Screening of the lipid-lowering probiotic  Lactiplantibacillus plantarum  SDJ09 and its anti-obesity mechanism

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Based on the evaluation of functional and prebiotic properties, Lactiplantibacillus plantarum SDJ09 was selected as a promising candidate. It gave a 48.16% cholesterol reduction and 33.73% pancreatic lipase inhibition in cells; exhibited high resistance to acid, bile salts, and gastrointestinal fluid; and had strong antibacterial activity and high adhesion capabilities. More importantly, the lipid-lowering effect of L. plantarum SDJ09 was also investigated using 3T3-L1 mature adipocytes and HepG2 nonalcoholic fatty liver disease models. L. plantarum SDJ09 effectively decreased triglyceride accumulation by more than 50% in both cell models, in which the expression of PPARγ, C/EBPα, aP2, and LPL in 3T3-L1 cells was significantly downregulated by L. plantarum SDJ09. L. plantarum SDJ09 also improved lipid metabolism by downregulating the expression of HMGCR, SREBP-1c, ACC, and FAS and upregulating the expression of CYP7A1 in HepG2 nonalcoholic steatohepatitis cells. Therefore, L. plantarum SDJ09 has the potential to effectively decrease obesity and non-alcoholic fatty liver disease (NAFLD) by inhibiting lipid accumulation, providing a prospective probiotic agent for anti-obesity. Lactiplantibacillus plantarum probiotic lipid-lowering lipid metabolism anti-obesity 1. Introduction As a chronic disease, obesity is caused by an imbalance between energy intake and energy expenditure and is related to various factors such as genetics, diet, and exercise [ 1 ]. More importantly, obesity can cause a series of metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus, hyperlipidemia, and atherosclerosis [ 2 ]. For example, the prevalence of NAFLD has been increasing along with obesity rates, resulting in a rise in the prevalence of NAFLD in obese children/adolescents from 20% (2005–2011) to 51% (2012–2019) [ 3 ]. According to a report from the World Health Organization in 2016, an estimated 650 million adults, or approximately 13% of global adult population, suffer from obesity [ 4 ]. Currently, the treatment of obesity poses a significant challenge. Bariatric surgery is the most effective approach, but it is not suitable for all patients, is not sufficiently scalable, and does not address the broader health concerns associated with obesity [ 5 ]. Due to the difficulty of maintaining a healthy weight, pharmacotherapy is a widely accepted treatment option for improving metabolic health parameters [ 6 ]. For instance, treatment with orlistat led to an average of 5% weight loss at one year and 3% weight loss at four years, but this drug had side effects, such as gastrointestinal discomfort, oily stools, and flatulence [ 7 ]. Recently, research demonstrated that human gut microbiota could modulate energy, glucose, and lipid metabolism in several organs (including the brain, liver, and muscle) via metabolites and signal transduction molecules [ 8 – 10 ]. Consequently, microbiota-based therapeutic strategies, such as probiotic therapy aimed at improving gut flora, have gained attention as a potential treatment option for obesity and metabolic syndrome. Probiotics—defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”—show promising preclinical potential to reduce obesity [ 11 ], NAFLD, diabetes [ 12 ], insulin resistance, and metabolic syndrome [ 13 ]. More importantly, the components, inactivated bacteria and metabolites of probiotics could also be used to regulate lipid metabolism, gut microbiota, and glucose metabolism [ 14 , 15 ]. Therefore, probiotics have been considered as a noninvasive yet potentially highly effective therapeutic approach against obesity and associated metabolic diseases [ 16 – 18 ]. Among of them, Lactobacillus and Bifidobacterium , the most common species of probiotics, have been used to treat obesity [ 19 ]. For example, L. plantarum LMT1-48 was shown to downregulate the expression of proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBPα), adipocyte protein 2 (aP2), and lipoprotein lipase (LPL), thereby inhibiting lipid accumulation in 3T3-L1 adipocytes [ 20 ]. Moreover, it was also found that L. plantarum LMT1-48 effectively improved body weight in obese mice and further reduced hepatic fat accumulation by modulating liver enzymes in humans [ 21 ]. However, more development of lipid-lowering lactic acid bacteria is needed, and the anti-obesity mechanism of lactic acid bacteria needs elucidation. In this study, several lactic acid bacteria with robust probiotic properties were selected from different fermented foods. Based on the investigation of probiotic potential, strain L. plantarum SDJ09 was further investigated for its anti-obesity probiotics by assessing its lipid-lowering effects. Furthermore, we gained mechanistic understanding by treating 3T3-L1 mature adipocytes and HepG2 non-alcoholic fatty liver cell models with cell-free extracts, cell metabolites, and heat-inactivated cells derived from L. plantarum SDJ09. 2. Materials and methods 2.1 Screening of lipid-lowering lactic acid bacteria (LAB) 2.1.1 Isolation of LAB strains Ten traditional fermented foods (including pickled peppers, sauerkraut, milk fans, ham, sour soup, chili sauce, sour beans, sour bamboo shoots, pickles, and soybean sauce) were purchased from southwest China, and then 25 g of each sample was placed in 225 mL of sterile 0.9% NaCl for 30 min. Subsequently, a 100 µL dilution was spread on de Man, Rogosa, and Sharpe (MRS) solid medium and incubated for 48 h at 37°C. Strains with similar color and morphology to LAB were purified and cultured three times in succession, and then individual colonies were selected for a catalase test and Gram staining. Gram-positive and catalase-negative bacteria were selected and stored in 50% glycerol at − 20°C [ 22 ]. Bacterial genomic DNA was extracted using a TIANamp bacteria DNA kit (Tiangen Biotech, Beijing, China) following the manufacturer's instructions and amplified with universal primers 27F and 1492R. PCR products were analyzed by 1% agarose gel electrophoresis and sent to Shanghai Bioengineering Company for sequencing (Shanghai, China). The 16S rRNA gene sequences were compared in the NCBI database ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ), and homology analysis was performed using BLAST. 