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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2022 Apr 13;60(3):1036–1044. doi: 10.1007/s13197-022-05445-w

Lemon fermented products prevent obesity in high-fat diet-fed rats by modulating lipid metabolism and gut microbiota

Chih-Chung Wu 1, Yu-Wen Huang 2, Chih-Yao Hou 2, Ya-Ting Chen 2, Cheng-Di Dong 3, Chiu-Wen Chen 3, Reeta Rani Singhania 3, Jie-Yin Leang 4, Shu-Ling Hsieh 2,✉
PMCID: PMC9998762  PMID: 36908372

Abstract

Citrus limon (lemon) possesses immunoregulatory, antioxidant, and lipid-lowering effects. Our previous study showed that lemon fermented products (LFP) which were lemon fermented with Lactobacillus OPC1 had the ability to avert obesity. However, the LFP effects on the pathway of lipid metabolism by gut microbiota were still unclear. This study was aimed to investigate the LFP effects on liver lipid metabolism and gut microbiota in a rat model of obesity caused by a high-calorie diet. LFP effectively reduced the total triglyceride (49.7%) and total cholesterol (53.3%) contents of the liver. Additionally, the mRNA levels of genes related to triglyceride metabolism (SREBP-1c, PPARγ, and ACC), cholesterol metabolism (HMG-CoA reductase, ACAT, and LCAT), and lipid β-oxidation (PPARα, and CPT-1) were regulated by LFP. Furthermore, LFP reduced the ratio of Firmicutes/Bacteroidetes and enhanced the ratio of Firmicutes Clostridia. Overall, these findings suggested that LFP might use as a potential dietary supplement for preventing obesity by modulating the lipid metabolism and improving the gut microbiota.

Keywords: Lemon fermented products, Liver lipid, Gut microbiota, F/B ratio, Lipid metabolism

Introduction

Obesity is described as an excess of fatty tissue in the body that raises the risk of a number of chronic diseases including diabetes mellitus type 2, hypertension, and cardiovascular disease (Bermano et al. 2020; Burhans et al. 2011). However, the regulation of obesity is a series of complex mechanisms. The massive secretion of insulin will promote triglyceride metabolism by stimulating the activity of sterol regulatory element-binding protein-1c (SREBP-1c), CCAAT/enhancer-binding proteins (C/EBP), and peroxisome proliferators activated receptor gamma (PPARγ). PPAR alpha (PPARα) regulates lipid and glucose homeostasis in adipocytes, hepatocytes, muscle cells, and epithelial cells. On the other side, its activation increased mitochondria β-oxidation promoting fatty acid metabolism which results in lipid content reduction. Carnitine palmitoyltransferase I (CPT-1) inhibit malonyl-CoA to reduce lipogenesis, controlling the β-oxidation process of long-chain fatty acid into mitochondria. In addition, cholesterol is one of the substances that make up cell membranes. When there is too much cholesterol in the body, it is easy to cause atherosclerosis. The liver is the main site of cholesterol metabolism and regulates cholesterol through the expression of HMG-CoA-reductase, Sterol O-acyltransferase (ACAT) and Lecithin-cholesterol acyltransferase (LCAT) metabolism (Javitt 1994).

