CN116349846A - 一种调节肠道菌群的组合物及其制备方法和应用 - Google Patents
一种调节肠道菌群的组合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种调节肠道菌群的组合物及其制备方法和应用。该组合物的成分包括龙眼肉,牡蛎,马鹿胎,菜花粉,制大黄,鳖甲,桑白皮,鲜芦根,赤小豆,菊苣。本发明提供的特定组分的组合物,试验证明其具有调节肠道菌群的功能,通过提高益生菌/致病菌的比值进而达到改善肠道健康的目的。并且本发明的原料易获取且制备方式简单,安全性极高无毒副作用,可单独使用或与其他可食用物质复配,均能有效改善肠道环境,提高有益菌的数量,降低致病菌的数量。
Description
技术领域
本发明涉及医疗保健食品技术领域,具体而言,涉及一种调节肠道菌群的组合物及其制备方法和应用。
背景技术
人体肠道中存在的数量庞大的微生物,这群微生物依靠人体的肠道生活,同时帮助人体完成多种生理生化功能。肠道微生物不仅在膳食和宿主之间起到了重要的桥梁作用,其本身以及代谢产物也能调节人体健康,因此肠道微生物与人体健康密切相关。肠道微生物通过与宿主之间的动态性生理作用,达到微生态平衡,有效防止肠道内细菌及内毒素易位。当正常的微生物群落受到宿主及外环境影响时,原有的平衡遭到破坏,肠道菌群失调,肠道正常菌群的种类、数量和比例发生异常变化,转变为病理性组合状态,进而导致宿主致病。
当机体免疫功能降低或者菌群状态发生改变时,肠道优势菌群变化会导致肠道微生物环境的失调。条件致病菌如大肠埃希氏菌能破坏肠道屏障,是导致全世界菌血症最普遍的病原体,并且手术前该菌超标会引起菌血症。人体肠道中许多有益菌能改善这些问题,例如双歧杆菌能产生短链脂肪酸(乳酸)、多种维生素、氨基酸,提高机体对钙离子的吸收,增强人体免疫机制,对人体健康具有生物屏障、营养作用、抗肿瘤作用、增强免疫作用、改善胃肠道功能抗衰老等生理功能。发酵乳杆菌能促进脂肪组织氧化磷酸化以防止饮食引起的肥胖,调节免疫反应和改变肠道微生物群来改善葡聚糖硫酸钠诱导的结肠炎。德氏乳杆菌可以添加到口服营养品,适用于慢性肠道疾病患者的治疗。
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调节肠道菌群的组合物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:
本发明提供了一种调节肠道菌群的组合物,其成分包括龙眼肉,牡蛎,马鹿胎,菜花粉,制大黄,鳖甲,桑白皮,鲜芦根,赤小豆,菊苣;
按重量份数计,组合物的成分包括25-45份龙眼肉、20-38份牡蛎、15-25份马鹿胎、10-20份菜花粉、10-15份制大黄、10-15份鳖甲、1-5份桑白皮、1-5份鲜芦根、0.1-5份赤小豆和0.1-1份菊苣。
在一些实施方式中,按重量份数计,组合物的成分包括25-35份龙眼肉、20-30份牡蛎、10-25份马鹿胎、10-20份菜花粉、10-15份制大黄、10-15份鳖甲、1-5份桑白皮、1-5份鲜芦根、0.1-5份赤小豆和0.1-1份菊苣。
在一些实施方式中,肠道菌群包括有益菌,有益菌包括双歧杆菌、发酵乳杆菌、德氏乳杆菌和罗伊氏乳杆菌中的至少一种。
在一些实施方式中,肠道菌群还包括条件致病菌,条件致病菌包括大肠埃希氏菌。
本发明提供了上述组合物的制备方法,按配比将各组分混合均匀后即得组合物。
本发明提供了上述组合物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,有益菌包括双歧杆菌、发酵乳杆菌、德氏乳杆菌和罗伊氏乳杆菌中的至少一种。
本发明提供了上述组合物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,条件致病菌包括大肠埃希氏菌。
本发明还提供了一种食品,其包括上述调节肠道菌群的组合物。
本发明具有以下有益效果:
