CN116694509A - 一株代谢l-色氨酸产吲哚衍生物且具有增强肠道屏障功能的植物乳杆菌及其应用 - Google Patents
一株代谢l-色氨酸产吲哚衍生物且具有增强肠道屏障功能的植物乳杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种代谢L‑色氨酸产吲哚衍生物且具有增强肠道屏障功能的植物乳杆菌及其筛选方法与应用，属于微生物技术领域。本发明的植物乳杆菌DPUL‑S164可以在体内外代谢L‑色氨酸产生ILA和IAId；可以缓解DSS诱导Balb/c小鼠体重下降，疾病活性指数增加，和结肠长度的缩短，通过下调血清促炎因子IL‑6和TNF‑α的水平，增加抗炎细胞因子IL‑10的水平，通过激活AhR信号通路，促进粘蛋白和紧密连接蛋白的增加，改善受损的肠屏障，具有很好的应用前景。
Description
技术领域
本发明属于微生物技术领域，具体涉及一株代谢L-色氨酸产吲哚衍生物且具有增强肠道屏障功能的植物乳杆菌及其作为功能食品或药品的应用。
背景技术
肠道屏障由单层柱状上皮和细胞间紧密连接的肠细胞组成，是抵抗肠腔内恶劣环境的重要防御机制。完整的肠道屏障可以不仅可以有效阻止细菌毒素和病原微生物等有害物质通过。然而，肠道屏障的损伤时，通常会增强肠道感染的风险，促进腔内细菌、毒素进入上皮组织，最后导致炎症反应和胃肠道疾病。
L-色氨酸(Trp)在维持肠道免疫耐受和肠道微生物群的平衡中发挥着至关重要的作用。近期研究发现，微生物群的变化通过调节色氨酸代谢来调节宿主免疫系统。研究发现，微生物色氨酸代谢产物在宿主健康和疾病中发挥着关键的作用。研究发现，肠道中多种微生物都具有代谢色氨酸产生吲哚衍生物的能力。不同细菌由于其代谢色氨酸产吲哚衍生物机制不同，因此可产生不同的吲哚衍生物，例如吲哚-3-乳酸、吲哚-3-甲醛、吲哚-3-丙酸、吲哚-3甲醇和吲哚-3-乙醛等吲哚衍生物，这些吲哚衍生物是有效的生物活性代谢物，通过激活芳基烃受体(AhR)影响小鼠肠道屏障完整性和免疫细胞。乳杆菌被报道具有增强肠道屏障功能，缓解肠道炎症的作用，并且乳杆菌也可代谢L-色氨酸产生吲哚衍生物。因此，乳杆菌产生的吲哚衍生物可能是其发挥增强肠道屏障功能，缓解肠道炎症的关键效应分子。因此，鉴于乳杆菌具有代谢L-色氨酸产生吲哚衍生物的优点，将代谢L-色氨酸产生吲哚衍生物的乳杆菌与L-色氨酸复配、作为一种具有增强肠道屏障功能的功能食品或药品具有非常广泛的应用前景。
发明内容
鉴于此，本发明的目的在于提供一株代谢L-色氨酸产吲哚衍生物且具有增强肠道屏障功能的植物乳杆菌及其应用。
为了达到上述目的，本发明提供如下技术方案：
一株代谢L-色氨酸产吲哚衍生物的植物乳杆菌(Lactobacillus plantarum)DPUL-S164，于2023年4月18日保藏于中国典型培养物保藏中心，菌株保藏号为CCTCC NO：M2023547。
基于上述技术方案，进一步地，所述的植物乳杆菌(Lactobacillus plantarum)DPUL-S164具有增强肠道屏障功能，在体内外能够代谢L-色氨酸(L-Trp)并高产吲哚-3-乳酸(ILA)和吲哚-3-甲醛(IAId)。
基于上述技术方案，进一步地，采用MRS培养基对该菌株进行培养，并配制含L-色氨酸MRS培养基(MRS-Trp)利用吲哚实验鉴定该植物乳杆菌代谢L-色氨酸的能力，进一步通过高效液相色谱法测定植物乳杆菌DPUL-S164利用L-色氨酸产吲哚衍生物水平，并探究对肠道屏障功能的影响评价其产吲哚衍生物并具有增强肠道屏障功能的效果。
基于上述技术方案，进一步地，通过吲哚实验和高效液相色谱法验证该植物乳杆菌高产吲哚衍生物的步骤为：利用吲哚实验初步验证其可代谢L-色氨酸产吲哚衍生物，使用Waters C18正相色谱柱测定该植物乳杆菌培养物样品与吲哚衍生物标准品在相同保留时间下的峰面积，根据测定的标准曲线求得样品中吲哚衍生物的含量，基于吲哚衍生物的含量评价植物乳杆菌DPUL-S164产吲哚衍生物的水平。
本发明还提供上述的植物乳杆菌(Lactobacillus plantarum)DPUL-S164在制备用于增强肠道屏障功能的功能食品或药品中的应用。