2.1.2 Evaluation of cholesterol-lowering To evaluate cholesterol-lowering, each of 39 lactic acid bacteria strains was inoculated (3% v/v) into MRS broth supplemented with cholesterol (100 µg/mL, Sigma, Shanghai, China) for culturing for 36 h at 37°C (using MRS broth supplemented with cholesterol as a control). The amount of cholesterol in the cell-free supernatant was analyzed using the phthalaldehyde method to draw the standard curve (y = 0.0078x + 0.0026, R 2 = 0.9982) [ 23 ]. The cholesterol-lowering rate was calculated using the following formula: $$cℎolesterol lowering rate\left(\%\right)=\frac{{A}_{0}-A}{{A}_{0}}\times 100\% cℎolesterol lowering rate\left(\%\right)=\frac{{A}_{0}-A}{{A}_{0}}\times 100\%$$ A 0 : Absorbance before fermentation; A: Absorbance after fermentation 2.1.3 Measurement of pancreatic lipase inhibition activity We followed the method of a previous study [ 24 ] with the following modifications. Strains were cultured without agitation for 18 h at 37°C in MRS broth (3% v/v), and then the broth was washed three time with PBS (pH = 7.2) and re-suspended with PBS to adjust the OD 600 to 0.5 ± 0.02 (10 8 –10 9 CFU/mL). Subsequently, a mixture was prepared by adding 12.5 µL of 5 mM 4-nitrophenyl octanoate (NPC, Solarbio, Beijing, China), 500 µL of Tris-HCl (pH = 8.5) buffer, and 125 µL of bacterial solution. After mixing, 12.5 µL of pancreatic lipase (5 mg/mL in Tris-HCL buffer) was added, mixed for 2 min, and then incubated for 30 min at 37°C. Finally, the absorbance was measured at 412 nm using a 96-well plate (n = 3). The blank sample contained LAB, but dimethyl sulfoxide (DMSO) replaced NPC. The control sample contained NPC but no LAB. The blank control contained no LAB, and DMSO replaced NPC. The inhibition activity was calculated using the following formula: $$Inℎibition activity\left(\%\right)=1-\left(\frac{C-{C}_{0}}{{C}_{b}-{C}_{a}}\right)\times 100\%Inℎibition activity\left(\%\right)=1-\left(\frac{C-{C}_{0}}{{C}_{b}-{C}_{a}}\right)\times 100\%$$ C 0 : absorbance of a blank of the sample; C : absorbance of a sample C b : absorbance of a blank of the control sample; C a : absorbance of a control sample 2.2 Assessment of probiotic characteristics 2.2.1 Tolerance to treatment After incubating for 18 h at 37°C, LAB cells were harvested (6,000 g, 10 min, 4°C) and resuspended to approximately 10 9 CFU/mL in sterile PBS and then inoculated at 3% into the MRS medium (pH = 2.0 and 0.3% bovine bile salt) and incubated for 3 h. An in vitro static digestion model was used to simulate gastrointestinal fluid [ 25 ], in which 3 g/L of pepsin (Coolaiber, Beijing, China) was dissolved in PBS (pH = 3.0) to simulate gastric fluid, and 1.0 g/L trypsin (Coolaiber Technology Co., Ltd, Beijing, China) was dissolved in PBS (pH = 8.0) to simulate intestinal fluid. Then, the viability was determined by counting the number of viable bacteria on the plates before and after treatment, and survival rates were calculated according to the following formula (n = 3): $$Relative survival ratio \left(\%\right)=\frac{N}{{N}_{0}}\times 100\%Relative survival ratio \left(\%\right)=\frac{N}{{N}_{0}}\times 100\%$$ N : CFU of viable cells survived; N 0 : CFU of initial viable cells inoculated 2.2.2 Antibacterial activity of LAB As the indicator strain, Escherichia coli and Staphylococcus aureus were adjusted to 10 7 CFU/mL with PBS and then evenly coated on the Luria Broth plate (OXOID, Beijing, China). Subsequently, the antibacterial ability of LAB cell-free supernatants (CFS) was evaluated with the Oxford cup method [ 26 ]. 2.2.3 Adhesion of LAB According to a previous study [ 27 ], the method of microbial adhesion to hydrocarbons was used to investigate the surface hydrophobicity of strains. After incubating for 18 h at 37°C, LAB cells were harvested (6,000 g, 10 min, 4°C) and washed twice with sterile PBS buffer, and then resuspended to adjust the OD 600 to 1.0 (A a ). Subsequently, 4 mL of bacterial suspension was added into 0.8 mL of xylene for vortexing for 2 min and then held without agitation at room temperature for 20 min. Finally, the absorbance of the aqueous phase was measured at 600 nm (A b ), and the surface hydrophobicity was calculated as follows: $$Surface Hydropℎobicity\left(\%\right)=\left(1-{\frac{{A}_{a}}{A}}_{b}\right)\times 100\%Surface Hydropℎobicity\left(\%\right)=\left(1-{\frac{{A}_{a}}{A}}_{b}\right)\times 100%$$ Furthermore, after the LAB cells were resuspended to an OD 600 of 0.5 ± 0.02 (A c ), 4 mL of the bacterial suspension was held without agitation for 2 h at room temperature, and then the absorbance of the supernatant was measured at 600 nm (A d ). The auto-aggregation capacity was calculated as follows: $$Auto-Aggregation\left(\%\right)=\left(1-\frac{{A}_{c}}{{A}_{d}}\right)\times 100\text{%}Auto-Aggregation\left(\%\right)=\left(1-\frac{{A}_{c}}{{A}_{d}}\right)\times 100\text{%}$$ 2.3 Safety determination of the LAB After incubating for 18 h at 37°C, LAB cells were harvested (6,000 g, 10 min, 4°C), washed twice with sterile PBS buffer, and then resuspended to 10 7 CFU/mL. Then, an antibiotic sensitivity test of LAB was performed using a drug-sensitive disk agar diffusion method (K-B method) [ 28 ]. Furthermore, the hemolytic activity was also detected by streaking the blood on agar plates (7% v/v sheep blood) [ 29 ] that were incubated for 24–48 h at 37°C, and then the zones of hemolysis around the colonies were observed. 2.4 Construction of cell culture model and cell experiment 2.4.1 Cells culture and treatment The mouse 3T3-L1 cell line was purchased from the Beinanchuanlian Institute of Biotechnology (Beijing, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, China) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, China) and 1% penicillin-streptomycin at 37°C under 5% CO 2 . To induce adipocyte differentiation, 3T3-L1 preadipocytes were grown to the contact inhibition stage, maintained in the post-fusion stage for 2 days, and then switched into DMEM supplemented with 10% FBS, 0.5 mM isobutyl methyl xanthine (Macklin, Shanghai, China), 0.5 µM dexamethasone (Solarbio, Beijing, China), and 10 µg/mL insulin (Macklin, Shanghai, China) for 2 days. Subsequently, the differentiation medium was replaced by 10% FBS-DMEM with 10 µg/mL insulin for 2 days. Finally, the induction medium was changed to growth medium, and the cells were cultured for 2 days. The HepG2 (human hepatocellular liver carcinoma) cell line was purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C under 5% CO 2 . After obtaining an 80% confluent monolayer, the cells were switched into serum-free media and then treated with various doses of oleic acid (OA, Solarbio, Beijing, China) for 24 h. 2.4.2 Preparation of L. plantarum SDJ09 samples Preparation of cell-free extracts (CFE): After incubating for 18 h at 37°C, LAB cells were harvested (6,000 g, 10 min, 4°C), washed twice with sterile water, resuspended to adjust the OD 600 to 0.5, and then sonicated in ice for 30 min (200 W, 5 s “on,” 8 s “off”). Subsequently, homogenized extracts were centrifuged for 15 min (8,000 g, 4°C), and then the supernatant was filtered with a 0.45 µL filter and lyophilized. For experiments, the powder was dissolved and adjusted to 30 µg/mL with sterile water [ 30 ]. Preparation of cell metabolites (CM): After incubating for 18 h at 37°C, LAB cells were harvested (6,000 g, 10 