Gut plays an important role in nutrition and energy intake as well as lipid metabolism, and it can help prevent metabolic disorders from developing (Tremaroli and Bäckhed 2012). Recent studies have found that both food and its functional components may change the composition of intestinal bacteria to regulate lipid metabolism and prevent obesity. Therefore, the regulation of intestinal bacteria may become a potential ability to control obesity (Martel et al. 2017). After high-fat diet-induced obesity in mice, the feces of obese mice had a reduced operational taxonomic unit (OTU), and also found that it can increase the intestinal flora. The ratio of Firmicutes/Bacteroides in bacteria indicates that obesity reduces the diversity of intestinal bacteria and modulates the composition of intestinal bacteria to make metabolic disorders. The fermentation of intestinal bacteria is a complex reaction. It involves the interaction of many bacterial species and the decomposition of dietary fiber, protein and carbohydrates to yield short-chain fatty acids (SCFAs), which includes butyric acid, propionic acid and acetic acid. SCFAs generate 5–10% of dietary energy from food, of which 95% is absorbed in the gut and only 5% is excreted in feces (Fernandes et al. 2014). Acetate and propionate are absorbed by the liver and promote adipogenesis and gluconeogenesis (Tremaroli and Bäckhed 2012). Butyric acid can synthesize vitamin K, biotin, folic acid, and provide an energy source of the colon mucosa, promote apoptosis, increase anti-inflammatory activity and improve insulin through activation of potent histone deacetylase inhibitor activity (Pryde et al. 2002). Previous studies have shown that the polyphenols of citrus can reduce the stimulation of oxidative stress in the body by changing the ecology of intestinal bacteria and promoting the increase of beneficial bacteria for intestinal health (Elavarasan et al. 2012). Our study has confirmed that lemon fermented products (LFP) can achieve anti-obesity effects by regulating the expression of lipid metabolism factors in the epididymal fat. However, the lipid metabolism pathway of the liver is still unclear, and it is not known whether the anti-obesity effect is achieved by regulating the changes in the gut microbiota.

In this study, the effectiveness and mechanism of LFP's effects on liver lipid metabolism (including triglyceride, cholesterol metabolism, and lipid β-oxidation) and gut microbiota a Wistar rats of obesity caused by a high-calorie diet were examined to establish its potent application in averting obesity.

Materials and methods

Chemicals and reagents

Chloroform was procured from Sigma-Aldrich (St. Louis, USA). Phosphate-buffered saline (PBS) and sodium bicarbonate were procured from Uni-onward (Taipei, Taiwan). Isopropanol was procured from J.T Baker (Pennsylvania, USA). 10 mM dNTPs were procured from Protech (Taipei, Taiwan). Commercial kits TC (CH202) and TG (TR212) to analyze the levels of total cholesterol were procured from RANDOX (Ireland, UK). RNAsin, M-MLV RT 5X buffer, M-MLV Reverse transcriptase and Oligo dT were procured from Promega (Madison, USA). 2X SYBR Frist MM and primer were procured from Topgen (Kaohsiung, Taiwan).

Preparation of LFP

LFP was prepared according to the procedure defined by previous study (Hsieh et al. 2020). Openmind Co. Ltd. gave lemon and Lactobacillus OPC1 (Kaohsiung, Taiwan). The lemon was then squeezed into juice using a citrus juicer equipment. Lemon juice (a pH of 2.5–2.8 and a °Brix of 7–10) was inoculated with Lactobacillus OPC1 (0.1 percent, powder, estimated by the weight of the lemon juice) at 25 °C to ferment. To get lemon fermented products (LFP), the sample was sterilized (80 °C for 1 min) after 90 days of fermentation.

Animals and treatments

The animal model was prepared according to the procedure established by our previous study (Wu et al. 2021). BioLASCO Taiwan Co., Ltd. provided male Wistar rats (six-week-old, 250 ± 20 g) (Taipei, Taiwan). All animal care and experimental techniques utilized in this study were approved by the Institutional Animal Care and Use Committees (IACUC) at the National Kaohsiung University of Science and Technology (Kaohsiung, Taiwan). 0109-AAAP-014 is the IACUC number. Rats were housed in a cage with a 12-h light/12-h dark cycle and a constant ambient temperature of 22 °C. The rats were divided into five groups (n = 5), after getting acclimatized in a week and fed a normal diet (ND), a high-calorie diet (HD), or HD combined with 0.58 g/kg LFP (the low dose of LFP; LLFP), HD combined with 1.73 g/kg medium dose of LFP (the medium dose of LFP; MLFP), and HD combined with 2.89 g/kg LFP (the high doses of LFP Oral gavage was used to deliver LFP. ND: 3.36 kcal/g, 13.5% lipid, 58.0% carbohydrate, 28.5% protein; HD: 8.48 kcal/g, 45.0% lipid, 45.0% carbohydrate, 10.0% protein.

