*3.2. Structure of the Three Different Granularity of OIDFs*

From Figure 1a, with regard to particle size analysis: the particle size distribution curves of the three OIDFs were all unimodal, and the particle size distribution of OIDF-10 is relatively concentrated, with a higher peak and a normal distribution. When the average particle size of OIDF is smaller and more uniform, the difference in its physical and chemical properties is noticeable.

From Figure 1b, as shown in the SEM figure, the surface of OIDF-100 processed by 10 MPa pressure processing is relatively smooth, the structure is compact and complete, but the particle size is not uniform, and there are large flake particles. The surface of OIDF-50 after high-pressure treatment at 50 MPa is rough, curled in many places, and the structure is partially damaged, which is related to the uneven force during grinding [24], The surface of the sample OIDF-10 is very different from OIDF-50 and OIDF-100, the particle size is significantly reduced, the surface is flocculent, becomes loose and porous, the internal structure is exposed, and there is a uniform accumulation phenomenon.

From Figure 1c, the FT-IR spectrum shows that the three particle sizes of OIDF have similar characteristic curve peaks, the broad peak at 3420 cm−<sup>1</sup> is the stretching vibration peak of hydroxyl (-OH), and hydrogen bonds in cellulose and hemicellulose [25] are a characteristic band of all cellulose. The absorption peak at 2927 cm−<sup>1</sup> is the stretching vibration peak on the carboxymethyl group and methylene group (-CH) in hemicellulose [26]. Characteristic absorption peaks at 1739 cm−<sup>1</sup> and 1630 cm−<sup>1</sup> are due to the asymmetric stretching vibrations of C=O of acetyl groups or esters, including methyl esterification or free carboxyl groups [27]. As the particle size decreases, the intensity of the absorption peak gradually weakens, which may be due to the destruction of some ester structures during the grinding process. There is also literature showing that the weakening of the absorption peak here will lead to a poor water holding capacity [28], which provides a theory for water holding research. The absorption peak at 1531 cm−<sup>1</sup> is the characteristic absorption peak of C=O in the imide bond; the absorption peak at 1245 cm−<sup>1</sup> is the vibration of the OH or -CO group of hemicellulose, and 1053 cm−<sup>1</sup> is the C-O-C stretching vibration peak. The absorption peak at 890 cm−<sup>1</sup> is the characteristic absorption peak of the β-configuration glycosidic bond [29]. To sum up, compared with the characteristic peaks of OIDF-50 and OIDF-100, the peak shape, position and number of OIDF-10 did not change significantly, indicating that the main components and structure of OIDF did not change substantially after DHPM treatment.

**Figure 1.** *Cont*.

**Figure 1.** Structural characterization of OIDF with three particle sizes: (**a**) Particle size characterization; (**b**) SEM characterization; (**c**) FI-IR characterization; (**d**) TG characterization; (**e**) XRD characterization). Figure 1b (**A**: OIDF-10, 1000×; **B**: OIDF-10, 8000×; **C**: OIDF-50, 1000×; **D**: OIDF-50, 8000×; **E**: OIDF-100, 1000×; **F**: OIDF-100, 8000×).

From Figure 1d, the OIDF thermogravimetric curves of the three particle sizes showed the same trend, mainly composed of four stages. The first stage is the drying stage. From the initial temperature of 25 ◦C to 110.25 ◦C, the thermogravimetric curves of the three particle sizes of OIDF all show a weight loss of about 3.9%, which is due to the evaporation of free water and crystal water inside the fiber. The second stage is the pre-carbonization stage, the temperature range is 110.25–208 ◦C, and the weight loss is 0.73%, which belongs to the process of slow decomposition of macromolecules; the third stage occurs at about 208.38–355.55 ◦C, which belongs to the carbonization stage. The decrease was significant, with weight loss as high as 62%, which may be due to thermal decomposition or degradation of cellulose and hemicellulose [30]. It is consistent with the conclusion obtained by Fourier transform infrared spectroscopic analysis of OIDF with different particle sizes. The final stage is the combustion stage. When the temperature exceeds 355 ◦C, the thermogravimetric curve trend is stable due to the gradual decomposition of the residual sample into carbon and ash. The retention rate is the percentage of the final residue of the sample to the original mass. The retention rate of OIDF-10 is 22.89%, the retention rate of OIDF-50 is 21.77%, and the retention rate of OIDF-100 is 20.35%.

