CN116458636A - 一种改善肠道微生物群的复合制剂及其制备方法和应用 - Google Patents
一种改善肠道微生物群的复合制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种改善肠道微生物群的复合制剂及其制备方法和应用。该复合制剂的成分包括藜麦、蚕蛹、耳叶牛皮消、桃仁、黄连、首乌藤、荞麦花粉、白果、玉米花粉和葡萄糖酸锌。本发明复合制剂通过采用特定的组分,为肠道益生菌提供食物,促进有益细菌的生长繁殖,同时抑制条件致病菌的生长与活性,维持肠道微生物的稳态平衡。其中,本发明的复合制剂能够显著地提高乳酸乳球菌/条件致病菌的比值,促进乳酸乳球菌增殖,进而改善由乳酸乳球菌等有益菌失调导致的肥胖和胰岛素抵抗,降低代谢疾病发生的风险。
Description
技术领域
本发明涉及医疗保健食品技术领域,具体而言,涉及一种改善肠道微生物群的复合制剂及其制备方法和应用。
背景技术
数以万亿的微生物存在于人体的肠道中,被统称为“肠道微生物群”。这些微生物通过母婴传播的方式从产前开始在肠道定植。基因测序数据显示,尽管在健康个体中发现了多种多样的细菌物种,但肠道宏基因组(即肠道微生物群落中的所有基因)都参与宿主的核心功能,如难以消化的营养物质的消化和降解,以及宿主免疫系统和消化道的发育和刺激。肠道微生物群也产生与宿主的代谢相互作用的信号分子,如短链脂肪酸(SCFA)是通过肠道细菌发酵膳食纤维而产生的,它们与G蛋白偶联受体(GPCR)的相互作用,影响脂肪细胞和外周器官中的胰岛素敏感性,从而调节能量代谢。
在整个生命过程中,肠道生态系统的瞬时变化可导致微生物-宿主共生关系的破坏。例如,长期摄入高脂肪食物会导致肠道菌群失调,使肠道中存在更高水平的乙酸,这种正向的调节机制使食量越来越大,从而促进肥胖,和产生胰岛素抵抗。肥胖发展过程中肠道优势菌群变化会促使肠道微生物环境进一步失调:肠道中有益菌(例:Faecalibacterium粪杆菌属;Lactococcus lactis乳酸乳球菌;Bifidobacteriumbifidum双歧杆菌)比重显著降低,而条件致病菌(例:Escherichia coli大肠埃希氏菌)比重显著增加,进而诱发炎症反应。
由于肠道生态系统在维持宿主生理方面的重要作用,它的改变会引发多种生理障碍,包括低度炎症、代谢紊乱、脂质过度积累和胰岛素敏感性丧失,进而增加了代谢性疾病发生的风险。因此,维持肠道有益菌与条件致病菌的平衡是非常必要的。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种改善肠道微生物群的复合制剂及其制备方法和应用,以改善上述问题。
本发明是这样实现的:
本发明提供了一种改善肠道微生物群的复合制剂,其成分包括藜麦、蚕蛹、耳叶牛皮消、桃仁、黄连、首乌藤、荞麦花粉、白果、玉米花粉和葡萄糖酸锌;
按重量份数计,复合制剂的成分包括:10-32份藜麦、10-32份蚕蛹、10-22份耳叶牛皮消、10-20份桃仁、6-11份黄连、6-11份首乌藤、5-10份荞麦花粉、5-10份白果、2-8份玉米花粉、0.5-5份葡萄糖酸锌。
在一些实施方式中,复合制剂的成分包括:32份藜麦、22份蚕蛹、10份耳叶牛皮消、10份桃仁、6份黄连、6份首乌藤、5份荞麦花粉、5份白果、2份玉米花粉、2份葡萄糖酸锌。
在一些实施方式中,肠道菌群包括有益菌;有益菌包括乳酸乳球菌、多形拟杆菌、单形拟杆菌和酸胺球菌属中的至少一种;
在一些实施方式中,有益菌为乳酸乳球菌。
在一些实施方式中,肠道菌群包括还包括条件致病菌;条件致病菌包括迟缓埃格特菌、大肠埃希氏菌、沙门氏菌属和弗氏志贺氏菌中的至少一种。
本发明还提供了上述复合制剂的制备方法,包括将按配比将各组分混合均匀后即得复合制剂。
本发明还提供了上述复合制剂在制备促进肠道有益菌的食品或药品中的应用。
在一些实施方式中,肠道有益菌包括乳酸乳球菌、多形拟杆菌、单形拟杆菌和酸胺球菌属中的至少一种;
在一些实施方式中,有益菌为乳酸乳球菌。
本发明还提供了上述复合制剂在制备抑制肠道致病菌的食品或药品中的应用。
在一些实施方式中,肠道致病菌包括迟缓埃格特菌、大肠埃希氏菌、沙门氏菌属和弗氏志贺氏菌中的至少一种。
本发明还提供了一种食品,其包括上述改善肠道微生物群的复合制剂。
本发明具有以下有益效果:
本发明复合制剂通过采用特定的组分,为肠道益生菌提供食物,促进有益细菌的生长繁殖,同时抑制条件致病菌的生长与活性,维持肠道微生物的稳态平衡。其中,本发明的复合制剂能够显著地提高乳酸乳球菌/条件致病菌的比值,促进乳酸乳球菌增殖,进而改善由乳酸乳球菌等有益菌失调导致的肥胖和胰岛素抵抗,降低代谢疾病发生的风险。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
