CN117441888A - 一种提高肠道中粪杆菌属与条件致病菌比值的组合物及其应用 - Google Patents
一种提高肠道中粪杆菌属与条件致病菌比值的组合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种提高肠道中粪杆菌属与条件致病菌比值的组合物及其应用。该组合物以乳香酸为主料，并添加了表儿茶素、玛咖、瓜拉纳提取物、垂柳、金银花提取物和川陈皮素，其能够提高肠道中有益菌的比重，并有效抑制条件致病菌的生长及繁殖，充分调节了肠道益生菌菌群的组成，减少机体低度炎症、代谢紊乱、脂质过度积累和胰岛素敏感性丧失的风险。
Description
技术领域
本发明涉及微生物技术领域，具体而言，涉及一种提高肠道中粪杆菌属与条件致病菌比值的组合物。
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；Roseburia罗斯氏菌属；Bifidobacteriumbifidum双歧杆菌)比重显著降低，而条件致病菌(例：Eggerthella lenta迟缓埃格特菌)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。因此提供一种调节肠道生态系统的方法至关重要。
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提高肠道中粪杆菌属与条件致病菌比值的组合物，该组合物能够提高肠道中有益菌的比重，降低条件致病菌的比重，维持肠道菌群平衡，进而降低代谢性疾病发生的风险。
本发明是这样实现的：
第一方面，本发明提供了一种提高肠道中粪杆菌属与条件致病菌比值的组合物，其原料按质量百分数计包括：乳香酸21％-64％，表儿茶素12％-22％，玛咖11％-21％，瓜拉纳提取物10％-20％，垂柳2％-8％，金银花提取物0.5％-5％和川陈皮素0.5％-5％。
在一些实施例中，上述表儿茶素来源于绿茶，在本发明中来源于绿茶的表儿茶素记为绿茶-表儿茶素。
在一些实施例中，上述川陈皮素来源于陈皮，在本发明中来源于陈皮的川陈皮素记为陈皮-川陈皮素。
在一些实施例中，上述组合物的制备方法包括按配比将原料采用缓冲液进行溶解，混合均匀后即得组合物。
第二方面，本发明提供了上述组合物在制备调节肠道菌群的食品或药品中的应用。
在一些实施例中，上述应用包括通过含提高肠道中粪杆菌属与条件致病菌比值的组合物的食品或药品提高有益菌的丰度并降低条件致病菌的丰度以调节肠道菌群。
在一些实施例中，上述有益菌包括粪杆菌属、乳杆菌、嗜酸乳杆菌和拟杆菌；优选地，有益菌为粪杆菌属。
在一些实施例中，上述条件致病菌包括大肠埃希氏菌和迟缓埃格特菌。
第三方面，本发明提供了一种食品，其包括食品学上可接受的辅料和上述组合物。
第四方面，本发明提供了一种药物，其包括药学上可接受的辅料和上述组合物。
本发明具有以下有益效果：
本发明提供了一种调控肠道菌群的组合物，以乳香酸为主料，并添加了表儿茶素、玛咖、瓜拉纳提取物、垂柳、金银花提取物和川陈皮素，其能够提高肠道中有益菌的比重，并有效抑制条件致病菌的生长及繁殖，充分调节了肠道益生菌菌群的组成，减少机体低度炎症、代谢紊乱、脂质过度积累和胰岛素敏感性丧失的风险。
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。
本发明提供了一种提高肠道中粪杆菌属与条件致病菌比值的组合物，其原料按质量百分数计包括：乳香酸21％-64％，绿茶-表儿茶素12％-22％，玛咖11％-21％，瓜拉纳提取物10％-20％，垂柳2％-8％，金银花提取物0.5％-5％和陈皮-川陈皮素0.5％-5％。
