CN115491334A - 一株鼠李糖乳杆菌及其应用 - Google Patents
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Abstract
本发明公开了一株鼠李糖乳杆菌及其应用,涉及微生物技术领域。本发明公开的鼠李糖乳杆菌(Lactobacillus rhamnosus),该菌株被命名为GforU‑32,已于2022年8月31日保藏在中国典型培养物保藏中心,其保藏编号为CCTCC No:M 20221355的鼠李糖乳杆菌。实验表明,GforU‑32具有抗衰老、提高细胞抗菌能力的功能,可用于制备食品、药品、化妆品等。
Description
技术领域
本发明属于微生物的技术领域,具体地涉及一株鼠李糖乳杆菌及其应用。
背景技术
人体的皮肤一般从25~30岁以后即随着年龄的增长而逐渐衰老,大约在35~40岁后逐渐出现比较明显的衰老变化。皮肤衰老也称为皮肤老化,是皮肤组织在内外环境的不断作用下细胞结构功能发生退化,出现皮肤质地改变(如弹性、张力、厚薄及表面纹理等)、色素改变、血管萎缩或增生的生理现象。皮肤衰老通常分为内源性老化(自然衰老)和外源性老化(光老化)。在衰老机制的研究中发现,减少细胞凋亡,降低细胞炎症,增加细胞外基质的合成以及减少其降解,提升细胞的抗氧化能力等都已被证实能够减缓皮肤细胞衰老。寻求针对不同皮肤老化途径并且提升皮肤细胞状态的抗衰老物质是目前重要的研究方向。
人体的皮肤表面由细菌、真菌、病毒等各种微生物,与皮肤组织、细胞以及各种分泌物、微环境等共同组成了一个微观的生态***,即皮肤微生态。皮肤上共生微生物的存在会导致其对皮肤上营养和空间的竞争,从而极大地影响病原微生物进入皮肤表面。这些常驻微生物可以直接或通过调控皮肤的角质形成细胞而间接分泌抗菌肽,以抵御外界病原菌定殖;还可分解角质细胞碎屑或脂质,辅助乳化皮脂膜,维持表皮酸性环境,以抵御外界病原菌定殖。正常情况下,皮肤表面各种微生物、宿主以及外环境三者互相作用,构成了微生态平衡,可以一定程度上抵御外界对皮肤的伤害,保持皮肤健康。
因此,提供一种利用微生物技术开发的皮肤微生态相关产品,在改善皮肤状态方面具有广大的应用价值。
发明内容
有鉴于此,本发明的目的是提供了一株鼠李糖乳杆菌及其应用。
本发明的其一目的是提供了鼠李糖乳杆菌(Lactobacillus rhamnosus),该菌株被命名为GforU-32,已于2022年8月31日保藏在中国典型培养物保藏中心,其保藏编号为CCTCC No:M 20221355的鼠李糖乳杆菌。
较佳地,采样于健康女童粪便,将样品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GforU-32;
在革兰氏染色镜检下菌株GforU-32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、表面光滑不透明的圆形菌落,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。
本发明的另一目的是提供了上述鼠李糖乳杆菌在制备改善肌肤状况的产品中的应用,其中,所述改善肌肤状况包括促进细胞增殖、抗衰老、提升皮肤抗菌能力中的至少一种。
一些实施方案中,所述抗衰老为上调细胞外基质相关基因的表达;所述细胞外基质相关基因由SPTSSA、TIMP1、LN、FN、COL1A1中的至少一种组成。
一些实施方案中,所述抗衰老为上调抑制细胞凋亡相关基因BCL-2的表达。
一些实施方案中,所述抗衰老为上调细胞抗氧化相关基因NRF2和/或SIRT-3的表达。
一些实施方案中,所述抗衰老为下调降解细胞外基质相关基因的表达;所述降解细胞外基质相关基因由P38MAPK、MMP家族中的至少一种组成。
一些实施方案中,所述抗衰老为下调细胞凋亡相关基因的表达;所述细胞凋亡相关基因由Caspase家族的至少一种组成。
一些实施方案中,所述提升皮肤抗菌能力为上调抗菌肽相关基因S100A7、S100A8、DEFB1和/或DEFB4的表达。
本发明的再一目的是提供了一种改善皮肤状况的产品,所述产品为食品、药品或化妆品,由包括上述鼠李糖乳杆菌原料制成,所述产品中的鼠李糖乳杆菌包括如下所示的至少一种:
