CN114672443B - 一株具有预防或改善面部泛红和i型玫瑰痤疮功能的植物乳植杆菌 - Google Patents
一株具有预防或改善面部泛红和i型玫瑰痤疮功能的植物乳植杆菌 Download PDFInfo
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- CN114672443B CN114672443B CN202210493268.8A CN202210493268A CN114672443B CN 114672443 B CN114672443 B CN 114672443B CN 202210493268 A CN202210493268 A CN 202210493268A CN 114672443 B CN114672443 B CN 114672443B
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- lactobacillus plantarum
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Abstract
本发明属于益生菌筛选与应用技术领域,具体涉及一株新的植物乳植杆菌(Lactiplantibacillus plantarum)及其应用。所提供的植物乳植杆菌分离自健康皮肤的表面,对柯氏棒状杆菌有较强的抑菌作用,对皮肤细胞的黏附性好,具有预防或改善面部皮肤泛红和I型玫瑰痤疮的功能,已于2021年5月24日保藏于中国武汉 武汉大学的中国典型培养物保藏中心,其保藏号为CCTCC NO:M2021593。
Description
技术领域
本发明属于益生菌筛选与应用技术领域,具体涉及一株具有预防或改善面部泛红和I型玫瑰痤疮功能的植物乳植杆菌及其应用。
背景技术
益生菌是一种活性微生物,当施以足够数量时能够给宿主带来健康益处。随着肠道微生物组研究的不断进展,肠道微生物组组成的改变与某些疾病之间的关系也已逐渐明确,并且越来越受到人们的重视,如某些变态反应性疾病、肠炎、自闭症、糖尿病等。上述进展对皮肤微生物组与皮肤健康的基础以及临床应用研究起到了至关重要的推动作用。
皮肤作为人体表面积最大的器官,同样栖息着众多微生物,皮肤表面微生物与皮肤表面各种细胞组织、分泌物及微环境构成的整体,维持着皮肤微生态平衡。健康的皮肤微生态系统具有重要的健康意义,其能够抵御外部不良因素侵扰,令皮肤屏障健康,而皮肤微生态失衡则会导致皮肤屏障功能受损。例如,特应性皮炎与葡萄球菌属增加及皮肤表面有益益生菌的减少呈现正关系;痤疮的发生与痤疮丙酸杆菌的增加和皮肤其它有益菌属的减少有关,等等。
柯氏棒状杆菌是一种革兰阳性短小棒状杆菌,具有亲脂生长的特点,国内外对柯氏棒状杆菌的研究甚少,以临床报道案例居多。近年来有研究发现面部泛红及玫瑰痤疮与皮肤逐渐增加的柯氏棒状杆菌有关。其中,此处玫瑰痤疮指I型玫瑰痤疮,也称为红斑毛细血管扩张型玫瑰痤疮,表现为以面中部为主的红斑,亦可累计面颊、前额及下颏。在此前,面部泛红或I型玫瑰痤疮的治疗方式主要是使用抗生素、异维A酸等,但效果不尽人意,尤其抗生素的使用会带来一些不可估计的副作用,包括胃肠道反应、肝损害、菌群失调等。皮肤微生态理念的提出及柯氏棒状杆菌的深入研究为面部泛红或I型玫瑰痤疮的治疗提供了新的思路。
医学或化妆品领域开展了皮肤微生态相关研究,试图通过调节皮肤微生物组的组成,保护益生菌群,减少致病菌,或提供促进皮肤有益共生菌生长的微环境等达到维持、改善或促进皮肤健康的目的。与食品中添加的益生菌不同,在化妆品中使用的大多是益生菌的碎片或溶胞产物,包含菌体及在发酵过程中合成的全部活性成分,含有肽聚糖、磷壁酸、蛋白质、肽、细菌素、短链脂肪酸和有机酸等,能够维护皮肤屏障,预防和治疗轻度皮肤感染。迄今为止的临床研究表明,外用益生菌可以通过影响皮肤微生物组的组成,起到改善皮肤健康的作用。CLR Berlin公司的护肤原料ProRenew Complex CLR™(乳球菌发酵溶胞物),经过早期的细胞生物学和临床研究证明该原料能够明显改善头皮的敏感性和干燥度。荷兰帝斯曼针对性地推出了益生素概念的护肤活性物OXY 229 PF,它通过独特的面包酵母菌株VdH2发酵而来,能降低油脂水平,还能最大程度地减少柯氏棒状杆菌水平,从而预防面部泛红。
目前,关于预防面部泛红和I型玫瑰痤疮的益生菌研究较少,因此本发明旨在筛选获得防治面部泛红和I型玫瑰痤疮效果突出,作用机制明确的益生菌株。
发明内容
本发明的目的是提供一株新的植物乳植杆菌(Lactiplantibacillus plantarum)及其应用;所提供的植物乳植杆菌分离自健康皮肤的表面,能够抑制柯氏棒状杆菌,具有预防或改善面部泛红和I型玫瑰痤疮的功能,效果显著。
本发明所提供的植物乳植杆菌,为植物乳植杆菌VHPribo O04(Lactiplantibaci llusplantarumVHPribo O04),已于2021年5月24日保藏于中国典型培养物保藏中心(地址:中国武汉武汉大学),其保藏号为CCTCC NO:M2021593。
