Plasma Adipokine Levels

A Leptin Mouse/Rat ELISA kit (Biovendor, Brno, Czech Republic) and Rat Adiponectin ELISA kit (Assaypro, Charles, MO, USA) were used to detect plasma leptin and adiponectin levels, respectively, following the manufacturer's instructions. The absorbance was read on a microplate reader (Molecular Devices) at 450 nm for all adipokines.


**Table 10.** Primers used for the quantitative polymerase chain reaction.

SREBP-1c, sterol response element-binding protein-1c; ACC, acetyl CoA carboxylase; SCD1, stearoyl coenzyme A desaturase 1; FAS, fatty acid synthase; PPARα, peroxisome proliferator-activated receptor α; CPT-1, carnitine palmitoyl transferase-1; ZO-1, zonula occludens-1.

### 4.4.3. IR Analysis

Plasma insulin levels were assayed with a Rat Insulin ELISA kit (Mercodia, Uppsala, Sweden). The fasting blood glucose level was detected via the Glucose Monitoring System (Abbott Diabetes Care, Oyl, UK). The HOMA-IRI was calculated with the following formula: HOMA-IRI = fasting blood glucose (mg/dL) × fasting insulin (mIU/L)/405.

### 4.4.4. Determination of Intestinal Damage

Intestinal Tight Junction Protein mRNA Levels

The intestinal tight junction protein mRNA levels were measured by quantitative (q)PCR methods. The method of ileum sample preparation was the same as that for the measurement of hepatic FA metabolism-related genes. Information on primers is given in Table 10.

### Fecal Microbiotic Analysis

The fecal microbiotic composition was analyzed using a 16S ribosomal (r)RNA Next Generation Sequencing (NGS) system. Fresh feces were collected into sterilized 2-mL Eppendorf tubes and stored at −80 ◦C for analysis. The amplified fecal DNA was purified with Agencourt AMPure XP Reagent beads (Beckman Coulter, Brea, CA, USA). A qPCR (KAPA SYBR FAST qPCR Master Mix) was used to quantify each library with the Roche LightCycler 480 system, and then pooled equally to 4 nM for the Illumina MiSeq NGS system (illumina, San Diego, CA, USA). More than 80,000 reads with paired-end sequencing (>250 bp\*2) were generated, and the QIIME2 workflow [60] classified organisms from the amplicons using the GreenGenes taxonomy database. MicrobiomeAnalyst [61] was used to perform statistical and visual analyses of the microbiome data.

### Fecal SCFA Concentrations

Fecal SCFAs were extracted according to the method described by García-Villalba et al. [62] with slight modification. Fresh feces were weighed and suspended in 1 mL of water with 0.5% phosphoric acid per 0.1 g of sample into sterilized 2-mL Eppendorf tubes and stored at −80 ◦C until analysis. The SCFA analysis was performed by gas chromatographic-mass spectrometric (GC-MS) system of Agilent 5977B coupled with a 7820A autoinjector (Agilent Technologies, Santa Clara, CA, USA). For GC-MS, a Nukol™ 30 m × 0.25 × 0.25 µm capillary column (24107, Supelco, Bellefonte, PA, USA) was used. Injection was made in the pulsed split mode with an injection volume of 1 µL and an injector temperature of 250 ◦C. GC separation was carried out at 90 ◦C initially, then increased to 150 ◦C at 15 ◦C /min, to 170 ◦C at 3 ◦C/min and finally to 200 ◦C at 50 ◦C/min (total time 16.267 min). The solvent delay was 3.2 min. The identification of SCFAs was based on the retention times of standard compounds using a commercial standard solution (469754.4.5. Statistical Analysis

**5. Conclusions** 

U, Supelco). Data were quantitated with MassHunter Quantitative Analysis software (Agilent Technologies). Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism vers. 8.0.1 (GraphPad Software, San Diego, CA, USA).

