CN116144521A - 沙克乳酸杆菌amp5104菌株及其用途 - Google Patents
沙克乳酸杆菌amp5104菌株及其用途 Download PDFInfo
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- CN116144521A CN116144521A CN202210320548.9A CN202210320548A CN116144521A CN 116144521 A CN116144521 A CN 116144521A CN 202210320548 A CN202210320548 A CN 202210320548A CN 116144521 A CN116144521 A CN 116144521A
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Abstract
本发明涉及一种沙克乳酸杆菌AMP5104菌株及其用途,所述菌株可有效地用于预防、改善或治疗皮肤相关状况或炎症相关状况。
Description
技术领域
本发明涉及一种沙克乳酸杆菌AMP5104菌株及其用途。
背景技术
皮肤是身体直接暴露于外部环境的一部分,用于保护身体免受外部刺激。人体皮肤组织中栖息着无数微生物群落(microflora),皮肤微生物具有有益微生物和有害微生物的平衡,从而增强皮肤免疫力。然而,当有害微生物因外部刺激、压力、激素变化而大量增殖以导致破坏皮肤微生物的平衡时,皮肤屏障的减弱会引起由有害微生物导致的原发性炎症反应,并有害微生物的超抗原等的有毒代谢产物会诱发继发性炎症反应,如发痒、加剧炎症反应等。由上述原因引起的皮肤问题,不仅在美观上有问题,炎症反应过程中产生的物质也会伴随皮肤色素沉着,促进皮肤弹力纤维分解,导致皮肤老化。
另一方面,益生菌(Probiotics)是韩国传统食品泡菜发挥功效的原因,所以对益生菌的研究十分活跃。尤其,由于乳酸菌分泌有机酸,因此具有优异的抗菌活性,通过抗菌活性维持微生物菌群的良好平衡。皮肤通过这些微生物菌群的平衡和皮肤免疫细胞因子的平衡来实现皮肤免疫。
因此,需要开发一种在皮肤免疫平衡中起作用的新型微生物菌株。
发明内容
本发明一方面提供一种属于乳杆菌属(Lactobacillus sp.)的沙克乳酸杆菌(Lactobacillus sakei)菌株。
本发明另一方面提供一种所述菌株的干物质、破碎物、培养液、培养液的提取物或裂解物。
本发明再一方面提供一种组合物,其包括所述菌株、其干物质、破碎物、培养液、培养液的提取物、裂解物或其混合物。
本发明一方面提供一种沙克乳酸杆菌(Lactobacillus sakei)菌株。
所述沙克乳酸杆菌(Lactobacillus sakei)菌株可以是以保藏号KCTC14319BP保藏的菌株。
所述沙克乳酸杆菌(Lactobacillus sakei)菌株可以是包括序列号1的16s rRNA的菌株。
所述菌株可以具有皮肤美容改善效果,例如抑制皮肤老化、皮肤抗菌、增强皮肤屏障、抑制皮肤炎症或调节皮肤免疫平衡习效果。
所述菌株可以增加β-防御素(defensins)(HBD)-2、抗菌肽(cathelicidin)(LL-37)和RNase7的表达。另外,所述菌株可以增加血红素氧合酶-1(Heme oxygenase-1,HO-1)、白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的表达。此外,所述菌株可以维持TNF-α和白介素-10(IL-10)表达的免疫稳态(Immunological homeostasis)。因此,所述菌株具有通过抗炎、抗菌、抗氧化作用和皮肤角质细胞保护作用来维持皮肤免疫平衡的功能。
本发明另一方面提供一种所述菌株的干物质、破碎物、培养液或培养液的提取物。
所述菌株的具体细节如上所述。