The contents of liver lipid assay

According to the method of Folch in 1957 (Folch, 1957), about 0.1 ~ 0.2 g of liver tissue weight was removed, and 500 µL of a mixture of chloroform and methanol (2:1, v/v) was added to grind. Dried at 60 °C for 1 h, centrifuge at 10,000 × g for 15 min. Aliquot 100 µL of supernatant, place in a 75 °C until completely dry, and store at – 20 °C. Before the analysis of triglycerides and total cholesterol, the prepared liver lipid samples were added to 100 µL of 95% ethanol. The mixture was uniformly mixed by ultrasonic vibration. The amounts of TG and TC were measured using commercial kits following the manufacturer's instructions.

mRNA levels

The TRIzol reagent was used to extract total RNA according to the manufacturer's instructions. An Epoch microvolume spectrophotometer (Bio-Tek, Vermont, USA) was used to measure the total RNA. The cDNA was generated using M-MLV reverse transcriptase after RNase treatment and RNA purification. SYBR green was used to perform a quantitative PCR analysis. Using a real-time PCR method (LightCycler® 96 Real-Time PCR System obtained from Roche Life Science, Basel, Switzerland), relative gene expression was measured. For all experiments, a denaturation step of 120 s at 95 °C was followed by 40 cycles (at 95 °C for 5 s and at 60 °C for 30 s). In the melting analysis, denaturing at 95 °C for 10 s, cooling to 65 °C for 60 s, and then heating to 97 °C. Specific primers were used to amplify each cDNA. The values of target genes' threshold cycles (Ct) were standardized to the Ct values of β-actin. The fold changes in gene expression were calculated using the comparative Ct technique. The genes (target) were PPARγ, SREBP-1c, ACC, HMG-CoA-R, ACAT, LCAT, PPARα, and CPT-1 (Table. S1), and the gene-specific primers utilized were β-actin as a control group. The experimental approach was prepared according to the procedure established by our previous study (Wu et al. 2021).

The contents of SCFAs

SCFAs were determined according to the validated protocol of Hsu et al. (Hsu et al. 2021). First of all 0.03 g of feces was taken and then 0.3 mL of ddH2O was added, homogenized, centrifuged at 4 °C, 14,100 × g for 15 min, then to 150 μL of supernatant 50 μL of sulfuric acid was added, 10 μL 2-ethyl butyric acid and 400 μL ethoxyethane after shaking with ether for 15 min, centrifuged at 10,600 × g for 15 min at 4 °C. The supernatant was analyzed by GS/MS (Agilent Technologies, USA).

Bacterial genomic DNA isolation of stool

The QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany) was used to isolate total genomic DNA from 200 mg fecal samples according to the manufacturer's instructions. The concentrations of DNA were determined using a NanoDrop2000 (Thermo Scientific, Waltham, MA, USA), and the samples were kept at 80 °C until they were used.

16S rRNA gene sequencing and data analysis

According to the Illumina 16S Metagenomic Sequencing Library Preparation guide, the V3-V4 region of the 16S rRNA gene was PCR-amplified with the primers 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3') to form amplicon libraries. The amplicons were then paired-end sequenced (PE 2 × 250) using an Illumina HiSeq 2000 platform, as per the manufacturer's instructions. After passing quality control, the paired forward and reverse reads were concatenated and mapped to the Silva database using the UPARSE technique to create operational taxonomic units (OTUs) with 97% identity (drive5, Tiburon, CA, USA). The data was analyzed using QIIME (Quantitative Insights Into Microbial Ecology). ChimeraSlayer was used to eliminate chimeric sequences. OTUs were allocated to sequences that were 97 percent similar. The genes were annotated using the Silva database. Weighted principal coordinate analysis (PCoA) was used to examine beta diversity.

