CN112933116A - 植物乳杆菌在制备血管保护剂中的应用 - Google Patents
植物乳杆菌在制备血管保护剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种植物乳杆菌CCFM639(保藏编号为CGMCC No.9664)在制备血管保护剂中的应用，所述血管保护剂为菌剂、食品或药物组合物。所述菌剂是将含有植物乳杆菌的菌液通过常规冷冻干燥工艺制备得到的粉剂，活性植物乳杆菌含量为109CFU/g以上。本发明首次提出植物乳杆菌CCFM639能够降低血管活性氧水平、增加NO水平、缓解血管舒张功能受损，提出了植物乳杆菌CCFM639在制备血管保护剂中的应用。
Description
技术领域
本发明涉及功能性微生物技术领域，尤其是涉及一种植物乳杆菌在制备血管保护剂中的应用。
背景技术
近几十年来，心脑血管疾病的发病率和死亡率在全球范围中占首位，严重威胁人们的生命健康。血管作为运送血液的一系列连续且相对密闭的管道系统，遍布于人体各个组织和器官，负责将心脏搏出的血液输送到全身各个组织器官，同时将代谢产物运回心脏，通过肾、肺等器官排出体外。血管的功能稳态与心脑血管疾病息息相关。血管内皮细胞是血管壁上的第一道屏障，具有选择性渗透屏障功能，能促进水和小分子物质的交换。血管内皮细胞在血管生理、病理生理和修复过程中都起着至关重要的作用，生理条件下，血管内皮细胞在接受一些理化刺激后能够合成和分泌多种血管活性物质，如内皮衍生的超极化因子、NO、抗凝物、前列腺素、血管舒缩因子、细胞黏附因子等，在血管稳态调控中发挥各自特有的生物学效应。同时，血管内皮细胞在不同的刺激下还可以通过各种途径合成和释放一些因子来调节血管的张力变化。常见的血管舒张保护剂如：卡托普利[中国药理学与毒理学杂志,2012,26(06):816-822]、银杏叶片[实用医药杂志,2005(11):50-52]等。血管内皮失调和损伤的因素有很多，如氧化应激导致的血管内皮损伤，活性氧(ROS)积累在机体或细胞中而引起的氧化损伤过程。
研究表明，肠道微生物生态失调可以促进动脉粥样硬化和高血压两大危险因素，从而促进心血管疾病的进展，心血管疾病患者的肠道菌群的组成比例及数量与健康人群明显不同。宿主-微生物相互作用的失衡可激活多种途径导致心血管疾病危险因素进展，导致血管纤维化和血管张力的改变进而促进高血压的发生[Nutrients.2017；10；9(8):859]。还有研究表明，膳食纤维细菌发酵的产物——短链脂肪酸，能扩张血管和降低血压；肉碱和胆碱在肠道细菌的代谢产物——三甲胺是循环系统的潜在毒性分子[Exp Biol Med(Maywood).2020；245(2):166-175]。因此，微生物代谢产物和肠道菌群可能是心血管疾病的潜在诊断和治疗靶点。
目前，许多常见的血管相关慢性疾病还不能根治，难以有效恢复器官血供，或是因可用的临床一线治疗药物作用靶点存在全身性的特点，不能实现器官的精准性，长期应用会引起诸多不良反应。肠道益生菌是一类通过进入肠道后改善肠道微生物平衡而对宿主产生有益健康影响的微生物。因此，从肠道微生物入手，寻找预防和/或治疗血管功能障碍疾病的新药物或新方法，或许能克服现有治疗药物以及治疗方法不良反应明显等的缺陷，利用益生菌相关膳食干预策略和途径为缓解血管损伤开辟出新的途径和解决方案。
发明内容
针对现有技术存在的上述问题，本发明申请提供了一种植物乳杆菌在制备血管保护剂中的应用。本发明首次提出植物乳杆菌CCFM639能够降低血管活性氧水平、增加NO水平、缓解血管舒张功能受损，提出了植物乳杆菌CCFM639在制备血管保护剂中的应用。
本发明的技术方案如下：
