CN107198250A - 改善肠道微生态预防慢性病组合物和均衡营养食品及应用 - Google Patents
改善肠道微生态预防慢性病组合物和均衡营养食品及应用 Download PDFInfo
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Abstract
本发明公开了一种改善肠道微生态预防慢性病的组合物,包括菊粉、低聚半乳糖和复合膳食纤维,其中菊粉、低聚半乳糖和复合膳食纤维配比为:1~75:1~75:5~95,较优配比为:10~50:10~50:25~50。本发明还公开了一种含该组合物的均衡营养食品,包括谷物粉,谷物粉与组合物的配比为:50~95:50~5,较优配比为:70~95:30~5。本发明通过普通聚糖维护非益生菌的适量生存,功能性聚糖益生元组合促进益生菌的生长,通过聚糖间的协同作用维持肠道微生物稳态平衡,降低肠通量、内毒素及炎症因子,增强免疫能力,预防慢性病。本发明均衡营养食品,提供超级生物体的均衡营养,改善亚健康体质,预防慢性病。
Description
技术领域
本发明涉及以肠道微生物为靶点的疾病预防及营养食品领域,特别是涉及一种改善肠道微生态预防慢性病的组合物、均衡营养食品及应用。
背景技术
人体是一个由人的细胞和所有共生微生物构成的“超级生物体”,也是一个非常复杂的生态系统。在人的身体内外生活的微生物细胞数量甚至是人体自身细胞数量的10倍,有百万亿之多。超过90%的共生微生物生活在人的体内,称为“肠道菌群”。成人肠道栖息着大约1014数量级的细菌,主要定植在结肠,形成一个约1.5kg重的菌群生态系统,包括1000~1150种细菌。21世纪以来,人类面临着“膳食纤维鸿沟”的巨大挑战,该“膳食纤维鸿沟”是指在现代饮食方式中,人们很难按照推荐标准吃到足够量的膳食纤维,以至于一些肠道微生物消失,肠道菌群被破坏,肠道通透性增加,机体长期处于低度炎症状态,免疫力降低,进而引起各种慢性疾病。
现在有观点认为,人体许多健康问题都是由体内菌群失衡引起,决定因素是微生态的平衡。如肠道菌群和肥胖有关,肠道菌群失衡引起肥胖的作用机制,主要有以下三方面:部分产丁酸的菌属能降解膳食中的多糖,转变成短链脂肪酸供身体吸收利用,导致宿主从饮食中吸收能量的能力增加;肠道菌群还能够调控能量储存组织基因的表达活性,主要与脂肪生成相关的基因有Fiaf及ChREBP/SREBP-1;高脂饮食通过改变肠道菌群结构,引起代谢性内毒素血症,然后通过LPS/CDl4信号通路引起慢性系统性炎症,导致肥胖、胰岛素抵抗等。还有研究表明:肠道菌群的代谢物与主要心血管不良事件发生率提高有关,肠道菌群与结直肠癌具有相关性。且还有观点认为肠道菌群产生的毒素是令人体衰老、得病的重要原因。
基于人们对肠道菌群新的认识,肠道菌群是多种慢病致病和疾病治疗的重要因素,并且具有可塑性,因而是理想的预防及治疗疾病的新靶点。现已有研究发现,调节肠道微生态的平衡,可实现缓解与治疗疾病的目的。比如腹泻、便秘、阴道感染等综合症状,国内外研究证明使用特定的经临床证实的益生菌可有效地进行治疗。还有研究报道通过使用低聚果糖益生元调节肠道菌群的结构,可以影响多种胃肠道激素的合成和分泌,如结肠中的GLP-1和GLP-2浓度增加,二肽基肽酶IV(DPP-4)浓度降低,从而改善代谢性内毒素血症;α糖苷酶抑制剂可以明显提高有益菌如双歧杆菌的数目,改善糖代谢作用,并且减少脂肪组织炎症。还有研究表明膳食纤维可减缓消化速度和加快胆固醇的排泄,降低患肠癌的风险,同时可吸收食物中有毒物质,可预防肥胖症、肠道疾病、心血管疾病、糖尿病、降低血压、抗乳腺癌、改变肠道系统中微生物群落组成以平衡菌群、提高人体免疫功能。
但现有的研究与应用仍存在诸多问题:1、没有解决21世纪人类面临的“膳食纤维鸿沟”的巨大挑战,以至于一些肠道微生物消失,肠道菌群被破坏,进而引起各种慢性疾病,占人类死亡原因85%的慢性疾病有增无减;2、没有有效足量的营养组合物以改善肠道菌群的失衡,单独添加食用功能聚糖组合物或普通聚糖组合物,对人体产生积极作用的同时,也会阶段性的造成个别有益菌过多或不足,肠通量增加等负向作用;3、缺乏有关将重要功能聚糖益生元组合与普通聚糖复合纤维素协同作用的研究及应用,以弥补简单聚糖组合物类型的缺点与不足;4、缺乏同时满足人体和肠道菌群(超级生物体)营养的主食,不能简便地进行一级预防,从根本上保证人类的健康问题。
