CN115386524B - 一种乳酸杆菌组合物及其在护肤品中的应用 - Google Patents
一种乳酸杆菌组合物及其在护肤品中的应用 Download PDFInfo
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Abstract
本发明公开了一种乳酸杆菌组合物,其包括至少一种选自由乳酸杆菌菌株A:罗伊氏乳杆菌Lactobacillus reuteri FPHC0010;乳酸杆菌菌株B:唾液乳杆菌Lactobacillus salivarius LS02;乳酸杆菌菌株C:鼠李糖乳杆菌Lactobacillus rhamnosus FPHC0021,组成的乳酸杆菌菌株。本发明中所述菌株的灭活细胞制剂,配置为护肤品或以其他适当的形式,施用在皮肤上,可抑制金葡对皮肤细胞的黏附并中断金葡的致病过程,减少金葡引起的不适感,降低与金葡相关的特应性皮炎的发生率,改善皮肤健康状态。
Description
技术领域
本发明涉及微生物及生物技术领域,具体涉及一种乳酸杆菌组合物及其在护肤品中的应用。
背景技术
作为最大的人体器官,皮肤是身体防止创伤,毒素、环境损害和致病生物入侵的第一道屏障,同时它也为包括细菌、真菌和病毒在内的共生微生物提供了栖息地。皮肤上中常驻微生物数量的确切数字很难获得,但根据培养结果估计皮肤上微生物的数量约为每平方厘米100000个以上。皮肤表面(角质层或表皮最外层)是一个由死亡的角化细胞组成的相对干燥的环境,在角质层之下的表皮部分由分化的角质细胞层组成,其中夹杂着其他细胞,如黑色素细胞、朗格汉斯细胞等。另外90%的皮肤由真皮层组成,真皮层包含其他结构,如血管、淋巴管、毛囊、皮脂腺和汗腺,所有这些都为微生物的繁殖提供了有利条件,汗液本身含有抗菌肽(AMP)、无机盐、尿素、短链脂肪酸等成分,而皮脂腺则分泌由甘油三酯、神经酰胺、甾醇、蜡等构成的皮脂,皮肤细胞、汗液、皮脂都可被皮肤微生物作为营养成分而加以利用。皮肤微生物组的复杂性和稳定性因具体的皮肤部位而异,它对保护身体免受环境病原体的侵害、调节免疫系统和分解天然产物都很重要。皮肤菌群在组成上具有极大的个体性差异,但仍能表现出明显的共性,凝固酶阴性葡萄球菌、棒状杆菌、丙酸菌、微球菌等是比例最高的细菌种类,真菌以嗜脂的马拉色菌类为主。皮肤菌群具有调节皮肤免疫系统发育和状态、维护皮肤pH、抑制致病菌入侵定殖、预防或减少皮肤损伤、产生体味等作用。正常的皮肤菌群在组成和数量上具有相当的稳定性,并对皮肤健康有良性作用。
葡萄球菌(Staphylococcus)是皮肤菌群中的主要类群,大多数菌株是对皮肤无害的常栖性种类,如表皮葡萄球菌、人葡萄球菌、头状葡萄球菌等。但在该属内,金黄色葡萄球菌(S.aureus)是一种典型致病菌,特别是凝固酶阳性的菌株。S.aureus常寄居在人体皮肤上,但正常/健康皮肤可以抵抗其定殖,因此在正常的光滑皮肤上检出率很低,但在鼻孔或皮肤褶皱等潮湿处检出率较高,约25-30%的成人鼻腔(美国调查数据)中可发现该菌,在会阴部及趾间等皮肤皱褶部位也可检出。
S.aureus是皮肤感染的主要致病菌之一,与特应性皮炎和湿疹的发病也密切相关。在分子水平上,S.aureus的毒力因子包括:
凝固酶(Coagulase,Coa):是致病性S.aureus的代表性特征,临床将 Coa阳性的S.aureus视为致病菌。Coa可激活凝血酶原使纤维蛋白原转变为纤维蛋白,使含有抗凝剂的动物血浆凝固,是S.aureus毒性的辅助因子。
PVL(Panton Valentine Leukocidin,杀白细胞素):细胞外毒素,双亚基蛋白形成环状结构聚合体,插入到巨噬细胞或中性粒细胞的细胞膜上,形成孔径约2nm的孔洞,使Ca2 +、Mg2+等大量内流,导致细胞坏死凋亡。
