CN111733071A - 一种体外模拟人体肠道菌群发酵的设备及方法 - Google Patents
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Abstract
本发明公开了一种体外模拟人体肠道菌群发酵的设备及方法，本发明提供了一种体外模拟人体肠道菌群发酵的设备及方法。该系统包括:系统控制器、速率控制系统、搅拌系统、气路系统、温度调节系统、pH调节系统和取样系统。该设备和方法包括控制系统参数设置和模拟发酵等步骤。本发明模拟了离体条件下的人体肠道菌群发酵，模拟真实，重现性好，系统采用智能控制，适用于营养学、食品学、生理学及微生物学等领域的研究。
Description
技术领域
本发明涉及人体肠道菌群发酵培养技术领域，尤其涉及一种体外模拟人体肠道菌群发酵的设备及方法。
背景技术
肠道微生态是目前微生物学、营养学和免疫学的研究热点。由于哺乳动物肠道菌群与人体肠道菌群差异较大，动物模型并不能很好地模拟人体情况，因而体外模拟人体肠道菌群发酵装置被不断推广，为食品学、营养学和微生物学的研究提供了巨大的方便，逐渐被学术界接受，某些模拟装置甚至可以作为生产设备，生产需要肠道环境或肠菌激活的某些活性物质。如SHIME系统采用5个反应罐，以A-B-C-D-E串联形式连接。余应新等人设计的系统在SHIME系统的基础上加入一个新的，模拟口腔的环节，同样是串联连接(F-A-B-C-D-E)。但是现有设备较为复杂，占地面积较大，成本高昂，不适用于小型实验室。
发明内容
为解决现有技术的缺点和不足，提供一种体外模拟人体肠道菌群发酵的设备及方法，为在体外条件下，真实地模拟人体肠道微生态对摄入食物的代谢和发酵，提供了一种较为便捷的实验方法。
为实现本发明目的而提供的一种体外模拟人体肠道菌群发酵的设备及方法，包括有肠道菌群发酵系统、气路系统、温度调节系统、pH调节系统、搅拌系统和系统控制器，其中肠道菌群发酵系统包括发酵罐、罐盖和多联磁力搅拌器，所述罐盖装有pH仪，pH仪通过数据线与系统控制器连接；所述罐盖上设置有气体进出口、酸液进口、碱液进口以及取样口，所述气体进出口通过铜管连接有气体存储罐；酸液进口连接蠕动泵，通过硅胶管与盐酸试剂瓶连接；碱液进口连接蠕动泵，通过硅胶管与碳酸氢钠试剂瓶连接；取样口通过硅胶管与注射器连接；所述发酵罐放在多联磁力搅拌器上，通过多联磁力搅拌器内部的转子搅拌发酵罐内的发酵液；所述气路系统为气罐及管线；所述温度调节系统为恒温培养箱，所述发酵罐及多联磁力搅拌器置于恒温培养箱中并且以模拟人体37℃恒温环境培养；所述pH调节系统为pH仪；所述搅拌系统为多联磁力；所述系统控制器包括人机交换界面和中央处理器，所述中央处理器通过数据线与多联磁力搅拌器、pH仪和蠕动泵连接。
作为上述方案的进一步改进，具体包括有控制系统参数设置以及肠道菌群模拟发酵两大步骤，
其中:控制系统参数设置具体如下:系统温度37℃，发酵罐的pH通过0.05M盐酸和0.5M碳酸氢钠保持在一定范围参数内，若模拟小肠菌群发酵设置pH参数6.0～6.5之间、若模拟升结肠菌群发酵设置pH参数5.7～5.9之间、若模拟横结肠菌群发酵设置pH参数6.3～6.5之间、若模拟降结肠菌群发酵设置pH参数6.7～6.9之间，发酵模拟时间为0-48h，发酵罐中培养基体积135ml，粪便稀释液体积15ml；