综上所述,近年来微生物组学、代谢组学、蛋白质组学、转录组学、宏基因组学关联分析等技术逐渐被应用于肠道微生物的研究中,对诠释肠道细菌和人体健康的关系,研究肠道细菌在疾病发生中的作用给予了极大的推动,研制改善肠道微生态的组合物,消除慢性炎症及免疫力低下等致病因素,改善躯体亚健康体质,预防心脑血管疾病、糖尿病、慢性神经退行性疾病及癌症等慢性疾病,研制同时满足人体及维护肠道微生态的食品,即研制超级生物体主食、解决“膳食纤维素鸿沟”均是重要的营养健康问题。本发明就是在此基础上研发一种新的维护肠道生态环境提供菌群营养的组合物及均衡营养食品,改善人类躯体亚健康体质,有效一级预防非感染类慢性疾病,实属当前重要研发课题之一。
发明内容
本发明要解决的技术问题是提供一种改善肠道微生态预防慢性疾病的组合物,促进有益菌的生长与繁殖,维护非益生菌的适量生存,抑制有害菌的生长与活性,维持肠道微生物稳态平衡,降低肠通量、血液内毒素及炎症因子,增强细胞代谢及体质免疫能力,代谢短链脂肪酸、支链氨基酸、维生素等营养代谢物,降低肠道PH值,促进钙铁镁等矿物质的吸收,消除慢性炎症及免疫力低下等致病因素,改善躯体亚健康体质,一级预防心脑血管疾病、糖尿病等慢性疾病。
为解决上述技术问题,本发明提供一种改善肠道微生态预防慢性病的组合物,包括菊粉、低聚半乳糖和复合膳食纤维,其中所述菊粉、低聚半乳糖和复合膳食纤维的配方比为:1~75:1~75:5~95。
作为本发明的一种改进,,所述菊粉、低聚半乳糖和复合膳食纤维的配方比为:10~50:10~50:25~50。
进一步改进,所述菊粉为低聚果糖、多聚果糖或低聚果糖与多聚果糖的混合物;所述复合膳食纤维为非益生元的可溶纤维与不溶纤维的混合物,其中,所述可溶纤维与不溶纤维的配方比为10~90:10~90。所述功能性聚糖菊粉、低聚半乳糖和普通聚糖复合膳食纤维的配方比为:25:25:50、25:50:25或35:15:50。
进一步改进,所述非益生元的可溶纤维为抗性糊精、聚葡萄糖、低聚异麦芽糖或其混合物;所述不溶纤维为不溶于水的纤维素、半纤维素、木质素或其混合物。
本发明还提供一种包含上述组合物的均衡营养食品,该均衡营养食品还包括谷物粉,所述谷物粉与所述组合物的配方比为:50~95:50~5。
进一步改进,所述谷物粉与所述组合物的配方比为:70~95:30~5。
进一步改进,所述谷物粉为小麦面粉、大米粉、玉米粉和马铃薯全粉中一种或多种的组合物,所述均衡营养食品被制备成复方大米、面粉、面条、意面、米线、面包、面包团或面包预拌粉的形式。
进一步改进,所述复方大米的制备方法为:将所述谷物粉与所述组合物混合,并采用压制工艺挤压成大米形态,其中所述组合物中的不溶纤维素采用微晶纤维素。
本发明还公开上述组合物在制备预防炎症性肠病的食品或药品中的应用。
本发明还公开上述组合物在制备改善胃肠动力和预防便秘的食品或药品中的应用。
本发明还公开上述组合物在制备预防糖尿病的食品或药品中的应用。
本发明还公开上述组合物在制备预防心脑血管疾病的食品或药品中的应用。
本发明还公开上述组合物在制备调节肠道菌群提高免疫系统的食品或药品中的应用。
采用这样的设计后,本发明至少具有以下优点:
本发明组合物通过包含功能性聚糖益生元组合,为肠道益生菌提供食物,促进有益细菌的生长繁殖,同时抑制有害菌的生长与活性,维持肠道微生物的稳态平衡,并且其包含的普通聚糖复合膳食纤维具有发酵性、吸水溶胀、梯度黏合、机械隔离、网孔吸附、离子交换及菌群调节的作用,为肠道内有益细菌的生长提供有利环境,且可减缓消化速度和加快胆固醇的排泄,吸收食物中有毒物质,维持非益生菌的适量生存。功能聚糖与普通聚糖的协同作用,消除单独使用功能聚糖或普通聚糖的负向作用,降低肠通量、血液内毒素及炎症因子,增强细胞代谢及体质免疫能力,代谢短链脂肪酸、支链氨基酸、维生素等营养代谢物,降低肠道PH值,促进钙铁镁等矿物质的吸收,消除慢性炎症及免疫力低下等致病因素,改善躯体亚健康体质,预防心脑血管疾病、糖尿病等慢性疾病。本发明组合物能良好的维持肠道微生物的稳态环境预防慢性疾病。
本发明包含上述组合物的均衡营养食品作为人们每天不可或缺的主食,不仅为人体本身提供营养成分,还为与人体共生的菌群提供环境及营养物质,维护人体这个超级生物体的均衡营养,有效改善人类亚健康体质,预防疾病,保障健康。
附图说明
上述仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,以下结合附图与具体实施方式对本发明作进一步的详细说明。
图1是本发明应用实施例中小鼠肠道内容物pH值的变化图。
图2是本发明应用实施例中小鼠血清生化指标DAO的变化图。
图3是本发明应用实施例中小鼠血清生化指标TMAO的变化图。
图4是本发明应用实施例中小鼠血清生化指标SCFA的变化图。
图5是本发明应用实施例中小鼠小肠绒毛的高度和隐窝切片变化图。
