*4.5. Microbiota Analysis in Feces*

DNA was extracted from fecal samples for microflora analyses using 16S rRNA high-throughput sequencing. For DNA extraction, the fecal microbiomes were analyzed from 20 mg fecal samples obtained from each animal in the normal as well as fucoidan diet group. For each mouse in either group, total DNA was extracted from a 100 mg fecal sample using QIAamp DNA Stool Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. The DNA concentration was determined using a NanoDrop (Scrum, Tokyo, Japan). Extracted fecal DNA was examined using 16S rDNA gene sequencing by MiSeq (Illumina, Tokyo, Japan). Library preparation, deep sequence, and data analysis were carried out using methods described by Inoue et al [39]. Data analysis was performed by Bio-Linux, a Linux computing platform customized for bioinformatics research [40].

#### *4.6. Quantification of Fecal Mucin and Cecum IgA*

Mucin was extracted from each 100 mg fecal sample. Fecal mucin contents were determined using a fecal mucin assay kit (Mucin Assay Kit, Cosmo Bio, Co., Ltd., Tokyo, Japan). A fluorometric assay discriminated O-linked glycoproteins (mucins) from N-linked glycoproteins. Fluorescence was measured using a SoftMax® Pro spectrometer (Molecular Devices, CA, USA). Total IgA was extracted from 50 mg of cecum samples and quantified using a mouse IgA ELISA quantitation kit (Immundiagnostik, Bensheim, Germany) as specified in the manufacturer's instructions. The reaction products were determined from absorbance at 450 nm using XFluor4 (Tecan, Zurich Switzerland), and IgA was qualified.