本发明所提供的植物乳植杆菌VHPribo O04株,其Riboprinter 指纹图谱如图3所示;其RAPD指纹图谱如图4所示,rep-PCR指纹图谱如图5所示;MALDI-TOF-MS图谱如图6所示。
本发明所提供的植物乳植杆菌VHPribo O04株在制备具有抗氧化功能的制品中的应用。
本发明所提供的植物乳植杆菌VHPribo O04株在制备具有降解胆固醇功能的制品中的应用。
本发明所提供的植物乳植杆菌VHPribo O04株在制备具有预防或改善面部泛红和I型玫瑰痤疮功能的制品中的应用。
所述的制品为保健品、药品或功能性护肤品。
所述的制品,优选为功能性护肤品。
本发明还提供了一种护肤品,包含植物乳植杆菌VHPribo O04株的溶胞产物。
所述溶胞产物的制备方法如下:
将活化后的植物乳植杆菌VHProbi O04按照1%的体积比接种到发酵培养基中,37℃静置发酵24h;发酵结束后,采用高压均质机对植物乳植杆菌VHProbi O04菌液进行破碎处理,压力为100MPa,重复均质3次;置于70℃水浴锅彻底灭活处理,制备成溶胞产物。
所述发酵培养基中各组分及质量百分比分别为:红糖2%,骨胶原蛋白肽3%,酵母粉0.3%,磷酸氢二胺0.25%,其余为水。
本发明从健康人皮肤表面筛选出的植物乳植杆菌VHProbi O04对常见的抗生素敏感,不产生溶血素,不能够溶解血细胞,具有良好的生物安全性;对人工肠胃液具有较强的耐受性。
所述植物乳植杆菌VHProbi O04能有效吸附黄曲霉毒素B1,吸附率为26.60%;该菌株具有较强的抗氧化能力,DPPH清除率达到18%,HRS清除率达到44.73%,菌体抗脂质过氧化抑制率菌体为21.61%、发酵液为40.97%;该菌株还能有效降解胆固醇,降解率达到20.39%。
所述植物乳植杆菌VHProbi O04对柯氏棒状杆菌有较好的抑菌作用,抑菌圈直径超过18mm;对皮肤细胞的黏附性较好,黏附能力为2.35。该菌株的溶胞产物对皮肤HACAT细胞基本无刺激性,能显著降低炎症因子水平,有效缓解柯氏棒状杆菌引起的炎症反应,且具有剂量依赖性趋势。本发明以植物乳植杆菌VHProbi O04溶胞产物为唯一功效成分制备得到精华乳液。面部泛红志愿者试用精华乳液8周后,皮肤血红素有不同程度下降,下降最大幅度为40.7%。交叉偏振光和五光谱拍照显示志愿者面部泛红或I型玫瑰痤疮有明显改善,取得了意料不到的效果。
本发明所述植物乳植杆菌VHProbi O04可广泛用于制备具有预防或改善皮肤泛红和I型玫瑰痤疮功能的保健品、药品或护肤品,应用前景广阔。
附图说明
图1为O04菌株抑制柯氏棒状杆菌效果图;
图2为O04菌株API 50CHL碳源代谢图;
图3为O04菌株Riboprinter 指纹图谱;
图4为O04菌株的RAPD指纹图谱;
图5为O04菌株的rep-PCR指纹图谱;
图6为O04菌株MALDI-TOF-MS蛋白质指纹图谱;
图7为不同组别HACAT细胞培养液中IL-6含量对比;
图8为志愿者使用含乳酸菌溶胞产物的精华乳液前后交叉偏振光局部照片;
图9为志愿者使用含乳酸菌溶胞产物的精华乳液前后五光谱皮肤镜照片。
具体实施方式
本发明提供的植物乳植杆菌VHProbi O04符合法规要求,可以作为一种食品原料来源使用,长期服用不会有副作用及过量的风险。经多相分类学鉴定,植物乳植杆菌VPHrobi O04为一株新发现的菌株。本发明提供的植物乳植杆菌VHProbi O04 具有预防或改善面部泛红和I型玫瑰痤疮功效,单独使用该菌株且无需与益生元和/或其它益生菌复配即可对面部泛红和I型玫瑰痤疮起到改善效果,具有重要的应用价值。
申请人于2021年5月24日将所述植物乳植杆菌VHProbi O04保藏于中国武汉武汉大学的中国典型培养物保藏中心,其保藏号为CCTCC NO:M2021593。
本发明所述筛选方法并不局限于实施例所述,已知的能够达到筛选目的的方法均可以,实施例的筛选说明只是对本发明的说明,并不是对本发明保护范围的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
下面结合具体实施例对本发明做详细的描述。
实施例1植物乳植杆菌VHProbi O04的分离筛选
1、菌株分离
配制MRS (Man Rogosa Sharpe) 肉汤:纯水1L,蛋白胨10g,牛肉浸取物10g,酵母提取物 5.0g, 乙酸钠5g,葡萄糖5g,磷酸二氢钾2g,吐温80 1.0mL,柠檬酸二胺 2.0g,碳酸钙20g,七水硫酸镁0.58g,七水硫酸锰0.25g,调pH 6.2-6.5。
配制MRS琼脂培养基1LMRS肉汤添加15g琼脂。
依据2019版《人类遗传资源库伦理规范》,与样本提供者签订项目承诺书和知情同意书后,按照生物样本库标准操作规范,用无菌棉签于14天内未使用过抗菌类或激素类面霜、药膏等护肤品、无皮肤疾病的健康人前额中央区约4cm2,以一定的力度来回摩擦50次,将棉签头用无菌剪刀剪下置于保护液中,反复震荡。取100μL混匀液梯度稀释,涂布于MRS琼脂培养基后于37℃厌氧培养48h,待平板长出单菌落镜检。根据镜检结果,申请人共筛选出20株(类)杆菌,并反复纯化,确定得到的是纯菌株,分别命名为O01、O02、……、O18、O19、O20。
2、抗柯氏棒状杆菌乳酸菌筛选
乳酸菌菌液制备:将上述分离得到的20株潜在乳酸菌分别接种至MRS肉汤,37℃静置氧培养48h。