本发明提供了特定组分的组合物,试验证明本其具有调节肠道菌群的功能,通过提高益生菌/致病菌的比值进而达到改善肠道健康的目的。并且本发明的原料易获取且制备方式简单,安全性极高无毒副作用,可单独使用或与其他可食用物质复配,均能有效改善肠道环境,提高有益菌的数量,降低致病菌的数量。
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。
下面对本发明实施例的调节肠道菌群的组合物及其制备方法与应用进行具体说明。
目前市场上常见的肠道微生物调节剂主要包括益生菌和益生元。补充益生菌虽然有益肠道健康,但外来的益生菌难以在肠道内长期定植及形成分布优势,因此效果不能持续。为了维护肠道微生态平衡,提高益生菌如双歧杆菌、发酵乳杆菌、德氏乳杆菌和罗伊氏乳杆菌等的丰度,进而通过肠道菌群的作用增强人体免疫力,改善胃肠道功能抗衰老,预防结肠炎以及其他肠道疾病,本发明的发明人经过大量的创造性劳动获得了一种能够肠道菌群的组合物,其成分如下:
按重量份数计,上述组合物的成分包括25-45份龙眼肉、20-38份牡蛎、15-25份马鹿胎、10-20份菜花粉、10-15份制大黄、10-15份鳖甲、1-5份桑白皮、1-5份鲜芦根、0.1-5份赤小豆和0.1-1份菊苣。
为了获得更优的调节效果,发明人在上述组合物的基础上进行了进一步地实验,优化后的能够肠道菌群的组合物的成分如下:
按重量份数计,组合物的成分包括25-35份龙眼肉、20-30份牡蛎、10-25份马鹿胎、10-20份菜花粉、10-15份制大黄、10-15份鳖甲、1-5份桑白皮、1-5份鲜芦根、0.1-5份赤小豆和0.1-1份菊苣。
肠道微生物丰度检测发现,在经过上述组合物干预后,肠道菌群中的有益菌丰度得到提高的包括双歧杆菌、发酵乳杆菌、德氏乳杆菌和罗伊氏乳杆菌中的至少一种。而肠道菌群中的条件致病菌丰度降低的包括大肠埃希氏菌。
本发明中调节肠道菌群的组合物的制备方法的步骤包括:按配比将各组分混合均匀后即得组合物。
其中,龙眼肉、牡蛎、马鹿胎、菜花粉、制大黄、鳖甲、桑白皮、鲜芦根、赤小豆和菊苣均购于西安奥赛生物科技有限公司。
本发明提供了上述组合物在制备促进肠道有益菌的食品或药品中的应用。此处的有益菌包括双歧杆菌、发酵乳杆菌、德氏乳杆菌和罗伊氏乳杆菌中的至少一种。
肠道菌群失调的关键因素之一在于肠道中有益菌的比值减少,本发明提供的组合物具有显著提高双歧杆菌、发酵乳杆菌、德氏乳杆菌和罗伊氏乳杆菌等有益菌的丰度。因此,本发明的组合物可以用于制备提高有益菌丰度,减少致病菌丰度的食品或药品,具体地,该组合物可以用于制备增强人体免疫力、改善胃肠道功能抗衰老、预防结肠炎以及其他肠道疾病的食品或药品。
本发明提供了上述组合物在制备抑制肠道致病菌的食品或药品中的应用。此处的条件致病菌包括大肠埃希氏菌。
肠道菌群失调的另一关键因素在于肠道中条件致病菌的比值增加,本发明提供的组合物具有显著降低大肠埃希氏菌等致病菌的丰度。因此,本发明的组合物可以用于制备降低条件致病菌的丰度的食品或药品,具体地,该组合物可以用于制备预防肠道疾病的食品或药品。
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1
本实施例提供一种调节肠道菌群的组合物,其成分如下:
龙眼肉300g,牡蛎270g,马鹿胎200g,菜花粉80g,制大黄60g,鳖甲20g,桑白皮20g,鲜芦根20g,赤小豆20g,菊苣10g。
实施例2
本实施例提供一种调节肠道菌群的组合物,其成分如下:
龙眼肉250g,牡蛎240g,马鹿胎180g,菜花粉100g,制大黄80g,鳖甲50g,桑白皮30g,鲜芦根30g,赤小豆30g,菊苣10g。
实施例3
本实施例提供一种调节肠道菌群的组合物,其成分如下:
龙眼肉330g,牡蛎200,马鹿胎120g,菜花粉120g,制大黄150g,鳖甲80g,桑白皮10g,鲜芦根10g,赤小豆5g,菊苣5g。
实施例4
本实施例提供一种调节肠道菌群的组合物,其成分如下:
