6 5 Vol. 6 No. 5 2015 5 Journal of Food Safety and Quality May., 2015 张芹, 周中凯 *, 任晓冲 (, 300457) 摘要 : 目的 方法 16 C57BL/6J, 1,, 5, 16S rrna V1-V3, Miseq, silva 结果 :, ; ; Firmicutes( ), ; Bacteroidetes( ) ;, Allobaculum, ; Lactobacillus S24-7_norank 结论, 关键词 : ; ; Comparision of intestinal microbiota in mice with normal and high-sugar diet using Miseq high-throughput sequencing ZHANG Qin, ZHOU Zhong-Kai *, REN Xiao-Chong (College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China) ABSTRACT: Objective To compare the composition of fecal microbiota in normal mice and high-sugar diet mice by using Miseq high-throughput sequencing technology. Methods Totally 16 C57BL/6J mice were randomly divided into 2 groups. The feces were collected after a 5-wk experimental period. The fecal bacterial composition was investigated by Miseq high-throughput sequencing of the V1-V3 region of the 16S rrna gene compared with silva databases. Results The results showed that the body weight and blood glucose of mice with high-sugar diet were significantly increased compared to the mice fed normal diet. Bacterial diversity and richness in normal group was higher than that in high-sugar mice. In the phylum level, the relative abundance of Firmicutes was somewhat higher, while the proportion of Bacteroidetes was lower in high-sugar group compared with the normal group. In the genus level, Allobaculum was the most abundant bacterial in both groups, however, there was a significant difference in the percentage of Lactobacillus and S24-7_norank. Conclusion The diet has significant effect on gastro-intestinal microbiota, so we can improve the microbiota 基金项目 : (31471701) (SQ2013ZOA100001) Fund: Supported by the National Natural Science Foundation of China (31471701), the Ministry of Science and Technology China-EU Cooperation Project (SQ2013ZOA100001) * 通讯作者 :,, E-mail: zkzhou@tust.edu.cn *Corresponding author: ZHOU Zhong-Kai, Professor, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China. E-mail: zkzhou@tust.edu.cn
5, : 1777 though regulating the structure of diet. KEY WORDS: high-sugar diet; microbiota; high-throughput sequencing 1 引言,,, [1,2],, [3,4],, [5,6,7],, 16S rrna V1-V3, silva,,,,, 2 材料与方法 2.1 材料 2.1.1 实验动物 C57BL/6J, SPF ( ),, 16.5±0.7 g, 2.1.2 小鼠饲料 : 24% 17% 10% 3% 17% 13% 10% 3% 2% 0.5% 0.5% : 14.2 MJ/kg, 18.84%, : 10% 90% 2.2 方法 2.2.1 样品采集 1 :, 2, 4, ( ) 5, 5,,, 2.2.2 小鼠粪便菌群的基因组 DNA 提取与 PCR 扩增 E.Z.N.A. DNA ( OMEGA ) DNA, DNA, 1% DNA 27F 5 -AGAGTTTGATCCTGGCTCAG-3, 533R 5 -TTAC CGCGGCTGCTGGCAC-3, 16S rrna V1-V3 PCR : 5 FastPfu 4 µl, 5 µmol/l 0.8 µl, 5 µmol/l 0.8 µl, DNA 10 ng, 2.5 mmol/l dntp 2 µl, FastPfu 0.4 µl, dd H 2 O 20 µl PCR : 95 3 min; 26 95 30 s, 55 30 s, 72 45 s; 72 5 min, 10 PCR 2%, 3 µl 2.2.3 生物信息学分析 PCR Illumina Miseq, : Miseq PE reads overlap, reads, reads merge, N polya/t ; barcode ; 200 bp >0 20 : (1) (operational taxonomic units, OTU) :, QIIME, silva 16S