其中，乳香酸属于五环三萜类化合物，可以有效抑制5-脂氧合酶的活性，降低炎症因子的形成，发挥抗炎作用；乳香酸可明显减轻药物对胃粘膜的损伤和应激性粘膜损伤，降低胃液酸度；乳香酸对肿瘤细胞有抗增殖、分化诱导和细胞凋亡等作用，在预防癌症发生的过程中具有更加良好的临床价值；此外乳香酸具有免疫调节活性，可降低淋巴細胞转化，抑制亢奋的免疫功能。
绿茶-表儿茶素作为黄酮物质具有诸多生理活性，如抗氧化、清除自由基、加强新陈代谢、调节免疫和抗肿瘤、降脂降糖、预防心血管疾病、抗炎、保护神经、抑菌等作用等功能，其中表儿茶素抗氧化能力被认为是分子中的酚羟基基团和自由基的结合达到清除自由基作用，而抗肿瘤则是影响肿瘤细胞的周期进程而抑制肿瘤细胞的周期生长，同时还有诸多光谱抑菌效果，被认为具有极大的疾病预防潜力。
玛咖中含有丰富的营养成分和独特的次生代谢产物,玛咖多糖对羟自由基具有显著的抑制作用，具有抗氧化作用；玛咖中富含3种支链氨基酸——缬氨酸、亮氨酸和异亮氨酸,它们具有抗中枢神经疲劳的作用,另外玛咖中含有的牛磺酸也具有抗疲劳作用；玛咖黄铜和玛咖芥子油苷对α-葡萄糖苷酶有抑制作用,且能够抑制糖异生代谢,是玛咖能够降低血糖的途径之一；玛咖中含有的苄基芥子油苷和苄基异硫氰酸是玛咖中对动物模型的化学致癌具抑制作用的成分,苄基异硫氰酸被证实为抗有丝分裂剂，芥子油苷和异硫氰酸苄酯等含硫有机化合物对胃癌、肺癌等有一定的作用。
瓜拉纳提取物具有抗疲劳、抗抑郁、止泻、镇痛、解热、抗菌、抗氧化、抗菌、促智活性等作用。例如，可以通过降低血乳酸水平，提高糖原储存，缓解因运动代谢产物堆积而产生的疲劳；调节多巴胺能信号通路，激活运动神经元驱动运动；多酚物质通过限制细胞内铁对脂肪细胞内脂质的活性。
垂柳中富含天然药用成分能消灭人体内的敏感菌和致病菌，并能阻止人体内炎症滋生，它对人类的肠胃炎症和咽喉炎症都有明显预防和缓解作用；垂柳树叶中含有的水阳甙，具有解热止痛的重要作用，进入人体后还能转化成具有超强抗菌能力的水杨酸。
金银花提取物主要含有黄酮类、三萜类、环烯醚萜类、有机酸以及挥发油等成分；具有宣散风热，清解血毒，提高免疫力的功效，对多种致病菌、病毒有抑制作用，如金银花提取物对金黄色葡萄球菌耐药植株的呼吸有显著刺激作用，常用于耐药菌株导致的内外科炎症，如治疗肺结核并发呼吸道感染、肺炎、急性细菌性痢疾、腹泻等。也有用于降低咽喉部带菌率。
陈皮-川陈皮素有抗血细胞凝集、抗血栓形成、抗癌、抗真菌、抗炎、抗过敏、抗胆碱酯酶和抗癫痫作用，是碳水化合物代谢促进剂。
本发明通过上述原料的组合，制备出一种既可以提高肠道中有益菌的丰度，又能降低条件致病菌的丰度，进而起到维持肠道微生物的稳态平衡的作用的组合物。
在本发明中，提高肠道中粪杆菌属与条件致病菌比值的组合物的制备方法为：将原料粉碎后采用缓冲液进行溶解，混合均匀即可，本发明中所选用的原料均是可溶性粉末，易于溶解。缓冲液的种类有多种，包括但不限于PBS、磷酸、柠檬酸中的一种或多种。
具体来说，本发明还提供了一种调节肠道中粪杆菌属与条件致病菌比值的组合物的培养基，其包括基础培养基以及上述调节肠道中粪杆菌属与条件致病菌比值的组合物，其制备方法是将组合物添加至基础培养基中。
其中，组合物经缓冲液溶解并配置成浓度为4-6％的溶解液后添加至基础培养基，溶解液与基础培养基的体积比为1：1-1.5。利用本发明提供的培养基对粪便样品进行培养，可以降低粪便样品中致病菌的比值。
通过上述方法获得的组合物可以同时提高有益菌的丰度，同时还可以降低有害菌的丰度。
其中，有益菌包括粪杆菌属、乳杆菌和拟杆菌。条件致病菌包括大肠埃希菌和迟缓埃格特菌。
粪杆菌(Faecalibacterium)属和拟杆菌(bacteroides)均广泛存在于人体肠道中，最近一篇文献报道了：包括粪杆菌属和拟杆菌在内的有益菌与2型糖尿病呈负相关，健康人群体内这些微生物比2型糖尿病患者体内相对数量更多。因此推测上述微生物有利于预防2性糖尿病的发生和发展。
