Abstract

This study investigated the therapeutic potential of fermented lemon peel (FLP07) in mitigating HFD-induced NAFLD, utilizing a mouse model to compare its efficacy against the hepatoprotective agent silymarin. While HFD-fed mice exhibited significant weight gain, dyslipidemia, and intervention with FLP07 effectively improved serum triglyceride (TG) and total cholesterol (TC) levels. Furthermore, FLP07 partially restored the AST/ALT ratio and significantly reduced hepatic lipid droplet accumulation, showing comparable efficacy to silymarin. These results suggest that FLP07 is a promising natural dietary supplement for managing NAFLD and dyslipidemia.

1. Introduction

Lemon peel, a primary by-product of citrus processing (Figure 1), accounts for approximately 50% of the fruit's total dry weight ¹. Given its substantial content of essential minerals and dietary fibers, lemon peel serves as a highly suitable substrate for probiotic cultivation ². Furthermore, the microbial bioconversion of agricultural by-products aligns with global sustainability goals by transforming waste into high-value functional ingredients ³. Fermentation facilitates the release and enhancement of bioactive constituents, thereby improving the biochemical profile and biological activity of the substrate ^{4, 5, 6, 7, 8}. Our preliminary investigations indicated that fermentation of citrus peel using lactic acid bacteria (LAB) significantly increased the concentration of aglycone flavonoids ⁹. Consistent with previous findings, LAB-mediated fermentation effectively liberates phenolic compounds and elevates antioxidant activity through enzymatic biotransformation. Specifically, our prior data demonstrated that fermented citrus products could reduce lipid accumulation by 42.4% in oleic acid-treated HepG2 cells ⁹. Building upon these findings, the present study aims to utilize fermentation technology to repurpose lemon by-products for human health applications. We will employ an animal model ^{10, 11, 12, 13} to evaluate the therapeutic efficacy of fermented lemon peel in Non-alcoholic fatty liver disease (NAFLD).

NAFLD has emerged as a predominant global public health concern, characterized by pathological triglyceride accumulation in hepatocytes in the absence of significant alcohol consumption ¹⁴. As the most common chronic liver disease worldwide, NAFLD affects approximately 25% of the adult population and is particularly prevalent in East Asia, including Taiwan ^{15, 16, 17}. The pathogenesis of NAFLD is inextricably linked to metabolic syndrome, encompassing obesity and insulin resistance. Clinically, it presents a spectrum of severity; progressive inflammation and hepatocellular injury can lead to non-alcoholic steatohepatitis (NASH), which is associated with increased mortality due to the risks of fibrosis, cirrhosis, and hepatocellular carcinoma ¹⁸.

Current therapeutic strategies prioritize dietary interventions and the identification of bioactive compounds capable of ameliorating hepatic steatosis and restoring metabolic homeostasis. Citrus fruits (Citrus spp., family Rutaceae), such as lemons, grapefruit, and mandarins, are renowned for their medicinal properties attributed to a diverse profile of bioactive constituents ^{18, 19}. Lemon peel, frequently discarded as an agro-industrial by-product, is a rich source of fermentable fibers, flavonoids, and polyphenols. These compounds have demonstrated significant hepatoprotective, antioxidative, and hypolipidemic activities ²⁰. Furthermore, fermentation processes have been shown to enhance the bioavailability and therapeutic efficacy of these plant-derived compounds by modifying chemical structures and increasing the yield of beneficial metabolites ²¹. Specifically, fermented lemon peel may facilitate the release of active polyphenols and stimulate the production of short-chain fatty acids (SCFAs), thereby offering a potential mechanism for hepatic lipid regulation. Despite this potential, the direct anti-steatotic effects of fermented lemon peel preparations remain underexplored.

Short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, are important metabolites produced by gut microbial fermentation of dietary fibers. These SCFAs have been shown to attenuate hepatic lipogenesis, enhance fatty acid oxidation, and activate AMP-activated protein kinase (AMPK) signaling, thereby exerting protective effects against lipid accumulation in the liver ^{22, 23}. Short-chain fatty acids (SCFAs) are a group of saturated aliphatic acids and produced through probiotic fermentation of dietary fibers that play an important role in gut health and energy metabolism. SCFAs involve glucose metabolism and insulin sensitivity via diverse pathways affecting the development of diabetes and NAFLD. Recently research have revealed that in the high-fat diet induced NAFLD hamster model, SCFAs significantly reduced total cholesterol ²⁴.