Statistical analysis

The data was examined by SSPS (SPSS, Inc., Chicago, IL, USA) for windows version 20.0. The importance of variations between the mean values was evaluated using Duncan's test. A statistically significant result was determined by a P value of < 0.05.

Results and discussions

Effect of LFP on the liver lipid of rats fed a high-calorie diet

To investigate the LFP effects on the liver lipid of rats fed a high-calorie diet (Fig. 1). After 9 weeks of feeding, the liver TG, and TC of the HD group were expressively higher than that of the other groups. When compared to the HD group, rats treated with MLFP, HLFP or LLFP, had significantly lower liver TG and TC (P < 0.05).

Fig. 1.

Fig. 1

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Effect of LFP on the liver lipid of rats fed a high-calorie diet. Duncan’s test was used to determine the significance of differences in mRNA levels of genes related to the liver lipid. The data is presented as means ± SD (n = 5). Different letters a−d denote statistically significant differences in liver lipids (P < 0.05). The groups are abbreviated as Normal diet (ND), High calories diet (HD), HD with low LFP (HD + LLFP), HD with medium LFP (HD + MLFP), HD with high LFP (HD + HLFP)

The liver is one of the most important metabolic organs. Excessive fat accumulation can cause hepatic steatosis. It can easily lead to fatty liver and other liver diseases. Therefore, reducing the accumulation of fat in the liver can reduce the occurrence of liver disease (Nguyen et al. 2008). Soybean meal fermented by Lactobacillus Plantarum FPS2520 (FPS) can reduce the body weight and liver TG and TC content of rats. Fermented ginseng root and ginseng fruit can reduce the body weight, fat cell area and liver TG and TC content of mice (Li et al. 2018). Based on the above, it was found that fermented plants can reduce the content of TG and TC in animal livers. In this study, it was also shown that LFP could reduce the content of TG and TC in rat liver.

Effect of LFP on mRNA levels of genes related to triglyceride metabolism in the liver of rats fed a high-calorie diet

Real-time RCR was used to look at the mRNA levels of genes associated to triglyceride metabolism in the liver of rats fed a high-calorie diet to learn more about the mechanism of LFP on triglyceride metabolism. The HD group had substantially greater mRNA levels of PPAR, SREBP-1c, and ACC (P < 0.05) than the ND group. The mRNA levels of PPAR, SREBP-1c, and ACC in the liver of the LLFP, MLFP, and HLFP treated groups were substantially lower than those of the HD group (P  < 0.05). (Fig. 2). FAS, ACC, and SREBP-1c mRNA levels can be reduced by Lactobacillus plantarum EM fermented cabbage-apple (FCA) (Park et al. 2020). Curcuma longa L. ethanol extract (FCE50) inhibits the mRNA levels of PPARγ, C/EBPα, FAS, and ACC (Kim et al. 2016). In this study, it was also shown that LFP could regulate the mRNA levels of genes related to triglyceride metabolism in rat liver (including PPAR, SREBP-1c, and ACC).

Fig. 2.

Fig. 2

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Effect of LFP on mRNA levels of genes related to triglyceride metabolism in the liver of rats fed a high-calorie diet. Duncan’s test was used to determine the significance of differences in mRNA levels of genes related to triglyceride metabolism. The data is presented as means ± SD (n = 5). Different letters a−c denote statistically significant differences in mRNA levels (P  < 0.05). Data are the means ± SD (n = 5). The groups are abbreviated as Fig. 1

LFP effects on mRNA levels of genes associated to cholesterol metabolism in the liver of rats fed a high-calorie diet

