CN113908166A - N-乙酰神经氨酸在制备用于促进Roseburia增殖的促进剂中的用途 - Google Patents
N-乙酰神经氨酸在制备用于促进Roseburia增殖的促进剂中的用途 Download PDFInfo
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Abstract
本发明公开了N‑乙酰神经氨酸在制备用于促进Roseburia增殖的促进剂中的用途,本发明通过N‑乙酰神经氨酸的干预实验,发现N‑乙酰神经氨酸具有调节肠道菌群,具体为促进罗氏菌属Roseburia增殖,增加罗氏菌属Roseburia丰度的作用,因此可作为增殖促进剂,在体外进行罗氏菌属Roseburia的大规模发酵培养以制成相应的益生菌剂,同时也可作为益生元药物、功能食品或保健食品作用于体内,从而有效预防和/或治疗肠道菌群紊乱,并辅助治疗与罗氏菌属Roseburia相关的包括克罗恩、痛风、肝硬化、便秘、抑郁症和2型糖尿病在内的多种疾病,即本发明首次发现了N‑乙酰神经氨酸的新用途,在医药保健和工业化生产中均具有极高的应用价值。
Description
技术领域
本发明属于益生菌增殖促进剂技术领域,具体涉及N-乙酰神经氨酸在制备用于促进Roseburia增殖的促进剂中的用途。
背景技术
N-乙酰神经氨酸,系唾液酸(N-酰基神经氨酸)在自然界分布最广的,主要是动物细胞膜或分泌液的糖蛋白、糖脂质或细菌荚膜物质等的组成糖。是一种能使唾液产生光滑感觉的负电荷离子。N-乙酰神经氨酸在糖蛋白或糖脂质中,由于2位的酮基形成糖苷键而使它位于糖侧链的非还原性末端,其阴电荷或特有的化学结构使其具有各种生理特异性。研究发现N-乙酰神经氨酸具有抗细菌、病毒,抗炎、提高免疫力等多种功能,是母乳低聚糖的组成之一,研究报道多种不同的单糖经排列组合形成母乳低聚糖后,可阻止病原体对肠粘膜的黏附,还具有调节肠道菌群,促进免疫系统成熟的作用。然而N-乙酰神经氨酸对肠道益生菌Roseburia的调节促进作用目前未见相关报道。
肠道菌群,作为寄居在人体肠道内微生物群落的总称,是近年来微生物学、医学、基因学等领域最引人关注的研究焦点之一。肠道菌群与人体健康休戚相关,其中最主要的就是用于消化吸收食物,肠道为菌群提供天然的厌氧环境作为住所,菌群则擅长分解复杂纤维和多糖,生成可供人体吸收的葡萄糖、维生素、脂肪、微量元素等。其次,大量的菌群黏附在肠壁上,为肠道穿上了一层天然的铠甲,避免肠壁与有害物质直接接触,保护肠道健康,同时共生菌群还可用于帮助消灭人体内的致病菌。此外,肠道菌群通过与体内多种物质共同作用,调节人体的生理和心理状态。很多科学家认为肠道菌群可以看作是人体的另一个器官,其与多种高发的代谢性疾病密切相关。
已有研究报道,一种重要的肠道益生菌罗氏菌Roseburia与多种疾病有密切关联,如文献:El Mouzan,et al.Microbiota profile in new-onset pediatric Crohn’sdisease:data from a non-Western population.Gut Pathogens,2018.中公开了包括Roseburia在内的多种益生菌丰度在克罗恩患者体内显著降低了;文献Guo,Zhuang etal.Intestinal Microbiota Distinguish Gout Patients from HealthyHumans.Scientific Reports,2016中报道了在痛风患者体内,其肠道微生物Roseburia等被几乎耗尽,并且肠道菌群变化特征与2型糖尿病患者和肝硬化类似。文献Liu,Tong etal.Microbial and metabolomic profiles in correlation with depression andanxiety co-morbidities in diarrhoea-predominant IBS patients.BMCMicrobiology,2020.20(1).中报道了抑郁症与肠道菌群变化相关,其中Roseburia、Faecalibacterium等肠道微生物与抑郁程度呈负相关。还有研究报道罗氏菌属Roseburia与便秘相关。
发明内容
本发明的目的在于提供了N-乙酰神经氨酸在制备用于促进Roseburia增殖的促进剂中的用途,通过模拟肠道环境并进行N-乙酰神经氨酸干预实验,首次验证了N-乙酰神经氨酸具有调节罗氏菌属Roseburia并增加其丰度的功效,不仅可作为Roseburia的增殖促进剂,用于体外Roseburia的大规模发酵培养,还可作为药物、功能食品或保健食品作用于体内,以调节肠道菌群,并辅助治疗与Roseburia相关的疾病,如:克罗恩、痛风、肝硬化、便秘、抑郁症和/或2型糖尿病等。
为实现上述目的,本发明采用的技术方案是:
本发明提供了N-乙酰神经氨酸在制备用于促进Roseburia增殖的促进剂中的用途。
进一步地,所述促进剂中还包括GAM培养基。
本发明还提供了N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备增加Roseburia丰度的药物、功能食品或保健食品中的用途。
