CN117551589B - 长双歧杆菌婴儿亚种的溶胞产物的应用 - Google Patents
长双歧杆菌婴儿亚种的溶胞产物的应用 Download PDFInfo
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- CN117551589B CN117551589B CN202410036411.XA CN202410036411A CN117551589B CN 117551589 B CN117551589 B CN 117551589B CN 202410036411 A CN202410036411 A CN 202410036411A CN 117551589 B CN117551589 B CN 117551589B
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Abstract
本发明涉及化妆品领域,尤其涉及长双歧杆菌婴儿亚种的溶胞产物的应用。本发明提供了菌株的溶胞产物,所述菌株为长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis),其保藏编号为:CGMCC NO.27851。本发明涉及的菌种的溶胞产物可在食品及美妆领域达到屏障修护和美白等功效,能在美容护肤领域有更好的发挥。
Description
技术领域
本发明涉及化妆品领域,尤其涉及长双歧杆菌婴儿亚种的溶胞产物的应用。
背景技术
黑色素是存在于每个人皮肤基底层的一种蛋白质。紫外线的照射会令黑色素产生变化,生成一种保护皮肤的物质,然后黑色素又经由细胞代谢的层层移动,到了肌肤表皮层,形成了我们所看到的色斑和肤色不匀等皮肤问题。
所以,美白也是一个由内而外的护理工程。首先要选择一套适合自己的美白产品,因为美白成分各不相同,所以理论上最好是使用同一品牌同一系列的产品,这样可以防止不同美白成分互相冲突产生的不良反应。市面上比较多的美白产品都是以烟酰胺为主要成分,能够帮助肌肤抑制和调节黑色素的转移,并减淡已形成的色斑。
发明内容
有鉴于此,本发明提供了长双歧杆菌婴儿亚种的溶胞产物的应用。本发明提供的菌种的溶胞产物可在美妆领域达屏障修护和美白的功效,且效果更显著。
本发明提供了菌株的溶胞产物,所述菌株为长双歧杆菌婴儿亚种(Bifidobacterium longum subsp. infantis),其保藏编号为:CGMCC NO.27851。
在本发明的一些实施方案中,所述溶胞产物包括:有机酸10~20%、氨基酸1~10%、无机盐0.5~5%、糖醇1~5%和单糖0.1~3%。
在本发明的一些实施方案中,所述溶胞产物包括:有机酸17%、氨基酸4%、无机盐2%、糖醇2%和单糖1%。
在本发明的一些实施方案中,所述溶胞产物包括:乙醇酸17%、焦谷氨酸4%、羟胺2%、脱氧赤藓糖醇2%和D-塔罗糖1%。
本发明还提供了上述溶胞产物的制备方法,包括如下步骤:
S1:取所述长双歧杆菌婴儿亚种(Bifidobacterium longum subsp. infantis)接种培养后,获得种子液;
S2:取所述种子液发酵培养后,破碎菌体,离心,收集沉淀后,获得所述溶胞产物。
在本发明的一些实施方案中,上述制备方法S1中所述接种的接种量为5%。
在本发明的一些实施方案中,上述制备方法S2中所述离心的转速为3000 rpm,时间为30 min。
本发明还提供了上述溶胞产物和/或上述制备方法获得的溶胞产物在制备屏障修护的个护产品中的应用。
本发明还提供了上述溶胞产物和/或上述制备方法获得的溶胞产物在制备美白的个护产品中的应用。
在本发明的一些实施方案中,上述个护产品包括:化妆品。
在本发明的一些实施方案中,上述个护产品的剂型包括:膏霜型、乳液型、粉剂和精华型中的一种或多种。
在本发明的一些实施方案中,上述个护产品包括:基础护理产品和/或彩妆产品。
本发明提供了菌株的溶胞产物,所述菌株为长双歧杆菌婴儿亚种(Bifidobacterium longum subsp. infantis),其保藏编号为:CGMCC NO.27851。
本发明提供的菌种分离自母乳喂养的健康婴儿,试验结果表明,本发明提供的菌种的溶胞产物具有屏障修护和美白的功效,且效果更显著。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示菌株形态图;
