CN116515666A - 一株具有治疗痤疮作用的瑞士乳杆菌及其应用 - Google Patents
一株具有治疗痤疮作用的瑞士乳杆菌及其应用 Download PDFInfo
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- CN116515666A CN116515666A CN202211298498.5A CN202211298498A CN116515666A CN 116515666 A CN116515666 A CN 116515666A CN 202211298498 A CN202211298498 A CN 202211298498A CN 116515666 A CN116515666 A CN 116515666A
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- lactobacillus helveticus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明涉及益生菌筛选与应用技术领域,具体提供了一株新的瑞士乳杆菌(Lactobacillus helveticus)及其应用。所提供的嗜酸乳杆菌分离自泡菜发酵液中,保藏编号为CCTCC NO:M2021595,能够减轻皮肤炎症,改善皮肤健康状况,预防及缓解痤疮症状。
Description
技术领域
本发明属于益生菌筛选与应用技术领域,具体涉及一株具有治疗痤疮作用的瑞士乳杆菌及其应用。
背景技术
痤疮是主要累及面部毛囊皮脂腺的慢性炎症性皮肤病。目前,痤疮的发病机制仍未完全阐明,痤疮的发生主要与皮脂分泌过多、毛囊皮脂腺导管堵塞、细菌感染等因素相关。皮脂分泌过多可导致痤疮丙酸杆菌繁殖增加,出现不同程度的炎症反应。而痤疮皮脂分泌过多与遗传和激素分泌异常有较大的相关性。
肠道微生物影响免疫系统,肠道菌群失调引起的炎症反应是许多皮肤疾病的发病原因之一。肠道上皮细胞通透性增加会激活T细胞中细胞因子的表达和调节性T细胞产生,导致全身炎症破坏皮肤稳态。促炎细胞因子会进一步增强小肠上皮细胞通透性并建立全身炎症的恶性循环。另外,某些肠道微生物产生的有害代谢产物如苯酚和对甲苯会通过血液循环进入身体各个部位,当它们在皮肤部位累积时就会对表皮细胞分化和皮肤屏障功能产生不利影响。
世界卫生组织/联合国粮农组织将益生菌定义为:活的微生物,当施以足够数量时能够对宿主带来健康益处。益生菌及其代谢物已被证明可与肠道相关淋巴组织存在相互作用,这种互动在帮助免疫系统应对病原体或过敏原或共生细菌方面至关重要。据报道,益生菌可抑制上皮细胞和角质形成细胞中的细胞因子IL-8,可以减少痤疮丙酸杆菌产生的炎症介质并减少血管扩张、水肿、肥大细胞脱颗粒和TNF-α释放。
在一项前瞻性的、随机、双盲安慰剂对照试验中,与对照组相比,鼠李糖乳杆菌GG组痤疮患者12周试验结束后痤疮得到显著改善,并且IGF-1基因表达水平下降以及FoxO1的基因表达水平上升,该研究表明益生菌或可使痤疮患者获益。另外,近些年科研人员发现,益生菌发酵完成经灭活后形成的后生元仍然可以调节宿主免疫反应,发挥健康益处。Seité等给特异性皮炎患者涂抹含线状透明颤菌的润肤剂后显著降低了 SCORAD指数。Park等发现使用乳酸杆菌润肤剂显著下调了特异性皮炎患者经皮水分丢失和视觉模拟评分。Gueniche等发现副干酪乳酸杆菌可加速受损皮肤屏障的恢复。Puch等干敏性皮肤女性使用24周的混合发酵乳制乳液后,其皮肤经皮水分丢失指标下降,角质屏障功能得到明显改善。因此,口服或外用益生菌后生元产品或可用于治疗痤疮。
发明内容
本发明的目的是提供一株新的瑞士乳杆菌(Lactobacillus helveticus)及其应用;所提供的瑞士乳杆菌分离自泡菜发酵液中,能够减轻皮肤炎症,改善皮肤健康状况,预防及缓解痤疮症状。
本发明所提供的瑞士乳杆菌,为瑞士乳杆菌VHPribo Y21(Lactobacillus helveticus VHPribo Y21),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:M2021595。