在本说明书中,术语“培养液”可以与“培养上清液”、“条件培养液”或“条件培养基”互换使用,并且可以指一种完整的培养基,其包括通过在可以提供营养素以使沙克乳酸杆菌菌株能够在体外生长和存活的培养基中培养所述菌株一定时间而获得的所述菌株、其代谢产物、额外营养素等。另外,所述培养液可以指从通过培养菌株而获得的菌体培养液中除去菌体的培养液。另一方面,将从所述培养液中除去了菌体的液体也称为“上清液”,这可以通过如下方法获得:将培养液静置一定时间,仅吸收除沉降到下层的部分以外的上层液体,或者通过过滤来除去菌体,或者通过离心分离培养液来除去下层的沉淀物并仅吸收上层液体。所述“菌体”是指本发明的菌株本身,其包括通过从皮肤样品等分离而选择的菌株本身,或通过培养所述菌株而从培养液分离出的菌株。所述菌体可以通过离心分离培养液并取出沉降到下层的部分来获得,或者由于菌体在重力作用下沉降到培养液的下层,因此可以将其静置一定时间后除去上层液体来获得。
所述培养液可以包括通过培养菌株而获得的培养液本身、其浓缩物或其冻结干燥物,也可以包括通过从培养液中去除菌株而获得的培养上清液、其浓缩物或冻结干燥物。
所述培养液可以通过将沙克乳酸杆菌菌株在培养基(例如,液态的乳酸杆菌(Lactobacilli)MRS肉汤(broth)培养基,乳酸菌培养基除外)中在高于10℃或低于40℃的任意温度下培养一定时间,例如4小时至72小时而获得。
在一具体实施例中,菌株的培养上清液可以通过离心分离或过滤菌株培养液来去除菌株的步骤而获得。
在另一实施例中,浓缩物可以通过将所述菌株培养液本身或在使用离心分离器或过滤器过滤所述培养液后获得的上清液浓缩来获得。
用于培养所述沙克乳酸杆菌菌株的培养基和培养条件可以由本领域普通技术人员适当选择或变更使用。
在本说明书中,术语“破碎物”可以是指通过化学力或物理学力破碎菌株自身的细胞壁而获得的产物,并可与“裂解物”互换使用。
在本说明书中,术语“培养液提取物”是指从所述培养液或其浓缩物中提取的,并且可以包括提取液、提取液的稀释液或浓缩液、通过干燥提取液而获得的干燥物,或这些粗提纯产物或提纯产物、通过将其分馏而获得的馏分。
本发明再一方面提供一种所述菌株、所述菌株的干物质、破碎物、培养液、培养液的提取物或裂解物的用途。具体地,提供一种组合物,其包括沙克乳酸杆菌菌株、其干物质、破碎物、培养液、其培养液的提取物或裂解物。
所述菌株的用途可以包括改善皮肤状况、改善皮肤美容、预防、改善或治疗皮肤疾病、预防、改善或治疗皮肤炎性疾病等。
所述改善皮肤状况或改善皮肤美容可以是抑制或改善皮肤老化,或增强皮肤屏障。
在本说明书中,术语“皮肤老化”是指随着年龄的增长皮肤上出现的有形和无形的变化的统称,例如,表皮厚度变薄、真皮细胞数量或血管数量、恢复DNA损伤的能力、细胞更换周期、伤口愈合、皮肤屏障功能、表皮的水分保持、汗液分泌、皮脂分泌、维生素D产生、物理损伤防护、化学物质去除能力、免疫反应、感觉功能和体温调节的减少。
所述菌株或其培养液可以用于改善由外源性因素或内源性因素引起的皮肤老化。所述外源性因素是指各种外在因素如紫外线(光),所述内源性因素是指主要由时间过去而引起的因素,也被称为时间因素。即,所述皮肤老化具体地不仅包括由紫外线、公害、香烟烟雾、化学物质等外部刺激引起的过早老化症状,还包括因年龄增长而减少皮肤细胞增殖所发生的自然老化现象,这是一个包含皱纹、弹性降低、皮肤耷拉和干燥现象等一切的概念。另外,皱纹包括由内源性因素的和外源性因素的的变化引起的刺激改变组成皮肤组织的成分而引起的皱纹。
所述老化可以是由紫外线、活性氧等引起的老化。在本说明书中,术语“光老化(Photoaging)”是指由外部环境因素引起的现象,紫外线是最典型的因素。紫外线导致蛋白酶的活化、基质蛋白质的链断裂以及如异常交联等的生物组分的损害,这种机制的重复导致在外观上明显的皮肤老化。所述“活性氧”导致皮肤细胞和组织损伤,通过破坏由抗氧化酶和非酶抗氧化剂组成的皮肤抗氧化防御网以使氧化剂和抗氧化剂之间的平衡转向氧化状态,从而对生物体组分造成损害,例如脂质过氧化、蛋白质氧化、破坏细胞间质成分的蛋白酶的活化、作为弹性纤维的胶原蛋白和弹性蛋白的链断裂和异常交联、透明质酸链断裂、促进黑色素生成反应和DNA氧化。另外,其引起皮肤发炎并抑制皮肤的免疫功能,从而增加细菌感染或癌症发生率。结果,加速以弹性减少、皱纹和瑕疵、雀斑等为特征的皮肤老化。
