,具体涉及米曲霉与结合酵母发酵复合液的保湿和/或皮肤屏障修护应用,以及米曲霉与结合酵母发酵复合液在制备具有保湿和/或皮肤屏障修护作用的皮肤外用剂、药品及保健食品中的应用。
背景技术
水通道蛋白(Aquaporin,AQP3)是水通道蛋白家族的一个亚族,是皮肤表达最多的水通道蛋白,在表皮和基底层表达,保持皮肤细胞间质和细胞内水合水平,在皮肤保湿中起关键作用。
丝聚蛋白(Filaggrin,FLG)是参与皮肤屏障的重要因子,主要存在于表皮颗粒层和透明层,主要功效是参与表皮细胞的分化及皮肤屏障的形成,FLG在表皮角质层降解形成的氨基酸小分子具有保湿功能。
透明质酸(HA)又称为玻尿酸,是一种酸性粘多糖,透明质酸分子中的羟基、羧基及其他极性基团能与水分子形成氢键而结合大量水分,因而具有良好的成膜性,可以在皮肤表面形成一层均匀的薄膜,减少皮肤表面水分的蒸发,从而能为皮肤提供一个良好的保湿环境。
本申请以HaCaT细胞为研究对象,通过免疫荧光技术检测细胞AQP3和FLG蛋白表达量情况,以及检测待测样品作用下对透明质酸变化情况来评价待测样品的保湿功效。
红米,原植物为禾本科稻属植物稻(Oryza sativa L.)去壳的种仁,因种皮呈棕红色,故得此名。现代研究表明,红米具有抗氧化、美白活性。
米曲霉(Aspergillus oryzae)作为丝状真菌中的一个常见种,属曲霉属,不同于黄曲霉,米曲霉不会分泌致癌的黄曲霉毒素,是世界卫生组织和美国官方认证的食品级公认安全菌株,其代谢产物也被广泛应用于医药、化妆品、农业等领域。
酵母(Saccharomyces)是一种单胞菌,在传统发酵食品工业中,可产酸产碱,代谢糖类物质,有时还可以产生芳香醇类和芳香醛类物质,常用于酱油、面团等食品的发酵。
例如,中国专利申请CN202010642140.4涉及一种红米发酵滤液及其制备方法与在制备化妆品中的应用。该文献公开了红米利用植物乳杆菌发酵工艺,且具有补水保湿、美白、抗衰老作用。又如,中国专利申请CN201810771431.6涉及一种抗皱保湿面膜液的制作方法。该文献公开了黑米经过米曲霉发酵后,再制作成面膜具有保湿功效。又如,“大米发酵滤液上调紧密连接蛋白与增强皮肤屏障效果研究”(《日用化学品科学》,2022年11期)报道了酵母菌发酵大米具有皮肤屏障修护作用。
然而,这些文献均未将米曲霉发酵滤液与结合酵母发酵滤液复配的相关报道,更没有指出这种米曲霉与结合酵母发酵复合液具有保湿、皮肤屏障作用。本发明通过米曲霉发酵滤液与结合酵母发酵滤液复配,意外地发现复配液具有优异的促进AQP3、FLG作用,且均优于单独菌种发酵滤液,同时还有促进HA的作用。
发明内容
一方面,本发明提供了米曲霉与结合酵母发酵复合液在皮肤保湿和/或皮肤屏障修护中的应用,所述米曲霉与结合酵母发酵复合液包含米曲霉发酵滤液和结合酵母发酵滤液,其中,米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:3至3:1。
在优选的实施方式中,米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:1。
在优选的实施方式中,所述米曲霉与结合酵母发酵复合液的体积浓度至少为0.1%,更优选至少为1%。
在优选的实施方式中,所述皮肤保湿通过促进透明质酸表达实现,所述皮肤屏障修护通过促进水通道蛋白和/或和丝聚蛋白的表达实现。
另一方面,本发明涉及米曲霉与结合酵母发酵复合液在制备具有皮肤保湿和/或皮肤屏障修护作用的皮肤外用剂、药品及保健食品中的应用,所述米曲霉与结合酵母发酵复合液包含米曲霉发酵滤液和结合酵母发酵滤液,其中,米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:3至3:1。
在优选的实施方式中,所述米曲霉与结合酵母发酵复合液在皮肤外用剂、药品及保健食品中的含量为0.25-20重量%,更优选1.25-5重量%。
在优选的实施方式中,所述皮肤外用剂选自:面霜、乳液、啫喱、化妆水、精华液、面膜、眼霜、气雾清洁泡、喷雾、沐浴露、或洗面奶。
在优选的实施方式中,所述米曲霉发酵滤液中总糖≥9.9wt%,其中所述总糖中葡萄糖的占比≥40%。
在优选的实施方式中,所述米曲霉发酵滤液中总蛋白≥0.27wt%,其中所述总蛋白中肽的占比≥80%。
附图简要说明
图1显示了AQP3含量测定实验结果。
图2显示了FLG含量测定实验结果。
图3显示了HA含量测定实验结果。
具体实施方式
除非另有定义,本文所用的所有技术和科学术语具有本发明所属领域普通技术人员共同理解的相同含义。虽然与本文所述相似或等同的任何方法和材料可用于实施或测试本发明,但本文描述的是优选的方法和材料。对于本发明的目的,下面定义了以下术语。
本文所用术语“约”指与参比品的数量、水平、数值、维度、大小或用量相比,差异可高达30%、20%或10%的数量、水平、数值、维度、大小或用量。本文所使用的百分含量,除非另有说明,均以重量计。
在全篇说明书和权利要求书中,除非另有要求,以下词语“包含”和其变体“含有”和“包括”应理解为意指包括所述的整体或步骤,或一组整体或步骤,但不排除任何其它整体或步骤,或其它一组整体或步骤。
如本文所用,“局部施用”意指例如利用手或诸如擦拭物、辊或喷雾器之类的施用装置,直接涂抹或散布在外部皮肤、头皮或毛发上。
米曲霉发酵滤液与结合酵母发酵滤液按比例复配后,具有保湿功效和皮肤屏障修护功效,效果优于单独使用发酵滤液。本发明涉及米曲霉与结合酵母发酵复合液的保湿和/或皮肤屏障修护作用,首次发现米曲霉与结合酵母发酵复合液可以作为功效添加剂,应用于皮肤外用剂、药品及保健食品,以帮助实现皮肤保湿和/或修护皮肤屏障功能,例如帮助改善皮肤屏障、干燥问题等。