致病菌活化:柯氏棒状杆菌(DSM109755,DSM44385)接种至含5%牛血清的营养肉汤中,37℃培养48h。
铺下层培养基,营养琼脂灭菌后倒入平板中,铺满平板即可。待琼脂凝固后,均匀放置无菌牛津杯。铺上层培养基,将上述活化后的柯氏棒状杆菌菌液按0.4%体积比加入营养肉汤半固体培养基中。菌与培养基混匀后,取14mL倾注到下层培养基上。待凝固后取出牛津杯,并取混匀的100μL乳酸菌发酵液加入牛津杯孔中。在37℃条件下,培养48h后观察有无抑菌圈。
结果显示,本发明分离得到的 20株潜在乳酸菌中有8株菌抑菌圈直径超过15mm,分别为O02、O03、O04、O07、O08、O10、O12、O17。其中O04菌株对柯氏棒状杆菌的抑菌圈直径最大,为18.67±0.47mm(见图1),抑菌效果最强。
实施例2 O04菌株鉴定
1、菌落形态鉴定
将O04菌株接种于MRS琼脂培养基上,37℃厌氧培养24h后,可见O04菌株的单菌落呈乳白色,菌落直径在1-3mm左右,表面光滑,显微镜下菌株形态为杆菌。
2、生理生化特性鉴定
本实施例中接种液的准备如下:在无菌条件下,取适量新鲜O04菌液,5000rpm/min离心5 min,用PBS缓冲液洗2次,再用同体积PBS缓冲液重菌体后稀释50倍,作为接种液。
2.1、盐度耐受性试验
在无菌条件下,向96孔板中分别加入190μL盐浓度为1%、2%、3%、4% 、5%、6%、7%、8%的BSM液体培养基,每个盐浓度做3个平行,然后再加入10μL接种液,不接菌的孔作为对照。每孔加入50μL高压灭菌过的石蜡油以防止培养过程中水分蒸发。置于37℃恒温培养,观察培养基是否变浑浊。结果显示O04菌株最大耐受盐浓度为8%。
2.2、温度耐受性试验
取适量新鲜菌液(24h,37℃),5000rpm 离心5 min ,用PSP溶液洗一次,再用同体积PSP溶液重悬后稀释50倍,作为接种液。
将接种液按10% 的接种量接种到10mL MRS液体培养基中,不接菌的5mL MRS液体培养基作为对照,分别置于15℃恒温培养箱培养7天,45℃恒温培养箱培养2天,观察菌液是否变浑浊。
结果显示,O04 菌株在15℃耐受性较好,45℃不耐受。
2.3、碳源代谢试验
利用API 50CHL试剂条测定O04菌株对49种碳源的代谢作用。试验方法及结果判读参照API 50CHL试剂盒说明书进行操作。
O04菌株鉴定结果为:%ID=99.9且T值=0.87,API结果为植物乳植杆菌,为非常好的鉴定。结果见图2。
2.4、葡萄糖产酸产气试验
本实施例中所用的培养基配方如下:
蛋白胨 0.5g;酵母提取物 0.3g;吐温 80 0.1mL;盐溶液 A 0.5ml;盐溶液 B0.5ml;乙酸钠 0.5g;葡萄糖 2.5g;2%溴甲酚绿(w/v) 0.05mL;蒸馏水 100ml;
pH6.8~7.0。
将配制好的培养基分装至含有倒置小试管的大试管中,3mL/管,121℃,高
压灭菌 15min。
盐溶液 A 成分:KH2PO4 10g、K2HPO4 1.0g,溶于蒸馏水,定容至 100mL。
盐溶液 B 成分:MgSO4·7H2O 11.5g、MnSO4·2H2O 2.4g、FeSO4·7H2O 0.68g,溶于蒸馏水,定容至 100mL。
在无菌条件下,将接种液按 10%的接种量接种培养基,不接菌的培养基作为对照,然后用 2mL 无菌液体石蜡封住顶部,置于 37℃培养24h,观察培养基颜色有无变化。
结果显示:37℃培养 24h后,培养基由绿色变为黄色,小倒管内无气体,说明 O04菌株发酵葡萄糖产酸不产气。
2.5、精氨酸产氨试验
在PY基础培养液中加入配制好的精氨酸溶液。
精氨酸溶液的成分及制备如下:
精氨酸1.5g;半胱氨酸(1g/10ml H2O) 0.05ml;蒸馏水10ml;
调pH至7.0,灭菌后加3滴至3ml培养基中。
取适量新鲜菌液(24h,37℃),5000rpm 离心5 min ,用PSP溶液洗一次,再用同体积PSP溶液重悬后稀释50倍,作为接种液。
将接种液按10% 的接种量接种于10mL含精氨酸的培养基中,并同时接种不含精氨酸的培养基作为对照。置适温培养1~3d。
取已生长好的培养液1滴于洁净载玻片表面,加入1~2滴奈氏试剂,若黄色变为棕色,为阳性反应,产氨。
结果显示:奈氏试剂检测结果为阴性,说明O04菌株不产氨。
3、分子生物学鉴定
3.1 16s rDNA 基因序列分析
3.1.1 基因组DNA提取
参照天根细菌基因组DNA提取试剂盒(目录号:DP302)操作。
3.1.2 16s rDNA 基因扩增
引物序列:
27F:AGAGTTTGATCCTGGCTCA;
1492R:GGTTACCTTGTTACGACTT。
通过测序获得O04菌株的16s rDNA序列SEQ ID NO:1,并将该序列在NCBI 数据库中进行比对,初步确定O04菌株为植物乳植杆菌。
3.2 Riboprinter指纹图谱
用一根取菌棒从琼脂培养基平板上沾取已纯化好的单菌落,将其放入有缓冲液的样品管中,用手持搅拌器搅拌使其在缓冲液中悬浮,然后将样品架放入加热器中灭活后放入Riboprinter 系统中,样品经过DNA制备、转膜、成像检测及数据处理后,得到细菌鉴定结果。鉴定结果显示,O04菌株为植物乳植杆菌,其Riboprinter指纹图谱结果见图3。