基于上述技术方案，进一步地，所述的植物乳杆菌(Lactobacillus plantarum)DPUL-S164与L-色氨酸(L-Trp)联合使用。
基于上述技术方案，进一步地，所述增强肠道屏障功能包括调节肠道免疫屏障、机械屏障和化学屏障。
基于上述技术方案，进一步地，所述增强肠道屏障功能包括促进杯状细胞和粘蛋白的增加，改善粘液层的损伤程度。
基于上述技术方案，进一步地，所述增强肠屏障功能包括促进AhR配体吲哚-3-乳酸和吲哚-3-甲醛的增加，激活AhR信号通路，促进结肠紧密连接蛋白的表达，进而发挥增强肠道屏障的作用。
基于上述技术方案，进一步地，所述增强肠屏障功能包括缓减肠道疾病活性指数增加、结肠长度缩短和改善结肠组织病变。
本发明的有益效果如下：
本发明筛选获得一株代谢L-色氨酸产吲哚衍生物的植物乳杆菌(Lactobacillusplantarum)DPUL-S164，所述的植物乳杆菌(Lactobacillus plantarum)DPUL-S164在体内外能够代谢L-Trp并高产ILA和IAId，可单独或与L-Trp联合使用用于制备增强肠道屏障功能的功能食品或药品，通过促进杯状细胞和粘蛋白的增加，改善粘液层的损伤程度，促进AhR配体ILA和IAId的增加，激活AhR信号通路，促进结肠紧密连接蛋白的表达等多种途径，进而发挥增强肠道屏障的作用，具有很好的应用前景。
附图说明
为了更清楚地说明本发明实施例，下面将对实施例涉及的附图进行简单地介绍。
图1为植物乳杆菌(Lactobacillus plantarum)DPUL-S164体外产生吲哚衍生物的种类及含量。
图2为植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障功能损伤小鼠病理指标的影响。
图3为植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障功能损伤小鼠免疫指标的影响。
图4为植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障功能损伤小鼠粘液层的影响。
图5为植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障功能损伤小鼠结肠上皮细胞层紧密连接蛋白的影响。
图6为植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障功能损伤小鼠结肠AhR表达的影响。
图7为植物乳杆菌(Lactobacillus plantarum)DPUL-S164体内产生吲哚衍生物的种类及含量。
具体实施方式
下述非限定性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1菌株分离与纯化
本发明的植物乳杆菌DPUL-S164在MRS培养基中进行培养，具体步骤如下：
菌株的培养方法：
(1)MRS培养基制备
按照20g/L葡萄糖，10g/L蛋白胨，5g/L酵母膏，10g/L牛肉膏，1mL吐温-80，2g/L磷酸氢二钾，2g/L柠檬酸二铵，5g/L乙酸钠，0.58g/L七水硫酸镁，0.25g/L四水硫酸锰，称取这些原料，混合均匀，将得到的混合物混溶于去离子水中，得到的溶液在温度121℃下灭菌20min，得到所述的MRS培养基。
(2)菌株的富集培养
将植物乳杆菌DPUL-S164接种于灭菌的MRS培养基，在37℃厌氧培养培养18h并对菌株连续活化3次，置于4℃条件下备用。
实施例2植物乳杆菌DPUL-S164产吲哚衍生物鉴定
使用Kovacs吲哚试剂盒检测吲哚的生产(Hopebio，青岛，中国)。接种2％实施例1纯化的植物乳杆菌DPUL-S164于新鲜的MRS培养液中，在37℃下培养18h，连续培养三代。将培养好的菌株离心(3500rpm，5min)去掉上清液，重悬于生理盐水中调整菌株OD600值为1，接种0.5mL乳杆菌到4.5mL的MRS-Trp(在MRS培养基中加入2g/LTrp得到MRS-Trp培养基)培养基中，培养48小时后，离心收集1mL上清液并向上清液中加入4滴Kovacs吲哚试剂，混匀后静置2周后观察乳杆菌上清液上层中是否有玫瑰红色环。若上清液中出现玫瑰红色环说明培养液中有吲哚衍生物。
