CN112402455B - 牦牛奶发酵提取物的应用 - Google Patents
牦牛奶发酵提取物的应用 Download PDFInfo
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- CN112402455B CN112402455B CN202010175249.1A CN202010175249A CN112402455B CN 112402455 B CN112402455 B CN 112402455B CN 202010175249 A CN202010175249 A CN 202010175249A CN 112402455 B CN112402455 B CN 112402455B
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- skin
- yak milk
- fermentation
- extract
- fermented
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Classifications
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- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
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- C12N1/16—Yeasts; Culture media therefor
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- C12N1/185—Saccharomyces isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
本发明属于天然提取物及日化领域,具体涉及牦牛奶发酵提取物的应用。牦牛奶经过植物乳杆菌的发酵,发酵液离心过滤后得到的牦牛奶发酵提取物,能够降低金黄色葡萄球菌/表皮葡萄球菌的比值、促进真皮成纤维细胞和表皮细胞增殖,从而实现调节皮肤菌群、维持和/或修复皮肤表面微生态,以及修复老化的皮肤及美白,因此,牦牛奶发酵提取物可用作制备皮肤外用剂,作为修复老化皮肤、皮肤美白、改善皮肤表面微生态的活性成分。
Description
技术领域
本发明属于植物提取领域及日化领域,具体涉及牦牛奶发酵提取物的应用。
背景技术
人类利用牛奶和奶制品进行美容护肤的记载并不少见,如古罗马人认为在牛奶中浸泡过的面包擦脸可以是皮肤白嫩光滑。牛奶中的牛乳蛋白连同脂肪可以附在皮肤表面,可以起到滋润皮肤的作用;牛奶中的酵素可促进皮肤表层角质的分解等。而牦牛奶中含有高含量的维生素、蛋白质、氨基酸和微量元素等。相比牛奶,其营养物质种类更为丰富,含量更高,如氨基酸、维生素A含量分别比牛奶中高15%、6%;钙含量是普通牛奶的1.13倍,锌含量是牛奶的3倍。已有报道显示,人们可利用乳品中的酪蛋白在化妆品中作为保湿剂、营养剂和调理剂,另有利用牦牛乳蛋白在护发素与洗发水中添加的案例。
大多数乳酸菌无毒、无害,并且乳酸菌菌体及其代谢产物具有较强的抗氧化活性。它的抗氧化活性主要是指对超氧阴离子自由基(O2-)、羟自由基(OH-)和1,1–二苯基–2–苦味肼基自由基(DPPH)的清除作用及抗脂质过氧化性。植物乳杆菌(Lactobacillus plantarum; L. plantarum)常用于制备酸奶等传统乳制品,同时能够调节人体的肠道微生物平衡,还具有很好的降血脂、降血糖等功效,是常见的益生菌食品和乳制品生产菌,另有报道其溶胞上清也对黑色素生成有抑制效果。同时可见乳杆菌对细菌性皮肤感染的主要致病菌(金黄色葡萄球菌和化脓性链球菌)具有抑制作用的研究。