3.3 RAPD和rep-PCR指纹图谱鉴定
3.3.1 RAPD指纹图谱鉴定
引物序列: GAGGGTGGCGGTTCT。
表1 RAPD 反应体系
反应成分 | 体积 |
TaqDNA 聚合酶(5U/μL) | 0.2 μl |
10×Buffer(含Mg2+) | 2 μl |
引物(10 uM) | 1 μl |
dNTPs(2.5 mM) | 0.8 μl |
DNA模板 | 2 μl |
无菌双蒸水 | 14 μl |
总体积 | 20 μl |
制备1.5%的琼脂糖凝胶板,DL2000DNA Marker 作为结果对照,稳压100V电80min,最后利用凝胶成像系统检测电泳图。
O04菌株的RAPD指纹图谱如图4所示。
3.3.2 rep-PCR指纹图谱
引物序列:CTACGGCAAGGCGACGCTGACG。
表2 rep-PCR 的反应体系
反应成分 | 体积 |
r TaqDNA聚合酶 | 0.2 μl |
10×Ex Taq DNA Buffer(含Mg2+) | 2 μl |
引物(10 uM) | 1 μl |
dNTPs(2.5 mM) | 2 μl |
DNA模板 | 2 μl |
无菌双蒸水 | 12.8 μl |
DL2000 DNA Marker 作为结果对照。电压100 V,电泳时间80min 检测扩增结果。O04菌株的的rep-PCR指纹图谱如图5所示。
3.4 MALDI-TOF-MS 检测菌株核糖体蛋白表达
按照 0.1%的接种量在 MRS 液体培养基中接种新鲜菌液,37℃,150rpm 培养 48h后,收集菌体,无菌水洗涤 4 次,晾干表面水分。然后取少量新鲜菌体以薄膜的形式均匀涂布于靶板上,加 1μL 溶胞产物覆盖样品,晾干后,再加 1μL 基质溶液覆盖样品,晾干后,将样品靶放入质谱仪进行鉴定。用激光照射样品与基质形成的共结晶薄膜,使样品中蛋白质电离,离子在 10~20KV 电场作用下加速飞过飞行管道,根据到达检测器的飞行时间不同检测蛋白质的分子量。利用Autofms 1000 分析软件 Autof Analyzer v1.0 获取蛋白质指纹图谱,O04 菌株主要核糖体蛋白的离子峰为:m/z5184.576、5403.998、5732.765、6111.272、6867.898、7285.449、7425.843、7852.539、8948.927、9485.074。鉴定结果见图6。
3.5全基因组测序
取新鲜O04菌液按照1%的体积比例接种到500 mL MRS肉汤培养基中,37 ℃培养20h,8000rpm离心10 min,收集菌体。菌体送到测序中心,得到该菌的全基因组序列,基因组序列已上传至NCBI基因数据库,GenBank 登录号为CP094950-CP094951,包含1个染色体和 1个质粒,GenBank登录号分别是CP094950和CP094951。
将O04菌株的菌落形态以及生理生化特性结果上传至网站http://www.tgw1916.net/bacteria_logare_desktop.htmL,同时结合文献De Clerck E,et al. Systematic andapplied microbiology,2004,27(1)50公布的结果,进行比对。综合分子生物学的鉴定结果,确定O04菌株为一株新的植物乳植杆菌,将其命名为植物乳植杆菌VHProbi O04(Lactiplantibacillus plantarumVHProbi O04)。
实施例3植物乳植杆菌VHProbi O04对人工胃液和人工肠液的耐受性试验
1、人工胃液的配制
分别称取蛋白胨 5g、酵母提取物 2.5g、葡萄糖1g和NaCl 2g,加入1000mL蒸馏水,用稀盐酸调pH3.0,然后115℃灭菌20min。然后使用前加入3.2g猪粘膜胃蛋白酶,摇匀溶解,置37℃水浴摇床中温水浴1h,以模拟人体温度。
2、人工肠液的配制
分别称取蛋白胨5g、酵母提取物2.5g、葡萄糖1g、KH2PO46.8g和牛胆盐 3.0g,加入77mL 的0.2mol/L的NaOH溶液,定容至1000mL,用稀盐酸或者氢氧化钠溶液调pH6.8±0.1,115℃灭菌20min。然后使用前加入1g胰酶,摇匀溶解,置37℃水浴摇床中温水浴1h,以模拟人体温度。
3、试验方法
取2mL新鲜菌液,5000rpm/min 离心5min 收集菌体,菌体用生理盐水洗涤3次,再用2mL 生理盐水重悬,作为接种液。取1mL接种液,加入到24mL人工胃液或肠液中,置于37℃水浴摇床(200rpm/min)3h,取样1mL,检测活菌量。
活菌计数方法按照国标《GB4789.35-2016-食品微生物检验乳酸菌检验》测定菌量,该菌株经过人工胃液或肠液消化后的活菌量(Log CFU/mL)见表3。
表3 人工胃肠液消化后的活菌量
消化前 | 人工胃液消化后 | 人工肠液消化后 |
7.85±0.01 | 7.74±0.00 | 7.23±0.03 |
从表3可知,本发明筛选到的植物乳植杆菌 VHProbi O04 经人工胃液和肠液消化后,存活率下降极小,说明该菌株对人工胃液和肠液具有很强的耐受性。