所述瑞士乳杆菌VHProbi Y21株,其16s rDNA序列为SEQ ID NO:1。
本发明所提供的瑞士乳杆菌 VHProbi Y21株,其MALDI-TOF核糖体蛋白分子量图谱如图2所示;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。
本发明所提供的瑞士乳杆菌可用于制备功能性食品、保健品、药品或化妆品。
本发明提供的瑞士乳杆菌可用于制备具有抗氧化功能的制品;
本发明提供的瑞士乳杆菌可用于制备预防或治疗痤疮的制品。
所述制品为功能性食品或化妆品。
本发明提供的瑞士乳杆菌VHProbi Y21对人工肠胃液具有很强的耐受性;该菌株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;该菌株具备抗氧化能力,菌体抗脂质过氧化抑制率菌体为9.7%、上清液为15.9%;DPPH清除率达到20.5%、 HRS清除率达到45.75%;胆固醇降解率为22.2%;另外,该菌株黄曲霉毒素B1吸附能力为15.30%;细胞表面疏水性为11.78%;皮肤细胞黏附力为3.78。
瑞士乳杆菌VHProbi Y21及其裂解液,能够有效缓解兔耳痤疮红肿程度,减轻兔耳痤疮部位的皮肤粗糙程度和硬度,改善兔耳痤疮部位的皮损症状,兔耳皮肤有较完整的表皮层和真皮层。瑞士乳杆菌VHProbi Y21还能够显著降低白兔体内促炎细胞因子水平,调节其免疫反应,减缓痤疮炎症的发展进程。瑞士乳杆菌VHProbi Y21缓解痤疮效果与化学药剂相当。
本发明提供的瑞士乳杆菌 VHProbi Y21,对机体无毒害作用,可以添加在化妆品中,用于治疗痤疮,具有广阔的应用前景。
附图说明
图1为Y21菌株结晶紫染色光镜照片和单菌落照片;
图2为Y21菌株MALDI-TOF核糖体蛋白指纹图谱;
图3为Y21菌株Riboprinter 指纹图谱;
图4为Y21菌株的RAPD指纹图谱;
图5为Y21菌株的rep-PCR指纹图谱;
图6为各组兔耳痤疮症状对比;
图7为各组细胞因子检测结果对比;
图8为各组兔耳病理切片结果。
具体实施方式
本发明提供的瑞士乳杆菌 VHProbi Y21符合法规要求,经多相分类学鉴定,瑞士乳杆菌VHProbi Y21为一株新发现的菌株。本发明提供的瑞士乳杆菌 VHProbi Y21 能够有效预防和缓解痤疮,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痤疮有缓解功效;具有重要的应用价值。
申请人于2021年5月24日将所述瑞士乳杆菌 VHProbi Y21保藏于武汉大学的中国典型培养物保藏中心,其保藏号为CCTCC NO:M2021595。
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 瑞士乳杆菌VHProbi Y21的分离筛选
1、初筛
配制MRS琼脂培养基调pH 6.2-6.5,121℃高压灭菌15 min。
取1g新鲜泡菜发酵液,经无菌生理盐水稀释后放入无菌样品袋中,用匀浆仪拍打混匀;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25株潜在乳酸杆菌,分别命名为Y01、Y02、……、Y20、Y21、Y22、Y23、Y24、Y25。
2、复筛
配制1L 的MRS液体培养基121℃高压灭菌15min,待培养基冷却后加入3.2g猪粘膜胃蛋白酶,摇匀溶解,置37℃水浴摇床中水浴1h制成耐酸性培养基。
将筛选得到的25株乳酸杆菌Y01、Y02、……、Y20、Y21、Y22、Y23、Y24、Y25按6%接种量分别接种于上述耐酸性培养基中,37℃条件下厌氧静置培养48h,取发酵液进行菌量计数。
结果显示,在所述25株乳酸杆菌发酵液中活菌量的对数值中,Y21菌株经耐酸性培养基复筛后活菌量最多,对数值高达9.50 Log CFU/mL。从而说明Y21菌株耐酸能力最高。
实施例2 菌株鉴定
1、菌落形态鉴定
将Y21菌株接种于MRS琼脂培养基上,37℃厌氧培养24h后,可见Y21单菌落呈乳白色,菌落直径在2-3mm左右,表面湿润光滑呈圆形,结晶紫染色后显微镜下呈短杆菌。
