CN117701455A - 坚强芽孢杆菌及其在制备改善皮肤状况的产品中的应用 - Google Patents
坚强芽孢杆菌及其在制备改善皮肤状况的产品中的应用 Download PDFInfo
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- Cosmetics (AREA)
Abstract
本发明公开了一种坚强芽孢杆菌及其在制备改善皮肤状况的产品中的应用,涉及微生物技术领域。本发明公开了一种坚强芽孢杆菌,该菌株为坚强芽孢杆菌NHNK‑602,已于2023年10月25日保藏在中国典型培养物保藏中心,其保藏编号为CCTCC NO:M 20232030。实验表明,NHNK‑602具有修护皮肤屏障、保湿、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
本发明涉及海洋微生物技术领域,特别涉及一种坚强芽孢杆菌及其在制备改善皮肤状况的产品中的应用。
背景技术
陆地河流和大气输入海洋的物质包括软泥沙、灰尘、动植物的遗骸、宇宙尘埃、以及人类活动中落入海底的东西等统称为海底沉积物。海底沉积物按照物质来源可以分为五种:陆源物质、生物物质、与岩浆活动有关的物质、海底岩石熔滤的物质、宇宙物质。
海洋沉积物中的微生物对全球生物量的贡献巨大,是地球系统的重要组成部分。海底沉积物既包括好氧微生物生态系统,也包括厌氧微生物生态系统,它们在不同的地质时期依靠着极低的生物可用能量生存。海洋微生物在特殊的环境中生存,产生的生物活性分子通常具有结构新颖、功能多样的特点。海洋生活条件竞争激烈,微生物的次生代谢物的化学多样性极其丰富。
微藻是当前化妆品研发的重要生物来源,利用乳酸菌对螺旋藻进行发酵处理,得到的螺旋藻发酵上清液和固形物提取液具备良好的抗氧化活性。酵母发酵物因含有丰富的活性成分(游离氨基酸、小分子肽、维生素等)被广泛应用于化妆品领域中,它们的主要功效有美白、保湿、延缓肌肤老化、调理皮肤的酸碱度和修复受损皮肤等。细菌发酵物能作为天然防腐剂应用于化妆品中,例如乳酸链球菌素是利用微生物发酵技术,经乳酸链球菌发酵后制备得到的一种纯天然、高效、安全的生物活性抗菌肽,通过干扰细胞膜的正常功能可有效抑制革兰氏阳性菌生长和繁殖。
因此,提供一种利用微生物技术开发的海洋产业相关产品用以改善皮肤状态具有广大的应用价值。
发明内容
有鉴于此, 本发明提供了一种坚强芽孢杆菌及其在制备改善皮肤状况的产品中的应用。
本发明提供了坚强芽孢杆菌(Bacillus firmus),该菌株为坚强芽孢杆菌NHNK-602,已于2023年10月25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 20232030的坚强芽孢杆菌)。
本发明的另一目的在于提供了上述坚强芽孢杆菌在制备改善皮肤状况的产品中的应用。
进一步的,所述改善皮肤状况为修护皮肤屏障、保湿、抗衰老、提高细胞抗菌能力中的至少一种。
在一些实施方式中,所述修复皮肤屏障为上调屏障修护相关基因表达包括FLG和/或OCLN中至少一种。
在一些实施方式中,所述保湿为上调保湿相关基因GBA的表达。
在一些实施方式中,所述抗衰老为如下a)~f)所示的至少一种:
a)、上调细胞外基质相关基因TIMP1、SMAD3和/或SPTSSA的表达;
b)、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包括MMP家族中的至少一种;
c)、下调细胞凋亡相关基因的表达;所述细胞凋亡相关基因包括BAX和/或Caspase家族的至少一种;
d)、上调细胞自噬相关基因LC3B的表达;
e)、上调细胞抗氧化相关基因NRF2的表达;
f)、上调免疫调节因子相关基因MOR的表达。
进一步的,所述提升皮肤抗菌能力为上调抗菌肽相关基因S100A7、S100A8、DEFB4的表达。
在一些实施方式中,所述产品为食品、药品或化妆品。
本发明的另一目的在于提供了一种改善皮肤状况的产品,由包括上述坚强芽孢杆菌的原料制成。
进一步的,所述产品中的坚强芽孢杆菌包括如下(1)或(2)所示的一种或两种:
(1)上述的坚强芽孢杆菌的菌群和/或菌剂;
(2)上述的坚强芽孢杆菌的培养物、外泌体、裂解物和/或提取物。
本发明公开的NHNK-602,其保藏编号为 CCTCC NO: M 20232030。实验表明,本发明提供的坚强芽孢杆菌具有修护皮肤屏障、保湿、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
坚强芽孢杆菌(Bacillus firmus)NHNK-602,于2023年10月25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 20232030。
具体实施方式
本发明提供了坚强芽孢杆菌及其在制备改善皮肤状况的产品中的应用。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。
本发明的坚强芽孢杆菌菌株NHNK-602,来源于海洋沉积泥,经16S rDNA鉴定为坚强芽孢杆菌(Bacillus firmus)。该菌株革兰氏阳性,显微镜下呈杆状;在BHI平板上生长,可形成乳白色至淡黄色圆形菌落,边缘有小锯齿,呈半透明;最适生长温度37℃。
坚强芽孢杆菌(Bacillus firmus)NHNK-602,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:2023年10月25日,保藏编号为 CCTCC NO: M 20232030。