本文所描述的米曲霉与结合酵母发酵复合液可任选为成品包装形式。在一个实施例中,所述包装为装有米曲霉与结合酵母发酵复合液的诸如塑料、金属或玻璃管或广口瓶之类的容器。该产品可另外具有用于储存该容器的诸如塑料或纸板盒之类的包装。在一个实施例中,该产品包含米曲霉与结合酵母发酵复合液并且具有引导使用者将米曲霉与结合酵母发酵复合液施用至皮肤以治疗如下文论述的皮肤老化迹象的说明书。这种说明书可印刷在容器上、标签插页或任何其它包装上。
米曲霉与结合酵母发酵复合液
米曲霉(Aspergillus oryzae)作为丝状真菌中的一个常见种,属曲霉属。
结合酵母(Zygosaccharomyces)是一种单胞菌,常用于传统发酵食品工业中。
本发明意外地发现,米曲霉发酵滤液与结合酵母发酵滤液按比例复配后,具有保湿和皮肤屏障修护功效。因此,米曲霉与结合酵母发酵复合液可以应用于化妆品中,实现皮肤保湿和皮肤屏障修护功效。
研究中发现与单独使用米曲霉发酵滤液、结合酵母发酵滤液相比,米曲霉发酵滤液与结合酵母发酵滤液按比例复配后,具有更好地促进人皮肤永生角质化细胞(HaCaT细胞)的水通道蛋白(Aquaporin3,AQP3)和丝聚蛋白(Filaggrin,FLG)的表达,同时还能更好促进HaCaT细胞的透明质酸(Hyaluronic acid,HA)表达。因此,米曲霉与结合酵母发酵复合液可作为功效成分加入到护肤品中使用,以辅助改善皮肤屏障、干燥问题等。
在一些实施方式中,米曲霉发酵滤液的制备过程包括以下步骤:
(a)提供红米,粉碎得到碎米,加水覆盖碎米;
(b)加入淀粉酶液化;
(c)加入糖化酶糖化;
(d)加入米曲霉发酵;
(e)过滤后得到米曲霉发酵滤液。
在优选的实施方式中,红米是摩梭红米。在优选的实施方式中,步骤(a)包括加热步骤,例如加热至40-60℃,优选50℃。在优选的实施方式中,步骤(b)的液化时间为1小时。在优选的实施方式中,步骤(c)包括待米浆冷却后加入糖化酶糖化,糖化时间1小时。在优选的实施方式中,步骤(d)的发酵时间为36-168小时。在优选的实施方式中,步骤(d)之后还包括灭菌,离心,过滤,活性炭脱色步骤。
在一些实施方式中,结合酵母发酵滤液的制备过程包括以下步骤:
(a)提供红米,粉碎得到碎米,加水覆盖碎米;
(b)加入淀粉酶液化;
(c)加入糖化酶糖化;
(d)加入结合酵母发酵;
(e)过滤后得到结合酵母发酵滤液。
在优选的实施方式中,步骤(a)包括加热步骤,例如加热至40-60℃,优选50℃。在优选的实施方式中,步骤(b)的液化时间为1小时。在优选的实施方式中,步骤(c)包括待米浆冷却后加入糖化酶糖化,糖化时间1小时。在优选的实施方式中,步骤(d)的发酵时间为36-168小时。在优选的实施方式中,步骤(d)之后还包括灭菌,离心,过滤,活性炭脱色步骤。
在一些实施方式中,本发明米曲霉与结合酵母发酵复合液中米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:3至3:1。在优选的实施方式中,本发明米曲霉与结合酵母发酵复合液中米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:1至3:1。在优选的实施方式中,本发明米曲霉与结合酵母发酵复合液中米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:3至1:1。
在一些实施方式中,米曲霉与结合酵母发酵复合液的体积浓度至少为0.1%,更优选体积浓度至少为1%。
在一些实施方式中,米曲霉与结合酵母发酵复合液的体积浓度优选小于10%,更优选小于5%。
在一些实施方式中,米曲霉与结合酵母发酵复合液的体积浓度为1%-3%,优选1.25%-3%。
皮肤外用剂
在一些实施方式中,米曲霉与结合酵母发酵复合液可用于皮肤外用剂的制备。所述皮肤外用剂优选为化妆品组合物,包括但不限于面霜、乳液、啫喱、化妆水、精华液、面膜、眼霜、气雾(清洁泡)、喷雾、沐浴露和洗面奶等剂型的产品。
米曲霉与结合酵母发酵复合液在皮肤外用剂中的含量为0.001-20重量%,优选0.005-10重量%,更优选0.01-5重量%。
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,例如可以是化妆品组合物。所述化妆品组合物中可以是基础化妆品、面部妆容化妆品、身体用化妆品、头发护理用化妆品等,对其剂型无特殊限制,根据不同目的可合理选择。所述化妆品组合物中根据剂型和目的的不同还含有不同的化妆品学层面允许的介质或基质赋形剂。
包含米曲霉与结合酵母发酵复合液的皮肤外用剂可局部施用至人皮肤和/或毛发。该皮肤外用剂还可包含美容上可接受的局部用载体,该局部用载体可为皮肤外用剂的约50重量%至约99.99重量%(如皮肤外用剂的约80重量%至约99重量%)。在本发明的一个优选的实施例中,美容上可接受的局部用载体包括水。美容上可接受的局部用载体可包括一种或多种选自润湿剂、润肤剂、油脂、湿润剂以及相似物质。在一个实施例中,美容上可接受的局部用载体包括诸如无纺布或膜材料之类的基材。
可将皮肤外用剂制备成多种产品类型,包括但不限于洗剂、霜剂、凝胶剂、棒剂、喷雾剂、油膏剂、清洁液体洗剂和固体皂剂、洗发剂和毛发调理剂、毛发固定剂、糊剂、泡沫剂、粉末剂、摩丝、剃须膏、擦拭物、贴剂、水凝胶、成膜产品、面膜和皮肤膜剂、膜剂和化妆品如粉底以及睫毛膏。这些产品类型可含有几种类型的美容上可接受的局部用载体,包括但不限于溶液、悬浮液、乳液(例如微乳液和纳米乳液)、凝胶、固体和脂质体。