肠道菌群的平衡与人类健康密切相关,已有文献报道:肠道中包括乳酸乳球菌在内的有益菌比重显著降低,条件致病菌比重显著增加,会促进肥胖,和产生胰岛素抵抗。鉴于此,本发明特提供一种调节乳酸乳球菌等有益菌与条件致病菌的平衡的复合制剂,该复合制剂的成分包括:藜麦、蚕蛹、耳叶牛皮消、桃仁、黄连、首乌藤、荞麦花粉、白果、玉米花粉和葡萄糖酸锌。
进一步地,按重量份数计,上述复合制剂的成分包括:10-32份藜麦、10-32份蚕蛹、10-22份耳叶牛皮消、10-20份桃仁、6-11份黄连、6-11份首乌藤、5-10份荞麦花粉、5-10份白果、2-8份玉米花粉、0.5-5份葡萄糖酸锌。
更优地,上述复合制剂的成分包括:32份藜麦、22份蚕蛹、10份耳叶牛皮消、10份桃仁、6份黄连、6份首乌藤、5份荞麦花粉、5份白果、2份玉米花粉、2份葡萄糖酸锌。
根据发明人的实验结果来看,本发明复合制剂能够调节的有益菌包括乳酸乳球菌(Lactococcus lactis)、多形拟杆菌(Bacteroides thetaiotaomicron)、单形拟杆菌(Bacteroides uniformis)和酸胺球菌属(Acidaminococcus)中的至少一种。更为优选地,上述有益菌为乳酸乳球菌。
根据发明人的实验结果来看,本发明复合制剂能够抑制的条件致病菌包括迟缓埃格特菌(Eggerthella lenta)、大肠埃希氏菌(Escherichia coli)、沙门氏菌属(Salmonella)和弗氏志贺氏菌(Shigella flexneri)中的至少一种。
在本发明中,上述复合制剂的制备方法包括将按配比将各组分混合均匀后即得复合制剂。
此外,本发明中的复合制剂还可以制备为一种食品,该食品具有改善肠道微生物群的功能,具体地,该食品可以提高有益菌/条件致病菌比值,抑制条件致病菌的生长。
本发明中的复合制剂还可以制备为一种药品,该药品具有改善肠道微生物群的功能,具体地,该药品可以提高有益菌/条件致病菌比值,促进有益菌的生长,抑制条件致病菌的生长。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例与对比例中采用的藜麦、蚕蛹、耳叶牛皮消、桃仁、黄连、首乌藤、荞麦花粉、白果、玉米花粉和葡萄糖酸锌均为市售商品,购于西安奥赛生物科技有限公司
实施例1
本实施例提供一种改善肠道微生物群的复合制剂,其成分如下:
320g藜麦、220g蚕蛹、100g耳叶牛皮消、100g桃仁、60g黄连、60g首乌藤、50g荞麦花粉、50g白果、20g玉米花粉、20g葡萄糖酸锌。
对比例1
本对比例与实施例1的区别在于成分不同,其成分如下:
220g蚕蛹、100g耳叶牛皮消、100g桃仁、60g黄连、60g首乌藤、50g荞麦花粉、50g白果、20g玉米花粉、20g葡萄糖酸锌。
对比例2
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、100g耳叶牛皮消、100g桃仁、60g黄连、60g首乌藤、50g荞麦花粉、50g白果、20g玉米花粉、20g葡萄糖酸锌。
对比例3
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、220g蚕蛹、100g桃仁、60g黄连、60g首乌藤、50g荞麦花粉、50g白果、20g玉米花粉、20g葡萄糖酸锌。
对比例4
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、220g蚕蛹、100g耳叶牛皮消、60g黄连、60g首乌藤、50g荞麦花粉、50g白果、20g玉米花粉、20g葡萄糖酸锌。
对比例5
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、220g蚕蛹、100g耳叶牛皮消、100g桃仁、60g首乌藤、50g荞麦花粉、50g白果、20g玉米花粉、20g葡萄糖酸锌。
对比例6
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、220g蚕蛹、100g耳叶牛皮消、100g桃仁、60g黄连、50g荞麦花粉、50g白果、20g玉米花粉、20g葡萄糖酸锌。
对比例7
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、220g蚕蛹、100g耳叶牛皮消、100g桃仁、60g黄连、50g荞麦花粉、60g首乌藤、50g白果、20g玉米花粉、20g葡萄糖酸锌。
对比例8
