Zircoprep mini kit

A Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) and a Superoxide Dismutase Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) were used to measure bacterial H 2 O 2 and SOD levels, respectively, in accordance with the manufacturer's instructions. In brief, the bacterial cell suspensions described above were treated with the NTAAP device for 5 min (S. mutans), 1 min (P. gingivalis), and 7 min (E. faecalis). The treatment distance was fixed at 1 mm. After NTAAP treatment, the bacteria were lysed with a ZircoPrep Mini Kit (Nippon Genetics Co., Ltd., Tokyo, Japan). For H 2 O 2 quantification, the H 2 O 2 detection solution was mixed with the bacterial lysate and incubated at room temperature for 30 min. Sample densities were measured using a spectrometer (TriStar LB 941, Berthold Technologies, Bad Wildbad, Germany) at 570 nm. For SOD quantification, the SOD detection solution was mixed with the bacterial lysate and incubated in a plate shaker for 20 min at room temperature. Absorbance was then measured at 405 nm using a spectrometer. The protein content of the bacterial lysate was also determined quantitatively using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), and both the H 2 O 2 and the SOD levels were normalized to the protein content.

DNA was extracted from fresh faeces of mice in the control, DSS and DSS + 10 % psyllium groups using the QIAamp DNA Stool mini kit (Qiagen) and the ZircoPrep mini kit (Nippon Genetics), according to the manufacturer's instructions. The DSS + 5 % psyllium group was omitted in this analysis because the 5 and 10 % psyllium diets showed similar protective effects on DSSinduced colitis. The V2-V3 region of the 16S rRNA gene from the DNA samples was amplified with universal primers: HAD-1 GC (CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T) and HAD-2 (GTA TTA CCG CGG CTG CTG GCA C). The denaturing gradient gel electrophoresis (DGGE) analysis was conducted as previously described (26) . The gel was silver-stained with Bio-Rad silver stain and densitograms of the DGGE bands were created via Image J software. Results of the hierarchical cluster analysis of the DGGE band patterns were displayed as a dendrogram via R software (The R Foundation for Statistical Computing). The squared distances between clusters were measured by Ward's method (27) .