Preliminary research demonstrated dietary SCFAs in fermented foods may have a direct influence on metabolic functions. Japan Scientist found that dietary SCFA intake suppressed the high-fat diet (HFD)-induced liver weight gain and hepatic TGs accumulation along with a change in hepatic lipid metabolism-related genes ²⁵. SCFAs supplementation improved hepatic metabolic functions via FFAR3 without influencing intestinal environment. These findings could help to promote the development of functional foods using SCFAs. Hence, SCFAs functional food is considered a promising strategy for the prevention of NAFLD ²⁵.

To the best of our knowledge, there is a paucity of research regarding the efficacy of fermented lemon peel, a source rich in short-chain fatty acids, in the amelioration of hepatic steatosis. This study aims to investigate the effects of fermented lemon peel, which is rich in short-chain fatty acids, on ameliorating high-fat diet-induced non-alcoholic fatty liver disease in mice.

2. Materials and Methods

Acetate (AA), propionate (PA), butyrate (BA), 3-Nitrophenylhydrazine hydrochloride (3NPH·HCl), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC·HCl), HPLC-grade water, HPLC-grade methoanl and MS-grade formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Eureka lemons (Citrus limon) peel purchased from organic farm (Pingtung County, Taiwan). Lemon peel was mixed with pure water and treated with Viscozyme (45°C, 2 h) for hydrolysis. To inactivate the enzymes, the reaction mixture was heated in a boiling water. Lactobacillus plantarum PM-A87 (BCRC910475) and Lactobacillus acidophilus PM-A0002 (BCRC 910308) are a self-isolating strain and stored at -80°C. Weigh an appropriate amount of lemon peel and glucose to prepare the fermentation culture medium. Add Lactic acid bacterial liquid pre-cultured for 24 hours, mix it with the fermentation culture medium, and co-fermentation at 35°C in an incubator for 168 hrs. Centrifuge the fermentation broth for 5 minutes at 3000 rpm to obtain the supernatant and then dried into a powder (fermented lemon peel, FLP07).

To analyze of 3-NPH-labeled SCFAs in LC-MS/MS, 3-NPH derivatization method was used ²⁶. Each standard compound was dissolved in 50% methanol to 0.2 M. FLP07 sample was added methanol and sonication 30 min, then at 2000 rpm for 5 min by centrifugation and suspension measure to 50 ml using a volumetric flask. Next, 50 μL of extracted suspension was mixed with 25 μL of 0.2 M 3-NPH and 25 μL of 0.2 M EDC on ice. The mixture was then incubation at 40 °C for 20 min. Finally, all the sample solutions were filtered through a 0.22 μm membrane filter and then analyzed by HPLC−MS/MS. HPLC-ESI-MS/MS analysis was performed using an Nexera XR-20A system (Shimadzu 8045, Kyoto, Japan) coupled to an API 4000 triple quadrupole tandem mass spectrometer (Applied Biosystem, Foster City, CA, USA). Chromatographic separation was performed on a C18 column (150×4.0 mm I.D, 5 μm, Agilent, USA). The mobile phase consisted of 0.1 % formic acid aqueous solution (solution A) and methanol (solution B) and a gradient elution program was set as follows: solution A, 90–60% (0–3.5 min), 60–40% (3.5–6 min), 40–0% (6–9 min), 0–40% (9–10min) and 40–60% (10–12min), 50–90% (12–15min). The column temperature was fixed at 30°C, the flow rate was set 0.5 mL/min, and injection volume was 2 μL. The electrospray negative mode was selected as an ion source detection. The quantification was performed in multiple reactions monitoring (MRM). The optimized ESI source parameters were as follows: ion spray voltage, -4500 V for negative mode and 4500 V for positive mode; nitrogen nebulizer gas pressure, 50 psi; nitrogen curtain gas pressure, 11 psi; heater temperature, 460°C; collisionally activated dissociation (CAD) gas, 11 psi. The precursor-to-product ion transitions were m/z 194.1/152, m/z 208.1/165.1 and m/z 222.1/137.1 for 3-NPH-AA, 3-NPH-PA and 3-NPH-BA, respectively. Their optimized declustering potentials (DP) and collision energies (CE) were listed on Table 1. All data acquisition and processing were performed using Analyst 1.7.3 software (AB SCIEX, Concord, ON, Canada). The peak area of each component in the FLP07 was acquired from its chromatogram and the abundance of each compound was calculated from its corresponding calibration curve. Experiments were conducted in triplicate and the resulting data was represented as mg/g.