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。
人体皮肤表面和肠道中贮存着大量的微生物,自出生开始就伴随着我们,与人类为共生关系。其中,皮肤表面菌群或表面微生态,作为人体皮肤物理屏障更外层的一种重要生物屏障,对皮肤的健康状态有着重要作用。表面菌群根据不同的性别、年龄、外部环境等具有明显的个体差异,而表面菌群的失衡,特别是某些条件致病菌在表面菌群中的比例异常时,往往会引起各种肌肤疾病。表皮葡萄球菌S. e(Staphylococcus epidermidis; S. epidermidis)是人体皮肤的常驻菌之一,而金黄色葡萄球菌S. a(Staphylococcus aureus; S. aureus)比例异常时,则会破坏皮肤表面屏障,或者引起炎症等皮肤问题。
微生物是维持皮肤微生态平衡的重要成员。皮肤微生物以细菌为主,皮肤微生物可分为常驻菌和暂驻菌。常驻菌群与皮肤健康息息相关,它们定植于皮肤表面形成生物膜,一方面具有占位保护效应,作为物理屏障阻挡外源性致病菌的入侵;另一方面还可通过产生抗菌肽而抑制或杀灭病原菌,主要包括表皮葡萄球菌、微球菌、棒状杆菌、丙酸杆菌、不动杆菌及马拉色菌等。暂驻菌包括金黄色葡萄球菌S. aureus、链球菌、大肠杆菌及白念珠菌等,它们是引起皮肤感染的主要病原菌。S. aureus是皮肤常见感染细菌之一。研究表明特异性皮炎患者(AD)患者皮损处S. aureus定植率达78%。对已有研究进行梳理发现,AD皮肤表面微生态处于失衡状态,这不仅表现为S. aureus数量的显著升高,更加体现在其他皮肤常驻菌相对丰度、菌群多样性及丰富度的减少。
《皮炎湿疹患者皮肤菌群的测定及其临床研究》(孟华,《临床医药文献电子杂志》,2018)的报道,皮肤菌群测定显示:观察组(各型皮炎湿疹患者40例)金黄色葡萄球菌(57.50%)、表皮葡萄球菌(32.50%),显著高于对照组(健康人40例)中金黄色葡萄球菌(0%)和表皮葡萄球菌(7.50%)。根据报道计算可知,病患皮肤中S. a/S. e的比值为1.77,而健康皮肤中S. a/S. e的比值为0%。
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。
因此,如果能够促进人皮肤成纤维细胞的增殖,则可以延缓和修复皮肤衰老,或者降低金黄色葡萄球菌/表皮葡萄球菌的比值即S. a/S. e值,可以改善皮肤表面菌群和表面微生态,使皮肤更为健康。若单一活性物质能够同时解决上述问题,则可以在成分简化的基础上,使皮肤外用剂具有多重功效。
同时,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。因此,在人体中酪氨酸酶是皮肤合成黑色素的关键酶。
而目前还未见有将牦牛奶经过发酵后的提取物用作皮肤外用剂的报道,也未见牦牛奶发酵提取物用以改善皮肤表面微生态或者修复老化皮肤,及通过抑制酪氨酸酶活性作为美白的活性成分的报道。
发明内容
本发明旨在提供牦牛奶发酵提取物在制备皮肤外用剂方面的应用。
本发明还提供了一种皮肤外用剂,含有牦牛奶发酵提取物。
技术方案为:牦牛奶发酵提取物用于制备皮肤外用剂。具体的,所述的牦牛奶发酵提取物作为维持和/或修复皮肤表面微生态的活性成分;或者作为修复老化的皮肤的活性成分;或者作为美白的活性成分。
所述的牦牛奶发酵提取物通过调节皮肤菌群来维持和/或修复皮肤表面微生态;所述的牦牛奶发酵提取物通过促进皮肤细胞增殖修复老化的皮肤;所述的牦牛奶发酵提取物通过抑制皮肤细胞酪氨酸酶活力,实现美白作用。