图2示菌株进化树;
图3示革兰氏染色图;
图4示无溶血环;
图5示菌株溶胞产物制备工艺;
图6示菌株溶胞产物在细胞划痕实验中促进细胞迁移的实物照片;
图7示菌株溶胞产物对SLS刺激损伤3D表皮皮肤模型组织形态的改善结果。
生物保藏说明
生物材料:YSGBifido001;分类命名:长双歧杆菌婴儿亚种(Bifidobacteriumlongum subsp. infantis);于2023年07月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心;地址:北京市朝阳区北辰西路1号院3号 中国科学院微生物研究所;保藏编号:CGMCC No.27851。
具体实施方式
本发明公开了长双歧杆菌婴儿亚种的溶胞产物的应用。
应该理解,表述“……中的一种或多种”单独地包括每个在所述表述后叙述的物体以及所述叙述的物体中的两者或更多者的各种不同组合,除非从上下文和用法中另有理解。与三个或更多个叙述的物体相结合的表述“和/或”应该被理解为具有相同的含义,除非从上下文另有理解。
术语“包括”、“具有”或“含有”,包括其语法同义语的使用,通常应该被理解为开放性和非限制性的,例如不排除其他未叙述的要素或步骤,除非另有具体陈述或从上下文另有理解。
应该理解,只要本发明仍可操作,步骤的顺序或执行某些行动的顺序并不重要。此外,两个或更多个步骤或行动可以同时进行。
本文中的任何和所有实例或示例性语言如“例如”或“包括”的使用,仅仅打算更好地说明本发明,并且除非提出权利要求,否则不对本发明的范围构成限制。本说明书中的任何语言都不应解释为指示任何未要求保护的要素对于本发明的实践是必不可少的。
此外,用以界定本发明的数值范围与参数皆是约略的数值,此处已尽可能精确地呈现具体实施例中的相关数值。然而,任何数值本质上不可避免地含有因个别测试方法所致的标准偏差。因此,除非另有明确的说明,应当理解本公开所用的所有范围、数量、数值与百分比均经过“约”的修饰。在此处,“约”通常是指实际数值在一特定数值或范围的正负10%、5%、1%或0.5%之内。
本发明的菌种来源:
微生物来源(细菌来源),DNA序列验证为独家菌种,CGMCC NO.27851。
本发明的菌种类别:
长双歧杆菌婴儿亚种(Bifidobacterium longum subsp. infantis),细菌。
本发明的菌种介绍:
YSGBifido001分离自4月龄广州母乳喂养的健康婴儿,经16S rDNA序列比对鉴定为长双歧杆菌婴儿亚种(Bifidobacterium longum subsp. infantis)。其在MRS培养基(固体/液体)中生长情况良好,厌氧条件下培养1天即能达到对数生长期。在MRS固体培养基上生长的菌株菌落形态为乳白色圆形,边缘整齐。菌株在血平板上生长无明显溶血环。革兰染色后在显微镜下菌体呈蓝紫色,杆状,中间细两端膨大,似“杠铃”。
下面结合实施例,进一步阐述本发明。
本发明实施例1~实施例2和验证例1~验证例4中,所用原料及试剂均可由市场购得。
实施例1 长双歧婴儿亚种菌株的分离纯化
(1)、实验方法
1)粪便收集:细胞培养瓶中加入细菌冻存液(20 mL),使用灭菌棉签收集本地母乳喂养婴儿的粪便样本于细胞培养瓶中,将培养瓶封入厌氧袋里,置于冰盒中保存和运输。
2)稀释涂布:震荡后,以1:9的比例混合粪便悬液和PBS缓冲液,使用移液枪吹打混匀后再吸取100 μL上述悬液至900 μL PBS缓冲液中,依次梯度稀释10次。各取20 μL每个梯度的稀释液,分别涂布于CBA、MRS、BHI平板上,37℃有氧或厌氧条件下培养48~72 h。
3)单菌落增菌培养:选择菌落数量在5~100个的平板,使用一次性接种环随机挑选单菌落于20 μL PBS缓冲液中,吹打混匀后取15 μL的菌液涂布于步骤2)的同种平板上,37℃有氧或厌氧条件下培养48~72 h,另取5 μL菌液留待革兰氏染色鉴定。
4)细菌传代:使用一次性接种环尽可能得刮取平板上生长的细菌,重悬于200 μL的PBS中,接着将200 μL菌液再涂布到同种平板上,37℃有氧或厌氧条件下培养48~72 h。
5)细菌冻存:细菌增菌在平板上培养到足量后,将细菌用接种环收集于由胎BHI、牛血清培养基、甘油混合而成的无菌冻存液中,吹打混匀,-80℃保存。同时留取部分细菌待提取细菌基因组DNA。