实施例4植物乳植杆菌VHProbi O04的溶血性及抗生素耐受性试验
1、溶血性试验
称取TBS基础培养基的各种组分,溶解,121℃高压灭菌15 min,等培养基冷却到50℃的时候加入5%的无菌脱纤维绵羊血,混匀,倒平板。将测试菌株划线接种于准备好的血细胞平板,37℃培养箱培养,24 ~ 48h观察测试菌是否有溶血现象。
结果显示:植物乳植杆菌 VHProbi O04不能生长,血细胞平板没有变化,说明植物乳植杆菌 VHProbi O04不产生溶血素,不能够溶解血细胞。
2、抗生素耐受性试验
微量肉汤稀释法测定抗生素对植物乳植杆菌 VHProbi O04的最小抑菌浓度MIC值具体结果见表4。
表4 植物乳植杆菌 VHProbi O04的抗生素MIC值
MIC单位μg/mL。
从表4的结果可以看出,本发明提供的植物乳植杆菌 VHProbi O04对红霉素等敏感,生物安全性良好。
实施例5植物乳植杆菌VHProbi O04的黄曲霉毒素B1吸附能力
1、配制浓度为1μg/mL的AFB1-PBS溶液。
2、接种吸附:取1mL新鲜菌液(24h,37℃),8000rpm离心5min,弃上清,用同体积PBS缓冲液清洗菌体2次,8000rpm离心5min,弃上清,然后将菌体重悬于1mL上述AFB1-PBS溶液,置于37℃恒温培养箱,1h后取出,8000rpm离心10min,取上清待测。
3、按照黄曲霉毒素B1检测试剂盒说明书测定上清液中黄曲霉毒素B1浓度。
测定前,将上清液用甲醇稀释100倍。
结果显示:本发明提供的植物乳植杆菌 VHProbi O04 的黄曲霉毒素B1吸附率为26.60%,标准差为0.06%
实施例6植物乳植杆菌VHProbi O04抗氧化功能测定
1、菌株清除DPPH(1,1-二苯基-2-三硝基苯肼)能力测定
取1mL待测菌株的PBS菌悬液,加入1mL 0.4 mM的现配的DPPH自由基溶液,混合均匀后然后置于室温温度下遮光反应30 min,然后测定样品在波长 517nm处的吸光度A样本,测3次平行。对照组样品以等体积PBS溶液和DPPH·乙醇混合液,并以等体积PBS菌悬液和乙醇混合液空白调零。清除率按下列公式计算:清除率%=[1-(A样品-A空白)/A对照]×100%。结果见表5。
表5 DPPH自由基清除率表
菌株 | 清除率% | 标准差 |
植物乳植杆菌 VHProbi O04 | 18.05% | 1.5% |
2、菌株清除HRS能力的测定
将100μL 5mM的水杨酸钠-乙醇溶液,100μL 5mM的硫酸亚铁,500μL去离子水和200μL乳酸菌PBS菌悬液混匀后加入100μL过氧化氢溶液(3mM),37℃水浴15min后在510nm波长处测量样品吸光度。羟自由基清除率按照下列公式进行计算。
清除率%=(A样品-A控制)/(A空白-A控制)×100%。
其中:A控制为去离子水替代样品,A空白为去离子水替代样品和H2O2。
具体结果见表6。
表6 HRS自由基清除率表
菌株 | 清除率% | 标准差 |
植物乳植杆菌 VHProbi O04 | 44.73% | 1.4% |
3、菌株抗脂质过氧化实验鉴定
亚油酸乳化液的制备:0.1mL亚油酸,0.2mL Tween 20,19.7mL去离子水。
0.5 mL的PBS溶液(pH 7.4)中加入1 mL亚油酸的乳化液, 1 mLFeSO4(1%),再加入0.5 mL样品,37℃水浴1.5 h,混合液加入0.2 mL TCA(4%),2 mL TBA(0.8%),100 ℃水浴30min,迅速冷却,4000 rpm/min离心15 min,收集上清液在532 nm下测吸光度即为A;对照组以0.5 mL蒸馏水代替样品即为A0。
抑制率/% =(A0-A)/ A0×100%。
其中,A为样品组吸光度;A0为对照组吸光度。具体结果见表7。
表7植物乳植杆菌 VHProbi O04抗脂质过氧化抑制率表
抑制率 | 标准差 | |
菌体 | 21.61% | 2.2% |
发酵上清液 | 40.97% | 4.59% |
实施例7植物乳植杆菌VHProbi O04体外胆固醇降解试验
1、胆固醇胶束溶液的配制
准确称取1g 胆固醇,溶于无水乙醇中,并定容至100 mL,在无菌条件下用 0.22 µm 微孔滤膜过滤除菌。
2、培养基配制
称取蛋白胨10.0 g、牛肉膏10.0 g、酵母膏 5.0 g、柠檬酸氢二铵2.0 g、葡萄糖20.0 g、吐温80 1.0 mL、乙酸钠5.0 g、硫酸镁0.1 g、硫酸锰 0.05 g、磷酸氢二钾2.0 g、胆盐1.0 g、蒸馏水1000mL,调节pH值7.3,115℃灭菌30min,然后加入胆固醇溶液使胆固醇终浓度为0.1%。
按照0.1%的体积比将植物乳植杆菌 VHProbi O04新鲜菌液接种至上述培养基中,37℃静止培养48h,然后取0.2mL菌液,加入1.8mL无水乙醇,混匀,静止10 min,3000转离心5min,取上清液测定胆固醇含量。胆固醇测定方法按照GB/T 5009.128-2003<食品中胆固醇的测定>。
结果显示:本发明提供的植物乳植杆菌 VHProbi O04对胆固醇的降解率达到20.39%(此为不含胆盐的数据)。
