CN113924357A - 新长双歧杆菌菌株或包含所述菌株的化妆品组合物 - Google Patents
新长双歧杆菌菌株或包含所述菌株的化妆品组合物 Download PDFInfo
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- CN113924357A CN113924357A CN201980095232.0A CN201980095232A CN113924357A CN 113924357 A CN113924357 A CN 113924357A CN 201980095232 A CN201980095232 A CN 201980095232A CN 113924357 A CN113924357 A CN 113924357A
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Abstract
本发明涉及新长双歧杆菌ATG‑F5(Bifidobacterium longum ATG‑F5)菌株。F5菌株是一种功能性有益菌,其免于抗生素抗性影响,通过对皮肤病原体痤疮丙酸杆菌(Cutibacterium aches)的抗菌活性、对自由基的抗氧化作用、皮肤屏障改善和抗炎作用来改善皮肤健康,以及通过皮肤美白功能、皱纹改善功能和干燥皮肤缓解功能来增强皮肤美容。所述菌株可用于化妆品组合物或健康功能性食品中。
Description
技术领域
本发明涉及新长双歧杆菌ATG-F5(Bifidobacterium longum ATG-F5,登记号.KCTC13828BP)菌株或包含所述菌株的化妆品组合物。
背景技术
在皮肤护理领域,多种护理技术随着其应用范围逐渐扩大而被使用。在多种技术中,人们更喜欢具有提亮皮肤的美白功能性物质的化妆品,或者具有创造看起来年轻的皮肤并促进皮肤弹性的皱纹改善和抑制功能的化妆品。最近,也出现了用作皮肤屏障和防止干燥皮肤的功能性化妆品,并且由于人们观念的转变,正在开发具有更多功能的美白和皱纹改善化妆品。
在这些技术中,皮肤美白化妆品是基于黑素活性抑制的原理,所述黑素活性抑制决定了人皮肤的颜色。黑素是存在于表皮基底层中并且阻断太阳的紫外(UV)线的色素。然而,当皮肤的生理功能由于过度合成或老化而变差时,黑素会沉淀到皮肤中,导致斑点、雀斑和多处色素。
黑素合成受酪氨酸酶、酪氨酸酶相关蛋白-1(tyrosinase related protein-1,TRP-1)、酪氨酸酶相关蛋白-2(tyrosinase related protein-2,TRP-2)、促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)(其为环腺苷酸(cyclic adenosinemonophosphate,cAMP)的诱导剂)、以及α-黑素细胞刺激激素(α-melanocyte stimulatinghormone,α-MSH)调节(Chan et al.,2011)。尤其是酪氨酸酶、酪氨酸酶相关蛋白-1(TRP-1)和酪氨酸酶相关蛋白-2(TRP-2)调节对黑素产生的刺激。通过促进羟基化反应,酪氨酸酶催化酪氨酸转化为3,4-二羟基苯丙氨酸(DOPA)以及DOPA转化为DOPA-醌。(Jang和Park,2017)。这种提高的DOPA-醌会导致黑素产生。因此,抑制酪氨酸酶活性通过抑制黑素产生在皮肤美白中发挥重要作用。
具有皱纹改善和抑制功能的化妆品基于涉及以皮肤弹性著称的胶原蛋白的原理。皮肤老化有两种类型:内致性和外致性。随着我们年龄的增长,内致性老化会自然发生,而外致性老化或光老化是由暴露于紫外(UV)光引起的。人皮肤由三层组织构成:表皮、真皮和皮下脂肪。表皮主要由角质形成细胞组成,以保护皮肤,而真皮与皮肤弹性和皱纹相关。真皮表现出纤维状蛋白质胶原蛋白和弹性蛋白。胶原蛋白是纤维状蛋白质,并且存在14种类型,其中主要存在作用于皮肤的1型胶原蛋白(Kim and Yoon.,2013)。基质金属蛋白酶(matrix metalloproteinase,MMP)是降解胶原蛋白的基质蛋白,并且已知有超过20种类型的MMP。当MMP-1(也称为胶原酶-1)降解胶原蛋白时,皮肤弹性下降并且皱纹形成(Lee etal.,2013)。因此,可通过调节作用于皮肤的蛋白质来降低皱纹并保持皮肤弹性。
除了以上与皮肤护理相关的功能之外,还有新出现的功能,例如皮肤屏障增强和干燥皮肤缓解。改善皮肤屏障功能涉及改善皮肤状况,同时增强闭合蛋白(occludin)和密封蛋白(claudin)(即当紧密连接蛋白参与作为皮肤组织的上皮细胞中的细胞与细胞接触时所需要的蛋白质)的表达(B.Tebbe et al.,2002)。干燥皮肤缓解功能涉及诱导透明质酸合酶2(hyaluronan synthase 2,HAS2)透明质酸(HA)合成以及因此的降低皮肤中的水分流失。HA在皮肤中发挥重要的生物学作用,例如保持水分、维持组织结构和弹性以及交换营养。
因此,本发明提出了以登记号KCTC13828BP保藏的长双歧杆菌ATG-F5菌株作为增强与皮肤美容相关的功能的方式,所述功能包括皮肤美白、皱纹改善、皮肤屏障增强、干燥皮肤缓解、抗炎和抗痤疮功能。
[相关技术文献]
[专利文献]
(专利文献1)韩国专利登记号.10-1589464(发明名称:新双歧杆菌菌株和包含所述菌株的用于促进生长的功能性食品组合物 申请人:Cell Biotech Co.,Ltd.,登记日期:2016年1月22日)
(专利文献2)欧洲专利登记号.2308566B1(发明名称:Use of orallyadministered probiotic bifidobacteria for human beauty benefits,申请人:TheProcter&Gamble Company等,登记日期:2011年4月13日)
(专利文献5)韩国专利公开号.10-2016-0092564(发明名称:包含酵母菌和乳酸菌的两阶段发酵裂解物的具有抗氧化、皱纹改善和保湿效果的化妆品组合物 申请人:Incos,公开日期:2016年8月5日)
(专利文献7)韩国登记专利号.10-1885195(发明名称:包含木苏发酵提取物作为活性成分的化妆品组合物 申请人:Koreana Cosmetics Co.,Ltd.,登记日期:2018年7月30日)
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发明内容
技术问题
本发明的目的是提供新长双歧杆菌ATG-F5菌株或包含所述菌株的化妆品组合物。
技术方案
本发明涉及新长双歧杆菌ATG-F5(登记号.KCTC13828BP)菌株。
所述菌株通过促进闭合蛋白(OCLN)和密封蛋白4(CLDN4)的基因表达以及通过促进透明质酸合酶2(HAS2)的基因表达缓解皮肤干燥来增强皮肤屏障。