6)革兰氏染色鉴定:在载玻片中央滴加挑选单菌落时制备的5 μL菌液,使用酒精灯间隔烘烤固定,滴加结晶紫染液覆盖1 min,流水洗净,然后滴加卢戈碘液染液1 min覆盖,流水洗涤,再用脱色酒精覆盖载玻片表面20-30 s,轻轻摇晃载玻片,流水洗净酒精后使用番红染液复染1 min覆盖,流水洗涤,待载玻片干燥后使用光学显微镜观察细菌形态。若发现菌落不纯,存在两种或以上的细菌形态,则重新对样本进行平板划线接种,培养后重新挑取单菌落。
7)细菌基因组DNA提取:使用细菌基因组DNA提取试剂盒(天根生化科技公司),按照说明书细菌基因组DNA提取,并用NanoDrop2000测定DNA浓度。
8)16S片段扩增:
<1>采用27F/1492R引物对,27F序列:3’-AGAGTTTGATCMTGGCTCAG-5’(如SEQ IDNO:1所示)
1492R序列:3’-GGTTACCTTGTTACGACTT-5’(如SEQ ID NO:2所示)。
反应体系如下所示:
表1
<2>PCR扩增程序如下:
表2
循环数为31;结束温度设为4℃。
9)琼脂糖凝胶电泳:配置25 mL含有1%(w/v)琼脂糖的1×TAE缓冲液,加热融化,加入2.5 μLGoldView,混匀后倒入制胶槽,插入梳子,冷却。待冷却后拔掉梳子,放入水平电泳槽中,每孔加入5 μL扩增后的DNA样本及3 μL DL2000 DNA marker,10 V恒压电泳20 min,紫外灯下观察扩增条带是否单一,大小是否在1500 bp左右。
10)16S rRNA测序与结果比对:将扩增后并经琼脂糖凝胶电泳确认无误的DNA 16S片段样本测序(上海生工公司),得到测序结果后,使用ChromasPro软件对序列拼接,并在NCBI网站上blast,初步鉴定细菌的类型。
(2)、实验结果
将分离鉴定的菌株命名为长双歧杆菌婴儿亚种(Bifidobacterium longumsubsp. Infantis) YSGBifido001。YSGBifido001的16S rDNA的核苷酸序列如SEQ ID NO:3所示。其16S rDNA与已知长双歧杆菌菌株对比(National Center for Biotechnology数据库)至少存在两个碱基突变位点,其亲缘关系最近的菌株为B. longum subsp. infantisstrain ATCC 15697。
序列:3’-TGCAAGTCGAACGGGATCCATCAGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGTTCCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGTGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGTCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGATCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCA-5’(如SEQ ID NO:3所示)。
实施例2 菌株溶胞生产流程
(1)、甘油管菌种制备
将原始冻干粉菌种活化后待单菌落长大后,进行活化,获得菌液(OD*10>0.260、pH<4.50);
将上述菌5%接种量(培养20~24小时后检测OD*10>0.260可判定培养合格)转接如表3所示的培养基中,培养20小时检测镜检达标后(OD*10>0.260、pH<4.50),菌种甘油管(保菌液与菌液的体积比为1:1)保种,-80℃保存期3个月,每3月传代一次保种,每次传代必须检验菌种是否污染。
将上述获得的菌株长双歧杆菌婴儿亚种,接种于培养基(培养基配方如表3所示),接种量为5%,于37℃培养20~24h,离心得到沉淀后,添加PBS缓冲液乳化均匀,细胞破壁,得到溶胞产物。获得的菌株溶胞产物的成分如表4所示。
表3
表4
验证例1 总酸、pH、总蛋白测定
对实施例2获得的溶胞产物检测。
(1)采用酸碱滴定法测定总酸含量。
(2)采用pH计测定pH。
(3)采用BCA蛋白浓度测定试剂盒(Biosharp公司)测定总蛋白含量。
实验结果如表5所示。
表5
验证例2