进一步,所述牦牛奶发酵提取物降低金黄色葡萄球菌/表皮葡萄球菌的比值来调节皮肤菌群,或者促进真皮成纤维细胞和/或表皮细胞增殖。
牦牛奶发酵提取物可以通过液体发酵方法获得,将牦牛奶与水混合,用进行发酵。优选的,所述为植物乳杆菌。发酵液经过离心取上清液、过滤后取滤液即得到牦牛奶发酵提取物。还可以进行稀释、浓缩,或者制成浸膏或干粉。
牦牛奶发酵提取物的制备方法为:(1)牦牛奶或者牦牛奶干奶粉与水混合制成培养基,接入植物乳杆菌(Lactobacillus plantarum; L. plantarum)发酵,在30~40℃、150~250rpm条件下恒温发酵16-48小时进行液体发酵;(2)发酵液在4~10℃、1000~8000rpm条件下离心10~60min,取上清液;(3)上清液调节pH至5.0~7.0,80~90℃加热30~60min后,1000~8000rpm离心10~30min,取上清液;(4)上清液用孔径0.05~0.22μm滤膜在1~5bar压力下过滤,取发酵滤液。培养基中牦牛奶固形物的含量为1wt%~5wt%,余量为水。优选的发酵温度为35~40℃。
所述的牦牛奶发酵提取物为牦牛奶发酵滤液,或者由所述牦牛奶发酵滤液制得的稀释液、浓缩液、浸膏或干燥物。
优选的,牦牛奶产自泛喜马拉雅地区。
一种皮肤外用剂,含有牦牛奶发酵提取物;所述的牦牛奶发酵提取物作为维持和/或修复皮肤表面微生态的活性成分,或者作为修复老化的皮肤的活性成分;或者作为美白的活性成分。
以干物质含量计,牦牛奶发酵提取物的含量为0.001wt%~5wt%;优选的,以干物质含量计,牦牛奶发酵提取物的含量为0.005wt%~5wt%。更优选的,以干物质含量计,牦牛奶发酵提取物的含量为0.01wt%~2wt%。
优选的,所述皮肤外用剂还含有以下成分中的至少一种:补水活性成分、美白活性成分、保湿活性成分、抗氧化活性成分、抗皱活性成分、祛斑活性成分、消痘活性成分、防晒活性成分、祛粉刺/痤疮活性成分、祛头屑活性成分、抗过敏活性成分或者皮脂腺抑制活性成分。
皮肤外用剂指以涂抹、喷洒或者其它类似方法,散布于人体表面的任何部位,以达到清洁、保养、美容、修饰、改变外观或者修正人体气味,保持良好状态的产品。皮肤外用剂可以是起到清洁、护理、美化或保养等作用的基础化妆品、面部化妆品、头部护理品、身体护理品。
优选的,上述皮肤外用剂为化妆品。
一种皮肤外用剂原料,以牦牛奶发酵提取物为活性成分,并含有4wt%~10wt%丁二醇、0.45 wt%~ 0.72wt%苯氧乙醇和0.05 wt%~ 0.08 wt%乙基己基甘油。
所述皮肤外用剂原料的制备方法为:(1)牦牛奶或者牦牛奶干奶粉经过液体发酵的发酵液在4~10℃、1000~8000rpm条件下离心10~60min,取上清液;(2)上清液调节pH至5.0~7.0,80~90℃加热30~60min后,1000~8000rpm离心10~30min,取上清液;(3)上清液用孔径0.05~0.22μm滤膜在1~5bar压力下过滤,取发酵滤液;(4)发酵滤液中依次加入丁二醇、苯氧乙醇和乙基己基甘油。
一种化妆水,其组分包括:以固含量计0.01wt%~3wt%的牦牛奶发酵提取物,以及:0~5wt%甘油、2 wt%~5wt%丁二醇、0~4 wt%羟乙基脲、0~2 wt%烟酰胺、0~0.02wt%透明质酸钠或透明质酸钾、0~0.2wt%甘草酸二钾、0~0.2wt%尿囊素、0~0.2wt%羟苯甲酯、0~0.1wt% EDTA二钠、0~0.5wt%苯氧乙醇、0~0.1wt%乙基己基甘油、0~可接受剂量香精,余量为去离子水。