min, 4°C) and washed twice with sterile PBS buffer. Then, the cell concentration was adjusted to 1 × 10 8 CFU/mL, serum-free DMEM was added, and the cells were incubated for 2 h at 37°C. After centrifuging for 10 min (6,000 g, 4°C), the supernatant was filtered through a 0.22 µL filter membrane to obtain the metabolite sample. Preparation of heat-inactivated LAB (HK): After incubating for 18 h at 37°C, the LAB cells were harvested (6,000 g, 10 min, 4°C), washed twice with sterile water, and re-suspended to adjust the OD 600 to 0.5. The LAB cells were sterilized and heat-inactivated at 121°C for 15 min. 2.4.3 Cell viability assay and Oil Red O Staining HepG2 cells were inoculated into a 96-well plate at a density of 1 × 10 5 cells/well and incubated for 24 h at 37°C, and then cells were treated with OA at different levels (0, 200, 400, 800, 1000 nmol/L) for 24 h at 37°C. After drug treatment, 100 µL of culture medium with 10% CCK-8 (MCE, New Jersey, USA) was added into each well to replace the supernatant, and the culture was continued for 1 h at 37°C. Subsequently, three components of L. plantarum SDJ09 (CFE, CM, and HK) were added into each well and cultured for 24 h at 37°C. Cells were washed twice with PBS buffer, and then fixed with Oil Red O fixative for 30 min. The plate was treated with 60% isopropanol for 5 min, and the isopropanol was replaced with newly prepared Oil Red O staining solution. The plate was treated for 15 min and then washed five times with water. Afterwards, Mayer’s hematoxylin staining solution was added and mixed for 2 min to remove the excess dye-free solution, and Oil Red O buffer was added and mixed for 1 min. Finally, cells were covered with double-distilled water, and images of the stained lipid droplets were visualized with an inverted microscope. 2.5 Western blot After homogenizing in lysis buffer for 20 min, cell samples were centrifuged (12,000 g, 4 o C, 5 min) and harvested. The concentration of total protein was measured with a BCA Protein Assay Kit (Beyotime, Shanghai, China). The target proteins were separated using a 8% or 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes with 120 mA current for 1.5 h. The membranes were blocked with 5% nonfat milk for 1h at room temperature, and then the polyvinylidene difluoride membranes were treated with primary antibodies to PPARγ (Boster, Wuhan, China), C/EBPα (Boster, Wuhan, China), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR, BBI, Shanghai, China), cholesterol 7α-hydroxylase (CYP7A1, BBI, Shanghai, China), sterol regulatory element binding protein-1c (SREBP-1c, BBI, Shanghai, China), or glyceraldehyde-3-phosphate dehydrogenase (GADPH, BBI, Shanghai, China), in 5% nonfat milk in TBST buffer overnight at 4°C. Secondary antibodies were incubated with the membranes at room temperature for 1 h at 25°C 5% nonfat milk in TBST buffer. The immunoreactive proteins were detected using enhanced chemiluminescence. The expression level of target proteins was determined by comparison to GADPH protein levels. 2.6 Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Cells were gently washed twice with cold PBS and lysed in RIPA buffer. Total RNA was isolated using an RNA extraction kit (Takara-Bio, China). cDNA was synthesized using a PrimeScript™ RT Reagent Kit with a gDNA Eraser (Takara-Bio, Shuzo, Japan). Real-time PCR was carried out using SYBR® Premix Ex Taq™ (Takara-Bio, Shuzo, Japan) on a LightCycler® 96 (Roche, Basel, Switzerland). Sequences used in this study are listed in Table S1 . 2.7. Statistical Analysis All experimental results were based on three replicates. Statistical analysis was conducted using SPSS software (version 19.0; IBM Corporation, Armonk, NY, USA). Results are expressed as mean ± standard deviation from three measurements. 3. Results 3.1 Screening of lipid-lowering LAB In this study, 39 colonies were screened with the MRS plates. They were opaque, white or light yellow, round, rod-shaped, catalase-negative, and Gram-positive. Each of the 39 strains was also taxonomically identified according to its 16S rDNA sequence. Based on the physiological and phylogenetic experiments, these strains belonged to L. plantarum (22), L. pentosus (8), L. paraplantarum (5), and L. fermentum (4) (Table 1 ). Meanwhile, the cholesterol-lowering ability of the 39 LAB was also evaluated. We found that different strains exhibited different physiological properties for degrading cholesterol, with degradation from 4.47–55.15% (Fig. 1 A). Among of them, there were ten strains that exhibited higher cholesterol degradation, including SS02 (44.12%), SS05 (51.96%), SS08 (52.10%), SS13 (49.76%), SDJ01 (46.70%), SDJ07 (55.15%), SDJ09 (48.16%), SDJ13 (53.30%), SC04 (42.75%), and SC20 (45.26%). These ten strains were further tested for their inhibition of pancreatic lipase, and five strains had higher inhibition: SDJ07 (34.24%), SDJ09 (33.73%), SS02 (32.45%), SC20 (32.01%), and SS05 (31.43%) (Fig. 1 B). Table 1 Lactic acid bacteria (n = 39) isolated from the ten fermented foods used herein Strain Closest species Isolation source Number of strain SS02 SS03 SS05 SS08 SS09 SS10 SS11 SS13 SDJ01 SDJ02 SDJ07 SDJ08 SDJ09 SDJ10 SDJ12 SDJ13 SC01 SC03 SC11 SC12 SC13 SC17 Lactiplantibacillus plantarum sour bamboo shoots milk fans sour bean sauerkraut pickles 5 3 8 4 2 SS01 SS04 SS06 SS07 SDJ14 SDJ15 SC19 SC20 Lactiplantibacillus pentosus sour bamboo shoots sour bean sauerkraut 4 2 2 SC04 SC06 SC07 SC09 SC16 Lactiplantibacillus paraplantarum sauerkraut pickles 4 1 SDJ03 SDJ04 SDJ06 SDJ11 Limosilactobacillus fermentum sour bean 4 3.2 Investigating the probiotic properties of LAB After a 3-h exposure to various conditions, including acid, bile, simulated gastric digestion, and intestinal fluid, the survival rates of the five strains were investigated in detail (Table 2 ). When cells were incubated at pH 2.0, the survival rate of the five strains ranged from 29.41–98.50%, in which strains SDJ07 and SDJ09 exhibited the highest survival rates, reaching 98.50% and 62.96%, respectively. Similarly, after incubation with 3 g/L of bile salt, the survival rates of the five strains were higher than that of the control strain LGG, and strains SDJ09 and SS05 had the highest survival rates of 61.9% and 42.35%, respectively. In artificial gastric fluid, the survival rates of the five strains were also higher than that of LGG, ranging from 62.75–96.61%. After incubation in simulated intestinal fluid, the survival rate of the five strains ranged from 36.44–67.77%, in which strains SC20 and SDJ09 exhibited the highest survival rates, reaching 67.77% and 61.38%, respectively. Furthermore, the bacteriostatic abilities of the five strains were also investigated and shown in Table 2 . These showed antibacterial properties against Staphylococcus aureus and Escherichia coli , and their inhibition zone diameters were greater than 10 mm. In