植物乳杆菌在制备血管保护剂中的应用。
所述植物乳杆菌为CCFM639，保藏编号为CGMCC No.9664。
所述血管保护剂为菌剂、食品或药物组合物。
所述菌剂是将含有植物乳杆菌的菌液通过常规冷冻干燥工艺制备得到的粉剂，活性植物乳杆菌含量为109CFU/g以上。
使用含有植物乳杆菌的发酵剂生产发酵食品。
所述发酵食品为乳制品、豆制品或果蔬制品。
所述药物组合物是由植物乳杆菌与在药学上可接受的载体组成；所述药学上可接受的载体为填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂中的一种或多种；药物组合物中活菌数109CFU/g以上。
所述血管保护剂具有耐受胃肠液并缓解血管损伤、改善血管功能障碍相关疾病、改善血管功能损伤、缓解改善血管功能损伤的作用。
一种用于保护血管的菌剂，所述菌剂的制备方法包括以下步骤:
(1)培养基的制备
使用以按培养基总质量计87.7％的水将10％酶水解脱脂乳、0.5％葡萄糖、1.5％胰蛋白胨与0.3％酵母浸膏溶解，然后调整其pH为6.8，得到培养基；
(2)保护剂的制备
将水与保护剂原料混合制备得到含有100g/L脱脂奶粉、30mL/L甘油、100g/L 麦芽糊精、150g/L海藻糖、10g/L L-谷氨酸钠的保护剂；
(3)将植物乳杆菌CCFM639菌种按照培养基的质量计2-4％的接种量接种到在温度110-120℃下灭菌8-12min后冷却的培养基中，然后在温度37℃下培养 18h，用pH7.2磷酸盐缓冲液清洗2-4次，再用步骤(2)所得的保护剂重悬使菌浓达到109CFU/ml；
(4)再将步骤(3)所得的悬浮液在温度37℃下预培养60min，再进行冷冻干燥得到所述菌剂。
一种用于保护血管的药物，所述药物组合物为颗粒剂、胶囊剂、片剂、丸剂或口服液剂型。
本发明有益的技术效果在于：
本发明植物乳杆菌CCFM639可增加血管内皮NO的产生，缓解氧化应激状态，提高血管舒张功能。因此，植物乳杆菌CCFM639菌株可以应用于制备含有生物活性的特殊产品，或作为可食用物质的成分或添加剂，且不具有药物的毒副作用，具有非常广泛的应用前景。
附图说明
图1植物乳杆菌CCFM639对肠系膜血管内皮细胞NO生成的影响。
图2植物乳杆菌CCFM639对肠系膜血管内皮细胞ROS生成的影响。
图3植物乳杆菌CCFM639对肠系膜血管舒张功能的影响。
具体实施方式
下面结合附图和实施例，对本发明进行具体描述。
具体实施方式所涉及的动物、实验仪器与试剂来源：
C57BL/6J小鼠：上海斯莱克实验动物有限责任公司
麦氏比浊仪：北京索莱宝科技有限公司
蔡司超分辨激光共聚焦显微镜：德国卡尔·蔡司股份有限公司
高盐饲料：南通特洛菲饲料科技有限公司
培养基成分：MRS肉汤(北京索莱宝科技有限公司)
NO试剂盒：DAR-4M AM，美国圣克鲁斯生物技术有限公司
ROS试剂盒：CM-H2DCFDA绿色荧光探针，赛默飞世尔科技(中国)有限公司
Wire myography血管张力检测仪：DMT 620M型仪器
植物乳杆菌CCFM639(Lactobacillus plantarum)为已公开菌株，已于2014 年09月15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，保藏地址为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，保藏编号为 CGMCC No.9664。
植物乳杆菌CCFM639具有下述已知生物学特性：
(1)菌体特征：呈革兰氏染色阳性，细胞呈中杆状，菌体约0.6-1.1μm宽， 2-6μm长，成单、成对或者成链，不形成芽孢，两端圆形。