(1)鼠李糖乳杆菌的活菌和/或灭活菌;
(2)鼠李糖乳杆菌的培养物、外泌体、裂解物和/或提取物。
本发明公开的鼠李糖乳杆菌(Lactobacillus rhamnosus)GforU-32,其保藏编号为CCTCC No:M 20221355的鼠李糖乳杆菌。实验表明,GforU-32具有抗衰老、提高细胞抗菌能力的功能,可用于制备食品、药品、化妆品等。
生物保藏说明
鼠李糖乳杆菌(Lactobacillus rhamnosus)GforU-32,于2022年8月31日保藏在中国典型培养物保藏中心(简称CCTCC,地址为:武汉市武昌区八一路299号,武汉大学,邮编430072),其保藏编号为CCTCC No:M 20221355。
具体实施方式
本发明提供了鼠李糖乳杆菌及其应用。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。
本发明的鼠李糖乳杆菌菌株GforU-32,来源于6岁健康女孩粪便,经16SrDNA鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)。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℃。
鼠李糖乳杆菌(Lactobacillus rhamnosus)GforU-32,于2022年8月31日保藏在中国典型培养物保藏中心(简称CCTCC,地址为:武汉市武昌区八一路299号,武汉大学,邮编430072),其保藏编号为CCTCC No:M 20221355。
进一步,本发明提供的鼠李糖乳杆菌GforU-32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、外泌体、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,本发明的鼠李糖乳杆菌GforU-32具有上调HaCaT细胞外基质相关的丝氨酸棕榈酰转移酶基因SPTSSA、组织金属蛋白酶抑制物1基因TIMP1和I型胶原α1链基因COL1A1,以及抗氧化相关的核因子E2相关因子2基因NRF2表达的作用,基因相对表达倍数1.13~1.66;具有下调降解细胞外基质相关的丝氨酸/苏氨酸蛋白激酶基因P38MAPK和基质金属蛋白酶家族基因MMP1,基因相对表达倍数0.22~0.81。
体外细胞实验表明,本发明的鼠李糖乳杆菌GforU-32具有促进人成纤维细胞HFF增殖的作用,增殖率115.74%~118.11%。
体外细胞实验表明,本发明的鼠李糖乳杆菌GforU-32具有上调HFF细胞外基质相关的丝氨酸棕榈酰转移酶基因SPTSSA、层粘连蛋白基因LN和纤维连接蛋白基因FN,抗氧化相关的Sirtuins蛋白家族基因SIRT-3,以及抑制细胞凋亡相关的B淋巴细胞瘤-2基因BCL-2表达的作用,基因相对表达倍数1.23~1.76;具有下调降解细胞外基质相关的基质金属蛋白酶家族基因MMP家族,细胞凋亡相关的半胱氨酸蛋白酶家族基因Caspase家族,基因相对表达倍数0.30~0.80。
体外细胞实验表明,本发明的鼠李糖乳杆菌GforU-32具有上调抗菌肽相关基因银屑病素基因S100A7、钙粒蛋白A基因S100A8、β防御素1基因DEFB1和β防御素4基因DEFB4表达的作用,基因相对表达倍数1.26~3.36。
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:GforU-32的分离
采样于6岁健康女孩粪便中采样。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GforU-32。
革兰氏染色镜检:菌株GforU-32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:GforU-32的核酸鉴定
1、16S rDNA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,12000r/min转速离心1min收集菌体,按照DNA提取试剂盒步骤进行操作。引物采用细菌通用引物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GforU-32菌株为鼠李糖乳杆菌(Lactobacillus rhamnosus)。
