CN113925854A - 绿原酸在制备Anaerostipes生长促进剂中的应用 - Google Patents
绿原酸在制备Anaerostipes生长促进剂中的应用 Download PDFInfo
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Abstract
本发明公开了绿原酸在制备Anaerostipes生长促进剂中的应用，通过将来源于咖啡豆提取物的绿原酸对肠道菌群进行干预实验，首次验证了绿原酸具有调节肠道菌群，促进益生菌丁酸弧菌Anaerostipes增殖的作用，因此不仅可将绿原酸制成相应的生长促进剂用于体外促进Anaerostipes增殖发酵，以实现工业化高效制备益生菌剂；还可制成相应药物或保健食品，直接作用于体内，以改善肠道菌群紊乱或辅助治疗与Anaerostipes相关的包括：脂肪肝、结肠癌、便秘和抑郁症等多种疾病，制成相应的辅助治疗药物、功能食品或保健食品，具有极高的应用价值。
Description
技术领域
本发明属于益生菌调节技术领域，具体涉及绿原酸在制备Anaerostipes生长促进剂中的应用。
背景技术
绿原酸(Chlorogenic acid)，是由咖啡酸(Caffeic acid)与奎尼酸(Quinicacid，1-羟基六氢没食子酸)生成的缩酚酸，是植物体在有氧呼吸过程中经莽草酸途径产生的一种苯丙素类化合物，来源于茜草科植物小果咖啡、中果咖啡及大果咖啡的种子，即绿咖啡豆的提取物。研究发现其具有广泛的抗菌、抗病毒作用，抗氧化、抗衰老作用，以及降压和保护心血管等作用。然而其对于肠道益生菌如Anaerostipes的调节作用，目前未见相关研究报道。
肠道菌群是寄居在人体肠道中数量庞大的微生物群落，其构成复杂，细菌种类繁多，包括共生菌、条件致病菌和致病菌群等类型，其不仅用于消化吸收食物，目前多项研究表明肠道菌群与多种疾病相关。很多科学家认为肠道菌群可以看作是人体的另一个器官，可作为疾病治疗靶点之一，通过调节肠道微生物的群落结构或额外补充益生菌剂以改善疾病。
文献“Zhuang,Min et al.Abundance of Probiotics and Butyrate-ProductionMicrobiome Manages Constipation via Short-Chain Fatty Acids Production andHormones Secretion.Molecular Nutrition&Food Research,2019”，“Francesco Strati,et al.Altered gut microbiota in Rett syndrome.Microbiome,2016”以及“LouiseBrunkwall et al.Self-reported bowel symptoms are associated with differencesin overall gut microbiota composition and enrichment of Blautia in apopulation-based cohort.Journal of Gastroenterology and Hepatology.2020”中报道了与便秘以及肠道疾病相关的多种肠道益生菌：Anaerostipes，Blautia以及Faecalibacterium。此外，文献“任士萌，非酒精性脂肪性肝病患者肠道菌群改变”中公开了非酒精性脂肪性肝病(NAFLD)患者体内的肠道菌群与健康人群相比有丰度变化，其中Anaerostipes等部分菌属与NAFLD以及相关的炎症反应相关。文献“Kobel etal.Effectiveness of Inpatient Psychotherapy for Patients With and WithoutMigratory Background:Do They Benefit Equally？.Frontiers in Psychiatry,2020”中公开了与抑郁症相关的肠道微生物Anaerostipes和Blautia。
发明内容
本发明的目的在于提供了绿原酸在制备Anaerostipes生长促进剂中的应用，通过将来源于咖啡豆提取物的绿原酸对肠道菌群进行干预实验，本发明首次验证了绿原酸具有调节肠道菌群，促进Anaerostipes生长增殖的作用，因此可制成生长促进剂或药物或保健食品，用于体外促进益生菌增殖发酵或体内改善肠道菌群紊乱，并可辅助治疗与Anaerostipes相关的疾病，如：脂肪肝、结肠癌、便秘和抑郁症等。