实施例3植物乳杆菌DPUL-S164产吲哚衍生物的高效液相色谱测定
接种2％实施例2的植物乳杆菌(Lactobacillus plantarum)DPUL-S164于新鲜的MRS培养液中，在37℃下培养18h，连续培养三代。将培养好的菌株离心(3500rpm，5min)去掉上清液，重悬于生理盐水中调整菌株OD600值为1，接种2mL乳杆菌到18mL的MRS-Trp培养基中，培养48小时后，将培养物以5000rpm的速度离心5分钟，弃掉菌体沉淀。将15毫升上清液与乙腈按1:1的比例混合。加入硫酸镁(0.2克/毫升)和乙酸钠(0.05克/毫升)，通过涡旋轻轻摇动混合10分钟，然后以3500rpm离心10分钟，取出10mL有机相，置于50mL离心管中，并在真空冷冻浓缩仪中干燥。然后用1mL水溶解样品，并用0.22μm的滤膜过滤除杂后通过高效液相色谱测定样品中所含有的吲哚衍生物。
将吲哚-3-乳酸(ILA)、吲哚-3-甲醛(IAId)和吲哚-3-乙酸(IAA)准品溶解于甲醇中，并将浓度稀释至500、100、50、10、1、0.1mg/L，进样前经0.22μm滤膜过滤。高效液相色谱条件：色谱柱为岛津C18(4.6mm×250mm，5μm)；0.1％甲酸水溶液(v：v)作为流动相A，0.1％甲酸乙腈(v：v)作为流动相B。柱温保持在40℃，样品用梯度洗脱：0-2分钟，5％B；2-22分钟，40％B；22-28分钟，100％B；28-32分钟，100％B；32-32.5分钟，5％B，然后在5％B下再洗脱3分钟，进行运行后平衡。整个运行过程中，流速保持在0.3mL/min。
结果如图1所示，与对照MRS-TBC(tryptophan broth culture，TBC)相比，植物乳杆菌DPUL-S164-TBC中ILA和IAId的含量明显增加。
实施例4植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障损伤小鼠的调节作用
1.动物分组
8周龄的雄性Balb/c小鼠适应性饲养一周后，将28只8周龄雄性Balb/C小鼠随机分为4组，先将DSS组、Trp组和植物乳杆菌组的小鼠用四联抗生素处理10d清除小鼠原有肠道中可代谢L-Trp的微生物。空白组小鼠实验期间灌胃0.2mL的PBS溶液。Trp组和乳杆菌组造模前7d，每天分别用0.2mL无菌Trp溶液(含有2mg Trp)或浓度为1.5×109CFU/mL的植物乳杆菌Trp混合溶液(含有2mg Trp)进行干预。选用2.5％葡聚糖硫酸钠(DSS)水溶液建立肠屏障功能缺陷小鼠，第8d后小鼠持续饮用2.5％DSS水溶液一周。在造模期间初步通过小鼠体重、粪便形状、便血程度判断模型是否建立成功。
2.植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障损伤小鼠病理的影响
通过记录小鼠从饮用2.5％DSS水后每一天的体重变化、粪便形状和便血情况，处死小鼠取小鼠结肠进行长度测量。结果如图2所示，结果显示，植物乳杆菌DPUL-S164联合色氨酸干预缓减小鼠体重下降，疾病活性指数增加、结肠长度缩短和结肠组织病变。
3.植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障损伤小鼠免疫指标的影响
处死小鼠前进行眼球取血，采用ELISA试剂盒法检测血清中IL-10、IL-6和TNF-α的表达水平。结果如图3所示，DSS处理小鼠后，明显增加了小鼠血清中促炎细胞因子TNF-α和IL-6的表达水平，降低了抗炎细胞因子IL-10的水平(p<0.05)。相比DSS组，Trp单独处理明显降低了血清IL-6的水平(p<0.05)。产吲哚衍生物的植物乳杆菌DPUL-S164联合Trp干预明显增加了抗炎因子IL-10的表达水平，降低了促炎细胞因子IL-6的表达水平。
4.植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障损伤小鼠粘液层的影响