所述菌株具有抑制引起痤疮的细菌痤疮丙酸杆菌(Cutibacterium acnes)生长的抗痤疮功能,以及具有提高白介素10(interleukin 10,IL-10)的抗炎活性。
所述菌株还具有通过抑制酪氨酸酶活性和控制黑素产生的皮肤美白功能和通过抑制MMP-1活性促进前胶原(pro-collagen)的皱纹改善功能。
在本发明的一个方面中,长双歧杆菌ATG-F5菌株可被替换为或包括选自以下的至少一种:菌株的细菌细胞;细胞裂解物;菌株培养物;其中从菌株培养物中去除细胞的培养物溶液;菌株的细胞提取物;菌株培养物的提取物;和从菌株培养物中去除细胞的培养物溶液的提取物。
因此,本发明提供了包含所述菌株并且具有皮肤屏障增强、干燥皮肤缓解、抗炎、抗痤疮、抗氧化和皱纹改善的作用的化妆品组合物。
化妆品组合物的制剂可选自皮肤洗剂皮肤软化剂紧肤剂收敛剂乳液洗剂保湿乳液滋养乳液按摩霜滋养霜保湿霜护手霜护足霜颈霜粉底精华液剥撕式面膜皂清洁泡沫清洁洗剂清洁霜身体洗剂洗发香波毛发处理剂护发素和身体清洁剂
在本发明的另一个方面中,提供了包含所述菌株并且具有功能例如皮肤屏障增强、干燥皮肤缓解、抗炎、抗痤疮、抗氧化和皱纹改善的健康功能性食品。
本发明还涉及包含所述菌株并且用于预防或治疗选自干燥皮肤、痤疮和炎性疾病的疾病的组合物。
炎性疾病可选自干燥皮肤、痤疮和选自以下的炎性疾病:炎性肠病、炎性胶原血管病、肾小球肾炎、炎性皮肤病、结节病、视网膜炎、胃炎、肝炎、肠炎、关节炎、扁桃体炎、咽炎、支气管炎、肺炎、胰腺炎、脓毒症、膀胱炎、肾炎和神经炎。
在下文中,将更详细地描述本发明。
本发明的长双歧杆菌ATG-F5菌株可在BL培养基(下文中称为BL)或包含0.3至0.7g/L L-半胱氨酸的MRS培养基(下文中称为MRS-cys)的液体或固体培养基(肉汤或琼脂)中培养,和在BL或MRS-cys肉汤中培养,长双歧杆菌ATG-F5菌株可培养至8.0×108至1.0×109CFU/ml的浓度。当向MRS培养基添加L-半胱氨酸时,优选添加0.6g/L。
优选地,菌株在30℃至37℃下培养16至24小时,并且用于培养的最佳温度为37℃,最低温度为15℃,最高温度为37℃,最佳pH为6.8,最低可培养pH为5.0,以及最高pH为7.3。最佳孵育时间为18小时,最低孵育时间为8小时,以及最高孵育时间为72小时。
本发明的菌株是对抗生素不具有抗性的安全菌株,所述抗生素至少一种选自氨苄青霉素(ampicillin)、万古霉素(vancomycin)、庆大霉素(gentamicin)、卡那霉素(kanamycin)、链霉素(streptomycin)、克林霉素(clindamycin)、红霉素(erythromycin)、四环素(tetracycline)和氯霉素(chloramphenicol)。
本发明提供了包含长双歧杆菌ATG-F5菌株的多种组合物,所述菌株即选自以下的一种或更多种:菌株的细菌细胞;细胞裂解物;菌株培养物;其中从菌株培养物中去除细胞的培养物溶液;菌株的细胞提取物;菌株培养物的提取物;和从菌株培养物中去除细胞的培养物溶液的提取物。
其中,F5菌株的细胞裂解物可在通过将菌株的培养物溶液离心而将细胞与培养物上清液分离、将其悬浮于PBS中、用溶菌酶进行处理以及进行声处理之后获得。当通过离心分离细胞时,可将细胞用缓冲液例如PBS进行洗涤并再次离心。离心可分别以3,000至5000rpm进行5至60分钟。优选地,对悬浮于PBS中的细胞的溶菌酶处理在30℃至37℃下以300至700μg/ml的浓度进行并且超声处理条件为50至90W,20至60秒,以及20至40个循环。此时,优选进行热处理以使细胞裂解物中可能存活的微生物死亡。可在50℃至70℃下热处理30至60分钟,并且最好用浴加热以使蛋白质变性最小化,而不是直接加热。另外,通过将pH调节至7至8并进行过滤(使用0.2μm孔过滤器),分离的培养物上清液可用作来源于多种培养物的组合物。
来源于菌株的每种组合物均可在没有另外处理的情况下使用,通过多种方法纯化或干燥,并添加至化妆品组合物、药物组合物或健康功能性食品。
来源于菌株的每种组合物可通过进行冷冻干燥来干燥,并且可包含在干燥常见菌株或来源于所述菌株的组合物时所使用的赋形剂。用于冷冻干燥的赋形剂可选自葡糖酸、海藻酸、海藻酸钠、羟丙基纤维素、羟丙基甲基纤维素、羧甲基纤维素钠、甲基纤维素、卡波姆、透明质酸、黄芪胶、刺梧桐胶、水溶性淀粉、果胶、明胶、聚乙烯醇、聚乙烯吡咯烷酮、寡糖、糖醇、葡萄糖酸钙、乳酸钙、聚甲基丙烯酸甲酯、小麦蛋白、大豆蛋白、甲基纤维素、aquacoat瓜尔胶、槐豆胶、黄原胶、结冷胶、阿拉伯胶、海藻糖等。
本发明可提供包含来源于所述菌株的组合物的化妆品组合物,该化妆品组合物可用作用于增强皮肤屏障、缓解皮肤干燥、抗炎、抗痤疮、抗氧化或皱纹改善效果的组合物。当化妆品组合物的总重量为基于按重量计100%时,可包含按重量计0.001%至20%的来源于所述菌株的多种组合物。化妆品组合物的制剂可优选地选自皮肤洗剂、皮肤软化剂、紧肤剂、收敛剂、乳液洗剂、保湿乳液、滋养乳液、按摩霜、滋养霜、保湿霜、护手霜、护足霜、颈霜、粉底、精华液、剥撕式面膜、皂、清洁泡沫、清洁洗剂、清洁霜、身体洗剂、洗发香波、毛发处理剂、护发素和身体清洁剂。
本发明的化妆品组合物可还包含选自以下的组分:水溶性维生素、油溶性维生素、聚合物肽、聚合物多糖、鞘脂和海藻提取物。
水溶性维生素可以是可在化妆品中共混的任意水溶性维生素,但优选维生素B1、维生素B2、维生素B6、吡哆醇、盐酸吡哆醇、维生素B12、泛酸、烟酸、烟酰胺、叶酸、维生素C、维生素H等。另外,其盐类(盐酸硫胺素、抗坏血酸钠等)及衍生物(抗坏血酸-2-磷酸钠盐、抗坏血酸-2-磷酸镁盐等)也包括在本发明中使用的水溶性维生素中。水溶性维生素可通过常规方法来获得,所述常规方法例如微生物转化方法、从微生物培养物中纯化、酶方法或化学合成方法。
油溶性维生素可以是可在化妆品组合物中共混的任意油溶性维生素,但优选维生素A、胡萝卜素、维生素D2、维生素D3、维生素E(d1-α生育酚、d-α生育酚、d-α生育酚)等及其衍生物(抗坏血酸棕榈酸酯、抗坏血酸硬脂酸酯、抗坏血酸二棕榈酸酯、dl-α生育酚乙酸酯、烟酸dl-α生育酚维生素E、DL-泛醇、D-泛醇、泛乙醚等)也包括在本发明中使用的油溶性维生素中。油溶性维生素可通过常规方法来获得,所述常规方法例如微生物转化方法、从微生物培养物中纯化、酶方法或化学合成方法。
聚合物肽可以是可在化妆品组合物中共混的任意聚合物肽,但优选地,例示了胶原蛋白、水解的胶原蛋白、明胶、弹性蛋白、水解的弹性蛋白、角蛋白等。聚合物肽可通过常规方法来纯化和获得,所述常规方法例如从微生物培养基中纯化的方法、酶方法或化学合成方法,或者其可在从天然产物例如猪和牛的真皮以及蚕的丝纤维中纯化之后使用。
聚合物多糖可以是可在化妆品组合物中共混的任意聚合物多糖,但优选地,可提及羟乙基纤维素、黄原胶、透明质酸钠、硫酸软骨素,或其盐(钠盐等)。例如,硫酸软骨素或其盐可在从哺乳动物或鱼类中纯化之后使用。