图6是本发明应用实施例中空白组小鼠21天肠道菌群在门水平上的组成图。
图7是本发明应用实施例中实验组A小鼠21天肠道菌群在门水平上的组成图。
图8是本发明应用实施例中实验组B小鼠21天肠道菌群在门水平上的组成图。
图9是本发明应用实施例中实验组C小鼠21天肠道菌群在门水平上的组成图。
图10是本发明应用实施例中四个实验组小鼠21天肠道菌群在属水平上的组成图。
具体实施方式
本发明改善肠道微生态预防慢性病的组合物包括:菊粉、低聚半乳糖和复合膳食纤维,其中该菊粉、低聚半乳糖和复合膳食纤维的配方比例为:1~75:1~75:5~95。
本发明的菊粉(Inulin)是指低聚果糖、多聚果糖或低聚果糖与多聚果糖的混合物;低聚半乳糖(Galactooligosaccharides,GOS)是一种具有天然属性的功能性低聚糖,其分子结构一般是在半乳糖或葡萄糖分子上连接1~7个半乳糖基,即Gal-(Gal)n-Glc/Gal(n为0-6);复合膳食纤维是指非益生元的可溶纤维和不溶纤维的混合物,其中,非益生元的可溶纤维是指抗性糊精、聚葡萄糖、低聚异麦芽糖或其混合物,不溶纤维(solkfloc)是指不溶于水的纤维素、半纤维素、木质素或其混合物,本实施例中非益生元可溶纤维与不溶纤维的配比为10~90:10~90。
本发明该菊粉、低聚半乳糖和复合膳食纤维的较优配方比例为:10~50:10~50:25~50。
本发明该菊粉、低聚半乳糖和复合膳食纤维的更优配方比例为:25:25:50、25:50:25或35:15:50。
为了使该改善肠道微生态的组合物更好的作用于人体,本发明还提供了包含上述组合物的均衡营养食品,该均衡营养食品包括谷物粉和上述组合物,其谷物粉与上述组合物的配方比为50~95:50~5。
其中谷物粉是指小麦面粉、大米(粳米、籼米、糯米)粉、玉米粉、马铃薯全粉或其组合物,则由该谷物粉和上述组合物形成的均衡营养食品形态主要有复合大米、面粉、面条(擀面条、干面条、半干面条、湿面条)、意面、米线、面包、面包团或面包预拌粉等等多种形式的食品,且不限于上述这些形式。
本发明该谷物粉和上述组合物的较优配方比例为:70~95:30~5。
本发明该谷物粉和上述组合物的更优配方比例为:70:30、85:15或88:12。
当然,本发明均衡营养食品中还可以包括矿物质、维生素以及低聚木糖、L-阿拉伯糖、水苏糖、瓜尔豆胶等营养强化剂及补充剂,其添加范围均符合国际食品法典委员会(CAC)《食品中必需营养素添加的通用原则》要求。
本发明中功能性聚糖菊粉、低聚半乳糖、水苏糖均属于益生元,能通过选择性的刺激一种或者几种有益细菌的生长和活性,抑制有害菌的生长与活性。该益生元组合给肠道中益生菌提供“食物”,促进有益细菌生长繁殖,抑制有害菌的生长与活性。本发明组合物中的普通聚糖组合维护适量的非益生菌的生存,复合不溶膳食纤维具有发酵性、吸水溶胀、梯度黏合、机械隔离、网孔吸附、离子交换及菌群调节的作用,为肠道内有益细菌的生长提供有利环境,且可减缓消化速度和加快胆固醇的排泄,吸收食物中有毒物质,降低患肠癌的风险。所以本发明均衡营养食品能良好的维持肠道微生物的稳态环境,降低肠道通透性,一级预防慢性疾病,保障健康。
产品实施例1:均衡营养大米
谷物粉采用粳米粉和糯米粉以80:20混合的大米粉混合物,菌群营养组合物采用菊粉:低聚半乳糖:聚葡萄糖:微晶纤维素为30:20:25:25的组分配比混合物,且谷物粉与菌群营养组合物的配比为85:15。
该均衡营养大米的制备方法如下:
1、将菌群营养组合物中各组分按比例置于搅拌器中充分混合,制成预混粉料,备用;
2、将粳米粉和糯米粉以80:20的比例混合成大米粉混合物,将该大米粉混合物与步骤1得到的预混粉料按80:20的比例混合,再加27~29%的水搅拌调质,得均衡营养大米半成品;
3、将步骤2得到的均衡营养大米半成品用双螺杆挤压机挤压造粒,然后用流化床干燥机干燥,再经过冷却、筛理,即得本实施例均衡营养大米。
本实施例均衡营养大米的成品形态似天然大米,品相美观。其口感特性:细腻滑润,没有颗粒感,不回生。其功能特性:满足了超级生物体(人体和肠道菌群)的均衡营养需求,升糖指数小于50,能改善便秘,增殖益生菌,降低肠道PH值,改善肠道微生态环境。
产品实施例2:均衡营养面粉
谷物粉采用普通面粉,菌群营养组合物采用菊粉:低聚半乳糖:聚葡萄糖:微晶纤维素为25:25:20:30的组分配比混合物,且谷物粉与菌群营养组合物的配比为90:10。
该均衡营养面粉的制备方法如下:
1、将菌群营养组合物中各组分按比例置于搅拌器中充分混合,制成预混粉料,备用;
2、将普通面粉与步骤1得到的预混粉料按90:10的比例混合,搅拌充分调质,筛理,即可本实施例均衡营养面粉。
