CN114634899B - 发酵乳杆菌及其应用 - Google Patents
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Abstract
本发明涉及微生物技术领域,尤其涉及发酵乳杆菌及其应用。本发明公开的发酵乳杆菌SEUNEU‑108寄存于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20211559。实验表明,SEUNEU‑108具有维持及修护皮肤屏障、治疗痤疮及改善敏感肌的功能,可用于制备药品、化妆品等。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及发酵乳杆菌及其应用。
背景技术
皮肤是人体内最大的器官,总重量大约占个体体重的16%,一方面维持机体稳定,另一方面也是抵御外界不良因素侵扰的第一道防线。有研究表明,如果外界环境导致皮肤屏障中的相关基因异常,就会诱发皮肤疾病。
皮肤屏障是由角质层的表皮形成细胞和角质间的脂质形成的结构性的屏障。皮肤屏障防止人体过多水分释放,并防止如化学物质或微生物的有害物质进入我们的身体。组成死亡的角质细胞的表面的角质细胞外皮在细胞间脂质的稳定性中起重要作用。皮肤屏障受损将引起皮肤干燥,皮肤老化、色素沉着异位性皮炎、湿疹、银屑病、鱼鳞病、日旋光性皮炎等皮肤敏感、刺激性皮炎、激素依赖性皮炎等皮肤油腻,皮脂溢出性疾病,如痤疮、酒糟鼻、脂溢性皮炎。
角质间结构性脂质神经酰胺,在基底层向角质分化过程中含量逐渐增加,到达角质层排至细胞间隙,构成防止水分丢失的屏障。角质细胞内含水量高,随着细胞向上代谢分化,角质细胞形状会逐渐变成扁平状,且细胞核及胞器开始退化萎缩,并在角质层形成不具细胞核与胞器的死细胞。角质层本身的亲水、屏障功能,以及角质层中所含有的天然保湿因子即氨基酸类,乳酸盐及糖类等作用,使得角质层通常含有10~30%的水分,这种环境成为了皮肤自身微生物菌落生长的摇篮。但随着年龄的增长,角质层的含水量会逐渐减少,当水分含量低于10%的时候,就会引起皮肤的各种问题。
益生菌用在化妆品上,可平衡皮肤表皮菌群,修复皮肤屏障。同时上调保湿基因的表达,有效增加肌肤对营养物质的吸收,增强肌肤的免疫力。
痤疮是一种常见的慢性炎症性皮肤疾病,主要与皮脂分泌过多、毛囊皮脂腺导管堵塞、细菌感染和炎症反应等因素密切相关。研究表明,痤疮丙酸杆菌(Propionibacteriumacne)被认为引发痤疮的主要病原菌,它可以诱导和活化痤疮炎症的起始环节,并将甘油转换为脂肪酸,导致炎症反应;同时产生蛋白酶、透明质酸酶及趋化因子,使毛囊过度角化,形成痤疮。另一方面,皮肤常驻的表皮葡萄球菌与痤疮丙酸杆菌能够相互拮抗竞争,增加表皮葡萄球菌的数量能够抑制痤疮丙酸杆菌增殖。因此,降低痤疮丙酸杆菌/表皮葡萄球菌的菌群比例,可以有效缓解痤疮炎症,从而通过调节皮肤微生态平衡来维持皮肤健康。
敏感肌通常是由于皮肤细胞受损而使皮肤免疫力下降,角质层变薄导致皮肤滋润度不够,最终导致肌肤的屏障功能过于薄弱,无法抵御外界刺激,从而易于产生泛红,发热,瘙痒、刺痛等不适现象的产生。而皮肤屏障的受损,则会进一步导致金黄色葡萄球菌(Staphylococcus aureus)的定植增殖,导致发炎红肿。皮肤常驻的表皮葡萄球菌(Staphylococcus epidermidis)与金黄色葡萄球菌相互拮抗,能够降低后者的增殖。因此降低金黄色葡萄球菌/表皮葡萄球菌的菌群比例,有助于建立皮肤菌群的平衡分布,从而改善敏感肌。因此,提供一种利用微生态技术开发的益生菌相关产品,具有重要的现实意义。
发明内容
有鉴于此,本发明提供了发酵乳杆菌及其应用。
本发明提供了保藏编号为CCTCC NO:M 20211559的发酵乳杆菌(Lactobacillusfermentum)。
本发明还提供了所述的发酵乳杆菌在制备改善肌肤状况的产品中的应用。
本发明中,所述改善肌肤状况包括修护皮肤屏障、保湿、抗炎、调节皮肤微生物菌群的比例中的至少一种。
一些实施方案中,所述修复皮肤屏障包括修复皮肤细胞和/或上调屏障修护相关基因的表达。