HL(Haemolysin,溶血毒素):强致病性的细胞穿孔素,根据抗原性分为 5种(α~ε),可导致细胞膜形成孔道,导致细胞内外离子失衡和细胞溶解,研究证实α-HL与皮肤感染的症状严重程度密切相关。
ET(Exfoliative,表皮剥脱毒素):一种导致严重的烫伤样皮肤综合征的外毒素,约5%的S.aureus菌株可以产生,ET会诱导自身抗体破坏角质形成细胞间的桥粒芯蛋白-1或作为超抗原诱导皮肤损伤。
SE(Enterotoxin,肠毒素):一种耐热耐消化酶的毒性蛋白,经消化道吸收入血后可刺激中枢神经导致呕吐,可刺激T细胞释放IL-2等免疫因子抑制体液和细胞免疫,使单核-巨噬细胞清除功能障碍。
TNase(耐热核酸酶):在S.aureus菌株中广泛存在的特征之一,可降解人体细胞死亡后释放的核酸并将其作为自身营养成分。
TSST-1:S.aureus分泌的导致中毒性休克综合征一种超抗原,可刺激Th1 CD4+ T细胞等释放IL-2、INF-γ、TNF-α等细胞因子并促进B细胞分化和分泌中和抗体,大量TSST-1可诱发T依赖性B细胞发生凋亡,抑制宿主对毒素的中和反应。
S.aureus及其毒力因子对皮肤感染和皮肤不适感的作用机制已有研究报道。在皮肤疼痛感上,S.aureus可产生N-formyl多肽,而皮肤的痛觉神经元具有感知该多肽的受体,结合后引发痛觉信号;α-HL可在感觉神经元末梢的细胞膜上形成孔洞,导致离子进入神经元触发该神经元释放疼痛信号。并进一步通过痛觉神经元相关的CGRP释放来抑制巨噬细胞产生TNF-α和减少T、B细胞向淋巴结的迁移来压制机体的免疫反应。
在产生慢性炎症/特应性皮炎/湿疹方面,已发现α-HL可直接引起角质形成细胞坏死,引起TNF-a表达增加并引发炎症。肠毒素具有超抗原特性,具有强大的免疫激活能力,能够直接作用于T淋巴细胞、抗原提呈细胞和MHC-11 类分子阳性细胞,刺激T、B淋巴细胞活化,分泌细胞因子和特异IgE,引发皮炎;超抗原可以损伤CD4+CD25+T细胞的免疫抑制功能,造成免疫反应异常;还可使机体对糖皮质激素的敏感性降低,降低外用糖皮质激素的疗效。SPA (Staphylococcal Protein A,细胞壁抗原,90%以上菌株含有)可以刺激角质形成细胞过度表达IL-18,后者可以介导自然型特应性皮炎。S.aureus产生的蛋白酶可导致皮肤屏障的破坏,增加抗原和刺激物的透过性。特应性皮炎患者血清中金黄色葡萄球菌毒素特异IgE浓度升高,且与疾病严重程度正相关。
在皮肤疾病和皮肤菌群组成上,已证实了皮肤菌群中S.aureus数量变化和特应性皮炎(atopic dermatitis,AD,也称作特应性湿疹atopic eczema,AE) 的相关性。多项研究已证实皮肤菌群中金黄色葡萄球菌比例升高和定殖与AD 发作有关。P Meylan等认为金葡的定殖与婴儿皮炎和湿疹的发作有关(Journal of Investigative Dermatology,2017(137):2497-2504);Kong等的研究也支持同样观点,儿童在AD发作期间皮肤菌群中金葡等致病菌比例会爆发性增长,AD 症状消退后皮肤菌群也随之恢复(Genome Research,2020(22):850–859)。对成人的流行病学调查显示,成人AD发生和严重程度与皮肤上金葡的检出呈正相关(Sci.Transl.Med.9,eaah4680,2017)。S.aureus可在AD患者皮肤表面形成生物膜,增强定殖能力(JAMA Dermatol.2014;150(3):260-265)。在小鼠等动物模型上也证实了S.aureus增多和AD等皮肤炎症的相关性(Immunity 42,2015,756–766;J InvestDermatol.2009,129(10):2435–2442)。