乳杆菌广泛分布于含有碳水化合物的动植物发酵产品中，也见于温血动物的口腔、阴道和肠道内。有研究证明乳杆菌可增强肝脏线粒体的健康，这意味着宿主代谢葡萄糖和脂质的方式得到了改善，通过调节肠道中乳杆菌的平衡，这为严重的代谢性疾病进行益生菌治疗打开了大门。
以上有益菌和条件致病菌在肠道中的丰度对机体健康有重要影响，因此维持上述有益菌和条件致病菌比值至关重要。经过试验验证，本发明的组合就能提高粪杆菌属、乳杆菌、嗜酸乳杆菌和拟杆菌等有益菌的丰度，降低大肠埃希菌和迟缓埃格特菌等条件致病菌的丰度。
基于此，本发明还提供了上述调节肠道中粪杆菌属与条件致病菌比值的组合物的多种应用。
具体地，上述应用包括：调节肠道中粪杆菌属与条件致病菌比值的组合物在制备促进粪杆菌属食品或药品中的应用；调节肠道中粪杆菌属与条件致病菌比值的组合物在制备抑制大肠埃希菌食品或药品中的应用，或是
调节肠道中粪杆菌属与条件致病菌比值的组合物在制备用于治疗或改善代谢性疾病的药物中的应用；调节肠道中粪杆菌属与条件致病菌比值的组合物在制备用于治疗或改善代谢性疾病的药物中的应用，或是
调节肠道中粪杆菌属与条件致病菌比值的组合物在定向发酵用于粪菌移植的特异供体菌群的方法中的应用。
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。
本实施例中所用的原料乳香酸、表儿茶素、玛咖、瓜拉纳提取物、垂柳、金银花提取物和川陈皮素购于西安奥赛生物科技有限公司。
实施例1
本实施例提供了一种提高肠道中粪杆菌属/条件致病菌比值的组合物，其原料包括：500g乳香酸；180g绿茶-表儿茶素、120g玛咖、120g瓜拉纳提取物提物，40g垂柳，20g金银花提取物，20g陈皮-川陈皮素。
实施例2
本实施例提供了一种提高肠道中粪杆菌属/条件致病菌比值的组合物，其原料包括：210g乳香酸；220g绿茶-表儿茶素、210g玛咖、200g瓜拉纳提取物提物，80g垂柳，40g金银花提取物，40g陈皮-川陈皮素。
实施例3
本实施例提供了一种提高肠道中粪杆菌属/条件致病菌比值的组合物，其原料包括：600g乳香酸，130g绿茶-表儿茶素，120g玛咖、100g瓜拉纳提取物提物，30g垂柳，10g金银花提取物，10g陈皮-川陈皮素。
实施例4
本实施例提供了一种提高肠道中粪杆菌属/条件致病菌比值的组合物，其原料包括：350g乳香酸，170g绿茶-表儿茶素，160g玛咖、150g瓜拉纳提取物提物，80g垂柳，50g金银花提取物，40g陈皮-川陈皮素。
对比例1
本对比例提供的组合物的原料包括：360g绿茶-表儿茶素，240g玛咖、240g瓜拉纳提取物提物，80g垂柳，40g金银花提取物，40g陈皮-川陈皮素。
对比例2
本对比例提供的组合物的原料包括：610g乳香酸，146g玛咖、146g瓜拉纳提取物提物，50g垂柳，24g金银花提取物，24g陈皮-川陈皮素。
对比例3
本对比例提供的组合物的原料包括：573g乳香酸；202g绿茶-表儿茶素、135g瓜拉纳提取物提物，45g垂柳，23g金银花提取物，22g陈皮-川陈皮素。
对比例4
本对比例提供的组合物的原料包括：578g乳香酸；195g绿茶-表儿茶素、136g玛咖、46g垂柳，23g金银花提取物，22g陈皮-川陈皮素。
对比例5
本对比例提供的组合物的原料包括：520g乳香酸；190g绿茶-表儿茶素、125g玛咖、124g瓜拉纳提取物提物，21g金银花提取物，20g陈皮-川陈皮素。
实验例
一、体外实验：
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
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、对比例1-5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。
其中混合液为：将实施例1-4和对比例1-5组合物用PBS缓冲液溶解至质量百分浓度为5％的混合液。