(1)、竞品:CLR 二裂酵母(Repair Complex CLR PF),1%、3%、5%;
方法:qPCR;
模型:HaCat;
指标:LOR。
(2)实验结果
总结:发酵溶胞产物对LOR基因的扩增倍数均有所提高。
1)LOR PCR
LOR的引物:F:5'-CTTGGAACATGGCCTAGCTC-3'(如SEQ ID NO:4所示);
R:5'-CATTCCCAGCACAACAGAGA-3'(如SEQ ID NO:5所示)。
实验结果如表6所示,通过与相同竞品CLR的对比可发现,溶胞产物对LOR基因表达的提升较高。溶胞产物可通过上调LOR基因可达到修护功效。且溶胞产物在低浓度下,对LOR的基因表达量更高;
表6 实验结果
其中:*表示差异显著,**表示差异极显著。
验证例3 修护-基于角质形成细胞的测试
(1)、细胞划痕
表7
其中:滤液+溶胞中,滤液占比50%,溶胞占比50%。滤液和滤液+溶胞为对比例。
1)、测试方法
细胞接种:细胞复苏后至铺板率达到60%左右时,接种细胞至6孔板,37℃、5%CO2孵育过夜。
给药:根据表7分组,待细胞铺板率达到70%~80%左右时,进行分组给药,每组设3个重复。样品组每孔加入2 mL含有相应浓度待测样品的培养液,空白对照组每孔加入2 mL培养液,阳性对照组每孔加入2 mL含有EGF的培养液。给药完成后将6孔板放置在CO2培养箱中继续培养24 h。
划痕:孵育结束后,划痕。
清洗:划痕结束后,用PBS缓冲液清洗细胞3次,加入细胞培养液,CO2培养箱培养24h。
拍照:划痕0h后在4倍镜下拍照,划痕24 h后,用PBS清洗一次,在4倍镜下拍照。
提升率计算:提升率=(样品组-空白对照组)/空白对照组×100%
结果统计分析:应用GraphPad Prism作图,结果表示为Mean±SD。各组间比较采用t-test统计分析。统计分析均为双尾。P<0.05认为具有显著差异,P<0.01认为具有极显著差异。
2)、测试结果
表8
实验结果如图6和表8所示,溶胞能够促进细胞迁移,相比BC组具有显著性差异。
(2)、基因表达量
1)、测试方法
细胞接种:复苏细胞至铺板率达到60%左右时,接种细胞至6孔板,CO2培养箱(37℃、5%CO2)孵育过夜。
给药:根据表9~表11测试分组,待6孔板中细胞铺板率达到40%~60%时,进行分组给药,每组设3个重复。阳性对照组每孔加入2 mL含有VE的培养液,空白对照组每孔加入2 mL培养液,样品组每孔加入2 mL含有相应浓度待测样品的培养液。给药完成后将6孔板放置在CO2培养箱中继续培养24 h。
收集细胞:培养24 h后,吸弃旧液,PBS清洗两次,每孔加入1 mL RNAiso Plus,吹打裂解细胞后,收取细胞。
基因表达检测:提取RNA,反转录后,荧光定量PCR检测,采用2-△△CT方法进行结果计算。
上调率计算:上调率=(样品组-空白对照组)/空白对照组×100%
结果统计分析:应用GraphPad Prism作图,结果表示为Mean±SD。各组间比较采用t-test统计分析。统计分析均为双尾。P<0.05认为具有显著差异,P<0.01认为具有极显著差异。
其中:滤液+溶胞中,滤液占比50%,溶胞占比50%。
滤液和滤液+溶胞为对比例。
2)、测试结果
①Claudin-4 PCR
Claudin-4的引物:F:5'-TCTCCTCTGTTCCGGGTAGG-3'(如SEQ ID NO:6所示);
R:5'-CGTCCATCCACTCTGCACTT-3'(如SEQ ID NO:7所示)。
表9
实验结果如表9所示,溶胞能够显著提高Claudin-4的表达。
②ABCA12 PCR
ABCA12的引物:F:5'-GCTGGAAACCAACAAGACTGCTTTA-3'(如SEQ ID NO:8所示);
R:5'-TATAGTGGAACCTTGGCTGCTGGTA-3'(如SEQ ID NO:9所示)。
表10
实验结果如表10所示,溶胞能够显著提高ABCA12的表达。
③ACACA PCR
ACACA的引物:F:5'-AGTGAGGATGGCAGCTCTGGA-3'(如SEQ ID NO:10所示);
R:5'-ATGAGATGTGGGCAGCATGAAC-3'(如SEQ ID NO:11所示)。
表11
实验结果如表11所示,溶胞能够显著提高ACACA的表达。