包含米曲霉与结合酵母发酵复合液的皮肤外用剂可配制成溶液剂。溶液剂通常包含水性溶剂或有机溶剂(例如,约50%至约99.99%或约90%至约99%的美容上可接受的水性溶剂或有机溶剂)。合适的有机溶剂的例子包括丙二醇、聚乙二醇、聚丙二醇、甘油、1,2,4-丁三醇、山梨醇酯、1,2,6-己三醇、乙醇以及它们的混合物。
皮肤外用剂可配制成包含润肤剂的溶液。此类皮肤外用剂优选包含约2%至约50%的一种或多种润肤剂。如本发明所用,“润肤剂”指用于预防或减轻干燥,例如通过防止皮肤水分经皮肤损失的物质。润肤剂的例子包括但不限于植物油、矿物油、脂族酯等。
洗剂可由这种溶液制备。洗剂通常含有约1%至约20%(例如,约5%至约10%)的一种或多种润肤剂和约50%至约90%(例如,约60%至约80%)的水分。
可以由溶液配制的另一类型的产品是霜剂。霜剂通常含有约5%至约50%(例如,约10%至约20%)的一种或多种润肤剂和约45%至约85%(例如,约50%至约75%)的水分。
尽管优选的是包含米曲霉与结合酵母发酵复合液的皮肤外用剂包含水,但作为另一种选择该皮肤外用剂可以是无水的或为不包含水而是有机和/或有机硅溶剂、油脂、类脂和蜡的油膏剂。油膏剂可含有动物或植物油或半固体烃的简单基料。油膏剂可含有约2%至约10%的一种或多种润肤剂和约0.1%至约2%的一种或多种增稠剂。
可将该皮肤外用剂配制为乳剂。如果局部用载体是乳液,则约1%至约10%(如约2%至约5%)的该局部用载体含有一种或多种乳化剂。乳化剂可以为非离子型、阴离子型或阳离子型。合适的乳化剂的例子包括个人护理和化妆品配方领域中通常鉴定为合适乳化剂的那些。
洗剂和霜剂可配制成乳剂。通常这类洗剂含有0.5%至约5%的一种或多种乳化剂。这种霜剂通常含有约1%至约20%(如约5%至约10%)的一种或多种润肤剂;约20%至约80%(如30%至约70%)的水;以及约1%至约10%(如约2%至约5%)的一种或多种乳化剂。
水包油和油包水型单乳液护肤制剂,例如洗剂和霜剂是化妆品领域所熟知的,并且可用于本发明。多相乳状液皮肤外用剂(例如水包油包水型和油包水包油型)也可用于本发明。通常,此类单相乳状液或多相乳状液含有水分、润肤剂和乳化剂作为其基本成分。
包含米曲霉与结合酵母发酵复合液的皮肤外用剂还可配制成凝胶剂(例如,使用合适的胶凝剂的水性凝胶、醇类凝胶、醇/水型凝胶或油性凝胶)。用于水性凝胶和/或醇类凝胶的适当胶凝剂包括但不限于天然树胶、丙烯酸和丙烯酸酯聚合物和共聚物以及纤维素衍生物(例如羟甲基纤维素和羟丙基纤维素)。用于油(例如矿物油)的适当胶凝剂包括但不限于氢化丁烯/乙烯/苯乙烯共聚物和氢化乙烯/丙烯/苯乙烯共聚物。这种凝胶剂通常含有约0.1重量%和5重量%之间的这类胶凝剂。
包含米曲霉与结合酵母发酵复合液的皮肤外用剂也可配制成固体制剂(例如蜡基棒剂、条皂、粉剂或含有粉剂的擦拭物)。
除了上述组分外,可用于本发明的皮肤外用剂还可含有多种在常规上以其技术领域确定的水平用于在皮肤和毛发上使用的皮肤外用剂中的其他油溶性物质和/或水溶性物质。
本发明的皮肤外用剂可以包含在皮肤护理组合物中通常能找到的附加组分,例如润肤剂、皮肤调节剂、乳化剂、防腐剂、抗氧化剂、香料、螯合剂等,只要它们与皮肤外用剂中其他的组分在物理上和化学上相容、且不影响本发明的米曲霉与结合酵母发酵复合液的效果即可。
在本发明的皮肤外用剂的一些实施方式中,可使用一种或多种防腐剂。适宜的防腐剂包括对羟基苯乙酮、C1-C4对羟基苯甲酸烷基酯和苯氧基乙醇。基于组合物总重量,防腐剂的用量为大约0.5至大约2重量%,优选大约0.5至1重量%。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种抗氧化剂。适宜的抗氧化剂包括丁化羟基甲苯(BHT)、抗坏血酸棕榈酸酯(BHA)、丁羟茴醚、苯基-α-萘基胺、氢醌、没食子丙酯、去甲二氢愈创木酸、维生素E或维生素E的衍生物、维生素C及其衍生物、泛酸钙、绿茶提取物和混合的多酚,以及上面所述的物质的混合物。所使用的抗氧化剂是组合物总重量的大约0.02至0.5重量%,更最优选的是从大约0.002至0.1重量%的用量范围。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种润肤剂,通过其保持在皮肤表面上或在角质层中的能力,起到润滑剂的作用,以减少剥落,改善皮肤的外观。典型的润肤剂包括脂肪酯、脂肪醇、矿物油、聚醚硅氧烷共聚物等等。适宜的润肤剂的例子非限定地包括,聚丙二醇(“PPG”)-15十八烷基醚,PPG-10十六烷基醚,Steareth-10,Oleth-8,PPG-4十二烷基醚,维生素E醋酸酯,羊毛脂,鲸蜡醇,鲸蜡硬脂醇乙基己酸酯,十六十八醇,甘油硬脂酸酯,羟基硬脂酸辛脂,二甲基聚硅氧烷,以及它们的组合。鲸蜡醇,鲸蜡硬脂醇乙基己酸酯,十六十八醇,甘油硬脂酸酯以及它们的组合是优选的。使用时,润肤剂是基于组合物总重量的大约0.1至大约30重量%,优选大约1至大约30重量%的用量范围。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种保湿剂。保湿剂也称为湿润剂,其有助于提高润肤剂的效果,减少剥落,刺激组成的鳞皮的去除和提高皮肤触感。可使用多元醇作为保湿剂,包括但并不限于,甘油,聚亚烷基二醇,亚烷基多元醇及其衍生物,包含丁二醇、丙二醇、双丙二醇,聚丙三醇,聚乙二醇及其衍生物,山梨醇,羟丙基山梨醇,己二醇,1,3-二丁二醇,1,2,6-己三醇,乙氧基化甘油,丙氧基化甘油,以及它们的组合。使用时,基于组合物的总重量,保湿剂的用量为大约0.1至大约20重量%,优选是大约1至大约15重量%。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种乳化剂。乳化剂可在有效稳定量的范围内使用。优选地,在组合物总重量的基础上,以大约1.0至大约10.0重量%,更优选的是大约3.0至大约6.0重量%的用量来使用乳化剂。可以使用任何与组合物中的组分相容的乳化剂。适宜的乳化剂包括硬脂酸,鲸蜡醇,甘油硬脂酸酯,卵磷脂,十八烷醇,Steareth-2,Steareth-20,丙烯酸(酯)类/C10-30烷醇丙烯酸酯交联聚合物,以及它们的组合。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种pH调节剂。本发明的皮肤外用剂中有益的pH调节剂包括氨丁三醇。使用时,基于组合物的总重量pH调节剂的用量为大约0.1至大约2重量%,优选是大约0.1至大约1重量%。