本实施例均衡营养面粉的成品形态似普通面粉,口感细腻,做各种面食均没有颗粒感。其功能特性:满足了超级生物体(人体和肠道菌群)的均衡营养需求,吸水特性增强,升糖指数小于50,能增殖益生菌,降低肠道PH值,改善便秘,改善肠道微生态环境。
产品实施例3:均衡营养面条
谷物粉采用普通面粉,菌群营养组合物采用菊粉:低聚半乳糖:聚葡萄糖:微晶纤维素为25:25:25:25的组分配比混合物,且谷物粉与菌群营养组合物的配比为70:30。
该均衡营养面条的制备方法如下:
1、将菌群营养组合物中各组分按比例置于搅拌器中充分混合,制成预混粉料,备用;
2、将普通面粉与步骤1得到的预混粉料按70:30的比例混合,再加水搅拌,加水至含水量50%左右,水温15~20℃为宜,搅拌10~15分钟;
3、熟化10~15分钟,熟化温度25℃左右,熟化后轧面、切条、干燥、冷风定条,含水量降至14%,即得本实施例均衡营养面条。
本实施例均衡营养面条口感细腻滑润、劲道。其功能特性:满足了超级生物体(人体和肠道菌群)的均衡营养需求,升糖指数小于50,能增殖益生菌,降低肠道PH值,改善便秘,改善肠道微生态环境。
产品实施例4:均衡营养面包
谷物粉采用中筋面粉,菌群营养组合物采用菊粉:低聚半乳糖:聚葡萄糖:微晶纤维素为25:20:25:30的组分配比混合物,且谷物粉与菌群营养组合物的配比为88:12。
该均衡营养面包的制备方法如下:
1、将菌群营养组合物中各组分按比例置于搅拌器中充分混合,制成预混粉料,备用;
2、将中筋面粉与步骤1得到的预混粉料按85:15的比例混合,再加水搅拌,按照传统面包工艺和面、第一次发酵、第二次调制面团处理、第二次发酵、整形、成型、烤前加工处理、烘烤、冷却、即得本实施例均衡营养面包条。该步骤由于菌群营养组合物组分的特性省去了传统方法制作面包的白砂糖、植物油等配料。
本实施例均衡营养面包口感细腻滑润、组织蓬松、柔软,不易塌陷。其功能特性:满足了超级生物体(人体和肠道菌群)的均衡营养需求,升糖指数小于50,能增殖益生菌,降低肠道PH值,改善便秘,改善肠道微生态环境。
本发明改善肠道微生态的组合物包含功能聚糖、普通聚糖膳食纤维,多种聚糖发生显著的协同作用,对宿主产生积极作用没有产生负向作用。对肠道有益菌、中性菌提供组合营养及利于其增殖的肠道环境,丰富了肠道菌群的代谢组物,包括对人体有益的初级代谢组物及次级代谢组物,改善了肠道的微生态,达到以改善肠道微生物稳态为靶点,预防慢性疾病的效果;本发明均衡营养食品还同时提供了肠道菌群及人体(超级生物体)的均衡营养,以其方便的主食为载体提供了维护肠道微生态稳态平衡的组合物,有效改善人类躯体亚健康体质,预防危及人类重大健康问题的慢性疾病。
应用实施例
本实验把聚糖类组合物分为三个类型,1)功能性聚糖组合物;2)普通聚糖组合物;3)包含多种功能性聚糖和普通聚糖的组合物;用于研究不同类型聚糖组合物的协同作用,这个协同作用的重点在于两个方面,一是适宜微生物丰度值及微生物生存环境的变化;二是对宿主生理指标的影响变化。具体试验步骤如下:
1.材料与设备
1.1试剂
DAO试剂盒,SCFA试剂盒,TMAO试剂盒,由上海酶联生物科技有限公司提供;
1.2实验动物
SPF级8周龄的BARBL/C小鼠,购买于军事医学科学院实验动物中心;
1.3实验饲料
按照上述聚糖类组合物的分类不同,制备四种实验饲料组:
空白组O基料100%,即小鼠一般饲料;
实验组A基料+功能聚糖组合物具体为:基料85%、低聚半乳糖7.5%、菊粉7.5%;
实验组B基料+普通聚糖组合物具体为:基料85%、不溶膳食纤维7.5%(麦麸提取)、聚葡萄糖7.5%;
实验组C基料+功能聚糖组合物+普通聚糖组合物具体为:基料85%、低聚半乳糖4%、菊粉3.5%、不溶膳食纤维4%、聚葡萄糖3.5%;上述试验饲料均由北京瑞千景科技发展有限公司提供。且上述配方基于要做成食品应用,按人均每日食用主食250~300克算,满足食用复合膳食纤维37~45克的用量做效果参考。
1.4主要仪器与设备
电子天平、pH计,梅特勒-托利多仪器(上海)有限公司;气相色谱质谱联用仪。
2实验方法
2.1实验设计
将小鼠随机分配到空白组O、实验组A、实验组B、实验组C四个组中(8只/组),饲喂于独立通风笼系统,严格控制12h光照/黑夜,环境温度设定在22℃;
本实验分4周,第1周为适应期,第2、3、4周为实验期。在适应期,四组小鼠均进食空白饲料(即基料100%);在实验期,空白组O小鼠进食空白饲料,实验组小鼠分别进食实验组A、实验组B、实验组C对应饲料;在实验期第二周和第三周结束时,随机抽取3只小鼠,摘眼球采血,脱臼法处死小鼠,取小肠,存放于福尔马林溶液,用于HE染色;收集结肠内容物样品,-80℃保存;并分析pH、细菌菌群组成等。血液收集后分离血清,采用试剂盒测血清中各项生化指标二胺氧化酶(DAO)、氧化三甲胺(TMAO)、短链脂肪酸(SCFA)。