(2)菌落特征：在MRS培养基上形成明显的菌落，直径在0.2-2.5mm之间，圆形，乳白色，半透明，表面湿润光滑，边缘整齐，不产生色素。
(3)生长特性：该菌株的最低生长温度为18℃，最高生长温度为40℃，在温度30-37℃下生长最佳，能耐受的最高和最低初始生长pH为9.0和2.5，最适生长初始pH为6.0，菌株的延迟期相对较短，4h左右开始进入对数生长期，12h 就达到稳定期。
下述实施例中涉及的检测方法如下:
活菌数的检测方法：采用国标《GB4789.35-2016食品安全国家标准食品微生物学检测乳酸菌检测》。
实施例1：
植物乳杆菌增加肠系膜血管内皮细胞NO的生成
取高盐饮食诱导的高血压小鼠16只，随机分为2组(对照组、CCFM639组)，每组8只。
使用MRS液体培养基在28℃的有氧环境下，培养扩增植物乳杆菌CCFM639 菌株，220rpm振荡培养2天至培养基完全浑浊。用离心管量取10ml菌液，离心，2400rpm，5min，倒掉上清，用生理盐水重悬，再用麦氏比浊仪计数，将菌体调配成浓度为108CFU/mL的菌液，用于CCFM639组。
对照组以0.2mL生理盐水/只/天进行灌胃处理，CCFM639组以0.2mL菌液/ 只/天灌胃处理，持续灌胃4周。
每组取出3只小鼠进行肠系膜原代动脉内皮细胞分离与培养。具体步骤为：将小鼠用CO2气体安乐死，固定好四肢，腹部喷洒75％乙醇进行消毒；用已消毒的剪刀剖开腹腔，暴露出肠子，将肠系膜分离开来；剪出肠系膜，在生物安全柜中将其剪碎，随后置于0.02％IA型胶原酶消化液中，于37℃的摇床水浴锅中消化45min；将消化好的肠系膜1200rpm离心5min，弃上清，将沉淀用 ECM培养基重悬，后转入共聚焦小皿，置于37℃5％CO2的培养箱中培养；2h 后，观察细胞贴壁情况，用PBS洗两遍，加入新的ECM培养基，置于37℃5％ CO2的培养箱中培养。第二天细胞可用于实验。用10μM DAR-4M AM染料与细胞共敷育15min，用NPSS缓冲液洗三遍，加入800μL NPSS缓冲液；激光共聚焦显微镜以594nm激发光激发DAR-4M AM，记录DAR-4M AM的信号变化，待荧光稳定后加入200μL GSK(500nM)，GSK终浓度为100nM。采用绿色波段激发光激发细胞产生红色荧光，选择合适的视野进行拍摄，用ZEN BLUE软件计算平均荧光值。
结果如图1所示：对照组的NO的平均荧光密度数值为3.252，CCFM639组 NO的平均荧光密度数值为8.745，是对照组的2.6倍左右，差异具有统计学意义(****P<0.0001)。结果说明植物乳杆菌CCFM639能够增加高盐饮食诱导的高血压小鼠肠系膜血管内皮细胞NO的产生。
实施例2：植物乳杆菌降低肠系膜血管内皮细胞ROS的产生
参考上述方法分离原代肠系膜内皮细胞后，参考CM-H2DCFDA的使用说明书，用200μM CM-H2DCFDA染料与细胞共敷育30分钟，用NPSS缓冲液洗三遍，加入800μL NPSS缓冲液；激光共聚焦显微镜以488nm激发光激发 CM-H2DCFDA，记录CM-H2DCFDA荧光变化，在蔡司超分辨激光共聚焦显微镜ZEN BLUE软件中记录ROS的平均荧光密度值。
结果如图2所示：对照组ROS平均荧光密度值为72.85，CCFM639组平均荧光密度值为21.33，是对照组的1/3左右，差异具有统计学意义 (****P<0.0001)。结果说明植物乳杆菌CCFM639能够减少高盐饮食诱导的高血压小鼠肠系膜内皮ROS的产生。
实施例3：植物乳杆菌增强肠系膜动脉血管舒张能力
