CN114574393A - 一种德氏乳杆菌seuneu-110及其在皮肤方面的应用 - Google Patents
一种德氏乳杆菌seuneu-110及其在皮肤方面的应用 Download PDFInfo
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Abstract
本发明提供一种德氏乳杆菌SEUNEU‑110,其分类名为Lactobacillus delbrueckii,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20211561,其菌株为经间歇灭菌的,其生理状态为活的或死的;所述德氏乳杆菌具有皮肤修护的功能,所述皮肤修护功能包括降低细胞ROS生成量抗氧化、清除自由基、上调皮肤屏障修复相关基因的表达、下调HaCaT细胞炎性因子基因的表达、降低Raw264.7细胞一氧化氮的生成量以及上调保湿相关基因的表达;所述德氏乳杆菌,其功能型菌株存在形式包括其培养上清液和灭活菌株,可应用领域包括食品、药品和化妆品。
Description
技术领域
本发明属于微生物技术领域,具体为一种德氏乳杆菌SEUNEU-110及其在皮肤方面的应用。
背景技术
在生命系统中,分子构建出细胞,细胞搭建起器官,器官终联系成系统;在每一层的架构之外都存在特殊的屏障,保证屏障以内能隔开外界,保证屏障以内可获取信息;皮肤是人体最大的器官,它覆盖于我们的整体架构之外,形成我们整体的第一道防线,维持我们整个内部系统的平衡状态。但由于皮肤长期暴露在外界环境中,会遭到外界各种因素的影响,皮肤本身就会逐渐失去原有的活力,甚至开始出现病变,因此需要寻找有效且安全的技术方法来解决这些问题。
益生菌一大类在我们生活中的应用逐渐广泛,对已存在菌种功能的挖掘以及对新菌种的开发工作也在不断取得进展,其作用范围包括食品、药品以及化妆品各个领域。近些年,益生菌作为化妆品对改善皮肤问题起到了重要作用,其有效作用形式多以组合物的方式呈现,且主要起发酵作用。
在中国专利CN202110300709.3中提供了一种益生菌组合物包括鼠李糖乳杆菌和德氏乳杆菌在皮肤方面的应用,其作用为降低IL-10Y和IL-6的表达以及提升皮肤的免疫功能;其发挥功能的有效成分为具有生物活性益生菌组合物整体,文件中涉及的单独菌体则无法实现同等效果。因此益生菌领域的开发,包括菌种多样性以及功能多样性的开发具有很大的实际意义。
发明内容
本申请发明人经大量实验从酸菜浆水中筛选到了一株具有优异益生菌功能的德氏乳杆菌SEUNEU-110,经大量实验证实,该德氏乳杆菌SEUNEU-110对皮肤修护相关因子的表达具有明显的调控作用。
因此,第一方面,本申请提供了一种德氏乳杆菌,其分类名为Lactobacillusdelbrueckii,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M20211561。
优选的,所述德氏乳杆菌其生理状态为活的、死的以及经间歇灭菌的。
第二方面,本申请提供了一种德氏乳杆菌具有皮肤修护的功能,应用领域包括食品、药品以及化妆品。
进一步的,所述皮肤修护功能包括降低细胞ROS生成量抗氧化、、清除自由基、上调皮肤屏障修复相关基因的表达、下调HaCaT细胞炎性因子基因的表达、降低Raw264.7细胞一氧化氮的生成量以及上调保湿相关基因的表达。
进一步的,所述德氏乳杆菌其功能性菌株存在形式包括其培养上清液和灭活菌株。
优选的,所述培养上清液制备方法包括:
(1)在MRS固体平板上接种培养德氏乳杆菌,获得生长均匀的菌株;
(2)从所得菌株中挑取单菌落菌株,将其置于MRS液体培养基中进行37℃恒温培养过夜,获得长势优良且生理特征明显的菌体培养产物;
(3)将所得培养产物用酶标仪检测OD600值,调整培养基浓度,至OD600=0.2;
(4)将调整后的培养基离心,即得德氏乳杆菌培养上清液。优选的,所述灭活菌株制备过程包括:
(1)在MRS固体平板上接种培养德氏乳杆菌,获得生长均匀的菌株;