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Agricultural Technology Research Institute (Approval No. 113103) and conducted in accordance with animal welfare regulations. Male C57BL/6 (B6) mice, aged 5 weeks and weighing approximately 20 g, were obtained from the National Laboratory Animal Center (Taiwan). A total of 24 mice were housed in open cages within the GLP-certified animal facility (rodent section) of our institute under controlled conditions (12-h light/dark cycle, positive-pressure ventilation, 23 ± 3°C, and 30–70% relative humidity). Feed intake was recorded three times per week, and water was provided ad libitum. After one week of acclimatization, mice were randomly assigned into four groups (n = 6 per group) at 7 weeks of age as follows: (1) CTL: Mice fed a standard chow diet (LabDiet 5058; LabDiet, St. Louis, MO, USA) and orally administered sterile water; (2) HFD: Mice fed a 60 kcal% fat diet (D12492; Research Diets, Inc., New Brunswick, NJ, USA) and orally administered sterile water; (3) HFD+FLP07: Mice fed a 60 kcal% fat diet (D12492) and orally administered lemon fermentation product (500 mg/kg body weight/day); (4) HFD+Sily: Mice fed a 60 kcal% fat diet (D12492) and orally administered a silymarin-based commercial drug (184.95 mg/kg body weight/day). FLP07 and Sily administration began 1 week prior to initiation of the high-fat diet and continued daily throughout the 16-week experimental period. The dose of the silymarin-based commercial product was calculated by converting the manufacturer’s recommended human daily dosage to the mouse equivalent using body surface area normalization, as described in Systematic Reviews in Pharmacy (2020). At the end of the 16-week experimental period, all mice were humanely euthanized in accordance with approved protocols.

Body weight was measured weekly from the start of treatment until sacrifice, and food intake was recorded two to three times per week to calculate the average weekly consumption. At sacrifice, liver and visceral fat weights were measured to calculate the liver index (liver weight/body weight × 100%) and visceral fat index (visceral fat weight/body weight × 100%). Animals were fasted for at least 8 h prior to blood collection, with free access to water. After collection, blood samples were clotted at room temperature for 30 min and centrifuged at 3,500 rpm for 15 min at 4°C. The obtained serum was stored at −70°C for biochemical assays. Serum biochemical parameters, including AST, ALT, TG, TC, and glucose (expressed in mg/dL), were analyzed using a fully automatic dry chemistry analyzer (FUJI DRI-CHEM 4000, Fujifilm Co., Tokyo, Japan). HDL-C and LDL-C levels were determined by outsourcing analysis to MedGine Biotechnology Co., Ltd. (Taichung, Taiwan), using standardized enzymatic colorimetric assays according to the manufacturer’s instructions. Hepatic triglycerides were extracted from liver tissue using isopropanol and quantified with the Micro Triglyceride (TG) Assay Kit (KTB2200). Samples were hydrolyzed with lipoprotein lipase, and glycerol was subsequently oxidized to produce a chromogenic compound measured at 505 nm. Hepatic TG content is reported as mg per g of liver tissue. For histopathology, liver samples were fixed in 10% neutral-buffered formalin for at least 24 h, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). To minimize bias, sections were consistently taken from the same region of the right hepatic lobe. Microscopic evaluation assessed hepatocyte injury, lipid accumulation, necrosis, and other features of chronic liver damage. Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS software (version 12.0; SPSS Inc., Chicago, IL, USA). Differences among groups were evaluated by one-way ANOVA followed by Duncan’s multiple range test. A p-value < 0.05 was considered statistically significant.

