CN114703108A - 一株发酵粘液乳杆菌及其在改善面部泛红和i型玫瑰痤疮中的应用 - Google Patents
一株发酵粘液乳杆菌及其在改善面部泛红和i型玫瑰痤疮中的应用 Download PDFInfo
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- CN114703108A CN114703108A CN202210493250.8A CN202210493250A CN114703108A CN 114703108 A CN114703108 A CN 114703108A CN 202210493250 A CN202210493250 A CN 202210493250A CN 114703108 A CN114703108 A CN 114703108A
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Abstract
本发明属于益生菌筛选与应用技术领域,具体涉及一株新的发酵粘液乳杆菌(Limosilactobacillus fermentum)及其应用。所提供的发酵粘液乳杆菌分离自健康成年女性口腔,能够显著抑制柯氏棒状杆菌,有效改善面部泛红和I型玫瑰痤疮的症状,已于2021年5月24日保藏于中国典型培养物保藏中心,其保藏号为CCTCC NO:M2021597。
Description
技术领域
本发明属于益生菌筛选与应用技术领域,具体涉及一株具有改善面部泛红和I型玫瑰痤疮功能的发酵粘液乳杆菌及其应用。
背景技术
皮肤作为人体表面积最大的器官承载着来自1000多种物种约1千亿个微生物,其中74%~80%为细菌,5%~10%为真菌,10%~20%为病毒。这些微生物主要存在于皮肤表面以及皮肤附属器中,如毛囊、汗腺及皮脂腺等,丙酸杆菌属、棒状杆菌属、葡萄球菌属和马拉色菌属等微生物共同组成皮肤微生物的多样性。不同的微生物和微生物之间、不同的皮肤环境和微生物之间共同维持着皮肤的动态平衡。
越来越多的证据表明,皮肤微生态失调与诸多皮肤问题相关。有研究证明了皮肤微生物组的变化与皮肤屏障之间存在联系,并确认了对三种最常见的皮肤类型干性、油性和中性皮肤影响最大的三种细菌,即痤疮丙酸杆菌能够影响皮脂生成、引起痤疮;表皮葡萄球菌是健康皮肤的基石;柯氏棒状杆菌是控制皮肤发红、玫瑰痤疮的新靶点。其中,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。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,其标志性特点有面中部红斑,发红,毛细管扩张。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。皮肤微生态及柯氏棒状杆菌的深入研究为面部泛红和I型玫瑰痤疮的治疗提供了新的思路。
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(例如双歧杆菌、乳酸杆菌)。保护皮肤的益生菌群,减少致病菌,增强皮肤免疫屏障是健康皮肤的基石。目前认为,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。与食品中添加的益生菌不同,在化妆品中添加的是益生菌发酵液或其溶胞产物成分。益生菌乳酸杆菌发酵产物含有水溶性活性物质,包括肽聚糖和脂磷壁酸,以及代谢物(包括肽、细菌素、短链脂肪酸和有机酸),能够预防和治疗轻度皮肤感染。研究显示每日 2 次外用含长双歧杆菌溶胞产物的润肤霜 29 d 可增强敏感性皮肤的皮肤屏障功能,进而改善皮肤干燥,缓解皮肤敏感。荷兰皇家帝斯曼取自酵母菌的护肤活性物OXY 229 PF,能降低油脂水平,还能最大程度地减少柯氏棒状杆菌水平,预防面部泛红。日本一丸公司的La· FloraEC-12美肌菌,是乳酸杆菌的发酵产物,也用于提升皮肤屏障机能。
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发酵粘液乳杆菌(Limosilactobacillusfermentum)及其应用;所提供的发酵粘液乳杆菌分离自健康成年女性口腔,能够显著抑制柯氏棒状杆菌,有效改善面部泛红和I型玫瑰痤疮的症状。
本发明所提供的发酵粘液乳杆菌,为发酵粘液乳杆菌VHProbi O48(Limosilacto bacillusfermentumVHProbi O48)株,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:M2021597。
本发明所提供的发酵粘液乳杆菌VHProbi O48株,其Riboprinter指纹图谱如图4所示;其RAPD指纹图谱如图5所示,rep-PCR指纹图谱如图6所示;MALDI-TOF-MS图谱如图7所示。
本发明所提供的发酵粘液乳杆菌VHPribO48株在制备具有抗氧化功能的制品中的应用。
本发明所提供的发酵粘液乳杆菌VHPribO48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、药品或护肤品。
所述的制品优选护肤品。
本发明还提供了一种护肤品,包含发酵粘液乳杆菌VHPribO48株的溶胞产物。
本发明筛选出的发酵粘液乳杆菌VHProbi O48对胃液具有很强的耐受性,对红霉素和氨苄西林等常见的抗生素敏感,不产生溶血素,不能够溶解血细胞,具有良好的生物安全性。该菌株具有较强的抗氧化能力,DPPH清除率达到17.72%,抗脂质过氧化抑制率为57.6%。