As shown in Figure 1e, any crystalline substance has its own unique X-ray diffraction pattern, and the position and shape of the characteristic diffraction peaks can be used to qualitatively and quantitatively analyze the substance, and to determine the crystal structure and degree of crystallinity. The results are shown in Figure 1e. The diffraction angles (2θ angles) are 15.42◦, 20.78◦, and 36.08◦, which are typical characteristic diffraction peaks of cellulose, indicating that the crystal types of the three OIDFs belong to the cellulose I type. It is a state in which two phases coexist in a crystalline region and an amorphous region [31]. According to the calculation, the crystallinity of OIDF-100 is 33.65%, the crystallinity of OIDF-50 is 30.81%, and the crystallinity of OIDF-10 is 27.08%, indicating that the crystallinity of cellulose in OIDF-10 is significantly reduced after high-pressure microfluidization. Ullah et al. [32] also found that insoluble dietary fiber in soybean dregs was treated with high-energy wet media, and the crystallinity of cellulose gradually decreased with the prolongation of treatment time. In addition, Kang and Lu et al. [33,34] also came to the same conclusion.

#### *3.3. Physicochemical Properties of the Three OIDFs*

The shear force is increased in the high-pressure micro-jet process, as illustrated in Figure 2, due to the mechanical effect of ultra-high pressure. Water holding capacity drops, oil holding capacity decreases, and swelling property rises when the OIDF particle size reduces, which might be related to an increase in the specific surface area as the particle size lowers. Ultra-high pressure damages the fiber structure, causing more hydroxyl groups of hydrophilic groups to be destroyed, resulting in a weakening of the fiber's hydrophilic ability and a loss in water holding capacity and moisture content [35]. For example, Jasim et al. [36] and Zheng et al. [37] discovered that following ultrafine pulverization, the water-holding and oil-holding capabilities of insoluble dietary fiber declined as the particle size dropped. This might be due to certain hydroxyl and ester structures being destroyed during processing, which is consistent with the findings of this study.

**Figure 2.** Effects of different particle sizes of OIDF on the water holding capacity (WHC), swelling capacity (WSC) and oil holding capacity (OHC).

As shown in Figure 3, the three particle sizes of dietary fibers have varied adsorption capabilities for cholesterol in the three pH conditions. The smaller the particle size of dietary fibers in the same pH environment, the greater the adsorption capability. The adsorption ability of three types of dietary fiber to cholesterol steadily declined as pH increased. This finding suggests that dietary fiber has good cholesterol adsorption capabilities in a stomach acid environment. Similarly, in three different acidic circumstances, dietary fiber exhibited a similar adsorption rule to sodium cholate and adsorbed cholesterol, indicating that dietary fiber had high adsorption qualities to cholate in the stomach acid environment.

**Figure 3.** The effect of different particle sizes of OIDF on the adsorption of cholesterol (CAC) and sodium cholate (SCAC).

#### *3.4. Effects of Different Particle Sizes of OIDF on Blood Lipid Levels in High-Fat Diet Rats*

As shown in Figure 4, a high-fat diet can significantly increase the content of TC and TG in blood. After OIDF intervention, there is a relief, and the group fed with OIDF-10 is the most significant. OIDF-50 and OIDF-100 groups have significant differences in reducing TC levels but no significant difference in reducing TG levels. This may be due to the large particle size, which leads to low intestinal utilization of OIDF, and thus, the reducing TG levels are not apparent. Regarding HDL-C, the OIDF-10 group significantly increased. HDL-C is mainly synthesized in the liver, which can promote the reverse transport of cholesterol, thereby achieving a lipid-lowering effect. Under a high-quality diet, the content of LDL-C in serum is higher, indicating that it is positively correlated with high blood lipids. OIDF-10 can significantly reduce LDL-C content, while the intervention effect of OIDF-50 and OIDF-100 is not significant. This is consistent with the findings of Zhou [38] and Hoang [39], but the exact reason is not clear.

**Figure 4.** Effects of different particle sizes of OIDF on blood lipid levels in rats (TC, TG, HDL-C, LDL-C). Different letters in the same column (a, b, c and d) are significantly different (*p* < 0.05). The results are expressed as mean ± SD (*n* = 3).

