2.5 Microbiome Assessment

Sampling time of intestinal contents was on the morning after 10-week intervention and overnight fasting and water restriction. The intestinal contents were collected from the middle section of colon after anesthesia and laparotomy. Fecal samples were collected from all groups for microbiota 16S rRNA analysis. The microflora were detected by Lianchuan Biotechnology Co., Ltd. (Hangzhou, China). Bacterial genomic DNA was obtained from frozen colon contents using the QIAamp DNA Stool Mini Kit (51504; Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions. Successful DNA isolation was confirmed by agarose gel electrophoresis. DNA samples were amplified by polymerase chain reaction (PCR) using bar‐coded primers flanking the V3–V4 region of the 16S rRNA gene. PCR was performed using a thermocycler under the following conditions: 1 pre-denaturation cycle at 94°C for 4 min, 25 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 45 s, elongation at 72°C for 30 s, and 1 post-elongation cycle at 72°C for 5 min. The PCR amplicon products were separated on 0.8% agarose gels and extracted. Only PCR products without primer dimers and contaminant bands were used for sequencing by synthesis. High‐throughput pyrosequencing of the PCR products was performed using the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA). Paired‐end reads of the original DNA fragments were excluded from the analysis if they did not well-match a 12‐base Golay barcode (one or no errors), the read overlap was < 35 bases, the overlapped region differed by more than 15%, or there were more than three base calls below Q20. Bacterial operational taxonomic units (OTUs) were created by clustering the reads at 97% identity in the Quantitative Insights into Microbial Ecology (QIIME; http://qiime.org/scripts/pick_otus.html) database.