进一步地,所述N-乙酰神经氨酸通过增加Roseburia的丰度,达到改善克罗恩、痛风、肝硬化、便秘、抑郁症和/或2型糖尿病的作用。因此可将其制成相应的辅助治疗药物。
本发明还提供了N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备辅助治疗克罗恩的药物、功能食品或保健食品中的用途。
本发明还提供了N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备辅助治疗痛风的药物、功能食品或保健食品中的用途。
本发明还提供了N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备辅助治疗抑郁症的药物、功能食品或保健食品中的用途。
本发明还提供了N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备辅助治疗2型糖尿病的药物、功能食品或保健食品中的用途。
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,所述药物为片剂、胶囊剂、粉剂、丸剂、颗粒剂或溶液剂。
本发明还提供了一种促进Roseburia增殖的方法,所述方法包括:配制GAM培养基,向其中加入用PBS缓冲液溶解的N-乙酰神经氨酸,使N-乙酰神经氨酸的终浓度为2%wt,得到N-乙酰神经氨酸-GAM培养基用于培养Roseburia。
与现有技术相比,本发明的有益效果是:本发明通过在体外模拟肠道环境,并收集人体的肠道菌群进行N-乙酰神经氨酸的干预实验,发现N-乙酰神经氨酸具有调节肠道菌群,具体为促进罗氏菌属Roseburia增殖,增加罗氏菌属Roseburia丰度的作用,因此可作为增殖促进剂,在体外进行罗氏菌属Roseburia的大规模发酵培养以制成相应的益生菌剂,同时也可作为益生元药物、功能食品或保健食品作用于体内,从而有效预防和/或治疗肠道菌群紊乱,并辅助治疗与罗氏菌属Roseburia相关的包括克罗恩、痛风、肝硬化、便秘、抑郁症和2型糖尿病在内的多种疾病,即本发明首次发现了N-乙酰神经氨酸的新用途,在医药保健和工业化生产中均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罗氏菌属Roseburia的丰度测定和显著性分析结果;
图2为本发明实施例1中经N-乙酰神经氨酸干预后与对照组的罗氏菌属Roseburia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
由于来源于人体的肠道菌群无法完全在小鼠体内进行定殖,因此取人体粪便经处理后作为肠道菌群样品,在体外模拟人体内的肠道环境,观察N-乙酰神经氨酸干预处理后对肠道菌群的作用,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。
用量筒量取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0.1M的HCl或NaOH调整PH=7.4±0.05。
将上述完全溶解并调节pH后的溶液用玻璃棒引流至1000ml容量瓶中,用少量超纯水冲洗烧杯后倒入容量瓶,重复3次,并用超纯水定容至刻度线,塞上盖子后上下颠倒10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,将黄色玻璃瓶盖子拧松后,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,将溶液存于4℃冰箱中,可保存6个月。
(2)粪便样品处理
以10g为例,最终浓度为5%。用百分之一的天平称量出粪便样品10g,用自动移液枪取适量上述PBS缓冲液加至离心管中,在振荡器上充分混匀。然后将完全混匀的样品平均分装至新50ml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,把稀释好的样品依次通过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,以终浓度为5%用上述PBS溶液定容,得到肠道菌群样品。
2、N-乙酰神经氨酸干预实验
(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。用电子分析天平称取60g改良GAM肉汤药品,倒入1000ml烧杯中。用量筒量取800ml超纯水倒入烧杯中,放入搅拌转子,并放到磁力加热搅拌器上搅拌至完全溶解。将上述完全溶解的溶液用玻璃棒引流至1000ml容量瓶,并用少量超纯水冲洗烧杯后倒入容量瓶,接着用超纯水定容至刻度线,塞上盖子后上下颠倒,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,每1000ml的瓶子内放500ml培养基,防止高压灭菌时喷出。将装有培养基的黄色盖子的玻璃瓶,瓶盖拧松,放入脉冲高压灭菌锅,使用液体程序,121℃,15分钟。灭菌完后,立即把瓶盖拧紧,冷却至室温。