2.2血清代谢参数的测定
小鼠眼球取血后,室温静置1h,3000rpm离心15min,分离上层血清,保存于-80℃。血清中生化指标,采用试剂盒测定,包括DAO、TMAO、SCFA;
2.3肠道内容物pH值的测定
称取一定100mg肠道内容物,按照15mL/g添加去离子水,充分分散,13000g离心2min,用pH计测定上清液的pH值。
2.4肠道内容物代谢组学的测定
秤取100mg样品,加入甲醇:水:氯仿(3:1:1)的溶液,漩涡震荡使其分散均匀,静置12小时,离心取上清。取提取液100μl,加入20μl0.2mg/ml的核糖醇作为内标物质,充分混匀后,在45℃下用N2吹干;向吹干的提取物中加入40μL20mg/mL的盐酸甲氧胺溶液,充分混匀,在30℃下,以130rpm转速下反应90min;充分反应后,再加入40μL BSTFA(含1%TMCS),充分混匀后,在37℃下气浴30min,取出后,在室温下放置120min,再在4℃下待测。
GC-MS色谱条件:
GC条件:Agilent 7890,串联LECO Pegasus 4D TOF/MS检测器;
Gerstel MPS进样系统;
色谱柱:DB-5MS 30m×250μm×0.25mm
程序升温:70℃,保持1min,以5℃/min升至280℃,保持10min
载气:He;流速:1mL/min;进样体积:1μL;分流比1:2;
质谱条件:
MS条件:EI源:质量扫描范围:50-800Da,扫描速度10/s;进样口、传输线和离子源温度分别为:250℃、250℃及220℃。
数据处理
色谱图中各化合物通过工作站Chroma TOF 4.50进行色谱峰对齐、去卷积、峰检索后(自带的NIST等质谱数据库及相关标准品进行鉴定),再利用核糖醇(内标物)进行归一化处理原始图谱经积分后(信噪比S/N>100),最终获得各色谱峰及代谢物名称、峰面积等相关数据。
2.5小肠绒毛高度和隐窝深度的变化测量
Image pro-plus6.0分析肠组织绒毛长度、隐窝深度及杯状细胞的方法:运用IPP软件的直线工具选取绒毛较伸展并具有完整肠绒毛的图片对每张切片的绒毛长度和隐窝深度进行测量。
2.6肠道内容物16sDNA的测定
微生物DNA的提取参照肠道内容物DNA提取试剂盒(Omega Bio-tek,Norcross,GA,U.S.)的说明书。提取的DNA质量通过1%琼脂糖凝胶电泳进行检测。DNA样品保存在-20°冰箱。采用Illumina MiSeq测序平台PE300通过高通量测序方法对细菌16S核糖体RNA基因的16S v3-v4高变区进行PCR扩增(扩增条件为:95℃解链2min,95℃30s,55℃30s,72℃30s,25个循环,再72℃延伸10min),所用引物为:正向引物(515F)5’-barcode-GTGCCAGCMGCCGCGG)-3’,反向引物(907R)5’-CCGTCAATTCMTTTRAGTTT-3’,且对于每个样本,分别加上8bp的标签序列。每个样品的扩增均做三次重复,反应体系为25μL,包括2.5μL的10×Pyrobest Buffer,2μL的2.5mM dNTPs,正反向引物各1μL(10μM),0.4U的DNA聚合酶(Pyrobest DNA Polymerase,TaKaRa),15ng的DNA模板,其余为ddH2O。从2%琼脂糖凝胶中回收扩增子,依照AxyPrep DNA Gel Extraction Kit说明书进行纯化(AxygenBiosciences,Union City,CA,U.S.),再用QuantiFluorTM-ST(Promega,U.S.)进行定量。将纯化后的扩增子等量混合,然后根据Illumina MiSeq测序平台的标准流程进行双端2×300bp测序。用QIIME(version 1.17)软件对原始fastq文件进行处理,处理标准如下:(i)以10bp为一个滑动窗口,如果窗口内平均质量值低于20,则从窗口开始截去后端碱基,并过滤掉小于50bp的序列。(ii)根据标签序列区分样本,标签序列允许的错配数为0,引物错配数为2,且去掉包含模糊碱基的序列。(iii)相互拼接上的序列之间,重叠区不得少于10bp,且去掉无法拼接的序列。用UPARSE(version 7.1http://drive5.com/uparse/)软件聚类生成操作分类单元(Operational Units,OTUs),相似度为97%。再用UCHIME软件鉴别嵌合体序列,并将之去除。分类学比对时,用silva(SSU115)16S核糖体RNA数据库,算法为RDPClassifier(http://rdp.cme.msu.edu/),置信阈值为70%。
