*2.3. Antioxidant Capacity of the Fermentation Samples*

The DPPH free-radical scavenging ability of the fermentation samples was determined according to a previous method [23].

The determination of ABTS radical cation scavenging activity was based on the method described before [24].

#### *2.4. Gas Volume and pH*

The measurement of gas volume during fermentation was based on a previous study [25]. This was done by emptying the gas in a syringe, and then inserting the syringe needle into the rubber stopper of the anaerobic bottle, allowing the gas produced in the anaerobic bottle to drain into the syringe. After fermentation, the volume of produced gas in each syringe was recorded.

The pH value was performed at 0, 4, 12, and 24 h after starting the colonic incubation, using a pH meter (PHS-3C, Leici, China) with a pH electrode (PY-P22-2S, Sartorius, Germany).

#### *2.5. Determination of Total Carbohydrate Contents*

Total carbohydrate content was determined according to the phenol–sulfuric acid method. We mixed 0.45 mL of 72% concentrated sulfuric acid and lyophilized 1 mL fermented sample in a test tube; then we heated the mixture at 30 ◦C for 1 h. We then added 4.95 mL of distilled water and heated the mixture at 100 ◦C for 3 h to obtain the hydrolyzed liquid for testing. The hydrolyzed sample was diluted at an appropriate dilution factor. A total of 200 μL of the diluted solution and 800 μL of a 2.5% phenol solution were mixed into the test tube. Then, 2.5 mL of concentrated sulfuric acid was added to the tube, and the mixture was mixed well and cooled to room temperature. The absorption value was read at 490 nm, and the total carbohydrate concentration of the fermentation liquid was calculated with a linear regression equation established for glucose.

#### *2.6. Analysis of C3G and Metabolites*

The High-Performance Liquid Chromatography (HPLC) system (Thermo U3000, USA) was used for the chromatographic separation of the C3G and its metabolites in all sample groups during fermentation. Reversed-phase chromatography was performed with a C18 column (4.6 × 250 mm, 5 μm; Thermo, Waltham, MA, USA). The mobile phase was a 2% formic acid aqueous solution (A) and acetonitrile (B). The gradient elution program was as follows: 0–34 min, 6–30% B; 34–35 min, 30–90% B; 35–40 min, 95–95% B; 40–41 min, 95–95% B; and 41–45 min, 6–6% B.

The Ultraviolet (UV) absorption intensity of the eluent was read at 280 and 520 nm. Compared with the peak area of standards for C3G and C3G metabolites, the contents of the C3G and C3G metabolite at different fermentation times were calculated.

#### *2.7. DNA Extraction and 16S rRNA Sequence*

Total genomic DNA was extracted by using a MagPure Soil DNA KF Kit (Catalogue No. D6356-02), and the manufacturer's instructions were followed. Quality and quantity of DNA were verified with NanoDrop and agarose gel. Then, the sample was diluted to 1 ng/μL and stored at −20 ◦C until further processing. Two primer pairs, 343F-5- -TACGGRAGGCAGCAG-3 and 798R-5- -AGGGTATCTAATCCT-3- , were conducted to amplify the V3/V4 region (343–798) of the 16S rRNA gene. For library construction, a 30-μL PCR mixture was prepared as follows: 10–50 ng of DNA template, 1 μL of the forward and reverse PCR primers (5 pmol/μL each), 15 μL 2× Gflex PCR buffer (Takara), and 0.6 μL of Tks Gflex DNA polymerase (1.25 U/μL, Takara). An appropriate volume of double-distilled water (ddH2O) was added to bring the volume up to 30 μL. The PCR program was as follows: initial denaturation at 94 ◦C for 5 min, followed by 26 cycles of denaturation at 94 ◦C for 30 s, annealing at 56 ◦C for 30 s, extension at 72 ◦C for 20 s, and a final extension at 72 ◦C for 5 min.


**2.8 CRABPs (cellular retinoic acid-binding proteins)**

CRABP1 (Cellular Retinoic Acid Binding Protein 1) and CRABP2 (Cellular Retinoic Acid Binding Protein 2) are paralogous genes that encode cellular RA binding proteins. These proteins transport RA to the nucleus and function to regulate the access of RA to the nuclear RA receptors. Specifically, CRABPs are cytosol-to-nuclear shuttling proteins, which facilitate RA binding to its cognate receptor complex and nuclear transfer. These activities in the retinoid signaling pathway play an important role in RA-mediated differentiation and proliferation processes. CRABPs are structurally similar to the cellular retinol-binding proteins, but CRABPs only bind RA, which contributes to RA-directed differentiation in epithelial tissue. Diseases associated with CRABPs include embryonal carcinomas.


**6. Research methodology**

The research methodology encompasses different techniques and procedures that are used to collect and analyze data for research. This is important, since it provides a better view of how the conclusion was made. The methodology covers the population of study, method of data collection and data analysis technique.