(2)从所得菌株中挑取单菌落菌株,将其置于MRS液体培养基中进行37℃恒温培养过夜,获得长势优良且生理特征明显的菌体培养产物;
(3)将所得液体培养基进行离心,离心后弃掉上清液;
(4)在弃掉上清液的培养基中加入PBS缓冲液,对培养基进行稀释,最终得到OD600=0.2的培养基;
(5)将培养基置于121℃中高压灭活30min,即得灭活菌体。
与现有技术相比,本发明具有下述有益技术效果:
本发明提供一种德氏乳杆菌SEUNEU-110,其分类名为Lactobacillusdelbrueckii,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20211561,其菌株生理状态包括活的、死的以及经间歇灭菌的;所述德氏乳杆菌具有皮肤修护的功能,所述皮肤修护功能包括降低细胞ROS生成量抗氧化,清除率可达21.5-28.9%;清除自由基DPPH清除率18%~55%、ABTS自由基清除率35%~105%;上调皮肤屏障修复相关基因FLG、IVL、OVOL1、LOR的表达,基因表达量上调1.1~3.53倍;下调HaCaT细胞炎性因子IL-6、IL-8、IL-22、环氧合酶-2基因COX-2、辣椒素受体1基因以及TRPV1基因的表达,基因表达量下调12.65%~72.39%;降低Raw264.7细胞NOD的生成量达55.03%~76.65%;上调保湿相关基因AQP3、GBA的表达,上调量为1.1~3.6倍;所述德氏乳杆菌,其功能型菌株存在形式包括其培养上清液和灭活菌株,可应用领域包括食品、药品和化妆品。
附图说明
图1为德氏乳杆菌SEUNEU-110下调炎症因子相关基因表达的效果图
图2为德氏乳杆菌SEUNEU-110上清液上调屏障修护相关基因表达的效果图
图3为德氏乳杆菌SEUNEU-110灭活菌体上调屏障秀相关基因表达的效果图
实施方式
以下结合实施例对本发明做进一步的说明,应当理解,本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,为了更好地说明本发明的内容,本领域技术人员应当理解,没有某些具体细节,本发明同样可以实施。在另外一些实施例中,对于本领域技术人员熟知的方法、手段未作详细描述,以便于凸显本发明的主旨。
实施例一:
菌种分离
本申请从酸菜浆水中采样,分离筛选了一种德氏乳杆菌,命名为德氏乳杆菌SEUNEU-110。
(1)从酸菜浆水中取样,将所得样品置于生理盐水中震荡混匀,使菌体细胞维持固有形态;
(2)从生理盐水上清液中取少量菌体划线于MRS固体培养基上进行培养,保持37℃恒温培养48h,保持菌体的不断繁殖生长;
(3)将所得菌体进行反复筛选,挑取状态完好的单个菌落,即为德氏乳杆菌SEUNEU-110。
(4)对菌体进行革兰氏染色镜检,结果为:菌株显示革兰氏染色阳性,在显微镜下呈杆状;当其在MRS固体培养基上进行培养时,形成的菌落为白色的表面光滑且不透明的圆形菌落,边缘整齐;其在MRS液体培养基中呈现浑浊生长,久置菌体成白色沉淀。
菌体核酸鉴定
(1)将所得德氏乳杆菌SEUNEU-110单菌落置于MRS液体培养基中进行37℃恒温培养过夜;
(2)将培养完成的液体培养基进行离心处理,收集菌体;
(3)按照DNA提取试剂盒的步骤对所得菌体进行PCR扩增和延伸反应;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;
(4)将菌体DNA的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)后得出德氏乳杆菌SEUNEU-110菌株为德氏乳杆菌Lactobacillusdelbrueckii。
德氏乳杆菌SEUNEU-110培养上清液的制备
(1)在MRS固体平板上接种培养德氏乳杆菌,获得生长均匀的菌株;
(2)从所得菌株中挑取单菌落菌株,将其置于MRS液体培养基中进行37℃恒温培养过夜;
(3)将所得培养产物用酶标仪检测OD600值,调整培养基浓度,至OD600=0.2;