#### *3.5. Effects of Different Particle Sizes of OIDF on Hepatic Steatosis in High-Fat Diet Rats*

After H&E staining, the morphological changes of hepatocytes and fat droplets can be visually observed by microscope observation. The morphological observation results of the liver tissue of rats in each group are shown in Figure 5A. It can be seen from the figure that the size of hepatocytes in the NC group was normal throughout the eight weeks of feeding, the cells had complete cytoplasm and obvious nuclei, and the cell boundaries were clear and neatly arranged, while in the other groups, different degrees of accumulation of fat droplets were observed. In histological section, the white circles indicate fat droplets, which may be due to the accumulation of lipids in the body due to a long-term high-fat diet and the lack of exercise in cage feeding. After circulation in the liver, aggregation

occurred. Different degrees of fat droplets were observed in other groups. Fat droplets accumulate. From the figure, we can clearly observe that in the HD group, after eight weeks of high-fat feeding, a large number of fat droplets appeared in the liver slices, and the liver was severely fatty. After eight weeks of intervention, compared with the HD group, the steatosis of liver cells in the HD-OIDF-50 and HD-OIDF-100 groups was slightly reduced, and the degree of lipid accumulation was not as fast as that in the HD group, but the morphological changes were different from those in the HD group, although not by much as the effect of inhibiting liver adipose is not as significant as the OIDF-10 group.

As shown in Figure 5B, RT-PCR was performed on the mRNA levels of three key genes of 3-hydroxy-3-methylglutaryl coenzyme (HMG-CoAr), Cholesterol 7α-hydroxylase (CYP7A1), and Low-density lipoprotein receptor (LDL-R) in rat liver. HMG-CoAr is a key rate-limiting enzyme for cholesterol synthesis. From the mRNA expression level of the gene, the relative expression of HMG-CoAr gene in the HD group was up-regulated in the NC group, indicating that a high-fat diet would increase the synthesis of cholesterol. This result was consistent with the results of lipid expression in serum and liver of HD group. As shown in Figure 5B,C, the effect of PCR was not significant. Combined with the analysis of the translation level Western Blot data, high-fat feeding in the HD group led to the high expression of HMG-CoAr gene, and OIDF intervention could reduce its expression, of which OIDF-10 had the most reduction effect. LDL-R can mediate the endocytosis of LDL and reduce the synthesis of hepatic cholesterol by increasing the reabsorption of LDL [40,41]. Therefore, the up-regulation of LDL-R in the OIDF-10 group may be due to the decrease in the blood lipid level of LDL-C. After eight weeks of OIDF dietary intervention, the expression level of CYP7A1 gene in the HD-OIDF-10 group was significantly higher than that in the HD group. Some researchers believe that FXR can activate bile acid synthesis by inducing the expression of CYP7A1 [42]. The high expression of the gene may promote the synthesis of bile acids, thereby promoting the metabolism of cholesterol.

**Figure 5.** *Cont*.

**Figure 5.** Effects of different particle sizes of OIDF on diet-induced hepatic steatosis in mice. Histological changes in liver sections were measured by H&E staining at 400× magnification: (**A**) Relative mRNA expression levels of CYP7A1, HMG-CoAr, LDL-R were determined by qRT-PCR; (**B**) The protein expressions of CYP7A1, HMG-CoAr and LDL-R in liver were determined by Western blotting; (**C**) Values are expressed as mean ± SD (*n* = 10). \* represents *p* < 0.05, \*\* represents *p* < 0.01, and \*\*\* represents *p* < 0.001, and \*\*\*\* represents *p* < 0.0001, compared to the HD group. NC, normal diet-fed group; HD, high-fat diet-fed group; HD-OIDF-10, HD-OIDF-50, HD-OIDF-100, representing three distinct particle sizes of OIDF.

The above three genes were confirmed by Western Blot, as shown in Figure 5C, and their protein level expression and mRNA expression trend were the same.

#### *3.6. Bacterial Composition Analysis between Different Populations*

By statistic on the ASV/OTU table after leveling, the specific composition table of the microbial community in each sample at each classification level can be obtained. Through this table, the number of taxa contained in different samples at each taxonomic level can be calculated first. Analysis software: QIIME2 (2019.4); Personal company self-compiled perl script. Analysis step: According to the results of the sequence species taxonomic annotation and the selected samples, we can count the number of taxa contained in each of the seven taxonomic levels of domain, phylum, class, order, family, genus, and species in the species annotation results of these samples. Some studies have used Firmicutes/Bacteroidetes ratios to explore the degree of obesity [43]. Compared with the HD group, the HD-OIDF-10 group was able to significantly reduce the ratio of Firmicutes/Bacteroidetes. As shown in Figure 6A and Table 3 (percentages shown in Table 3 are the mean value), at the phylum level, Firmicutes NC group accounted for 80.32%, HD group accounted for 80.67%, HD-OIDF-10 group accounted for 88.25%, HD-OIDF-50 group accounted for 93.04%, HD-OIDF-100 group accounted for 90.64%. Firmicutes are the dominant beneficial bacteria in the