进一步,本发明提供的坚强芽孢杆菌NHNK-602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。
体外细胞实验表明,本发明的坚强芽孢杆菌NHNK-602具有上调皮肤屏障修护相关因子丝聚合蛋白(filaggrin)FLG、人紧密连接蛋白(Human Occludin)OCLN表达的作用,基因表达量上调1.27~2.68倍。
体外细胞实验表明,本发明的坚强芽孢杆菌NHNK-602具有上调保湿因子葡萄糖脑苷脂酶基因GBA表达的作用,基因表达量上调1.19~1.26倍。
体外细胞实验表明,本发明的坚强芽孢杆菌NHNK-602具有上调HaCaT细胞外基质相关的组织金属蛋白酶抑制物1基因TIMP1、丝氨酸棕榈酰转移酶基因SPTSSA和信号转导蛋白SMAD3基因,细胞自噬相关的微管相关蛋白1轻链3β基因LC3B,抗氧化核因子E2相关因子2基因NRF2基因表达的作用,基因相对表达倍数1.22~2.07;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MMP1表达的作用,基因相对表达倍数0.43~0.58。
体外细胞实验表明,本发明的坚强芽孢杆菌NHNK-602具有上调免疫调节因子相关的β-内啡肽受体基因MOR表达的作用,基因相对表达倍数2.57~2.84倍;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、,细胞凋亡相关的BCL2-Associated X蛋白基因BAX和半胱氨酸蛋白酶家族基因Caspase表达的作用,基因相对表达倍数0.21~0.76。
体外细胞实验表明,本发明的坚强芽孢杆菌NHNK-602具有上调抗菌肽相关基因S100A7、钙粒蛋白A基因S100A8、β防御素4基因DEFB4表达的作用,基因相对表达倍数4.58~20.99。
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 NHNK-602的分离
从山东大学实验室获得的海洋沉积泥中秤取3g,以PBS清洗两次后取适当量水样以低速离心去除杂质,取上清划线于BHI固体平板,37℃恒温培养16h后,挑取菌落反复接种筛选,直至得到均匀的单个菌落,命名为NHNK-602。
革兰氏染色镜检:菌株NHNK-602为革兰氏染色阳性,显微镜下呈杆状,芽孢位于菌体中央;在BHI平板上生长,可形成圆形半透明菌落,菌落边缘不平整,呈乳白色或淡黄色;在BHI液体培养基中呈均匀浑浊生长,久置菌体呈白色或浅黄色沉淀。
实施例2 NHNK-602的核酸鉴定
1、16SrDNA基因序列分析:
挑取单菌落置BHI液体培养基中,37℃摇床培养过夜后,12000转离心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NHNK-602菌株为坚强芽孢杆菌(Bacillus firmus)。
实施例3:NHNK-602促进HaCaT屏障修护相关基因表达实验
1、NHNK-602上清液制备:
挑取坚强芽孢杆菌NHNK-602单菌落于BHI液体培养基,37℃摇床培养16~18h,酶标仪检测并以BHI稀释调整OD600=0.2,121℃,30min高压灭活,12000转离心2min,经0.22μm滤膜过滤为上清液。
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(2ml/孔,内含5×105细胞)至6孔板,5%二氧化碳培养箱37℃过夜培养至细胞贴壁。分别加入上清液5%(V/V),对照组以等体积BHI替代上清液,培养24h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FLG和OCLN基因的表达。以添加等体积BHI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。
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。
结果见下表:
结果显示NHNK-602具有促进皮肤屏障修护的作用。
实施例4:NHNK-602上调HaCaT保湿相关基因表达实验
1、NHNK-602上清液制备:
制备方法参照实施例3。
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(2ml/孔,内含5×105细胞)至6孔板,5%二氧化碳培养箱37℃过夜培养至细胞贴壁。加入上清液5%(V/V)(对照组以等体积BHI替代),培养24h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GBA基因的表达。以等体积BHI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NHNK-602具有促进皮肤保湿的作用。
实施例5:NHNK-602调节光老化HaCaT细胞外基质/细胞自噬/抗氧化相关基因表达实验
1、NHNK-602上清液制备:
制备方法参照实施例3。
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、NHNK-602添加
将上清液5%(V/V)加入刺激过的HaCaT细胞(对照组以等体积BHI替代上清液)。每组3平行,37℃培养过夜。