所述“增强皮肤屏障”可以指增强位于皮肤最外侧并防止水分和营养流失的皮肤屏障的功能的任何作用。
术语“抗炎症”可以与“抑制或改善炎症”互换使用,并且可以指减轻免疫反应以抑制NO产生的任何作用。
所述“皮肤疾病”可以是由皮肤屏障功能损伤引起的疾病、皮肤老化、皮肤伤口、皮肤疤痕或皮肤炎症。术语“预防”包括抑制疾病的发生。术语“治疗”包括抑制、减轻或消除疾病的发生。
所述皮肤屏障功能损伤可以指由于皮肤屏障功能的降低或损伤而皮肤上出现的所有变化。例如,可以包括皮肤皱纹增加、干燥、皮肤炎、特应性皮肤炎、过敏性皮肤炎、痤疮等。
所述皮肤炎性疾病可以为选自皮肤伤口、皮炎、特应性皮炎、瘙痒症、湿疹性皮肤病、干性湿疹、红斑、荨麻疹、牛皮癣、轻度皮疹和痤疮中的任一种。
在一实施例中,确认了所述菌株和乳杆菌属戊糖乳杆菌的裂解物混合液具有显着的皮肤抗菌活性、解毒和改善老化效果、平衡皮肤免疫效果。因此在另一具体实施例中,所述菌株可以与属于具有皮肤改善效果的乳杆菌的另一种菌株,即第二乳杆菌属菌株一起使用以显示协同作用。所述第二乳杆菌属菌株例如可以包括戊糖乳杆菌(Lactobacilluspentosus)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、嗜酸乳杆菌(Lactobacillusacidophilus)、保加利亚乳杆菌(Lactobacillus bulgaricus)、植物乳杆菌(Lactobacillus plantarum)、德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisubsp.Bulgaricus)、发酵乳杆菌(Lactobacillus fermentum)、短乳杆菌(Lactobacillusbrevis)、副干酪乳杆菌(Lactobacillus paracasei)、干酪乳杆菌(Lactobacilluscasei)、旧金山乳杆菌(Lactobacillus sanfranciscensis)、加氏乳杆菌(Lactobacillusgasseri)、泡菜乳杆菌(Lactobacillus kimchi)、罗伊氏乳杆菌(Lactobacillus reuteri)等,但不限于此。
基于所述组合物的总重量,所述组合物可以包括0.001%至80%,例如,0.01%至60%、0.01%至40%、0.01%至30%、0.01%至20%、0.01%至10%、0.01%至5%、0.05%至60%、0.05%至40%、0.05%至30%、0.05%至20%、0.05%至10%、0.05%至5%、0.1%至60%、0.1%至40%、0.1%至30%、0.1%至20%、0.1%至10%、0.1%至5%的菌株、其干物质、破碎物、培养液、培养液的提取物或裂解物。
术语“作为有效成分包括”是指本说明书中的菌株、其干物质、破碎物、培养液、培养液的提取物或裂解物以能够显示出上述效果的程度添加,并且包括为了递送和稳定药物将各种成分作为辅助成分添加并配制成(formulation)各种形态的含义。
所述组合物可以为化妆品组合物。
所述化妆品组合物可以是本领域通常制备的剂型,例如溶液、悬浮液、乳浊液、糊剂、凝胶、霜剂、乳液、粉末、香皂、含表面活性剂清洁剂、油、粉末粉底、乳浊液粉底、蜡粉底、喷雾剂、杆状等,但不限于此。更具体地,可以制备成如下剂型:柔软化妆水或营养化妆水等的化妆水;面部乳液、身体乳液、头皮精华素、头发乳液等的乳液;营养霜、保湿霜、眼霜等的乳霜;精华素;化妆软膏;喷雾剂;凝胶;面膜;防晒霜;妆前底乳;液体型、固体型或喷雾型等的粉底;和粉剂等。此时,所述化妆品组合物可以含有赋形剂,其包括乳化剂、皂酸、溶剂、着色剂、保存剂、抗氧化剂、消泡剂、抗菌剂、抗再沉积剂、酶、植物油或矿物油、脂肪、荧光物质、杀真菌剂、向水性诱导物质、保湿剂、芳香剂、芳香剂载体、蛋白质、有机硅、增溶剂、糖衍生物、防晒剂、维生素、植物提取物和蜡等,但不限于此。