鞘脂可以是可在化妆品组合物中共混的任意鞘脂,并且优选地提及了神经酰胺、植物鞘氨醇、鞘糖脂等。鞘脂通常通过常规方法从哺乳动物、鱼类、水生有壳动物、酵母菌、植物等中纯化,或者可通过化学合成来获得。
海藻提取物可以是可在化妆品组合物中共混的任意海藻提取物,但优选地,例示了褐藻提取物、红藻提取物、绿藻提取物等。从这些海藻提取物中提纯的角叉聚糖、海藻酸、海藻酸钠和海藻酸钾也包括在本发明中使用的海藻提取物中。海藻提取物可通过从海藻中通过常规方法纯化而获得。
本发明的化妆品组合物可还包含在常规化妆品组合物中共混的其他成分。
可添加的其他成分包括脂肪和油、保湿剂、润肤剂、表面活性剂、有机和无机着色剂、有机粉末、紫外线吸收剂、防腐剂、杀真菌剂、抗氧化剂、植物提取物、pH调节剂、醇、着色剂、芳香剂、血液循环促进剂、清凉剂止汗剂和纯净水。
油和脂肪成分包括酯油、烃油、硅脂、基于氟的脂肪、动物脂肪和植物脂肪。
油和脂肪成分包括酯油、烃油、硅脂、基于氟的脂肪、动物脂肪和植物脂肪。酯油的一些实例包括:甘油基三(2-乙基己酸酯)、2-乙基己酸十六烷基酯、肉豆蔻酸异丙酯、肉豆蔻酸丁酯、棕榈酸异丙酯、硬脂酸乙酯、棕榈酸辛酯、异硬脂酸异十六烷基酯、硬脂酸丁酯、亚油酸乙酯、亚油酸异丙酯、油酸乙酯、肉豆蔻酸异十六烷基酯、肉豆蔻酸异硬脂酯、棕榈酸异硬脂酯、肉豆蔻酸辛基十二烷基酯、异硬脂酸异十六烷基酯、癸二酸二乙酯、己二酸二异丙酯、新戊酸异烷基酯、三(辛酸、癸酸)甘油、三羟甲基丙烷2-乙基己酸酯、三羟甲基丙烷三异硬脂酸酯、四(2-乙基己酸酯)季戊四醇、辛酸十六烷基酯、月桂酸癸酯、月桂酸己酯、肉豆蔻酸癸酯、肉豆蔻酸肉豆蔻酯、肉豆蔻酸十六烷基酯、硬脂酸硬脂酸酯、油酸癸酯、蓖麻油酸十六烷基酯、月桂酸异硬脂酸酯、肉豆蔻酸异十三烷基酯、棕榈酸异十六烷基酯、硬脂酸辛酯、硬脂酸异十六烷基酯、油酸异癸酯、油酸辛基十二烷基酯、亚油酸辛基十二烷基酯、异硬脂酸异丙酯、鲸蜡硬脂基2-乙基己酸酯、硬脂基2-乙基己酸酯、异硬脂酸己酯、乙二醇二辛酸酯、乙二醇二油酸酯、丙二醇二癸酸、丙二醇二(辛酸,癸酸)、丙二醇二辛酸酯、新戊二醇二癸酸、新戊二醇二辛酸酯、三辛酸甘油酯、甘油十三烷酸、三异棕榈酸甘油酯、三异硬脂酸甘油酯、新戊酸辛基十二烷基酯、辛酸异硬脂酸酯、异壬酸辛酯、新癸酸己基癸酯、新癸酸辛基十二烷基酯、异硬脂酸异十六烷基酯、异硬脂酸异硬脂酸酯、异硬脂酸辛基癸酯、聚甘油油酸酯、聚甘油异硬脂酸酯、柠檬酸三异十六烷基酯、柠檬酸三异烷基酯、柠檬酸三异辛酯、乳酸月桂酯、乳酸肉豆蔻酯、乳酸十六烷基酯、乳酸辛基癸酯、柠檬酸三乙酯、柠檬酸乙酰基三乙酯、柠檬酸乙酰基三丁酯、柠檬酸三辛酯、苹果酸二异硬脂酸酯、2-乙基己基羟基硬脂酸酯、琥珀酸二2-乙基己基酯、己二酸二异丁酯、癸二酸二异丙酯、癸二酸二辛酯、硬脂酸胆固醇酯、异硬脂酸胆固醇酯、羟基硬脂酸胆固醇酯、油酸胆固醇酯、油酸二氢胆固醇酯、植物甾醇异硬脂酸酯、植物甾醇油酸酯、12-硬脂酰羟基硬脂酸酯异十六烷基、12-硬脂酰羟基硬脂酸硬脂酸酯和12-硬脂酰羟基硬脂酸酯异硬脂酸基。
烃油可包括角鲨烯、液体石蜡、α-烯烃寡聚物、异构烷烃、地蜡、石蜡、液体异构烷烃、聚丁烯、微晶蜡和矿脂。
硅脂可包括聚甲基硅酮、甲基苯基硅酮、甲基环聚硅氧烷、八甲基聚硅氧烷、十甲基聚硅氧烷、十二甲基环硅氧烷、二甲基硅氧烷-甲基十六烷基氧基硅氧烷共聚物、二甲基硅氧烷-甲基硬脂基氧基硅氧烷共聚物、经烷基修饰的硅油和经氨基修饰的硅油。
基于氟的脂肪可包括全氟聚醚。
动物和植物脂肪可包括鳄梨油杏仁油橄榄油、芝麻油、米糠油鸟花油大豆油、玉米油、菜籽油、杏桃核仁油棕榈仁油棕榈油蓖麻油、葵花籽油葡萄籽油棉籽油棕榈油夏威夷坚果油小麦胚芽油米胚芽油乳木果油月见草油澳洲坚果油白芒花油卵黄油牛脂马油貂油橙酒椰油荷荷巴油小烛树蜡巴西棕榈蜡液体羊毛脂 和氢化蓖麻油
保湿剂可包括水溶性低分子保湿剂、脂溶性分子保湿剂、水溶性聚合物和脂溶性聚合物。
水溶性低分子保湿剂可包括丝氨酸、谷氨酰胺、山梨糖醇、甘露糖醇、吡咯烷酮羧酸钠、甘油、丙二醇、1,3-丁二醇、乙二醇、聚乙二醇B(聚合度n=2或更高)、聚丙二醇(聚合度n=2或更高)、聚甘油B(聚合度n=2或更高)、乳酸和乳酸盐/酯。
脂溶性分子保湿剂可包括胆固醇和胆固醇酯。
水溶性聚合物可包括羧乙烯基聚合物、聚天冬氨酸、黄芪胶、黄原胶、甲基纤维素、羟甲基纤维素、羟乙基纤维素、羟丙基纤维素、羧甲基纤维素、水溶性甲壳质、壳聚糖和糊精。
脂溶性聚合物可包括聚乙烯吡咯烷酮-二十碳烯共聚物、聚乙烯吡咯烷酮-十六碳烯共聚物、硝酸纤维素、糊精脂肪酸酯和硅酮。
润肤剂可包括长链酰基谷氨酸胆固醇酯、羟基硬脂酸胆固醇酯、12-羟基硬脂酸、硬脂酸、松香酸和羊毛脂脂肪酸胆固醇酯。
表面活性剂可包括非离子型表面活性剂、阴离子型表面活性剂、阳离子型表面活性剂、两性表面活性剂。
非离子型表面活性剂可包括自乳化单硬脂酸甘油酯、丙二醇脂肪酸酯、甘油脂肪酸酯、聚甘油脂肪酸酯、脱水山梨糖醇脂肪酸酯、聚氧乙烯(POE)脱水山梨糖醇脂肪酸酯、POE山梨糖醇脂肪酸酯、POE甘油脂肪酸酯、POE烷基醚、POE脂肪酸酯、POE氢化蓖麻油、POE蓖麻油、聚氧乙烯-聚氧丙烯(POE-POP)共聚物、POE-POP烷基醚、经聚醚修饰的硅酮、月桂酸烷醇酰胺、烷基胺氧化物和氢化大豆磷脂。
阴离子型表面活性剂可包括脂肪酸皂、α酰基磺酸盐、烷基磺酸盐、烷基烯丙基磺酸盐、烷基萘磺酸盐、烷基硫酸盐、POE烷基醚硫酸盐、烷基酰胺硫酸盐、烷基磷酸盐、POE烷基磷酸盐、烷基酰胺磷酸盐、烷基酰基烷基牛磺酸盐、N-酰基氨基酸盐、POE烷基醚羧酸盐、烷基磺基琥珀酸盐、烷基磺基乙酸钠、酰化水解胶原肽盐和全氟烷基磷酸酯。
阳离子型表面活性剂可包括烷基三甲基氯化铵、硬脂基三甲基氯化铵、硬脂基三甲基溴化铵、鲸蜡硬脂基三甲基氯化铵、二硬脂基二甲基氯化铵、硬脂基二甲基苄基氯化铵、山嵛基三甲基溴化铵、苯扎氯铵、二乙基氨基乙基硬脂酸酯、硬脂酸二甲基氨基丙酰胺和羊毛酯衍生物季铵盐。
两性表面活性剂可包括羧基甜菜碱型、酰胺甜菜碱型、磺基甜菜碱型、羟基磺基甜菜碱型、酰胺磺基甜菜碱型、磷酸甜菜碱型、氨基羧酸盐型、咪唑啉衍生物型和酰胺胺型。
有机和无机着色剂可包括:无机着色剂例如硅酸、硅酸酐、硅酸镁、滑石、绢云母、云母、高岭土、三氧化二铁黏土、膨润土、钛涂覆云母、氯氧化铋、氧化锆、氧化镁、氧化锌、氧化钛、氧化铝、硫酸钙、硫酸钡、硫酸镁、碳酸钙、碳酸镁、氧化铁、群青、氧化铬、氢氧化铬、炉甘石及其化合物;以及有机着色剂例如聚酰胺、聚酯、聚丙烯、聚苯乙烯、聚氨酯、乙烯树脂、尿素树脂、酚醛树脂、氟树脂、硅树脂、丙烯酸树脂、三聚氰胺树脂、环氧树脂、聚碳酸酯树脂、苯乙烯-二乙烯基苯共聚物、丝粉、纤维素、CI颜料黄和CI颜料橙;以及有机和无机复合着色剂。