To investigate the LFP effects on mRNA levels of genes related to cholesterol metabolism in the liver of rats fed a high-calorie diet (Fig. 3). The HLFP, LLFP and MLFP treated groups showed expressively lower mRNA levels of HMG-CoA-R and ACAT in the liver than the HD group. (P  < 0.05). In comparison to the HD group, the LLFP, MLFP, and HLFP treated groups had significantly higher mRNA levels of LCAT in the liver (P  < 0.05). The liver is the central organ of cholesterol metabolism, it can break down dietary cholesterol, thereby inhibiting cholesterol synthesis (Fraser et al. 1978). Fermented garlic can reduce the gene expression of HMG-CoA-R and ACAT in the liver (Irfan et al. 2019). Degrace et al. (2016) used male C57BL/6 mice orally administered 0.57 g ethanol/kg (containing 5.9% Mild beer or Ethanol-free beer), and the results showed that Mild beer and Ethanol- Free beer reduces LACT gene expression in the liver. Previous research has found that it can lower liver lipid levels by influencing the expression of genes related to cholesterol metabolism, especially HMG-CoA-R, ACAT, and LCAT (Irfan et al. 2019; Degrace et al. 2016).

Fig. 3.

Fig. 3

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Effect of LFP on mRNA levels of genes related to cholesterol metabolism in the liver of rats fed a high-calorie diet. Duncan’s test was used to determine the significance of differences in mRNA levels of genes related to cholesterol metabolism. The data is presented as means ± SD (n = 5). Different letters a−d denote statistically significant differences in mRNA levels (P  < 0.05). The groups are abbreviated as Fig. 1

LFP effects on mRNA levels of genes related to lipid β-oxidation in the liver of rats fed a high-calorie diet

Furthermore, we investigated the mechanism of LFP on lipid β-oxidation in rats given a high-calorie diet by using real-time RCR to evaluate the mRNA levels of genes related to lipid β-oxidation in the liver. The HD group had significantly lower mRNA levels of PPARα and CPT-1 than the ND group (P  < 0.05). When compared to the HD group, the LLFP, MLFP, and HLFP treated groups had substantially higher mRNA levels of PPARα and CPT-1 in the liver (P  < 0.05) (Fig. 4). Curcuma longa L. ethanol extract (FCE50) increases the mRNA levels of CPT-1 to regulate the lipid β-oxidation in white adipose tissue of high-fat diet-induced obese rats (Kim et al. 2016). The Hakka stir-fried tea decreased the levels of serum and liver triglyceride and increased the phosphorylation of AMP-activated protein kinase (AMPK) and its direct downstream proteins ACC, and CPT-1 (Li et al. 2020). It can be seen from the above that it can promote the role of lipid β-oxidation by regulating the expression of PPAR and CPT-1, thereby inhibiting the accumulation of lipids.

Fig. 4.

Fig. 4

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Effect of LFP on mRNA levels of genes related to β-oxidation in the liver of rats fed a high-calorie diet. Duncan’s test was used to determine the significance of differences in mRNA levels of genes related to β-oxidation. The data is presented as means ± SD (n = 5). Different letters a−d denote statistically significant differences in mRNA levels (P  < 0.05). The groups are abbreviated as Fig. 1

LFP effects on SCFAs and microbial structure in rats fed with a high-calorie diet

The changes in SCFAs and microbial structure of rats fed with a high-calorie diet with LFP are given in Table 1. The content of acetic acid in MLFP, and HLFP was significantly lower than that in the HD group. The content of propionic acid in the HD group was expressively higher than that in ND group. The HLFP, LLFP, and MLFP treated groups displayed expressively decreased content of propionic acid on comparison with the HD group, the effect of HLFP is better. The butyric acid content of the HD group was significantly lower than that of the ND group. The butyric acid content of middle-dose LFP was significantly higher than that of the HD group. In addition, the Actinobacteria and F/B ratio of the HD group were significantly higher than those of the ND group. The MLFP treated groups exhibited significantly decreased Actinobacteria and F/B ratio compared with the HD group (P  < 0.05). It can be seen from the above that LLFP, MLFP, and HLFP can reduce the content of propionic acid in the feces of high-calorie diet-induced obese rats, while the MLFP and HLFP can reduce the content of acetic acid, and the MLFP can reduce the intestinal flora Actinobacteria and F/B ratio to achieve the purpose of regulating lipid metabolism.