在本发明的一个具体实施方式中,皮肤外用剂包含丙烯酸(酯)类/C10-30烷醇丙烯酸酯交联聚合物、甘油、对羟基苯乙酮、甘油硬脂酸酯和卵磷脂、十六/十八醇、鲸蜡硬脂醇乙基己酸酯、氨丁三醇或它们的组合。
另外的美容活性剂
在一些实施方式中,皮肤外用剂还可包含另外的美容活性剂。如本文所用,“美容活性剂”是对皮肤或毛发具有美容或治疗效果的化合物(如合成化合物或从天然来源或天然提取物分离的化合物),包括但不限于抗痤疮剂、控油剂、抗微生物剂、抗炎剂、抗真菌剂、抗寄生虫剂、外用止痛剂、防晒剂、光防护剂、抗氧化剂、角质层分离剂、表面活性剂、保湿剂、营养剂、维生素、能量增强剂、防汗剂、收敛剂、除臭剂、固化剂、抗硬角质剂和用于毛发和/或皮肤调理的试剂。
在一个实施例中,这些美容活性剂选自(但不限于):羟基酸、过氧化苯甲酰、D-泛醇、甲氧基肉桂酸辛酯、二氧化钛、水杨酸辛酯、胡莫柳酯、阿伏苯宗、类胡萝卜素、自由基清除剂、自旋捕获剂、胺、诸如视黄醇和视黄基棕榈酸酯之类的类视色素、神经酰胺、多不饱和脂肪酸、必需脂肪酸、酶、酶抑制剂、矿物质、诸如雌激素之类的激素、诸如氢化可的松之类的类固醇、2-二甲基氨乙醇、诸如氯化铜之类的铜盐、诸如Cu:Gly-His-Lys之类的含铜肽、辅酶Q10、肽、诸如脯氨酸之类的氨基酸、维生素、乳糖酸、乙酰基-辅酶A、烟酸、核黄素、硫胺素、核糖、诸如NADH和FADH2之类的电子传递物质和诸如芦荟、小白菊、麦片之类的其他植物提取物以及它们的衍生物和混合物。以本发明皮肤外用剂的重量计,美容活性剂通常以约0.001%至约20%,例如约0.005%至约10%如约0.01%至约5%的量存在。
维生素的例子包括但不限于维生素A、维生素B(例如维生素B3、维生素B5和维生素B12)、维生素C、维生素K和维生素E的不同形式(如α、β、γ或Δ生育酚)或它们的混合物以及它们的衍生物。
羟基酸的例子包括(但不限于)乙醇酸、乳酸、苹果酸、水杨酸、柠檬酸和酒石酸。
抗氧化剂的例子包括但不限于:水溶性抗氧化剂例如巯基化合物和它们的衍生物(如,焦亚硫酸钠和N-乙酰基-半胱氨酸)、硫辛酸和二氢硫辛酸、白藜芦醇、乳铁蛋白和抗坏血酸以及抗坏血酸衍生物(如抗坏血酸棕榈酸酯和抗坏血酸多肽)。适用于本发明的皮肤外用剂中的油溶性抗氧化剂包括(但不限于)丁基化羟基甲苯、类视色素(如,视黄醇和视黄基棕榈酸酯)、生育酚类(如,生育酚乙酯)、生育三烯酚类和泛醌。含有适用于本发明皮肤外用剂的抗氧化剂的天然提取物包括但不限于:含有黄酮类和异黄酮类以及它们的衍生物(如,染料木素和二联玉米蛋白)的提取物、含有白藜芦醇的提取物等。这种天然提取物的例子包括葡萄籽、绿茶、松树皮和蜂胶。
使用方法
可将本发明的皮肤外用剂局部施用至需要处理如上所述的一种或多种皮肤老化迹象的哺乳动物皮肤。在一个实施例中,可将皮肤外用剂施用至需要处理细纹和皱纹和/或弹性损失的皮肤。可根据合适的处理方案,如每月、每周、每隔一天、每天、每天两次等,将皮肤外用剂施用至需要这种处理的皮肤。
在某些实施例中,本发明的皮肤外用剂还可用用于处理与皮肤相关的其他需要。例如,本发明的皮肤外用剂可用于处理发炎后色素沉着过度、用于减少毛孔大小、用于减少皮脂产生以及用于缓和疤痕。在某些其他实施例中,可将本发明的皮肤外用剂与机械或物理表皮剥脱处理(例如微晶磨皮处理)同时施用或在该处理的数小时内施用,或与化学剥脱剂或角质层分离剂例如水杨酸同时施用。在某些其他实施例中,可将本发明的皮肤外用剂施用至粘膜或其它组织例如阴道组织、口腔组织或眼组织。在某些其他实施例中,可将本发明的皮肤外用剂施用至轻度伤口或术后部位以促进愈合、施用至昆虫叮咬处、施用至毒藤皮肤病症或相似皮肤状况,或通常用于减轻发痒。
实施例
下面结合具体实施例,以进一步阐述本发明。有必要在此指出的是,实施例只用于对本发明进行进一步的说明,不能理解为对本发明保护范围的限制,该领域的技术熟练人员可以根据上述本发明的内容做出一些非本质的改进和调整。下列实施例中未注明具体条件的试验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非另有说明,所有的百分比和份数按重量计。
实施例1:红米米曲霉发酵滤液制备
称取摩梭红米(市售,丽江宁蒗益康农产品开发有限公司)100g粉碎(大德药机DFY-600C),得到碎米。用400g水覆盖碎米,加热至50℃,搅拌后加入0.04g淀粉酶(隆科特240000u/g)液化1h。待米浆冷却后加入0.03g糖化酶(隆科特290000u/g)糖化1h,每隔20min搅拌一次。在糖化醪中接入5%米曲霉(江苏恒顺集团有限公司提供)摇床(上海智城分析仪器制造有限公司,ZWY-2102C)发酵7天。灭菌,在3500rpm条件下离心(卢湘仪,DL-5M)15min,滤纸(新星牌定性滤纸,规格60*60cm)过滤。滤液加入1.5%活性炭粉(国药集团化学试剂有限公司)于50℃200rpm条件下摇床1h,滤纸过滤。滤液再用0.45μm微孔滤膜(上海兴亚净化材料厂)过滤。得到红米米曲霉发酵滤液,备用。