综上所述,S.aureus是皮肤健康和皮肤状态的不利因素,应抑制S.aureus 在皮肤表面的定殖和致病过程,减少AD发病率和严重程度,促进皮肤健康和状态的改善。常规的致病菌控制措施为使用抗生素成分,但会带来产生抗药性、无选择性杀死有益菌、导致菌群失调等问题。通过微生物间相互作用来控制致病菌是一种对人体和人体菌群更加友好的方式。S.aureus致病过程第一步是与人体细胞接触并黏附在人体细胞上,阻断该步骤可终止S.aureus的致病过程。我们发现乳酸杆菌的一些菌株在人角质形成细胞HaCaT模型中可以抑制S. aureus的黏附,表明可以开发为促进皮肤健康、改善皮肤状态的成分。
发明内容
本发明所要解决的技术问题是:提供一种具有皮肤护理作用的乳酸杆菌组合物。
为解决上述技术问题,采用的技术方案为:一种乳酸杆菌组合物,其包括至少一种选自由乳酸杆菌菌株A:罗伊氏乳杆菌Lactobacillus reuteri FPHC0010,保藏编号为CGMCC No.24673;乳酸杆菌菌株B:唾液乳杆菌 Lactobacillus salivarius LS02,保藏编号为CGMCC No.24667;乳酸杆菌菌株C:鼠李糖乳杆菌Lactobacillus rhamnosusFPHC0021,保藏编号为CCTCC M2017640组成的乳酸杆菌菌株。
所述的FPHC0010,其保藏信息如下:
分类命名:罗伊氏乳杆菌
拉丁文名称:Lactobacillus reuteri;
保藏该生物材料样品的单位全称:中国微生物菌种保藏管理委员会普通微生物中心;
地址:北京市朝阳区北辰西路1号院3号;
保藏日期:2022年04月14日保藏中心收到,并登记入册;经保藏中心于2022年04月14日检测,结果:存活;
保藏编号:CGMCC No.24673。
所述的LS02,其保藏信息如下:
分类命名:唾液乳杆菌
拉丁文名称:Lactobacillus salivarius;
保藏该生物材料样品的单位全称:中国微生物菌种保藏管理委员会普通微生物中心;
地址:北京市朝阳区北辰西路1号院3号;
保藏日期:2022年04月11日保藏中心收到,并登记入册;经保藏中心于2022年04月11日检测,结果:存活;
保藏编号:CGMCC No.24667。
所述的鼠李糖乳杆菌FPHC0021,其保藏信息如下:
分类命名:鼠李糖乳杆菌
拉丁文名称:Lactobacillus rhamnosus;
保藏该生物材料样品的单位全称:中国典型培养物保藏中心;
地址:中国,武汉,武汉大学;
保藏日期:2017年10月30日保藏中心收到,并登记入册;经保藏中心于 2017年11月20日检测,结果:存活;
保藏编号:CGMCC No.M2017640。
所述的一种乳酸杆菌组合物,其中的菌株选自乳酸杆菌菌株A、乳酸杆菌菌株B和乳酸杆菌菌株C中任意两种组成的组。
所述的一种乳酸杆菌组合物,其中的菌株选自乳酸杆菌菌株A、乳酸杆菌菌株B和乳酸杆菌菌株C组成的组。
按质量份数,所述的乳酸杆菌为以下乳酸杆菌菌株中任意二种或三种的组合物:乳酸杆菌菌株A:1~99份,乳酸杆菌菌株B:1~99份,乳酸杆菌菌株 C:1~99份。
进一步优选的,按质量份数,所述的乳酸杆菌为以下乳酸杆菌菌株中任意二种或三种的组合物:乳酸杆菌菌株A:20~40份,乳酸杆菌菌株B:20~40 份,乳酸杆菌菌株C:20~40份。
本申请还保护一种乳酸杆菌组合物在护肤品中的应用。
所述的护肤品,按照质量百分含量,其组成为:水88.7%,甘油3.0%,甜菜碱1.0%,黄原胶0.3%,1,3-丁二醇3.0%,所述的乳酸杆菌组合物的灭活细胞制剂2.0%,PE9010(苯氧乙醇、乙基己基甘油)1.0%,香精1.0%。