Hepatic steatosis (fatty liver changes): The degree of hepatocellular steatosis was graded into five levels (0–4) following the guidelines of Evaluation methods for hepatoprotective effects of health foods (2016). At 200×total magnification, ten random fields per section were examined, each including at least 95% hepatocytes and containing a central vein. Scores were assigned as follows: 0= normal (0%), 1= slight (<10%), 2= moderate (10–33%), 3= severe (33–66%), and 4= very severe (66–100%). Hepatocellular degeneration and necrosis: Graded on a 0–4 scale according to Mann et al. (2012), where 0= normal, 1= minimal, 2= mild, 3= moderate, and 4 = severe. Only scores ≥2 were considered pathologically relevant. Ten random fields per section were examined at 200×, including at least 95% hepatocytes with a central or portal vein.

After feeding mice with samples, approximately 1g of feces was collected. DNA was extracted from the feces. Before sample DNA extraction, the reagents for the Presto™ DNA Extraction Kit (Taipei, Taiwan) were prepared. Alcohol was added to the Wash Buffer as instructed, ensuring thorough mixing. If there was precipitate at the bottom of the ST1 Buffer, it was heated to 37°C to dissolve. During DNA extraction, the dry bath was preheated to 70°C and the Elution Buffer was heated. 200μg of sample was placed in a tube containing Beating Beads, 800μL of ST1 Buffer and 10μL of Proteinase K were added, mixed thoroughly, and heated at 70°C for 5 minutes. Next, the sample was vortexed for 2 minutes and centrifuged at 8,000xg for 2 minutes. 500μL of supernatant was transferred to a new tube, 150μL of ST2 Buffer was added, and the mixture was vortexed. The mixture was then incubated at 4°C for 5 minutes. The supernatant was passed through an Inhibitor Removal Column and centrifuged at 16,000 x g for 1 minute. If precipitation occurred, the supernatant was discarded. 800 μL of ST3 Buffer was added and vortexed at high speed to mix. 650 μL was then transferred to a GD Column for filtration and centrifugation. The filtrate was discarded, and this step was repeated. Subsequently, the GD Column was washed twice with ST3 and Wash Buffer to ensure sample purity. Finally, 60 μL of preheated Elution Buffer was added to the center of the GD Column, allowed to stand for 2 minutes, and then centrifuged at 16,000 xg for 2 minutes to obtain the DNA extract. Further sample library preparation (Illumina DNA Prep) was performed. The amplified samples were sequenced using MiSeq Reagent and analyzed using an Illumina MiSeq (Illumina Inc., San Diego, CA, USA). The analysis was then performed using the Illumina® DRAGEN Metagenomics pipeline.

All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test (Minitab LLC, State College, PA, USA). A p-value of < 0.05 was considered statistically significant, and p < 0.01 was considered highly significant.

3. Results

A High-Performance Liquid Chromatography Electro-Spray Ionization Mass Spectrometry with multiple reaction monitoring method was carried out for analysis the SCFA compounds from FLP07. The RP-C18 column (150×4.0 mm I.D, 5 μm; Agilent, USA) was selected for HPLC -ESI-MS analysis based on its good separation ability and within 15 min.

With the ESI source of the mass spectrometer, 3-NPH-AA, 3NPH-PA and 3-NPH-BA showed better sensitivity in the negative ion mode (Figure 2). The precursor-to-product ion transitions were m/z 194.1/152, m/z 208.1/165.1 and m/z 222.1/137.1 for 3-NPH-AA, 3NPH-PA and 3-NPH-BA, respectively. Their optimized declustering potential (DP) and collision energies (CE) were list in Table 1.

The quantitative analytical results (Table 2) indicated their contents distributed in different samples. The contents of acetate, propionate and butyrate in the samples were in the range of 0.07-27.7 mg/g. In this study, the SCFA compounds significantly increased after fermentation.