有机粉末可包括:金属皂例如硬脂酸钙;烷基磷酸金属盐例如鲸蜡酸锌钠、月桂酸锌和月桂酸钙;酰基氨基酸多价金属盐例如N-月桂酰基-β-丙氨酸钙、N-月桂酰基-β-丙氨酸锌和N-月桂酰基甘氨酸钙;酰胺磺酸多价金属盐例如N-月桂酰基-牛磺酸钙和N-棕榈酰基-牛磺酸钙;N-酰基碱性氨基酸例如N-ε-月桂酰基-L-赖氨酸、N-ε-棕榈酰肼、N-α-帕里托酰鸟氨酸N-α-月桂酰基精氨酸和N-α-氢化牛脂肪酸酰基精氨酸;N-酰基多肽例如N-月桂酰基甘氨酰基甘氨酸;α-氨基脂肪酸例如α-氨基辛酸和α-氨基月桂酸;以及聚乙烯、聚丙烯、尼龙、聚甲基丙烯酸甲酯、聚苯乙烯、苯乙烯-二乙烯基苯共聚物和四氟化乙烯。
紫外线吸收剂可包括:对氨基苯甲酸、对氨基苯甲酸乙酯、对氨基苯甲酸戊酯、对氨基苯甲酸辛酯、水杨酸乙二醇酯、水杨酸苯酯、水杨酸辛酯、水杨酸苄酯、水杨酸丁苯酯、水杨酸高薄荷酯、肉桂酸苄酯、对甲氧基肉桂酸-2-乙氧基乙基、对甲氧基肉桂酸辛酯、双对甲氧基肉桂酸单-2-乙基己烷甘油酯、对甲氧基肉桂酸异丙酯、二异丙基肉桂酸酯混合物、尿刊酸、尿刊酸乙酯、羟基甲氧基二苯甲酮、羟基甲氧基二苯甲酮磺酸,及其盐形式;以及二羟基甲氧基二苯甲酮、二羟基甲氧基二苯甲酮二磺酸钠、二羟基二苯甲酮、四羟基二苯甲酮、4-叔丁基-4′-甲氧基二苯甲酰甲烷、2,4,6-三苯胺-p-(碳-2′-乙基己基-1′-氧基)-1,3,5-三嗪和2-(2-羟基-5-甲基苯基)苯并三唑。
pH调节剂可包括柠檬酸、柠檬酸钠、苹果酸、苹果酸钠、富马酸盐、富马酸钠、琥珀酸、琥珀酸钠、氢氧化钠和磷酸一氢钠。
醇可包括高级醇,例如鲸蜡醇。
共混成分不限于上述数种,并且在不损害本公开内容的目的和效果的范围内,可使用任何上述成分进行共混。然而,将优选相对于化妆品组合物的总重量,以按重量计0.01%至5%,更优选按重量计0.01%至3%的共混比例共混所述成分。
该实施方案的化妆品组合物可采用溶液剂、混悬剂或黏性混合物的形式。
该实施方案的化妆品组合物包含上述成分作为活性成分,但也可包含通常用于化妆品的其他成分。例如,可包含辅料和载体例如稳定剂、增溶剂、维生素、着色剂和芳香剂。
当制剂为散剂或喷雾剂的形式时,载体可以是乳糖、滑石、二氧化硅、氢氧化铝、硅酸钙或聚酰胺粉末。尤其是在喷雾剂的情况下,可添加抛射剂例如氯氟烃、丙烷-丁烷或二甲醚。
当制剂为溶液剂或混悬剂的形式时,载体可以是溶剂、溶剂化剂或乳化剂。即,可使用水、乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苄醇、苯甲酸苄酯、丙二醇、1,3-丁基乙二醇油、甘油脂肪族酯、聚乙二醇或脱水山梨糖醇脂肪酸酯。
当制剂为混悬剂的形式时,以下可用作载体:液体稀释剂例如水、乙醇、丙二醇;助悬剂例如乙氧基化异硬脂醇、聚氧乙烯山梨糖醇酯、聚氧乙烯脱水山梨糖醇酯、微晶纤维素、偏氢氧化铝、膨润土、琼脂和tracanth。
当制剂为含表面活性剂的清洁剂的形式时,以下可用作载体:脂肪醇硫酸盐、脂肪醇醚硫酸盐、磺基琥珀酸单酯、羟乙基磺酸盐咪唑啉衍生物、牛磺酸甲酯、肌氨酸盐、脂肪酸酰胺醚硫酸盐、烷基酰胺甜菜碱、脂肪醇、脂肪酸甘油酯、脂肪酸二乙醇酰胺、植物油、羊毛脂衍生物和乙氧基化甘油脂肪酸酯。
另外,本发明提供了包含来源于长双歧杆菌ATG-F5菌株的多种组合物的药物组合物。来源于长双歧杆菌ATG-F5菌株的组合物可以以按重量计0.001%至30%的量添加至本发明的药物组合物。
所述药物组合物可以是用于预防或治疗选自干燥皮肤、痤疮和炎性疾病的疾病的组合物,并且炎性疾病可以是选自以下的炎性疾病:炎性肠病、炎性胶原血管病、肾小球肾炎、炎性皮肤病、结节病、视网膜炎、胃炎、肝炎、肠炎、关节炎、扁桃体炎、咽炎、支气管炎、肺炎、胰腺炎、脓毒症、膀胱炎、肾炎和神经炎。
根据常规方法,药物组合物可以以经口剂型的形式配制和使用,例如散剂、颗粒剂、片剂、胶囊剂、混悬剂、混悬剂、糖浆剂、气雾剂等,外用制剂,栓剂等。可包含在药物组合物中的载体、赋形剂和稀释剂包括乳糖、右旋糖、蔗糖、山梨糖醇、甘露糖醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯胶、褐藻胶、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油。就制剂而言,使用通常使用的稀释剂或赋形剂例如填充剂、增量剂、黏合剂、润湿剂、崩解剂和表面活性剂进行制备。用于经口施用的固体制剂包括片剂、丸剂、散剂、颗粒剂、胶囊剂等,并且这些固体制剂通过将至少一种赋形剂例如淀粉、碳酸钙、蔗糖或乳糖、明胶等与本发明的菌株混合来制备。除了简单的赋形剂之外,还使用润滑剂,例如硬脂酸镁和滑石。用于经口使用的液体制剂包括混悬剂、液体溶液剂、混悬剂、糖浆剂等。除了水和液体石蜡(为常用的简单稀释剂)外,还可包括多种赋形剂,例如润湿剂、甜味剂、芳香剂和防腐剂。用于肠胃外施用的制剂包括无菌水性溶液剂、非水性溶液剂、混悬剂、混悬剂、冻干制剂、栓剂和阴道栓剂。作为非水性溶剂或助悬剂,可使用丙二醇、聚乙二醇、植物油(例如橄榄油)和可注射酯(例如油酸乙酯)。作为栓剂的基质,可使用witepsol聚乙二醇吐温61、可可脂、月桂酸酯甘油明胶等。
本发明的药物组合物的剂量将根据待治疗的对象的年龄、性别和体重,待治疗的具体疾病或病理学状况,疾病或病理学状况的严重程度,施用途径以及开处方者的判断而有所不同。基于这些因素的剂量确定在本领域技术人员的水平内,并且剂量通常为0.01mg/kg/天至约2000mg/kg/天。更优选的剂量为1mg/kg/天至500mg/kg/天。施用可每天一次施用或可将施用分为数次。上述剂量不以任何方式限制本发明的范围。
本发明的药物组合物可通过多种途径施用于哺乳动物,例如小鼠、家畜和人。由于本发明的菌株具有小的毒性和副作用,因此其是可安全使用的药物,即使为了预防目的而长时间服用时也是如此。
此外,本发明提供了具有增强皮肤屏障、缓解皮肤干燥、抗炎、抗痤疮、抗氧化或改善皱纹的作用的健康功能性食品,其含有来源于长双歧杆菌ATG-F5的组合物。可将来源于长双歧杆菌ATG-F5的组合物添加至本发明的健康功能性食品中,添加量为按重量计0.001%至50%。本发明的健康功能性食品包括例如片剂、胶囊剂、丸剂或液体剂的形式,并且可添加来源于本发明菌株的组合物的食品包括例如肉、香肠、面包、糖果、零食、面条、冰淇淋、乳制品、发酵乳、汤、离子饮料、饮料、酒精饮料、口香糖、茶和维生素复合物。
有益效果
本发明涉及新长双歧杆菌ATG-F5菌株。F5菌株是一种功能性有益菌,其免于抗生素抗性影响,通过对皮肤病原体痤疮丙酸杆菌的抗菌活性、对自由基的抗氧化作用、皮肤屏障改善和抗炎作用来改善皮肤健康,以及通过皮肤美白功能、皱纹改善功能和干燥皮肤缓解功能来增强皮肤美容。所述菌株可用于化妆品组合物或健康功能性食品中。
附图简述
图1显示了本发明的长双歧杆菌ATG-F5菌株的16S rRNA核苷酸序列;
图2显示了证实本发明的长双歧杆菌ATG-F5菌株针对两种引起痤疮的细菌(痤疮丙酸杆菌)的抗菌特性的结果;