所述发酵粘液乳杆菌VHProbi O48对柯氏棒状杆菌有较强的抑制作用,抑菌圈直径达到17.33mm;其溶胞产物对皮肤HACAT细胞基本无刺激性,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。本发明以发酵粘液乳杆菌VHProbi O48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,皮肤胆红素有不同程度下降,下降幅度最大为24.9%,效果显著;交叉偏振光和五光谱拍照显示志愿者面部泛红有明显改善,取得了意料不到的效果。
所述发酵粘液乳杆菌VHProbi O48可广泛用于制备具有改善皮肤泛红和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O48菌株抑菌圈图;
图2为O48菌株API 50CHL碳源代谢图;
图3为O48菌株胆盐酶活性图;
图4为O48菌株Riboprinter 指纹图谱;
图5为O48菌株的RAPD指纹图谱;
图6为O48菌株的rep-PCR指纹图谱;
图7为O48菌株MALDI-TOF-MS鉴定图谱;
图8为不同组别HACAT细胞培养液中IL-6含量对比;
图9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10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
本发明提供的发酵粘液乳杆菌VHProbi O48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,发酵粘液乳杆菌VPHrobi O48为一株新发现的菌株。本发明提供的发酵粘液乳杆菌VHProbi O48 具有防治面部泛红和I型玫瑰痤疮的功效,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型玫瑰痤疮起到防治功效,具有重要的应用价值。
申请人于2021年5月24日将所述发酵粘液乳杆菌VHProbi O48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:M2021597。
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 发酵粘液乳杆菌VHProbi O48的分离筛选
1、初筛
配制MRS (Man Rogosa Sharpe) 肉汤培养基: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,选择2个月内未使用抗生素的健康成年女性,用无菌棉签擦拭舌背中心1cm2 5秒,将棉签头用无菌剪刀剪下置于保护液中,反复震荡。取100ml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50株(类)杆菌,并反复纯化,确定得到的是纯菌株,分别命名为O1、O2、……、O48、O49、O50。
2、抗柯氏棒状杆菌筛选
乳酸菌菌液制备:将分离得到的50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:将柯氏棒状杆菌(DSM109755)接种至营养肉汤+5%牛血清培养基中,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,将培养48h的柯氏棒状杆菌菌液按0.4%的体积比加入营养肉汤半固体培养基中。菌液与培养基混匀后,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24h后,观察有无抑菌圈。
结果显示,本发明初筛获得的50株乳酸菌中抑菌圈直径超过15mm的有10株菌,分别为O02、O13、O20、O21、O33、O37、O46、O47、O48、O50。其中,O48菌株的抑菌圈直径最大,为17.33±1.07mm(见图1),对柯氏棒状杆菌的抑菌效果最强。
实施例2 O48菌株鉴定
1、菌落形态鉴定
将O48菌株接种于MRS琼脂培养基上,37℃厌氧培养24h后,可见O48单菌落呈乳白色,菌落直径在1-3mm左右,表面光滑,隆起,边缘整齐,菌体短杆状居多,单个或几个成串。
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O48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O48菌株最大耐受盐浓度为5%。
2.2、碳源代谢试验
利用API 50CHL试剂条测定O48菌株对49种碳源的代谢作用,如表2所示。按照API50CHL试剂盒说明书进行操作。
O48菌株鉴定结果为:%ID=99.6且T值=0.87,API结果为发酵粘液乳杆菌,为非常好的鉴定,结果见图2。
2.3、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,小倒管内充满气体,说明O48菌株发酵葡萄糖产酸产气。
2.4胆盐酶活性定性试验
在新鲜配制的MRS液体培养基中添加0.2%TCA、0.2%的巯基乙酸钠、0.37 g/LCaCl2和1.5%琼脂。121℃ 15min灭菌,倒入平板中,直至平板中MRS凝固倒置放入厌氧罐中备用。把无菌滤纸片均匀的放入制作好的平板中,用移液枪在滤纸片上滴加10 μL O48新鲜培养的菌液,平板再次正放入厌氧罐中,37 ℃培养72 h后观察结果。
结果如图3所示,滤纸片周围出现钙圈,说明O48菌株胆盐酶活性为阳性。
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O48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O48菌株为发酵粘液乳杆菌。
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,O48菌株为发酵粘液乳杆菌,其Riboprinter指纹图谱如图4所示。