4、qPCR法检测细胞外基质/细胞自噬/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TIMP1、SMAD3、SPTSSA,细胞自噬相关基因LC3B,抗氧化相关基因NRF2,以及降解细胞外基质相关基因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、SMAD3、SPTSSA,细胞自噬相关基因LC3B,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:
结果显示加入NHNK-602具有促进HaCaT细胞外基质合成、细胞自噬、抗氧化、减少细胞外基质降解的抗衰老作用。
实施例6:NHNK-602调节氧化损伤HFF细胞外基质/细胞凋亡/免疫调节因子相关基因表达实验
1、NHNK-602上清液制备:
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、NHNK-602添加
将上清液5%(V/V)加入刺激过的HFF细胞(对照组以等体积BHI替代上清液)。每组3平行,37℃培养过夜。
4、qPCR法检测细胞外基质/细胞凋亡/免疫调节因子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检测免疫调节因子相关基因MOR;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家族和BAX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。
上清液上调免疫调节因子相关基因MOR,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家族和BAX,结果见下表:
结果显示加入NHNK-602具有减少细胞凋亡、增加细胞免疫调节因子、减少细胞外基质降解的抗衰老作用。
实施例7: NHNK-602上调HaCaT抗菌肽相关基因表达实验
1、NHNK-602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、NHNK-602添加及LPS刺激
将上清液5%(V/V)加入培养过夜的HaCaT细胞中(对照组以等体积BHI替代上清液),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、DEFB4基因的表达。以等体积BHI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NHNK-602上清液具有促进抗菌肽基因表达的作用。
实施例8: NHNK-602上清液精华水制备实例
NHNK-602上清液精华水的制备,组方如下表所示
制备工艺:将较难溶解的透明质酸钠充分搅拌溶解,配置成1%溶液;称取卡波姆充分搅拌溶解后,将三乙醇胺、EDTA-2Na、甘油、丙二醇、丁二醇、透明质酸(1%)、实施例3制备的NHNK-602上清液、苯氧乙醇等分别按照处方比例加入,最后补足剩余去离子水,充分搅拌至均匀即可。
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. 一种坚强芽孢杆菌(Bacillus firmus),该菌株为坚强芽孢杆菌NHNK-602,已于2023年10月25日保藏在中国典型培养物保藏中心,其保藏编号为CCTCC NO: M 20232030。
2.权利要求1所述的坚强芽孢杆菌在制备改善皮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,所述改善皮肤状况为修护皮肤屏障、保湿、抗衰老、提高细胞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,所述修复皮肤屏障为上调屏障修护相关基因表达包括FLG和/或OCLN中至少一种。
5.根据权利要求3所述的应用,其特征在于,所述保湿为上调保湿相关基因GBA的表达。
6.根据权利要求3所述的应用,其特征在于,所述抗衰老为如下a)~f)所示的至少一种:
a)、上调细胞外基质相关基因TIMP1、SMAD3和/或SPTSSA的表达;
b)、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包括MMP家族中的至少一种;
c)、下调细胞凋亡相关基因的表达;所述细胞凋亡相关基因包括BAX和/或Caspase家族的至少一种;
d)、上调细胞自噬相关基因LC3B的表达;
e)、上调细胞抗氧化相关基因NRF2的表达;
f)、上调免疫调节因子相关基因MOR的表达。
7.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、DEFB4的表达。
8.根据权利要求1-7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所述坚强芽孢杆菌的原料制成。
10.根据权利要求9所述的产品,其特征在于,所述产品中的坚强芽孢杆菌包括如下(1)或(2)所示的一种或两种:
(1)权利要求1所述的坚强芽孢杆菌的菌群和/或菌剂;
(2)权利要求1所述的坚强芽孢杆菌的培养物、外泌体、裂解物和/或提取物。
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