实施例2:红米结合酵母发酵滤液制备
称取摩梭红米(市售,丽江宁蒗益康农产品开发有限公司)100g粉碎(大德药机DFY-600C),得到碎米。用400g水覆盖碎米,加热至50℃,搅拌后加入0.04g淀粉酶(隆科特240000u/g)液化1h。待米浆冷却后加入0.03g糖化酶(隆科特290000u/g)糖化1h,每隔20min搅拌一次。在糖化醪中接入5%结合酵母(江苏恒顺集团有限公司提供)摇床(上海智城分析仪器制造有限公司,ZWY-2102C)发酵7天。灭菌,在3500rpm条件下离心(卢湘仪,DL-5M)15min,滤纸(新星牌定性滤纸,规格60*60cm)过滤。滤液加入1.5%活性炭粉(国药集团化学试剂有限公司)于50℃200rpm条件下摇床1h,滤纸过滤。滤液再用0.45μm微孔滤膜(上海兴亚净化材料厂)过滤。得到红米结合酵母发酵滤液,备用。
实施例3:复配发酵滤液制备
将实施例1发酵物:实施例2发酵物按照重量比1:1混合均匀,备用。
实施例4:复配发酵滤液制备
将实施例1发酵物:实施例2发酵物按照重量比1:3混合均匀,备用。
实施例5:复配发酵滤液制备
将实施例1发酵物:实施例2发酵物按照重量比3:1混合均匀,备用。
实施例6:红米发酵滤液对水合相关蛋白、透明质酸表达性能评价
1.毒性测试实验方法
细胞接种:人皮肤永生化角质细胞按1×104个/孔的接种密度接种细胞至96孔板,培养箱(Thermo,160I)(37℃、5%CO2)中孵育过夜。
试验分组:试验设置调零组、溶剂对照组、阳性对照组与实施例1~5发酵物。每组实施例设置8个浓度梯度,每个浓度梯度下设置3个复孔。
配液:按下表方式配制溶液。
表1
给药:待96孔板中细胞铺板率达到40%~60%时进行给药。溶剂对照组每孔加入200μL含10%PBS培养液(Gibco);阳性对照组每孔加入200μL含10% DMSO(国药试剂)培养液;实施例组每孔加入200μL含有相应浓度实施例的培养液;调零组无细胞接种,仅加入200μL DMEM细胞培养液(VivaCell)。给药完后将96孔板放置在培养箱中培养24小时。
检测:细胞孵育培养24小时后,弃掉上清,加入0.5mg/mL MTT(Sigma),37℃避光孵育4小时,孵育结束后,弃掉上清,每孔加150μL DMSO,在490nm处读取OD值并计算细胞存活率。结果表示为Mean±SD。
实验结果:
表2
根据上述MTT结果,认为实施例1发酵物基于角质细胞,在5%(v/v)的浓度范围内未表现出明显的细胞毒性;实施例2发酵物基于角质细胞,在10%(v/v)的浓度范围内未表现出明显的细胞毒性;实施例3发酵物基于角质细胞,在10%(v/v)的浓度范围内未表现出明显的细胞毒性;实施例4发酵物基于角质细胞,在10%(v/v)的浓度范围内未表现出明显的细胞毒性;实施例5发酵物基于角质细胞,在10%(v/v)的浓度范围内未表现出明显的细胞毒性。
2.对水合相关蛋白含量测试方法
细胞接种:按1×105个/孔的接种密度接种细胞至24孔板,培养箱(37℃、5%CO2)中孵育过夜。
配液:按下表方式配制溶液。
表3
给药:待24孔板中细胞铺板率达到40%~60%时进行给药。每孔加样1mL,每组设3个复孔。给药完后将24孔板放置在培养箱中培养24小时。
收样固定:培养24h后,吸弃旧液,加入甲醇(国药试剂)固定细胞,用PBS润洗3次。
封闭:每孔加入1mL 1% BSA(爱必信)封闭1小时。
一抗孵育:弃掉封闭液,滴加一抗工作液(abcam),4℃孵育过夜,PBS缓冲液清洗3次。
二抗孵育:滴加二抗工作液(abcam),孵育2小时,PBS缓冲液清洗3次。
DAPI染色:每孔加入1.43μM DAPI(爱必信),孵育15分钟,PBS缓冲液清洗3次。
检测:荧光显微镜拍摄荧光图像(倒置荧光显微镜Leica DMi8,放大倍数200倍),对AQP3和FLG荧光强度进行定量分析。所有结果以Mean±SD表示。各组间比较采用t-test统计分析。P<0.05认为具有显著差异,P<0.01认为具有极显著差异,显著性以*表示。
AQP3含量测定结果:
表4
与空白对照组相比,米曲霉发酵滤液在体积浓度3%时,AQP3含量上调71%;在体积浓度1.25%时,上调28%。结合酵母发酵滤液在体积浓度3%时,AQP3含量上调68%;在体积浓度1.25%时,上调64%;在体积浓度0.5%时,上调40%。米曲霉发酵滤液:结合酵母发酵滤液(1:1)在体积浓度3%时,AQP3含量上调80%;在体积浓度1.25%时,上调14%。米曲霉发酵滤液:结合酵母发酵滤液(1:3)在体积浓度3%时,AQP3含量上调59%;在体积浓度1.25%时,上调30%。米曲霉发酵滤液:结合酵母发酵滤液(3:1)在体积浓度3%时,AQP3含量上调120%;在体积浓度1.25%时,上调98%;在体积浓度0.5%时,AQP3含量上调50%。说明米曲霉发酵滤液、结合酵母发酵滤液及其复配能够很好的促进AQP3表达,达到保湿效果。且针对上述结果,说明不同复配发酵滤液,保湿效果不同,复配发酵滤液(3:1)保湿效果最优,体积浓度为3%时,复配发酵滤液(3:1)>复配发酵滤液(1:1)>米曲霉发酵滤液>结合酵母发酵滤液>复配发酵滤液(1:3),说明复配发酵滤液促进AQP3效果优于单独发酵滤液。
FLG含量测定结果:
表5