3结果与结论
3.1肠道内容物的pH
参照附图1所示,在空白组O小鼠中,肠道内容物的pH值在14天和21天分别为8.23和8.17。而实验组A、B、C三组小鼠的肠道内容物pH值急剧下降至6.0左右,并且实验组C组数值是最稳定的。这是因为细菌分解寡糖,产生短链脂肪酸,从而降低了pH值。
肠道pH值是肠道微生态的重要参数,pH值参数变化显著,从该实验可知,实验组C配方饲料最为稳定理想。
3.2小鼠血清生化指标
表1实验组饲料干预后小鼠血清生化指标的变化
由表1和附图2至4可以看出,实验组小鼠血清各项指标同空白组比较结果为:
实验组A:DAO指标变化不稳定,TMAO数值负项变化(变大),SCFA变化不显著;
实验组B:DAO指标变化不显著,TMAO21天数值负项变化大(变大),SCFA变化波动大;
实验组C:DAO变化显著,TMAO变化显著,SCFA变化显著。
由于二胺氧化酶(DAO)是肠道黏膜细胞的内酶,血液中DAO数值的高低,直观表达了肠道黏膜的损伤程度,肠通量的高低。通常认为,低聚糖会选择性地促进肠道内有益菌的增殖,从而对宿主健康具有促进作用。然而,也有报道称:低聚果糖会增加大鼠肠道的通透性,在高剂量时甚至会增加Salmonella的异位,过量食用低聚糖也会引起肠道不适,如腹鸣、胀气。由表1可以看出,在实验期第二周结束时,A组小鼠的DAO值最低,但在实验期第三周结束时,反而升高;这可能是由于过量的功能性低聚糖组合物增加了小鼠的肠道通透性,导致DAO值升高所致;而C组小鼠呈现出了一个逐渐降低的过程,说明C组配方的协同作用弥补了A、B两组配方的重要缺陷,是理想的配方组合物。
依赖肠道微生物的胆碱代谢产物氧化三甲胺(TMAO)在心血管疾病发生发展中起着重要作用,其中宿主TMAO水平升高与心血管疾病呈明显正相关;肠道微生物将摄入的卵磷脂、胆碱、肉碱等营养素作为碳能量来源,肠道微生物具有三甲胺裂解(酶哺乳动物是没有这种酶的),可以使这些营养素中C-N键断裂三甲胺即作为代谢废物释放,通过门脉循环进入肝脏,在肝脏经过黄素单氧化酶,主要是(FMO3)氧化生成TMAO。
在实验期第二周和第三周结束时,A、B两组数值甚至高于空白组O,出现负向作用,说明功能性聚糖组合物和普通性聚糖组合物虽然对肠道微生态有影响变化,但对宿主会阶段性产生负向作用;C组小鼠的血清TMAO值最低,这说明C组饲料既能够促进有益菌的生长,又能平衡微生物稳态,并抑制能生成氧化三甲胺前体的微生物。
短链脂肪酸(SCFA)对于维持人体肠道健康有着重要的作用。尤其是丁酸,作为结肠粘膜的生长诱导剂和炎症的抑制剂,能诱导癌细胞凋亡,防止结肠癌变的发生。肠道中SCFA的含量可以反映细菌活性,影响肝脂质与碳水化合物的调控。细菌利用低聚糖产生短链脂肪酸,结合图1,实验组A、B、C三组小鼠的肠道内容物pH值均显著低于空白组,但从实验期第二周血清的检测结果分析发现,血清中的SCFA空白组小鼠最高;在实验期第3周结束时的血清结果来看,C组变化不明显,该结果和肠道黏膜增厚的通透性降低有关,与C组血清DAO最低相吻合。
综上所述,实验组A、B、C三组饲料代表了三种类型的配方,C组配方是最理想的一类配方,C组中各组份的协同作用对于肠道微生态稳态平衡及宿主产生了积极作用而没有产生副作用;A、B两组配方在对肠道微生态稳态平衡及宿主产生了积极作用的同时,均阶段性的造成了一定的副作用。这在后面的试验结果中也达到了进一步的印证。
3.3对小肠绒毛的高度和隐窝的影响
表2实验组饲料干预后小肠绒毛高度及隐窝深度的变化
从表2和附图5可以看出,实验组A、B、C与空白组O小鼠在喂养14天和21天的指标变化。小肠是机体营养素消化、吸收和转运的主要部位,因此良好的小肠粘膜结构对于完善消化生理功能,促进机体的生长发育尤为重要。小肠绒毛作为小肠的重要组成部分,不但对营养物质的吸收起着重要作用,而且有害菌群的定殖还会遭到小肠绒毛强有力摆动的破坏。益生菌对肠道形态功能的影响可能不仅仅是对肠绒毛形态的改变,还有更多细胞水平上的改变。绒毛高度与隐窝深度的比值反映了小肠的功能状态,当比值下降,表明黏膜受损,消化吸收功能下降,动物生长发育受阻等喂养14天后,发现A组和B组的绒毛高度与隐窝深度的比值是高于对照组的,而C组与之相差不明显;21天后,B组和C组的绒毛高度与隐窝深度的比值高于对照组,且C组表现最佳;可能是由于它促进了小鼠胃肠道正常微生物区系的建立,维持消化道内环境稳定,改善肠绒毛的存在环境,这也在血液生化检验C组二胺氧化酶DAO数值中得到了验证。