(3)、基于SLS刺激3D表皮皮肤模型(EpiKutis®)的测试
表12
其中:滤液+溶胞中,滤液占比50%,溶胞占比50%。
滤液和滤液+溶胞为对比例。
1)、测试方法
工作液配制:
1)0.1% SLS工作液配置:称取0.006g SLS溶于6 mL PBS缓冲液中,0.22 µm过滤,配制成0.1% SLS工作液。
2)阳性对照组(50 μM WY14643)工作液配置:吸取10 µL 30 mM WY14643母液溶于6 mL培养液中,配置成50 μM WY14643。
模型给药:
<1>根据表12测试分组,将模型转移到6孔板中(提前添加0.9mL EpiGrowth培养液),在6孔板上标注测试组编号。
<2>PC组、NC组和样品组表面添加25 μL 0.1%的SLS工作液,孵育30 min。
<3>孵育结束后,PC组液下添加相应浓度的工作液,样品组将样品工作液均匀添加于模型表面,置于CO2培养箱中孵育24 h。
<4>孵育结束后,用无菌PBS缓冲液清洗模型表面残留的受试物,用无菌棉签拭去模型内、外剩余液体。
组织形态测试:
取用于组织形态检测的模型,用4%的多聚甲醛进行固定处理,固定24 h后,进行H&E染色检测,显微镜下拍照观察,采集图片并分析。
结果统计分析:
应用GraphPad Prism作图,结果表示为Mean±SD。各组间比较采用 t-test 统计分析。统计分析均为双尾。P<0.05认为具有显著差异,P<0.01认为具有极显著差异。
2)、测试结果
如图7所示,组织形态检测结果显示,与BC组相比,NC组表皮模型活细胞层受损,有空泡出现,角质层疏松,说明SLS刺激条件有效。
与NC组相比,PC组活细胞层细胞排列紧凑,空泡减少,活细胞受损现象明显改善,说明本次阳性对照检测有效。
与NC组相比,溶胞-5%组空泡减少,活细胞受损现象明显改善,说明溶胞-5%对模型组织形态具有修复作用。
验证例4 美白
(1)、PCR美白基因检测
1)测试方法
细胞接种:复苏细胞至铺板率达到60%左右时,接种细胞至6孔板,37℃、5%CO2孵育过夜。
给药:根据表13~23分组,待6孔板细胞铺板率达到50%~60%时,分组给药,每组设3个重复孔。阳性对照组每孔加入2 mL含有曲酸的培养液,空白对照组每孔加入2 mL培养液,样品组每孔加入2 mL含有相应浓度待测样品的培养液。给药完成后将上述孔板放置在CO2培养箱中继续培养24h。
收集细胞:弃旧液,PBS缓冲液清洗两次,每孔加入1 mL RNAiso Plus,吹打裂解细胞后,收取细胞。
基因表达检测:提取RNA后反转录,荧光定量PCR检测,采用2-△△CT方法
进行结果计算。
下调率计算:下调率=(空白对照组-样品组)/空白对照组×100%
结果统计分析:应用GraphPad Prism作图,结果表示为Mean±SD。各组间比较采用t-test统计分析。统计分析均为双尾。P<0.05认为具有显著差异,P<0.01认为具有极显著差异。
其中:滤液+溶胞中,滤液占比50%,溶胞占比50%。
2)测试结果
①TYR PCR
TYR的引物:F:5'-TGCGGTGGGAACAAGAAA-3'(如SEQ ID NO:12所示);
R:5'-GACAATCTGCCAAGAGGAGAAGA-3'(如SEQ ID NO:13所示)。
表13
实验结果如表13所示,溶胞可以显著下调TYR基因的表达。
②Rab27a PCR
Rab27a的引物:F:5'-GAATGGAACGGTGTGTGGAC-3'(如SEQ ID NO:14所示);
R:5'-CCCCTTTCTCCTTTTCTTCACTT-3'(如SEQ ID NO:15所示)。
表14
实验结果如表14所示,溶胞对黑素小体转运相关基因Rab27a的表达均具有显著的下调率,且高于对比例。
③Myo5a PCR
Myo5a的引物:F:5'- GTGAGCGAGGAGCTTGATGT-3'(如SEQ ID NO:16所示);
R:5'- TCATCCTTGGGTTGGATGGC-3'(如SEQ ID NO:17所示)。
表15
实验结果如表15所示,只有溶胞对黑素小体转运相关基因Myo5a具有较高的下调率。
④MLPH PCR
MLPH的引物:F:5'-CAGCGACCAGACAGATGAGG-3'(如SEQ ID NO:18所示);
