CN114736831A - 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ProfMIC‑206寄存于中国典型培养物保藏中心,保藏编号为CCTCC NO:M20211533。实验表明,ProfMIC‑206具有维持及修护皮肤屏障、抗炎、抗氧化、改善敏感肌等功能,对鼻窦炎也具有一定的疗效,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。
益生菌用在化妆品上,可平衡皮肤表皮菌群,修复皮肤屏障。正如文献中报道的,益生菌外用可以治疗特异性皮炎,促进皮肤屏障的修复。同时研究发现益生菌能通过调节巨噬细胞的免疫功能以及吞噬能力来预防和治疗各种炎症疾病,并能有效增加肌肤对营养物质的吸收,抗自由基,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。
鼻窦炎是耳鼻咽喉科学中的常见疾病,主要由呼吸道变态反应、鼻-鼻窦粘膜清洁功能障碍、呼吸道微生物感染、鼻局部解剖结构异常等因素引发。研究表明流感嗜血杆菌(Haemophilus influenzae)是鼻窦炎的重要病原菌,而清酒乳杆菌(Lactobacillussakei)对于维持鼻腔正常功能有积极作用。因此,降低流感嗜血杆菌/清酒乳酸菌的菌群比例,有助于鼻窦炎的治疗。
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:M20211533的发酵乳杆菌(Lactobacillusfermentum),命名为ProfMIC-206。
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、抗氧化、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为FLG和/或OVOL1。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,所述抗氧化为清除超氧阴离子自由基和/或ABTS自由基。
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的生长和/或促进表皮葡萄球菌的增殖。
本发明提供的发酵乳杆菌ProfMIC-206具有调节皮肤微生物菌群的作用,从而改善敏感肌,并对鼻窦炎具有一定的疗效。具体地,发酵乳杆菌ProfMIC-206通过抑制流感嗜血杆菌的生长,降低流感嗜血杆菌/清酒乳杆菌的比例,从而有效治疗鼻窦炎;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,所述产品为食品、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ProfMIC-206,其保藏编号为CCTCC NO:M20211533。实验表明,ProfMIC-206具有维持及修护皮肤屏障、抗氧化、抗炎、改善敏感肌的功能,可用于制备食品、药品、化妆品等。
生物保藏说明
发酵乳杆菌ProfMIC-206(Lactobacillus fermentum ProfMIC-206),于2021年12月3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:M20211533。
附图说明
图1为ProfMIC-206促进HaCaT细胞修复。
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ProfMIC-206,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)ProfMIC-206,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3日,保藏编号为CCTCC NO:M20211533。
进一步,本发明提供的发酵乳杆菌ProfMIC-206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ProfMIC-206具有促进表皮细胞修复的作用,ProfMIC-206提升十二烷基硫酸钠(SDS)损伤的HaCaT细胞存活率达111.69%~136.52%。
体外细胞实验表明,本发明的发酵乳杆菌ProfMIC-206具有上调皮肤屏障修护相关因子丝聚合蛋白基因FLG和OVO样转录因子1基因OVOL1表达的作用,基因表达量上调1.20~1.54倍。
体外细胞实验表明,本发明的发酵乳杆菌ProfMIC-206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31.29%~41.72%。
体外细胞实验表明,本发明的发酵乳杆菌ProfMIC-206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13%~20%。
体外实验表明,本发明的发酵乳杆菌ProfMIC-206具有清除自由基的功能,自由基清除率24.22%~81.87%。
体外实验表明,本发明的发酵乳杆菌ProfMIC-206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ProfMIC-206具有降低流感嗜血杆菌/清酒乳杆菌的比例,从而治疗鼻窦炎的功能。
体外实验表明,本发明的发酵乳杆菌ProfMIC-206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。