(2)N-乙酰神经氨酸-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N-乙酰神经氨酸溶液(购于武汉中科光谷绿色生物技术有限公司,纯度98%,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,N-乙酰神经氨酸终浓度是2%。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 N-乙酰神经氨酸溶液+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(N-乙酰神经氨酸-GAM培养基)以及对照组(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,分别测定试验组和对照组中的肠道菌群的丰度,包括:Roseburia和Blautia,即各菌种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。
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;*表示P<0.05,即丰度变化有显著差异。
表1试验组和对照组的菌群丰度变化
结果显示,经N-乙酰神经氨酸干预后,肠道菌群中罗氏菌属Roseburia丰度相比于对照组有极显著增多,而部分肠道微生物如布劳特氏菌属Blautia则有减少趋势,但未与对照组达到显著性差异。上述结果说明,N-乙酰神经氨酸具有促进罗氏菌属Roseburia增殖的作用,通过极显著上调罗氏菌属Roseburia,可改善肠道菌群紊乱,并辅助治疗克罗恩、痛风、肝硬化、抑郁症和2型糖尿病等相关疾病;并且还可用于体外罗氏菌属Roseburia的大规模增殖培养。
2、生长曲线绘制
为观察N-乙酰神经氨酸对于罗氏菌属Roseburia的生长曲线的影响,测量并绘制相应的生长曲线图,测定方法为:
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。
添加有N-乙酰神经氨酸的GAM培养液为试验组,未添加N-乙酰神经氨酸的GAM培养液作为对照组,分别将试验组和对照组中接种罗氏菌属Roseburia,并每隔一段时间转移至厌氧箱内取样,测定菌液的OD600值。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,待取样完成后,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:
系统导出的OD600值数据如表2所示。
表2 N-乙酰神经氨酸对Roseburia生长曲线的影响
根据表2的数据绘制相应的生长曲线,结果如图2所示。结果显示,试验组和对照组内的Roseburia在4-6h开始进入对数生长期,8-28h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N-乙酰神经氨酸的培养下,Roseburia生长得更加旺盛,说明,N-乙酰神经氨酸对Roseburia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 (10)
1.N-乙酰神经氨酸在制备用于促进Roseburia增殖的促进剂中的用途。
2.根据权利要求1所述的用途,其特征在于,所述促进剂中还包括GAM培养基。
3.N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备增加Roseburia丰度的药物、功能食品或保健食品中的用途。
4.根据权利要求3所述的用途,其特征在于,所述N-乙酰神经氨酸通过增加Roseburia的丰度,达到改善克罗恩、痛风、肝硬化、便秘、抑郁症和/或2型糖尿病的作用。
5.N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备辅助治疗克罗恩的药物、功能食品或保健食品中的用途。
6.N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备辅助治疗痛风的药物、功能食品或保健食品中的用途。
7.N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备辅助治疗抑郁症的药物、功能食品或保健食品中的用途。
8.N-乙酰神经氨酸或包含N-乙酰神经氨酸的组合物在制备辅助治疗2型糖尿病的药物、功能食品或保健食品中的用途。
9.根据权利要求3-8任一项所述的用途,其特征在于,所述药物中还包括药学上可接受的辅料。
10.一种促进Roseburia增殖的方法,其特征在于,所述方法包括:配制GAM培养基,向其中加入用PBS缓冲液溶解的N-乙酰神经氨酸,使N-乙酰神经氨酸的终浓度为2%wt,得到N-乙酰神经氨酸-GAM培养基用于培养Roseburia。
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