CN112980712A - 一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用 - Google Patents
一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用 Download PDFInfo
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Abstract
本发明涉及一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用。本发明研究了高原汉族人群和平原汉族人群肠道微生物菌群的差异,发现Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis四种菌种在高原汉族人群和平原汉族人群之间差异显著,并且这四种菌种均与和人群情绪有关的临床指标密切相关,因此本发明提供了一种包含上述四种菌种的微生物复合物,通过施用该微生物复合物,可针对性的稳定高原汉族人群的情绪和肠道微生态环境。
Description
技术领域:
本发明涉及一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用,属于生物医药技术领域。
背景技术:
人体肠道中存在大量的共生菌群,其数量超过1000万亿个,大约是人体细胞总数的10倍。同时肠道内微生物基因的数量约为300万个,大约是人类基因组基因数量的100多倍,如此海量的微生物形成了与人体密不可分的共生关系,能够帮助人群适应多变的环境。人体内微生态环境与人体健康密切相关,微生态环境的改变会影响人体的临床指标。维持人体内微生态环境的稳定有利于维护人体的身体健康。可以通过补充益生菌来帮助维护肠道微生态环境的稳定,预防和治疗疾病,提高人体机能。
文献“Colicins produced by the Escherichia fergusonii strains closelyresemble colicins encoded by Escherichia coli”报道Escherichia fergusonii菌种有助于分泌大肠杆菌素,而大肠杆菌素有助于调节人体肠道菌群数量。
迄今为止,尚未见肠道中同时补充Escherichia fergusonii,LactobacillusPlantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis四种菌种稳定高原人群情绪及肠道菌群的报道。
发明内容:
针对现有技术的不足,本发明提供了一种调节高原人群情绪及肠道菌群稳定的微生物组合物及其应用,本发明中的微生物组合物用于帮助高原人群稳定情绪和肠道的微生态环境。
术语说明:
Escherichia fergusonii:费格森埃希菌,
Lactobacillus plantarum:植物乳杆菌,
Bifidobacterium animalis subsp.Lactis:动物双歧杆菌乳亚种,
Enterococcus faecalis:粪肠球菌。
本发明的技术方案如下:
一种调节高原人群情绪及肠道菌群稳定的微生物组合物,所述微生物组合物包括安全有效剂量的Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacteriumanimalis subsp.lactis和Enterococcus faecalis四种菌种。
根据本发明优选的,所述微生物组合物还包括食品上或药学上可接受的载体。
根据本发明优选的,所述高原人群为汉族人群。
根据本发明优选的,所述微生物组合物为口服制剂。
根据本发明优选的,所述微生物组合物为粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、舌下含片、或其组合。
上述微生物组合物在调节高原人群情绪及肠道菌群稳定中的应用。
根据本发明优选的,所述高原人群为汉族人群。
一种调节高原人群情绪及肠道菌群稳定的方法,所述方法为给高原人群施用上述微生物组合物。
根据本发明优选的,所述高原人群为汉族人群。
有益效果:
本发明研究了高原汉族人群和平原汉族人群肠道微生物菌群的差异,发现Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalissubsp.lactis和Enterococcus faecalis四种菌种在高原汉族人群和平原汉族人群之间差异显著,并且这四种菌种均与和人群情绪有关的临床指标密切相关,因此本发明提供了一种包含Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalissubsp.lactis和Enterococcus faecalis四种菌种的微生物复合物,通过施用该微生物复合物,可针对性的稳定高原汉族人群的情绪和肠道微生态环境。
附图说明
图1为三组人群肠道中Escherichia fergusonii菌种丰度的箱式图;图中,Han1000为平原汉族人群,H4A4为一周内的高原汉族人群,H4LB为两个月以上的高原汉族人群;