取上述分组小鼠各4只，分离肠系膜血管二级动脉，具体步骤如下：通过CO2气体安乐死小鼠，打开腹腔，取出肠组织，将其放入预冷的科氏溶液中，并在冰上分离小鼠肠系膜二级动脉，分离过程中不要拉扯血管，尽可能把血管周围的脂肪组织去除干净；将血管剪成2mm的血管段。用40μm细丝将血管段固定在DMT myograph槽中(尽量避免碰到血管内壁，槽中放入Krebs溶液)，后将槽置于DMT myoraph仪器上，连接好，通入95％O2；血管张力0mN维持30min，后将张力调至2mN，平衡60min，过程中15min换一次温度在 37℃氧饱和的Krebs溶液，并一直通95％O2。槽中换入60mM高钾溶液，5 min后换成37℃氧饱和的Krebs溶液，两者来回处理三次。曲线平稳在2mN，加入Phe(1μM)收缩血管，张力在最大值时，依次加入终浓度为10nM，100nM， 1μM和10μM的ACh舒张血管，用Lab Chart软件记录并分析数据。
通过血管舒张率的计算，结果(如图3)发现，对照组小鼠血管舒张最大至 50％，而CCFM639组小鼠的血管舒张显著提高，最大至80％，两组数据具有统计学差异(****P<0.0001)。
虽然本发明己以较佳实施例公开如下，这些实施例仅具有说明性，本发明不受这些实施例的限制，凡在本发明实质内容上所作的任何修改、等同替换和简单改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.植物乳杆菌在制备血管保护剂中的应用。
2.根据权利要求1所述的应用，其特征在于，所述植物乳杆菌为CCFM639，保藏编号为CGMCC No.9664。
3.根据权利要求1所述的应用，其特征在于，所述血管保护剂为菌剂、食品或药物组合物。
4.根据权利要求3所述的应用，其特征在于，所述菌剂是将含有植物乳杆菌的菌液通过常规冷冻干燥工艺制备得到的粉剂，活性植物乳杆菌含量为109CFU/g以上。
5.根据权利要求3所述的应用，其特征在于，使用含有植物乳杆菌的发酵剂生产发酵食品。
6.根据权利要求5所述的应用，其特征在于，所述发酵食品为乳制品、豆制品或果蔬制品。
7.根据权利要求3所述的应用，其特征在于，所述药物组合物是由植物乳杆菌与在药学上可接受的载体组成；所述药学上可接受的载体为填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂中的一种或多种；药物组合物中活菌数109CFU/g以上。
8.根据权利要求1所述的应用，其特征在于，所述血管保护剂具有耐受胃肠液并缓解血管损伤的作用。
9.一种用于保护血管的菌剂，其特征在于，所述菌剂的制备方法包括以下步骤:
(1)培养基的制备
使用以按培养基总质量计87.7％的水将10％酶水解脱脂乳、0.5％葡萄糖、1.5％胰蛋白胨与0.3％酵母浸膏溶解，然后调整其pH为6.8，得到培养基；
(2)保护剂的制备
将水与保护剂原料混合制备得到含有100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖、10g/L L-谷氨酸钠的保护剂；
(3)将植物乳杆菌CCFM639菌种按照培养基的质量计2-4％的接种量接种到在温度110-120℃下灭菌8-12min后冷却的培养基中，然后在温度37℃下培养18h，用pH7.2磷酸盐缓冲液清洗2-4次，再用步骤(2)所得的保护剂重悬使菌浓达到109CFU/ml；
(4)再将步骤(3)所得的悬浮液在温度37℃下预培养60min，再进行冷冻干燥得到所述菌剂。
10.一种用于保护血管的药物，其特征在于，所述药物组合物为颗粒剂、胶囊剂、片剂、丸剂或口服液剂型。
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