As shown in Table 3, final body weights of rats in the HFD group were higher than those of rats in the CTL group. In addition, higher weight gain was observed in HFD rats than in CTL rats. Compared with HFD feeding, administration of HFD+FLP07 and HFD+Sily both slightly inhibited body weight gain.

The AST/ALT ratio reflects liver injury and recovery. The CTL group exhibited a ratio of 2.27 ± 0.49. The HFD group showed a significant reduction to 1.78 ± 0.36 (p<0.05), with ALT elevation (30.83→44.50 U/L) exceeding that of AST (68.50→76.67 U/L), indicating hepatocellular damage. In contrast, the HFD+FLP07 and HFD+Sily groups recovered to 2.15 ± 0.85 and 2.29 ± 1.11, respectively, which were significantly higher than the HFD group (p<0.05) and approached CTL levels. ALT levels were reduced to 33.17 U/L (HFD+ FLP07) and 37.83 U/L (HFD+Sily), suggesting that both treatments attenuated ALT elevation and restored the AST/ALT ratio, thereby indicating hepatoprotective effects. Data are shown in Figure 3.

TG levels in CTL (82.50 ± 4.68 mg/dL) and HFD (87.33 ± 4.32 mg/dL) were significantly higher than in HFD+ FLP07 (69.67 ± 7.50 mg/dL) and HFD+Sily (71.33 ± 6.83 mg/dL) (p<0.05). For TC, the order was: HFD (168.67 ± 6.06 mg/dL) > HFD+Sily (156.83 ± 6.11 mg/dL) > HFD+ FLP07 (151.00 ± 18.53 mg/dL) > CTL (103.50 ± 2.17 mg/dL). HFD+ FLP07 showed significantly lower TC compared with HFD (p<0.05). These results indicate that the lemon fermentation product effectively reduces serum TG and TC. Notably, TG levels in CTL were also higher than in the treatment groups, which may reflect differences in chow versus HFD composition and lipid distribution (Figure 4). Serum glucose levels were significantly higher in HFD (154.67 ± 17.28 mg/dL), HFD+ FLP07 (146.50 ± 26.99 mg/dL), and HFD+Sily (146.00 ± 17.94 mg/dL) compared with CTL (80.67 ± 9.37 mg/dL) (p<0.05). While no significant differences were observed among the HFD-fed groups (p>0.05), both HFD+ FLP07 and HFD+Sily showed lower glucose values compared with HFD, suggesting a potential glucose-lowering trend. Although these differences were not statistically significant, the downward trend suggests a potential glucose-lowering effect of FLP07 (Figure 4).

Liver TG content in HFD (57.51 ± 2.61 mg/g) was significantly higher than in CTL (50.79 ± 0.88 mg/g), HFD+ FLP07 (50.07 ± 3.03 mg/g), and HFD+Sily (50.33 ± 3.73 mg/g) (p<0.05). No significant differences were observed among the latter three groups (p>0.05). These findings indicate that both FLP07 and silymarin prevented excessive hepatic TG accumulation, maintaining levels similar to CTL. Data are shown in Figure 5.

In normal mice fed a standard diet, hepatic lobular architecture appeared intact, with orderly sinusoidal structures. Occasional small vacuolar swelling of hepatocytes was observed around the central vein (CV), which is considered a common background change related to postprandial glycogen accumulation (Figure 6A). Mice fed an HFD exhibited hepatic lipid accumulation (lipidosis) following absorption of dietary fat via the portal vein (PV) to the central vein. Hepatic cords were disrupted due to hepatocyte swelling, and large lipid droplets displaced nuclei to the cell periphery. However, no additional necrosis was observed (Figure 6B). Mice treated with FLP07 showed remarkable improvement compared with HFD. Hepatocyte swelling was alleviated, and hepatic cord architecture was restored. Although some livers still contained small to moderate lipid droplets, the overall histological integrity was similar to CTL, supporting a protective effect of FLP07 (Figure 6C). Mice treated with the silymarin-based drug showed notable histological improvement, with reduced hepatocyte swelling and restored hepatic cord structure. However, some livers still displayed localized vacuolar lipid droplets, particularly around the portal vein (Figure 6D). Histopathological evaluation demonstrated that severe lipid droplet accumulation (33–66%) was significantly increased in HFD (p<0.001), with a relative risk more than 20-fold higher than that of CTL, confirming successful induction of fatty liver. Administration of FLP07 or silymarin significantly reduced the incidence of steatosis (p<0.001) as assessed by histopathological scoring. Collectively, HFD+ FLP07 demonstrated significant improvements in hepatic steatosis comparable to HFD+Sily. Importantly, HFD did not induce significant hepatocellular necrosis, and no additional degeneration or necrosis was observed in mice treated with FLP07 at doses up to 500 mg/kg, indicating a safety profile comparable to HFD+Sily.