图3显示了证实本发明的长双歧杆菌ATG-F5菌株的抗氧化功能的结果,其中抗氧化功能作为ABTS清除活性进行评价;
图4显示了证实与本发明的长双歧杆菌ATG-F5菌株的美白功能相关的提高黑素产生的作用(图4A)以及长双歧杆菌ATG-F5菌株对酪氨酸酶活性的抑制作用的结果(图4B);
图5显示了证实本发明的长双歧杆菌ATG-F5菌株的与皱纹改善功能相关的前胶原合成能力和MMP-1抑制效力的结果;
图6显示了证实提高本发明的长双歧杆菌ATG-F5菌株的与皮肤屏障增强功能相关的闭合蛋白(OCLN)和密封蛋白4(CLDN4)的基因表达的作用的结果;
图7显示了证实本发明的长双歧杆菌ATG-F5菌株提高与干燥皮肤缓解功能相关的透明质酸合酶2(HAS2)的基因表达的作用的结果;以及
图8显示了本发明的长双歧杆菌ATG-F5菌株的抗炎作用(白介素10/IL-10增加)。
具体实施方式
在下文中,将详细地描述本发明的一些优选实施方案。然而,本发明不限于本文中所描述的一些实施方案,并且可以以其他形式实施。提供这些实施方案以使得本公开内容将是透彻且完整的,并且这些实施方案将向本领域技术人员充分地传达本发明的精神。
<实施例1.用于功能性评价的微生物培养>
长双歧杆菌(以下简称F5)是从捐赠的新生儿粪便(2018年出生的婴儿,Daejeon,Korea)中分离的。为此,通过10倍连续稀释法用0.9%(w/v)盐水稀释新生儿粪便,将所得溶液涂抹在双歧杆菌选择性(BS,MBcell Seoul,Korea)固体培养基上,并在37℃下孵育细菌48小时。用显微镜观察BS培养基中产生的细菌菌落。通过观察,选择未表现出过氧化氢酶反应的细菌和芽孢杆菌(bacillus)型细菌并命名为ATG-F5菌株(以下简称为F5)。对于实验,首先将F5菌株在BL或MRS-cys琼脂固体培养基中培养,然后将纯分离的菌落接种在肉汤液体培养基中。微生物在37℃下培养过夜(即持续16至20小时)。
<实施例2.F5菌株特性的证实>
实施例2-1.F5菌株16S rRNA测序
应请求,Solgent Co.,Ltd.(位于Dajeon,Korea)对F5菌株进行了16S rRNA序列分析。用于测序分析的引物为:
27F(5′-AGA GTT TGA TCC TGGCTC AG-3′),518F(5′-CCA GCA GCC GCG GTA ATAC-3′),907R(5′-CCGTCA ATT CMT TTR AGT TT-3′),1492R(5′-GGT TAC CTT GTT ACG ACTT-3′),并且共读取核苷酸序列4次。使用国家生物技术信息中心(NCBI)的BLAST在线工具(https://blast.ncbi.nlm.nih.gov/Blast.cgi)分析通过每次读取的核苷酸序列比对得出的重叠群核苷酸序列。
将通过16S rRNA测序获得的SEQ ID NO:1的核苷酸序列(见图1)与NCBI的BLAST数据库进行比较。作为结果,长双歧杆菌菌株IRT与16S rRNA序列表现出99.9%的匹配,从而表明当通过分子分类学进行分类时,受试序列属于长双歧杆菌。
因此,在2019年3月18日,本发明的菌株保藏在韩国生物资源研究所,登录号为:KCTC13828BP。
实施例2-3.F5菌株的糖发酵模式
此外,进行了稍加修改的API50 CHL测试(BioMerieux,France),以确定糖发酵模式。简而言之,将L-半胱氨酸以0.5μg/ml的浓度添加至10mL API 50CHL培养基(BioMerieux,France),并将pH调节至约6.7。接下来,将纯培养的F5菌株混悬至OD600吸光度约为0.5,并将混悬培养溶液接种至API 50CH测试条的每个杯形托中,并在37℃下培养。在接种之后48小时和72小时时检查糖发酵结果。
如表1所示,使用API试剂盒的糖发酵评价的结果揭示,F5菌株降解L-阿拉伯糖、核糖、半乳糖、葡萄糖、果糖、甘露糖、甘露糖醇、山梨糖醇、甲基-αD-吡喃甘露糖苷、N-乙酰葡糖胺、苦杏仁苷、熊果苷、七叶苷、水杨苷、纤维二糖、麦芽糖、乳糖、蜜二糖、海藻糖、松三糖、棉子糖和松二糖。F5菌株也弱降解D-阿拉伯糖、甲基-αD-吡喃葡萄糖苷、龙胆二糖、L-岩藻糖和葡萄糖酸盐/酯。
[表1]
<实施例3.F5菌株抗菌活性的评价>
为了在与皮肤刺激相关的多种功能性评价项目中检查F5菌株的抗菌活性,通过盘式测试(disc test)对F5菌株针对总共两种类型的感染性或机会性细菌,具体是引起痤疮的细菌(痤疮丙酸杆菌,KCTC5012和KCTC3314)的抗菌活性进行评价。通过盘式测试,鉴定了透明区。将在BL肉汤(MBcell Seoul,Korea)板培养基中培养过夜的两种类型的细菌以约0.8的OD600吸光度各自混悬在1X磷酸盐缓冲盐水(PBS)中。每种混悬液用无菌棉签吸收,在琼脂培养基上铺开并干燥,其中将BL和MRS以1∶1的比例混合用于抗菌活性测试,并将8mm纸盘(Advantec,Japan)附着至干燥的琼脂培养基以用于测试。将在BL肉汤中培养18至20小时的F5细菌溶液以每个纸盘35μl接种在纸盘上,干燥约3分钟,并在37℃下孵育。测量孵育后产生的透明区直径,并从测量的透明区直径中减去8mm以获得最终值。
关于抗菌活性,使用痤疮丙酸杆菌(KCTC 5012和3314)菌株评价F5菌株的抗菌活性。F5菌株分别对KCTC5012和KCTC3314表现出9至10mm透明区的抗菌活性和5至6mm透明区的抗菌活性,对于痤疮丙酸杆菌菌株,它显示出高达10mm和6mm的透明区(见表2和图2)。
[表2]
<实施例4.F5菌株的抗生素抗性安全性>
使用包括氨苄青霉素、万古霉素、庆大霉素、卡那霉素、链霉素、克林霉素、红霉素、四环素和氯霉素在内的9种抗生素的E-测试条(BioMerieux,France)或MIC测试条(Liofilchem,Italy)进行抗生素测试,并获得最低抑菌浓度(minimum inhibitoryconcentration,MIC)值。
为此,将F5菌株混悬至OD600吸光度为约0.8,并使用无菌棉签涂抹在BL固体培养基上。将其上涂抹有F5菌株的固体培养基干燥约3分钟,安装E-测试条或MIC测试条,并在37℃下孵育24至48小时。
参考了欧洲食品安全局(EFSA)发布的指南的抗生素类型和可被认为安全的最低抑菌浓度(EFSA关于动物饲料中使用的添加剂和产品或物质的专家组,2012)。
如表3中所示,发现9种抗生素的敏感性测量结果与EFSA提供的指南的标准值一致。表3中每个数值的单位为μg/ml。
[表3]
<实施例5.F5菌株细胞裂解物的制备>
为了评价每个功能,制备了10倍浓缩的无细胞上清液(CFCS)。