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Y21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、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,Y21菌株在15℃不生长,45℃大量繁殖。
2.2、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,Y21菌株最大耐受盐浓度为2%。
2.2、碳源代谢试验
本实施例中所用的基础培养基配方如下:
蛋白胨1.5g;酵母提取物0.6g;吐温80 0.1g;盐溶液0.5mL;酚红18mg;蒸馏水100mL;pH7.4±0.2。盐溶液成分:MgSO4·7H2O 11.5g,MnSO4·4H2O 2.8g,蒸馏水100mL。
配制10g/100mL的糖、醇和苷类碳水化合物溶液,并用0.22μm的无菌过滤器进行过滤。在无菌条件下,向96孔板中加入20μL除菌后的碳水化合物溶液,每种碳水化合物4个平行,然后加入170μL灭菌后含酚红的基础培养基,再加入10μL 接种液,不接菌反应孔作为对照。每孔加入 50μL 液体石蜡以防止培养过程中水分蒸发。37℃厌氧培养,以酚红为指示剂,观察培养基颜色变化;具体结果见表 1。
表 1 Y21菌株碳源代谢结果表
纤维二糖 | 蜜二糖 | 棉子糖 | 甘露醇 | 苦杏仁苷 |
+ | - | - | - | + |
半乳糖 | 乳糖 | 麦芽糖 | 甘露糖 | 水杨苷 |
+ | + | + | + | + |
阿拉伯糖 | 葡萄糖酸钠 | 松三糖 | 核糖 | 山梨醇 |
- | - | - | - | - |
蔗糖 | 海藻糖 | 木糖 | 鼠李糖 | / |
+ | + | - | + | / |
注:“+”阳性反应;“-”阴性反应。
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,小倒管内无气体,说明 Y21菌株发酵葡萄糖产酸,不产气。
2.4、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Y21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Y21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Y21菌株为瑞士乳杆菌。
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,Y21菌株为瑞士乳杆菌,其Riboprinter指纹图谱结果见图2。
3.3 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裂解液覆盖样品,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,Y21菌株主要核糖体蛋白的离子峰为:m/z2599.217、3124.057、3457.129、4399.116、5197.056、5325.093、6246.762、6947.835、7638.294。鉴定结果如图3所示。
3.4 RAPD和rep-PCR指纹图谱鉴定
3.4.1、RAPD指纹图谱鉴定
1)引物序列: GAGGGTGGCGGTTCT。
2)RAPD 反应体系
表2 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 |
3)电泳
制备 1.5 %的琼脂糖凝胶板,DL2000 DNA Marker 作为结果对照,稳压 100V 电泳80min,最后利用凝胶成像系统检测电泳图。Y21 菌株的 RAPD 指纹图谱如图 4 所示。
3.4.2、rep-PCR指纹图谱
1)引物序列:CTACGGCAAGGCGACGCTGACG。
2)rep-PCR 的反应体系
表3 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 |