intestinal tract, indicating that OIDF with different particle sizes can increase the abundance of the Firmicutes after the intervention of a high-fat diet. Regarding Bacteroidetes, the NC group accounted for 1.27%, the HD group 0.03%, the HD-OIDF-10 group 4.44%, the HD-OIDF-50 group 2.34%, and the HD-OIDF-100 group 2.06%. Bacteroidetes are engaged in colonic metabolism, including glucose fermentation and bile acid and steroid biotransformation, and their numbers have grown. In the HD group, a high-fat diet resulted in a significant reduction in Bacteroidetes. The proportion of Bacteroidetes rose after OIDF intervention, notably in the HD-OIDF-10 group. The OIDF-10 intervention might dramatically reduce the rise in *Actinobacteria* and *Proteobacteria* produced by a highfat diet when compared to the NC group. At the genus level, as shown in Figure 6B, Oscillospira showed the largest quantitative difference among the groups, and Oscillospira was able to produce short-chain fatty acids (SCFAs) such as butyrate, suggesting that it may play an essential role in various aspects of human function and health [44]. At the same time, studies have shown that Oscillospira can ferment complex plant carbohydrates [44]. After the intervention of three particle sizes of OIDF, the content of Oscillospira in the HD-OIDF-10 group was the highest at 20.25%, which was significantly higher than that in the HD group by 2.4 times. It shows that the bacteria in the gut can obtain carbon sources by decomposing the plant polysaccharide OIDF. As shown in Figure 6C, the elements of the phylogenetic tree graph mainly include: the phylogenetic tree graph, coloring ASV characteristic sequences or OTU representative sequences (tips in the graph) according to the taxonomic level. The first column is the sequence ID, and the second column is the relative abundance of the sequence in each sample of the grouping scheme. From left to right are the HD group, the NC group, the OIDF-10 group, the OIDF-50 group, and the OIDF-100 group, with six samples in each group. As shown in Table 3, at the genus level, OIDF was also able to modulate gut microbiota composition, enabling the development of gut microbiota towards a healthy state, reducing the abundance in *Allobaculum* bacteria in the gut of rats on a high-fat diet. *Ruminococcus*, ruminant digestive tract flora, is fermented to produce lactic acid, and the OIDF was able to increase *Ruminococcus* abundance, of which OIDF-10 had the most significant effect, possibly through the production of lactate, which is produced by gut bacteria, making a valuable contribution to colon health. It helps promote gut health and the bacteria that produce it protect against disease.


**Table 3.** Ratio of microbiota (top10) at the phylum and genus levels (%) (*n* = 6).

**Figure 6.** Bacterial composition analysis between different populations: (**A**) t phylum level; (**B**) genus level; (**C**) Species composition analysis.

#### *3.7. Alpha Diversity Index and Beta diversity PCoA Analysis*

As can be seen from Figure 7, the chao1 index of intestinal flora in the HD group was the smallest, which is a measure of its richness. The emphasis on rare species was the lowest, which was significantly different from the HD-OIDF-10 group. The Shannon index of intestinal flora in the HD group was significantly lower than that in the NC and HD-OIDF-10 groups (*p* < 0.01), that is, the intestinal microecological diversity of the HD group was significantly lower than that of the NC and OIDF intervention groups. The reason may be due to the high-fat diet changes the intestinal ecology, and the excessive reproduction of harmful bacteria that reduces the diversity of intestinal flora [40]. The richness and diversity of the intestinal flora of the HD-OIDF-10 group were closer to those of the NC group, and the results of other types of the analysis showed the same trend, indicating that the species richness between HD and NC, OIDF-10, and OIDF-100 and diversity are significantly different.

As the picture shows, β-diversity analysis can be used to study the similarity and difference of the bacterial community structure in different samples. Commonly used analysis methods include sample hierarchical cluster analysis, principal coordinate analysis, and multi-dimensional scaling analysis [45]. This study focuses on principal coordinate analysis (PCoA). As shown in Figure 7, the HD group and the NC group were clustered separately. The difference was obvious, that is, the microflora structure of the high-fat diet rats was different from that of the normal mice. In contrast, the coordinates of the OIDF dietary intervention group were shifted to the NC group, indicating that OIDF was able to modulate gut microbiota composition in rats fed a high-fat diet. Figure 7 shows that HD-OIDF-10 intervention in high-fat diet rats has a significant difference with HD group rats (*p* < 0.05), which indicates that OIDF-10 can change the intestinal flora structure of high-fat diet rats. These results suggest that OIDF can modulate the gut microbiota structure in rats fed a high-fat diet.