以上结果表明,ProfMIC-206具有维持及修护皮肤屏障、抗炎、抗氧化、改善敏感肌等功能,对鼻窦炎具有一定的疗效,可用于制备药品、化妆品等。
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皮肤菌群菌液的体积百分比进行添加,例如,将上清液5%、灭活菌体10%分不同组加入培养过夜的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1 ProfMIC-206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ProfMIC-206。
革兰氏染色镜检:菌株ProfMIC-206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2 ProfMIC-206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ProfMIC-206菌株为发酵乳杆菌(Lactobacillus fermentum)。
实施例3 ProfMIC-206促进SDS诱导的HaCaT细胞损伤修复实验
1、ProfMIC-206菌液制备:
将活化的发酵乳杆菌ProfMIC-206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,离心取沉淀为灭活菌体,灭活发酵液经0.22μm滤膜过滤为灭活上清液。
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,加入5%上清液,孵育24h。加入10μl的CCK-8溶液,孵育4h,检测450nm处吸光值A。
细胞存活率=实验组A/对照组A×100%。
结果:
表1 ProfMIC-206促进HaCaT细胞修复
ProfMIC-206上清液及灭活菌体均对HaCaT细胞SDS损伤具有修复作用。ProfMIC-206促进HaCaT细胞修复结果见图1。
实施例4 ProfMIC-206促进HaCaT屏障修复相关基因表达实验
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。加入菌株上清液5%,灭活菌体10%,分别培养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和OVOL1基因的表达。以不添加菌株上清液和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:
表2 ProfMIC-206上清液上调屏障修护相关基因表达
表3 ProfMIC-206灭活菌体上调屏障修护相关基因表达
结果显示,加入ProfMIC-206具有促进皮肤屏障修护的作用。
实施例5 ProfMIC-206降低Raw264.7细胞NO生成量
1、ProfMIC-206菌液制备
将ProfMIC-206用MRS培养基培养过夜,检测OD600,调整菌液浓度至OD600=0.2,离心后,菌体121℃高压灭菌30min得菌体,离心上清用0.22μm滤膜过滤得上清液。
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、ProfMIC-206添加及LPS刺激
将上清液5%、灭活菌体10%分不同组加入培养过夜的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:
表4ProfMIC-206降低Raw264.7细胞NO生成量
结果显示ProfMIC-206具有抗炎作用,能够降低LPS诱导的Raw264.7细胞NO生成量,与LPS对照组相比上清液降低31.29%,灭活菌体降低41.72%。
实施例6 ProfMIC-206下调HaCaT细胞炎性因子的表达
1、ProfMIC-206样品制备
将ProfMIC-206用MRS培养过夜,检测OD600,调整菌液浓度至OD600=0.2,离心后,菌体121℃高压灭菌30min得菌体,离心上清用0.22μm滤膜过滤得上清液。
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,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、ProfMIC-206样品添加
将ProfMIC-206上清液以5%加入金黄色葡萄球菌刺激过的HaCaT细胞中,每组3个复孔,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,以不添加菌株上清液的处理组为对照,其基因表达量为1,计算炎症因子基因相对表达倍数F。
公式:F=2-ΔΔCT
结果:
表5 ProfMIC-206下调炎症因子基因的表达
结果显示ProfMIC-206能够下调金黄色葡萄球菌诱导的HaCaT炎性因子IL-6、TRPV1基因表达量降低,IL-6表达量降低20%,TRPV1表达量降低13%。因此,ProfMIC-206具有抗炎作用。
实施例7 ProfMIC-206自由基清除能力检测
1、发酵乳杆菌ProfMIC-206菌液制备:
将活化的发酵乳杆菌ProfMIC-206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滤膜过滤获得待测样品。
2、羟自由基清除能力检测