3)电泳
DL2000 DNA Marker 作为结果对照。电压 100 V,电泳时间 80min 检测扩增结果。Y21 菌株的的 rep-PCR 指纹图谱如图 5 所示。
3.4.3、rep-PCR指纹图谱
3.5 全基因组测序鉴定
取新鲜菌液按照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,得到该菌的全基因序列,基因序列上传至NCBI基因数据库,GenBank登录号为CP103857-CP103859。
综上,根据Y21 菌 株 的 菌 落 形 态、 生 理 生 化 特 性 结以及分子生物学的鉴定结果,可以得出结论,Y21菌株为一株新的瑞士乳杆菌,将其命名为瑞士乳杆菌VHProbi Y21。
实施例3 瑞士乳杆菌VHProbi Y21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、KH2PO4 6.8g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,该菌株经过人工肠液消化后的活菌量(Log CFU/mL)见表4。
表4 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 |
7.81±0.00 | 7.91±0.00 | 6.68±0.05 |
从表 4 可知,本发明筛选到的瑞士乳杆菌 VHProbi Y21 经人工胃液消化后,活菌量有少量上升,说明该菌株能耐受人工胃液并能萌发。在经过人工肠液下降1.23Log值,对人工肠液耐受能力较好。
实施例4 瑞士乳杆菌VHProbi Y21的抗生素耐受性实验
1、抗生素配制:氨苄青霉素、克林霉素、红霉素、庆大霉素、链霉素、四环素、万古霉素均配制成 2048 μg/mL 的贮存液,-20℃保存备用。使用时将贮存液用 BMS 液体培养基进行 2 倍系列梯度稀释成使用液,梯度稀释浓度为 1~1024μg /mL 共 11 个梯度。
2、接种液制备:接种液的准备:取适量新鲜菌液(24~48h,40℃培养),5000rpm 离心 5 min ,用无菌生理盐水洗一次,再用同体积生理盐水重悬菌体后稀释 50 倍,作为接种液。
3、微量肉汤稀释法测定抗生素对瑞士乳杆菌 VHProbi Y21 的最小抑菌浓度MIC。
a. 96 孔板第 1 列次加入不含抗生素的 BMS 液体培养基,作为阴性对照,向第2~12列依次加入 190μL 含不同浓度抗生素的 BMS 液体培养基,然后分别接种 10μL 上述接种液,做 3 个平行孔,并以 1 个孔不加菌液作为空白。
b. 加入 50μL 石蜡油覆盖防止水分蒸发。
c. 将 96 孔板于 40℃振荡培养 48h 后取出,测定 OD600 值,用 48h 的结果统计抗生素对菌株的 MIC 值,具体结果见表 5。
表5 瑞士乳杆菌 VHProbi Y21 的抗生素MIC值
MIC单位μg/mL
从表5的结果可以看出,本发明提供的瑞士乳杆菌 VHProbi Y21 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 瑞士乳杆菌VHProbi Y21抗氧化功能测定
1、菌株清除DPPH(1,1-二苯基-2-三硝基苯肼)和羟基自由基(HRS)能力测定
菌株清除DPPH自由基能力的测定
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6。
表6 DPPH自由基清除率表
菌株 | 清除率% | 标准差 |
瑞士乳杆菌 VHProbi Y21 | 20.50% | 0.6% |
3)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,结果见表7。
表7 HRS自由基清除率表
菌株 | 清除率% | 标准差 |
瑞士乳杆菌 VHProbi Y21 | 45.75% | 2.3% |
2、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30 min,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%
