6N mice fed high-fat diet

Journal of Functional Foods xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff

Lactobacillus plantarum LP104 ameliorates hyperlipidemia induced by AMPK pathways in C57BL/6N mice fed high-fat diet Yue Tenga, Yu Wanga, Yuan Tiana, Yi-ying Chena, Wu-yang Guana, Chun-hong Piaoa,b,c,d, ⁎ Yu-hua Wanga,b,c,d, a

College of Food Science and Engineering, Jilin Agricultural University, Changchun, China Jilin Province Innovation Center for Food Biological Manufacture, Jilin Agricultural University, Changchun, China c National Processing Laboratory for Soybean Industry and Technology, Changchun, China d National Engineering Laboratory for Wheat and Corn Deep Processing, Changchun, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Lactobacillus plantarum LP104 Hyperlipidemia AMPK Liver injury

Lactobacillus plantarum LP104 was isolated with excellent antioxidant properties from kimchi. In order to evaluate the role of LP104 in improving hyperlipidemia in high-fat-diet mice, C57BL/6N mice were randomly assigned into three groups treated with different diets: normal chow (Control), HFD, and HFD with L. plantarum LP104. After eight weeks, L. plantarum LP104 treatment significantly reduced HFD-induced body weight gain and the levels of serum or liver TC, TG, LDL, ALT, AST, LPS and TNF-α, and elevated liver HDL levels. The liver lipid accumulation was decreased in L. plantarum LP104 group. L. plantarum LP104 could activate the AMPK/ Nrf2/CYP2E1 related pathway and regulate expressions of related proteins by using western blotting. Therefore, L. plantarum LP104 will improve hyperlipidemia, liver metabolic disorders, and liver oxidative stress response.

1. Introduction Hyperlipidemia is a pathological condition characterized by the increased concentrations of serum triglycerides (TG), total cholesterol (TC), free fatty acids (FFA) and low-density lipoprotein cholesterol (LDL-C), along with the excessive accumulation of TG in the liver (Zhang, Wang et al., 2017, Zhang, Wu et al., 2017). The increased concentrations of blood cholesterol are strongly associated with an elevated risk of atherosclerosis and cardiovascular diseases (CVDs). The WHO predicted that up to 40% of deaths would be related to CVD by 2030, affecting approximately 23.6 million people worldwide (Chanet et al., 2012; Kim et al., 2017). The excessive accumulation of TG by hypercholesterolemia in the liver is also considered an essential contributor to nonalcoholic fatty liver disease (NAFLD) (Wang et al., 2019). It has been estimated that the overall global prevalence of NAFLD was around 25.43% (Younossi et al., 2016). Lifestyle modification consisting of diet, exercise, and weight loss has been advocated to treat patients with NAFLD (Xia et al., 2019). However, reduced compliance restricted the interventions (Del Ben, Polimeni, Baratta, Pastori, & Angelico, 2016). Thus, reducing the lipid levels in blood and liver became an important method that can be applied to prevent the formation of hyperlipidemia. To date, pharmacological treatments (such as statins) are the most widely used treatment for hypercholesterolemia. ⁎

However, some of these drugs are expensive and have harmful side effects (Rajesh, Sunita, & Virender Kumar, 2011; Stefano, Rodolfo, & Alberto, 2004; Vaughan, Murphy, & Buckley, 1996). Therefore, the development of functional and nutritious foods will play an essential role in the treatment of hyperlipidemia. Probiotics are “living microorganisms that, when administered in adequate amounts confer a health benefit on the host” (Zhang, Wang et al., 2017, Zhang, Wu et al., 2017) and it have been reported that they also exert other health-promoting effects on a variety of diseases, including hypertension (Siokkoon & Mintze, 2010), cancer (Kumar et al., 2010), allergy symptoms (Weston, Halbert, Richmond, & Prescott, 2005), arthritis (Baharav, Mor, Halpern, & Weinberger, 2004), obesity (Miyoshi, Ogawa, Higurashi, & Kadooka, 2014), etc. In particular, the hypolipidemic efficacy of probiotics has also been extensively studied in animal and clinical studies. Lactobacillus acidophilus lowered blood cholesterol by decomposing cholesterol and deconjugating bile salts (Song et al., 2019). Another study showed that Bifidobacterium longum reduced serum TC, LDL-C, and TG and increased high-density lipoprotein cholesterol (HDL-C) in humans (Nobili et al., 2018). Also, probiotics improved the activity of hepatic enzyme and decreasing the serum TC and LDL-C levels in the treatment of NAFLD (Nabavi, Rafraf, Somi, Homayouni-Rad, & Asghari-Jafarabadi, 2014). Therefore, application of probiotic interventions has attracted widespread attention (Devaraj,

Corresponding author at: College of Food Science and Engineering, Jilin Agricultural University, Changchun, China. E-mail address: [email protected] (Y.-h. Wang).

https://doi.org/10.1016/j.jff.2019.103665 Received 27 August 2019; Received in revised form 27 October 2019; Accepted 1 November 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Yue Teng, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103665

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

(n = 10): normal-fat diet (NFD) feeding group, high-fat diet (HFD) feeding group as well as in HFD + LP104 group, LP104 culture broth (109 CFU/mouse per day) was added to the random diet for 8 weeks. The composition of the HFD consisted of 78.8% normal diet, 10% egg yolk, 10% lard, 1% cholesterol and 0.2% bile salt. Food intake was recorded daily, the body weight was recorded every week, and the body length was finally measured to calculate Lee’s index. At the end of the experimental period, all mice were sacrificed after fasted for 12 h. The organs and fats were carefully removed. Portions of the liver were immersed in 10% formaldehyde for histological analysis, and the other portions were frozen immediately in liquid nitrogen and stored at −80 °C for immunoblot analysis. The abdominal and perirenal fat indexes were defined as the adipose tissue mass divided by the BW. Lee’s index was calculated according to formulas:

Reddy, & Xu, 2019). It was reported that the critical targets for hyperlipidemia prevention and treatment interventions should be exercise and diet, which are two health-related physiological regulators activate AMP-activated protein kinase (AMPK), a common regulatory mechanism (McCrabb et al., 2019). Probiotics can alter the expression of proteins upstream of AMPK, thereby improving the synthesis of cholesterol and fatty acids. Therefore, this study was aimed to evaluate the potential of L. plantarum LP104 to improve hyperlipidemia in mice fed HFD and investigate its effects on the mechanism of lipid metabolic disorders, offering theoretical evidence for developing functional foods based on LP104. 2. Materials and methods

Lee's index = body weight (g) 1/3 × 1000/body length (cm).

