CN113999792B - 一种双歧杆菌增殖促进剂及其制备方法和应用 - Google Patents
一种双歧杆菌增殖促进剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种双歧杆菌增殖促进剂及其制备方法和应用，其中所述增殖促进剂中包括：包含牡蛎提取物的GAM培养基。本发明通过牡蛎提取物‑GAM培养基对肠道微生物的干预实验，首次验证了包含牡蛎提取物的GAM培养基具有调节肠道微生物的作用，对于双歧杆菌Bifidobacterium的增殖和丰度增加具有极显著的促进作用，因此不仅可用于体外大规模培养益生菌Bifidobacterium以工业化高效生产相应的益生菌剂，同时还可作为药物或功能保健食品，改善肠道菌群紊乱所导致的肠道疾病，并辅助治疗与双歧杆菌Bifidobacterium缺失导致的相关的疾病，如结肠癌、便秘和抑郁症等，在工业生产和医药保健领域都具有广阔的市场前景和应用价值。
Description
技术领域
本发明属于双歧杆菌促进剂技术领域，具体涉及一种双歧杆菌增殖促进剂及其制备方法和应用。
背景技术
双歧杆菌属Bifidobacterium是一种十分重要的肠道益生菌，近年来研究发现，肠道微生物除了用于消化吸收食物外，还具有调节人体生理心理状态的作用，与多种临床疾病相关。通过调节肠道微生物群落结构，有望对某些疾病治疗提供帮助。已有多篇研究报道，肠道菌群中双歧杆菌属Bifidobacterium与多种肠道疾病或精神疾病相关。如文献：“Fahmy,Cinderella A et al.Bifidobacterium longum Suppresses Murine ColorectalCancer through the Modulation of oncomiRs and Tumor SuppressormiRNAs.Nutrition and Cancer,2019.”中报道了双歧杆菌属中的长双歧杆菌具有抑制结肠癌的作用。文献“Adhesive Bifidobacterium Induced Changes in Cecal MicrobiomeAlleviated Constipation in Mice”中报道了双歧杆菌属和乳杆菌属均可有效缓解便秘。同时文献“Lactobacillus reuteri NK33 and Bifidobacterium adolescentis NK98Alleviate Escherichia coli-Induced depression and Gut Dysbiosis in Mice”中报道了双歧杆菌属可用于缓解抑郁。
牡蛎(ostrea gigas tnunb)俗称海蛎子、蚝等，隶属软体动物门，双壳纲，珍珠贝目，是世界上第一大养殖贝类，是人类可利用的重要海洋生物资源之一。不仅肉鲜味美、营养丰富,而且具有独特的保健功能和药用价值，是一种营养价值很高的海产珍品。牡蛎提取物(JCOE)是以全牡蛎为原料，采用特殊方法分离提取的一种混合物的简称，研究发现牡蛎提取物具有多种药理作用，包括：增强免疫力、抗疲劳、保护肝脏、抗肿瘤抗菌以及保护心血管等。然而牡蛎提取物在肠道微生物调节，肠道益生菌生长促进中的相关作用还未见报道。
发明内容
本发明的目的在于提供了一种双歧杆菌增殖促进剂及其制备方法和应用，其中所述增殖促进剂中包括：包含牡蛎提取物的GAM培养基。本发明通过牡蛎提取物-GAM培养基对肠道微生物的干预实验，首次发现了包含牡蛎提取物的GAM培养基具有调节肠道微生物的作用，对于双歧杆菌Bifidobacterium增殖和丰度增加具有极显著的促进作用，因此可用于体外培养益生菌以工业化高效生产益生菌剂，同时还可作为药物或功能保健食品，改善肠道菌群紊乱所导致的肠道疾病，并辅助治疗与双歧杆菌Bifidobacterium缺失导致的相关的疾病，如结肠癌、便秘和抑郁症等。
为实现上述目的，本发明所采用的技术方案为：
本发明的目的之一在于提供了一种双歧杆菌Bifidobacterium增殖促进剂，所述增殖促进剂中包括：包含牡蛎提取物的GAM培养基。
进一步地，所述牡蛎提取物的质量浓度为2％。