同时还设置有对照组，分组具体如下：
实验组：2ml GAM培养基+1mL混合液+1mL肠道微生物样品+2ml PBS缓冲液；
对照组：2ml GAM培养基+1mL肠道微生物样品+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。
表1部分肠道菌群的丰度
注：*P<0.05，##P<0.01。
从体外实验结果看：在本发明的组合物范围内均可以达到调节目的，粪杆菌、乳杆菌丰度显著增加，拟杆菌也有明显的增加，而迟缓埃格特菌、大肠埃希氏菌、显著性降低。而对比例任何单一组分的方案并没有这样的效果。
二、迟缓埃格特菌分析
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)迟缓埃格特菌的培养
迟缓埃格特菌是一种专性厌氧革兰阳性杆菌，生长缓慢，培养5d内可形成肉眼可见菌落。将迟缓埃格特菌种接种于血平板上，加入厌氧包在37℃和5％ CO2箱厌氧培养5d后，取菌落进行后续鉴定。
(2)迟缓埃格特菌的鉴定
将原始菌分纯扩大培养后提取原始菌基因组DNA。
正向引物：27F(5'-AGAGTTTGATCATGGCTCAG-3')
反向引物：1492R(5'-TACGGCTACCTTGTACGACTT-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，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迟缓埃格特菌培养液+1mL PBS缓冲液；
条件对照组：3mL GAM培养基+1mL迟缓埃格特菌培养液+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迟缓埃格特菌丰度，其中迟缓埃格特菌的丰度测定结果如表2所示。并对迟缓埃格特菌干预前后的丰度变化进行显著性分析。
表2迟缓埃格特菌的丰度测定结果
对比空白组*P＜0.01，对比条件对照组#P＜0.01。
结果表明迟缓埃格特菌(Eggerthella lenta)经过组合物的干预后，丰度是有显著性的降低的。
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)
1.一种提高肠道中粪杆菌属与条件致病菌比值的组合物，其特征在于，其原料按质量百分数计包括：乳香酸21％-64％，表儿茶素12％-22％，玛咖11％-21％，瓜拉纳提取物10％-20％，垂柳2％-8％，金银花提取物0.5％-5％和川陈皮素0.5％-5％。
2.根据权利要求1所述的组合物，其特征在于，所述表儿茶素来源于绿茶。
3.根据权利要求2所述的组合物，其特征在于，所述川陈皮素来源于陈皮。
4.根据权利要求3所述的组合物，其特征在于，所述组合物的制备方法包括按配比将原料采用缓冲液进行溶解，混合均匀后即得所述组合物。
5.如权利要求1-4任一项所述的组合物在制备调节肠道菌群的食品或药品中的应用。
6.根据权利要求5所述的应用，其特征在于，所述应用包括通过含所述提高肠道中粪杆菌属与条件致病菌比值的组合物的食品或药品提高有益菌的丰度并降低条件致病菌的丰度以调节肠道菌群。
7.根据权利要求6所述的应用，其特征在于，所述有益菌包括粪杆菌属、乳杆菌、嗜酸乳杆菌和拟杆菌；优选地，所述有益菌为粪杆菌属。
8.根据权利要求7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和迟缓埃格特菌。
9.一种食品，其特征在于，包括食品学上可接受的辅料和如权利要求1-4任一项所述的组合物。
10.一种药物，其特征在于，包括药学上可接受的辅料和如权利要求1-4任一项所述的组合物。
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