(4)将调整后的培养基离心,即得德氏乳杆菌培养上清液。
德氏乳杆菌SEUNEU-110灭活菌体的制备
(1)在MRS固体平板上接种培养德氏乳杆菌,获得生长均匀的菌株;
(2)从所得菌株中挑取单菌落菌株,将其置于MRS液体培养基中进行37℃恒温培养过夜;
(3)将所得液体培养基进行离心,离心后弃掉上清液;
(4)在弃掉上清液的培养基中加入PBS缓冲液,对培养基进行稀释,最终得到OD600=0.2的培养基;
(5)将培养基置于121℃中高压灭活30min,即得灭活菌体。
德氏乳杆菌SEUNEU-110溶胞物的制备
(1)将德氏乳杆菌SEUNEU-110置于锥形瓶中静置培养至OD600=8.0;
(2)将所得OD600=8.0的菌液分装至50ml离心管中,保持4℃恒温进行6000rpm离心5min;
(3)离心后用灭菌的生理盐水洗涤两次,再用生理盐水重悬至60-70ml,调整至OD600=0.8;
(4)将菌悬液高压均质1min,需接入循环水降温;
(5)将均质后的悬液4℃,6000rpm离心5min,取上清液,-20℃冻存备用。
德氏乳杆菌SEUNEU-110降低细胞ROS生成量的抗氧化实验
(1)用碧云天生物技术研究所所产SOD酶活力检测试剂盒和CAT酶活力检测试剂盒对以上所得胞溶物进行检测,测定德氏乳杆菌菌体胞溶物的超氧化物歧化酶和过氧化氢酶的酶活力,检测结果见表1;
(2)将HaCaT细胞接种于黑底96孔的培养板上,培养24h;
(3)培养完成,弃去培养基进行洗涤,加入50μg/ml十二烷基硫酸钠SDS培2h,十二烷基硫酸钠SDS诱导细胞产生活性氧簇ROS造成细胞氧化损伤;
(4)培养完成后加入30%浓度德氏乳杆菌功能性菌株进行共同培养,同时设置三组平行对照,提高数据准确度;
(5)培养结束后去掉上清液进行洗涤,在每个孔中各加入100μL的DCFH-DA溶液,在培养箱中孵育培养30min,去掉上清液洗涤3次。
(6)洗涤后在每个孔中各加入100μL的ABAP溶液,于荧光酶标仪中进行0~60min的动力学检测,发射波长535nm,激发波长485nm,结果见表2。
表1:
SEUNEU-110抗氧化酶活力检测
表2:
SEUNEU-110降低HaCaT细胞ROS生成量
根据表1数据可知,所述德氏乳杆菌SEUNEU-110具有较高的SOD酶和CAT酶活性;根据表2数据可知,所述德氏乳杆菌SEUNEU-110能够降低十二烷基硫酸钠SDS诱导的HaCaT细胞ROS生成量,降低活性氧簇ROS对HaCaT细胞造成的氧化损伤,具有抗氧化作用。
实施例二:
德氏乳杆菌SEUNEU-110的分离、核酸鉴定、上清液的制备、灭活菌体的制备以及其胞溶物的制备,实验操作均与实施例一中的方法相同,操作结束即获得具有皮肤修复能力的乳酸片球菌的培养上清液、灭活菌株和具备SOD酶和CAT酶活性的溶胞物。
德氏乳杆菌SEUNEU-110清除自由基的实验
(1)将DPPH固体溶于甲醇,溶解后调整溶液至200μl时酶标仪下517nm吸光度为1.6;取1.5ml离心管,分别加入200μl的25%、50%以及100%的德氏乳杆菌胞溶物和200μlDPPH溶液,避光反应25min后8000rpm离心5min;离心后取上清液在酶标仪517nm下对其吸光度进行测定,吸光度水平的降低表明抗氧化性的增加,从而用来评价所述德氏乳杆菌的抗氧化能力。
(2)称取6.6mg过硫酸钾溶液和10ml水,再取2ml过硫酸钾加入7.7mg ABTS,进行混合,放置16h;将上述ABTS溶液用去离子水稀释,调至酶标仪734nm处吸光度为0.9;分别在96孔培养板加入25%、50%以及100%的德氏乳杆菌胞溶物50μl,再各加入150μl ABTS;避光反应15min后在酶标仪734nm下对其吸光度进行测定,吸光度水平的降低表明抗氧化性的增加,从而用来评价所述德氏乳杆菌的抗氧化能力;
(3)德氏乳杆菌SEUNEU-110对两种自由基的清除效果如下表。
SEUNEU-110自由基清除率%(n=3)