注: A为样品组吸光度;A0为对照组吸光度,结果见表8。
表8 抗脂质过氧化抑制率表
抑制率 | 标准差 | |
菌体 | 9.7% | 0.37% |
发酵上清液 | 15.9% | 0.8% |
实施例 6 瑞士乳杆菌 VHProbi Y21 体外胆固醇降解实验
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 100mL,在无菌条件下用 0.22 µm 微孔滤膜过滤除菌。
2.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 硫酸镁 0.1 硫酸锰 0.05 ,磷酸氢二钾 2.0g ,胆盐 1g,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%的接种量接种新鲜菌液,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 分钟,3000 转离心 5 分钟,取上清液用于测定胆固醇含量。胆固醇测定方法按照 GB/T5009.128-2003 <食品中胆固醇的测定>。
结果显示:本发明提供的瑞士乳杆菌 VHProbi Y21 对胆固醇的降解率达到22.02%(此为不含胆盐的数据)。
实施例7 瑞士乳杆菌VHProbi Y21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:本发明提供的瑞士乳杆菌VHProbi Y21的黄曲霉毒素B1吸附能力为15.30%,标准差为0.02%。
实施例8 瑞士乳杆菌VHProbi Y21的疏水性细胞表面测试
1、待测菌液制备:挑取纯化好的瑞士乳杆菌VHProbi Y21菌落接种于新配制的MRS液体培养基中,于37℃培养24~48h。再按1%(V/V)的接种量接至MRS液体培养基中于37℃继续培养24~48h后6000×g离心10min,收集菌体后用无菌生理盐水冲洗2次,再用灭菌0.1MKNO3 1mL溶液重悬菌体,作为待测菌液。
2、表面疏水性测定:吸取50μL上述菌悬液加入2450μL的0.1M KNO3并记录OD600为A0,取1.5mL菌悬液与500μL二甲苯混匀后在室温下静置10min(此时形成两相体系)。将两相体系涡旋振荡2min后再静置20min, 重新形成水相和有机相。小心吸取水相(不要吸到有机相)在600nm处测量吸光度A1。细胞疏水性按公式Hydrophobicity%=(A0-A1)/A1×%计算,测三次实验取平均值。
结果显示:本发明提供的瑞士乳杆菌VHProbi Y21细胞表面疏水性为11.78%,标准差为0.57%。
实施例9 瑞士乳杆菌VHProbi Y21的皮肤细胞黏附力试验
Hacat细胞以2×106cells/孔的接种量接种于六孔板,二氧化碳培养箱培养24h,用于细胞粘附实验;
将稳定期的菌株用MRS培养基重悬至5×107CFU/mL;
取1mL上述菌株加入已有细胞贴壁的六孔板,二氧化碳培养箱培养2h;
PBS反复洗涤3次,去除未粘附的细菌;
加入500ul胰酶消化3分钟,再加入1.5mL细胞培养液终止消化,反复吹打,并将所得溶液收集至无菌EP管中,并将收集的溶液进行10倍,100倍,1000倍,10000倍梯度稀释,涂板计数。同时对空白组的细胞进行计数。根据以下公式计算供试菌株的粘附能力:
粘附能力(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:本发明提供的瑞士乳杆菌 VHProbi Y21的细胞黏附力为3.78,标准差为0.20%。
实施例10 瑞士乳杆菌VHProbi Y21及其裂解液在缓解兔耳痤疮中的应用
1 实验动物处理及模型构建
1.1实验耗材
表9 实验耗材
名称 | 厂家 | 级别 | 规格/批号 |
NaCl | 江苏勤奋药业 | 药用级 | 500g/瓶 |
阿达帕林 | Sigma | 药用级 | 500KU |
水合氯醛 | 阿拉丁 | AR | C104202-500g |