2.1. Isolation and strain identification

2.5. Serum samples and liver homogenate preparation

Korean kimchi (nature fermentation) was soaked in sterilized MRS medium (BD-Difco Corporation, US), and then was cultured at 37 °C for 24 h. The strains were passaged three times, inoculated into 100 mL of MRS medium according to the inoculation amount of 3% of the culture solution, and cultured at 37 °C for 24 h, and the concentration of the cells was adjusted to 109 CFU/mL. Third generation strain was inoculated onto an MRS agar plate and incubated at 37 °C for 24 h. When grown on MRS agar plates, the colonies were milky white with a smooth round surface. The images generated by optical microscopy showed that the cells were Gram-positive, short rod-shaped, flagellafree, and spore-free. Based on the observation of colony morphology and microscopic examination, the pure strain was obtained by repeated steps of separation and purification. The pure strain was identified via 16S rDNA sequencing. DNA was extracted using the Ezup column of a bacterial genomic DNA extraction kit (Ezup Column Bacteria Genomic DNA Purification Kit, SK8255, Sangon Biotech Shanghai Co. Ltd, China). After PCR amplification and purification, the samples were sequenced by Shanghai Biotechnology Engineering Co. Ltd. The resulting 16S rDNA sequencing results were submitted to GenBank and were input to Blast on the NCBI website for homology comparison to which used the NJ (Neighbor-Joining) method, building a phylogenetic tree.

At the end of the experiment, whole blood was collected from the inferior vena cava after mice were anesthetized with Avertin. Serum was separated by centrifuging the samples at 2000g for 10 min at 4 °C and then stored in Eppendorf tubes at −80 °C. Samples of 0.1 g liver tissue were accurately weighed and then added into 0.9 mL normal saline and homogenized using a homogenizer (BioSpec Products, WI, USA). The liver tissue homogenate was then centrifuged at 3000g for 10 min, and the supernatant was used for further study. 2.6. Biochemical assays The levels of TG, TC, low-density lipoprotein (LDL), high-density lipoprotein (HDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutathione peroxidase (GSH-Px) in liver or serum were determined by a commercial kit (Jian-Cheng Institute, Nanjing, China). Serum or liver lipopolysaccharides (LPS), tumor necrosis factor-α (TNF-α) and FFA levels were determined by mouse ELISA kits (RD, USA). All data were measured using a microplate reader (Tecan, Switzerland). 2.7. Histological analysis

2.2. Preparation of strain fermentation supernatant and bacterial cells Pieces of fixed liver tissue were embedded in paraffin for histological analysis. The embedded tissues were sectioned at 5 μm thickness, stained with hematoxylin and eosin, and examined under a light microscope (Leica, Germany).

LP104 was passaged three times. A part of the fermentation broth was spared, and another part of the fermentation broth was centrifuged at 4 °C, 4000 r/min for 10 min, then the fermentation supernatant was separated for utilization; the cells were washed twice with 0.85% physiological saline and suspended in an equal volume of physiological saline to obtain bacterial cell fluid.

2.8. Western blot analysis Sliced liver tissues were placed in RIPA cell lysis buffer (Solarbio, Beijing, China) and homogenized in homogenates. The liver lysate was centrifuged at 12000 rpm for 10 min at 4 °C after 30 min on ice. The supernatant was removed and the total protein content was measured by using the BCA Protein Assay Kit (Beyotime Biotechnology, USA). 10–20 μg of protein were loaded onto 8%, 10% or 12% SDS-PAGE, proteins were transferred to PVDF membranes, the membranes, which were blocked with 5% skim milk for 1 h at room temperature, and the blocked membranes were incubated with antibodies against AMP-activated protein kinase (AMPK), phosphor-AMPK (pThr172), acetyl-CoA carboxylase (ACC), phosphor-ACC (pSer79), sterol regulatory elementbinding protein (SREBP-1), Stearoyl-CoA desaturase 1 (SCD1), nuclear factor-like 2 (Nrf2), β-actin (Cell Signaling Technology, Danvers, MA, US), Acyl Coenzyme A Oxidase 1 (ACOX1), Anti-Cytochrome P450 2E1 (CYP2E1), peroxisome proliferation-activated receptor alpha (PPARα) as well as microsomal triglyceride transfer protein (MTP) (Abcam, Cambridge, MA, US). The proteins were then visualized by enhanced chemiluminescent by using horseradish peroxidase-conjugated

2.3. In vitro antioxidant activities assays The DPPH assay followed the method used by Brand-Williams, Cuvelier, and Berset (1995). Superoxide anion free radical scavenging (%O2−) and hydroxyl progressive inhibition (%OH−) ability were measured using a·O2− kit and a %OH− kit (Jian-Cheng Institute, Nanjing, China), respectively. 2.4. Animal groups and feeding The animal experiment was approved by the Institutional Animal Care and Use Committee of Jilin Agricultural University (SCXK-20160006). C57BL6/N male mouse (9 weeks old) were obtained from Charles River (Beijing). Animals were maintained in a controlled environment at a temperature of 20 ± 2 °C and a humidity of 50 ± 5% under a 12-h dark/12-h light cycle and acclimated for a week before the experiments. The animals were randomly divided into three groups 2

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

The order of O2-clearing ability of each strain component was fermentation supernatant > fermentation liquid > bacterial cells, and the O2-clearing ability of the fermentation supernatant was 137.59 U/L (Fig. 1C). The order of the components and %OH scavenging ability of the strains was as follows: bacterial cells > fermentation broth > fermentation supernatant, and the %OH scavenging capacity of the cells was 777.1 U/mL (Fig. 1D), indicating that LP104 has a relatively high scavenging ability.

antibody (Cell Signaling Technology, Danvers, MA, US). The protein bands were visualized by image scanner (iBright CL1000, Thermo Fisher Scientific, USA). Protein levels were normalized to β-actin. The density of protein bands was quantified using Image J. 2.9. Statistical analysis Experimental data were pressed as means ± S.E.M for replicate analysis. Between the experimental groups, the differences P < 0.05 were considered significant and significant differences were assayed by the one-way ANOVA (Prism 7.0 software package, USA).