为此,将F5菌株的培养基以4,000rpm离心25分钟,以便将培养基分离为F5菌株和培养基上清液。将上清液的pH调节至7至8,并使用0.2μm孔注射器过滤器(Satorious,Germany)进行过滤。将经过滤的溶液储存在-20℃下。将细胞混悬在1X PBS中,在旋涡的情况下洗涤,在4℃和4,000rpm下离心5分钟以去除剩余的培养基,然后再次混悬在1X PBS中。将溶菌酶(Sigma-Aldrich,Germany)以500μg/ml的浓度分配到混悬的细胞中,并将所得混悬液在37℃下的孵育箱中孵育2小时,然后将其引入声波仪(70W、30秒和30个周期)中以经历超声波细胞裂解。作为结果,制备了裂解物。接下来,在60℃下对裂解物进行45分钟的热处理,以杀伤裂解物中可能仍然存活的细菌。在水浴中进行60℃热处理,以使蛋白质变性最小化。
此后,将细胞裂解物储存在-20℃下并解冻以用于实验。该细胞裂解物用于所有后续实验。此外,测量该混悬液的含水量以在使用期间以相对于固体含量的不同浓度分配混悬液,并计算固体含量以用于稀释。
<实施例6.F5菌株的抗氧化功能的证实>
计划使用ABTS([2,2’-联氮基-双(3-乙基苯并噻唑啉-6-磺酸)],Sigma-Aldrich,Germany)进行自由基清除实验以测量F5菌株的抗氧化功能。这是使用了当ABTS的阳离子自由基通过与抗氧化材料反应被去除时颜色从蓝绿色变色为无色的原理的实验(参考Nam etal.,2015)。
在本实验中,使用ABTS溶液测量了F5菌株裂解物的抗氧化能力。为了准备ABTS实验,通过以1∶1的比例混合14mM ABTS储备溶液和4.9mM过硫酸钾来制备混合溶液,并进行过夜暗反应以使混合溶液变成蓝-绿色。之后,通过稀释溶液来制备工作溶液以在OD734波长下表现出约0.7的吸光度。接下来,将10%(v/v)的F5菌株裂解物样品添加至工作溶液,并在黑暗中反应10分钟。然后,在734nm的波长下测量所得溶液的吸光度,并通过以下公式计算每个测量值。
ABTS自由基清除活性(%)={1-(OD样品/OD对照)}×100
使用该方法,通过ABTS自由基去除率测量的长双歧杆菌ATG-F5裂解物的抗氧化活性在图3中显示。
参考图3,可以看出,自由基清除活性模式从菌株裂解物的500μg/ml浓度开始显著提高,并且抗氧化功能从1mg/ml开始显著增强。
<实施例7.使用F5菌株的酪氨酸酶抑制剂的黑素抑制和美白功能的评价>
为了评价美白功能,使用了小鼠模型的细胞系B16F10黑素瘤细胞。B16F10是用于评价美白功能的代表性细胞系。使用这样的机制来评价美白功能:在细胞信号传导途径中,L-酪氨酸从酪氨酸酶激活途径开始并在最后反应点提高黑素产生。
对于本评价,将B16F10在75cm2烧瓶(Thermo fisher,USA)中并在如下制备的培养基中培养:通过将10%(v/v)胎牛血清(Sigma-Aldrich, Germany)和1%(v/v)青霉素/链霉素混合物(Sigma-Aldrich,Germany)添加至杜氏改良eagle培养基(DMEM,Gibco,USA)。然后,当细胞生长约80%时,收获细胞并传代培养。将细胞以2X106个细胞/ml的浓度接种在6孔板中,然后在5%CO2条件下在孵育箱中孵育24小时。
之后,去除培养基并使用杜氏磷酸盐缓冲盐水(DPBS,Gibco,USA)洗涤板。然后,制备对照组(未经处理)和用α-黑素细胞刺激激素(α-MSH,Sigma-Aldrich,USA)处理的测试组。α-MSH提高黑素的产生,从而引起皮肤变色。对于测试组,以200nmol/ml浓度分配α-MSH和以50、100和500μg/ml浓度分配细胞裂解物。α-MSH和菌株细胞裂解物的处理时间为24小时,并且细胞在5%C2环境中在孵育箱中在37℃下孵育。
24小时之后,去除每个孔中的上清液,并使用DPBS进行洗涤。使用微管将添加有1%(v/v)triton X-100(Daejung,Korea)的500μl 0.1M PBS分配到洗涤过的孔中以获得细胞。将细胞离心,将上清液单独保存,并收集沉淀物。然后,使用收集的细胞沉淀物评价黑素的产生。将1N NaOH添加至纯的沉淀物。将细胞沉淀物在80℃水浴中融化约1小时,然后放入冰中冷却,并在405nm波长下进行吸光度测量。通过相对比较进行分析。
因此,当使用小鼠B16F10黑素瘤细胞通过α-MSH刺激酪氨酸酶活性诱导黑素产生时,根据F5菌株细胞裂解物的处理浓度,黑素产生降低,如图5A所示。特别地,即使在每种菌株处理的过程中用α-MSH刺激黑素的产生,黑素的产生也会进一步受到抑制,表明存在显著的美白作用。
收集细胞沉淀物之后,将单独收集的上清液用于酪氨酸酶抑制剂测量。将40μl的上清液和160μl以2μg/ml溶解于0.1M PBS中的二羟基苯丙氨酸(L-DOPA,Sigma,USA)在孵育箱中在37℃下混合并反应1小时。之后,使用ELISA读取仪(BioTek,USA)测量475nm下的吸光度,以显示酪氨酸酶抑制剂的相对抑制能力。
因此,如图4B的结果所示,证实了在上清液的处理之后获得了酪氨酸酶活性抑制,这可以证明黑素产生被抑制,因为酪氨酸酶活性被抑制。
<实施例8.通过F5菌株对MMP-1的抑制评价前胶原的提高和皱纹改善功能>
为了评价皱纹改善功能,使用了人模型细胞系人真皮成纤维细胞(Human DermalFibroblast,HDF)细胞系。使用补充有10%(v/v)胎牛血清(Sigma-Aldrich,Germany)和1%(v/v)青霉素/链霉素混合物(Sigma-Aldrich,Germany)USA)的杜氏改良eagle培养基(DMEM,Gibco,Germany)培养细胞。此外,对于细胞培养,使用了75cm2的烧瓶(Thermofisher,USA)。当细胞在75cm2的烧瓶中生长约80%时,收获细胞并传代培养。
将用于实验的细胞以2X105个细胞/ml的浓度接种在24孔板中,然后使用孵育箱在5%CO2条件下培养24小时。之后,去除培养基,并使用杜氏磷酸盐缓冲盐水(DPBS,Gibco,USA)洗涤板。制备对照组(未经处理组)和用10ng/ml的TNF-α(人肿瘤坏死因子α,Sigma-Aldrich,USA)处理的测试组。TNF-α提高了MMP-1以产生皮肤皱纹。
对于测试组,在各自的浓度下分配F5菌株的50、100和500μg/ml的细胞裂解物以及10ng/ml的TNF-α。将TNF-α和F5菌株的裂解物处理24小时,并使用孵育箱在5%CO2条件下在37℃下孵育。
24小时之后,收集每个孔中的上清液,并进行基质金属蛋白酶-1(MMP-1)和1型胶原蛋白的酶联免疫吸附试验(ELISA)。使用人Pro-MMP-1 Quantikine ELISA试剂盒(R&Dsystems,USA)对MMP-1进行相对定量,使用人前胶原I型C肽(PIP)EIA试剂盒(Takara,JAPAN)对1型胶原蛋白进行相对定量。
结果,如图5A所示,当用TNF-α处理浓度为500μg/ml的F5菌株细胞裂解物时,证实了前胶原提高。前胶原提高至可通过对照(未经处理组)表现出的水平。也就是说,与单独用TNF-α处理相比,该提高是相当大的。