This study characterized the shifts in murine intestinal microbiota following an 12-week intervention across four experimental groups: a standard control diet, a high-fat diet (HFD), lemon fermentation peel (FLP07), and Silymarin (a positive control). Consistent with typical mammalian gut profiles, Firmicutes and Bacteroidetes constituted the predominant phyla across all cohorts, collectively accounting for over 80% of the total microbial abundance. In the control group, the taxonomic profile was characterized by a dominance of Bacteroidetes (75%), followed by Firmicutes (15%), Proteobacteria (3%), and Tenericutes (2%). Chronic HFD consumption induced a profound dysbiosis, significantly altering the phylum-level distribution. Notably, Firmicutes became the most prevalent taxon (69%), while Bacteroidetes decreased to 16%, accompanied by an emergence of Deferribacteres (6%). Subsequent administration of FLP07 and Silymarin appeared to modulate these HFD-induced shifts. In these intervention groups, while Firmicutes remained the leading phylum (62% and 55%, respectively), their relative abundance was significantly attenuated compared to the HFD group—decreasing by approximately 7% and 14%. Conversely, Bacteroidetes populations exhibited a restorative increase of 7% and 12%, respectively (Figure 7). These results suggest that both FLP07 and These results suggest that both FLP07 and Silymarin exert a regulatory effect on the Firmicutes-to-Bacteroidetes (F/B) ratio, a key indicator of metabolic health.

4. Discussion

Under normal physiological conditions, the gut microbiota maintains a eubiotic (balanced) state, characterized by inter-microbial antagonism, stable metabolic functions, and colonization resistance against pathogenic bacteria, thereby sustaining host health. However, significant alterations in the species diversity, composition, or proportions of the gut microbiota lead to dysbiosis, which is associated with various disease states. The gut microbiota of humans and animals is primarily composed of two dominant bacterial phyla: Firmicutes and Bacteroidetes. The absolute quantity and specific types of gut microbiota exhibit considerable inter-individual variability, influenced by factors such as host lifestyle and dietary habits ²⁷.Studies consistently indicate that the Firmicutes/Bacteroidetes ratio (F/B ratio) is a potential biomarker associated with several diseases and may serve as a preliminary diagnostic criterion for certain conditions. Obese individuals demonstrate an increased capacity for dietary energy extraction, which is linked to a higher abundance of Firmicutes in their gut, while a corresponding decrease in Bacteroidetes suggests that obesity is characterized by a shift in the F/B ratio ²⁸. Furthermore, individuals of normal weight typically exhibit greater diversity and abundance of Bacteroides (which are instrumental in breaking down plant starch and fiber for energy), whereas obese individuals show reduced Bacteroides diversity alongside an increased Firmicutes population ²⁹. A high-fat diet (HFD) can induce significant gut microbiota imbalance, promoting an increase in the types and quantities of various potentially harmful bacteria, including specific species within the Firmicutes, Bacteroidetes, Proteobacteria, Deferribacteres, and Clostridium phyla. Research indicates that elevated fat intake stimulates the growth of Proteobacteria and is positively correlated with total dietary fat consumption ³⁰. An HFD-induced obesity model experiment further revealed significant correlations between specific bacterial taxa and obesity phenotypes, noting an increased abundance of Deferribacteres and a decreased abundance of Porphyromonadaceae in affected subjects ^{30 31}. Collectively, these studies suggest that a sustained HFD promotes an increase in the abundance of specific bacteria from the Firmicutes, Bacteroidetes, Proteobacteria, and Deferribacteres phyla. These microbial changes, acting through different mechanisms, contribute to host obesity factors, such as enhanced lipogenesis and visceral fat accumulation in animal models.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive liver fat accumulation. Short-chain fatty acids (SCFAs), like acetate, propionate, and butyrate, are produced by microbes fermenting dietary fibers. These SCFAs can help manage NAFLD through the gut-liver axis by influencing lipid metabolism, barrier function, and inflammation. In previous study ²⁵, Hidenori Shimizu found Fermented foods constitute a vital component of the human diet, offering numerous health benefits. Among their key bioactive compounds are short-chain fatty acids (SCFAs)—typically produced from dietary fiber via gut microbial fermentation, but also found enriched directly in fermented products. Beyond serving as energy sources, SCFAs function as critical signaling molecules through G-protein coupled receptors, specifically FFAR2 and FFAR3 ²⁵. In this study, we demonstrate that dietary SCFA supplementation protects mice against high-fat diet-induced NAFLD. Such insights underscore the potential for developing SCFA-enriched functional foods to target metabolic disorders.