所述化妆品组合物中包括的成分除了作为活性成分的所述组合物外,还可以包括化妆品组合物中常用的成分,例如稳定剂、增溶剂、维生素、颜料及香料等的常规助剂和载体。
另外,所述组合物可以为皮肤外用剂组合物。
所述皮肤外用剂可以为乳膏、凝胶、软膏、皮肤乳化剂、皮肤悬浮液、经皮传递性贴片、含药绷带、乳液或其组合。所述皮肤外用剂可以根据需要适当混合通常用作化妆品或药物等的皮肤外用剂的成分,例如,水性成分、油性成分、粉末成分、醇类、保湿剂、增稠剂、紫外线吸收剂、美白剂、防腐剂、抗氧化剂、表面活性剂、香料、着色剂、各种皮肤营养剂或其组合。所述皮肤外用剂可以适当混合乙二胺四乙酸二钠、乙二胺四乙酸三钠、柠檬酸钠、多聚磷酸钠、偏磷酸钠、葡萄糖酸等的金属螯合剂、咖啡因、单宁、戊脉安、甘草提取物、光甘草定、大蓟果实的热水提取物、各种生药、生育酚乙酸酯、甘草酸、氨甲环酸及其衍生物或其盐等的药剂、维生素C、抗坏血酸磷酸酯镁、抗坏血酸葡糖苷、熊果苷、曲酸、葡萄糖、果糖、海藻糖等的糖类等,但不限于此。
在另一具体实施例中,所述组合物可以为药物组合物。
所述药物组合物还可以包括药学上可接受的稀释剂或载体。所述稀释剂可以是乳糖、玉米淀粉、大豆油、微晶纤维素或甘露醇,助流剂可以是硬脂酸镁、滑石粉或其组合。所述载体可以是赋形剂、崩解剂、结合剂、助流剂或其组合。所述赋形剂可以是微晶纤维素、乳糖、低取代羟基纤维素或其组合。所述崩解剂可以是羧甲基纤维素钙、羟基乙酸淀粉钠、无水磷酸一氢钙或其组合。所述结合剂可以是聚乙烯吡咯烷酮、低取代羟丙基纤维素、羟丙基纤维素或其组合。所述助流剂可以是硬脂酸镁、二氧化硅、滑石粉或其组合。
所述药物组合物可以配制成口服或非口服剂型。口服剂型可以是颗粒剂、散剂、溶液剂、片剂、胶囊剂、干糖浆剂或其组合。非口服剂型可以是注射剂,但不限于此。
所述组合物可以是食品组合物或保健功能食品组合物。
所述食品组合物或保健功能食品组合物可以通过单独使用所述菌株或其培养液,或者将其与其他食品或食品成分组合来制备,并且可以根据常规方法适当使用。活性成分的混合量可根据使用目的(预防、保健或治疗)适当确定。通常,当制备食品或饮料时,基于原料,本说明书的组合物可以以15重量份或更少的量添加。所述保健功能食品的种类没有特别限制。在保健功能食品的种类中,饮料组合物可以像常规饮料一样含有各种甜味剂或天然碳水化合物作为附加成分。所述天然碳水化合物包括:葡萄糖、果糖等的单糖;麦芽糖、蔗糖等的二糖;糊精、环糊精等的多糖;和木糖醇、山梨糖醇、赤藓糖醇等糖醇。所述调味剂可以包括:牛磺酸、甜菊提取物等的天然调味剂;或糖精、阿斯巴甜等的合成调味剂等。所述保健功能食品组合物还可以包括营养剂、维生素、电解质、调味剂、着色剂、果胶酸及其盐、海藻酸及其盐、有机酸、保护性胶体增稠剂、pH调节剂、稳定剂、防腐剂、甘油、酒精、碳酸饮料中使用的碳酸化剂或其组合,但不限于此。所述保健功能食品组合物还可以含有用于制备天然果汁、果汁饮料、蔬菜饮料的果肉,或者其组合。
另外,本发明另一方面提供一种预防、改善或治疗个体的状况的方法,其包括:对有需要的个体进行处理或施用有效量的所述组合物。
所述个体的状况可以是与皮肤相关状况或与炎症相关状况。
术语“施用”、“引入”和“植入”可互换使用并且可以是指通过一种方法或途径将根据一具体实施例的组合物放置到受试者体内,该方法或途径导致根据一具体实施例的组合物至少部分地定位到所需位点。
施用可以通过本领域已知的方法施用。施用可以通过任何方式直接施用于个体,例如静脉内、肌肉内、口服、透皮(transdermal)、粘膜、鼻内(intranasal)、气管内(intratracheal)或皮下施用。所述施用可以是全身或局部的。
所述个体可以是哺乳动物,例如人、牛、马、猪、狗、绵羊、山羊或猫。所述个体可以是需要改善皮肤美容效果的个体,例如皮肤保湿、增强皮肤屏障、抑制皮肤炎症和改善皮肤皱纹。