为实现上述目的，本发明采用的技术方案是：
本发明提供了绿原酸在制备Anaerostipes生长促进剂中的应用。
本发明还提供了一种促进Anaerostipes增殖的方法，所述方法包括：将绿原酸按照质量浓度5％加入至GAM培养基中，制成绿原酸-GAM培养基，用所述绿原酸-GAM培养基培养益生菌。
进一步地，所述绿原酸来源于咖啡豆提取物。
本发明还提供了绿原酸或包含绿原酸的组合物在制备改善肠道菌群紊乱药物、功能食品和/或保健食品中的应用。
进一步地，所述绿原酸通过促进丁酸弧菌Anaerostipes的增殖，达到改善脂肪肝、结肠癌、便秘和/或抑郁症的作用。
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，所述药物的剂型为颗粒剂、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：固体饮料、液体饮料、饼干、糕点食品、罐头、乳制品、口服液。
与现有技术相比，本发明的有益效果是：本发明通过体外模拟肠道环境，并以健康人体粪便中收集的肠道菌群作为样本进行绿原酸干预实验，首次发现验证了来源于咖啡豆提取物的绿原酸具有调节肠道菌群，促进益生菌丁酸弧菌Anaerostipes增殖的作用，因此不仅可将绿原酸制成相应的生长促进剂用于体外促进益生菌增殖发酵，以实现工业化高效制备益生菌剂；还可制成相应药物或保健食品，直接作用于体内，以改善肠道菌群紊乱或辅助治疗与Anaerostipes相关的包括：脂肪肝、结肠癌、便秘和抑郁症等多种疾病，制成相应的辅助治疗药物、功能食品或保健食品，具有极高的应用价值。
附图说明
图1为本发明实施例1中试验组和对照组中菌群的丰度测定和显著性分析结果；
图2为本发明实施例1中试验组和对照组的Anaerostipes的生长曲线图。
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
考虑到来源于人体的肠道菌群无法完全在小鼠体内定殖，因此直接取人体粪便经处理后作为肠道菌群样品，在体外模拟人体内肠道环境，并用来源于咖啡豆提取的绿原酸进行干预处理，观察经绿原酸干预处理后的肠道菌群变化，具体操作过程如下：
1、粪便样品处理
(1)PBS缓冲液配制
以1000ml为例，用电子分析天平分别称取下列药品，倒入1000ml烧杯中，其中包括：KH2PO4 0.24g，Na2HPO4·12H2O 2.90g，NaCl 8.00g，KCl 0.20g。
量取800ml超纯水倒入烧杯中，搅拌至完全溶解，然后用pH计测量上述溶液的pH，用HCl或NaOH调整溶液的pH值为7.4±0.05。
接着用玻璃棒将上述溶液引流至1000ml容量瓶中，用少量超纯水冲洗烧杯后内残留的溶液倒入容量瓶，重复3次，最后用超纯水定容至刻度线，塞上盖子后上下颠倒以确保溶液充分混匀。
将定容后的溶液倒入洁净黄色盖子的玻璃瓶，盖子拧松后，放入高压灭菌锅，121℃灭菌15分钟。取出后迅速拧紧盖子，待降至室温后，储存于4℃冰箱中待用。
(2)肠道菌群样品的制备
以10g为例，用天平称量10g粪便样品，取上述PBS缓冲液加至离心管中，在振荡器上充分混匀。然后将完全混匀的样品平均分装至新的离心管中，每管再加适量PBS缓冲液。混匀后在生物安全柜内进行过滤，将稀释后的样品依次通过20目、50目、100目、200目的滤网。收集滤液至离心管中，将离心管放入离心机，配平，6000G，4℃离心15min，弃上清。将沉淀称量后，用上述PBS缓冲液重悬，并按照终浓度5％定容，得到肠道菌群样品。
2、咖啡豆提取物-绿原酸干预实验
(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。称取60g改良GAM肉汤药品溶解于超纯水中，搅拌至完全溶解。如果用玻璃棒引流至1000ml容量瓶，并用少量超纯水冲洗烧杯并后倒入容量瓶中避免残留，重复3次。超纯水定容至刻度线，塞上盖子后上下颠倒至充分混匀。
将定容后的溶液倒入洁净黄色盖子的玻璃瓶，每1000ml的瓶子内放500ml培养基，防止高压灭菌时喷出，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)绿原酸-GAM培养基配制