试剂配制和检测方法按照索莱宝羟自由基清除能力检测试剂盒说明书进行。测定各样品536nm吸亮度,求平均值并计算各样品清除率,公式如下:
羟自由基清除率D%=(A测-A对)÷(A空-A对)×100%
3、超氧阴离子自由基清除能力检测
试剂配制和检测方法按照索莱宝超氧阴离子清除能力检测试剂盒说明书进行。测定各样品530nm吸亮度,求平均值并计算各样品清除率,公式如下:
超氧阴离子自由基清除率D%=(A对照-A测定)÷A对照×100%
4、ABTS自由基清除能力检测
试剂配制和检测方法按照索莱宝ABTS自由基清除能力检测试剂盒说明书进行。测定各样品405nm吸亮度,求平均值并计算各样品清除率,公式如下:
ABTS自由基清除率D%=[A空白-(A测定-A对照)]÷A空白×100%
结果:
表6 ProfMIC-206羟自由基清除率
表7 ProfMIC-206超氧自由基清除率
表8 ProfMIC-206ABTS自由基清除率
结果显示,ProfMIC-206对羟自由基、超氧阴离子自由基和ABTS自由基均具有清除作用,因此ProfMIC-206具有抗氧化功效。
实施例8 ProfMIC-206改变菌群比例治疗痤疮的作用
1、发酵乳杆菌ProfMIC-206菌液制备:
将活化的发酵乳杆菌ProfMIC-206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、添加上清液影响痤疮相关菌群生长实验
将灭活上清液以10%分别加入两种菌液中,痤疮丙酸杆菌37℃培养16h,表皮葡萄球菌37℃培养2h,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。
4、结果
表9 ProfMIC-206对痤疮相关菌群生长影响
结果显示ProfMIC-206对痤疮丙酸杆菌有显着抑制作用,而对表皮葡萄球菌有一定促进增殖作用。ProfMIC-206能够明显改变皮肤菌群比例,从而达到治疗痤疮的目的。
实施例9 ProfMIC-206改变菌群比例治疗鼻窦炎的作用
1、发酵乳杆菌ProfMIC-206菌液制备:
将活化的发酵乳杆菌ProfMIC-206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、鼻腔菌群菌液制备:
将流感嗜血杆菌ATCC 49766以BHI培养基、清酒乳杆菌以MRS培养基37℃培养18h,检测并调整OD600=0.2。
3、添加上清液影响鼻腔菌群生长实验
将灭活上清液以10%分别加入两种菌液中,37℃培养3h,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(对照=100%),以流感嗜血杆菌/清酒乳杆菌相对浓度比值评价对鼻腔菌群生长影响。
4、结果
表10 ProfMIC-206对鼻窦炎相关菌群影响
结果显示ProfMIC-206对流感嗜血杆菌有显着抑制作用,而对清酒乳杆菌有一定促进增殖作用。ProfMIC-206能够明显改变鼻窦炎相关菌群比例,从而有效治疗鼻窦炎。
实施例10 ProfMIC-206改变菌群比例改善敏感肌的作用
1、发酵乳杆菌ProfMIC-206菌液制备:
将活化的发酵乳杆菌ProfMIC-206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、敏感肌相关菌群菌液制备:
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,37℃培养2h,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
表11 ProfMIC-206对敏感肌相关菌群生长影响
结果显示ProfMIC-206对皮肤主要病原菌金黄色葡萄球菌有显着抑制作用,而对表皮葡萄球菌有一定促进增殖作用。ProfMIC-206能够改变皮肤菌群比例,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 (10)
1.保藏编号为CCTCC NO:M20211533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。
3.根据权利要求2所述的应用,其特征在于,所述改善肌肤状况包括修护皮肤屏障、抗氧化、抗炎、调节皮肤微生物菌群的比例中的至少一种。
4.根据权利要求3所述的应用,其特征在于,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;所述屏障修护相关基因为FLG和/或OVOL1。
5.根据权利要求3所述的应用,其特征在于,所述抗氧化为清除超氧阴离子自由基和/或ABTS自由基。
6.根据权利要求3所述的应用,其特征在于,所述调节皮肤微生物菌群的比例为抑制金黄色葡萄球菌的生长和/或促进表皮葡萄球菌的增殖。
7.根据权利要求3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。
8.根据权利要求2~7任一项所述的应用,其特征在于,所述产品为食品、药品或化妆品。
9.一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。
10.根据权利要求9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活菌和/或灭活菌;
(2)权利要求1所述的发酵乳杆菌的培养物、代谢物、裂解物和/或提取物。
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