Table 1.

Effect of LFP on SCFAs and microbial structure in rats fed a high-calorie diet

ND HD HD + LLFP HD + MLFP HD + HLFP
Acetic acid (μmol/g) 50.44 ± 7.98a 50.41 ± 8.17a 41.89 ± 2.42ab 36.25 ± 5.45bc 29.61 ± 3.68c
Propionic acid (μmol/g) 4.81 ± 0.68b 8.56 ± 2.38a 4.61 ± 1.40bc 3.96 ± 0.50c 2.89 ± 0.16d
Butyric acid (μmol/g) 2.71 ± 0.41a 1.76 ± 0.37b 1.88 ± 0.75b 3.14 ± 0.26a 1.67 ± 0.08b
Firmicutes (%) 60.12 ± 6.04a 59.19 ± 1.28a 61.06 ± 6.06a 60.28 ± 6.51a 59.28 ± 0.23a
Bacteroidetes (%) 36.35 ± 4.66a 32.42 ± 1.29a 32.11 ± 3.57a 34.74 ± 2.08a 33.18 ± 4.08a
Actinobacteria (%) 0.16 ± 0.04b 5.17 ± 0.45a 5.30 ± 0.82a 0.68 ± 0.16b 4.78 ± 0.16a
F/B ratio 1.47 ± 0.25c 1.85 ± 0.08a 1.84 ± 0.19a 1.48 ± 0.08bc 1.76 ± 0.06ab
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The data is presented as means ± SD (n = 5). Different letters a−d denote statistically significant differences in mRNA levels (P < 0.05). The groups are abbreviated as Fig. 1

Gut microbiota composition

In the main coordinate analysis of intestinal flora, it was found that the trend distribution of intestinal flora was different between ND group and HD group (Fig. 5a). In the bacterial phase heat map, it was found that the color from blue to red indicates that the content of the flora is from the least to the most. The contents of Ruminococcaceae, Lactobacillus, and Eutactus in the ND group are higher than those in the HD group. The contents of these flora are thought to be linked to the improvement of obesity (Fig. 5b). According to the relative abundance analysis of ''order'', the content of Firmicutes Clostridiales in the HD group was lower than that in the ND group; the Firmicutes Clostridiales in the middle dose of LFP was higher than that in the HD group (Fig. 5c). Firmicutes Clostridiales can be used as an indicator in animal models of high-calorie diet-induced obesity. From the above, it is known that the middle dose of LFP can improve the distribution of fecal intestinal flora and increase the proportion of Firmicutes Clostridiales in high-calorie diet-induced obese rats, so as to achieve the purpose of regulating lipid metabolism.

Fig. 5.

Fig. 5

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Gut microbiota composition. a Beta diversity is indicated by weighted principal co-ordinates analysis (PCoA); b Heatmap analysis; c The relative abundance of order (P < 0.05). n = 5. The groups are abbreviated as Fig. 1

