【作者】 王瑞；

【导师】 葛武鹏；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2018， 硕士

【摘要】 肠道菌群对婴幼儿发育及免疫系统构建至关重要,与成人健康及疾病亦关系密切。婴幼儿期（初生1000 d）是肠道菌群构建定殖的关键时期,此阶段肠道菌群的定殖多样性影响着婴幼儿免疫系统构建及未来成长的健康状况。本研究采集陕西关中地区健康婴幼儿（Ⅰ组（0⁶月龄）、Ⅱ组（6¹2月龄）、Ⅲ组（12³6月龄））粪便样品,通过高通量16S rRNA、末端限制性片段长度多态性分析技术（T-RFLP）、实时荧光定量PCR技术（QPCR）分析婴幼儿肠道微生物多样性。旨在探究初生婴幼儿肠道菌群多样性、及以双歧杆菌和乳杆菌为代表的优势菌种群构成及差异,为揭示生命早期肠道微生态与健康之间的关系、婴幼儿健康评价及婴配食品的开发提供参考依据。主要结论如下:（1）高通量16S rRNA探究婴幼儿肠道菌群多样性结果:（1）36份样品中,Ⅰ、Ⅱ、Ⅲ组在门水平上即出现差异,Ⅰ、Ⅱ组样品在门、纲、目、科及属分类水平上的优势菌均相同,组间对应水平下的优势菌丰度均无显著性差异（p>0.05）,在属分类水平的优势菌为双歧杆菌属Bifidobacterium（56.32%、58.62%）;Ⅲ组样品菌群在属分类水平的优势菌为双歧杆菌属Bifidobacterium（25.09%）和柔嫩菌属Faecalibacteri-um（19.22%）;（2）在Ⅰ、Ⅱ、Ⅲ组中组间比较,差异菌群构成不尽相同;（3）在门水平上不同性别、不同分娩方式的Ⅰ组及Ⅱ组婴幼儿肠道菌群无显著性差异（p>0.05）;而不同性别的Ⅲ组（顺产）幼儿和不同分娩方式的Ⅲ组（女）幼儿肠道菌群在门水平上存在显著性差异的菌群均是拟杆菌门,且其在女婴组丰度显著高于男婴组（p<0.05）,在顺产组丰度显著高于剖宫产组（p<0.05）。（2）T-RFLP与QPCR技术相结合评价双歧杆菌种群多样性表明:（1）T-RFLP结果表明Ⅰ、Ⅱ、Ⅲ组中均包括长双歧杆菌（B.longum）、短双歧杆菌（B.breve）、两歧双歧杆菌（B.bifidum）、动物双歧杆菌（B.animalis）及假链状双歧杆菌（B.pse-udocatenulatum）5种菌种,在Ⅲ组样品中还有青春双歧杆菌（B.adolescentis）;QPCR结果表明三组样品中该6种双歧杆菌均被检出;（2）Ⅰ、Ⅱ、Ⅲ组样品中双歧杆菌属的DNA含量均在10¹⁰ copies/g水平,且Ⅰ组样品中的含量(10^10.44 copies/g)显著高于Ⅱ、Ⅲ组样品（p<0.05）;（3）Ⅰ、Ⅱ组样品中B.longum和B.breve的丰度较高,Ⅰ组中对应的丰度分别为39.81%、30.90%,Ⅱ组中对应的丰度分别为35.48%、40.07%,Ⅲ组中丰度前3位的分别是B.longum、B.breve和B.bifidum,分别为28.84%、27.54%、34.67%。（3）QPCR探究婴幼儿肠道乳杆菌种群多样性表明:（1）不同阶段婴幼儿样品中均包括乳杆菌属（Lactobacillus）、干酪乳杆菌种（Lactobacillus casei）和短乳杆菌种（Lactobacillus brevis）,Ⅰ组中检测到同种乳杆菌种的样品量级少于Ⅱ、Ⅲ组;（2）三组样品中Lactobacillus的含量均在10⁸.5.5 copies/g水平,三组间无显著性差异（p>0.05）,罗伊氏乳杆菌（Lactobacillus reutei）和L.casei均在三组样品中含量较高。（3）Ⅰ、Ⅱ、Ⅲ组中优势乳杆菌均为L.casei和L.reuter,Ⅰ组中对应的丰度分别为67.61%、27.54.%,Ⅱ组分别为48.98%、43.65%,Ⅲ组分别为33.88%、56.23%。（4）Ⅰ、Ⅱ、Ⅲ组婴幼儿肠道菌群多样性及分布特征存在显著性差异（p<0.05）,双歧杆菌和乳杆菌的种群组间构成差异不明显,但丰度表达存在一定差异（p<0.05）。更多还原