### 4.4.5. Statistical Analysis Student's *t*-test was used to determine statistical differences between the NC and HF

*Plants* **2022**, *11*, x FOR PEER REVIEW 24 of 28

Fecal SCFAs were extracted according to the method described by García-Villalba et al. [62] with slight modification. Fresh feces were weighed and suspended in 1 mL of water with 0.5% phosphoric acid per 0.1 g of sample into sterilized 2-mL Eppendorf tubes and stored at −80 °C until analysis. The SCFA analysis was performed by gas chromatographic-mass spectrometric (GC-MS) system of Agilent 5977B coupled with a 7820A autoinjector (Agilent Technologies, Santa Clara, CA, USA). For GC-MS, a Nukol™ 30 m x 0.25 x 0.25 μm capillary column (24107, Supelco, Bellefonte, PA, USA) was used. Injection was made in the pulsed split mode with an injection volume of 1 μL and an injector temperature of 250 °C. GC separation was carried out at 90 °C initially, then increased to 150 °C at 15 °C /min, to 170 °C at 3 °C/min and finally to 200 °C at 50 °C/min (total time 16.267 min). The solvent delay was 3.2 min. The identification of SCFAs was based on the retention times of standard compounds using a commercial standard solution (46975-U, Supelco). Data were quantitated with MassHunter Quantitative

Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism vers. 8.0.1 (GraphPad Software, San Diego, CA, USA). Student's *t*-test was used to determine statistical differences between the NC and HF groups. A one-way analysis of variance (ANOVA), followed by Fisher's post hoc test was used to determine statistical differences among the HF, HF + 1% FRB, and HF + 5% FRB groups. Statistical significance was assigned at the *p* < 0.05 level. groups. A one-way analysis of variance (ANOVA), followed by Fisher's post hoc test was used to determine statistical differences among the HF, HF + 1% FRB, and HF + 5% FRB groups. Statistical significance was assigned at the *p* < 0.05 level. It was found that the water extract of fermented rice bran (by *Aspergillus kawachii*)

### **5. Conclusions** had a higher anti-oxidative ability. The current results demonstrated that after 8 weeks of

It was found that the water extract of fermented rice bran (by *Aspergillus kawachii*) had a higher anti-oxidative ability. The current results demonstrated that after 8 weeks of FRB treatment, rats in the HF + 5% FRB group had a significantly lower NAFLD score and hepatic IL-1β level, a decreasing trend of final BW and plasma leptin level, as well as an increase in beneficial bacteria in the gut microbiota. In summary, it was suggested that FRB showed the potential for alleviating liver damage induced by a HF diet, possibly through regulating the imbalanced adipokines and altering the gut microbiotic composition (Figure 15). FRB treatment, rats in the HF + 5% FRB group had a significantly lower NAFLD score and hepatic IL-1β level, a decreasing trend of final BW and plasma leptin level, as well as an increase in beneficial bacteria in the gut microbiota. In summary, it was suggested that FRB showed the potential for alleviating liver damage induced by a HF diet, possibly through regulating the imbalanced adipokines and altering the gut microbiotic composition (Figure 15).

**Figure 15.** Effects of the water extract of fermented rice bran (FRB) on liver damage and intestinal injury in aged rats with high-fat (HF) diet feeding. In this study, it was indicated that FRB ameliorated liver damage induced by the HF diet which was represented as a lower non-alcoholic fatty liver disease (NAFLD) score and hepatic interleukin (IL)-1β level in rats. The protective effects of FRB against liver damage may have been due to regulating the plasma adipokines and maintaining homeostasis of the gut microbiota. Solid blue arrow: levels of analytical items were significantly increased or decreased in the HF + 5% FRB group than in the HF group; dotted blue arrow: levels of analytical items in the HF + 5% FRB group showed an increasing or decreasing trend compared to the HF group.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11050607/s1, Table S1: LC/MS analysis of the water extract of fermented rice bran (FRB), Table S2: Amino acid compositions of the water extracts of rice bran (RB) and fermented RB (FRB), Table S3: Fatty acid compositions of the water extracts of rice bran (RB) and fermented RB (FRB), Table S4: Components in the water extracts of rice bran (RB) and fermented RB (FRB).

**Author Contributions:** Conceptualization, S.-C.Y. and H.S.; formal analysis, T.-Y.C., Y.-L.C. and W.- T.L.; data curation, T.-Y.C. and Y.-L.C.; writing—original draft preparation, T.-Y.C.; writing—review and editing, S.-C.Y.; supervision, W.-C.C., C.-L.Y., Y.-T.T., Y.-L.C. and S.-C.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Innovative Translational research, Ministry of Education (DP2-109-21121-01-O-03-03) and Formosa Produce Corporation, Taipei, Taiwan (109-6202-029-300).

**Institutional Review Board Statement:** The study was approved by the Institutional Animal Care and Use Committee of Taipei Medical University (LAC-2020-0143).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank the Laboratory Animal Center at Taipei Medical University, Taiwan for technical support in the animal experiment.

**Conflicts of Interest:** The authors declare that there are no conflict of interest regarding the publication of this paper.

### **Abbreviations**



### **References**