本发明的目的之二在于提供了所述双歧杆菌Bifidobacterium增殖促进剂的制备方法，所述方法包括：配制GAM培养基，同时将牡蛎提取物用PBS缓冲液溶解得到牡蛎提取物溶液，将所述牡蛎提取物溶液加入至GAM培养基中，使牡蛎提取物的终质量浓度为2％，即为所述增殖促进剂。
本发明的目的之三在于提供了所述双歧杆菌Bifidobacterium增殖促进剂的应用，所述增殖促进剂可用于制备促进双歧杆菌Bifidobacterium增殖的药物、功能食品和/或保健食品。
进一步地，所述增殖促进剂通过上调双歧杆菌Bifidobacterium的丰度，达到改善结肠癌、便秘和/或抑郁症的作用。
本发明的目的之四在于提供了所述双歧杆菌Bifidobacterium增殖促进剂在制备结肠癌辅助治疗药物、功能食品和/或保健食品中的应用。
本发明的目的之五在于提供了所述双歧杆菌Bifidobacterium增殖促进剂在制备便秘辅助治疗药物、功能食品和/或保健食品中的应用。
本发明的目的之六在于提供了所述双歧杆菌Bifidobacterium增殖促进剂在制备抑郁症辅助治疗药物、功能食品和/或保健食品中的应用。
进一步地，所述药物中还包括药学上可接受的辅料。
进一步地，所述药物的剂型包括：颗粒剂、片剂、胶囊剂、粉剂、丸剂或溶液剂。
进一步地，所述功能食品或保健食品包括：饮料、饼干、糕点、罐头、乳制品、口服液。
与现有技术相比，本发明的有益效果是：本发明提供了一种双歧杆菌Bifidobacterium增殖促进剂，具体为包含牡蛎提取物的GAM培养基，本发明通过模拟肠道环境，并取人体粪便中的肠道微生物进行牡蛎提取物-GAM培养基干预实验，首次验证了包含牡蛎提取物的GAM培养基具有调节肠道微生物的作用，对于双歧杆菌Bifidobacterium的增殖和丰度增加具有极显著的促进作用，因此不仅可用于体外大规模培养益生菌Bifidobacterium以工业化高效生产相应的益生菌剂，同时还可作为药物或功能保健食品，改善肠道菌群紊乱所导致的肠道疾病，并辅助治疗与双歧杆菌Bifidobacterium缺失导致的相关的疾病，如结肠癌、便秘和抑郁症等，在工业生产和医药保健领域都具有广阔的市场前景和应用价值。
附图说明
图1为本发明实施例1中试验组和对照组中双歧杆菌Bifidobacterium的丰度测定和显著性分析结果；
图2为本发明实施例1中增殖促进剂对于双歧杆菌Bifidobacterium生长曲线的影响。
具体实施方式
下面将结合本发明中的实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1
由于来源于人体的肠道菌群无法完全在小鼠体内定殖，为避免测定结果不准确，因此体外模拟人体内肠道环境，取人体粪便经处理后作为肠道菌群样品，观察经牡蛎提取物-GAM培养基干预后的肠道菌群变化，具体操作过程如下：
1、粪便样品处理
(1)PBS缓冲液配制
以1L PBS缓冲液为例，用电子分析天平称取下列药品，倒入1000ml烧杯中，包括：KH2PO4 0.24g，Na2HPO4·12H2O 2.90g，NaCl 8.00g，KCl 0.20g。用800ml超纯水溶解，然后用pH计测量上述溶液的pH，调节溶液的pH值至pH＝7.4±0.05。
接着将溶液引流至1000ml容量瓶中，冲洗烧杯后倒入容量瓶，并用超纯水定容至刻度线，充分混匀。将定容后的溶液倒入另一洁净玻璃瓶中，于高压灭菌锅中121℃灭菌15分钟。降至室温后，储存于4℃冰箱备用。
(2)肠道微生物样品的制备
称量10g粪便样品，用移液枪取适量上述PBS缓冲液加至离心管中，在振荡器上充分混匀。然后平均分装至新50ml离心管中，每管再加适量PBS缓冲，混匀后在生物安全柜内，将样品依次通过20目、50目、100目、200目的滤网进行过滤。收集滤液至离心管中，6000G，4℃离心15min，弃上清。将沉淀称量后，用上述PBS缓冲液重悬沉淀，并配制成浓度5％的肠道菌群样品。
2、牡蛎提取物-GAM培养基干预实验
(1)基础培养基配制
配制用于培养肠道菌群的基础培养基-GAM培养基，以1000ml为例。用电子分析天平称取60g改良GAM肉汤药品，倒入1000ml烧杯中，用超纯水完全溶解，接着用玻璃棒引流至1000ml容量瓶，并用少量超纯水冲洗烧杯后倒入容量瓶，重复3次。用超纯水定容至刻度线，上下颠倒数次，确保溶液充分混匀。