所述施用可以为每天将根据本发明一具体实施例的组合物按照以下量施用于个体:0.1mg至1000mg,例如0.1mg至500mg、0.1mg至100mg、0.1mg至50mg、0.1mg至25mg、1mg至1000mg、1mg至500mg、1mg至100mg、1mg至50mg、1mg至25mg、5mg至1000mg、5mg至500mg、5mg至100mg、5mg至50mg、5mg至25mg、10mg至100 0mg、10mg至500mg、10mg至100mg、10mg至50mg或10mg至25mg。然而,施用量可以根据诸如制剂方法、施用方法、患者的年龄、体重、性别、病理状况、饮食、施用时间、施用途径、排泄速度及反应灵敏性等的因素来不同地处方,本领域技术人员将理解,可以考虑这些因素以适当调整施用量。施用次数可以为每天1次或在临床上可接受的副作用范围内2次以上,关于施用部位,可以在一处或两处以上施用,并且每天或每2天至5天为间隔的总施用天数可以是在一次治疗时施用1天至30天。如有需要,可在适当的时间后重复相同的治疗。对于除人以外的动物,可以以每kg施用量与人相同的施用量来施用,或者以目标动物与人的器官(心脏等)的体积比(例如,平均值)等换算所述施用量的量来施用。
根据一方面的新型沙克乳酸杆菌菌株,可以有效地用于预防、改善或治疗皮肤相关状况或炎症相关状况。
附图说明
图1示出了维持皮肤免疫平衡的因素。
具体实施方式
下面将提供优选实施例以帮助理解本发明。然而,提供以下实施例仅是为了更容易理解本发明,并且本发明的内容不受以下实施例的限制。
【实施例】
实施例1、菌株的分离
将10g济州岛传统泡菜放入100ml无菌水中,在37℃静置1小时,然后用生理盐水稀释。接下来,对于每个稀释步骤,将100μl稀释液分配到乳酸杆菌(Lactobacilli)MRS肉汤(broth)固体培养基(BD公司,288130)中并平铺,然后将所平铺的固体培养基在37℃下培养2天。接下来,在将所形成的菌落等分后,使用已知引物27F、338F、518F、785F、518R、805R、907R和1492R进行16s rRNA基因序列鉴定。PCR扩增以95℃1分钟、55℃1分钟、75℃1分30秒的30个循环进行,最后在72℃下处理8分钟,然后在4℃下保存。在PCR反应完成后,使用ABI-3730XL(ABI,美国)确定所分离培养的物种的DNA序列。将所分离培养的微生物菌落中确定的16S rRNA区域的碱基序列与使用美国国家生物信息中心(NCBI,National Center forBiotechnology Information)中提供的BLAST程序注册的其他菌株进行比较和分析。仅选择使用同源性为97%或更小的具有新颖性的物种,其中选择同源性为94%或更小的新型微生物(以下简称“AMP5104”)。AMP5104具有序列号1(互补DNA)的16s rRNA序列。然后,所述菌株于2020年9月23日保藏在韩国生命工学研究院生物资源中心,其保藏号为KCTC14319BP。
实施例2、沙克乳酸杆菌AMP5104裂解物
制备所述实施例1中鉴定的沙克乳酸杆菌AMP5104菌株的裂解物。具体地,将实施例1的菌株接种到乳酸杆菌(Lactobacilli)MRS肉汤(broth)(BD公司,288130)中,在35℃至37℃下培养1天至2天,然后用匀浆器(homogenizer)破碎所述菌株的细胞壁。
实施例3、沙克乳酸杆菌AMP5104培养液
制备从所述实施例1中鉴定的沙克乳酸杆菌AMP5104菌株的培养液中去除菌株的培养液。具体地,将实施例1的菌株接种到乳酸杆菌MRS肉汤(BD公司,288130)中,在35℃至37℃下培养1天至2天,然后使用离心分离器从所述培养液中除去菌株。
实施例4、沙克乳酸杆菌AMP5104与戊糖乳杆菌HB-8023的混合裂解物
除了使用戊糖乳杆菌HB-8023菌株(KCTC13322BP)之外,以与所述实施例2相同的方法制备菌株裂解物。然后,将其与所述实施例2中制备的沙克乳酸杆菌AMP5104裂解物以1:1的比例混合。
【比较例】
比较例1、沙克乳酸杆菌L35裂解物