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。
O48菌株的RAPD指纹图谱如图5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。O48菌株的rep-PCR指纹图谱如图6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 获取蛋白指纹图谱,O48 菌株主要核糖体蛋白的离子峰为:m/z4714.574,结果如图7所示。
3.5全基因组测序
按照 0.1%的接种量在 MRS 液体培养基中接种新鲜菌液,37 ℃培养20 h,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CP095385。
将O48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 and applied 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O48菌株为一株新的发酵粘液乳杆菌,将其命名为发酵粘液乳杆菌VHProbi O48。
实施例3 发酵粘液乳杆菌VHProbi O48对人工胃液的耐受性试验
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、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消化后的活菌量(Log CFU/mL)见表3。
表3 人工胃液消化后的发酵粘液乳杆菌 VHProbi O48活菌量
消化前 | 人工胃液消化后 |
8.11±0.01 | 8.09±0.03 |
从表3的结果可知,本发明筛选到的发酵粘液乳杆菌 VHProbi O48 对胃液具有很强的耐受性。
实施例4 发酵粘液乳杆菌VHProbi O48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°C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,发酵粘液乳杆菌 VHProbi O48不能生长,血细胞平板没有变化,说明发酵粘液乳杆菌 VHProbi O48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发酵粘液乳杆菌 VHProbi O48的最小抑菌浓度MIC值具体结果见表4。
表4 发酵粘液乳杆菌 VHProbi O48的抗生素MIC值
MIC单位μg/mL。
从表4的结果可知,本发明提供的发酵粘液乳杆菌 VHProbi O48对红霉素和氨苄西林等常见抗生素敏感,生物安全性良好。
实施例5 发酵粘液乳杆菌VHProbi O48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%。
具体结果见表5。
表5 DPPH自由基清除率表
菌株 | 清除率% | 标准差 |
发酵粘液乳杆菌 VHProbi O48 | 17.72% | 4.57% |
2、菌株抗脂质过氧化试验鉴定
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6。
表6 发酵粘液乳杆菌 VHProbi O48抗脂质过氧化抑制率表
抑制率 | 标准差 | |
发酵上清液 | 57.6% | 0.3% |
实施例8 发酵粘液乳杆菌VHProbi O48菌体及其溶胞产物抗柯氏棒状杆菌的皮肤细胞实验
1、发酵粘液乳杆菌VHProbi O48灭活菌体溶液制备:
发酵粘液乳杆菌VHProbi O48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,得到灭活菌液。
2、发酵粘液乳杆菌VHProbi O48溶胞产物制备:
将活化好的发酵粘液乳杆菌VHProbi O48按照1%的体积比加入发酵培养基(红糖2%,骨胶原蛋白肽3%,酵母粉0.3%,磷酸氢二胺0.25%)中,37℃静置发酵24h;取新鲜培养的菌液采用高压均质机进行破碎处理,压力为100MPa,重复均质3次后,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、细胞实验方法:
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,将发酵粘液乳杆菌 VHProbi O48灭活菌体溶液或溶胞产物透析液分别以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)发酵粘液乳杆菌 VHProbi O48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 O48灭活菌体(20ul);D组添加柯氏棒状杆菌(20ul)+ VHProbi O48灭活菌体(20ul);E组添加柯氏棒状杆菌(20ul)+VHProbi O48灭活菌体(50ul);
(2)发酵粘液乳杆菌 VHProbi O48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 O48溶胞产物(20ul);D组添加柯氏棒状杆菌(20ul)+ VHProbi O48溶胞产物(20ul);E组添加柯氏棒状杆菌(20ul)+VHProbi O48溶胞产物(50ul)。
培养24小时后,取上清液,ELISA试剂盒检测促炎症反应因子IL-6的含量。
4、结果
白细胞介素(IL)是一种细胞因子,主要具有免疫调节的作用,能增强免疫细胞的杀伤作用,从而增强机体的免疫功能。其中IL-6、IL-1α、IL-8是主要的促炎症反应因子,且IL-6是炎症发生时最早升高的标志物。