特别地,如图5B所示,与单独的TNF-α处理组相比,在用F5菌株的细胞裂解物和TNF-α一起处理的测试组中,MMP-1的表达恢复到对照组(未经处理组)的水平。这证明,即使通过TNF-α攻击诱导了MMP-1的增加,F5菌株的细胞裂解物也会影响前胶原恢复(或减少)到与对照相对应的量,也证明F5菌株的裂解物具有显著增强1型胶原蛋白产生能力的作用。
<实施例9.F5菌株增强皮肤屏障或缓解皮肤干燥功能的评价>
使用人角质形成细胞HaCaT细胞系进行皮肤功能评价。以与上述相同的方式培养细胞。当细胞在75cm2的烧瓶中生长约80%时,收获细胞并传代培养。将细胞以5X106个细胞/ml的浓度接种在10cm圆形板上,然后使用孵育箱在5%CO2条件下培养24小时。此后,去除培养基,并用杜氏磷酸盐缓冲盐水(DPBS,Gibco,USA)洗涤板。将F5菌株的细胞裂解物以50、100和500μg/ml的浓度分配。对照组根本不处理。将F5菌株的裂解物处理24小时,并使用孵育箱在5%CO2条件下在37℃下培养。
接下来,洗涤板,并使用试剂(Ambion,USA)分离细胞的全部RNA。在对分离的RNA定量以合成cDNA之后,使用SuperScriptTM IV第一链合成系统(Invitrogen,USA)将其合成为cDNA。
之后,为了鉴定与皮肤相关的功能性基因,使用与皮肤屏障相关的闭合蛋白(OCLN)和密封蛋白4(CLDN4)的寡聚引物以及与皮肤干燥相关的透明质酸合酶2(HAS2)的寡聚引物进行定量实时PCR(qRT-PCR)。对于PCR分析,使用了快速实时PCR系统AppliedBiosystems 7500。在这种情况下,β-肌动蛋白被用作持家基因。实验中使用的引物列表在表4中显示。
[表4]
因此,如图6和7所示,当用浓度为50、100和500μg/ml的F5菌株的裂解物处理HaCaT细胞时,证实了与对照(未经处理组)相比,在浓度为500μg/ml的情况下,OCLN(闭合蛋白)提高了CLDN4(密封蛋白4)和透明质酸合酶2(HAS2)的表达。
因此,由于OCLN和CLDN4表达的提高,本发明的F5菌株的裂解物通过参与皮肤细胞之间的接触而具有显著增强皮肤屏障的功能。同样,可以证实F5菌株的细胞裂解物刺激透明质酸合酶2(HAS2)的表达以提高皮肤中透明质酸(HA)的产生,从而缓解皮肤干燥。
<实施例10.F5菌株抗炎功能的评价>
为了评价抗炎功能,检查了IL-10的表达水平。使用鼠巨噬细胞RAW264.7细胞系。关于培养方法,使用上述HDF细胞培养方法。当细胞在75cm2的烧瓶中生长至约80%时,收获细胞并传代培养。将细胞以1X106个细胞/ml的浓度接种在24孔板中,然后在5%CO2条件下在孵育箱中孵育24小时。首先,关于对照组,制备未经处理的细胞。关于阳性对照组,制备仅用LPS(脂多糖,Sigma-Aldrich,USA)处理的细胞。此外,还制备了单独用F5菌株细胞裂解物处理的组和用100μg/ml的F5菌株细胞裂解物和1μg/ml的LPS处理的组。24小时之后,收集每个孔中的上清液,并使用白介素10(IL-10,R&D,USA)进行相对定量。
结果在图8中显示。在由LPS诱导炎性应答的RAW264.7细胞的情况下,与当仅用LPS处理细胞时相比,当用LPS和F5菌株的裂解物二者处理细胞时,IL-10(抗炎细胞因子)的产生更显著地提高。
<化妆品制剂实施例1.紧肤剂-1的制备>
使用下表5的组成,根据常规方法制备含有长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物的紧肤剂(100g)。
[表5]
原材料 | 含量(g) |
长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物 | 3.0 |
丁二醇 | 2.0 |
丙二醇 | 2.0 |
聚氧乙烯(60)氢化蓖麻油 | 1.0 |
乙醇 | 10.0 |
三乙醇胺 | 0.1 |
防腐剂 | 痕量 |
着色剂 | 痕量 |
香料 | 痕量 |
纯化水 | 剩余部分 |
<化妆品制剂实施例2.保湿乳液的制备>
用70%(v/v)乙醇水溶液提取长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物之后,获得无溶剂浓缩物,并根据常规方法制备表6所示的含有浓缩物的保湿乳液(100g)。
[表6]
<化妆品制剂实施例3.营养霜的制备>
使用下表7的组成,根据常规方法制备含有长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物的营养霜(100g)。
[表7]
<化妆品制剂实施例4.精华液的制备>
使用下表8的组成,根据常规方法制备含有长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物的精华液(100g)。
[表8]
<化妆品制剂实施例5.粉底的制备>
使用下表9的组成,根据常规方法制备含有长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物的粉底(100g)。
[表9]
<化妆品制剂实施例6.洗发香波的制备>
使用下表10的组成,根据常规方法制备含有长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物的洗发香波(100g)。
[表10]
原材料 | 含量(g) |
长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物 | 3.0 |
花生四烯葡糖苷 | 4.5 |
乙醇 | 2.0 |
丁二醇 | 2.0 |
柠檬酸 | 0.1 |
苯氧乙醇 | 0.02 |
纯化水 | 剩余部分 |
<制剂实施例1.药物制剂>
制剂实施例1-1.片剂的制备
将200g的本发明的长双歧杆菌ATG-F5菌株细胞裂解物的冻干产物与175.9g乳糖、180g马铃薯淀粉和32g胶体硅酸混合。在向该混合物添加10%(w/v)明胶溶液之后,将所得产物研磨并使其通过14目筛。干燥剩余固体,并向其添加160g马铃薯淀粉、50g滑石和5g硬脂酸镁。将所得混合物制备成抗炎片剂。
制剂实施例1-2.软膏剂的制备
将1g本发明的长双歧杆菌ATG-F5菌株的细胞裂解物的冻干产物与99g矿脂混合以制备抗炎软膏剂。
<制剂实施例2.食物产品的制备>
制剂实施例2-1.烹饪调味品的制备
通过将本发明的长双歧杆菌菌株的细胞裂解物的冻干粉末以按重量计1%的量添加到烹饪调味品中来制备健康功能性烹饪调味品。
制剂实施例2-2.乳制品的制备
将本发明的长双歧杆菌菌株的细胞裂解物的冻干粉末以按重量计0.1%的量添加到乳中,并使用乳制备多种乳制品,例如黄油和冰淇淋。
制剂实施例2-5.蔬菜汁的制备