**Figure 7.** Alpha Diversity Index and Beta diversity PCoA analysis (G: HD group; K: NC group; S: HD-OIDF-10 group; M: HD-OIDF-50 group; L: HD-OIDF-100 group. \* represents *p* < 0.05, \*\* represents *p* < 0.01).

#### *3.8. Alpha Diversity Index and Beta Diversity PCoA Analysis*

As shown in Figure 8A, cluster analysis was performed between each group, and the top 20 bacterial species were obtained, which were *Ruminococcus*, *Clostridium*, *Desulfovibrio*, *Brevibacterium*, *Oscillospira*, *Ruminococcus*, *Coprococcus*, *Bacteroides*, *Yaniella*, *Blautia*, *Sporosarcina*, *Jeotgalicoccus*, *Corynebacterium*, *Lactobacillus*, *Dorea*, *Adlercreutzia*, *Allobaculum*, *Turicibacter*, *Akkermansia*, *Facklamia*. Compared with the HD group, HD-OIDF-10 group

increased the abundance of *Brevibacterium*, *Oscillospira*, *Ruminococcus*, *Coprococcus*, *Bacteroides*, *Yaniella*, and HD-OIDF-50 increased the abundance of *Ruminococcus*, *Clostridium*, HD-OIDF-100 compared with HD group increased the abundance of *Ruminococcus*, *Clostridium*, *Desulfovibrio*, *Oscillospira*, *Ruminococcus*, *Bacteroides* compared with the HD group, indicating that the OIDF can improve the dysbiosis caused by a high-fat diet. As shown in Figure 8B, in the PCA analysis, PC1 = 52.7%, PC2 = 22.2%, and the differences in PCA analysis between groups were significant. The HD-OIDF-10 group and the HD group with extremely significant differences were selected for MetagenomeSeq analysis, and the occurrence frequency of ASV was greater than or equal to 0.3 as the condition, as shown in Figure 8C. The sample groups were compared using the metagenomeSeq method. This method avoids the influence of data sparse (Rarefaction) process on the accuracy of results, and is especially suitable for sparse microbial composition data. The dotted line separated the significant difference (above) and the insignificant ASV, significant difference. The points are marked by colored dots or circles, gray circles indicate the insignificant ones and the significantly up-regulated ones in this group are indicated by colored solid circles. The color of the dots identifies its phylum level name and is marked at the bottom of the figure; for the top 10 genera with significantly up-regulated points, a grayscale background is added, and the final analysis shows that the difference ASV number is 62, and after classification, it is concentrated in *Actinobacteria*, *Bacteroidetes*, *Firmicutes*, *Proteobacteria*.

**Figure 8.** *Cont*.

(**C**) (between HD-OIDF-10 and HD)

**Figure 8.** Species differences and marker analysis: (**A**) Species composition heatmap; (**B**): PCA analysis; (**C**): MetagenomeSeq analysis. (G: HD group; K: NC group; S: HD-OIDF-10 group; M: HD-OIDF-50 group; L: HD-OIDF-100 group).

#### **4. Conclusions**

In this study, three distinct particle sizes of OIDF were prepared from high-purity okara insoluble dietary fiber, and their structures and physicochemical properties were characterized, such as OIDF-10 having the higher WSC, OHC, CAC and SCAC. Using three distinct particle sizes of OIDF to interfere with the lipid-lowering effect of highfat diet rats, we observed that OIDF supplementation could reduce blood lipid levels, alleviate hepatic steatosis, and regulate mRNA expression and protein of genes related to fat metabolism in the liver. The OIDF supplementation may provide a theoretical basis for OIDF with different particle sizes as functional food or dietary supplements by improving lipid metabolism and intestinal flora function in rats induced by a high-fat diet.

**Author Contributions:** H.Y. contributed to the conceptualization and supervision. Y.Z. contributed to the resources. H.F., S.W., M.S.S., J.F., J.L. and J.Z. contributed to the investigation, formal analysis, and writing. All the authors critically reviewed and contributed to the editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by China Agriculture Research System of MOF and MARA (CARS-04); Young and Middle-Aged Technological Innovation Outstanding Talent (Team) Project (Innovation), grant number (20210509015RQ).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University (no. 20210422001, 22 April 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Raw data can be provided by the corresponding author on request.

**Acknowledgments:** The authors acknowledge Shanghai Personal Biotechnology Co., Ltd., (Shanghai, China). For their support to the 16S rDNA Sequencing.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