油酸 | Macklin | AR | 0815202-500mL |
痤疮丙酸杆菌 | CGMCC | / | CGMCC15003 |
IL-10 | eBiosource | / | MBS2700946 |
TNF-α | eBiosource | / | MBS2732969 |
IFN-γ | eBiosource | / | MBS2705225 |
戊二醛 | Sigma-aldrich | / | 158127-500g |
HE 染色试剂盒 | Promega | / | 500T |
1.2 新鲜菌液及裂解液制备
将瑞士乳杆菌VHProbi Y21接种至新鲜培养的MRS培养基中,37℃厌氧培养12h后用作灌胃使用。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,制备成裂解液。
1.3 实验动物处理
选用清洁级新西兰大白兔,雄性,24只,随机分为4组,每组6只,由青龙山动物繁殖中心提供。实验兔专用颗粒饲料饲喂,饲养于清洁级动物房,温度18-20℃,相对湿度40%,分笼饲养,每笼随机放入1只,自由摄食饮水。
大白兔适应性饲养完成后分别为空白组、建模组、阳性组、益生菌组。其中,空白组不做任何处理,全程灌胃和涂抹生理盐水;建模组、阳性组和益生菌组采用痤疮丙酸杆菌和油酸构建痤疮模型,建模组和阳性组在建模后分别采用生理盐水和阿达帕林凝胶治疗,益生菌组采用瑞士乳杆菌VHProbi Y21及其裂解液治疗。整个实验期间严格依照动物伦理法照看并处理动物。
具体的处理方法如下:
分别于兔耳左右外耳道上,用0.25mL涂布器均匀涂布25%油酸0.1mL,隔天1次,连续涂布50天。将培养后的痤疮丙酸杆菌计数,调整浓度为106个/mL备用。在涂布油酸第20天,皮内注射痤疮杆菌50ul/耳,并在注射处做标记总计注射一次。
(1)空白组:适应性结束后每天灌胃10mL生理盐水,外耳道涂抹生理盐水1mL, 早晚各一次至第50天试验结束。不涂布油酸;
(2)建模组:每天灌胃10mL生理盐水,外耳道涂抹生理盐水1mL覆盖外耳道痤疮处,早晚各一次至第50天试验结束。涂布油酸,注射痤疮丙酸杆菌;
(3)阳性组:每天灌胃10mL生理盐水,涂抹阿达帕林凝胶1g覆盖外耳道痤疮处,早晚各一次至第50天试验结束。涂布油酸,注射痤疮丙酸杆菌;
(4)益生菌组:每天灌胃1×109CFU/mL瑞士乳杆菌VHProbi Y21菌液10mL,涂抹瑞士乳杆菌VHProbi Y21裂解液1mL覆盖外耳道痤疮处,早晚各一次至第50天试验结束。涂布油酸,注射痤疮丙酸杆菌;
2指标检测
第50天试验结束后,观察兔儿肿胀情况,采用痤疮皮损计数法来评估益生菌涂抹治疗效果,从兔儿硬度、厚薄、粗糙等方面观察兔儿皮肤情况。
表10 痤疮模型观察评分标准
厚薄及评分 | 硬度及评分 | 粗糙程度及评分 |
无增厚0分 | 未变硬0分 | 光滑0分 |
轻度增厚1分 | 轻度变硬1分 | 轻度粗糙1分 |
中度增厚2分 | 中度变硬2分 | 中度粗糙2分 |
显著增厚3分 | 显著变硬3分 | 显著粗糙3分 |
抽取静脉血ELISA法检测白兔血清IL-6、TNF-α和IL-1β 细胞因子的含量。
兔耳组织取材,制成5μm厚的石蜡切片进行HE染色。依据实验性痤疮组织病理学判定分级标准,根据模型动物的表皮增厚情况、毛囊口扩张程度以及所产生的角化物量,判断标本组织的病理学变化。
3数据分析
使用SPSS 22.0统计软件进行数据分析,结果以平均值±标准误的方式表示。两组间比较采用独立样本t检验,多组间差异比较采用单因素方差分析,方差齐时再进一步用最小显著差数法进行两两比较,方差不齐时采用秩和检验。p<0.05表示差异具有显著性,p<0.01表示差异具有极显著性。使用GraphPad Prism 8.0进行作图。
4. 实验结果
4.1终末期各组白兔一般状态观察
各组别典型兔耳观察结果见图6,各组别兔耳外观观察评分结果见表11。