3.3. Effect of LP104 on the basic characteristics As illustrated in Fig. 2, after 8-week feeding, HFD-fed mice showed significantly higher final body weight compared to control mouse. L. plantarum LP104 supplemented to HFD significantly reduced final body weight in mice. There were no significant differences in the daily food intake among the mouse of all groups. Lee's index of the HFD group was significantly higher than the regular control group; In contrast, Lee's index of the LP104 group relative to the HFD group decreased by 7.9%. The abdominal fat coefficient and perirenal fat coefficient were significantly greater in the HFD group than the control group. After intervention with L. plantarum LP104, the abdominal fat and perirenal fat coefficient decreased by 29.34% and 40.4%, respectively.

3. Result 3.1. Identification of strain The strains isolated from kimchi were identified by colony morphological observation, cell morphology observation, and 16S rDNA sequence analysis, which isolated from kimchi. Homology comparison was performed in the GenBank database using Blast analysis. The 16S rDNA sequence of the pure strain is highly homologous to that of the L. plantarum strain, and their high identities are up to 99%, so it was identified as L. plantarum (Fig. 1A) and named LP104 (CCTCC No. 2019084).

3.4. Effect of LP104 on serum lipid profiles As shown in Table 1, the serum TC, TG and LDL levels were markedly increased in the HFD group than the control group (P < 0.01). LP104-supplemented significantly reduced the increase of serum TC, TG and LDL levels caused by a high-fat diet. Serum HDL levels in the control group were lower than the high-fat diet group (P < 0.05), but the LP104 strain did not demonstrate apparent influence on the HDL-C levels. The L. plantarum LP104 significantly lowered FFA levels induced by HFD. The atherogenic indexes significantly increased in the HFD group, but L. plantarum LP104 intervention effectively attenuated the elevation (P < 0.05).

3.2. Antioxidant activity in vitro of LP104 DPPH free radical is a common and effective method for screening and evaluating antioxidant activity (He et al., 2017). In this study, the fermentation supernatant, bacterial cell, and fermentation broth of LP104 were separately added to the reaction system with the purpose to analyze their ability of scavenging DPPH free radicals. Furthermore, the order of DPPH scavenging ability of each strain component was fermentation supernatant > fermentation liquid > bacterial cells, and the clearance rate of the fermentation supernatant was 92.1% (Fig. 1B).

Fig. 1. The identification of strains by the 16S rDNA sequence. The 16S rDNA sequence of L. plantarum LP104 (A). Effect of L. plantarum LP104 on anti-oxidative enzyme activities in vitro (B). Data represent the mean ± SD of each group. 3

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

Fig. 2. Effect of L. plantarum LP104 on the basic characteristics of high fat diet treated mouse. Food intake (A) body weight changes (B) LEE’s index (C) the abdominal fat index (D) and the perirenal fat index (E) of mouse in the three groups. Control: Normal chow group; HFD: High fat group; HFD + LP104: high fat diet with LP104. Values are represented as mean ± SEM (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicate the significant differences between different groups.

and liver, activities of antioxidative enzymes, i.e., GSH-Px were significantly decreased in the control group compared to the HFD group (P < 0.01). Compared with the HFD group, a significant increase (P < 0.05) was seen in the activity of GSH-Px enzymes in the mouse fed HFD + LP104 group (Fig. 4D and E). These results suggested that L. plantarum LP104 inhibited the production of GSH-Px and thereby reduced oxidative stress.

3.5. LP104 ameliorated liver fat accumulation Hepatic lipid levels, including liver TC, TG, LDL and HDL were also measured. The liver TC, TG and LDL levels were remarkably increased in the HFD group (P < 0.01), but these increases were significantly reduced by intervention with L. plantarum LP104 (P < 0.05) (Fig. 3A–C). In addition, the levels of liver HDL were considerably decreased by HFD feeding, and this decrease was blocked by L. plantarum LP104 supplementation (P < 0.05) (Fig. 3D). Also, hematoxylin-eosin staining showed substantial fat accumulation in mouse fed a high-fat diet compared to the control group mouse. Additionally, the liver tissue cell boundary was blurred, and the integrity was destroyed in the mice fed a high-fat diet. The treatment of the L. plantarum LP104 ameliorated liver lipid accumulation and effectively relieved HFD-induced hepatic steatosis (Fig. 3E).

3.7. Effect of LP104 on inflammatory cytokines After eight weeks of treatment, the levels of serum and liver LPS and TNF-α in the high-fat diet mouse were significantly higher than those fed the regular diet (P < 0.05). The L. plantarum LP104 supplement considerably reduced serum and liver LPS and TNF-α levels compared to the HFD group (P < 0.05, Fig. 5). These results suggested that treatment with L. plantarum LP104 could attenuate liver injury and improve liver function.

3.6. Effect of LP104 on liver injury and lipid peroxidation

3.8. Effect of LP104 on hepatic AMPK signaling

ALT and AST are sensitive markers of liver injury. In this experiment, compared with a control group, serum or liver ALT and AST levels were dramatically increased in the HFD group (P < 0.01), but these increases were significantly inhibited by treatment with the L. plantarum LP104 (P < 0.05, Fig. 4A–C). Furthermore, in both serum

The AMPK signaling pathway is a classical pathway associated with metabolic function, such as lipid metabolism (Qu et al., 2016). Therefore, we examined several critical proteins within this signaling

Table 1 Effect of LP104 on serum lipid profiles of high fat diet mice.

Control HDF HDF + LP104

TC (mmol/L)

TG (mmol/L)

LDL (mmol/L)

c

c

b

3.37 ± 0.62 7.62 ± 1.10a 5.71 ± 0.10b

0.48 ± 0.01 0.60 ± 0.05a 0.46 ± 0.06b

0.74 ± 0.12 1.56 ± 0.50a 0.83 ± 0.19b

HDL (mmol/L) c

3.28 ± 0.19 4.86 ± 0.46a 4.48 ± 0.40a

FFA (μmol/L)

AI b

86.88 ± 10.17 104.78 ± 12.53a 89.36 ± 10.63b

1.14 ± 0.13b 1.56 ± 0.12a 1.28 ± 0.11b

Control: Normal chow group; HFD: High fat group; HFD + LP104: high fat diet with LP104. Values were expressed as mean ± SEM (n = 10). Mean values in the same column with different superscripts (a, b, c) are significantly different (p < 0.05). 4

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

Fig. 3. Effect of L. plantarum LP104 on liver TC (A), TG (B), LDL (C) and HDL (D) levels. The histological changes of liver sections were measured by H&E staining at 20x magnification (E). Control: Normal chow group; HFD: High fat group; HFD + LP104: high fat diet with LP104. Values are represented as mean ± SEM (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicate the significant differences between different groups.