结果如图8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,而发酵粘液乳杆菌 VHProbi O48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,从而说明发酵粘液乳杆菌 VHProbi O48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。
实施例9 发酵粘液乳杆菌VHProbi O48溶胞产物对脸部皮肤泛红的改善效果
前期体外实验已证明,发酵粘液乳杆菌VHProbi O48对柯氏棒状杆菌有较显著的抑制作用,其灭活菌体和溶胞产物均对皮肤HACAT细胞无刺激性,同时溶胞产物能有效缓解柯氏棒状杆菌引起的炎症反应。
经过动物实验已证明,发酵粘液乳杆菌VHProbi O48能有效缓解柯氏棒状杆菌感染的大鼠皮肤炎症状态,降低机体炎症因子水平,对皮肤损伤、泛红程度具有一定的改善作用。
1、发酵粘液乳杆菌VHProbi O48溶胞产物制备
将活化好的发酵粘液乳杆菌VHProbi O48按照1%的体积比加入发酵培养基(红糖2%,骨胶原蛋白肽3%,酵母粉0.3%,磷酸氢二胺0.25%)中,37℃静置发酵24h;取新鲜培养的菌液采用高压均质机进行破碎处理,压力为100MPa,重复均质3次后,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、发酵粘液乳杆菌VHProbi O48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上述发酵粘液乳杆菌VHProbi O48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。将加有溶胞产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
20名受试者使用溶胞产物均未产生可疑反应,说明本发明提供的发酵粘液乳杆菌VHProbi O48溶胞产物具有安全性,不会给人体带来不良反应。
3、发酵粘液乳杆菌VHProbi O48溶胞产物化妆品人体测试
3.1 配制精华乳液
按照表7所述配方比例配制精华乳液,其中发酵粘液乳杆菌VHProbi O48溶胞产物为唯一功效成分,其质量百分比为为8%。
表7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 O48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。在试验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
在进行皮肤检测之前,志愿者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S0O483血红素测试仪,检测毛细血管中血红素含量,通过检测红血丝严重部位的血红素下降的程度判断红血丝改善问题。
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 五光谱皮肤检测仪观察整体红血丝严重者改善情况。
3.4、结果
3.4.1、血红素测试
结果如表8所示,使用含发酵粘液乳杆菌VHProbi O48 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3%-24.9%,改善效果较好。
表8 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 | 51.4 | 53.6 | 46.2 | 41.3 | 52.0 | 48.6 | 51.3 | 46.2 |
使用后数值 | 38.6 | 51.0 | 39.7 | 35.6 | 46.9 | 47.1 | 44.2 | 39.3 |
下降百分比 | 24.9% | 4.9% | 14.1% | 13.8% | 9.8% | 3.1% | 13.8% | 14.9% |
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9所示,志愿者使用含发酵粘液乳杆菌VHProbi O48 溶胞产物的精华乳液后,面部红血丝有减轻趋势。
3.4.3、五光谱皮肤镜筛选照片
如图10所示,使用含发酵粘液乳杆菌VHProbi O48 溶胞产物的精华乳液后,志愿者面部泛红或红血丝均有不同程度的改善。
综上所述,本发明提供的发酵粘液乳杆菌VHProbi O48 对常见抗生素敏感,不产溶血素,生物安全性良好。所述发酵粘液乳杆菌VHProbi O48 溶胞产物对皮肤HACAT细胞无刺激性,能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。化妆品人体测试结果显示,所述发酵粘液乳杆菌VHProbi O48 溶胞产能有效改善皮肤泛红和I型玫瑰痤疮的症状,可广泛用于制备具有改善皮肤泛红和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> 一株发酵粘液乳杆菌及其在改善面部泛红和I型玫瑰痤疮中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1423
<212> DNA
<213> 发酵粘液乳杆菌(Limosilactobacillus fermentum)
<400> 1
ttaccccacc gactttgggt gttacaaact ctcatggtgt gacgggcggt gtgtacaagg 60