1778 6, 97%, OUT; (2) : OTU, alpha beta, Ace Chao1 Simpson Shannon Ace Chao1 ; Simpson Shannon ; (3) : R (the R project for statistical computing and graphics) 97%, ; (5) : R OTU silva, 80% OTU, OTU silva OTU ; (6) :, 3 结果与分析 3.1 正常组与高糖组体重和血糖值比较 1, 1,, 0.43, 1,, 5,, 3.06 g,,, 2,, 2,, 3.2 小鼠粪便菌群测序质量及多样性统计分析 16S rrna PCR, 3 3, 10860, 97% 167 OTUs, (coverage ) 0.9958 11441, 97% 87 OTUs, 0.9959 OUT,, 0.99, 2 97% 3 Ace Chao1 187 184, Table 1 表 1 正常组与高糖组体重比较 (g) Comparison of body weight between normal group and high-sugar group (g) 1 2 3 4 5 18.38±0.96 19.16±1.12 19.91±0.91 20.27±1.44 21.08±1.09 21.08±1.29 17.95±0.86 19.67±1.12 19.97±0.63 22.84±2.63 23.44±1.89 24.14±2.27 Table 2 表 2 正常组与高糖组餐后血糖值比较 Blood glucose level of normal group and high-sugar group 1 2 3 4 5 8.23±1.24 7.14±0.51 9.69±2.12 9.08±1.56 6.31±0.72 7.41±0.84 7.81±0.71 9.93±0.76 9.38±0.76 11.5±2.87 8.04±1.09 9.09±0.92 表 3 正常组与高糖组微生物丰度及多样性比较 Table 3 High-throughput sequencing data summary OTUs Ace Chao1 Simpson Shannon Coverage 10860 167 187 184 2.43 0.301 0.9958 11441 87 121 113 2.1 0.188 0.9959
5, : 1779 121 113, Ace Chao1,, ; Simpson 2.43, Shannon 0.301, 2.1 0.188, Shannon,, 3.3 微生物群落结构分析 1, Phylum( ), Firmicutes( ) Bacteroidetes ( ), Proteobacteria ( ) Deferribacteres ( ),, 96.1%, 64.14%,,, 2, Genus( ), Allobaculum S24-7_norank Lactobacillus,, 3 85.39%,, 95.21% Blautia Ruminococcaceae_uncultured Lachnospiraceae_uncultured Lachnospiraceae_incertae_ sedis Ruminococcaceae_incertae_sedis, S24-7_norank Blautia Ruminococcaceae_ uncultured Bacteroides Alistipes Desulfovibrio Fig. 1 1 Relative abundance of bacterial phyla in microbiota of normal group and high-sugar group. Others represent the unclassified bacteria
1780 食品安全质量检测学报 Fig. 2 4 讨 第6卷 图 2 正常组和高糖组小鼠粪便菌群在属水平上的分布 Relative abundance of bacterial genus in microbiota of normal group and high-sugar group. Others represent the unclassified bacteria 物从门的水平上可分为 6 大类 Firmicutes(厚壁菌门), 论 Bacteroidetes(拟杆菌门), Proteobacteria(变形菌门), 在本研究中, 采用 10%蔗糖代替基础饲料喂养 Actinobacteria(放线菌门), Fusobacteria(梭杆菌门)和 小鼠, 发现尽管未形成显著差异, 小鼠体重和血糖相 Verrucomicrobia(疣微菌门)组成[10,11], 其中 90%以上 比较全部采食基础饲料的小鼠有明显增加 该研究与 由厚壁菌门(Firmicutes)和拟杆菌门( Bacteroides)构 的报道一致 表明高能 成[12] 本研究中, 正常组在门水平上主要包含厚壁菌 饲料可导致小鼠血糖升高, 并具有诱发肥胖的危险 门 拟杆菌门从微生物的结构来看, 在门(phyla)的分 从粪便微生物群落丰度和结构上来看, 正常小 类水平上, 高糖组厚壁菌门数量显著高于正常饮食 Baxendale 等和 Kuroda 等 [8,9] 鼠粪便菌群与高糖小鼠的粪便菌群有着显著的差异 小鼠; 而拟杆菌门显著低于正常小鼠 本研究与 Ley 正常组小鼠粪便中微生物的丰度和多样性较高糖小 等[3]的研究结果一致, Ley 等对肥胖小鼠和非肥胖小 鼠粪便菌群高 粪便菌群可代表整个消化道菌群的状 鼠肠道内 5000 多种细菌的 16s rrna 基因序列进行分 况, 本研究结果说明正常小鼠肠道菌群的物种总数 析发现, 肥胖小鼠拟杆菌门的数量少 50, 同时具 远高于高糖小鼠的肠道菌群 有更多的厚壁菌门细菌, 同时他们也对肥胖和非肥 肠道微生物种类繁多, 健康的人类肠道中微生 胖的人的肠道菌群做了研究, 通过对 12 名肥胖患者
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1782 6 2003, 69: 4743 4752. 作者简介 [15] Resta SC. Effects of probiotics and commensals on intestinal epithelial physiology: implications for nutrient handling [J]. J Physiol, 2009, 587: 4169 4174. 张芹, 助理研究员, 主要研究方向为动物营养 E-mail: zhangq8201@tust.edu.cn [16] Attene-Ramos MS, Nava GM, Muellner MG, et al. DNA damage and toxicogenomic analyses of hydrogen sulfi de in human intestinal epithelial FHs 74 Int cells [J]. Environ Mol Mutagen, 2010, 51: 304 314. 周中凯, 教授, 主要研究方向为食品营养与加工 E-mail: zkzhou@tust.edu.cn ( 责任编辑 : 白洪健 ) 现代发酵工程在食品工业中的应用 专题征稿,,,,,,,,,,,,,, :,,,,,,,, 2015 7,, 2015 6 15 E-mail, 投稿方式 : : www.chinafoodj.com E-mail: jfoodsq@126.com 食品安全质量检测学报 编辑部