于无菌操作台上，用1ml的移液枪取上述GAM培养基分装至玻璃管中，每管2ml。将黑色盖子放在瓶口上微掩。接着用1ml移液器取绿原酸溶液(咖啡豆提取物-绿原酸购于陕西慧科生物，绿原酸50％，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。同时还设置有空白对照组，每组3个重复，分组具体如下：
试验组：2ml GAM培养基+2ml绿原酸溶液+1mL PBS缓冲液；
对照组：2ml GAM培养基+3mL PBS缓冲液；
确认好每个玻璃管盖拧紧后，转移至厌氧箱传递箱中，放置之前需用84消毒液消毒处理。放入厌氧箱后，拧松瓶盖，置换O2，需置换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(绿原酸-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。
接着分别将试验组和对照组样品于10000rpm离心3min，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菌群如Anaerostipes和Parasutterella的丰度，即不同菌群在检测到的所有菌种中所占的百分比，丰度测定结果如表1所示。
并对绿原酸干预前后的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绿原酸干预前后的菌群丰度
结果显示，经绿原酸干预后，肠道益生菌丁酸弧菌属Anaerostipes的丰度相比于对照组有极显著增多，而部分肠道微生物如：毛螺旋菌属Parasutterella与对照组则无明显差异。上述结果说明，绿原酸具有促进Anaerostipes增殖的作用，因此可制成Anaerostipes生长促进剂，不仅可用于体外大量培养益生菌Anaerostipes以制成益生菌剂，还可制成药物或保健食品，直接作用于体内的益生菌以改善肠道菌群紊乱并辅助治疗脂肪肝、结肠癌、便秘和/或抑郁症等多种疾病。
3.生长曲线测定
观察经绿原酸处理前后，Anaerostipes的生长曲线变化，测定方法具体如下：
准备100μl无菌枪头，需在高压灭菌锅内进行115℃、20mins的灭菌。移液枪、无菌96孔板事先用酒精消毒后放入生物安全柜进行30分钟的紫外灭菌。再转移至厌氧箱内进行取样测定OD600值。
采用绿原酸对Anaerostipes进行培养作为试验组，以未添加绿原酸的培养液作为对照组，定时取样测定。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***:Tecan i-control,2.0.10.0，设定***数值如下表：
根据***导出的数据如表2所示。
表2绿原酸对Anaerostipes的作用
根据上述数据，以测点时间为X轴，测定OD600值的百分数为Y值，绘制试验组和对照组相应的生长曲线图，测定结果如图2所示，结果显示，试验组和对照组内的Anaerostipes在两小时后开始迅速生长，8-12h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，在绿原酸的培养下，Anaerostipes生长得更加旺盛，说明，绿原酸对Anaerostipes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 (7)
1.绿原酸在制备Anaerostipes生长促进剂中的应用。
2.一种促进Anaerostipes增殖的方法，其特征在于，所述方法包括：将绿原酸按照质量浓度5％加入至GAM培养基中，制成绿原酸-GAM培养基，用所述绿原酸-GAM培养基培养Anaerostipes。
3.绿原酸或包含绿原酸的组合物在制备改善肠道菌群紊乱药物、功能食品和/或保健食品中的应用。
4.根据权利要求3所述的应用，其特征在于，所述绿原酸通过促进Anaerostipes的增殖，达到改善脂肪肝、结肠癌、便秘和/或抑郁症的作用。
5.根据权利要求3所述的应用，其特征在于，所述药物中还包括药学上可接受的辅料。
6.根据权利要求5所述的应用，其特征在于，所述药物的剂型为颗粒剂、片剂、胶囊剂、粉剂、丸剂或溶液剂。
7.据权利要求3所述的应用，其特征在于，所述功能食品或保健食品包括：固体饮料、液体饮料、饼干、糕点食品、罐头、乳制品。
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