lipid biosynthesis to alleviate hepatic steatosis, while L. plantarum LP104 regulates liver lipid metabolism by the AMPK pathway to some extent.

pathway to study the mechanism regulating lipid metabolism. The phosphorylation levels of AMPK and ACC were observed. Phosphorylation of AMPK was increased in the HFD + LP104 group treated with L. plantarum LP104 when that was significantly reduced in the HFD group. Phosphorylated AMPK signaling is known to inhibit fat synthesis by inhibiting phosphorylated ACC expression (Stefanovic-Racic et al., 2008). High fat diet-induced mice exhibited higher levels of ACC phosphorylation compared to the control groups. Down-regulation of ACC phosphorylation was performed in the HFD + LP104 group compared to the HFD group (Fig. 6B). Furthermore, expression of lipogenic proteins such as sterol regulatory element-binding protein 1c (SREBP1c) and stearoyl-CoA desaturase 1 (SCD-1) in liver tissue was upregulated in the HFD group compared with the control group. However, these lipogenic proteins were significantly down-regulated in the LP104 group compared to the HFD group (P < 0.05, Fig. 6C). Furthermore, activated AMPK signaling enhanced fatty acid oxidation by increasing PPARα expression. In the liver, lacking PPARα related genes are impaired in transcription, resulting in excess fatty acids. These fatty acids are usually derived from lipolysis and are transported to the liver to accumulate in the form of triglycerides, which ultimately leads to obesity (França et al., 2019). Compared with the HFD group, the expression of PPARα was significantly increased in the HFD + LP104 group, accompanied by an increase in the interpretation of ACOX and the lipid transporter gene MTP (Fig. 7B). MTP is involved in the aggregation of very-low-density lipoprotein (VLDL), which plays an essential role in the transport of lipids from the liver to extrahepatic tissues (Leung, Rivera, Furness, & Angus, 2016). These data indicate that L. plantarum LP104 can increase fatty acid oxidation and reduce

3.9. LP104 reduced the oxidative stress The induction of a high-fat diet leads to the occurrence of oxidative stress in the body. We also analyzed the effect of L. plantarum LP104 on oxidative stress in high-fat diet mouse. Nrf2 pathway was adaptively activated to reduce cellular damage when exposed to oxidative stress. The expression of Nrf2 was significantly down-regulated in the HFD group compared with the control group. In comparison with the HFD group, the expression of Nrf2 was increased significantly in the HFD + LP104 groups. Furthermore, intervention with L. plantarum LP104 significantly reduced the increase in CYP2E1 expression levels induced by the high-fat diet compared to the HFD group (Fig. 7C). A high-fat diet can cause oxidative stress in the body, and L. plantarum LP104 achieves the goal of alleviating lipid metabolism by relieving oxidative stress. 4. Discussion Probiotic is a novel, safe, and practical approach to reverse the metabolic abnormalities observed in hyperlipidemia and become a specific treatment (Salinas-Rubio, Tovar, & Noriega, 2018). L. plantarum LP104 fermentation liquid was combined in the diet homogeneously in advance to simulate the natural feeding and absorption process in humans. It was also found that a high-fat diet would result in 5

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

Fig. 4. Effect of L. plantarum LP104 on serum ALT (A), liver AST (B), serum AST (C), serum GSH-Px (D) and liver GSH-Px (E) levels. Control: Normal chow group; HFD: High fat group; HFD + LP104: high fat diet with LP104. Values are represented as mean ± SEM (n = 10). *p < 0.05, **p < 0.01 and ***p < 0.001 indicate the significant differences between different groups.

Fig. 5. Effect of L. plantarum LP104 on serum LPS (A), liver LPS (B), serum TNF-α (C) and liver TNF-α (D) levels. Control: Normal chow group; HFD: High fat group; HFD + LP104: high fat diet with LP104. Values are represented as mean ± SEM (n = 10). *p < 0.05, **p < 0.01 and ***p < 0.001 indicate the significant differences between different groups. 6

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

Fig. 6. Effect of L. plantarum LP104 on hepatic AMPK signaling. p-AMPK, AMPK, p-ACC, ACC, SREBP-1 and SCD1 protein expressions in liver. β-actin was used as a control for the protein blots (A). Quantification of AMPK and ACC phosphorylation (B). Relative protein levels of SREBP-1 and SCD1 (C). Data represent the mean ± SD of each group. *p < 0.05 and **p < 0.01verus HFD group.

HFD group was significantly higher than that of the HFD + LP 104 group (Fig. 2C). It suggested that the intervention of L. plantarum LP104 may effectively inhibit the obesity induced by a high-fat diet. It is well established that increased serum TC, TG, and LDL levels are primary features of hyperlipidemia induced by an HFD (Jeun et al., 2010; Nan et al., 2018). In our present study, high-fat diet induction significantly reduced HDL levels and increased TC, TG, and LDL levels, leading to lipid metabolism disorders. This might be due to the increased FFA accumulation in serum (Table 1). Meanwhile, H&E staining confirmed the effect of L. plantarum LP104 on HFD-induced hepatic steatosis (Fig. 3E). In the HFD + LP104 group, L. plantarum LP104 intake could significantly lower serum TC, TG, and LDL levels. Consistent with the study by Jeun et al., L. plantarum used single or mixed with L.

a significant increase in body weight, abdominal fat, and perirenal fat coefficient (Fig. 2D and E). Excessive abdominal adipocytes in the body are called hypertrophy and considered to be one of the characteristics of obesity (Schetz et al., 2019), which is closely related to hyperlipidemia, a chronic disease that causes liver injury (Choi et al., 2019). L. plantarum LMT1-48 also showed similar anti-obesity effects, including body weight loss and reduction of abdominal fat volume (Wang et al., 2019). Consistent with previous researches, in this study, L. plantarum LP104 significantly reduced HFD-induced body weight and increased abdominal and perirenal fat index. Our data showed that the food intake of the HFD group was lower than that of the HFD + LP 104 group, while the weight of the HFD group was significantly higher than that of HFD + LP 104 group (Fig. 2A, B). At the same time, Lee's index of the

Fig. 7. Effect of L. plantarum LP104 on adipogenesis and oxidative proteins in liver tissues. PPARα, ACOX, MTP, CYP2E1 and Nrf2 protein expressions in liver. β-actin was used as a control for the protein blots (A). Relative protein levels of PPARα, ACOX and MTP (B). Relative protein levels of CYP2E1 and Nrf2 (C). Data represent the mean ± SD of each group. *p < 0.05, **p < 0.01 and ***p < 0.001verus HFD group. 7