以上乳酸杆菌菌株的细胞在灭活细胞形式下在黏附抑制的置换、排阻、竞争模型中均表现出良好的抑制S.aureus对HaCaT细胞黏附的效果。
乳酸杆菌的拮抗S.aureus黏附分子机制为细胞表面的蛋白质(特别是转运蛋白和S-Layer蛋白)、肽聚糖、磷壁酸、胞壁酸等分子与人体细胞表面受体发生相互作用,占据细胞表面结合位点。除完整细胞外,使用超声波、超高压、反复冻融、溶菌酶等方法获得的破碎的乳酸杆菌细胞处理物(化妆品相关法规中称为溶胞产物)也具有相同或近似效果。因此可使用完整的菌体细胞或溶胞产物,统称为灭活细胞制剂。本发明中通过比较试验优选出的三个菌株,在不同模型上的效果有所差别,为使乳酸杆菌灭活细胞制剂获得较好的金葡抑制效果,可将三个菌株混合使用,在灭活处理前,每个菌株的菌落计数在总菌落计数中的比例为1~99份,优选为20~40份。
将制得的灭活细胞制剂与其他护肤品原料根据工艺和使用需要复配后,施用在人体皮肤上,起到减少S.aureus黏附和定殖、减少皮肤疾病发生、改善皮肤健康和状态的作用。
有益效果:本发明中所述菌株的灭活细胞制剂,配置为护肤品或以其他适当的形式,施用在皮肤上,可抑制金葡对皮肤细胞的黏附并中断金葡的致病过程,减少金葡引起的不适感,降低与金葡相关的特应性皮炎的发生率,改善皮肤健康状态。
附图说明
图1未处理组(无任何处理的HaCaT细胞照片);
图2阳性对照(只加入SA菌悬液的HaCaT细胞);
图3置换模型(先加入SA菌悬液后加入乳酸杆菌灭活细胞制剂的HaCaT细胞);
图4排阻模型(先加入乳酸杆菌灭活细胞制剂后加入SA菌悬液的HaCaT细胞);
图5竞争模型(同时加入SA菌悬液后和乳酸杆菌灭活细胞制剂的 HaCaT细胞)。
具体实施方式
下面结合实施例对本发明的方法予以进一步地说明,但并不因此而限制本发明。
乳酸杆菌灭活细胞制剂的制备
不同的乳酸杆菌菌株分别在MRS液体培养基、37℃静置培养48hr后离心收集菌体,在生理盐水中稀释至1.0E8CFU/ml,80℃保温10min进行灭活处理。可使用超声波破碎仪对菌体细胞进行破碎处理,超声波破碎步骤为可选步骤。4℃冷藏备用。
S.aureus黏附抑制率测试
S.aureus ATCC35556菌株在营养肉汤液体培养基、37℃震荡培养24hr后离心收集菌体,在生理盐水中稀释至1.0E8CFU/ml,4℃冷藏备用,下文中称为SA菌悬液。
人角质形成细胞HaCaT细胞株在含10%胎牛血清的DMEM培养基,37℃, 5%CO2气氛下在多孔板中培养至贴壁后,进行如下操作:
置换模型:HaCaT细胞吸去培养液,每孔加入SA菌悬液0.2ml,37℃下抚育30min后吸去上液,0.2ml生理盐水冲洗一次后,每孔加入乳酸杆菌灭活细胞制剂0.2ml,37℃下抚育30min,吸去上液,0.2ml生理盐水冲洗一次后,每孔加入0.2ml生理盐水吹打,吸取上液进行梯度稀释后在MSA平板(S.aureus 选择性平板)进行培养计数,得到不同乳酸杆菌菌株的S.aureus计数结果。
排阻模型:HaCaT细胞吸去培养液,每孔加入乳酸杆菌灭活细胞制剂 0.2ml,37℃下抚育30min后吸去上液,0.2ml生理盐水冲洗一次后,每孔加入SA菌悬液0.2ml,37℃下抚育30min,吸去上液,0.2ml生理盐水冲洗一次后,每孔加入0.2ml生理盐水吹打,吸取上液进行梯度稀释后在MSA平板(S. aureus选择性平板)进行培养计数,得到不同乳酸杆菌菌株的S.aureus计数结果。
竞争模型:HaCaT细胞吸去培养液,每孔加入SA菌悬液0.1ml和乳酸杆菌灭活细胞制剂0.1ml,37℃下抚育30min后吸去上液,0.2ml生理盐水冲洗一次后,每孔加入0.2ml生理盐水吹打,吸取上液进行梯度稀释后在MSA平板 (S.aureus选择性平板)进行培养计数,得到不同乳酸杆菌菌株的S.aureus计数结果。