实施例3:GforU-32调节光老化HaCaT细胞外基质/细胞自噬/炎症因子相关基因表达实验
1、GforU-32上清液及灭活菌体制备:
挑取鼠李糖乳杆菌GforU-32单菌落于MRS液体培养基,37℃培养箱静置培养16h~18h,酶标仪检测并以PBS稀释调整OD600=0.2,121℃,30min、0.28Mpa灭活,12000r/min转速离心2min,经0.22μm滤膜过滤为上清液。离心沉淀以适量PBS重悬,稀释调整OD600=0.2,为灭活菌体。
2、HaCaT细胞制备及紫外线损伤
将HaCaT细胞消化后以0.5ml/孔(内含2×105细胞)接种至24孔板,5%二氧化碳培养箱37℃培养过夜。对孔内细胞进行总剂量为2J/cm2的紫外线UVB照射损伤。
3、GforU-32添加
将上清液5%(V/V)、灭活菌体10%(V/V)分别加入刺激过的HaCaT细胞(对照组分别以等体积PBS替代上清液/灭活菌体)。每组3平行,37℃培养过夜。
4、qPCR法检测细胞外基质/细胞自噬相关基因相对表达倍数
将上述细胞弃去培养基后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度后反转录为cDNA,以GAPDH为内参基因,采用实时qPCR检测细胞外基质相关基因SPTSSA、TIMP1和COL1A1,抗氧化相关基因NRF2,以及降解细胞外基质相关基因P38MAPK和MMP1的表达。以对照组基因相对表达倍数F=1,利用2-ΔΔCT法计算各样品F值。
公式:F=2-ΔΔCT,其中:
△CT实验=CT实验-CT内参(实验);
△CT对照=CT对照-CT内参(对照);
△△CT=△CT实验-△CT对照。
上清液上调细胞外基质相关基因TIMP1,抗氧化相关基因NRF2;下调降解细胞外基质相关基因MMP1。结果见下表:
灭活菌体上调细胞外基质相关基因SPTSSA、TIMP1和COL1A1;下调降解细胞外基质相关基因P38MAPK和MMP1。结果见下表:
结果显示加入GforU-32具有促进HaCaT细胞外基质合成、增加细胞抗氧化和减少细胞外基质降解的抗衰老作用。
实施例4:GforU-32促进人成纤维细胞HFF的增殖
1、GforU-32上清液制备:
制备方法参照实施例3。
2、HFF细胞制备及GforU-32添加
接种以DMEM培养的HFF细胞,以100ul/孔(每孔含3×104细胞)转接至96孔板,过夜培养至细胞贴壁。配制200μM H2O2,每孔加入100μL,5%二氧化碳培养箱37℃培养1h。弃掉原培养基,PBS清洗两次,加入100μL新培养基,各孔分别添加灭活菌体10%(V/V),(对照组分别以等体积PBS替代上清液/灭活菌体)。培养24h。向每孔加入10μL的CCK-32溶液,培养4h,检测450nm处吸亮度A。
计算公式及结果如下表:
结果显示GforU-32具有促进HFF细胞增殖的作用。
实施例5:GforU-32调节氧化损伤HFF细胞外基质/细胞凋亡/抗氧化相关基因表达实验
1、GforU-32上清液及灭活菌体制备:
制备方法参照实施例3。
2、HFF细胞制备及H2O2诱导氧化损伤
将DMEM培养的HFF细胞消化后以0.5ml/孔(内含2×105细胞)接种至24孔板,5%二氧化碳培养箱37℃培养过夜。每孔加入终浓度为200μM的H2O2进行刺激,37℃静置1h。
3、GforU-32添加
将上清液5%(V/V)、灭活菌体10%(V/V)分别加入刺激过的HFF细胞(对照组分别以等体积PBS替代上清液/灭活菌体)。每组3平行,37℃培养过夜。
4、qPCR法检测细胞外基质/细胞凋亡/抗氧化相关基因相对表达倍数
将上述细胞弃去培养基后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度后反转录为cDNA,以GAPDH为内参基因,采用实时qPCR检测细胞外基质相关基因SPTSSA、LN和FN,抑制细胞凋亡相关基因BCL-2,抗氧化相关基因SIRT-3;以及降解细胞外基质相关基因MMP家族,细胞凋亡相关基因Caspase家族的表达。以对照组基因相对表达倍数F=1,利用2-ΔΔCT法计算各样品F值。