本发明的有益效果在于,将牦牛奶用植物乳杆菌发酵,发酵液离心过滤后得到的牦牛奶发酵提取物,能够降低金黄色葡萄球菌/表皮葡萄球菌的比值、促进真皮成纤维细胞和表皮细胞增殖,从而实现调节皮肤菌群、维持和/或修复皮肤表面微生态,以及修复老化的皮肤。因此,牦牛奶发酵提取物可以用作皮肤外用剂,尤其是化妆品的活性成分;可以用于制备皮肤外用剂,尤其是调节皮肤菌群,维持修复皮肤表面微生态及修复老化皮肤及美白作用的皮肤外用剂,具有良好的应用前景。
附图说明
图1为牦牛奶发酵产物滤液对表面菌群,S. a/S. e比值的影响
图2为牦牛奶发酵产物滤液对Fibroblast的生长影响。
图3为牦牛奶发酵产物滤液对HaCaT的生长影响如图3所示。
具体实施方式
实施例1 制备牦牛奶发酵产物滤液
本发酵产物的基质原料为牦牛奶或者牦牛奶干奶粉,产自泛喜马拉雅地区。将牦牛奶与去离子水混合,发酵培养基/培养液中牦牛奶固形物的含量为4wt%;高温高压灭菌后冷却用植物乳杆菌(Lactobacillus plantarum)在35~40℃、150~250rpm条件下发酵36小时。
牦牛奶发酵滤液的制备:
(1)发酵产物在4~10℃、6000rpm条件下离心10~30min,取上清液;
(2)调节pH至5.0~7.0,80~90℃水浴30~60min后,1000~4000rpm离心10~30min,取上清液;
(3)选用孔径为0.05、0.10或0.22μm的滤膜,滤膜预浸泡于75%(v/v)乙醇中;过滤滤器及器材用75%(v/v)乙醇清洗,70℃烘箱烘干后使用;
(5)膜过滤装置先使用0.5% NaOH水溶液循环和浸泡5~10min后,改用0.5%NaHSO3水溶液循环或浸泡5~10 min,然后用去离子水清洗。将膜片装入设备后,接入灭菌水清洗后将水排空;
(6)接入步骤(2)中的上清液进行过滤,通过调节压力(≤5bar)来控制过滤速度(1.0-2.0L/h),过滤后的发酵滤液装在无菌容器中储存。
所得到的牦牛奶发酵滤液中,干物质含量为1.5wt%。
实施例2 牦牛奶发酵提取物对表面菌群的影响
(一) 实验材料
1. 实验菌种
表皮葡萄球菌S. e(Staphylococcus epidermidis)、金黄色葡萄球菌S. a(Staphylococcus aureus)。
2. 培养基
LB培养基:0.5%粉,1.0%胰蛋白胨,1.0%NaCl,余量为水,所述百分比为质量百分比。121℃,20min高温高压灭菌后冷却备用。
3.样品信息
空白样品(Control):无菌生理盐水:0.9%NaCl,余量为水,所述百分比为质量百分比。121℃,20min高温高压灭菌后冷却备用。
实验样品:实施例1所获得的牦牛奶发酵产物滤液,其中固含量为1.5wt%。
(二)实验步骤
1. 将保藏的S. a和S. e(单菌落或冻存管菌液)接入无菌LB液体培养基中活化复苏(1 CFU/4 mL或50 μL/mL);摇床37℃,150-250rpm培养8-24小时;LB培养基可维持S. a和S. e的正常生长代谢,培养16h后,两者均可达到生长稳定期;
2. 2000~6000rpm离心移除培养基,生理盐水逐步稀释重悬菌液(C0)至1/2C0、1/4C0和1/8C0,测定各浓度两种重悬菌液的OD600,确定两者的菌体浓度比值,并根据各自的OD600值按比例稀释,重新获得等OD600菌悬液。
3.利用S. a和S. e的等OD菌悬液,分别加入不同浓度的牦牛奶发酵产物滤液到无菌LB培养基中,获得质量百分数为0.015%、0.15%、0.3%和0.45%的牦牛奶发酵滤液(干重)培养基,测试体系总体积4mL,并按体积比0.2%~2%接种。培养8-24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。p<0.05视为显著性差异,*p<0.05,**p<0.01,***p<0.001。