cccgggaacg tattcaccgc ggcatgctga tccgcgatta ctagcgattc cgacttcgtg 120
caggcgagtt gcagcctgca gtccgaactg agaacggttt taagagattt gcttgccctc 180
gcgagttcgc gactcgttgt accgtccatt gtagcacgtg tgtagcccag gtcataaggg 240
gcatgatgat ctgacgtcgt ccccaccttc ctccggtttg tcaccggcag tctcactaga 300
gtgcccaact taatgctggc aactagtaac aagggttgcg ctcgttgcgg gacttaaccc 360
aacatctcac gacacgagct gacgacgacc atgcaccacc tgtcattgcg ttcccgaagg 420
aaacgcccta tctctagggt tggcgcaaga tgtcaagacc tggtaaggtt cttcgcgtag 480
cttcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc ctttgagttt 540
caaccttgcg gtcgtactcc ccaggcggag tgcttaatgc gttagctccg gcactgaagg 600
gcggaaaccc tccaacacct agcactcatc gtttacggca tggactacca gggtatctaa 660
tcctgttcgc tacccatgct ttcgagtctc agcgtcagtt gcagaccagg tagccgcctt 720
cgccactggt gttcttccat atatctacgc attccaccgc tacacatgga gttccactac 780
cctcttctgc actcaagtta tccagtttcc gatgcacttc tccggttaag ccgaaggctt 840
tcacatcaga cttagaaaac cgcctgcact ctctttacgc ccaataaatc cggataacgc 900
ttgccaccta cgtattaccg cggctgctgg cacgtagtta gccgtgactt tctggttaaa 960
taccgtcaac gtatgaacag ttactctcat acgtgttctt ctttaacaac agagctttac 1020
gagccgaaac ccttcttcac tcacgcggtg ttgctccatc aggcttgcgc ccattgtgga 1080
agattcccta ctgctgcctc ccgtaggagt atgggccgtg tctcagtccc attgtggccg 1140
atcagtctct caactcggct atgcatcatc gccttggtag gccgttaccc caccaacaag 1200
ctaatgcacc gcaggtccat ccagaagtga tagcgagaag ccatctttta agcgttgttc 1260
atgcgaacaa cgttgttatg cggtattagc atctgtttcc aaatgttgtc ccccgcttct 1320
gggcaggtta cctacgtgtt actcacccgt ccgccactcg ttggcgacca aaatcaatca 1380
ggtgcaagca ccatcaatca atgggccaac gcgttcgact tgc 1423
Claims (8)
1.一种发酵粘液乳杆菌,其特征在于,所述发酵粘液乳杆菌的保藏号为CCTCC NO:M2021597。
2.如权利要求1所述的发酵粘液乳杆菌,其特征在于,所述发酵粘液乳杆菌的16s rDNA序列如SEQ ID NO:1所示。
3.如权利要求1所述的发酵粘液乳杆菌,其特征在于,所述发酵粘液乳杆菌的Riboprinter 指纹图谱如图4所示;其RAPD指纹图谱如图5所示,rep-PCR指纹图谱如图6所示,MALDI-TOF-MS图谱如图7所示。
4.权利要求1所述的发酵粘液乳杆菌在制备具有抗氧化功能的制品中的应用。
5.权利要求1所述发酵粘液乳杆菌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。
6.如权利要求4或5所述的应用,其特征在于,所述的制品为保健品、药品或护肤品。
7.如权利要求6所述的应用,其特征在于,所述的制品为护肤品。
8.一种护肤品,其特征在于,所述的护肤品包含权利要求1所述发酵粘液乳杆菌的溶胞产物。
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CN114107430A (zh) * | 2021-11-23 | 2022-03-01 | 浙江省农业科学院 | 基于傅里叶变换红外光谱的微生物分型系统在乳酸菌分型或乳酸菌类益生菌筛选中的用途 |
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CN117736942A (zh) * | 2024-02-20 | 2024-03-22 | 山东中科嘉亿生物工程有限公司 | 改善皮肤衰老的发酵粘液乳杆菌jylf-315及其后生元制剂和应用 |
CN117736942B (zh) * | 2024-02-20 | 2024-05-31 | 山东中科嘉亿生物工程有限公司 | 改善皮肤衰老的发酵粘液乳杆菌jylf-315及其后生元制剂和应用 |
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