一些具体实施例中,所述屏障修护相关基因包括FLG、IVL、OVOL1和LOR中的至少一种。一些具体实施例中,所述修复皮肤细胞为提高SDS诱导的细胞的存活率。具体地,所述细胞为人角质形成细胞。
一些实施方案中,所述保湿为上调保湿相关基因AQP3和/或GBA的表达。
一些实施方案中,所述调节皮肤微生物菌群的比例为:
抑制金黄色葡萄球菌和/或丙酸杆菌的生长,和/或
促进或不影响表皮葡萄球菌的生长。
本发明的发酵乳杆菌SEUNEU-108具有调节皮肤微生物菌群的作用,从而治疗痤疮、改善敏感肌。具体地,通过降低痤疮丙酸杆菌/表皮葡萄球菌的比例,从而治疗痤疮;通过降低金黄色葡萄球菌/表皮葡萄球菌的比例,从而改善敏感肌。
一些实施方案中,所述抗炎为:
下调细胞炎症相关因子IL-6和/或TRPV1基因的表达,和/或
降低NO的释放水平。
本发明中,所述抗炎中的炎症为表皮细胞的炎症,具体为金黄色葡萄球菌和/或脂多糖(LPS)诱导的炎症。
本发明中,所述产品为药品或化妆品。
本发明还提供一种改善皮肤状况的产品,由包括本发明发酵乳杆菌的原料制成。
一些实施方案中,所述皮肤状况的产品,包括如下(1)或(2)所示的一种或两种:
(1)本发明所述的发酵乳杆菌的活菌和/或灭活菌;
(2)本发明所述的发酵乳杆菌的培养物、裂解物和/或提取物。
本发明公开的发酵乳杆菌SEUNEU-108,其保藏编号为CCTCC NO:M20211559。实验表明,SEUNEU-108具有维持及修护皮肤屏障、保湿、抗炎、治疗痤疮及改善敏感肌的功能,可用于制备药品、化妆品等。
生物保藏说明
发酵乳杆菌SEUNEU-108(Lactobacillus fermentum SEUNEU-108),于2021年12月6日保藏在中国典型培养物保藏中心,地址为:中国.武汉.武汉大学,保藏编号为CCTCCNO:M20211559。
附图说明
图1为SEUNEU-108促进HaCaT细胞修复;
图2为SEUNEU-108灭活菌体上调屏障修护相关基因表达。
具体实施方式
本发明提供了发酵乳杆菌及其应用。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明的发酵乳杆菌菌株SEUNEU-108,来源于豆汁,经16S rRNA鉴定为发酵乳杆菌(Lactobacillus fermentum)。该菌株革兰氏阳性,显微镜下呈杆状;在MRS平板上生长,可形成表面光滑不透明的圆形菌落,白色,边缘整齐;在MRS液体培养基中呈均匀浑浊生长,久置菌体呈白色沉淀,最适生长温度37℃。
发酵乳杆菌(Lactobacillus fermentum)SEUNEU-108,保藏单位:中国典型培养物保藏中心,地址:湖北省武汉市武昌区八一路299号武汉大学校内,保藏日期:2021年12月6日,保藏编号为CCTCC NO:M20211559。
进一步,本发明提供的发酵乳杆菌SEUNEU-108在本发明所述的应用或产品中,存在形式是活的或死的或经间歇灭菌的,或为裂解物和/或提取物的形式,或为细菌产物的形式或为上清液形式或衍生物形式,所述衍生物形式优选地选自:代谢产物、代谢生物产物、益生素、细胞壁及其成分、胞外多糖,和含有免疫原性成分的化合物,优选地选自:上清液、灭活菌体。
体外细胞实验表明,本发明的发酵乳杆菌SEUNEU-108具有促进表皮细胞修复的作用,SEUNEU-108提升十二烷基硫酸钠(SDS)损伤的HaCaT细胞存活率达111.99%~115.55%。
体外细胞实验表明,本发明的发酵乳杆菌SEUNEU-108具有上调保湿相关基因AQP3和GBA表达的作用,基因表达量上调1.45~2.41倍。
体外细胞实验表明,本发明的发酵乳杆菌SEUNEU-108具有上调皮肤屏障修护相关因子丝聚合蛋白基因FLG、外皮蛋白基因IVL、OVO样转录因子1基因OVOL1和兜甲蛋白基因LOR表达的作用,基因表达量上调1.16~1.68倍。
体外细胞实验表明,本发明的发酵乳杆菌SEUNEU-108具有降低炎症因子释放的作用,能够降低脂多糖(LPS)诱导小鼠巨噬细胞Raw264.7的一氧化氮(NO)生成量35.93%~42.48%。