结果显示:第50天给药结束后,空白组兔耳外观光滑柔软,兔耳透光,没有角栓形成,毛细血管清晰可见;建模组兔耳可见明显红肿和皮损,厚度增加,有结痂、干燥脱皮现象,表明建模成功;阳性组兔耳出现角化,兔耳变厚,毛囊变粗,有黑头出现。而益生菌组兔耳透光度改善,没有皮损和脓包出现,红肿程度减轻,黑头减少,硬度和粗糙程度缓解。从而说明,本发明提供的瑞士乳杆菌VHProbi Y21及其裂解液能有效缓解兔耳痤疮的严重程度,改善痤疮皮肤状态。
表11 兔耳外观评分量表
4.2终末期各组别白兔血清ELISA检测结果
实验结束后,血清中三种促炎细胞因子水平变化如图7所示。
结果显示,与空白组相比,建模组白兔血清TNF-α、IL-6和IL-1β细胞因子水平升高,差异具有极显著性(P<0.001),说明模型组建模成功。与建模组相比,阳性组白兔血清TNF-α细胞因子水平降低,差异具有显著性(P<0.05);IL-6细胞因子水平降低,差异不显著;IL-1β细胞因子水平降低,差异具有极显著性(P<0.001)。与建模组相比,益生菌组白兔血清TNF-α细胞因子水平降低,差异具有显著性(P<0.05);IL-6细胞因子水平降低,差异不显著;IL-1β细胞因子水平降低,差异具有极显著性(P<0.001)。从而说明,本发明提供的瑞士乳杆菌VHProbi Y21能有效降低血清中促炎细胞因子的水平,减缓痤疮的炎症进程,效果与化学药剂阿达帕林凝胶基本相当。
4.3各组别兔耳病理观察
光学显微镜下观察各组兔耳HE切片染色结果,如图8所示。
空白组兔耳表皮层较薄,毛囊以及真皮和表皮交界处清晰,真皮内可见稀疏单核细胞浸润,真皮内稀疏单一结构;建模组光镜下可见表皮层及真皮层不完整,真皮过度角化,漏斗部扩大,过度角化,真皮内有炎性细胞浸润,毛囊上皮的颗粒层、棘层增生明显;阳性组兔耳表皮层较薄,真皮和表皮交界处以及毛囊清晰,真皮内可见单核细胞浸润,有疏松的角化物质充填。光镜下,益生菌组兔耳表皮层较薄,毛囊以及真皮和表皮交界处清晰,真皮内稀疏可见单核细胞浸润,真皮层可见疏松的角化物。从而说明,本发明提供的瑞士乳杆菌VHProbi Y21及其裂解液有助于促进兔耳皮肤表皮层和真皮层的结构恢复完整,减轻角化现象,有效缓解炎症程度,效果与化学药剂阿达帕林凝胶相当。
本发明提供的瑞士乳杆菌VHProbi Y21对于模拟人工肠胃液具有很强的耐受能力,有较强的细胞表面疏水性,这对于益生菌顺利经过胃肠道并在结肠处定植及发挥益生功能奠定了基础。同时,溶血性实验证实瑞士乳杆菌VHProbi Y21不产溶血素,对常见抗生素敏感,生物安全性良好。瑞士乳杆菌VHProbi Y21能够清除DPPH和HRS等自由基,具有一定的抗氧化功效。此外,瑞士乳杆菌VHProbi Y21能吸附黄曲霉毒素B1以及较强的抗脂质过氧化能力。经兔耳痤疮实验证实,灌胃瑞士乳杆菌VHProbi Y21及同期涂抹其裂解液能够缓解痤疮严重程度,调节其免疫反应水平。瑞士乳杆菌是属于传统乳酸菌属益生菌,具有悠久的使用历史,其益生功效表明可以开发成后生元产品,应用于食品和护肤品当中。
Claims (10)
1.一种瑞士乳杆菌,其特征在于,所述的瑞士乳杆菌的保藏编号为CCTCC NO:M2021595。
2.如权利要求1所述的瑞士乳杆菌,其特征在于,所述的瑞士乳杆菌的16s rDNA序列为SEQ ID NO:1。
3.如权利要求1所述的瑞士乳杆菌,其特征在于,所述的瑞士乳杆菌的MALDI-TOF核糖体蛋白分子量图谱如图2所示,Riboprinter 指纹图谱如图3所示,RAPD指纹图谱如图4所示,rep-PCR指纹图谱如图5所示。
4.权利要求1所述的瑞士乳杆菌在制备食品、保健品、药品或化妆品中的应用。
5.权利要求1所述的瑞士乳杆菌在制备具有抗氧化功能的制品中的应用。
6.权利要求1所述的瑞士乳杆菌在制备用于预防或治疗痤疮的制品中的应用。
7.一种益生菌制品,其特征在于,所述的益生菌制品中包含有权利要求1所述的瑞士乳杆菌的活菌和/或其发酵产物。
8.一种益生菌制品,其特征在于,所述的益生菌制品中包含有权利要求1所述的瑞士乳杆菌的裂解液。
9.如权利要求7或8所述的益生菌制品,其特征在于,所述的制品为功能性食品或保健品或药品。
10.如权利要求8所述的益生菌制品,其特征在于,所述的制品为化妆品。
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