【Abstract】 Intestinal flora is crucial for the development of infants and it’s immune system construction.It is also closely linked to adult health and disease.Childhood(within 1000 days after birth)is a key period for colonization and establishment of intestinal flora.The colonization diversity of the intestinal flora at this stage influences the establishment of the infant’s immune system and the health status in the future.This study collected the stool samples of healthy infants divided into 3 stages(group Ⅰ(0~6 months),group Ⅱ(6~12 months),and group Ⅲ(12~36 months)to analyze microbial diversity by high throughput 16 S r RNA,T-RFLP and QPCR technology.The purpose of this study was to explore the diversity of intestinal flora and the composition and diversity of dominant bacteria represented by Bifidobacterium and Lactobacillus of infants,which would provide a reference for revealing the relationship between intestinal microecology and health in early life,assessment of infant health and the development of infant formulae.The main conclusions were as follows:(1)The results of the intestinal microbiota diversity in the infants with different months by high throughput 16 S r RNA sequencing technology showed that:(1)In 36 infant samples,Ⅰ,Ⅱ,and Ⅲ groups showed differences at the level of the phylum,and the dominant bacteria at the level of phylum,class,order,family,and genus were same in the group Ⅰ and Ⅱ whose dominant genus was Bifidobacterium(56.32%,58.62%).In addition,there was no significant difference in the abundance of dominant bacteria at the corresponding classification level between the two groups(p>0.05).The predominant bacteria at the the level of genus were Bifidobacterium(25.09%)and Faecalibacterium(19.22%)in the group Ⅲ;(2)The bacterias whose abundance caused differences between the three groups were varied;(3)There was no significant differences in gut microbiota at the level of phylum between group Ⅰ and group Ⅱ with different genders and delivery methods(p>0.05),however,there were significant differences in the intestinal flora of group Ⅲ(vaginal delivery method)infants with different sex and those of group Ⅲ(female)infants with different delivery methods,and the different floras were all Bacteroidetes whose abundance in the girl group was significantly higher than that of the boy group and in the vaginal delivery group was significantly higher than that of the caesarean section group(p<0.05).(2)Using T-RFLP and QPCR techniques to evaluate the population diversity of Bifidobacterium showed that:(1)The result of T-RFLP showed that there were Bifidobacterium longum,Bifidobacterium breve,Bifidobacterium bifidum,Bifidobacterium animalis,and Bifidobacterium pseudocatenulatum in all groups,and there was also Bifidobacterium adolescentis in the group Ⅲ.However,the result of QPCR showed that there were 6 species in the three groups of samples;(2)The DNA content of Bifidobacterium in theⅠ,Ⅱand Ⅲ groups were about 1010 copies/g,and the content of group Ⅰ(1010.44 copies/g)was significantly higher than that of group Ⅱ and Ⅲ(p<0.05);(3)The abundance of Bifidobacterium longum and Bifidobacterium breve in the samples of Ⅰ and Ⅱ groups were higher,and the corresponding abundance in the Ⅰ and Ⅱ group were 39.8 1%,30.90% and 35.48%,27.54% respectively.The top three abundance of species in the group Ⅲ were Bifidobacterium longum,Bifidobacterium breve and Bifidobacterium bifidum,which were 28.84%,27.54% and 34.67% respectively.(3)The results about the diversity of Lactobacillus of infants’ samples determined by QPCR showed that:(1)Lactobacillus,Lactobacillus breve and Lactobacillus casei were detected in all of infants’ samples.The number of same Lactobacillus species detected in the group Ⅰ samples was lower than that in the group Ⅱ and Ⅲ;(2)The contents of Lactobacillus in the three groups were all around 108.5 copies/g,and there was no significant difference between the three groups(p<0.05).The contents of both Lactobacillus reuteri and Lactobacillus casei were higher in the three groups;(3)The Lactobacillus casei and Lactobacillus reuteri were the most prevalent Lactobacillus species in the group Ⅰ,Ⅱ and Ⅲ.The corresponding abundances in the group Ⅰ were 67.61%,and 27.54%,in the group Ⅱ were 48.98% and 43.65%,in the group Ⅲ were 33.88% and 56.23%.(4)There were significant differences in the diversity and distribution of intestinal microflora in infants aged 0~6 months,6~12 months and 12~36 months(p<0.05).The population composition of Bifidobacterium and Lactobacillus were not significantly different,however,there was a certain difference in abundance expression(p<0.05).更多还原