图2为三组人群肠道中Lactobacillus Paraplantarum菌种丰度的箱式图;图中,Han1000为平原汉族人群,H4A4为一周内的高原汉族人群,H4LB为两个月以上的高原汉族人群;
图3为三组人群肠道中Bifidobacterium animalis subsp.lactis菌种丰度的箱式图;图中,Han1000为平原汉族人群,H4A4为一周内的高原汉族人群,H4LB为两个月以上的高原汉族人群;
图4为三组人群肠道中Enterococcus faecalis菌种丰度的箱式图;图中,Han1000为平原汉族人群,H4A4为一周内的高原汉族人群,H4LB为两个月以上的高原汉族人群;
图5为三组人群临床指标CK(肌酸激酶)的箱式图;图中,Han1000为平原汉族人群,H4A4为一周内的高原汉族人群,H4LB为两个月以上的高原汉族人群;
图6为三组人群临床指标GLU(血糖)的箱式图;图中,Han1000为平原汉族人群,H4A4为一周内的高原汉族人群,H4LB为两个月以上的高原汉族人群。
具体实施方式:
下面结合实施例对本发明作进一步说明,但本发明的保护范围并不仅限于此。
本发明中涉及的微生物均为现有菌种,可从市场购得,不涉及微生物保藏。
研究对象:根据STROBE声明设计病例对照研究,纳入某年某月至某年某月在中国某部队的45个在海拔3500米高原以上服役一周以内的汉族男性士兵为实验组1,22个在海拔3500米高原以上服役两个月以上的汉族男性士兵为实验组2,根据实验组士兵年龄匹配96个平原汉族士兵进行对照。严格控制三组人群进行相同的中国部队混合饮食,保持相同的训练环境和训练强度,戒烟、酒,持续3个月。排除有慢性炎性疾病、口服抗生素、急性感染和胃肠道疾病的士兵。
每一个研究对象在纳入后均收到一个马桶粪便收集器,在排出大便后将大便装入粪便收集管,即刻保存在-20℃冰箱,次日将粪便标本转运至-80℃冰箱保存,统一进行检测。
实施例1平原汉族人群和高原汉族人群差异菌群的确定
1.1粪便样品DNA提取
吸取1000μL CTAB裂解液至2.0mL EP管中,加入溶菌酶,将500μL的粪便样品加入裂解液中,65℃水浴20min,期间颠倒混匀数次,以使样品充分裂解。离心取上清,加等体积苯酚(pH8.0):氯仿:异戊醇(25:24:1体积比)混合液,颠倒混匀,12000rpm离心10min,取上清,加等体积氯仿:异戊醇(24:1体积比)混合液,颠倒混匀,12000rpm离心10min,吸取上清至1.5mL离心管中,加入0.8倍体积的异丙醇,上下摇晃,-20℃沉淀过夜。12000rpm离心10分钟,倒出液体,注意不要倒出沉淀。用1mL 75%乙醇洗涤沉淀2次,超净工作台吹干或者室温晾干。加入ddH2O溶解DNA样品,加RNase A 1μL消化RNA,37℃放置15min。之后利用琼脂糖凝胶电泳检测DNA的纯度和浓度,取适量样本DNA于离心管中,使用无菌水稀释样本至1ng/μL。
1.2PCR扩增、混样和纯化
以稀释至1ng/μL的基因组DNA为模板,根据测序区域16S V3-V4,选择使用带Barcode的特异引物515F和806R,New England Biolabs公司的High-FidelityPCR Master Mix with GC Buffer,和高效高保真酶进行PCR,确保扩增效率和准确性。
PCR产物使用2%的琼脂糖凝胶进行电泳检测;根据PCR产物浓度进行等量混样,充分混匀后使用1×TAE、2%的琼脂糖凝胶电泳纯化PCR产物,切胶回收目标条带。产物纯化试剂盒使用的是Thermo Scientific公司GeneJET胶回收试剂盒回收产物。
1.3文库构建和上机测序
使用Thermo fisher公司的Ion Plus Fragment Library Kit 48rxns建库试剂盒进行文库的构建,构建好的文库经过Qubit定量和文库检测合格后,使用Thermo fisher的Ion S5TMXL进行上机测序。
1.4数据分析
1.4.1测序数据处理
使用Cutadapt(V1.9.1,http://cutadapt.readthedocs.io/en/stable/)先对reads进行低质量部分剪切,再根据Barcode从得到的reads中拆分出各样品数据,截去Barcode和引物序列初步质控得到原始数据,经过以上处理后得到的Reads需要进行去除嵌合体序列的处理,Reads序列通过与物种注释数据库进行比对,检测嵌合体序列,并最终去除其中的嵌合体序列,得到最终的有效数据。
1.4.2OTU聚类和物种注释
利用Uparse软件(Uparse v7.0.1001,http://www.drive5.com/uparse/)对所有样品的全部有效数据进行聚类,默认以97%的一致性将序列聚类成为OTUs(OperationalTaxonomic Units),同时会选取OTUs的代表性序列,依据其算法原则,筛选的是OTUs中出现频数最高的序列作为OTUs的代表序列。对OTUs序列进行物种注释,用Usearch方法与rdp_16s_sp.udb数据库进行物种注释分析(设定阈值为0.8~1),获得分类学信息并分别在属水平统计各样本的群落组成。
1.4.3菌属丰度计算和差异比较
用the Chao1估计量表示菌属丰度,绘制箱图对比三组人群中相关菌属/菌种丰度的差异,差异比较因为样本非正态分布采用Kruskal-Wallis检验,P<0.05为差异有统计学意义。
