*2.2. The Fermentation of Okara-HPIDF*

The preparation method of simulated intestinal fluid (SIF) was used following the United States Pharmacopeia (USP) [19]. Enzyme-free SIF was prepared in a sterile environment and sterilized at 121 °C for 20 min.

The collection method of feces was similar to the previous study [16] with minor changes: 10 g of feces was collected from C57BL/6 female mice (Beijing Vital River Laboratory Animal Technology Co. Ltd., Beijing, China), which was dissolved in 50 mL phosphate buffer solution (0.1 mol/L, pH = 6.5). After complete mixing, the residue was removed

via filtration with four layers of gauze. The suspension was stored at −20 °C in a sterile centrifuge (finished in 1 h).

After the preparation of SIF and the extraction of colonic microorganisms, they were mixed in a ratio of 7:2 (*v*/*v*). The simulated intestinal fluid with a colonic microorganism (SIF-CM) was used as the fermentation environment for HPIDF. The SIF-CM solution was prepared just before use.

#### *2.3. Bacterial 16S rDNA Sequencing*

Feces collected in 2.2 were used for 16S rDNA sequencing. DNA extraction from feces was carried out using the QIAamp® DNA Stool Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions. The quality of isolated DNA was evaluated on agarose gel electrophoresis and then DNA concentration was precisely measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA).

The V3-V4 region of the 16S rRNA gene was amplified from the purified DNA of each sample. The primers used for the PCR amplification are as follows: the forward primer 515F (5- -ACTCCTACGGGAGGCAGCA-3- ) and the reverse primer 907R (5- -GGACTACHVGGGTWTCTAAT-3'). The concentrations of PCR products were measured by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Afterward, all PCR products were pooled in equal amounts, and paired-end sequencing was performed using the Illumina MiSeq platform operated by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

#### *2.4. Simulated Fermentation of Okara-HPIDF In Vitro*

After mixing SIF-CM and HPIDF (9:1, *v*/*m*) under sterile conditions, the samples were cultured under anaerobic conditions at 37 °C for 16 h. After fermentation, the samples were filtered, the IDF was taken to determine the contents of the composition of monosaccharide after sterilization. The fermented-HPIDF (F-HPIDF) was used for the follow-up experiments after sterilization and freeze-drying.

#### *2.5. Structure of Okara-HPIDF before/after Fermentation*

#### 2.5.1. Scanning Electron Microscopy (SEM)

Merlin Compact scanning electron microscope (SEM; Carl Zeiss Jena GmbH, Jena, Germany) was used to observed the microstructure of HPIDF/F-HPIDF after the spray gold treatment. The scanning images were captured at accelerating voltages of 5.00 kV. All images were recorded at magnifications of 300× (low magnification) and 3000× (high magnification).

#### 2.5.2. Fourier Infrared Spectrum (FT-IR)

The polysaccharide functional group composition of HPIDF/F-HPIDF were performed using a Nicolet iS5 FT-IR spectrometer (Thermo Fisher, Waltham, MA, USA). The spectra were read over the range of 4000–400 cm−<sup>1</sup> with a resolution of 4 cm−<sup>1</sup> after the samples were mixed with KBr (1:100, *w*/*w*)