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、220g蚕蛹、100g耳叶牛皮消、100g桃仁、60g黄连、50g荞麦花粉、60g首乌藤、20g玉米花粉、20g葡萄糖酸锌。
对比例9
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、220g蚕蛹、100g耳叶牛皮消、100g桃仁、60g黄连、60g首乌藤、50g荞麦花粉、50g白果、20g葡萄糖酸锌。
对比例10
本对比例与实施例1的区别在于成分不同,其成分如下:
320g藜麦、220g蚕蛹、100g耳叶牛皮消、100g桃仁、60g黄连、60g首乌藤、50g荞麦花粉、50g白果、20g玉米花粉。
实验例1
本实验例是针对实施例和对比例1-10的复合制剂进行的体外实验,具体步骤如下:
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,包括:KH2PO4 0.24g,Na2HPO4·12H2O 2.90g,NaCl 8.00g,KCl 0.20g。用校准后的PH计测量上述溶液的PH,用0.1M的HCl或NaOH调整PH=7.4±0.05。然后放入高压灭菌锅,121℃,15分钟。待降至室温后,将溶液需存于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10%。
用百分之一的天平称量出10g粪便样品,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。将沉淀称量之后,以终浓度为5%用PBS溶液定容,得到肠道微生物样品。
2、基础培养基配制
配制用于培养肠道微生物的基础培养基: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,定容至1000ml。121℃,15分钟。灭菌完后,立即把瓶盖拧紧,冷却至室温。
3、混合液的制备
将实施例1以及对比例1-10的组合物用PBS缓冲液溶解至质量百分浓度为5%的混合液。
4、分组和干预
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。接着用1ml移液器取实施例1与对比例1-10复合制剂的混合液,加入上述装有GAM培养基的玻璃管中,每管2ml,作为实验组。同时还设置有对照组,分组具体如下:
实验组:2ml GAM培养基+2ml混合液+1ml PBS缓冲液;
对照组:2ml GAM培养基+2ml混合液+1ml 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,置换氧气,需置换12个小时。
将上述处理后得到的肠道微生物样品分别接种至试验组(复合制剂-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,分别测定试验组和对照组中的肠道菌群丰度,其中部分肠道菌群的丰度测定结果如表1所示。并对部分肠道菌群干预前后的丰度变化进行显著性分析。分析结果如表1所示:
表1部分肠道菌群干预前后的丰度变化分析
注:*P<0.05,##P<0.01。
从表1的分析结果来看,乳酸乳球菌(Roseburia)和拟杆菌属(Lactococcuslactis)丰度显著增加,酸胺球菌属(Acidaminococcus)也有一定丰度的增加,迟缓埃格特菌(Eggerthella lenta)和大肠埃希氏菌(Escherichia coli)显著性降低,因此可以证明经过本发明复合制剂干预的乳酸乳球菌(Roseburia)丰度显著富集,条件致病菌数量减少,肠道的调节能力得到了改善。
实验例2
本实验例验证复合制剂对条件致病菌-大肠埃希氏菌的抑制作用,具体情况如下:
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,包括:KH2PO4 0.24g,Na2HPO4·12H2O 2.90g,NaCl 8.00g,KCl 0.20g。用校准后的PH计测量上述溶液的PH,用0.1M的HCl或NaOH调整PH=7.4±0.05。然后放入高压灭菌锅,121℃,15分钟。待降至室温后,将溶液需存于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10%。
用百分之一的天平称量出10g粪便样品,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。将沉淀称量之后,以终浓度为5%用PBS溶液定容,得到肠道微生物样品。
2、基础培养基配制