其中：肠道菌群模拟发酵步骤如下:
第一步：开启恒温培养箱，设置温度37℃，使系统温度保持在37℃；
第二步：发酵罐内加入135ml培养基以及15ml制备好的10％粪便稀释液体积；
第三步：打开气罐，开启多联磁力搅拌器，开启pH调节系统使发酵罐内pH稳定在设置好的参数范围内，在模拟发酵过程中，通过控制注射器可定时采样。
作为上述方案的进一步改进，所述10％粪便稀释液的制备步骤如下:取近3个月未接受抗生素治疗、未服用益生菌或益生元等功能食品的健康人的新鲜粪便，用0.1％蛋白胨溶液稀释成10％粪水悬液，通过拍打式无菌均质器4次/秒混匀，纱网过滤，取15ml粪便稀释液，加入发酵罐。
作为上述方案的进一步改进，所述培养基中各组分的配比如下：蛋白胨：酵母提取物：氯化钠：磷酸氢二钾：磷酸二氢钾：七水硫酸镁：六水氯化钙：碳酸氢钠：吐温80：氯高铁血红色：维生素K：L-半胱氨酸HCL：猪胆盐之间的比例为：2:2:0.1:0.04:0.04:0.01:0.01:2:2:0.05:0.01:0.5:0.5。
本发明的有益效果是：
1.本发明的体外模拟人体肠道菌群发酵的实验方法实现了离体条件下模拟人体肠道菌群发酵的过程，全系统只需通过人机交换界面输入运行参数，由电脑自动控制完成发酵过程，模拟逼真，具有很好的稳定性，平行性，及可重现性。
2.本发明体外模拟人体肠道菌群发酵的实验方法适用于营养学、毒理学、生理学、食品学和微生物学等领域的研究，如营养学研究中对食物营养成分、微生态制剂(益生菌、益生元、合生元)等的营养性、有效性、安全性的评价；毒理学研究中对外源药物、抗生素、毒物等食入成分的代谢研究；生理学研究中通过代谢组学研究肠道菌群代谢产物对机体产生的影响；微生物学研究中肠道菌群的动力学研究等。
5.本发明中代谢产物可实现在线检测，如结肠菌群代谢培养物质后产气、产酸等、可直接通过气相、液相设备检测。
附图说明
以下结合附图对本发明的具体实施方式作进一步的详细说明，其中：
图1为本发明结构示意主视图；
图2为本发明发酵罐的连接示意图。
具体实施方式
如图1-图2所示，人体外模拟肠道菌群的发酵具体操作如下：
准备系统控制器(人机交换界面、数据处理器模块、设备控制器模块)，数据线、输液管道、发酵罐、多联磁力搅拌器、pH仪、气体存储罐、气体输入管道、蠕动泵、恒温培养箱。
发酵罐的气体进出口通过铜管与气体储存罐连接，将发酵罐置于多联磁力搅拌器上，多联磁力搅拌器通过数据线与系统控制器连接。
实验开始时，开启系统控制器，输入肠道菌群发酵模拟系统的pH参数，发酵罐的pH通过盐酸(0.05M)和碳酸氢钠(0.5M)的添加保持在参数范围内(若模拟小肠菌群发酵设置pH参数6.0～6.5之间，若模拟升结肠菌群发酵设置pH参数5.7～5.9之间、若模拟横结肠菌群发酵设置pH参数6.3～6.5之间，若模拟降结肠菌群发酵设置pH参数6.7～6.9之间)，发酵模拟时间为0-48h；发酵罐中培养基体积135ml，粪便稀释液体积15ml。
肠道菌群模拟发酵步骤如下:开启恒温培养箱，设置温度37℃，使系统温度保持在37℃；发酵罐内加入135ml培养基以及制备好的10％粪便稀释液体积15ml，打开气罐；开启多联磁力搅拌器，开启pH调节系统使发酵罐内pH稳定在设置好的参数范围内，在模拟发酵过程中通过控制注射器可定时采样。