体外细胞实验表明,本发明的发酵乳杆菌SEUNEU-108具有下调金黄色葡萄球菌诱导的HaCaT细胞炎症相关因子IL-6和TRPV1基因表达的作用,基因表达量下调40%~51%。
体外实验表明,本发明的发酵乳杆菌SEUNEU-108具有降低痤疮丙酸杆菌/表皮葡萄球菌的比例,从而治疗痤疮的功能。
体外实验表明,本发明的发酵乳杆菌SEUNEU-108具有降低金黄色葡萄球菌/表皮葡萄球菌的比例,从而改善敏感肌的功能。
本发明具体实施例中,在添加灭活上清液、灭活菌体时,均按照灭活上清液、灭活菌体占细胞培养体系或占皮肤菌群菌液的体积百分比进行添加。例如,将灭活上清液5%、灭活菌体10%分别加入到培养过夜的Raw264.7细胞中,即,上清液占Raw264.7细胞培养体系的体积百分比为5%,灭活菌体占Raw264.7细胞培养体系的体积百分比为10%,按照以上比例进行添加。
本发明采用的试材皆为普通市售品,均可市售购买得到,下面结合实施例,进一步阐述本发明:
实施例1SEUNEU-108的分离
于豆汁中采样。将样品适当处理后于生理盐水中震荡混匀,取上清划线于MRS固体平板,37℃恒温培养48h后,挑取白色菌落反复接种筛选,直至得到均匀的单个菌落,命名为SEUNEU-108。
革兰氏染色镜检:菌株SEUNEU-108为革兰氏染色阳性,显微镜下呈杆状;在MRS平板上生长,可形成白色、表面光滑圆润不透明圆形小菌落,边缘整齐;在MRS液体培养基中呈均匀浑浊生长,久置菌体呈白色沉淀。
实施例2SEUNEU-108的核酸鉴定
1、16s rRNA基因序列分析:
挑取单菌落置MRS液体培养基中,37℃培养过夜后,离心收集菌体,按照DNA提取试剂盒步骤进行操作。引物用细菌通用引物27F,1492R,PCR扩增体系为50μL体系,95℃预变性5min;94℃15s,57℃15s,72℃40s,35个循环;72℃延伸10min。
2、结果
PCR产物测序结果与GenBank中已发表的标准序列进行同源性比较(BLASTN)后得出SEUNEU-108菌株为发酵乳杆菌(Lactobacillus fermentum)。
实施例3SEUNEU-108促进SDS诱导的HaCaT细胞损伤修复实验
1、SEUNEU-108上清液及灭活菌体制备:
将活化的发酵乳杆菌SEUNEU-108菌液以MRS液体培养基培养于37℃培养箱静置培养16~18h,检测并调整OD,121℃,30min灭活,12000转离心2min,上清液经0.22μm滤膜过滤为灭活上清液。离心所得沉淀以PBS重悬调整OD600=0.2,为灭活菌体。
2、促进HaCaT细胞修复实验
接种HaCaT细胞(5×104cells/孔)至96孔板,过夜培养至细胞贴壁。配制50μg/mlSDS,每孔加入100μl,孵育8h,分别加入5%上清液,10%灭活菌体,孵育24h。加入10μl的CCK-8溶液,孵育4h,检测450nm处吸亮度A。
细胞存活率=实验组A/对照组A×100%。
以SDS诱导组为阴性对照,以其细胞存活率为100%,按照如上公式计算实验组的细胞存活率,结果见表1:
表1 SEUNEU-108促进HaCaT细胞修复
SEUNEU-108促进HaCaT细胞修复结果见图1。
表1和图1的结果可知,SEUNEU-108上清液及灭活菌体均对HaCaT细胞SDS损伤具有修复作用。
实施例4SEUNEU-108促进HaCaT屏障修护相关基因表达实验
1、SEUNEU-108灭活菌体制备:
将活化的发酵乳杆菌SEUNEU-108菌液以MRS液体培养基培养于37℃培养箱静置培养16~18h,检测并调整OD,121℃,30min灭活,12000转离心2min,所得沉淀以PBS重悬调整OD600=0.2,为灭活菌体。
2、促进HaCaT屏障修护相关基因表达实验
接种人永生化角质形成细胞HaCaT(5×105cells/孔)至6孔板,过夜培养至细胞贴壁。加入菌株灭活菌体10%,培养24h后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度,将所有样品调整至1μg,并反转录为cDNA,进行qPCR检测FLG、IVL、OVOL1和LOR基因的表达。以不添加SEUNEU-108灭活菌体处理组为对照,其基因表达量为1,根据公式进行计算表达变化倍数。