1.5测序结果
平原汉族人群和高原汉族人群粪便样本中Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcusfaecalis 4种菌种丰度差异较大。
三组人群中Escherichia fergusonii和Bifidobacterium animalissubsp.lactis菌种的五分位数结果如图1和图2所示,比较中位数发现,Escherichiafergusonii和Bifidobacterium animalis subsp.lactis菌种都是在平原汉族人群中含量最高,上高原后降低,Kruskal-Wallis数值显示三组数据有明显差异;
三组人群中Lactobacillus Plantarum和Enterococcus faecalis菌种的五分位数结果如图3和图4所示,Lactobacillus Plantarum和Enterococcus faecalis菌种相对丰度的中位数在三组人群中没有明显差别,但是均值是在汉族平原人群中最高,上高原后显著降低,Kruskal-Wallis数值显示三组数据有明显差异。
实施例2平原汉族人群和高原汉族人群临床指标的确定
临床指标是由血液样本测得。血液样本是用EDTA-K2采集晨空腹静脉血(8mL)。血样以4000r/min离心10min,分离血浆,-80℃保存至301医院(北京,海拔约40米)测定。使用血液学分析仪(Roche-cobas6000,美国)测定76项临床试验项目,使用2毫升血浆。此外,1.5mL血浆用于11种酶联免疫吸附试验(ELISA)(Expand Bio,China)。临床数据共涵盖76项指标。
图5-图6显示了临床指标中CK(肌酸激酶)和GLU(血糖)的五分位数结果。根据临床研究,CK(肌酸激酶)与人体中的血清素的水平有关,而血清素的异常会导致抑郁症等精神疾病,而GLU(血糖)水平降低也会导致情绪低落。Kruskal-Wallis数值显示,CK(肌酸激酶)和GLU(血糖)水平在三组人群中均有明显差异。CK(肌酸激酶)在刚上高原的人群中出现降低,在上高原一段时间后的人群体内出现回升,可能与高原适应有关;上高原后的汉族人群体内的GLU(血糖)水平会下降,相对平原汉族人体中的血糖水平表现出不稳定。
实施例3菌群与临床指标之间的关系
采用R软件进行spearman关联分析,研究Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcusfaecalis4种菌种与临床指标之间的关联关系,研究结果如表1所示。
表1. 4种菌种与临床指标的spearman关联分析结果
菌种 | CK(肌酸激酶) | GLU(葡萄糖) |
Escherichia fergusonii | 正相关 | 正相关 |
Lactobacillus Plantarum | 正相关 | 正相关 |
Bifidobacterium animalis subsp.lactis | 正相关 | 正相关 |
Enterococcus faecalis | 正相关 | 正相关 |
以上结果显示,Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis 4种菌种与CK(肌酸激酶)和GLU(葡萄糖)之间有较强关联关系,并且都呈正相关。因此补充这四种菌种可以稳定上高原后CK(肌酸激酶)和GLU(葡萄糖)水平,并帮助调节人体的精神状态。
结合实施例2和实施例3可知,Escherichia fergusonii,LactobacillusPlantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis四种菌种的组合物有助于稳定人体内肠道微生态环境稳定,并进一步帮助稳定临床指标,帮助高原汉族人群稳定情绪。
Claims (9)
1.一种调节高原人群情绪及肠道菌群稳定的微生物组合物,其特征在于,所述微生物组合物包括安全有效剂量的Escherichia fergusonii,Lactobacillus Plantarum,Bifidobacterium animalis subsp.lactis和Enterococcus faecalis四种菌种。
2.如权利要求1所述的微生物组合物,其特征在于,所述微生物组合物还包括食品上或药学上可接受的载体。
3.如权利要求1所述的微生物组合物,其特征在于,所述高原人群为汉族人群。
4.如权利要求1所述的微生物组合物,其特征在于,所述微生物组合物为口服制剂。
5.如权利要求1所述的微生物组合物,其特征在于,所述微生物组合物为粉末剂、散剂、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂、舌下含片、或其组合。
6.权利要求1所述的微生物组合物在调节高原人群情绪及肠道菌群稳定中的应用。
7.如权利要求6所述的应用,其特征在于,所述高原人群为汉族人群。
8.一种调节高原人群情绪及肠道菌群稳定的方法,其特征在于,所述方法为给高原人群施用权利要求1所述的微生物组合物。
9.如权利要求8所述的方法,其特征在于,所述高原人群为汉族人群。
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