上清液上调抑制细胞凋亡基因BCL-2,抗氧化相关基因SIRT-3;下调降解细胞外基质相关基因MMP家族,结果见下表:
灭活菌体上调细胞外基质相关基因SPTSSA、LN和FN;下调降解细胞外基质相关基因MMP家族,细胞凋亡相关基因Caspase家族。结果见下表:
结果显示加入GforU-32具有促进HFF细胞外基质合成、减少细胞凋亡、增加抗氧化、减少细胞外基质降解的抗衰老作用。
实施例6GforU-32上调HaCaT抗菌肽相关基因表达实验
1、GforU-32上清液制备:
制备方法参照实施例3。
2、HaCaT细胞制备
将HaCaT细胞消化后以0.5ml/孔(内含2×105细胞)接种至24孔板,5%二氧化碳培养箱37℃培养过夜。
3、GforU-32添加及LPS刺激
将上清液5%(V/V)、灭活菌体10%(V/V)分别加入培养过夜的HaCaT细胞中(对照组分别以等体积PBS替代上清液/灭活菌体),2h后添加0.5ml浓度为0.2μg/ml的LPS溶液,诱导细胞发炎,5%二氧化碳培养箱37℃培养20h。
4、qPCR法检测抗菌肽相关基因相对表达倍数
将上述细胞弃去培养基后,加入裂解液,提取细胞总RNA,检测RNA浓度及纯度后反转录为cDNA,以GAPDH为内参基因,采用实时qPCR检测S100A7、S100A8、DEFB1和DEFB4基因的表达。以等体积PBS处理组为对照(基因相对表达倍数F=1),利用2-ΔΔCT法计算各样品F值。
结果见下表:
结果显示GforU-32上清液具有促进抗菌肽基因表达的作用。
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)
1.一株鼠李糖乳杆菌(Lactobacillus rhamnosus),该菌株被命名为GforU-32,已于2022年8月31日保藏在中国典型培养物保藏中心,其保藏编号为CCTCC No:M 20221355的鼠李糖乳杆菌。
2.根据权利要求1所述的鼠李糖乳杆菌,其特征在于,采样于健康女童粪便,将样品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GforU-32;
在革兰氏染色镜检下菌株GforU-32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、表面光滑不透明的圆形菌落,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。
3.权利要求1~2任一项所述的鼠李糖乳杆菌在制备改善肌肤状况的产品中的应用,其中,所述改善肌肤状况包括促进细胞增殖、抗衰老、提升皮肤抗菌能力中的至少一种。
4.根据权利要求3所述的应用,其特征在于,所述抗衰老为上调细胞外基质相关基因的表达;所述细胞外基质相关基因由SPTSSA、TIMP1、LN、FN、COL1A1中的至少一种组成。
5.根据权利要求3所述的应用,其特征在于,所述抗衰老为上调抑制细胞凋亡相关基因BCL-2的表达。
6.根据权利要求3所述的应用,其特征在于,所述抗衰老为上调细胞抗氧化相关基因NRF2和/或SIRT-3的表达。
7.根据权利要求3所述的应用,其特征在于,所述抗衰老为下调降解细胞外基质相关基因的表达;所述降解细胞外基质相关基因由P38MAPK、MMP家族MMP1、MMP2、MMP7、MMP8、MMP10、MMP11中的至少一种组成。
8.根据权利要求3所述的应用,其特征在于,所述抗衰老为下调细胞凋亡相关基因的表达;所述细胞凋亡相关基因由Caspase家族Caspase-3、Caspase-9、Caspase-32的至少一种组成。
9.根据权利要求3所述的应用,其特征在于,所述提升皮肤抗菌能力为上调抗菌肽相关基因S100A7、S100A8、DEFB1和/或DEFB4的表达。
10.一种改善皮肤状况的产品,其特征在于,所述产品为食品、药品或化妆品,由包括权利要求1~2任一项所述的鼠李糖乳杆菌原料制成,所述产品中的鼠李糖乳杆菌包括如下所示的至少一种:
(1)鼠李糖乳杆菌的活菌和/或灭活菌;
(2)鼠李糖乳杆菌的培养物、外泌体、裂解物和/或提取物。
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