通过表中数据可知德氏乳杆菌SEUNEU-110对DPPH自由基和ABTS自由基的清除率呈剂量依赖性,浓度为100%时效果最佳,德氏乳杆菌SEUNEU-110添加浓度在100%浓度时基本可完全清除ABTS。
实施例三:
德氏乳杆菌SEUNEU-110的分离、核酸鉴定、上清液的制备、灭活菌体的制备以及其胞溶物的制备,实验操作均与实施例一中的方法相同,操作结束即获得具有皮肤修复能力的乳酸片球菌的培养上清液、灭活菌株和具备SOD酶和CAT酶活性的溶胞物。
德氏乳杆菌SEUNEU-110上调HaCaT细胞屏障修护相关基因表达的实验
(1)接种人永生化角质形成细胞HaCaT(5×105/孔)至6孔培养板,过夜培养至细胞贴壁;
(2)将培养所得细胞产物与5%菌株上清液和10%灭活菌体分别混合培养24h,同时对两类实验各设置三组平行试验作为对照,提高实验数据的准确度;
(3)在所得培养产物中加入裂解液,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、LOR、IVL和OVOL1基因的表达,计算表达变化倍数F=2-ΔΔCT,结果见下表。
SEUNEU-110灭活菌体上调修复基因的表达
通过表中数据可知,德氏乳杆菌SEUNEU-110对皮肤屏障修复相关基因FLG、LOR、IVL和OVOL1的表达的表达具有促进作用,能提高皮肤自身的修复功能。
实施例四:
德氏乳杆菌SEUNEU-110的分离、核酸鉴定、上清液的制备、灭活菌体的制备以及其胞溶物的制备,实验操作均与实施例一中的方法相同,操作结束即获得具有皮肤修复能力的乳酸片球菌的培养上清液、灭活菌株和具备SOD酶和CAT酶活性的溶胞物。
德氏乳杆菌SEUNEU-110下调HaCaT细胞炎症因子相关基因的表达
(1)在24孔培养板上接种一定量经过细胞消化后的HaCaT细胞,将其置于5%二氧化碳培养箱中保持37℃恒温培养过夜;
(2)将金黄色葡萄球菌接种至营养肉汤培养基上,设置37℃摇床培养过夜;
(3)在所得金黄色葡萄球菌菌液中加入MEM无血清培养基,调整其浓度至OD600=6;
(4)在培养过后的HaCaT细胞培养板上各孔各添加100μl的OD600=6的金黄色葡萄球菌菌液,培养3h,刺激HaCaT细胞产生炎症因子;
(5)培养完成后弃掉细胞培养基,用PBS缓冲液清洗所得HaCaT细胞培养产物5次,每孔重新加入1ml的MEM无血清培养基;
(6)在所得HaCaT细胞培养液中加入5%的乳酸片球菌SEUNEU-106培养上清液,每组3个复孔,37℃恒温培养过夜。
(7)将所得培养产物弃掉培养基,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,用反转录试剂盒和SYBRGreen qPCR试剂盒进行RT-PCR及qPCR,计算炎症因子IL-6、IL-8、IL-22、COX-2、TRPV1基因相对表达倍数F=2-ΔΔCT,结果如下表。
SEUNEU-110下调炎症相关因子的表达
通过表中数据可知,德氏乳杆菌SEUNEU-110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IL-6、IL-8、IL-22、COX-2、TRPV1的表达,表达量下调10.85%~78.53%。因此,SEUNEU-110具有抵抗皮肤炎症的作用。
实施例五:
德氏乳杆菌SEUNEU-110的分离、核酸鉴定、上清液的制备、灭活菌体的制备以及其胞溶物的制备,实验操作均与实施例一中的方法相同,操作结束即获得具有皮肤修复能力的乳酸片球菌的培养上清液、灭活菌株和具备SOD酶和CAT酶活性的溶胞物。
德氏乳杆菌SEUNEU-110降低Raw264.7细胞NO生成量的实验
(1)在24孔细胞培养板上接种一定量经过细胞消化后的Raw264.7细胞,将其置于5%二氧化碳培养箱中保持37℃恒温培养过夜。
(2)将培养后的Raw264.7细胞与5%上清液混合培养2h,同时设置三组平行试验作为对照,提高实验数据的准确度;