龙眼肉250,牡蛎200,马鹿胎150g,菜花粉150g,制大黄100g,鳖甲50g,桑白皮50g,鲜芦根25g,赤小豆20g,菊苣5g。
对比例1
本对比例提供一种调节肠道菌群的组合物,其成分如下:
龙眼肉200,牡蛎150,马鹿胎120g,菜花粉120g,制大黄120g,鳖甲120g,桑白皮60g,鲜芦根50g,赤小豆25g,菊苣5g。
对比例2
本对比例提供一种调节肠道菌群的组合物,其成分如下:
龙眼肉400g,牡蛎220g,马鹿胎180g,菜花粉70g,制大黄50g,鳖甲20g,桑白皮20g,鲜芦根20g,赤小豆15g,菊苣5g。
对比例3
本对比例提供一种调节肠道菌群的组合物,其成分如下:
桑葚300g,牡蛎270g,马鹿胎200g,菜花粉80g,制大黄60g,鳖甲20g,桑白皮20g,鲜芦根20g,赤小豆20g,菊苣10g。
实验例1
本实验例是针对实施例1-4和对比例1-3的组合物进行的体外实验,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1-4以及对比例1-3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1-4和对比例1-3组合物的混合液,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+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试验组(组合物-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,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
双歧杆菌属 | 发酵乳杆菌 | 德氏乳杆菌 | 罗伊氏乳杆菌 | 大肠埃希氏菌 | |
对照组 | 1.3±0.8 | 0.4±0.3 | 0.25±0.1 | 1.2±0.05 | 1.2±0.05 |
实施例1 | 7.2±0.3* | 16.7±0.3 | 19.7±0.4## | 5.5±0.3* | 0.02±0.05## |
实施例2 | 6.9±0.4## | 13.2±0.8## | 20.4±0.3* | 5.0±0.2 | 0.09±0.06* |
实施例3 | 7.3±1.1 | 15.1±0.5## | 17.7±0.6 | 4.9±0.3 | 0.1±0.1* |
实施例4 | 6.0±0.4## | 14.0±0.9 | 18.5±0.2* | 5.3±0.2 | 0.05±0.06* |
对比例1 | 5.7±0.2* | 10.5±1.2* | 10.9±0.7 | 2.5±0.4 | 0.4±0.7 |
对比例2 | 4.5±0.7 | 9.7±1.0 | 11.5±0.1 | 3.1±0.5 | 0.3±0.0 |
对比例3 | 4.0±0.1* | 11.1±0.9* | 12.7±0.3 | 3.2±0.1* | 0.3±0.1 |
注:*P<0.05,##P<0.01。
从表1的结果来看,利用本发明的组合物干预肠道菌群,会对部分肠道菌群的丰度产生影响,其中双歧杆菌属(Bifidobacterium)、发酵乳杆菌(Limosilactobacillusfermentum)、德氏乳杆菌(Lactobacillus delbrueckii)和罗伊氏乳杆菌(Lactobacillusreuteri)均有一定程度增加,大肠埃希氏菌(Escherichia coli)显著性降低。而对比例1-2的结果表明,超出本发明的保护范围的其他方案并没有这样的效果;将实施例1与对比例3对比,对比例3组合物中,双歧杆菌属(Bifidobacterium)、发酵乳杆菌(Limosilactobacillus fermentum)丰度有所增加,但不如实施例1中的效果显著;大肠埃希氏菌(Escherichia coli)也有所降低,但不如实施例1中的效果显著;说明本发明组合物的成分龙眼肉在组合物中具有不可替代性。