The changes in the intestinal flora and the content of SCFAs can directly reflect the balance of energy metabolism in the organism. Therefore, the changes in the intestinal flora and the content of SCFAs are one of the trends in the research on metabolic-related diseases in recent years (Zhao 2013). Zhang et al. (2020) found that Chenpi reduced the F/B ratio in feces (Zhang et al. 2020). Fernandes et al. (2014) divided healthy subjects into a lean group (BMI⩽25) and an obese group (BMI > 25) with 11 healthy subjects, and their feces were analyzed. The experimental results showed that the obese group was more than the lean group. There is more acetic acid in the feces (Fernandes et al. 2014). A mixture of agave and banana powder reduced serum TG and TC levels and decreased fecal acetic acid levels in obese mice (Alvarado-Jasso et al. 2020). Lactobacillus plantarum fermented Momordicacharantia juice (FMCJ) could reduce body weight, serum TG and TC levels, and increase fecal butyrate content in obese/diabetic rats (Gao et al. 2019). Based on past studies, it is found that fermented plants or citrus can regulate lipid metabolism by regulating changes in fecal intestinal bacteria or reducing F/B ratio, while lactic acid bacteria can increase the proportion of Clostridium in intestinal bacteria. The results of human experiments showed that the content of acetic acid in the feces of obese people will be higher, and the results of animal experiments showed that plants can regulate lipid metabolism by reducing the content of acetic acid or increasing the content of butyric acid in the feces of obese mice. The results of this study show that LFP can reduce the fecal F/B ratio and acetic acid content of obese rats, and increase the fecal butyric acid content and the proportion of Clostridium, so as to achieve the effect of regulating lipid metabolism.

Conclusion

According to this study, lemon fermented product (LFP) can inhibit lipid accumulation by reducing the mRNA levels of PPARγ, SREBP-1c, ACC, and ACAT in the liver, and improving the mRNA levels of HMG-CoA-R, LCAT, PPARα, and CPT-1 in the liver. In addition, LFP regulated the contents of SCFAs in gut microbiota and modulated the change of gut microbiota (F/B ratio). We assumed that the regulation in the content of Firmicutes Clostridiales was the reason. These findings suggest that LFP might be used as a potential dietary supplement for preventing obesity by modulating the triglyceride, cholesterol metabolism, lipid β-oxidation, and changing the gut microbiota.

Acknowledgements

All the authors acknowledge Openmind Co. Ltd. for providing lemon fermented goods for this project.

Abbreviations

PPARγ

Peroxisome proliferators activated receptor gamma

PPARα

Peroxisome proliferators activated receptor alpha (PPARα)

SREBP-1c

Sterol regulatory element-binding protein-1c

ACC

Acetyl CoA carboxylase

HMG-CoA-R

3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase

ACAT

Sterol O-acyltransferase

LCAT

Lecithin-cholesterol acyltransferase

CPT-1

Carnitine palmitoyltransferase I

SCFAs

Short-chain fatty acid

OUT

Operational taxonomic unit

Real-time PCR

Real-time reverse transcriptase polymerase chain reaction

PCoA

Principal coordinate analysis

Authors' contributions

Conceptualization, C.-C.W. and S.-L.H.; methodology, C.-C.W.; validation, C.-C.W. and Y.-W.H.; investigation, Y.-W.H., C.-Y.H. and Y.-T.C.; data curation, Y.-W.H.; writing-original draft, Y.-W.H.; formal analysis, C.-Y.H.; writing-review & editing, C.-D. D., C.-W.C. and R.-R.S.; resources, C.-Y.H. and J.-I.L.; supervision, S.-L.H.; project administration, S.-L.H. All authors read and agreed to the published version of the manuscript.

Funding

This research was funded by Council of Agriculture, Executive Yuan, Republic of China (Grant Numbers 110 Agriculture-14.1.1-science-aD).

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethics approval

The work has not been published before and it is not under consideration for publication elsewhere.

Consent to participate

The MS has been approved by all authors.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Chih-Chung Wu, Email: wuccmail@gmail.com.

Yu-Wen Huang, Email: may2377234@gmail.com.

Chih-Yao Hou, Email: chihyaohou@gmail.com.

Ya-Ting Chen, Email: melodyyu.chen@gmail.com.

Cheng-Di Dong, Email: cddong@nkust.edu.tw.

Chiu-Wen Chen, Email: cwchen@nkust.edu.tw.

Reeta Rani Singhania, Email: reetasinghania@nkust.edu.tw.

Jie-Yin Leang, Email: dllm7117@yahoo.com.tw.

Shu-Ling Hsieh, Email: slhsieh@nkust.edu.tw.

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