3.4肠道内容物代谢组学的测定
表3.试验组部分有机酸的数值变化
从表3可以看出,不同饲料喂养,小鼠肠道内容物中异亮氨酸,亮氨酸,缬氨酸,酪氨酸丁二酸、乙二酸,戊酸以及异戊酸含量的变化。糖尿病导致部分氨基酸代谢异常,有两大特征:1.血浆总氨基酸含量、生糖氨基酸含量在糖尿病时降低,而且与血糖呈显著负相关;2.不论血糖控制的好坏,支链氨基酸的含量及其占总氨基酸的比例均是增加。
但是在糖尿病发生前的5-10年前,部分氨基酸含量是升高的,尤其是血液中异亮氨酸、亮氨酸、缬氨酸、酪氨酸和苯丙氨酸等5种氨基酸水平就出现了异常升高的现象。该实验很清楚的显示,尤其是以21天的结果更为明显,这5种氨基酸随着饲料配比的合理化组合发生了明显的改变,基本以显著降低为主。对于防治与降低糖尿病的发病率具有显著性意义。
评估这些氨基酸的指标不但对于预测糖尿病发病风险,而且对于心脏病、肿瘤(肾癌、胰腺癌)也具有实际意义,针对性的及早干预和预防,起到精准性的一级预防作用。
细菌利用低聚糖产生短链脂肪酸,其含量增加,造成肠道内pH值下降。乙酸主要是由拟杆菌门产生的,但丁酸盐主要是由厚壁菌门产生的。丁酸是结肠细胞的主要能量来源,增加肠道健康和潜在的降低肠道通透性和预防代谢性内毒素血症等。喂养14天时,C组分别与A、B小鼠比较发现,丁二酸、乙二酸的含量上升,戊酸的含量下降;喂养21天时发现,乙二酸含量上升,丁二酸和异戊酸含量下降。
3.5肠道内容物16sDNA测定
本实验在小鼠肠道内容物样品中,检测到四个门的细菌,其中拟杆菌门,厚壁菌门和变形菌门是丰度最大的三个门。从图6至9显示的小鼠21天肠道菌群在门水平上的组成,可以看出,在空白组0小鼠中,可以看出拟杆菌门丰度达到65.6%,厚壁菌门31.66%,变形菌门1.44%;
在实验组A组小鼠中,三者丰度分别达到87.84%、2.98%、8.77%;
在实验组B组小鼠中,三者丰度分别达到90.72%,5.93%,2.75%;
在实验组C组小鼠中,三者丰度分别达到85,65%,7.00%,6.45%。
实验组A、B、C与空白组比较发现,拟杆菌门和变形菌门丰度增加;厚壁菌门丰度降低;由此可见,小鼠摄入低聚糖后,促进了拟杆菌门和变形菌门的增殖,厚壁菌门的生长则受到抑制。
本实验在小鼠肠道内容物中,共检测到14个属的细菌比例在0.1%以上;Bacteriodes、Alloprevotella(普雷沃菌属)、Parabacteriodes、Lachnospiraceae(毛螺科菌)、Parasutterella是肠道内容物样品中的主要属;从附图10显示的小鼠21天肠道菌群在属水平上的组成,可以看出:
小鼠摄入实验组A饲料后,肠道内容物中的Bacteriodes显著增加,而B组小鼠肠道内容物中Bacteriodes显著降低,C组Bacteriodes变化则不明显,说明功能性低聚糖组合物具有促进Bacteriodes增殖的作用,普通低聚糖则没有此作用;
B、C组小鼠肠道内容物样品中Alloprevotella、Parabacteriodes增加,A组则降低,说明普通低聚糖有促进Alloprevotella、Parabacteriodes增殖的能力;
与空白组相比,A、B、C三组样品中Lachnospiraceae都降低,而Parasutterella都有所升高;低聚半乳糖和菊粉作为益生元,不被机体消化吸收而直接进入大肠,对调整肠道菌群、维持肠道正常环境、调节肠道功能、提高机体健康水平具有重要作用。聚葡萄糖作为一种水溶性膳食纤维,可以缩短食物在胃内的排空时间,有效改善肠道功能,被摄入人体后,只是到了胃肠道的下半部分被发酵,产生短链脂肪酸如丁酸等,降低了肠道pH值,可以帮助抵抗感染等。从麸皮中提取的不溶性膳食纤维,不能溶解于水又不能被大肠中微生物酵解,仅能减少排泄物在肠道的停留时间,增加肠道内容物的体积,起到了改善微生物繁殖环境和润肠通便的作用。
本实验重点验证功能聚糖和普通聚糖组合物的协同作用,这个协同作用的重点在于两个方面,一是适宜微生物丰度值及微生物生存环境的变化;二是平衡的微生物营养影响宿主生理指标的变化。实验结果表明:各类型聚糖组合物均对小鼠肠道微生态及生理指标有显著的影响变化,其中,包含功能聚糖、普通聚糖组合物的协同作用改善了宿主肠道微生物稳态平衡,对宿主产生了积极作用而没有产生副作用;单独添加功能聚糖组合物或普通聚糖组合物的实验组,对宿主产生积极作用的同时,均阶段性的造成了一定的副作用。
3.6试验结论及分析:
