*2.9. Western Blot Analyses*

RIPA lysis buffer combined with a protease inhibitor cocktail was used to lyse liver samples (Beyotime Biotechnology, Shanghai, China). The lysates were centrifuged for 20 min at 12,000 rpm at 4 ◦C, the supernatants were collected, and the protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). SDS-PAGE was used to separate the protein lysates, which were then transferred to PVDF membranes. The blotted membranes were blocked for 2 h with 5% skimmed milk, then washed and incubated overnight at 4 ◦C with antibodies against CYP7A1, HMG-CoA, LDL-R. The bands were seen using an enhanced chemiluminescence reagent and assessed in an iBright CL1000 imaging system after incubation with the relevant primary and secondary antibodies (Invitrogen, Singapore). The expression of β-actin was used as a control for normalizing protein expressions.

#### *2.10. Gut Microbiota Sequencing Analysis*

For the sequencing of cecal contents for microflora sequencing, the QIAamp® DNA Stool Mini Kit (QIAGEN, Hilden, Germany) was used to extract DNA from feces according to the manufacturer's instructions. The quality of extracted DNAs was assessed using an agarose gel electrophoresis and subsequently a NanoDrop NC2000 spectrophotometer to precisely measure the DNA concentration (Thermo Fisher, Waltham, MA, USA).

Each sample's pure DNA was used to amplify the V3–V4 regions of the 16S rRNA gene. With forward primers, 338F (5- -ACTCCTACGGGAGGCAGCA-3- ) and the reverse primer 806R (5- -GGACTACHVGGGTWTCTAAT-3- ) were utilized in the PCR amplification. The Data science PicoGreen dsDNA Assay Kit was used to determine the concentrations of PCR products (Invitrogen, Carlsbad, CA, USA). Following that, all of the PCR results were pooled in equal proportions, and paired-end sequencing was conducted using the Illumina MiSeq platform, which is run by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

#### *2.11. Statistical Analysis*

The trials were conducted three times, and the findings were presented as mean, standard deviations (SD). GraphPad Prism 6 was used for statistical analysis (GraphPad Software, Inc., California, CA, USA). Using IBM SPSS 25.0 (SPSS Inc., Chicago, IL, USA), were evaluated using one-way analysis of variance (ANOVA), and *p* < 0.05 was considered to be statistically significant.

#### **3. Results**

#### *3.1. Determination of the Basic Components of the Three OIDFs*

As shown in Table 2, the protein level of OIDF reduced from 1.85% to 1.63% after it was digested by DHPM. The TDF content of OIDF-10 increased, as did the TDF and SDF (soluble dietary fiber) content, which could be attributed to the fact that after high-pressure treatment, some insoluble hemicellulose, cellulose, and pectin, etc., link bonds are broken, the structure is destroyed, and the molecules are converted into soluble short-chain small

molecules. This could be owing to the DF's intricate structure. DF and protein particles will agglomerate to form a whole in most cases [23]. They are subjected to various strong effects such as pressure, shear force, and friction force during the high-pressure microfluidic process, resulting in part of the protein being converted into a free state and released, so it gradually increases with the increase in pressure. During the particle size reduction process, the gliadin protein is lost, and the protein content is reduced.



Different letters in the same column (a, b, c) are significantly different (*p* < 0.05). The results are expressed as mean ± SD (*n* = 3).