实施例8植物乳植杆菌VHProbi O04的皮肤细胞黏附性测试
将HACAT 细胞培养至近汇合,使用胰酶消化为单细胞状态;取适量细胞使用血球计数板进行细胞计数。使用MRS培养基将菌体重悬备用。将待测菌悬液与HACAT 细胞共同孵育2h后,PBS洗去未黏附的细菌。加入胰酶消化细胞,收集液体涂布计数。
黏附能力(CFU/cells)=每个培养孔内粘附的细菌总数/每个培养孔的总细胞数。
结果显示:植物乳植杆菌 VHProbi O04黏附能力为2.35。
实施例9植物乳植杆菌VHProbi O04灭活菌体及溶胞产物抗柯氏棒状杆菌的皮肤细胞试验
1、植物乳植杆菌VHProbi O04灭活菌体溶液制备:
植物乳植杆菌 VHProbi O04使用MRS肉汤培养至稳定期,离心,PBS清洗3次,使用同体积PBS重悬,70℃水浴热灭活15min,得到灭活菌体溶液。
2、植物乳植杆菌VHProbi O04溶胞产物制备:
配制发酵培养基,其各组分及质量百分比分别为:红糖2%,骨胶原蛋白肽3%,酵母粉0.3%,磷酸氢二胺0.25%,其余为水。
将活化后的植物乳植杆菌VHProbi O04按照1%的体积比接种于上述发酵培养基中,37℃静置发酵24h;发酵结束后,采用高压均质机对植物乳植杆菌VHProbi O04菌液进行破碎处理,压力为100MPa,重复均质3次;置于70℃水浴锅彻底灭活处理,制备成溶胞产物。
使用3000D透析袋将上述溶胞产物进行透析处理,透析液为PBS缓冲液,透析过程一共更换3次透析液,每次更换透析液间隔时间为8-16h,获得溶胞产物透析液。
3、细胞试验:
将HACAT细胞复苏、培养至所需量,培养基为10%FBS的1640培养基。HACAT细胞密度生长至近汇合,胰酶消化成单细胞悬液,血球计数板计数,接种于12孔板,接种密度为4×105cells/孔;每孔培养基的添加量为1ml。细胞在孔板中继续培养24h后,更换新鲜培养液。
将柯氏棒状杆菌培养24h后,调节其浓度为1×109CFU/ml,上述每个细胞板孔添加量为20μl,同时,将植物乳植杆菌 VHProbiO04灭活菌体溶液或溶胞产物以20μl或50μl的添加量加入到细胞培养液中。具体分组及处理方式如下:
(1)植物乳植杆菌 VHProbiO04灭活菌体实验分组:A组为空白对照,不添加任何物质;B组只添加柯氏棒状杆菌(20ul);C组只添加VHProbi O04灭活菌体(20ul);D组添加柯氏棒状杆菌(20ul)+ VHProbi O04灭活菌体(20ul);E组添加柯氏棒状杆菌(20ul)+ VHProbiO04灭活菌体(50ul);
(2)植物乳植杆菌 VHProbiO04溶胞产物实验分组:A组为空白对照,不添加任何物质;B组只添加柯氏棒状杆菌(20ul);C组只添加VHProbi O04溶胞产物(20ul);D组添加柯氏棒状杆菌(20ul)+ VHProbi O04溶胞产物(20ul);E组添加柯氏棒状杆菌(20ul)+ VHProbiO04溶胞产物(50ul)。
培养24小时后,取上清液,利用ELISA试剂盒检测IL-6含量。
4、结果
白细胞介素(IL)是由多种细胞产生并作用于多种细胞的一类细胞因子,在传递信息,激活与调节免疫细胞,介导T、B细胞活化、增殖与分化及在炎症反应中起重要作用。IL-1α、IL-6、IL-8是主要的炎症反应促发剂,且IL-6是炎症发生时最早升高的标志物。
结果如图7所示,与空白对照A组相比,添加柯氏棒状杆菌的B组培养液中IL-6含量明显升高,添加灭活菌体或溶胞产物的C组IL-6含量未有明显变化,说明柯氏棒状杆菌刺激HACAT细胞产生了炎症反应,而植物乳植杆菌 VHProbiO04灭活菌体及其溶胞产物对HACAT细胞基本无刺激性;
与只添加柯氏棒状杆菌的B组相比,同时添加灭活菌体的D组和E组(左图)培养液中IL-6含量未有明显变化,而添加溶胞产物的D组和E组(右图)IL-6含量明显下降,且随着溶胞产物添加量的增加,IL-6含量下降幅度加大,从而说明植物乳植杆菌 VHProbiO04灭活菌体对缓解炎症反应的效果不明显,但其溶胞产物能显著降低促炎症反应因子的含量,有效缓解柯氏棒状杆菌引起的炎症反应,且具有剂量依赖性趋势,取得了意料不到的技术效果。
实施例10植物乳植杆菌VHProbi O04溶胞产物对面部皮肤泛红改善效果
从前期体外实验结果可知,植物乳植杆菌VHProbi O04对柯氏棒状杆菌有显著的抑制作用,其灭活菌体和溶胞产物均对皮肤HACAT细胞无刺激性,且溶胞产物能有效缓解柯氏棒状杆菌引起的炎症反应。
动物实验结果也已证实,植物乳植杆菌VHProbi O04能显著降低机体炎症因子水平,缓解柯氏棒状杆菌感染的大鼠皮肤炎症状态,对皮肤损伤、泛红程度具有一定的改善作用。
1、植物乳植杆菌VHProbi O04 溶胞产物制备
1.1、植物乳植杆菌VHProbi O04溶胞产物制备:
配制发酵培养基,其各组分及质量百分比分别为:红糖2%,骨胶原蛋白肽3%,酵母粉0.3%,磷酸氢二胺0.25%,其余为水。
将活化后的植物乳植杆菌VHProbi O04按照1%的体积比接种于上述发酵培养基中,37℃静置发酵24h;发酵结束后,采用高压均质机对植物乳植杆菌VHProbi O04菌液进行破碎处理,压力为100MPa,重复均质3次;置于70℃水浴锅彻底灭活处理,制备成溶胞产物。