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

lipogenesis factors (Fig. 6C). Fatty acids were transported into the mitochondria for β-oxidation by PPARα, and AMPK prompted its activity by inhibiting ACC (Hardie & Pan, 2002). Our results have shown that L. plantarum LP104 significantly increased the expression of PPARα protein in mouse liver. MTP reduces liver triglyceride levels by fatty acid oxidation, which is closely related to energy expenditure (Martin et al., 2015). We observed that LP104 significantly up-regulated the expression of PPARα, ACOX, and MTP proteins (Fig. 7B), which was associated with lipid oxidation. Thus, we suspected that the significant decrease of TG levels in the liver is related to the PPARα pathway. Therefore, our results indicated that L. plantarum LP104 supplementation is mediated by AMPK pathway to phosphorylate ACC, suppress SREBP-1/SCD1 signaling and up-regulation of PPARα, MTP, as well as ACOX protein expression to achieve the effect of lipid-lowering. Therefore, L. plantarum LP104 could increase fatty acid oxidation and reduce the lipid biosynthesis to alleviate hepatic steatosis. Oxidative stress plays a vital role in the pathogenesis of hyperlipidemia, which can lead to tissue damage through an imbalance of antioxidants (Zhang et al., 2016). If the DPPH could be removed, it would indicate that the test strain has the effect of reducing the chain reaction of hydrogen peroxide, peroxidic free radicals or lipid radicals. L. plantarum LP104 has significant DPPH free radicals, superoxide anion and hydroxyl radical scavenging ability in vitro (Fig. 1B–D). This could be due to the attribution of some cell surface-active compounds such as protein or polysaccharides and lipoteichoic acid, which were observed in L. plantarum C88 and Bifidobacteria documented by the works of respectively (Li, 2012; Yi, Fu, Li, Gao, & Zhang, 2009). The in vitro antioxidant activity of Lactobacillus plantarum MA2 fermentation supernatant is associated with the production of extracellular polysaccharide (EPS) (Tang, Xing, Li, Wang, & Wang, 2017). Lactobacillus lactis ATCC19435 contains a significant concentration of extracellular pyruvate, which non-enzymatically scavenges the H2O2 generated by NADH oxidase, increasing Antioxidant capacity (Van Niel, Hofvendahl, & Hahn-Hägerdal, 2002). GSH-Px was located in the cytoplasm that plays a crucial role in increasing the antioxidant capacity against peroxide free radicals and oxygen (Zhao et al., 2018). In this study, L. plantarum LP104 inhibited oxidative stress produced by excess cholesterol via increasing antioxidant enzyme activity GSH-Px (Fig. 4D and E). Similar to our findings, L. fermentum MTCC was found to suppress streptozotocin-induced oxidative damage in diabetic rats by inhibiting the lipid peroxidation and preserving the activity of GSH-Px enzymes (Yadav et al., 2018). Also, a study found that L. casei supplementation significantly increased the activity of antioxidant enzymes in serum and liver of hyperlipidemic rats (Zhang, Du, Wang, & Zhang, 2010). In the present study, HFD-induced high levels of TNF-α and LPS resulted in the development of oxidative stress, which was consistent with an increase in liver antioxidant enzymes (GSH-Px). Thus, a liver injury could be prevented by reducing oxidative stress induced by HFD. CYP2E1 is the most critical factor in producing oxidative stress and belongs to the most crucial enzyme in the microsomal alcohol oxidation system. It has been reported that there is an increase in the level of CYP2E1 due to the addition of alcohol, which can accelerate the excessive production of ROS (Wang et al., 2013). In this study, L. plantarum LP104 up-regulated the expression of the CYP2E1 protein (Fig. 7C). Nrf2 plays a pivotal role in cell defense against oxidative stress by regulating the expression of antioxidants and detoxification enzymes (Chen et al., 2017). In this study, L. plantarum LP104 intervention up-regulated the expression of Nrf2 (Fig. 7C), which was down-regulated in the HFD group. Similar results indicate that B. amyloliquefaciens SC06 is a probiotic that can increases CAT levels to activate the Nrf2/Keep1 signaling pathway, elevating antioxidant status (Wang et al., 2017). Therefore, we suggested that L. plantarum LP104 could activate the CYP2E1 and Nrf2 signaling pathways to attenuate oxidative stress in lipid metabolism. Taken together, we suggested that hyperlipidemia could be mitigated by treatment with L. plantarum LP104 through regulating lipid metabolism and oxidative stress, which are the roles of AMPK, Nrf2, and