(3)在所得各混合液中分别加入0.5ml的0.2μg/ml的LPS溶液培养20h,诱导Raw264.7细胞发炎;
(4)培养完成后通过NO含量检测试剂盒测定所得细胞培养上清液的NO含量,每次3个复孔,共进行三批实验,结果如下表。
SEUNEU-110降低Raw264.7细胞NO生成量
通过表中数据可知,德氏乳杆菌SEUNEU-110具有抵抗皮肤炎症的作用,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66.10%。
实施例六:
德氏乳杆菌SEUNEU-110的分离、核酸鉴定、上清液的制备、灭活菌体的制备以及其胞溶物的制备,实验操作均与实施例一中的方法相同,操作结束即获得具有皮肤修复能力的乳酸片球菌的培养上清液、灭活菌株和具备SOD酶和CAT酶活性的溶胞物。
德氏乳杆菌SEUNEU-110上调HaCaT细胞保湿相关基因表达的实验
(1)接种人永生化角质形成细胞HaCaT(5×105/孔)至6孔培养板,过夜培养至细胞贴壁;
(2)将培养所得细胞产物与5%菌株上清液和10%灭活菌体分别混合培养24h,同时对两类实验各设置三组平行试验作为对照,提高实验数据的准确度;
(3)在所得培养产物中加入裂解液,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AQP3、GBA基因的表达,计算表达变化倍数F=2-ΔΔCT,结果见下表。
SEUNEU-110上清液上调保湿基因的表达
SEUNEU-110灭活菌体上调保湿基因的表达
通过表中实验数据可知,德氏乳杆菌SEUNEU-110能够上调保湿相关基因AQP3、GBA的表达,上调量为1.1~3.6倍。
以上对本发明具体实施方式的描述并不限制本发明,本领域技术人员可以根据本发明作出各种改变或变形,只要不脱离本发明的精神,均应属于本发明权利要求保护的范围。
Claims (7)
1.一种德式乳杆菌,其分类名为Lactobacillus delbrueckii,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20211561。
2.根据权利要求1所述的一种德氏乳杆菌,其特征在于:所述德氏乳杆菌为经间歇灭菌的,其生理状态为活的或死的。
3.一种德式乳杆菌在皮肤方面的应用,其特征在于:所述德氏乳杆菌具有皮肤修护的功能,应用领域包括食品、药品以及化妆品。
4.根据权利要求3所述的一种德式乳杆菌在皮肤方面的应用,其特征在于:所述皮肤修护功能包括降低细胞ROS生成量抗氧化,和/或清除自由基,和/或上调皮肤屏障修复相关基因的表达,和/或下调HaCaT细胞炎性因子基因的表达,和/或降低Raw264.7细胞一氧化氮的生成量,和/或上调保湿相关基因的表达。
5.根据权利要求3所述的一种德式乳杆菌在皮肤方面的应用,其特征在于:所述德氏乳杆菌其功能性菌株存在形式包括其培养上清液和灭活菌株。
6.根据权利要求5所述的一种德氏乳杆菌在皮肤方面的应用,其特征在于:所述培养上清液制备方法包括:
(1)在MRS固体平板上接种培养德氏乳杆菌,获得生长均匀的菌株;
(2)从所得菌株中挑取单菌落菌株,将其置于MRS液体培养基中进行37℃恒温培养过夜;
(3)将所得培养产物用酶标仪检测OD600值,调整培养基浓度,至OD600=0.2;
(4)将调整后的培养基离心,即得德氏乳杆菌培养上清液。
7.根据权利要求5所述的一种德氏乳杆菌在皮肤方面的应用,其特征在于:所述灭活菌株制备过程包括:
(1)在MRS固体平板上接种培养德氏乳杆菌,获得生长均匀的菌株;
(2)从所得菌株中挑取单菌落菌株,将其置于MRS液体培养基中进行37℃恒温培养过夜;
(3)将所得液体培养基进行离心,离心后弃掉上清液;
(4)在弃掉上清液的培养基中加入PBS缓冲液,对培养基进行稀释,最终得到OD600=0.2的培养基;
(5)将培养基置于121℃中高压灭活30min,即得灭活菌体。
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