除了使用从与实施例1相同的济州岛传统泡菜中分离的沙克乳酸杆菌L35菌株之外,以与所述实施例2相同的方法制备菌株裂解物。
比较例2、沙克乳酸杆菌L42裂解物
除了使用从与实施例1相同的济州岛传统泡菜中分离的沙克乳酸杆菌L42菌株之外,以与所述实施例2相同的方法制备菌株裂解物。
【实验例】
细胞毒性和细胞增殖
为了确认根据一方面的沙克乳酸杆菌菌株的细胞活性,进行了CCK-8(wst-8)测定。具体地,在将人角质形成细胞细胞系(Human HaCaT)以5×104/孔分配到48孔板中后,在各种浓度(0.3%、0.6%、2.5%、5%和10%)下处理所述实施例2和比较例1至比较例2。然后,使用细胞计数试剂盒-8(cell counting kit-8,Munich,德国)评估细胞毒性,并使用分光光度计(spectrophotometer)在450nm处测量吸光度。
【表1】
结果,如表1所示,与比较例1至比较例2相比,可以确认实施例2在所有浓度下具有优异的细胞活性。
即,根据一方面的沙克乳酸杆菌AMP5104菌株具有最优异的细胞活性能力,并且没有细胞毒性,从而其可以用作化妆品、药物、食品等。
皮肤抗菌活性和皮肤屏障的改善
确认了根据一方面的沙克乳酸杆菌菌株的皮肤抗菌活性和皮肤屏障改善效果。具体地,在将人角质形成细胞细胞系(Human HaCaT)以7.5×105分配到6孔板中后,在37℃和5%CO2条件下的培养箱中培养24小时。接下来,在除去培养基并更换为未添加胎牛血清(FBS)的DMEM:F12(3:1)培养基后,用所述实施例2至实施例4和比较例1至比较例2处理,然后进一步培养24小时。接下来,在使用RNeasy微型试剂盒(Hilden,德国)从每个样品的细胞中分离RNA后,使用纳米微滴(nanodrop)在260nm处对RNA进行定量,并分别使用2μg RNA在扩增仪中合成cDNA(RT SuperMix试剂盒,美国)。以所合成的cDNA为模板,针对靶基因抗菌肽(cathelicidin)(LL-37)、RNase7和β-防御素(HBD)-2,将SYBR绿(SYBRGreen,Luna Universal qPCR Master Mix,New England BioLabs,美国)与引物和cDNA一起添加,并在实时(real-time)PCR仪(BMS公司MIC2仪器,昆士兰州)中进行实时聚合酶链反应。最后通过校正RPL13A基因来分析基因的表达水平,其结果见下表2。
【表2】
结果,如表2所示,可以确认实施例2至实施例4中抗菌肽的表达显着高于对比例1至对比例2。尤其,在与戊糖乳杆菌HB-8023菌株的混合裂解物中,抗菌肽的表达水平显著高。
即,根据一方面的沙克乳酸杆菌菌株增加抗菌肽如LL37、RNase7和HBD-2在角质形成细胞中的表达,从而表现出皮肤抗菌活性以预防、治疗或改善炎性皮肤疾病如特应性皮炎等,并且可以改善由所述炎性皮肤疾病引起的皮肤屏障功能。
皮肤解毒和老化
确认了根据一方面的沙克乳酸杆菌菌株的皮肤解毒和老化改善效果。具体地,除了使用血红素氧合酶-1(HO-1)引物外,以与上述相同的方法分析抗氧化酶的表达水平,其结果如下表3所示。
【表3】
结果,如表3所示,可以确认与比较例1至比较例2相比,实施例2至实施例4的HO-1表达显着增加。
即,根据一方面的沙克乳酸杆菌菌株具有通过去除因紫外线、活性氧等而积累的毒素来改善皮肤老化的效果。
皮肤炎症
确认了根据一方面的沙克乳酸杆菌菌株的皮肤炎症减轻和免疫增强效果。具体地,除了使用肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)和白介素-1β(IL-1β)引物外,以与上述相同的方法分析细胞因子的表达水平,其结果如下表4所示。
【表4】
由结果可以确认,如表4所示,对实施例2至实施例4而言,与比较例1至比较例2相比,炎性细胞因子的表达显着增加,并且LDH的表达显着减少。
即,根据一方面的沙克乳酸杆菌菌株不仅可以保护皮肤而不损伤皮肤细胞,还可以预防、治疗和改善皮肤炎性疾病。