不同浓度牦牛奶发酵产物滤液(干重含量)对S. a/S. e的调节作用如图1所示。实验结果显示,牦牛奶发酵产物滤液可以影响S. a/S. e比值,选择性影响S. a和S. e生长,能够降低S. a/S. e值,帮助人体表面微生物菌群的稳态维持,或对失衡的皮肤表面微生态进行调整修复。
如图1,四种浓度下,S. a/S. e的值相比于空白对照的1.660,分别下降5.24%、33.31%、49.64和65.84%。这表明牦牛奶发酵产物能显著地降低生物量比值,并且随着浓度的增加而逐渐降低;牦牛奶发酵产物具有将皮肤表面微生物菌群中S. a和S. e的比例调整至更加健康状态的潜力。
实施例3 牦牛奶发酵产物的细胞增殖作用
(一)实验材料
细胞:真皮成纤维细胞Fibroblast、人永生化表皮细胞HaCaT。
实验样品:牦牛奶发酵产物滤液用去离子水配制成不同浓度溶液,在不同细胞生长所需的无血清DMEM或MEM培养基中加入各浓度的牦牛奶发酵产物滤液,使培养基中牦牛奶发酵产物滤液干物质含量为0.015%、0.075%和0.15%,所述百分比为质量百分比。
(二)实验方法
牦牛奶发酵产物滤液用去离子水配制成不同浓度溶液,加入到人成纤维细胞培养液中,以不含样品的去离子水为空白对照,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,评估对人成纤维细胞的增殖作用。p<0.05视为显著性差异,*p<0.05,**p<0.01。无血清培养基中牦牛奶发酵产物滤液的干物质含量分别为0.015%、0.075%和0.15%,所述百分比为质量百分比。
(三)实验结果
牦牛奶发酵产物滤液对Fibroblast的生长影响如图2所示,对HaCaT的生长影响如图3所示。
如图2、3结果所示,不同浓度牦牛奶发酵产物滤液(干重含量)均对成纤维细胞和表皮细胞有增殖作用,并且随着浓度的增加,对成纤维细胞和表皮细胞的生长促进作用逐渐增强,0.15 wt%牦牛奶发酵产物滤液能够使成纤维细胞和HaCaT的最高细胞活力分别达到143.72%和140.03%。
因此,牦牛奶发酵产物滤液即牦牛奶发酵提取物能够促进人皮肤细胞增殖,从而修复老化的皮肤,具有抗衰老的效果。
实施例4 牦牛奶发酵产物的细胞增殖作用
(一) 实验材料
细胞:小鼠黑色素瘤细胞B16
实验样品:无菌牦牛奶发酵产物滤液原液,加入B16细胞培养基后所含牦牛奶发酵产物滤液的干物质含量为0.015%,所述百分比为质量百分比。
(二) 实验方法
采用L-Dopa氧化法测定样品对酪氨酸酶的抑制作用。B16黑色素细胞以1×105密度培养在96孔板中,经24h后,将无菌牦牛奶发酵产物滤液以0.015%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 0.2 mg/mL的L-DOPA,37℃处理3h后再测定490 nm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/实验组细胞密度)/(对照组OD值/对照组细胞密度)]×100%。同时用MTT法测定样品对B16细胞的活力影响。
(三) 实验结果
实验结果根据表1显示,0.015%的牦牛奶发酵产物滤液对B16细胞活力没有显著影响,同时能够明显抑制酪氨酸酶活力。
表1小鼠B16黑色素瘤细胞酪氨酸酶抑制结果
测试浓度 | B16细胞活力(%) | B16酪氨酸酶抑制率(%) | |
Control | - | 100 | 0 |
Arbutin(阳性对照) | 0.1mg/mL | 99 | 25 |
牦牛奶发酵产物滤液 | 0.015% | 98 | 35 |
实施例5 牦牛奶发酵产物滤液用于制备化妆品/皮肤外用剂原料