paracasei exhibited a lowering of blood cholesterol in rats fed a high fat and cholesterol diet (Lay-Gaik & Min-Tze, 2010). Our data suggested that L. plantarum LP104 has the potential to improve dyslipidemia, thereby reducing the risk of atherosclerosis and cardiovascular disease caused in a high-fat diet. HDL can transport excess TC from peripheral tissues or cells to the liver for catabolism through blood circulation. Compared with the control group, serum HDL levels in the HFD group and HFD + LP 104 group were significantly higher, which may be a consequence of the body's stress response to lower serum cholesterol levels (Rinella, 2015). Compared with the HFD group, serum HDL levels in the HFD + LP 104 group did not change significantly, indicating that L. plantarum LP104 had little effect on serum HDL, but L. plantarum LP104 significantly inhibited serum AI levels in mice. Besides, abnormal increases in liver TC and TG levels indicate that liver lipid metabolism disorders have occurred, which could diagnose liver damage status (Lv et al., 2014). Our data showed that L. plantarum LP104 was effectively reduced liver TC, TG, and LDL levels due to increased high-fat diet (Fig. 3). ALT and AST levels are essential markers for predicting the degree of liver injury (Moreira, Texeira, Ferreira, Peluzio, & Alfenas, 2012). L. plantarum LP104 significantly decreased ALT and AST levels (Fig. 4), indicating that L. plantarum LP104 could attenuate HFD-induced liver injury and protect liver function. Previous studies have shown that LPS could spread from the gut to the circulatory system to cope with high fat intake (Lee et al., 2013) and our results further confirmed this conclusion. After eight weeks of HFD treatment, serum and liver LPS levels were significantly higher than those fed a regular diet. Supplementation with L. plantarum LP104 significantly reduced serum and liver LPS levels in the high fat-fed mouse. TNF-α is a crucial mediator of liver injury caused by LPS (Lee et al., 2016). In this study, the trend of TNF-α levels was consistent with LPS levels, L. plantarum LP104 significantly inhibited the increase in serum and liver TNF-α levels induced by a high-fat diet (Fig. 5). In conclusion, L. plantarum LP104 ameliorated hyperlipidemia, liver inflammation, and liver damage caused by a high-fat diet. The liver is the center of lipid metabolism and is closely related to metabolic diseases. Probiotics regulated lipid metabolism in the liver by activating AMPK, reducing lipid biosynthesis, and promoting fatty acid oxidation (Hardie et al., 2002). Once activated, AMPK would modulate fat acid oxidation and switch off anabolic pathways by phosphorylating multiple downstream substrates to conserve ATP levels (Smith et al., 2016). Interestingly, AMPK phosphorylation could block ACC dimerization, which in turn reduces the ACC activity, thereby inhibiting de novo fat lipogenesis and switches on fatty acid oxidation in mitochondria (Luo et al., 2019). Therefore, we believe that AMPK activation could be considered as a therapeutic target for alleviating the effects of hyperlipidemia (Liu et al., 2019). Acetyl-CoA carboxylase (ACC) is a rate-limiting enzyme in fatty acid biosynthesis, and upon phosphorylation by the AMP-activated protein kinase (AMPK), it will inhibit fatty acid synthesis (Quan et al., 2013). In this study, we found that the HFD group significantly attenuated AMPK phosphorylation compared to the control group. As expected, L. plantarum LP104 intervention increased the level of attenuated AMPK phosphorylation. These data suggested that HFD induces the removal of AMPK phosphorylation (Fig. 6B), resulting in increased levels of serum FFA by de novo fat lipogenesis. Treatment with L. plantarum LP104 could increase AMPK activity and block ACC activity, which can improve HFD-induced hyperlipidemia by suppressing de novo lipogenesis and increasing fatty acid oxidation. Furthermore, it is well documented that AMPK activation inhibits SREBP-1, resulting in the attenuation of hyperlipidemia (Shimomura & Bashmakov, 1999). SREBP-1 protein is a vital liver transcription factor controlling many genes involved in cholesterol and another lipid metabolism (Luo et al., 2019). SREBP-1 and SCD1 are capable of promoting the expression of lipogenic proteins, mediating lipogenesis, and lipid accumulation in tissues (Wang et al., 2019). In the present study, LP104 reduced the levels of TC and LDL (Fig. 3) in the liver by down-regulating the above-described adipogenesis and 8

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

CYP2E1 pathways, respectively. In conclusion, our findings in the present study suggest that L. plantarum LP104 effectively prevented the hyperlipidemia and liver injury induced by consumption of a high-fat diet. We propose that the beneficial effects of treatment with L. plantarum LP104 are partly related to the activation of the AMPK/Nrf2/CYP2E1 pathway, which will significantly improve hepatic lipid metabolic disorders and ameliorate hepatic oxidative stress response.

