Analysis of fecal microbiota
Brushes and sheets for stool collection were distributed to the participants, and stool samples were collected before, at week 4, and at week 8 of the intervention. Stool samples were refrigerated, and bacterial DNA was extracted within 3 weeks after collection. Metagenomic analyses of 16S rRNA of the extracted DNA samples were performed using a next-generation sequencer (MiSeq; Illumina K.K., Tokyo, Japan). Bacterial DNA extraction from feces, library preparation, and deep sequencing were performed as previously described [