实验例2
本实验例验证组合物对条件致病菌-大肠埃希氏菌的抑制作用,具体情况如下:
1、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
将购买的菌种接于牛肉膏蛋白胨培养基平板中活化,37℃恒温培养48到72小时,因为接种时和具体培养条件的不同,其生长一般都在2天外才能长出明显的菌落。培养基如下:牛肉膏3g,蛋白胨10g,NaCl 5g,琼脂15~20g,水1000mL,PH7.4~7.6。
(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F(5'-TGT TCA GTGGCA AGA GTT-3')和反向引物R(5'-TAA TCG ATA TAC CCG CTC-3')进行16S rRNA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。试剂盒、引物及测序服务均由爱康健公司完成。测序后与GeneBank数据库中大肠埃希氏菌(Escherichia coli)的16S rRNA基因进行比对。
(1)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合物的混合液,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组合物混合液+1ml大肠埃希氏菌1ml PBS缓冲液;
条件对照组:3ml GAM培养基+1ml大肠埃希氏菌+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。分析结果如表2所示:
表2大肠埃希氏菌干预前后的丰度变化分析
对比空白组*P<0.01,对比条件对照组#P<0.01。
从表2的分析结果表明大肠埃希氏菌经过组合物的干预后,其丰度有显著性的降低的,证明本发明的组合物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)
1.一种调节肠道菌群的组合物,其特征在于,其成分包括龙眼肉,牡蛎,马鹿胎,菜花粉,制大黄,鳖甲,桑白皮,鲜芦根,赤小豆,菊苣;
按重量份数计,所述组合物的成分包括25-45份龙眼肉、20-38份牡蛎、10-25份马鹿胎、10-20份菜花粉、10-15份制大黄、10-15份鳖甲、1-5份桑白皮、1-5份鲜芦根、0.1-5份赤小豆和0.1-1份菊苣。
2.根据权利要求1所述的组合物,其特征在于,按重量份数计,所述组合物的成分包括25-35份龙眼肉、20-30份牡蛎、10-25份马鹿胎、10-20份菜花粉、10-15份制大黄、10-15份鳖甲、1-5份桑白皮、1-5份鲜芦根、0.1-5份赤小豆和0.1-1份菊苣。
3.根据权利要求1所述的组合物,其特征在于,所述肠道菌群包括有益菌,所述有益菌包括双歧杆菌、发酵乳杆菌、德氏乳杆菌和罗伊氏乳杆菌中的至少一种。
4.根据权利要求3所述的组合物,其特征在于,所述肠道菌群还包括条件致病菌,所述条件致病菌包括大肠埃希氏菌。
5.如权利要求1-4任一项所述的组合物的制备方法,其特征在于,按配比将各组分混合均匀后即得所述组合物。
6.如权利要求1-4任一项所述的组合物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,所述有益菌包括双歧杆菌、发酵乳杆菌、德氏乳杆菌和罗伊氏乳杆菌中的至少一种。
8.如权利要求1-4任一项所述的组合物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,所述条件致病菌包括大肠埃希氏菌。
10.一种食品,其特征在于,其包括权利要求1-4任一项所述的调节肠道菌群的组合物。
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