addition, the surface hydrophobicity of the five strains was also evaluated based on the adhesion to the hydrocarbon phase in solutions containing xylene. As shown in Fig. 2 , strains SDJ07 and SDJ09 had better hydrophobicity towards xylene, and the hydrophobicity value reached 89.50% and 91.57%, respectively. More interestingly, strain SDJ09 exhibited the highest auto-aggregation ability, 67.01% at 2 h, suggesting that strain SDJ09 had higher adhesion than that of other LAB strains. In addition, we tested the five strains for their susceptibility to 10 antibiotics. As shown in Table S2 , the five strains were resistant to vancomycin and resistant or intermediate to polymyxin but were susceptible or intermediate to eight or more antibiotics. More importantly, when the five strains were grown on amniotic blood plates, all strains exhibited γ-hemolysis on plates without green rings, clear rings, or hemolytic phenomena (Fig. 2 C), indicating that the five strains were safe at the cellular level. Table 2 Comparison of acid tolerance, bile tolerance, simulated gastric digestion tolerance, simulated intestinal fluid tolerance, and bacteriostatic abilities of the five strains * Strains Acid Tolerance (%) Bile tolerance (%) Simulated gastric digestion tolerance (%) Simulated intestinal fluid tolerance (%) Diameter of inhibition zone (mm) S. aureus E. coli SS02 29.41 ± 1.49d 12.66 ± 1.29c 73.04 ± 1.80c 44.35 ± 4.15cd 12.30 ± 0.75 13.43 ± 0.54 SS05 45.45 ± 0.06c 42.35 ± 2.86b 96.61 ± 0.71a 36.44 ± 0.25d 10.60 ± 0.90 11.90 ± 0.91 SDJ07 98.50 ± 1.73a 40.60 ± 3.54b 90.91 ± 0.06ab 50.00 ± 0.62c 11.20 ± 1.44 10.60 ± 0.30 SDJ09 62.96 ± 5.70b 61.90 ± 3.17a 86.90 ± 1.98b 61.38 ± 4.81b 11.20 ± 0.10 12.00 ± 1.76 SC20 33.91 ± 2.12c 16.06 ± 0.84c 92.56 ± 8.77ab 67.77 ± 3.37a 12.33 ± 1.11 15.3 ± 0.2 GG 39.88 ± 1.51c - 62.75 ± 1.10d 40.20 ± 0.48d - - * Results are expressed as the mean ± SD tested in triplicate. Different letters represent statistically significant differences (P < 0.05) between groups. 3.4 Effect of L. plantarum SDJ09 on lipid accumulation in 3T3-L1 adipocytes To assess the potential of SDJ09 to inhibit lipid accumulation in adipocytes, mature 3T3-L1 adipocytes were used as an in vitro model. As shown in Fig. 3 I, we found that the lipid area (red) of the 3T3-L1 mature adipocytes was effectively reduced by CFE, CM, and HK from SDJ09 according to the result of Oil Red O staining. Similarly, triglyceride (TG) content in mature 3T3-L1 cells also significantly decreased after treatment with CFE, CM, and HK from SDJ09 from 0.33 mg/mg protein to 0.13, 0.08, and 0.12 mg/mg protein, respectively (Fig. 3II). Meanwhile, to further investigate the physiological mechanism of inhibiting lipid accumulation, we assessed the effect of CFE, CM, and HK from L. plantarum SDJ09 on the expression of PPARγ and C/EBPα in 3T3-L1 cells. As shown in Fig. 4 A, CFE and CM decreased PPARγ expression by 12% and 11%, respectively, while HK decreased C/EBPα expression by 70%. Therefore, strain SDJ09 probably had its lipid-lowering effect through inhibiting the expression of PPARγ and C/EBPα in mature 3T3-L1 cells. Correspondingly, the qRT-PCR results also showed that these three components of SDJ09 influenced the expression of PPARγ, C/EBPα, LPL, and aP2 genes to different degrees. As shown in Fig. 4 B, CFE significantly decreased the expression of PPARγ (P < 0.01), C/EBPα (P < 0.01), and aP2 (P < 0.05) by 17%, 24%, and 15%, respectively, but did not affect the expression of LPL. However, CM did not affect the expression of C/EBPα but significantly inhibited the expression of PPARγ, LPL, and aP2 (all P < 0.01) by 47%, 22%, and 42%, respectively. HK significantly decreased the expression of PPARγ and C/EBPα by 27% and 32%, respectively (both P < 0.01), but did not affect the expression of LPL or aP2. 3.5 Effect of L. plantarum SDJ09 on lipid accumulation in HepG2 nonalcoholic steatosis cells Oil Red O staining results showed (Fig. 5 I) that normal cells grew in a long rhomboid shape, with a complete nuclear membrane and a clear edge, and only a few red lipid droplets were seen inside the cells (a), while in the treated group (b–e), many red lipid droplets surrounded the nucleus. With an increase in oleic acid concentration, the red lipid droplets also increased. High doses of oleic acid had toxic effects on the cells (Fig. 5 II). At oleic acid concentrations lower than 400 µmol/L, there was no significant effect on the cell survival rate. The TG content in the liver steatosis model resulting from 400 nmol/L OA treatment was 2.4 times that of the normal cells (Fig. 5 III), indicating that the liver steatosis cell model was successfully established. We found that 24-h treatment with CFE, CM, and HK from SDJ09 decreased the TG accumulation by 56%, 51%, and 61%, respectively, in 400 nmol/L OA-treated HepG2 cells. Furthermore, to investigate the effect of strain SDJ109 on the inhibition of fat accumulation and cholesterol pathway mechanisms in HepG2 cells, the expression levels of several enzymes (including SREBP-1c, FAS, ACC, HMGCR, and CYP7A1) were analyzed by western blot and RT-PCR. As shown in Fig. 6 A, CFE, CM, and HK from SDJ09 significantly decreased the expression of HMGCR: CFE (60%), CM (59%), and HK (50%). Similarly, the CFE and HK slightly decreased the expression of SREBP-1c by 16% and 17%, respectively, but the CM did not affect the expression of SREBP-1c. However, CFE, CM, and HK significantly enhanced the expression of CYP7A1 with increases of 42%, 46%, and 56%, respectively. Meanwhile, qRT-PCR was used to verify the results of western blot at the mRNA level, in which both CFE and CM effectively decreased the expression of SREBP-1c (71% and 82%), HMGCR (59% and 79%), ACC (6% and 7%), and FAS (50% and 61%) (P < 0.01), and significantly increased the expression of CYP7A1 (100% and 30%) (P < 0.01) (Fig. 6 B). Correspondingly, HK significantly (P < 0.01) decreased the expression of SREBP-1c (59%), HMGCR (57%), and ACC (16%) but did not affect the expression of CYP7A1 or FAS. Therefore, L. plantarum SDJ09 effectively decreased the hepatic lipid accumulation by inhibiting the expression of SREBP-1c and HMGCR and promoting the expression of CYP7A1. 