通过向1,000ml番茄汁或胡萝卜汁中添加0.5g本发明的长双歧杆菌菌株的细胞裂解物冻干粉末来制备健康功能性蔬菜汁。
制剂实施例2-6.果汁的制备
通过向1,000ml苹果汁或葡萄汁中添加0.1g本发明的长双歧杆菌菌株的细胞裂解物冻干粉末来制备健康功能性果汁。
[保藏机构]
机构名称:韩国典型培养物保藏中心
登记号:KCTC13828BP
保藏时间:2019年3月18日
<110> AtoGen Co., Ltd
<120> 新长双歧杆菌菌株或包含所述菌株的化妆品组合物
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 1398
<212> DNA
<213> 未知
<220>
<223> 长双歧杆菌(Bifidobacterium longum)
<400> 1
catgcagtcg aacgggatcc atcaagcttg cttggtggtg agagtggcga acgggtgagt 60
aatgcgtgac cgacctgccc catacaccgg aatagctcct ggaaacgggt ggtaatgccg 120
gatgttccag ttgatcgcat ggtcttctgg gaaagctttc gcggtatggg atggggtcgc 180
gtcctatcag cttgacggcg gggtaacggc ccaccgtggc ttcgacgggt agccggcctg 240
agagggcgac cggccacatt gggactgaga tacggcccag actcctacgg gaggcagcag 300
tggggaatat tgcacaatgg gcgcaagcct gatgcagcga cgccgcgtga gggatggagg 360
ccttcgggtt gtaaacctct tttatcgggg agcaagcgtg agtgagttta cccgttgaat 420
aagcaccggc taactacgtg ccagcagccg cggtaatacg tagggtgcaa gcgttatccg 480
gaattattgg gcgtaaaggg ctcgtaggcg gttcgtcgcg tccggtgtga aagtccatcg 540
cttaacggtg gatccgcgcc gggtacgggc gggcttgagt gcggtagggg agactggaat 600
tcccggtgta acggtggaat gtgtagatat cgggaagaac accaatggcg aaggcaggtc 660
tctgggccgt tactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc 720
tggtagtcca cgccgtaaac ggtggatgct ggatgtgggg cccgttccac gggttccgtg 780
tcggagctaa cgcgttaagc atcccgcctg gggagtacgg ccgcaaggct aaaactcaaa 840
gaaattgacg ggggcccgca caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa 900
gaaccttacc tgggcttgac atgttcccga cgatcccaga gatggggttt cccttcgggg 960
cgggttcaca ggtggtgcat ggtcgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1020
ccgcaacgag cgcaaccctc gccccgtgtt gccagcggat tgtgccggga actcacgggg 1080
gaccgccggg gttaactcgg aggaaggtgg ggatgacgtc agatcatcat gccccttacg 1140
tccagggctt cacgcatgct acaatggccg gtacaacggg atgcgacgcg gcgacgcgga 1200
gcggatccct gaaaaccggt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagg 1260
cggagtcgct agtaatcgcg aatcagcaac gtcgcggtga atgcgttccc gggccttgta 1320
cacaccgccc gtcaagtcat gaaagtgggc agcacccgaa gccggtggcc taaccccttg 1380
tgggatggag ccgtctaa 1398
Claims (13)
1.新长双歧杆菌ATG-F5(Bifidobacterium longum ATG-F5,登记号.KCTC13828BP)菌株,其具有皮肤屏障增强功能。
2.权利要求1所述的长双歧杆菌ATG-F5菌株,其中所述菌株具有干燥皮肤缓解功能。
3.权利要求1所述的长双歧杆菌ATG-F5菌株,其中所述菌株具有抗痤疮功能。
4.权利要求1所述的长双歧杆菌ATG-F5菌株,其中所述菌株具有抗氧化功能。
5.权利要求1所述的长双歧杆菌ATG-F5菌株,其中所述菌株具有皮肤美白功能。
6.权利要求1所述的长双歧杆菌ATG-F5菌株,其中所述菌株具有皱纹改善功能。
7.权利要求1所述的长双歧杆菌ATG-F5菌株,其中所述菌株具有抗炎功能。
8.包含权利要求1所述菌株的化妆品组合物,其具有功能例如皮肤屏障增强、干燥皮肤缓解、抗炎、抗痤疮、抗氧化和皱纹改善。
9.权利要求8所述的化妆品组合物,其中所述化妆品组合物的制剂选自皮肤洗剂、皮肤软化剂、紧肤剂、收敛剂、乳液洗剂、保湿乳液、滋养乳液、按摩霜、滋养霜、保湿霜、护手霜、护足霜、颈霜、粉底、精华液、剥撕式面膜、皂、清洁泡沫、清洁洗剂、清洁霜、身体洗剂、洗发香波、毛发处理剂、护发素和身体清洁剂。
10.包含权利要求1所述菌株的健康功能性食品,其具有功能例如皮肤屏障增强、干燥皮肤缓解、抗炎、抗痤疮、抗氧化和皱纹改善。
11.权利要求10所述的健康功能性食品,其中所述健康功能性食品选自肉、香肠、面包、糖果、零食、面条、冰淇淋、乳制品、发酵乳、汤、离子饮料、饮料、酒精饮料、口香糖、茶和维生素复合物。
12.用于预防或治疗选自干燥皮肤、痤疮和炎性疾病的疾病的组合物,其包含权利要求1所述的菌株。
13.权利要求12所述的用于预防或治疗选自干燥皮肤、痤疮和炎性疾病的疾病的组合物,其中所述炎性疾病选自炎性肠病、炎性胶原血管病、肾小球肾炎、炎性皮肤病、结节病、视网膜炎、胃炎、肝炎、肠炎、关节炎、扁桃体炎、咽痛、支气管炎、肺炎、胰腺炎、血液中毒、膀胱炎、肾炎和神经炎。
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