处死小鼠后取小鼠结肠，用4％多聚甲醛进行固定、包埋、切片以及AB/PAS染色。实验结果如图4所示，相比空白组，DSS处理后粘液层出现明显的破坏，结肠杯状细胞明显消失，粘蛋白的表达显著下降，而植物乳杆菌DPUL-S164联合Trp明显增加了杯状细胞数量和粘蛋白的表达，明显改善了小鼠肠道粘液层的受损程度。
5.植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障损伤小鼠上皮细胞层的影响
处死小鼠后取小鼠结肠，用4％多聚甲醛进行固定、包埋、切片以及免疫组化染色。通过免疫组化检测了产吲哚衍生物植物乳杆菌DPUL-S164对结肠上皮细胞紧密连接蛋白表达的影响。从图5中可以看出，DSS处理后明显下调了ZO-1和Occudin蛋白的表达，但植物乳杆菌DPUL-S164联合Trp干预小鼠后明显上调了ZO-1和Occudin蛋白的表达(p<0.05)。
6.植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障损伤小鼠AhR信号通路的影响
处死小鼠后取小鼠结肠，用4％多聚甲醛进行固定、包埋、切片以及免疫组化染色。通过用免疫组化检测产吲哚衍生物乳杆菌对结肠AhR信号通路的影响。从图6中可以看出，DSS诱导明显下调了AhR和Cyp1a1蛋白的表达，但植物乳杆菌DPUL-S164联合Trp干预小鼠明显上调了AhR和Cyp1a1蛋白的表达。
7.植物乳杆菌(Lactobacillus plantarum)DPUL-S164对肠屏障损伤小鼠色氨酸代谢的影响
小鼠粪便解冻后，称重50mg样品，用500uL甲醇提取，提取液旋流3min，-20℃冰箱保存30min，4℃12000r/min离心10min，取250μL上清液，上清液再次在4℃下12000r/min离心5min，取150μL上清液进行LC-MS分析。如图7所示，植物乳杆菌DPUL-S164在体内外均能代谢L-色氨酸产生ILA和IAId。
Claims (8)
1.一株代谢L-色氨酸产吲哚衍生物的植物乳杆菌(Lactobacillus plantarum)DPUL-S164，于2023年4月18日保藏于中国典型培养物保藏中心，菌株保藏号为CCTCC NO：M2023547。
2.根据权利要求1所述的植物乳杆菌(Lactobacillus plantarum)DPUL-S164，其特征在于，所述的植物乳杆菌(Lactobacillus plantarum)DPUL-S164在体内外能够代谢L-色氨酸(L-Trp)并高产吲哚-3-乳酸(ILA)和吲哚-3-甲醛(IAId)。
3.权利要求1或2所述的植物乳杆菌(Lactobacillus plantarum)DPUL-S164在制备用于增强肠道屏障功能的功能食品或药品中的应用。
4.根据权利要求3所述的应用，其特征在于，所述的植物乳杆菌(Lactobacillusplantarum)DPUL-S164与L-色氨酸(L-Trp)联合使用。
5.根据权利要求3或4所述的应用，其特征在于，所述增强肠道屏障功能包括调节免疫屏障、机械屏障和化学屏障。
6.根据权利要求5所述的应用，其特征在于，所述增强肠道屏障功能包括促进杯状细胞和粘蛋白的增加，改善粘液层的损伤程度。
7.根据权利要求3或4所述的应用，其特征在于，所述增强肠屏障功能包括促进芳基烃受体(AhR)的配体ILA和IAId的增加，激活AhR信号通路，促进结肠紧密连接蛋白的表达，进而发挥增强肠道屏障的作用。
8.根据权利要求3或4所述的应用，其特征在于，所述增强肠屏障功能包括缓减肠道疾病活性指数增加、结肠长度缩短和改善结肠组织病变。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117645965A (zh) * | 2024-01-29 | 2024-03-05 | 潍坊康地恩生物科技有限公司 | 一株具有降解肠道吲哚、缓解慢性肾衰功能的鼠李糖乳酪杆菌 |
| CN117645965B (zh) * | 2024-01-29 | 2024-05-10 | 青岛蔚蓝生物集团有限公司 | 一株具有降解肠道吲哚、缓解慢性肾衰功能的鼠李糖乳酪杆菌 |
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