4. Discussion At present, excessive cholesterol and triglycerides in the daily diet and blood have been shown to be the main risk factors for obesity and metabolic diseases [ 6 ]. Probiotics can effectively reduce serum triglycerides and cholesterol, and thus are promising for attenuating obesity and related metabolic diseases [ 14 ]. However, although probiotics have been widely screened for anti-obesity treatment, it is still necessary to screen strains with good therapeutic effects and strong probiotic properties. For probiotics, strong resistance to gastrointestinal stress conditions (including acidity, bile, pepsin, and trypsin), antimicrobial activity against enteric pathogenic microorganisms, good adhesion, and safety are the initial requirements. In this study, L. plantarum SDJ09 was selected from several fermented foods and exhibited high tolerance to the gastrointestinal environment with a survival rate of 62.96% at pH 2.0 for 3 h, 42.35% after a 3-h exposure to 3 g/L bile salt, 86.98% after a 3-h exposure to 3 g/L pepsin (pH = 3.0), and 61.38% after an 8-h exposure to 1 g/L trypsin (pH = 8.0). More importantly, L. plantarum SDJ09 had strong cholesterol-lowering and pancreatic lipase inhibition activity, showing a 48.16% reduction in cholesterol and 33.73% pancreatic lipase inhibition, which was higher than that of L. plantarum SMFM2017-NK2 (10.95 ± 5.87%), L. fermentum SMFM2017-NK3 (14.43%), and P. pentosaceus SMFM2017-GK1 (18.92%) [ 31 ]. As one of the important indicators of adhesion to the intestine, hydrophobicity is considered to be a key factor for adhesion to intestinal epithelial cells. Generally, a strain with a hydrophobicity greater than 60% is considered to be highly hydrophobic [ 32 ]. Furthermore, strains with strong auto-aggregation ability can achieve high cell density in the gastrointestinal tract, which is conducive to adhering to the intestine and avoiding colonization by pathogenic microorganisms [ 33 ]. L. plantarum SDJ09 showed higher hydrophobicity (91.57%) than that of L. plantarum Lb37 (57.40% ± 1.19%) and L. plantarum S57 (67.4% ± 1.3%) [ 34 , 35 ] and had a significant auto-aggregation ability at 2 h, with a self-aggregation ability of 67.01%. Lipid accumulation occurs when preadipocytes differentiate into mature adipocytes, which is regulated by several transcription factors. Among them, PPARγ and C/EBPα work synergistically to drive the terminal differentiation of preadipocytes and then control the transcriptional activation of mature adipocyte markers [ 36 , 37 ]. For example, lipoprotein lipase (LPL) is a key enzyme involved in TG clearance and attaches to the luminal surfaces of capillary endothelial cells in muscle and adipocytes to hydrolyze TG [ 38 ], and aP2 is a soluble fatty acid carrier and promotes the removal of fatty acids from adipocytes. Both LPL and aP2 are regulated by PPARγ and C/EBPα [ 39 ]. In this study, three fractions of L. plantarum SDJ09 effectively decreased the accumulation of TG in differentiated 3T3-L1 cells and impacted expression of PPARγ, C/EBPα, LPL, and aP2. CEF significantly upregulated PPARγ and C/EBPα and downregulated aP2 expression but did not significantly affect LPL. CM did not significantly affect C/EBPα but significantly downregulated LPL and aP2 gene expression. Therefore, L. plantarum SDJ09 has a multipronged effect on lipid accumulation through multiple mechanisms, including the downregulation of the expression of PPARγ, C/EBPα, LPL, and aP2, providing an effective way to combat obesity and a potential candidate for an anti-obesity drug [ 40 ]. Currently, antilipidemic drugs include statins, PCSK9 inhibitors, bile acid chelators, and cholesterol absorption inhibitors, in which statins are the most effective lipid-lowering drugs through inhibiting cholesterol synthase HMGCR [ 41 ], and bile acid chelators can promote bile acid synthesis and deplete cholesterol in the body through increasing the activity of CYP7A1 [ 42 ]. As a new drug target, acetyl CoA carboxylase (ACC) can reduce liver triglyceride content by simultaneously reducing fatty acid synthesis and stimulating fatty acid oxidation [ 43 ]. ACC and FAS are important lipogenic enzymes for fatty acid synthesis and are typically regulated by SREBP-1c [ 44 ]. More interestingly, the three fractions of L. plantarum SDJ09 significantly decreased the expression of HMGCR protein and increased the expression of CYP7A1 protein, indicating that L. plantarum SDJ09 effectively reduced hepatic lipid accumulation by inhibiting the expression of SREBP-1c and HMGCR and promoting the expression of CYP7A1. L. plantarum SDJ09 downregulated the expression of lipid synthesis genes (e.g., ACC and FAS) by reducing the expression of SREBP-1c, inhibiting the expression of cholesterol synthase HMGCR, and promoting bile acid synthesis through upregulating the expression of CYP7A1, thereby decreasing lipid accumulation in steatotic HepG2 cells. 5. Conclusion In this study, based on the investigation of probiotic properties, we found that L. plantarum SDJ09 exhibits exceptional tolerance to treatment that simulates the GI tract, inhibitory effects against pathogens, strong adhesion, and safety. More importantly, L. plantarum SDJ09 effectively reduced lipid synthesis by inhibiting the expression of transcription factors and genes associated with lipid synthesis. Furthermore, L. plantarum SDJ09 also promoted the expression of lipolysis-related genes, facilitating fat metabolism and decreasing TG content, suggesting that L. plantarum SDJ09 has the potential to serve as a probiotic candidate strain in functional foods targeted towards anti-obesity and NAFLD. Declarations Funding This work was funded by the National Natural Science Foundation of China (grant number: grant number: 32160558), Key Projects in Guangxi (grant number: 2019GXNSFDA245008), and the “Bagui Young Scholars” Special Project. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Contributions The first draft of the manuscript was written by Baoxin Yang and all authors commented on previous versions of the manuscript. Data collection was performed by Wenxuan Wang,Cuiwen Jian and Beibei Lv. Formal analysis was performed by Hailin He and Miao Wang. Funding acquisition and material preparation were performed by Shubo Li. The manuscript was revised by Yuan Guo. All authors read and approved the final manuscript. Corresponding author Correspondence to Yuan Guo. Ethical Approval Not applicable. Consent to participate Not applicable. Consent to Publish Not applicable. Availability of data and materials Authors will make availability of data and materials on reasonable request. References Suk, L. E., Ji, C. S., Don, K. H., Hye, K. M., YunJeong, J., GeumSoog, K., & Yeong, J. G. (2022). Anti-Obesity Activity in 3T3-L1 Cells of Cornus officinalis Fruits Harvested at Different Times. Processes , 10(10). H MSD, H. K. (2006). The medical complications of obesity. QJM: monthly journal of the Association of Physicians , 99(9). Evangelos, C., Ioanna, P., Margarita, P., Emmanouil, E. M. 