皮肤免疫平衡
确认了根据一方面的沙克乳酸杆菌菌株的皮肤免疫平衡效果。具体地,除了使用肿瘤坏死因子-α(TNF-α)和白介素-10(IL-10)引物外,以与上述相同的方法分析细胞因子的表达水平,其结果如下表5所示。
【表5】
由结果可以确认,如表5所示,对实施例2至实施例4而言,与比较例1至比较例2相比,促炎性细胞因子(pro-inflammatory)肿瘤坏死因子-α和抗炎性细胞因子(anti-inflammatory)白介素-10的表达显着增加。尤其,针对抗炎性细胞因子的促炎性细胞因子的表达比率(肿瘤坏死因子-α/白介素-10)显示接近1的值,从而可以确认与比较例1至比较例2相比,免疫平衡(Immunological homeostasis)的维持能力很优异。
即,根据一方面的沙克乳酸杆菌菌株可以维持免疫平衡状态,从而有效预防、治疗和改善过敏性皮肤疾病,例如特应性皮炎、荨麻疹、瘙痒症、皮炎等。
由紫外线引起的抗氧化效果
确认了根据一方面的沙克乳酸杆菌菌株的由紫外线诱导的细胞内抗氧化活性。具体地,将人角质形成细胞细胞系(Human HaCaT)以1.0×106个细胞/mL的浓度分配到96孔板中,在37℃下培养16小时。接下来,加入所述实施例2至实施例4和比较例1至比较例2并在37℃下进一步培养1小时,然后将细胞暴露于UVB(30mJ/cm2)并在37℃下进一步培养24小时。接下来,每孔加入DCF-DA溶液(25μM),将细胞在黑暗中放置10分钟,然后使用珀金埃尔默LS-5B荧光分光计(PerkinElmer,沃尔瑟姆,马萨诸塞州,美国)检测由细胞内活性氧物质(ROS)产生的2',7'-二氯荧光素(DCF)产物的荧光。绿茶提取物用作对照组。
【表6】
对照组 | 实施例2 | 实施例3 | 实施例4 | 比较例1 | 比较例2 | |
细胞内抗氧化活性(%) | 53.17 | 49.45 | 47.72 | 51.32 | 32.25 | 28.51 |
结果,如表6所示,可以确认实施例2至实施例4的细胞内抗氧化活性水平显着高于比较例1至比较例2。具体地,可以确认实施例2至实施例4显示出与已知具有优异抗氧化活性的绿茶提取物相似水平的抗氧化活性。
即,根据一方面的沙克乳酸杆菌菌株通过减弱因紫外线而增加的皮肤中ROS水平来具有优异的抗氧化活性,从而对预防和改善皮肤老化有效。
由自由基清除能力引起的抗氧化效果
确认了根据一方面的沙克乳酸杆菌菌株的由自由基清除能力引起的抗氧化活性。具体地,在将实施例2至实施例4和比较例1至比较例2以一定容量加入96孔板的各孔中后,加入由100μM甲醇溶液制备的DPPH(2,2-Di(4-tert-octylphenyl)-1-picrylhydrazylfree radical)以使溶液的总体积为200μg。接下来,在37℃下放置30分钟后,使用分光光度计(spectrophotometer)在560nm处测量吸光度。绿茶提取物用作对照组。
【表7】
对照组 | 实施例2 | 实施例3 | 实施例4 | 比较例1 | 比较例2 | |
自由基清除活性(%) | 86.91 | 84.72 | 84.65 | 85.24 | 83.33 | 81.17 |
结果,如表7所示,可以确认实施例2至实施例4的自由基清除活性显着优异于比较例1至比较例2。具体地,可以确认实施例2至实施例4显示出与已知具有优异抗氧化活性的绿茶提取物相似水平的自由基清除活性。
即,根据一方面的沙克乳酸杆菌菌株具有优异的清除自由基的抗氧化活性,从而可以用于预防或改善皮肤老化。
【保藏号】
保藏机构名称:韩国生命工学研究院
保藏号:KCTC14319BP
保藏日:20200923
本发明的上述描述仅用于例示,本发明所属领域的普通技术人员将理解,在不改变本发明的技术精神或基本特征的情况下,可以容易地将其修改为其他具体形式。因此,应当理解,上述实施例在所有方面都是示例性的,而不是限制性的。
序列表
<110> (株)伊思韩佛(IT'S HANBUL CO., LTD.)