Non-alcoholic fatty liver disease (NAFLD) is closely associated with high-fat dietary intake, characterized by weight gain, dyslipidemia, and pathological changes in the liver. In this study, a mouse model of NAFLD was successfully established using a high-fat diet, enabling evaluation of the protective effects of fermented lemon peel in comparison with the commercial hepatoprotective agent silymarin ³². The high-fat diet significantly increased body weight, hepatic lipid accumulation, accompanied by a decreased AST/ALT ratio, abnormal serum lipid profile.

Although neither fermented lemon extract nor silymarin significantly suppressed HFD-induced weight gain, both interventions effectively improved serum TG and TC abnormalities, partially restored the AST/ALT ratio, and reduced hepatic TG accumulation. Notably, TG levels in the CTL group were higher than in the HFD+ FLP07 groups, which may reflect differences in dietary composition between chow and high-fat diets as well as distinct lipid distribution patterns. Histopathological analysis further confirmed that hepatocyte swelling and lipid droplet accumulation observed in the HFD group were markedly alleviated by fermented lemon peel, with improvements comparable to those seen with silymarin.

In addition, no mortality, abnormal clinical signs, or histopathological evidence of hepatocellular necrosis was observed in mice treated with fermented lemon extract, supporting a safety profile comparable to the positive control. Taken together, these findings suggest that fermented lemon extract mitigates high-fat diet–induced dyslipidemia and hepatic injury, demonstrating both hepatoprotective effects and favorable safety. Fermented lemon extract may therefore represent a promising candidate for development as a natural dietary supplement for the management of NAFLD, although further studies are needed to validate its efficacy and clarify the underlying mechanisms.

5. Conclusion

Bioactive compounds of fermented lemon peel act as a bridge between the host and gut microbiota. By modulating the gut microbiome and restore intestinal homeostasis, these natural compounds effectively alleviate the symptoms of NAFLD. Future clinical guidelines are expected to increasingly recommend lemon-based nutraceuticals as a strategic approach to managing metabolic liver diseases. This study shows the therapeutic potential of fermented citrus peel in metabolic fatty liver disease by in vitro model. However, further work is still needed in the future, that is, to verify the effect through human clinical trials.

Author Contributions: Ho-shin Huang and Chi-Yu Yang carried out all the experiments; Pei-Chun Lin, Chun-Mei Lu, Ting-Yuan Hsu designed all the experiments and analyzed the data; Ho-shin Huang and Pei-Chun Lin wrote the manuscript.

Funding: This research was supported by Agricultural Technology Park Administration Center of Taiwan (114-ATP-1.6.2- -01).

Acknowledgments: We extend a special thanks to members from Agricultural Technology Research Institute for their support throughout the preparation of this manuscript.

Conflicts of Interest: The authors declare that there are no conflicts of interest.

References