处理工艺为:实施例1所得到的过滤液中先后加入丁二醇、苯氧乙醇和乙基己基甘油,充分搅拌使之混合均匀,混合后的样品中,丁二醇含量为4wt%~10wt%,苯氧乙醇含量为0.45 wt%~ 0.72wt%,乙基己基甘油含量为0.05 wt%~ 0.08 wt%。最后将样品室温或者4℃避光保存。待生产时,按照本领域常规的化妆水/精华液/乳液/霜的制备方法添加。上述配方产物也可以直接用作化妆水。
实施例6 含牦牛奶发酵提取物的化妆水
取实施例1的牦牛奶发酵产物滤液,用于制备具有调节皮肤微生态菌群和修复作用的化妆水,按照本领域常规的化妆水的制备方法,化妆水所含的组分如表1所示:
表2
。
Claims (7)
1.牦牛奶发酵提取物在制备皮肤外用剂方面的应用,所述的牦牛奶发酵提取物通过调节皮肤菌群来维持和/或修复皮肤表面微生态;或者通过促进皮肤细胞增殖修复老化的皮肤;或者通过抑制皮肤细胞酪氨酸酶活力,实现美白作用;所述牦牛奶发酵提取物通过如下方法制备得到:
(1)牦牛奶或者牦牛奶干奶粉与水混合制成培养基,接入植物乳杆菌(Lactobacillusplantarum; L. plantarum)发酵,在 30~40°C、150~250rpm 条件下恒温发酵 16-48 小时进行液体发酵;
(2)发酵液在 4~10°C、1000~8000rpm 条件下离心 10~60min,取上清液;
(3)上清液调节 pH 至 5.0~7.0,80~90°C加热 30~60min 后,1000~8000rpm 离心10~30min,取上清液;
(4)上清液用孔径 0.05~0.22μm 滤膜在 1~5bar 压力下过滤,取发酵滤液,培养基中牦牛奶固形物的含量为 1wt%~5wt%,余量为水。
2. 如权利要求 1 所述的应用,其特征在于,所述发酵温度为 35~40°C。
3. 如权利要求 1 所述的应用,其特征在于,所述牦牛奶发酵提取物降低金黄色葡萄球菌/表皮葡萄球菌的比值来调节皮肤菌群,或者促进真皮成纤维细胞和/或表皮细胞增殖。
4. 如权利要求 1 所述的应用,其特征在于,所述的牦牛奶发酵提取物为牦牛奶经植物乳杆菌发酵的发酵滤液,或者由所述发酵滤液制得的稀释液、浓缩液、浸膏或干燥物。
5.一种皮肤外用剂,其特征在于,所述皮肤外用剂含有牦牛奶发酵提取物;所述的牦牛奶发酵提取物通过调节皮肤菌群来维持和/或修复皮肤表面微生态;或者通过促进皮肤细胞增殖修复老化的皮肤,或者作为美白的活性成分;所述牦牛奶发酵提取物通过如下方法制备得到:
(1)牦牛奶或者牦牛奶干奶粉与水混合制成培养基,接入植物乳杆菌(Lactobacillusplantarum; L. plantarum)发酵,在 30~40°C、150~250rpm 条件下恒温发酵 16-48 小时进行液体发酵;
(2)发酵液在 4~10°C、1000~8000rpm 条件下离心 10~60min,取上清液;
(3)上清液调节 pH 至 5.0~7.0,80~90°C加热 30~60min 后,1000~8000rpm 离心10~30min,取上清液;
(4)上清液用孔径 0.05~0.22μm 滤膜在 1~5bar 压力下过滤,取发酵滤液,其中,培养基中牦牛奶固形物的含量为 1wt%~5wt%,余量为水。
6. 如权利要求 5 所述的皮肤外用剂,其特征在于,以固含量计,牦牛奶发酵提取物的含量为 0.005wt%~5wt%。
7. 一种皮肤外用剂活性成分原料,其特征在于,以权利要求5所述制备方法得到的牦牛奶发酵提取物为活性成分,并含有 4wt%~10wt%丁二醇,0.45 wt%~ 0.72wt%苯氧乙醇和 0.05 wt%~ 0.08 wt%乙基己基甘油。
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