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Autochthonous Lactic Acid Bacteria Isolated From Dairy Cow Feces Exhibiting Promising Probiotic Properties and in vitro Antibacterial Activity Against Foodborne Pathogens in Cattle. Front Vet Sci , 7 , 239–239. Kotzamanidis, C., Kourelis, A., Litopoulou-Tzanetaki, E., Tzanetakis, N., & Yiangou, M. (2010). Evaluation of adhesion capacity, cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains. International Journal of Food Microbiology , 140 (2), 154–163. Somashekaraiah, R., Shruthi, B., Deepthi, B. V., & Sreenivasa, M. Y. (2019). Probiotic Properties of Lactic Acid Bacteria Isolated From Neera: A Naturally Fermenting Coconut Palm Nectar. Frontiers In Microbiology , 10 , 1382. Yadav, R., Puniya, A. K., & Shukla, P. (2016). Probiotic Properties of Lactobacillus plantarum RYPR1 from an Indigenous Fermented Beverage Raabadi. Frontiers In Microbiology , 7 , 1683. SukJin, K., SooIm, C., Miran, J., Yulah, J., ChangHo, K., & GunHee, K. (2020). Anti-adipogenic effect of Lactobacillus fermentum MG4231 and MG4244 through AMPK pathway in 3T3-L1 preadipocytes. Food science and biotechnology , 29(11). Doyeon, K., Yukyung, C., Sejeong, K., Jimyeong, H., Hyemin, O., Yewon, L., Yujin, K., Yeongeun, S., Eunyoung, P., Joohyun, K. (2021). Lactobacillus fermentum SMFM2017-NK4 Isolated from Kimchi Can Prevent Obesity by Inhibiting Fat Accumulation. Foods , 10(4). Hernández-Alcántara, A. M., Wacher, C., Llamas, M. G., López, P., & Pérez-Chabela, M. L. (2018). Probiotic properties and stress response of thermotolerant lactic acid bacteria isolated from cooked meat products. LWT , 91 , 249–257. I, R. O. O. (2015). SA, R A. Probiotic potentials of yeasts isolated from some cereal-based Nigerian traditional fermented food products. Journal of applied microbiology , 119(3). R-S, S., F-P, P., & Ll, S. S. C. P. (2021). PM. Selection of probiotic Lactobacillus strains with antimicrobial activity to be used as biocontrol agents in food industry. LWT , 143. Mahsa, S., Bahman, P., Atefeh, M., Amin, H. M., & Yousef, N. (2022). Screening of potential probiotic lactic acid bacteria with antimicrobial properties and selection of superior bacteria for application as biocontrol using machine learning models. LWT , 162. Yadav, A. K., & Jang, B-C. (2021). Inhibition of Lipid Accumulation and Cyclooxygenase-2 Expression in Differentiating 3T3-L1 Preadipocytes by Pazopanib, a Multikinase Inhibitor. International Journal of Molecular Sciences , 22(9). Jiaqi, Z., Ailin, Z., & Wei, Q. (2022). The Potential to Fight Obesity with Adipogenesis Modulating Compounds. International Journal of Molecular Sciences , 23(4). Su, X., Kong, Y., & Peng, D. (2018). New insights into apolipoprotein A5 in controlling lipoprotein metabolism in obesity and the metabolic syndrome patients. Lipids in Health and Disease , 17(1). Yim, M. J., Hosokawa, M., Mizushina, Y., Yoshida, H., Saito, Y., & Miyashita, K. (2011). Suppressive effects of Amarouciaxanthin A on 3T3-L1 adipocyte differentiation through down-regulation of PPARgamma and C/EBPalpha mRNA expression. Journal Of Agriculture And Food Chemistry , 59 (5), 1646–1652. Cong, L., XiaoHong, Z., PiMin, G., HaiYue, N., LinZheng, L., YiFan, W., Xue, H., & LanWei, Z. (2021). Lactiplantibacillus plantarum H-87 prevents high-fat diet-induced obesity by regulating bile acid metabolism in C57BL/6J mice. Food & function , 12(10). Demyen, M., Alkhalloufi, K., & Pyrsopoulos, N. T. (2013). Lipid-Lowering Agents and Hepatotoxicity. Clinics in Liver Disease , 17 (4), 699–714. Ian, J., Jonathan, N., & F WJR. (2011). S PS,. New insights into bile acid malabsorption. Current gastroenterology reports , 13(5). Pradeep, K., Nancy, R., M, R. T., Subramaniam, P., & Farsad, A. (2022). Acetyl Co-A Carboxylase Inhibition Halts Hyperglycemia Induced Upregulation of De Novo Lipogenesis in Podocytes and Proximal Tubular Cells. Metabolites , 12(10). Li, Y., Xu, S., Mihaylova, M. M., Zheng, B., Hou, X., Jiang, B., Park, O., Luo, Z., Lefai, E., Shyy John, Y. J., et al. (2011). AMPK Phosphorylates and Inhibits SREBP Activity to Attenuate Hepatic Steatosis and Atherosclerosis in Diet-Induced Insulin-Resistant Mice. Cell Metabolism , 13 (4), 376–388. Supplementary Files Highlights.docx TableS1S2.docx SDJ09.tif Cite Share Download PDF Status: Published Journal Publication published 02 Aug, 2024 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted Reviewers agreed at journal 28 Mar, 2024 Reviewers invited by journal 28 Mar, 2024 Editor invited by journal 22 Mar, 2024 First submitted to journal 22 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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05:05:16","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":30595396,"visible":true,"origin":"","legend":"","description":"","filename":"SDJ09.tif","url":"https://assets-eu.researchsquare.com/files/rs-4115321/v1/59780f260e76825cde4ca472.tif"}],"financialInterests":"","formattedTitle":"Screening of the lipid-lowering probiotic Lactiplantibacillus plantarum SDJ09 and its anti-obesity mechanism","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs a chronic disease, obesity is caused by an imbalance between energy intake and energy expenditure and is related to various factors such as genetics, diet, and exercise [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. More importantly, obesity can cause a series of metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus, hyperlipidemia, and atherosclerosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For example, the prevalence of NAFLD has been increasing along with obesity rates, resulting in a rise in the prevalence of NAFLD in obese children/adolescents from 20% (2005\u0026ndash;2011) to 51% (2012\u0026ndash;2019) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. According to a report from the World Health Organization in 2016, an estimated 650\u0026nbsp;million adults, or approximately 13% of global adult population, suffer from obesity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the treatment of obesity poses a significant challenge. Bariatric surgery is the most effective approach, but it is not suitable for all patients, is not sufficiently scalable, and does not address the broader health concerns associated with obesity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to the difficulty of maintaining a healthy weight, pharmacotherapy is a widely accepted treatment option for improving metabolic health parameters [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For instance, treatment with orlistat led to an average of 5% weight loss at one year and 3% weight loss at four years, but this drug had side effects, such as gastrointestinal discomfort, oily stools, and flatulence [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recently, research demonstrated that human gut microbiota could modulate energy, glucose, and lipid metabolism in several organs (including the brain, liver, and muscle) via metabolites and signal transduction molecules [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consequently, microbiota-based therapeutic strategies, such as probiotic therapy aimed at improving gut flora, have gained attention as a potential treatment option for obesity and metabolic syndrome.