本实验通过研究了不同聚糖组合物对小鼠肠道微生态系统的影响;采用了四种饲料配方喂养小鼠:O组、A组、B组和C组;通过宏基因组分析小鼠肠道内容物菌群发现:不同的膳食饮食对肠道菌群影响很大,低聚半乳糖+菊粉(A组)会导致拟杆菌属显著升高,普雷沃菌属显著降低,肠道菌群的多样性降低;双歧杆菌虽然显著增加,但所占比例较小;聚葡萄糖+不溶性膳食纤维(B组)饲养小鼠,拟杆菌属显著降低,普雷沃菌属和多形拟杆菌属显著升高;低聚半乳糖+菊粉+聚葡萄糖+复合不溶纤维素(C组)饲养小鼠,拟杆菌属、普雷沃菌属等菌属有波动,但变化不显著,说明不同的膳食饮食对肠道菌群影响很大。还通过结肠内容物的pH和血液中SCFA数据表明,低聚糖和膳食纤维发酵产生大量短链脂肪酸,使得pH下降;血清中DAO,TMAO含量和小肠绒毛数据分析发现,C组小鼠表现最佳,说明对正常小鼠来说,聚糖、膳食纤维的多样性有助于肠道微生态系统的健康。
综上,各类型聚糖组合物均对小鼠肠道微生态及生理指标有显著的影响变化,其中,包含功能聚糖、普通聚糖组合物的协同作用改善了宿主肠道微生物稳态平衡,对宿主产生了积极作用而没有产生副作用;单独添加功能聚糖组合物或普通聚糖组合物的实验组,对宿主产生积极作用的同时,均阶段性的造成了一定的副作用。表明平衡膳食不能简单的添加功能聚糖或普通聚糖等营养强化剂及补充剂,而食用包含功能聚糖和普通聚糖的配方组合物的均衡营养才是维护肠道微生态稳态、保障健康的关键。
本发明改善肠道微生态的组合物包含功能聚糖、普通聚糖复合膳食纤维,多种聚糖发生显著的协同作用,对宿主产生积极作用且没有产生负向作用。对肠道有益菌、中性菌提供组合营养及利于其增殖的肠道环境,丰富了肠道菌群的代谢组物,包括对人体有益的初级代谢组物及次级代谢组物,改善了肠道的微生态,达到以改善肠道微生物稳态为靶点,预防慢性疾病的效果;本发明均衡营养食品还同时提供了肠道菌群及人体(超级生物体)的均衡营养,以其方便的主食为载体提供了维护肠道微生态稳态平衡的组合物,有效改善人类躯体亚健康体质,预防危及人类重大健康问题的慢性疾病。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,本领域技术人员利用上述揭示的技术内容做出些许简单修改、等同变化或修饰,均落在本发明的保护范围内。
Claims (13)
1.一种改善肠道微生态预防慢性病的组合物,其特征在于,包括菊粉、低聚半乳糖和复合膳食纤维,其中所述菊粉、低聚半乳糖和复合膳食纤维的配方比为:1~75:1~75:5~95。
2.根据权利要求1所述的改善肠道微生态预防慢性病的组合物,其特征在于,所述菊粉、低聚半乳糖和复合膳食纤维的配方比为:10~50:10~50:25~50。
3.根据权利要求1所述的改善肠道微生态预防慢性病的组合物,其特征在于,所述菊粉为低聚果糖、多聚果糖或低聚果糖与多聚果糖的混合物;所述复合膳食纤维为非益生元的可溶纤维与不溶纤维的混合物,其中,所述可溶纤维与不溶纤维的配方比为10~90:10~90。
4.根据权利要求3所述的改善肠道微生态预防慢性病的组合物,其特征在于,所述非益生元的可溶纤维为抗性糊精、聚葡萄糖、低聚异麦芽糖或其混合物;所述不溶纤维为不溶于水的纤维素、半纤维素、木质素或其混合物。
5.一种包含上述权利要求1至4任一项所述组合物的均衡营养食品,其特征在于,还包括谷物粉,所述谷物粉与所述组合物的配方比为:50~95:50~5。
6.根据权利要求5所述的均衡营养食品,其特征在于,所述谷物粉与所述组合物的配方比为:70~95:30~5。
7.根据权利要求5所述的均衡营养食品,其特征在于,所述谷物粉为小麦面粉、大米粉、玉米粉和马铃薯全粉中一种或多种的组合物,所述均衡营养食品被制备成复方大米、面粉、面条、意面、米线、面包、面包团或面包预拌粉的形式。
8.根据权利要求7所述的均衡营养食品,其特征在于,所述复方大米的制备方法为:将所述谷物粉与所述组合物混合,并采用压制工艺挤压成大米形态,其中所述组合物中的不溶纤维素采用微晶纤维素。
9.根据权利要求1至4任一项所述的组合物在制备预防炎症性肠病的食品或药品中的应用。
10.根据权利要求1至4任一项所述的组合物在制备改善胃肠动力和预防便秘的食品或药品中的应用。
11.根据权利要求1至4任一项所述的组合物在制备预防糖尿病的食品或药品中的应用。
12.根据权利要求1至4任一项所述的组合物在制备预防心脑血管疾病的食品或药品中的应用。
13.根据权利要求1至4任一项所述的组合物在制备调节肠道菌群提高免疫能力的食品或药品中的应用。
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