公式:F=2-ΔΔCT
结果见表2:
表2 SEUNEU-108灭活菌体上调屏障修护相关基因表达
结果显示加入SEUNEU-108具有促进皮肤屏障修护的作用。
SEUNEU-108灭活菌体上调HaCaT屏障修护相关基因表达结果见图2。
实施例5SEUNEU-108上调HaCaT保湿相关基因表达实验
1、SEUNEU-108灭活菌体制备:
将活化的发酵乳杆菌SEUNEU-108菌液以MRS液体培养基培养于37℃培养箱静置培养16~18h,检测并调整OD,121℃,30min灭活,12000转离心2min,所得沉淀以PBS重悬调整OD600=0.2,为灭活菌体。
2、上调HaCaT保湿相关基因表达实验
接种人永生化角质形成细胞HaCaT(5×105cells/孔)至6孔板,过夜培养至细胞贴壁。按照体积比加入菌株灭活菌体10%,培养24h后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度,将所有样品调整至1μg,并反转录为cDNA,进行qPCR检测保湿相关基因AQP3、GBA的表达。以不添加SEUNEU-108灭活菌体的处理组为对照,其基因表达量为1,根据公式进行计算表达变化倍数。
公式:F=2-ΔΔCT
结果见表3:
表3 SEUNEU-108灭活菌体上调保湿相关基因表达
结果显示加入SEUNEU-108具有促进皮肤保湿的作用。
实施例6:SEUNEU-108降低Raw264.7细胞NO生成量
1、SEUNEU-108上清液及灭活菌体制备:
将活化的发酵乳杆菌SEUNEU-108菌液以MRS液体培养基培养于37℃培养箱静置培养16~18h,检测并调整OD,121℃,30min灭活,12000转离心2min,上清液经0.22μm滤膜过滤为灭活上清液。离心所得沉淀以PBS重悬调整OD600=0.2,为灭活菌体。
2、Raw264.7细胞制备
将Raw264.7细胞消化后以2×105cells/孔接种至24孔板,5%二氧化碳培养箱37℃培养过夜。
3、SEUNEU-108添加及LPS刺激
将灭活上清液5%、灭活菌体10%分别加入到培养过夜的Raw264.7细胞中,2h后添加0.5ml浓度为0.2μg/ml的LPS溶液,诱导Raw264.7细胞发炎,20h后取细胞培养上清液,按照碧云天NO检测试剂盒所述方法进行标准曲线绘制,计算样品中NO的浓度及抑制率。
结果见表4:
表4 SEUNEU-108降低Raw264.7细胞NO生成量
结果显示SEUNEU-108具有抗炎作用,能够降低LPS诱导的Raw264.7细胞NO生成量,与LPS对照组相比降低35.94%~42.48%。
实施例7:SEUNEU-108下调HaCaT细胞炎症因子相关基因的表达
1、SEUNEU-108上清液制备:
将活化的发酵乳杆菌SEUNEU-108菌液以MRS液体培养基培养于37℃培养箱静置培养16~18h,检测并调整OD,121℃,30min灭活,12000转离心2min,上清液经0.22μm滤膜过滤为灭活上清液。
2、HaCaT细胞制备
将HaCaT细胞消化后以2×105cells/孔接种至24孔板,5%二氧化碳培养箱37℃培养过夜。
3、金黄色葡萄球菌制备及添加
金黄色葡萄球菌接入营养肉汤培养基,37℃摇床培养过夜,用MEM无血清培养基调整菌液浓度至OD600=6.0,每孔100μl添加入培养过夜的HaCaT细胞中,刺激细胞产生炎症因子,3h后弃去细胞培养基,PBS清洗5次,每孔重新加入1ml的MEM无血清培养基。
4、SEUNEU-108上清液添加
将SEUNEU-108上清液5%的比例加入到金黄色葡萄球菌刺激过的HaCaT细胞培养液中,每组3个复孔,37℃培养过夜。
5、qPCR法检测细胞炎症因子mRNA相对表达倍数
将上述细胞弃去培养基后,用RNA提取试剂盒提取RNA,检测RNA浓度及纯度,将所有样品调整至1μg,用反转录试剂盒、SYBRGreen qPCR试剂盒进行RT-PCR及qPCR,以不添加SEUNEU-108上清液的处理组为对照,其基因表达量为1,计算炎症因子基因IL-6和TRPV1的相对表达倍数F。