与空白对照组相比,米曲霉发酵滤液在体积浓度3%时,FLG含量上调51%;在体积浓度1.25%时,上调26%;在体积浓度0.5%时,上调20%。结合酵母发酵滤液在体积浓度3%时,FLG含量上调68%;在体积浓度1.25%时,上调31%。米曲霉发酵滤液:结合酵母发酵滤液(1:1)在体积浓度3%时,FLG含量上调82%;在体积浓度1.25%时,上调31%;在体积浓度0.5%时,上调17%。米曲霉发酵滤液:结合酵母发酵滤液(1:3)在体积浓度3%时,FLG含量上调80%;在体积浓度1.25%时,上调43%。米曲霉发酵滤液:结合酵母发酵滤液(3:1)在体积浓度3%时,FLG含量上调55%;在体积浓度1.25%时,上调37%。说明米曲霉发酵滤液、结合酵母发酵滤液及其复配能够很好的促进FLG表达,达到皮肤屏障修护效果。且针对上述结果,说明不同复配发酵滤液,皮肤屏障修护效果不同,复配发酵滤液(1:1)皮肤屏障修护效果最优,体积浓度为3%时,复配发酵滤液(1:1)>复配发酵滤液(1:3)>结合酵母发酵滤液>复配发酵滤液(3:1)>米曲霉发酵滤液,说明复配发酵滤液促进FLG效果优于单独发酵滤液。
3.对HA含量测试方法
细胞接种:按1×105个/孔的接种密度接种细胞至24孔板,培养箱(37℃、5%CO2)中孵育过夜。
配液:按下表方式配制溶液。
表6
给药:待24孔板中细胞铺板率达到40%~60%时进行给药。每孔加样1mL,每组设3个复孔。给药完后将24孔板放置在培养箱中培养24小时。
收样:培养24h后,吸弃旧液,PBS清洗3次。
ELISA检测:根据HA ELISA试剂盒(CUSABIO)的操作说明书进行检测。
结果统计分析:结果表示为Mean±SD。
HA含量测定结果:
表7
与空白对照组相比,米曲霉发酵滤液在体积浓度3%时,HA含量上调61%;在体积浓度1.25%时,上调57%;在体积浓度0.5%时,上调52%。结合酵母发酵滤液在体积浓度3%时,HA含量上调63%;在体积浓度1.25%时,上调42%;在体积浓度0.5%时,上调37%。米曲霉发酵滤液:结合酵母发酵滤液(1:1)在体积浓度3%时,HA含量上调31%;在体积浓度1.25%时,上调30%;在体积浓度0.5%时,上调25%。米曲霉发酵滤液:结合酵母发酵滤液(1:3)在体积浓度3%时,HA含量上调20%;在体积浓度1.25%时,上调9%;在体积浓度0.5%时,上调5%。米曲霉发酵滤液:结合酵母发酵滤液(3:1)在体积浓度3%时,HA含量上调30%;在体积浓度1.25%时,上调12%;在体积浓度0.5%时,上调9%。说明米曲霉发酵滤液、结合酵母发酵滤液及其复配能够很好的促进HA表达,达到保湿效果。且针对上述结果,说明不同复配发酵滤液,保湿效果不同,米曲霉保湿效果最优,体积浓度为1.25%时,米曲霉发酵滤液>结合酵母发酵滤液>复配发酵滤液(1:1)>复配发酵滤液(3:1)>复配发酵滤液(1:3)。
实施例7:米曲霉发酵滤液的成分比较研究
1.游离氨基酸含量测定(参考GB/T 30987-2020)
1.1标准溶液制备
称取L-谷氨酸(上海安谱实验科技股份有限公司)和L-酪氨酸(上海安谱实验科技股份有限公司)各0.01mg,用水溶解配制成混合溶液,各氨基酸浓度为1μg/mL备用。
1.2样品前处理
取实施例1发酵滤液5g于10mL容量瓶中,加入0.02mol/L盐酸(国药集团)定容,超声20min后,6000rpm条件下离心5min,取上清液2.5mL。加入1.5mL 0.02mol/L盐酸过柱。取100μL样品于15mL离心管中,60℃干燥至恒重。加入50μL乙醇:异硫氰酸苯酯(CNW):水:三乙胺(国药集团)(7:1:1:1),现用现配。加入流动相A 0.45mL,混匀,过0.45μm滤膜,备用。
1.3含量测定
色谱柱:C18 SHISEIDO(4.6mm*250mm*5μm)
进样量:10μL
柱温:40℃
流动相:流动相A:0.1mol/L无水乙酸钠(国药集团):乙腈(Merck)(97:3),混匀后调PH至6.5;流动相B:乙腈:水(80:20)
洗脱:按下表方式梯度洗脱
表8
检测波长:254nm
氨基酸含量计算公式:
其中:W-样品中各氨基酸含量(mg/kg);C-样品中目标物浓度(mg/L);C0-空白对照中目标物浓度(mg/L);V-体积(mL);N-稀释倍数;m-样品取样量(g)。
2.蛋白质含量测定(参考GB 5009.5-2016)
2.1甲基红乙醇溶液及溴甲酚绿乙醇溶液的配制
称取0.1g甲基红(国药集团),用95%乙醇稀释至100mL,备用;称取0.1g溴甲酚绿用95%乙醇稀释至100mL,备用。
2.2自动凯氏定氮仪法
称取实施例1发酵滤液10g于消化管中。加入0.4g硫酸铜(国药集团)、6g硫酸钾(国药集团)及12mL硫酸(国药集团)于消化炉进行消化,当消化炉温度达到420℃之后,此时消化管中液体呈蓝绿色。等澄清透明后,继续消化1h。取出冷却后加入20mL水,于自动凯氏定氮仪(济南海能仪器股份有限公司K9840)上蒸馏7min,接收瓶中加1-2滴甲基红乙醇溶液:溴甲酚绿乙醇溶液(1:5),现用现配。再加入10mL 20g/L硼酸(国药试剂)溶液,接收蒸馏液到200mL。用0.1mol/L盐酸滴定,终点为浅灰红色,同时做试剂空白。
蛋白质含量计算公式:
其中:W-样品中蛋白质含量(g/100g);V1-样品中消耗盐酸溶液体积(mL);V2-试剂空白消耗盐酸溶液体积(mL);V3-吸取消化液体积(mL);C-盐酸溶液浓度(mol/L);m-样品取样量(g);F-氮换算陈蛋白质系数(米换算系数为5.95)。
3.总多酚含量测定(参考T/AHFIA 005-2018)
3.1标准曲线制备
没食子酸(上海源叶生物科技有限公司)溶液:移取1mL、2mL、3mL、4mL、5mL的100mg/L没食子酸标准储备液于100mL容量瓶中,用水定容至刻度,摇匀,备用。