\u003c/p\u003e \u003cp\u003eProbiotics\u0026mdash;defined as \u0026ldquo;live microorganisms which when administered in adequate amounts confer a health benefit on the host\u0026rdquo;\u0026mdash;show promising preclinical potential to reduce obesity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], NAFLD, diabetes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], insulin resistance, and metabolic syndrome [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. More importantly, the components, inactivated bacteria and metabolites of probiotics could also be used to regulate lipid metabolism, gut microbiota, and glucose metabolism [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, probiotics have been considered as a noninvasive yet potentially highly effective therapeutic approach against obesity and associated metabolic diseases [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Among of them, \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e, the most common species of probiotics, have been used to treat obesity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For example, \u003cem\u003eL. plantarum\u003c/em\u003e LMT1-48 was shown to downregulate the expression of proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBPα), adipocyte protein 2 (aP2), and lipoprotein lipase (LPL), thereby inhibiting lipid accumulation in 3T3-L1 adipocytes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, it was also found that \u003cem\u003eL. plantarum\u003c/em\u003e LMT1-48 effectively improved body weight in obese mice and further reduced hepatic fat accumulation by modulating liver enzymes in humans [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, more development of lipid-lowering lactic acid bacteria is needed, and the anti-obesity mechanism of lactic acid bacteria needs elucidation.\u003c/p\u003e \u003cp\u003eIn this study, several lactic acid bacteria with robust probiotic properties were selected from different fermented foods. Based on the investigation of probiotic potential, strain \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 was further investigated for its anti-obesity probiotics by assessing its lipid-lowering effects. Furthermore, we gained mechanistic understanding by treating 3T3-L1 mature adipocytes and HepG2 non-alcoholic fatty liver cell models with cell-free extracts, cell metabolites, and heat-inactivated cells derived from \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1 Screening of lipid-lowering lactic acid bacteria (LAB)\u003c/h2\u003e\n\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n\u003ch2\u003e2.1.1 Isolation of LAB strains\u003c/h2\u003e\n\u003cp\u003eTen traditional fermented foods (including pickled peppers, sauerkraut, milk fans, ham, sour soup, chili sauce, sour beans, sour bamboo shoots, pickles, and soybean sauce) were purchased from southwest China, and then 25 g of each sample was placed in 225 mL of sterile 0.9% NaCl for 30 min. Subsequently, a 100 \u0026micro;L dilution was spread on de Man, Rogosa, and Sharpe (MRS) solid medium and incubated for 48 h at 37\u0026deg;C. Strains with similar color and morphology to LAB were purified and cultured three times in succession, and then individual colonies were selected for a catalase test and Gram staining. Gram-positive and catalase-negative bacteria were selected and stored in 50% glycerol at \u0026minus;\u0026thinsp;20\u0026deg;C [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBacterial genomic DNA was extracted using a TIANamp bacteria DNA kit (Tiangen Biotech, Beijing, China) following the manufacturer's instructions and amplified with universal primers 27F and 1492R. PCR products were analyzed by 1% agarose gel electrophoresis and sent to Shanghai Bioengineering Company for sequencing (Shanghai, China). The 16S rRNA gene sequences were compared in the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003c/span\u003e), and homology analysis was performed using BLAST.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n\u003ch2\u003e2.1.2 Evaluation of cholesterol-lowering\u003c/h2\u003e\n\u003cp\u003eTo evaluate cholesterol-lowering, each of 39 lactic acid bacteria strains was inoculated (3% v/v) into MRS broth supplemented with cholesterol (100 \u0026micro;g/mL, Sigma, Shanghai, China) for culturing for 36 h at 37\u0026deg;C (using MRS broth supplemented with cholesterol as a control). The amount of cholesterol in the cell-free supernatant was analyzed using the phthalaldehyde method to draw the standard curve (y\u0026thinsp;=\u0026thinsp;0.0078x\u0026thinsp;+\u0026thinsp;0.0026, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9982) [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The cholesterol-lowering rate was calculated using the following formula:\u003c/p\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equa\" class=\"mathdisplay\"\u003e$$cℎolesterol lowering rate\\left(\\%\\right)=\\frac{{A}_{0}-A}{{A}_{0}}\\times 100\\% cℎolesterol lowering rate\\left(\\%\\right)=\\frac{{A}_{0}-A}{{A}_{0}}\\times 100\\%$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eA\u003csub\u003e0\u003c/sub\u003e: Absorbance before fermentation; A: Absorbance after fermentation\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n\u003ch2\u003e2.1.3 Measurement of pancreatic lipase inhibition activity\u003c/h2\u003e\n\u003cp\u003eWe followed the method of a previous study [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] with the following modifications. Strains were cultured without agitation for 18 h at 37\u0026deg;C in MRS broth (3% v/v), and then the broth was washed three time with PBS (pH\u0026thinsp;=\u0026thinsp;7.2) and re-suspended with PBS to adjust the OD\u003csub\u003e600\u003c/sub\u003e to 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (10\u003csup\u003e8\u003c/sup\u003e\u0026ndash;10\u003csup\u003e9\u003c/sup\u003e CFU/mL). Subsequently, a mixture was prepared by adding 12.5 \u0026micro;L of 5 mM 4-nitrophenyl octanoate (NPC, Solarbio, Beijing, China), 500 \u0026micro;L of Tris-HCl (pH\u0026thinsp;=\u0026thinsp;8.5) buffer, and 125 \u0026micro;L of bacterial solution. After mixing, 12.5 \u0026micro;L of pancreatic lipase (5 mg/mL in Tris-HCL buffer) was added, mixed for 2 min, and then incubated for 30 min at 37\u0026deg;C. Finally, the absorbance was measured at 412 nm using a 96-well plate (n\u0026thinsp;=\u0026thinsp;3). The blank sample contained LAB, but dimethyl sulfoxide (DMSO) replaced NPC. The control sample contained NPC but no LAB. The blank control contained no LAB, and DMSO replaced NPC. The inhibition activity was calculated using the following formula:\u003c/p\u003e\n\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equb\" class=\"mathdisplay\"\u003e$$Inℎibition activity\\left(\\%\\right)=1-\\left(\\frac{C-{C}_{0}}{{C}_{b}-{C}_{a}}\\right)\\times 100\\%Inℎibition activity\\left(\\%\\right)=1-\\left(\\frac{C-{C}_{0}}{{C}_{b}-{C}_{a}}\\right)\\times 