公式:F=2-ΔΔCT
结果见表5:
表5 SEUNEU-108下调炎症相关因子的表达
结果显示SEUNEU-108能够下调金黄色葡萄球菌诱导的HaCaT细胞炎症因子相关基因表达,表达量下调40%~51%。因此,SEUNEU-108具有抗炎作用。
实施例8SEUNEU-108改变菌群比例治疗痤疮的作用
1、SEUNEU-108上清液制备:
将活化的发酵乳杆菌SEUNEU-108菌液以MRS液体培养基培养于37℃培养箱静置培养16~18h,检测并调整OD600=2.0,121℃,30min灭活,离心取上清液,0.22μm滤膜过滤,得待测样品。
2、痤疮相关菌群菌液制备:
将痤疮丙酸杆菌CGMCC 1.5003和表皮葡萄球菌CGMCC 1.4260分别以BHI培养基37℃培养18h,检测并调整OD600=0.2。
3、添加上清液影响皮肤菌群生长实验
将灭活上清10%,分别加入到两种菌液中,培养至对数期,分别测定两种菌液浓度(OD600),以未添加上清液的菌液为对照,计算两种菌液的相对浓度,以痤疮丙酸杆菌/表皮葡萄球菌相对浓度比值评价对皮肤菌群生长影响。
表6 SEUNEU-108对痤疮相关菌群生长影响
4、结果见表6:
结果显示SEUNEU-108对痤疮丙酸杆菌有显着抑制作用,而对表皮葡萄球菌无抑制作用。SEUNEU-108能够改变痤疮相关菌群比例,从而达到治疗痤疮的目的。
实施例9SEUNEU-108改变菌群比例改善敏感肌的作用
1、SEUNEU-108上清液制备:
将活化的发酵乳杆菌SEUNEU-108菌液以MRS液体培养基培养于37℃培养箱静置培养16~18h,检测并调整OD600=2.0,121℃,30min灭活,离心取上清液,0.22μm滤膜过滤,得待测样品。
2、皮肤菌群菌液制备:
将金黄色葡萄球菌CGMCC 1.8721和表皮葡萄球菌CGMCC 1.4260分别以BHI培养基37℃培养18h,检测并调整OD600=0.2。
3、添加上清液影响敏感肌相关菌群生长实验
将灭活上清液以10%分别加入两种菌液中,培养至对数期,分别测定两种菌液浓度(OD600),以未添加上清液的菌液为对照,计算两种菌液的相对浓度,以金黄色葡萄球菌/表皮葡萄球菌相对浓度比值评价对敏感肌相关菌群生长影响。
4、结果见表7
表7 SEUNEU-108对敏感肌相关菌群生长影响
结果显示SEUNEU-108对皮肤主要病原菌金黄色葡萄球菌有显着抑制作用,而对表皮葡萄球菌无抑制作用。SEUNEU-108能够改变菌群比例,从而有效改善敏感肌。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.保藏编号为 CCTCC NO: M 20211559的发酵乳杆菌(Lactobacillus fermentum)。
2.权利要求1所述的发酵乳杆菌在制备改善肌肤状况的产品中的应用;
所述改善肌肤状况为修护皮肤屏障、保湿、抗炎、调节皮肤微生物菌群的比例中的至少一种;
所述调节皮肤微生物菌群的比例为:
抑制金黄色葡萄球菌和/或痤疮丙酸杆菌的生长,和
不影响表皮葡萄球菌的生长;
所述产品为药品或化妆品。
3.根据权利要求2所述的应用,其特征在于,所述修护皮肤屏障包括修复皮肤细胞和/或上调屏障修护相关基因的表达;所述屏障修护相关基因包括FLG、IVL、OVOL1和LOR中的至少一种。
4.根据权利要求2所述的应用,其特征在于,所述保湿为上调保湿相关基因AQP3和/或GBA的表达。
5.根据权利要求2所述的应用,其特征在于,所述抗炎为:
下调细胞炎症相关因子IL-6和/或TRPV1基因的表达,和/或降低NO的释放水平。
6.一种改善皮肤状况的产品,其特征在于,由包括权利要求1所述发酵乳杆菌的原料制成。
7.根据权利要求6所述的产品,其特征在于,包括如下(1)或(2)所示的一种或两种:
(1)权利要求1所述的发酵乳杆菌的灭活菌;
(2)权利要求1所述的发酵乳杆菌的灭活上清液。
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