培养基(1L)包括以下成分：
以上实施例不局限于该实施例自身的技术方案，实施例之间可以相互结合成新的实施例。以上实施例仅用以说明本发明的技术方案而并非对其进行限制，凡未脱离本发明精神和范围的任何修改或者等同替换，其均应涵盖在本发明技术方案的范围内。
Claims (4)
1.一种体外模拟人体肠道菌群发酵的设备，其特征在于：包括有肠道菌群发酵系统、气路系统、温度调节系统、pH调节系统、搅拌系统和系统控制器，其中肠道菌群发酵系统包括发酵罐、罐盖和多联磁力搅拌器，所述罐盖装有pH仪，pH仪通过数据线与系统控制器连接；所述罐盖上设置有气体进出口、酸液进口、碱液进口以及取样口，所述气体进口通过铜管连接有气体存储罐；酸液进口连接蠕动泵，通过硅胶管与盐酸试剂瓶连接；碱液进口连接蠕动泵，通过硅胶管与碳酸氢钠试剂瓶连接；取样口通过硅胶管与注射器连接；所述发酵罐放在多联磁力搅拌器上，通过多联磁力搅拌器内部的转子搅拌发酵罐内的发酵液；所述气路系统为气罐及管线；所述温度调节系统为恒温培养箱，所述发酵罐及多联磁力搅拌器置于恒温培养箱中并且以模拟人体37℃恒温环境培养；所述pH调节系统为pH仪；所述搅拌系统为多联磁力；所述系统控制器包括人机交换界面和中央处理器，所述中央处理器通过数据线与多联磁力搅拌器、pH仪和蠕动泵连接。
2.一种体外模拟人体肠道菌群发酵的方法，其特征在于：具体包括有控制系统参数设置以及肠道菌群模拟发酵两大步骤，
其中:控制系统参数设置具体如下:系统温度37℃，发酵罐的pH通过0.05M盐酸和0.5M碳酸氢钠保持在一定范围参数内，若模拟小肠菌群发酵设置pH参数6.0～6.5之间、若模拟升结肠菌群发酵设置pH参数5.7～5.9之间、若模拟横结肠菌群发酵设置pH参数6.3～6.5之间、若模拟降结肠菌群发酵设置pH参数6.7～6.9之间，发酵模拟时间为0-48h，发酵罐中培养基体积135ml，粪便稀释液体积15ml；
其中：肠道菌群模拟发酵步骤如下:
第一步：开启恒温培养箱，设置温度37℃，使系统温度保持在37℃；
第二步：发酵罐内加入135ml培养基以及15ml制备好的10％粪便稀释液体积；
第三步：打开气罐，开启多联磁力搅拌器，开启pH调节系统使发酵罐内pH稳定在设置好的参数范围内，在模拟发酵过程中，通过控制注射器可定时采样。
3.根据权利要求2所述的一种体外模拟人体肠道菌群发酵的方法，其特征在于：所述10％粪便稀释液的制备步骤如下:取近3个月未接受抗生素治疗、未服用益生菌或益生元等功能食品的健康人的新鲜粪便，用0.1％蛋白胨溶液稀释成10％粪水悬液，通过拍打式无菌均质器4次/秒混匀，纱网过滤，取15ml粪便稀释液，加入发酵罐。
4.根据权利要求3所述的一种体外模拟人体肠道菌群发酵的方法，其特征在于：所述培养基中各组分的配比如下：蛋白胨：酵母提取物：氯化钠：磷酸氢二钾：磷酸二氢钾：七水硫酸镁：六水氯化钙：碳酸氢钠：吐温80：氯高铁血红素：维生素K：L-半胱氨酸HCL：猪胆盐之间的比例为：2:2:0.1:0.04:0.04:0.01:0.01:2:2:0.05:0.01:0.5:0.5。
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