所得溶胞产物,外观为浅棕色至棕褐色,pH值5.0±0.2,可溶性固含物含量5-10%,菌落总数小于10CFU/ml,无致病菌检出,重金属砷未检出,符合化妆品卫生标准GB7916-87的质量要求。
按5%体积比将1,2-己二醇添加至溶胞产物中,备用。
2、植物乳植杆菌VHProbi O04溶胞产物安全性检测
选择合适的年龄范围在18-60岁志愿者20人,进行皮肤斑贴试验。
2.1 试验方法
取0.02ml-0.025ml上述溶胞产物滴加在滤纸片上,再将滤纸片置于斑试器内。每个样品均设置空白对照。将加有受试物的斑试器用低致敏胶带贴敷于受试者的前臂曲侧,用手掌轻压使之均匀地贴敷于皮肤上,持续24h。24h后去除斑试器,静置30min后,等待压痕消失,观察皮肤的反应。如果试验结果为阴性,则需要在斑贴试验后24h和48h分别再观察一次。
2.2 试验结果
发现20名受试者使用上述溶胞产物均未产生可疑反应,说明本发明提供的植物乳植杆菌VHProbi O04溶胞产物具有安全性,不会给人体带来不良反应。
3、植物乳植杆菌VHProbi O04溶胞产物化妆品人体测试
3.1、植物乳植杆菌VHProbi O04溶胞产物精华乳液制备
按照表8所述配方比例配制精华乳液,其中植物乳植杆菌VHProbi O04溶胞产物为唯一功效成分,其质量百分比为为8%。
表8 植物乳植杆菌VHProbi O04溶胞产物精华乳液配方
配方比例 | g/份 | 配方比例 | g/份 |
去离子水 | To 100 | 霍霍巴籽油 | 0.6 |
VHProbi O04溶胞产物 | 8 | 山梨坦橄榄油酸酯 | 0.6 |
聚二甲基硅氧烷(5cst) | 5 | 鲸蜡硬酯醇 | 0.4 |
甘油 | 4 | 1,2-己二醇 | 0.4 |
辛酸/癸酸甘油三酯 | 3 | 聚丙烯酸酯交联聚合物-6 | 0.35 |
异十六烷 | 2.5 | 对羟基苯乙酮 | 0.3 |
鲸蜡硬脂醇橄榄油酸酯 | 1.5 | 苯氧乙醇 | 0.2 |
聚二甲基硅氧烷(350cst) | 1 | 透明质酸钠 | 0.1 |
牛油果树果脂 | 0.8 | EDTA二钠 | 0.05 |
3.2、招募年龄范围在18-60岁的面部泛红或Ⅰ型玫瑰痤疮志愿者8名。在开始前检测志愿者皮肤以下各项指标,志愿者每天早晚使用精华乳液各一次,使用8周后再次进行指标检测。
3.3检测指标
志愿者进行皮肤检测之前需洗脸后在室温静坐30min。
3.3.1、血红素测试
使用德国CK MC1000多功能测试仪S0043血红素测试仪,检测毛细血管中血红素含量,通过检测面部泛红严重部位的血红素下降的程度判断泛红或红血丝改善问题。
3.3.2、交叉偏振光局部照片
使用德国CK VisioScope PC35显微镜拍摄皮肤下层的微观红血丝及血管状态,较为直观观察红血丝改善情况。
3.3.3、五光谱皮肤镜照片
使用德国DJM 五光谱皮肤检测仪整体观察面部泛红或I型玫瑰痤疮改善情况。
3.4、结果
3.4.1、血红素测试
如表9所示,使用含植物乳植杆菌VHProbi O04溶胞产物的精华乳液8周,8位志愿者面部血红素均有下降,下降幅度9.7%-40.7%,改善效果较好。
表9 志愿者面部血红素检测数值
志愿者序号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
使用前数值 | 40.8 | 67.8 | 47 | 41.5 | 47.7 | 46.6 | 46.7 | 45.2 |
使用后数值 | 35.2 | 40.2 | 39.8 | 36.5 | 38.8 | 42.1 | 40.1 | 40.7 |
下降百分比 | 13.7% | 40.7% | 15.3% | 12.0% | 18.5% | 9.7% | 14.1% | 10.0% |
3.4.2、交叉偏振光显微照片对比
拍摄部位选取红血丝集中区域,如图8所示,使用含植物乳植杆菌VHProbi O04溶胞产物的精华乳液后,志愿者面部红血丝有减轻趋势,尤其其中一位I型玫瑰痤疮志愿者改善效果明显。
3.4.3 五光谱皮肤镜筛选照片
如图9所示,使用含植物乳植杆菌VHProbi O04溶胞产物的精华乳液后,志愿者脸部泛红或红血丝明显改善,皮肤状态也较使用前有所改善。
上述实验结果表明,本发明提供的植物乳植杆菌VHProbi O04 溶胞产物对皮肤泛红和I型玫瑰痤疮具有较为显著的改善作用。
综上所述,本发明提供的植物乳植杆菌VHProbi O04 对常见抗生素敏感,不产溶血素,生物安全性良好。所述植物乳植杆菌VHProbi O04溶胞产物对于皮肤HACAT细胞无刺激性,且能显著降低炎症因子水平,有效缓解因柯氏棒状杆菌引起的炎症反应。化妆品人体测试结果显示,植物乳植杆菌VHProbi O04溶胞产物对改善皮肤泛红和I型玫瑰痤疮具有较为显著的作用,可广泛用于制备具有预防或改善皮肤泛红和I型玫瑰痤疮功能的保健品、药品或护肤品。
序列表
<110> 山东百沃生物科技有限公司
<120> 一株具有预防或改善面部泛红和I型玫瑰痤疮功能的植物乳植杆菌
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1433