空白对照为以上各模型中只加入SA菌悬液抚育、吹打后的S.aureus计数结果。
各模型的抑制率计算公式为:
抑制率(Inhibition Rate,%)=(空白对照S.aureus计数结果-乳酸杆菌菌株的S.aureus结果)÷空白对照S.aureus计数结果×100%。
平均抑制率为三种模型抑制率的算术平均值。
不同乳酸杆菌菌株的抑制率计算结果见下表:
从黏附抑制率数据可知,某些乳酸杆菌菌株的灭活细胞对S.aureus对 HaCaT细胞的黏附没有任何抑制效果,有效菌株在不同模型中的抑制率也各有差异,表明具有菌株特异性,需要通过测试获得特定菌株。L.salivarius LS02、 L.reuteri FPHC0010、L.rhamnosus FPHC0021在该测试中具有最高的平均抑制率,分别为39.94%、41.22%、35.09%。
观察以上三个乳酸杆菌菌株在不同模型中的黏附抑制率,认为L.salivariusLS02在排阻和竞争模型中表现较好,L.reuteri FPHC0010在置换和竞争模型中表现较好,L.rhamnosus FPHC0021在三种模型中性能较为均衡。认为可使用以上菌株组合来获得更好的黏附抑制效果。将三个菌株按照灭活后细胞数量比为 1:1:1,即100份中各占33.3份的比例,混合后为复合乳酸杆菌灭活细胞制剂,按照上文所述相同方法在置换、排阻、竞争模型中测试对S.aureus的黏附抑制效果,所得抑制率为51.84%、67.29%、64.71%,平均抑制率为61.28%,明显高于三个菌株单独使用时的抑制率,表明与单独使用相比,乳酸杆菌细胞的组合使用可更好地抑制金黄色葡萄球菌黏附到角质形成细胞上。
以L.salivarius LS02菌株为例,不同模型中的细胞照片见下式,均为革兰氏染色。未处理组为单独的HaCaT细胞,阳性对照为只黏附S.aureus菌体,置换、排阻、竞争为各模型中L.salivarius LS02菌体和S.aureus菌体混合黏附在HaCaT细胞的照片。
含有乳酸杆菌灭活细胞制剂的护肤品的配制,按如下配方制备护肤产品:
操作工艺为:A相混合并搅拌溶解,加热至约80℃,搅拌下加入预先混匀的B相,搅拌溶解后,降温至60-65℃,搅拌下加入C相、D相,搅拌混合均匀。冷却至室温后为成品。
Claims (5)
1.一种乳酸杆菌组合物,其特征在于:其中的菌株选自乳酸杆菌菌株A、乳酸杆菌菌株B和乳酸杆菌菌株C组成的组;
所述的A:Lactobacillus reuteri FPHC0010,保藏编号为CGMCC No.24673;所述的乳酸杆菌菌株B:Lactobacillus salivarius LS02,保藏编号为CGMCC No.24667;所述的乳酸杆菌菌株C:Lactobacillus rhamnosus FPHC0021,保藏编号为CCTCC M2017640。
2.根据权利要求1所述的一种乳酸杆菌组合物,其特征在于:按质量份数,乳酸杆菌菌株A:1~99份,乳酸杆菌菌株B:1~99份,乳酸杆菌菌株C:1~99份。
3.根据权利要求2所述的一种乳酸杆菌组合物,其特征在于:按质量份数,乳酸杆菌菌株A:20~40份,乳酸杆菌菌株B:20~40份,乳酸杆菌菌株C:20~40份。
4.权利要求1的一种乳酸杆菌组合物在制备护肤品中的应用。
5.根据权利要求4所述的一种乳酸杆菌组合物在制备护肤品中的应用,所述的护肤品,按照质量百分含量,其组成为:水88.7%,甘油3.0%,甜菜碱1.0%,黄原胶0.3%,1,3-丁二醇3.0%,权利要求1-3任一项所述的乳酸杆菌组合物的灭活细胞制剂2.0%,苯氧乙醇和乙基己基甘油1.0%,香精1.0%。
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