obesity effects of black ginseng extract in high fat diet-fed mice. Journal of Ginseng Research, 37, 308–349. Lee, H. S., Lim, W. C., Lee, S. J., Lee, S. H., Lee, J. H., ... Cho, H. Y. (2016). Anti-obesity effect of garlic extract fermented by lactobacillus plantarum BL2 in diet-induced obese mice. Journal of Medicinal Food, 19, 823–829. Leung, C., Rivera, L., Furness, J. B., & Angus, P. W. (2016). The role of the gut microbiota in NAFLD. Nature Reviews Gastroenterology & Hepatology, 13(7), 412. https://doi.org/ 10.1038/nrgastro.2016.85. Li, X. C. (2012). Improved pyrogallol autoxidation method: A reliable and cheap superoxide-scavenging assay suitable for all antioxidants. Journal of Agricultural and Food Chemistry, 60(25), 6418–6424. Liu, C., Guo, Y., Sun, L., Lai, X., Li, Q., Zhang, W., ... Cao, F. (2019). Six types of tea reduce high-fat diet-induced fat accumulation in mice by increasing lipid metabolism and suppressing inflammation. Food & Function, 10(4), 2061–2074. https://doi.org/10. 1039/c8fo02334d. Luo, J., Qi, J., Wang, W., Luo, Z., Liu, L., Zhang, G., ... Peng, X. (2019). Anti-obesity effect of flaxseed polysaccharide via inducing satiety due to leptin resistance removal and promoting lipid metabolism through the AMP-activated protein kinase (AMPK) signaling pathway. Journal of agricultural and food chemistry, 67, 7040–7049. Lv, L. X., Hu, X. J., Qian, G. R., Zhang, H., Lu, H. F., Zheng, B. W., ... Li, L. J. (2014). Administration of Lactobacillus salivarius LI01 or Pediococcus pentosaceus LI05 improves acute liver injury induced by d-galactosamine in rats. Applied Microbiology & Biotechnology, 98, 5619–5632. Martin, M. A., Ramos, S., Mateos, R., Marais, J. P., Bravo-Clemente, L., Khoo, C., & Goya, L. (2015). Chemical characterization and chemo-protective activity of cranberry phenolic powders in a model cell culture. Response of the antioxidant defenses and regulation of signaling pathways. Food Research International, 71, 68–82. McCrabb, S., Lane, C., Hall, A., Milat, A., Bauman, A., Sutherland, R., ... Wolfenden, L. (2019). Scaling-up evidence-based obesity interventions: A systematic review assessing intervention adaptations and effectiveness and quantifying the scale-up penalty. Obesity Reviews.. Miyoshi, M., Ogawa, A., Higurashi, S., & Kadooka, Y. (2014). Anti-obesity effect of lactobacillus gasseri sbt2055 accompanied by inhibition of pro-inflammatory gene expression in the visceral adipose tissue in diet-induced obese mice. European Journal of Nutrition, 53, 599–606. Moreira, A. P. B., Texeira, T. F. S., Ferreira, A. B., Peluzio, M. D. C. G., & Alfenas, R. D. C. G. (2012). Influence of a high-fat diet on gut microbiota, intestinal permeability and metabolic endotoxaemia. British Journal of Nutrition, 108, 801–809. Nabavi, S., Rafraf, M., Somi, M. H., Homayouni-Rad, A., & Asghari-Jafarabadi, M. (2014). Effects of probiotic yogurt consumption on metabolic factors in individuals with nonalcoholic fatty liver disease. Journal of Dairy Science, 97, 7386–7393. Nan, B., Liu, Y. L., You, Y., Li, W. C., Fan, J. J., Wang, Y. S., ... Wang, Y. H. (2018). Protective effects of enhanced minor ginsenosides in Lactobacillus fermentum KP-3fermented ginseng in mice fed a high fat diet. Food & Function, 9. https://doi.org/10. 1039/c8fo01056k. Nobili, V., Putignani, L., Mosca, A., Del Chierico, F., Vernocchi, P., Alisi, A., ... Drago, L. (2018). Bifidobacteria and lactobacilli in the gut microbiome of children with nonalcoholic fatty liver disease: Which strains act as health players. Archives of Medical Science, 14, 81–87. Qu, L. L., Yu, B., Li, Z., Jiang, W. X., Jiang, J. D., & Kong, W. J. (2016). Gastrodin ameliorates oxidative stress and proinflammatory response in nonalcoholic fatty liver disease through the AMPK/Nrf2 pathway. Phytotherapy research, 30(3), 402–411. Quan, H. Y., Kim, S. J., Jo, H. K., Kim, G. W., & Chung, S. H. (2013). Betulinic acid alleviates non-alcoholic fatty liver by inhibiting SREBP1 activity via the AMPKmTOR-SREBP signaling pathway. Biochemical Pharmacology, 85, 1330–1340. Rajesh, K., Sunita, G., & Virender Kumar, B. (2011). Hypocholesterolaemic effect of dietary inclusion of two putative probiotic bile salt hydrolase-producing Lactobacillus plantarum strains in Sprague-Dawley rats. British Journal of Nutrition, 105, 561–573. Rinella, M. E. (2015). Nonalcoholic fatty liver disease: A systematic review. JAMA, 313, 2263–2273. Salinas-Rubio, D., Tovar, A. R., & Noriega, L. G. (2018). Emerging perspectives on branched-chain amino acid metabolism during adipocyte differentiation. Current Opinion in Clinical Nutrition and Metabolic Care, 21(1), 49–57. Schetz, M., De Jong, A., Deane, A. M., Druml, W., Hemelaar, P., Pelosi, P., ... Jaber, S. (2019). Obesity in the critically ill: A narrative review. Intensive Care Medicine, 1–13. https://doi.org/10.1007/s00134-019-05594-1. Shimomura, I., & Bashmakov, Y. J. (1999). Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. Journal of Biological Chemistry, 274, 30028–30032. Siokkoon, Y., & Mintze, L. (2010). Angiotensin I-converting enzyme inhibitory activity and bioconversion of isoflavones by probiotics in soymilk supplemented with prebiotics. International Journal of Food Sciences & Nutrition, 61, 161–181. Smith, B. K., Marcinko, K., Desjardins, E. M., Lally, J. S., Ford, R. J., & Steinberg, G. R. (2016). Treatment of non-alcoholic fatty liver disease (NAFLD): Role of AMPK. American Journal of Physiology Endocrinology & Metabolism, 311(4), E730–E740. Song, Z., Cai, Y., Lao, X., Wang, X., Lin, X., Cui, Y., ... Li, J. (2019). Taxonomic profiling and populational patterns of bacterial bile salt hydrolase (BSH) genes based on worldwide human gut microbiome. Microbiome, 7(1)https://www.ncbi.nlm.nih.gov/ pmc/articles/pmid/30674356/. Stefano, B., Rodolfo, P., & Alberto, C. (2004). Safety of statins: Focus on clinical pharmacokinetics and drug interactions. Circulation, 109, III50-57. Stefanovic-Racic, M., Perdomo, G., Mantell, B. S., Sipula, I. J., Brown, N. F., & O'Doherty, R. M. (2008). A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels. American Journal of Physiology-Endocrinology and Metabolism, 294(5), E969–E977. Tang, W., Xing, Z., Li, C., Wang, J., & Wang, Y. (2017). Molecular mechanisms and in

Ethics of statement All procedures used in this study comply with the Guide for the Institutional Animal Care and Use Committee of Jilin Agricultural University. Efforts were made to minimize animal suffering and reduce the number of subjects used. Declaration of Competing Interest The authors declare that they have no conflicts of interest (financial or otherwise) related to the data presented in this manuscript. Acknowledgements This work was supported by the National Key Research and Development Program of China (2017YFE0105400). References Baharav, E., Mor, F., Halpern, M., & Weinberger, A. (2004). Lactobacillus GG bacteria ameliorate arthritis in Lewis rats. Journal of Nutrition, 134, 1964. https://doi.org/10. 1093/jn/134.8.1964. Brand-Williams, W., Cuvelier, M. E., & Berset, C. L. W. T. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food science and Technology, 28(1), 25–30. Chanet, A., Milenkovic, D., Deval, C., Potier, M., Constans, J., Mazur, A., ... Bérard, A. M. (2012). Naringin, the major grapefruit flavonoid, specifically affects atherosclerosis development in diet-induced hypercholesterolemia in mice. The Journal of Nutritional Biochemistry, 23, 469–477. Chen, Q., Wang, T., Li, J., Wang, S., Qiu, F., Yu, H., ... Wang, T. (2017). Effects of natural products on fructose-induced nonalcoholic fatty liver disease (NAFLD). Nutrients, 9, 96. https://www.mdpi.com/2072-6643/9/2/96. Choi, W. J., Dong, H. J., Jeong, H. U., Jung, H. H., Kim, Y. H., & Kim, T. H. (2019). Antiobesity effects of lactobacillus plantarum LMT1-48 accompanied by inhibition of enterobacter cloacae in the intestine of diet-induced obese mice. Journal of Medicinal Food. https://www.liebertpub.com/doi/abs/10.1089/jmf.2018.4329?rfr_dat=cr_ pub%3Dpubmed&url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org& journalCode=jmf. Del Ben, M., Polimeni, L., Baratta, F., Pastori, D., & Angelico, F. (2016). The role of nutraceuticals for the treatment of non-alcoholic fatty liver disease. British Journal of Clinical Pharmacology, 83, 88–95. Devaraj, R. D., Reddy, C. K., & Xu, B. (2019). Health-promoting effects of konjac glucomannan and its practical applications: A critical review. International Journal of Biological Macromolecules, 126, 273–281. França, L. M., Coêlho, C. F. F., Freitas, L. N. C., Souza, I. L. S., Chagas, V. T., Debbas, V., ... Paes, A. M. D. A. (2019). Syzygium cumini leaf extract reverts hypertriglyceridemia via downregulation of the hepatic XBP-1s/PDI/MTP axis in monosodium L-glutamateinduced obese rats. Oxidative Medicine and Cellular Longevity. https://www.hindawi. com/journals/omcl/2019/9417498/. Hardie, D. G., & Pan, D. A. (2002). Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase. Biochemical Society Transactions, 30(1064)https:// portlandpress.com/biochemsoctrans/article-lookup/doi/10.1042/bst0301064. He, L., Zhou, X., Huang, N., Li, H., Tian, J., Li, T., ... Yin, Y. (2017). AMPK regulation of glucose, lipid and protein metabolism: Mechanisms and nutritional significance. Current Protein and Peptide Science, 18(6), 562–570. Jeun, J., Kim, S., Cho, S. Y., Jun, H. J., Park, H. J., Seo, J. G., ... Lee, S. J. (2010). Hypocholesterolemic effects of Lactobacillus plantarum KCTC3928 by increased bile acid excretion in C57BL/6 mice. Nutrition, 26(3), 321–330. Kim, S. J., Park, S., Sin, H. S., Jang, S. H., Lee, S. W., Kim, S. Y., ... Yang, D. (2017). Hypocholesterolemic effects of probiotic mixture on diet-induced hypercholesterolemic rats. Nutrients, 9, 293. https://www.mdpi.com/2072-6643/9/3/293. Kumar, M., Kumar, A., Nagpal, R., Mohania, D., Behare, P., Verma, V., ... Jain, S. (2010). Cancer-preventing attributes of probiotics: An update. International Journal of Food Sciences & Nutrition, 61, 473–496. Lay-Gaik, O., & Min-Tze, L. (2010). Cholesterol-lowering effects of probiotics and prebiotics: A review of in vivo and in vitro findings. International Journal of Molecular Sciences, 11, 2499–2522. Lee, M. R., Kim, B. C., Kim, R., Oh, H. I., Kim, H. K., Choi, K. J., & Sung, C. K. (2013). Anti-