配制用于培养肠道微生物的基础培养基: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,定容至1000ml。121℃,15分钟。灭菌完后,立即把瓶盖拧紧,冷却至室温。
3、分组和干预
(1)大肠埃希氏菌的培养
大肠埃希氏菌是一种兼性厌氧革兰阴性杆菌,培养2d内放可形成肉眼可见菌落,从公司购买的大肠埃希氏菌,将其在接种于LST培养基上,培养2d后,取菌落进行后续鉴定。
(2)大肠埃希氏菌的鉴定
将原始菌分纯扩大培养后提取原始菌基因组DNA
正向引物:(5'-TGTGATATCTACCCGCTTCGC-3')
反向引物:(5'-AGAACGGTTTGTGGTTAATCAGGA-3')
进行16SrRNA基因PCR扩增实验。反应程序如下:96℃3min、96℃30s、58℃30s、72℃1min,此条件下35个循环、72℃10min,PCR反应结束后,1%的琼脂糖鉴定并使用凝胶回收试剂盒回收所需PCR产物片段。测序后与GeneBank数据库中已知菌的16S rRNA基因进行比对。
(3)操作步骤
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。接着用1ml移液器取实施例1复合制剂的混合液,加入上述装有GAM培养基的玻璃管中,每管2ml,作为实验组,还设有条件对照组和空白组,做三组平行,分组具体如下:
实验组:3ml GAM培养基+1ml复合制剂混合液+1ml大肠埃希氏菌培养液+1ml PBS缓冲液;
条件对照组:3ml GAM培养基+1ml大肠埃希氏菌培养液+2ml PBS缓冲液;
空白组:3ml 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,置换氧气,需置换12个小时。
将上述处理后得到的肠道微生物样品分别接种至实验组、条件对照组以及空白对照组中,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丰度,即不同肠道微生物在检测到的所有菌种中所占的百分比,其中部分肠道菌群的丰度测定结果如表2所示。并对部分肠道菌群干预前后的丰度变化进行显著性分析。
表2大肠埃希氏菌干预前后的丰度变化分析
注:*P<0.05,##P<0.01。
从表2的分析结果表明大肠埃希氏菌(Escherichia coli)经过复合制剂的干预后,三组平行实验中大肠埃希氏菌丰度均是有显著性的降低的,证明本发明的复合制剂对大肠埃希氏菌是有抑制作用的。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种改善肠道微生物群的复合制剂,其特征在于,其成分包括藜麦、蚕蛹、耳叶牛皮消、桃仁、黄连、首乌藤、荞麦花粉、白果、玉米花粉和葡萄糖酸锌;
按重量份数计,所述复合制剂的成分包括:10-32份藜麦、10-32份蚕蛹、10-22份耳叶牛皮消、10-20份桃仁、6-11份黄连、6-11份首乌藤、5-10份荞麦花粉、5-10份白果、2-8份玉米花粉、0.5-5份葡萄糖酸锌。
2.根据权利要求1所述的复合制剂,其特征在于,按重量份数计,所述复合制剂的成分包括:32份藜麦、22份蚕蛹、10份耳叶牛皮消、10份桃仁、6份黄连、6份首乌藤、5份荞麦花粉、5份白果、2份玉米花粉、2份葡萄糖酸锌。
3.根据权利要求1所述的复合制剂,其特征在于,所述肠道菌群包括有益菌;
所述有益菌包括乳酸乳球菌、多形拟杆菌、单形拟杆菌和酸胺球菌属中的至少一种;
优选地,所述有益菌为乳酸乳球菌。
4.根据权利要求1所述的复合制剂,其特征在于,所述肠道菌群包括还包括条件致病菌;
所述条件致病菌包括迟缓埃格特菌、大肠埃希氏菌、沙门氏菌属和弗氏志贺氏菌中的至少一种。
5.如权利要求1-4任一项所述的复合制剂的制备方法,其特征在于,将按配比将各成分加入缓冲溶液中,溶解、混合均匀后即得所述复合制剂。
6.如权利要求1-4任一项所述的复合制剂在制备促进肠道有益菌的食品或药品中的应用。
7.根据权利要求6所述的应用,其特征在于,所述肠道有益菌包括乳酸乳球菌、多形拟杆菌、单形拟杆菌和酸胺球菌属中的至少一种;
优选地,所述有益菌为乳酸乳球菌。
8.如权利要求1-4任一项所述的复合制剂在制备抑制肠道致病菌的食品或药品中的应用。
9.根据权利要求8所述的应用,其特征在于,所述肠道致病菌包括迟缓埃格特菌、大肠埃希氏菌、沙门氏菌属和弗氏志贺氏菌中的至少一种。
10.一种食品,其特征在于,其包括权利要求1-4任一项所述的改善肠道微生物群的复合制剂。
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