取没食子酸溶液1mL于10mL试管中。加入5mL 10%福林酚(国药集团),摇匀,反应5min。加入4mL 75%碳酸钠溶液(国药集团),加水定容至刻度,摇匀,静置60min。用比色皿在760nm波长下用紫外分光光度计(Agilent 8453)测定吸光度,绘制标准曲线。
3.2样品测定
取10g实施例1发酵滤液于离心管中,加入70℃预热的60%乙醇溶液5mL,搅拌均匀,置70℃浸提10min。取出冷却至室温,4000rpm条件下离心10min,取上清液至10mL容量瓶,残渣重复一次,合并提取液,过0.45μm滤膜,按上述方法进行总多酚含量测定。
总多酚含量计算公式:
其中:W-样品中总多酚含量(g/100g);A-样品溶液吸光度;A0-空白对照溶液吸光度;V-体积(mL);N-稀释倍数;k-没食子酸标曲斜率;m-样品取样量(g)。
4.总糖及单糖含量测定(参考GB/T 15672-2009)
4.1总糖含量测定
4.1.1标准曲线制备
葡萄糖(上海源叶生物科技有限公司)溶液:移取0.2mL、0.4mL、0.6mL、0.8mL、1.0mL的100mg/L葡萄糖标准储备液于10mL具塞试管中,用水定容至1mL。加入1ml 5%苯酚(国药集团)和5mL浓硫酸(国药集团),摇匀,静置10min。再将试管置于30℃反应20min。用比色皿在490nm处测定吸光度,绘制标准曲线。
4.1.2样品测定
取10g样品于250mL锥形瓶中,加入50mL水和15mL浓盐酸(莱阳康德化工有限公司),置于100℃水解3h。冷却至室温后过滤,用水定容至250mL,按上述方法进行总糖含量测定。
总糖含量计算公式:
其中:W-样品中总糖含量(g/100g);m1-样品中葡萄糖质量(mg);V1-样品总体积(mL);V2-测定时溶液体积(mL);m-样品取样量(g)。
4.2单糖含量测定
4.2.1样品前处理
取实施例1发酵滤液2g于水解管中,加入1mL水和1mL 4mol/L三氟氯酸(国药试剂),110℃干燥箱(泰斯特101-OAB)水解5h。取出后冷却至室温,取0.1mL水解液于4mL离心管中,60℃干燥箱中干燥2h。加入0.05mL 0.3mol/L的氢氧化钠溶液(国药集团)和0.05mL1-苯基-3-甲基-5-吡唑啉酮(阿拉丁PMP)甲醇溶液,70℃反应60min。取出后冷却至室温,加入0.05mL 0.3mol/L盐酸、0.75mL水和1.5mL氯仿(ACS),摇匀静置分层。取上层溶液,反复三次,过0.45μm滤膜,备用。
4.2.2含量测定
色谱柱:C18 Agilent(4.6mm*250mm*5μm)
进样量:10μL
柱温:25℃
流速:1.0mL/min
流动相:0.1mol/L磷酸二氢钾(调节PH至6.8):乙腈(82:18)
检测波长:254nm
葡萄糖含量计算公式:
其中:W-样品中单糖含量(mg/kg);C-样品中单糖浓度(mg/L);C0-空白对照溶液中单糖浓度(mg/L);V-体积(mL);N-稀释倍数;m-样品取样量(g)。
5.γ-氨基丁酸含量测定(参考QB/T 4587-2013)
5.1样品前处理
取实施例1发酵滤液10g,加入25mL 60%乙醇30℃水浴3h,收集上清液,重复一次。合并上清液浓缩至浸膏,用60%乙醇定容至5mL容量瓶中。取120μL样品,加入邻苯二甲醛衍生液600μL(10mg邻苯二甲醛(CNW)用0.5mL甲醇溶解,加入PH为10.2的0.4mol/L硼酸缓冲液(CNW)2mL和2-巯基乙醇(国药集团)30μL,混匀),衍生10min,备用。
5.2含量测定
色谱柱:C18 Agilent(4.6mm*250mm*5μm)
进样量:10μL
柱温:40℃
流动相:流动相A:25mmol/L乙酸钠(国药集团),用4%乙酸调PH至5.9;流动相B:乙腈
洗脱:按下表方式梯度洗脱
表9
检测波长:332nm
γ-氨基丁酸含量计算公式:
其中:W-样品中γ-氨基丁酸含量(mg/kg);C-样品中目标物浓度(mg/L);C0-空白对照中目标物浓度(mg/L);V-体积(mL);N-稀释倍数;m-样品取样量(g)。
6.VB3含量测定(参考GB 5009.89-2016)
6.1样品前处理
取实施例1发酵滤液2g于50mL离心管中,加入25mL水和0.25g淀粉酶酶解30min,先调PH至1.7,再调至4.5。超声提取10min,定容至50mL。离心,过0.45μm滤膜,备用。
6.2含量测定
色谱柱:C18 SHISEIDO(4.6mm*250mm*5μm)
进样量:10μL
柱温:20℃
流速:1.0mL/min
流动相:取甲醇70mL,异丙醇20mL,庚烷硫酸钠1g,用910mL水溶解混匀,用高氯酸调PH至2.1,过0.45μm滤膜
检测波长:261nm
VB3含量计算公式:
其中:W-样品中VB3含量(μg/kg);C-样品中目标物浓度(μg/L);C0-空白对照中目标物浓度(μg/L);V-体积(mL);N-稀释倍数;m-样品取样量(g)。
7.神经酰胺含量测定
实验原理:本试剂盒应用双抗体夹心法测定标本中植物神经酰胺(Ceramide)水平。用纯化的Ceramide抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加Ceramide,再与HRP标记的Ceramide抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的Ceramide呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),通过标准曲线计算样品中Ceramide浓度。