<212> DNA
<213> 植物乳植杆菌(Lactiplantibacillus plantarum)
<400> 1
ggctggttcc taaaaggtta ccccaccgac tttgggtgtt acaaactctc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta 120
gcgattccga cttcatgtag gcgagttgca gcctacaatc cgaactgaga atggctttaa 180
gagattagct tactctcgcg agttcgcaac tcgttgtacc atccattgta gcacgtgtgt 240
agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca 300
ccggcagtct caccagagtg cccaacttaa tgctggcaac tgataataag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420
atccatgtcc ccgaagggaa cgtctaatct cttagatttg catagtatgt caagacctgg 480
taaggttctt cgcgtagctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcag ccttgcggcc gtactcccca ggcggaatgc ttaatgcgtt 600
agctgcagca ctgaagggcg gaaaccctcc aacacttagc attcatcgtt tacggtatgg 660
actaccaggg tatctaatcc tgtttgctac ccatactttc gagcctcagc gtcagttaca 720
gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 780
acatggagtt ccactgtcct cttctgcact caagtttccc agtttccgat gcacttcttc 840
ggttgagccg aaggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca 900
ataaatccgg acaacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960
gtggctttct ggttaaatac cgtcaatacc tgaacagtta ctctcagata tgttcttctt 1020
taacaacaga gttttacgag ccgaaaccct tcttcactca cgcggcgttg ctccatcaga 1080
ctttcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtttg ggccgtgtct 1140
cagtcccaat gtggccgatt accctctcag gtcggctacg tatcattgcc atggtgagcc 1200
gttaccccac catctagcta atacgccgcg ggaccatcca aaagtgatag ccgaagccat 1260
ctttcaaact cggaccatgc ggtccaagtt gttatgcggt attagcatct gtttccaggt 1320
gttatccccc gcttctgggc aggtttccca cgtgttactc accagttcgc cactcactca 1380
aatgtaaatc atgatgcaag caccaatcaa taccagagtt cgttcgactg cat 1433
Claims (6)
1.一种植物乳植杆菌(Lactiplantibacillus plantarum),其特征在于,所述植物乳植杆菌的保藏号为CCTCC NO:M2021593。
2.权利要求1所述的植物乳植杆菌在制备有助于抗氧化的保健品中的应用。
3.权利要求1所述的植物乳植杆菌在制备用于降低胆固醇的药品中的应用。
4.权利要求1所述的植物乳植杆菌在制备具有改善面部泛红功能的护肤品中的应用,其特征在于,所述植物乳植杆菌为灭活菌体或其溶胞产物。
5.一种护肤品,其特征在于,所述的护肤品包含权利要求1所述植物乳植杆菌的溶胞产物;所述溶胞产物的制备方法为:将活化后的权利要求1所述植物乳植杆菌按照1%的体积比接种于发酵培养基中,37℃静置发酵24h;发酵结束后,采用高压均质机对植物乳植杆菌菌液进行破碎处理,压力为100MPa,重复均质3次;置于70℃水浴锅彻底灭活处理,制备成溶胞产物。
6.如权利要求5所述的护肤品,其特征在于,所述的发酵培养基中各组分及其质量百分比分别为:红糖2%,骨胶原蛋白肽3%,酵母粉0.3%,磷酸氢二胺0.25%,其余为水。
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