9

Journal of Functional Foods xxx (xxxx) xxxx

Y. Teng, et al.

org/10.1039/c8fo01452c. Yadav, R., Dey, D. K., Vij, R., Meena, S., Kapila, R., & Kapila, S. (2018). Consumption of probiotic lactobacillus fermentum MTCC: 5898-fermented milk attenuates dyslipidemia, oxidative stress, and inflammation in male rats fed on cholesterol-enriched diet. Probiotics & Antimicrobial Proteins, 11(2), 509–518. Yi, Z. J., Fu, Y. R., Li, F. M., Gao, K. S., & Zhang, X. G. (2009). Effect of LTA isolated from bifidobacteria on D-galactose-induced aging. Experimental Gerontology, 44(12), 760–765. Younossi, Z. M., Koenig, A. B., Abdelatif, D., Fazel, Y., Henry, L., & Wymer, M. (2016). Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology, 64, 73–84. Zhang, Y., Du, R., Wang, L., & Zhang, H. (2010). The antioxidative effects of probiotic Lactobacillus casei Zhang on the hyperlipidemic rats. European Food Research & Technology, 231, 151–158. Zhang, Y., Wang, Z., Jin, G., Yang, X., & Zhou, H. (2017). Regulating dyslipidemia effect of polysaccharides from Pleurotus ostreatus on fat-emulsion-induced hyperlipidemia rats. International Journal of Biological Macromolecules, 101, 107–116. Zhang, X. L., Wu, Y. F., Wang, Y. S., Wang, X. Z., Piao, C. H., Liu, J. M., ... Wang, Y. H. (2017). The protective effects of probiotic-fermented soymilk on high-fat diet-induced hyperlipidemia and liver injury. Journal of Functional Foods, 30, 220–227. Zhang, Z., Zhou, Z., Li, Y., Zhou, L., Ding, Q., & Xu, L. (2016). Isolated exopolysaccharides from Lactobacillus rhamnosus GG alleviated adipogenesis mediated by TLR2 in mice. Scientific Reports, 6, 36083. https://doi.org/10.1038/srep36083. Zhao, H., Zhang, J., Liu, X., Yang, Q., Dong, Y., & Jia, L. (2018). The antioxidant activities of alkalic-extractable polysaccharides from Coprinus comatus on alcohol-induced liver injury in mice. Scientific Reports, 8, 11695. https://www.nature.com/articles/ s41598-018-30104-6.

vitro antioxidant effects of lactobacillus plantarum ma2. Food Chemistry, 221, 1642–1649. Van Niel, E. W., Hofvendahl, K., & Hahn-Hägerdal, B. (2002). Formation and conversion of oxygen metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under different growth conditions. Applied and Environmental Microbiology, 68(9), 4350–4356. Vaughan, C. J., Murphy, M. B., & Buckley, B. M. (1996). Statins do more than just lower cholesterol. Lancet, 348(9034), 1079–1082. Wang, G., Huang, W., Xia, Y., Xiong, Z., & Ai, L. (2019). Cholesterol-lowering potentials of Lactobacillus strain overexpression of bile salt hydrolase on high cholesterol dietinduced hypercholesterolemic mice. Food & Function, 10. https://doi.org/10.1039/ c8fo02181c. Wang, K., Liao, M., Zhou, N., Bao, L., Ma, K., Zheng, Z., ... Liu, S. J. (2019). Parabacteroides distasonis alleviates obesity and metabolic dysfunctions via production of succinate and secondary bile acids. Cell Reports, 26, 222–235. Wang, Y., Liu, Y., Kirpich, I., Ma, Z., Wang, C., Zhang, M., ... Feng, W. (2013). Lactobacillus rhamnosus gg reduces hepatic TNF-α production and inflammation in chronic alcohol-induced liver injury. The Journal of Nutritional Biochemistry, 24, 1609–1615. Wang, Y., Wu, Y., Wang, Y., Fu, A., Gong, L., Li, W., & Li, Y. (2017). Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2/Keap1 signaling pathway and decreasing ROS production. Applied Microbiology and Biotechnology, 101, 3015–3026. Weston, S., Halbert, A., Richmond, P., & Prescott, S. L. (2005). Effects of probiotics on atopic dermatitis: A randomised controlled trial. Child, 90(9), 892–897. Xia, S. F., Qiu, Y. Y., Chen, L. M., Jiang, Y. Y., Huang, W., Xie, Z. X., ... Sun, J. (2019). Myricetin alleviated hepatic steatosis by acting on microRNA-146b/thyroid hormone receptor b pathway in high-fat diet fed C57BL/6J mice. Food & Function. https://doi.

10