将定容后的溶液倒入洁净黄色盖子的玻璃瓶，瓶盖拧松后放入脉冲高压灭菌锅，使用液体程序，121℃灭菌15分钟。灭菌完后，立即把瓶盖拧紧，冷却至室温备用。
(2)牡蛎提取物-GAM培养基配制
于无菌操作台上取上述GAM培养基分装至玻璃管中，每管2ml。接着用移液器取牡蛎提取物溶液(其中牡蛎提取物购于西安奥赛生物，用PBS缓冲液溶解至质量百分浓度为：5％)，加入上述装有GAM培养基的玻璃管中，每管2ml，作为试验组，其中牡蛎提取物的终浓度为质量分数2％。同时还设置有对照组，每组3个重复，分组具体如下：
试验组：2ml GAM培养基+2ml牡蛎提取物溶液+1mL PBS缓冲液；
对照组：2ml GAM培养基+3mL PBS缓冲液；
将玻璃管盖拧紧后转移至厌氧箱传递箱中(放置之前需用84消毒液消毒处理)，拧松瓶盖，置换O2，置换12个小时。
将上述处理后得到的肠道菌群样品分别接种至试验组(牡蛎提取物-GAM培养基)以及空白对照组(GAM培养基-PBS缓冲液)中，每管1ml。
于厌氧箱中培养72小时后，观察菌群的生长状况，拍照留存。
接着分别将试验组样品和对照组样品，10000rpm，3min离心，弃上清，将沉淀用液氮处理，并送至武汉艾康健生物科技有限公司进行检测，分别测定试验组和对照组中的肠道菌群如Bifidobacterium和Anaerostipes的丰度，即各个菌群在检测到的所有菌种中所占的百分比，测定结果如表1所示。
并对经牡蛎提取物-GAM培养基干预前后的菌群丰度变化进行显著性分析，分析结果如图1所示，其中**表示P＜0.01，即丰度变化有极显著差异。
表1试验组和对照组的菌群丰度
结果显示，经牡蛎提取物-GAM培养基干预后，肠道益生菌中的重要益生菌双歧杆菌Bifidobacterium的丰度有极显著提高，且丰度相对较高。然而部分肠道微生物如Anaerostipes的丰度则与对照组并无明显差异。结果说明，牡蛎提取物-GAM培养基可作为双歧杆菌Bifidobacterium的增殖促进剂，不仅可用于体外以工业化高效生产益生菌剂，同时还可作为药物或功能保健食品，改善肠道菌群紊乱所导致的肠道疾病，并辅助治疗与双歧杆菌Bifidobacterium缺乏导致的如结肠癌、便秘和抑郁症等相关疾病。
3、牡蛎提取物-GAM培养基对双歧杆菌Bifidobacterium生长曲线的影响
为进一步观察所述增殖促进剂即牡蛎提取物-GAM培养基对于双歧杆菌Bifidobacterium生长曲线的影响，以上述制备得到的牡蛎提取物-GAM培养基作为试验组，以GAM培养基作为对照组，分别用于培养双歧杆菌Bifidobacterium并定时取样测定菌液的OD600值变化。培养液充分摇匀再进行取样，每个培养瓶取三次100μl样品的量，分别纵向添加，再横向稀释三至五组连续的梯度，每个培养瓶使用一根无菌枪头。以此类推，对所有的培养液进行测定。
使用系统:Tecan i-control,2.0.10.0，设定系统数值如下表：
系统测定得出的数据如表2所示。
表2牡蛎提取物-GAM培养基对Bifidobacterium生长的影响
根据上述数据绘制相应的生长曲线图，如图2所示，结果显示，试验组和对照组内的Bifidobacterium在4-12h开始进入对数生长期，12-28h为稳定期，28h后进入衰亡期。对比试验组和对照组的生长曲线可以得出，在牡蛎提取物-GAM培养基的培养下，Bifidobacterium生长得更加旺盛，说明，包含有牡蛎提取物的GAM培养基对Bifidobacterium的生长有促进作用。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。
Claims (1)
1.双歧杆菌Bifidobacterium增殖促进剂在制备促进双歧杆菌Bifidobacterium增殖产品中的应用，其特征在于，所述增殖促进剂为包含牡蛎提取物的GAM培养基；
所述增殖促进剂的制备方法为：配制GAM培养基，同时将牡蛎提取物用PBS缓冲液溶解得到牡蛎提取物溶液，将所述牡蛎提取物溶液加入至GAM培养基中，使牡蛎提取物的终质量浓度为2％，即为所述增殖促进剂。
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