7.1组织匀浆的制备
取组织块0.2g,用PBS漂洗,擦干,置于5mL匀浆管中,加入1.8mL0.05mol/L Tris-HCl,匀浆,制成10%匀浆液。3000rpm条件下离心15min,取上清液,备用。
7.2神经酰胺含量测定
标准品加样:设置标准品孔和样品孔,标准品孔各加不同浓度标准品50μL。
加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余步骤相同)、实施例孔。在酶标包被板上实施例孔中先加样品稀释液40μL,再加分别加实施例1发酵滤液10μL。
加酶:每孔加入酶标试剂100μL,空白孔不加。
温育:用封板膜封板后至37℃水浴60min。
配液:将20倍浓缩洗涤液用水20倍稀释后备用。
洗涤:小心揭开封板膜,弃去液体,甩干,每孔加满洗涤液,静置30s,弃去,重复5次,甩干。
显色:每孔先加显色剂A液50μL,再加入显色剂B液50μL,混匀,37℃避光显色15min。
终止:每孔加入终止液50μL,此时溶液蓝色转黄色。
测定:以空白孔调零,450nm波长测定吸光度,并计算测试液中神经酰胺浓度。
神经酰胺含量计算公式:
其中:W-样品中神经酰胺含量(pg/mL);C-样品目标物浓度(ng/L);C0-空白对照中目标物浓度(ng/L);V-体积(mL);N-稀释倍数;m-样品取样量(g)。
成分比较研究结果:
表10
红米米曲霉发酵滤液谷氨酸含量为4.21mg/L;酪氨酸含量为1.30mg/L;总蛋白含量为0.27%,其中肽的占比为81.4%;总多酚含量为0.01%;总糖含量为9.9%,其中葡萄糖的占比为40.7%;γ-氨基丁酸含量为13.21mg/L;VB3含量为17.61mg/L;神经酰胺含量为330.66pg/mL。
米曲霉发酵滤液与结合酵母发酵滤液复配可用于皮肤外用剂的制备。所述皮肤外用剂优选为化妆品组合物,例如化妆水、精华液、乳霜等。在皮肤外用剂中的重量百分比为0.25%-20%(w/w)。优选的重量百分比为0.5%-10%(w/w)。更优选的重量百分比为1.25%-5%(w/w)。最优选的重量百分比为3%-5%(w/w)。
以下是红米发酵滤液在皮肤外用剂中的具体应用的实施例,及其这些剂型的配方和制备方法。以下各表中“-”表示无添加。
应用例1:面霜的制备
应用例2:乳液的制备
应用例3:啫喱的制备
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应用例4:化妆水的制备
应用例5:精华液的制备
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应用例6:面膜的制备
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应用例7:眼霜的制备
应用例8:气雾(清洁泡)的制备
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应用例9:喷雾的制备
应用例10:沐浴露的制备
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应用例11:洗面奶的制备
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Claims (12)
1.米曲霉与结合酵母发酵复合液在皮肤保湿和/或皮肤屏障修护中的应用,所述米曲霉与结合酵母发酵复合液包含米曲霉发酵滤液和结合酵母发酵滤液,其中,米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:3至3:1。
2.如权利要求1所述的应用,其特征在于,米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:1。
3.如权利要求1所述的应用,其特征在于,所述米曲霉与结合酵母发酵复合液的体积浓度至少为0.1%。
4.如权利要求3所述的应用,其特征在于,所述米曲霉与结合酵母发酵复合液的体积浓度至少为1%。
5.如权利要求1-4中任一项所述的应用,其特征在于,所述皮肤保湿通过促进透明质酸表达实现,所述皮肤屏障修护通过促进水通道蛋白和/或和丝聚蛋白的表达实现。
6.米曲霉与结合酵母发酵复合液在制备具有皮肤保湿和/或皮肤屏障修护作用的皮肤外用剂、药品及保健食品中的应用,所述米曲霉与结合酵母发酵复合液包含米曲霉发酵滤液和结合酵母发酵滤液,其中,米曲霉发酵滤液与结合酵母发酵滤液的重量比为1:3至3:1。
7.如权利要求6所述的应用,其特征在于,所述皮肤保湿通过促进透明质酸表达实现,所述皮肤屏障修护通过促进水通道蛋白和/或和丝聚蛋白的表达实现。
8.如权利要求6所述的应用,其特征在于,所述米曲霉与结合酵母发酵复合液在皮肤外用剂、药品及保健食品中的含量为0.25-20重量%。
9.如权利要求8所述的应用,其特征在于,所述米曲霉与结合酵母发酵复合液在皮肤外用剂、药品及保健食品中的含量为1.25-5重量%。
10.如权利要求6所述的应用,其特征在于,所述皮肤外用剂选自:面霜、乳液、啫喱、化妆水、精华液、面膜、眼霜、气雾清洁泡、喷雾、沐浴露、或洗面奶。
11.如权利要求1-10任一项所述的应用,其特征在于,所述米曲霉发酵滤液中总糖≥9.9wt%,其中所述总糖中葡萄糖的占比≥40%。
12.如权利要求1-10中任一项所述的应用,其特征在于,所述红米米曲霉发酵滤液中总蛋白≥0.27wt%,其中所述总蛋白中肽的占比≥80%。
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