R:5'-GACCTTGAGGCTGAGTGGAG-3'(如SEQ ID NO:19所示)。
表16
实验结果如表16所示,溶胞对黑素小体转运相关基因MLPH的表达具有较高的下调率,且好于滤液+溶胞。
⑤SLC45A2 PCR
SLC45A2的引物:F:5'-CCTCAATGGGGCTACTGTTGT-3'(如SEQ ID NO:20所示);
R:5'-GCCCATCAATGAAGTCGGCA-3'(如SEQ ID NO:21所示)。
表17
实验结果如表17所示,溶胞可以显著下调黑色素合成相关基因SLC45A2的表达,且下调率高于其他对比例。
MITF PCR
MITF的引物:F:5'-AATTGTCCATCTGCCTCTGAGTAG-3'(如SEQ ID NO:22所示);
R:5'-GTATGACCAGGTTGCTTGTATGC-3'(如SEQ ID NO:23所示)。
MITF参与黑素合成的转录因子。
表18
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实验结果如表18所示,溶胞可显著下调黑色素合成相关基因MITF的表达。
TRP-1 PCR
TRP-1的引物:F:5'-GTGCCACTGTTGAGGCTTTG-3'(如SEQ ID NO:24所示);
R: 5'-ATGGGGATACTGAGGGCTGT-3'(如SEQ ID NO:25所示)。
TRP-1参与到酪氨酸合成黑素的过程中。
表19
实验结果如表19所示,溶胞对黑色素合成相关基因TRP-1的表达的下调率高达37%。
TRP-2 PCR
TRP-2的引物:F:5'-AGGATATACACCCCTAATGGAGACA-3'(如SEQ ID NO:26所示);
R:5'-AAGCAAGCAAAGCGGAAACTAC-3'(如SEQ ID NO:27所示)。
TRP-2参与到酪氨酸合成黑素的过程中。
表20
实验结果如表20所示,溶胞对TRP-2的表达的下调率高于其他对比例。
Pmel17 PCR
Pmel17的引物:F:5'-AACAGCAGTGCAGATGACGA-3'(如SEQ ID NO:28所示);
R:5'-CGGTTGACCCTTCAGGTGTT-3'(如SEQ ID NO:29所示)。
表21
实验结果如表21所示,溶胞可显著下调黑素小体转运相关基因Pmel17的表达。
⑩Kinesin PCR
Kinesin的引物:F:5'-GGGGACTCCGTAAAACAGGG-3'(如SEQ ID NO:30所示);
R:5'-TCCTCGTGCACGCTTAAGTT-3'(如SEQ ID NO:31所示)。
Kinesin与Rab1、SKIP构成转运复合体,参与微管介导的黑素小体转运。
表22
实验结果如表22所示,溶胞可显著下调黑素小体转运相关基因Kinesin的表达。
①①CDC42PCR
CDC42的引物:F:5'-ATTACGACCGCTGAGTTATCCAC-3'(如SEQ ID NO:32所示);
R:5'-TCAGGCACCCACTTTTCTTTC-3'(如SEQ ID NO:33所示)。
表23
实验结果如表23所示,溶胞可显著下调黑素小体转运相关基因CDC42的表达。
以上所述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 (4)
1.菌株的溶胞产物的制备方法,其特征在于,包括如下步骤:
S1:取长双歧杆菌婴儿亚种(Bifidobacterium longum subsp. infantis)接种培养后,获得种子液;所述长双歧杆菌婴儿亚种(Bifidobacterium longum subsp. infantis)的保藏编号为:CGMCC NO.27851;
S2:取所述种子液发酵培养后,破碎菌体,离心,收集沉淀后,获得所述溶胞产物。
2.根据权利要求1所述的制备方法,其制备得到的溶胞产物包括:乙醇酸17%、焦谷氨酸4%、羟胺2%、脱氧赤藓糖醇2%和D-塔罗糖1%。
3.如权利要求1或2所述方法制备得到的溶胞产物在制备屏障修护的个护产品中的应用。
4.如权利要求1或2所述的方法制备得到的溶胞产物在制备美白的个护产品中的应用。
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