<120> 沙克乳酸杆菌AMP5104菌株及其用途
<130> PX066652CN
<150> KR10-2021-0161382
<151> 2021-11-22
<160> 1
<170> PatentIn version 3.2
<210> 1
<211> 1534
<212> DNA
<213> 人工的(Artificial)
<220>
<223> 沙克乳酸杆菌AMP 5104(Lactobacillus sakei AMP 5104)
<400> 1
tggctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgaacgc actctcgttt 60
agattgaagg agcttgctcc tgattgataa acatttgagt gagtggcgga cgggtgagta 120
acacgtgggt aacctgccct aaagtggggg ataacatttg gaaacagatg ctaataccgc 180
ataaaaccta acaccgcatg gtgtagggtt gaaagatggt ttcggctatc actttaggat 240
ggacccgcgg tgcattagtt agttggtgag gtaaaggctc accaagaccg tgatgcatag 300
ccgacctgag agggtaatcg gccacactgg gactgagaca cggcccagac tcctacggga 360
ggcagcagta gggaatcttc cacaatggac gaaagtctga tggagcaacg ccgcgtgagt 420
gaagaaggtt ttcggatcgt aaaactctgt tgttggagaa gaatgtatct gatagtaact 480
gatcaggtag tgacggtatc caaccagaaa gccacggcta actacgtgcc agcagccgcg 540
gtaatacgta ggtggcaagc gttgtccgga tttattgggc gtaaagcgag cgcaggcggt 600
ttcttaagtc tgatgtgaaa gccttcggct caaccgaaga agtgcatcgg aaactgggaa 660
acttgagtgc agaagaggac agtggaactc catgtgtagc ggtgaaatgc gtagatatat 720
ggaagaacac cagtggcgaa ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag 780
catgggtagc aaacaggatt agataccctg gtagtccatg ccgtaaacga tgagtgctag 840
gtgttggagg gtttccgccc ttcagtgccg cagctaacgc attaagcact ccgcctgggg 900
agtacgaccg caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 960
atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctttgaccac 1020
tctagagata gagctttccc ttcggggaca aagtgacagg tggtgcatgg ttgtcgtcag 1080
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat tactagttgc 1140
cagcatttag ttgggcactc tagtgagact gccggtgaca aaccggagga aggtggggac 1200
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggatggtac 1260
aacgagttgc gagaccgcga ggtttagcta atctcttaaa accattctca gttcggattg 1320
taggctgcaa ctcgcctaca tgaagccgga atcgctagta atcgcggatc agcatgccgc 1380
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgagag tttgtaacac 1440
ccaaagccgg tgaggtaacc ccttcggggg agccagcccg tctaaagtgg ggacagatga 1500
attaagggtg aagtcgtaag aggggggacc ccgt 1534
Claims (11)
1.一种沙克乳酸杆菌AMP5104菌株,其保藏号为KCTC14319BP。
2.根据权利要求1所述的沙克乳酸杆菌AMP5104菌株,其中,所述菌株具有包括由序列号1所示的核酸序列的16s rRNA。
3.一种沙克乳酸杆菌AMP5104菌株的干物质、破碎物、培养液、培养液的提取物或裂解物,其保藏号为KCTC14319BP。
4.一种化妆品组合物,其包括权利要求1所述的菌株、其干物质、破碎物、培养液、培养液的提取物、裂解物或其混合物。
5.根据权利要求4所述的化妆品组合物,其还包括第二乳杆菌属菌株、其干物质、破碎物、培养液、培养液的提取物、裂解物或其混合物。
6.根据权利要求4或5所述的化妆品组合物,其用于改善皮肤状况、皮肤老化、皮肤抗菌、皮肤炎症、皮肤免疫或皮肤屏障。
7.一种皮肤外用剂组合物,其包括权利要求1所述的菌株、其干物质、破碎物、培养液、培养液的提取物、裂解物或其混合物,并且用于改善皮肤状况、皮肤老化、皮肤抗菌、皮肤炎症、皮肤免疫或皮肤屏障。
8.一种药物组合物,其包括权利要求1所述的菌株、其干物质、破碎物、培养液、培养液的提取物、裂解物或其混合物,并且用于预防或治疗皮肤抗菌、皮肤免疫、皮肤炎性疾病。
9.根据权利要求8所述的药物组合物,所述皮肤炎性疾病为选自皮肤伤口、皮炎、特应性皮炎、瘙痒症、湿疹性皮肤病、干性湿疹、红斑、荨麻疹、牛皮癣、轻度皮疹和痤疮中的任一种。
10.一种保健功能食品组合物,其包括权利要求1所述的菌株、其干物质、破碎物、培养液、培养液的提取物、裂解物或其混合物,并且用于改善皮肤状况、皮肤老化、皮肤抗菌、皮肤炎症、皮肤免疫或皮肤屏障。
11.一种食品组合物,其包括权利要求1所述的菌株、其干物质、破碎物、培养液、培养液的提取物、裂解物或其混合物,并且用于改善皮肤状况、皮肤老化、皮肤抗菌、皮肤炎症、皮肤免疫或皮肤屏障。
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