100\\%$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eC\u003c/em\u003e \u003csub\u003e \u003cem\u003e0\u003c/em\u003e \u003c/sub\u003e: absorbance of a blank of the sample; \u003cem\u003eC\u003c/em\u003e: absorbance of a sample\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC\u003c/em\u003e \u003csub\u003e \u003cem\u003eb\u003c/em\u003e \u003c/sub\u003e: absorbance of a blank of the control sample; \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e: absorbance of a control sample\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2 Assessment of probiotic characteristics\u003c/h2\u003e\n\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n\u003ch2\u003e2.2.1 Tolerance to treatment\u003c/h2\u003e\n\u003cp\u003eAfter incubating for 18 h at 37\u0026deg;C, LAB cells were harvested (6,000 g, 10 min, 4\u0026deg;C) and resuspended to approximately 10\u003csup\u003e9\u003c/sup\u003e CFU/mL in sterile PBS and then inoculated at 3% into the MRS medium (pH\u0026thinsp;=\u0026thinsp;2.0 and 0.3% bovine bile salt) and incubated for 3 h. An in vitro static digestion model was used to simulate gastrointestinal fluid [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], in which 3 g/L of pepsin (Coolaiber, Beijing, China) was dissolved in PBS (pH\u0026thinsp;=\u0026thinsp;3.0) to simulate gastric fluid, and 1.0 g/L trypsin (Coolaiber Technology Co., Ltd, Beijing, China) was dissolved in PBS (pH\u0026thinsp;=\u0026thinsp;8.0) to simulate intestinal fluid. Then, the viability was determined by counting the number of viable bacteria on the plates before and after treatment, and survival rates were calculated according to the following formula (n\u0026thinsp;=\u0026thinsp;3):\u003c/p\u003e\n\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equc\" class=\"mathdisplay\"\u003e$$Relative survival ratio \\left(\\%\\right)=\\frac{N}{{N}_{0}}\\times 100\\%Relative survival ratio \\left(\\%\\right)=\\frac{N}{{N}_{0}}\\times 100\\%$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eN\u003c/em\u003e: CFU of viable cells survived; \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e: CFU of initial viable cells inoculated\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n\u003ch2\u003e2.2.2 Antibacterial activity of LAB\u003c/h2\u003e\n\u003cp\u003eAs the indicator strain, \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e were adjusted to 10\u003csup\u003e7\u003c/sup\u003e CFU/mL with PBS and then evenly coated on the Luria Broth plate (OXOID, Beijing, China). Subsequently, the antibacterial ability of LAB cell-free supernatants (CFS) was evaluated with the Oxford cup method [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n\u003ch2\u003e2.2.3 Adhesion of LAB\u003c/h2\u003e\n\u003cp\u003eAccording to a previous study [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e], the method of microbial adhesion to hydrocarbons was used to investigate the surface hydrophobicity of strains. After incubating for 18 h at 37\u0026deg;C, LAB cells were harvested (6,000 g, 10 min, 4\u0026deg;C) and washed twice with sterile PBS buffer, and then resuspended to adjust the OD\u003csub\u003e600\u003c/sub\u003e to 1.0 (A\u003csub\u003ea\u003c/sub\u003e). Subsequently, 4 mL of bacterial suspension was added into 0.8 mL of xylene for vortexing for 2 min and then held without agitation at room temperature for 20 min. Finally, the absorbance of the aqueous phase was measured at 600 nm (A\u003csub\u003eb\u003c/sub\u003e), and the surface hydrophobicity was calculated as follows:\u003c/p\u003e\n\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equd\" class=\"mathdisplay\"\u003e$$Surface Hydropℎobicity\\left(\\%\\right)=\\left(1-{\\frac{{A}_{a}}{A}}_{b}\\right)\\times 100\\%Surface Hydropℎobicity\\left(\\%\\right)=\\left(1-{\\frac{{A}_{a}}{A}}_{b}\\right)\\times 100%$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eFurthermore, after the LAB cells were resuspended to an OD\u003csub\u003e600\u003c/sub\u003e of 0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (A\u003csub\u003ec\u003c/sub\u003e), 4 mL of the bacterial suspension was held without agitation for 2 h at room temperature, and then the absorbance of the supernatant was measured at 600 nm (A\u003csub\u003ed\u003c/sub\u003e). The auto-aggregation capacity was calculated as follows:\u003c/p\u003e\n\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Eque\" class=\"mathdisplay\"\u003e$$Auto-Aggregation\\left(\\%\\right)=\\left(1-\\frac{{A}_{c}}{{A}_{d}}\\right)\\times 100\\text{%}Auto-Aggregation\\left(\\%\\right)=\\left(1-\\frac{{A}_{c}}{{A}_{d}}\\right)\\times 100\\text{%}$$\u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3 Safety determination of the LAB\u003c/h2\u003e\n\u003cp\u003eAfter incubating for 18 h at 37\u0026deg;C, LAB cells were harvested (6,000 g, 10 min, 4\u0026deg;C), washed twice with sterile PBS buffer, and then resuspended to 10\u003csup\u003e7\u003c/sup\u003e CFU/mL. Then, an antibiotic sensitivity test of LAB was performed using a drug-sensitive disk agar diffusion method (K-B method) [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, the hemolytic activity was also detected by streaking the blood on agar plates (7% v/v sheep blood) [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] that were incubated for 24\u0026ndash;48 h at 37\u0026deg;C, and then the zones of hemolysis around the colonies were observed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4 Construction of cell culture model and cell experiment\u003c/h2\u003e\n\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.1 Cells culture and treatment\u003c/h2\u003e\n\u003cp\u003eThe mouse 3T3-L1 cell line was purchased from the Beinanchuanlian Institute of Biotechnology (Beijing, China) and cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM, Thermo Fisher Scientific, China) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, China) and 1% penicillin-streptomycin at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. To induce adipocyte differentiation, 3T3-L1 preadipocytes were grown to the contact inhibition stage, maintained in the post-fusion stage for 2 days, and then switched into DMEM supplemented with 10% FBS, 0.5 mM isobutyl methyl xanthine (Macklin, Shanghai, China), 0.5 \u0026micro;M dexamethasone (Solarbio, Beijing, China), and 10 \u0026micro;g/mL insulin (Macklin, Shanghai, China) for 2 days. Subsequently, the differentiation medium was replaced by 10% FBS-DMEM with 10 \u0026micro;g/mL insulin for 2 days. Finally, the induction medium was changed to growth medium, and the cells were cultured for 2 days.\u003c/p\u003e\n\u003cp\u003eThe HepG2 (human hepatocellular liver carcinoma) cell line was purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. After obtaining an 80% confluent monolayer, the cells were switched into serum-free media and then treated with various doses of oleic acid (OA, Solarbio, Beijing, China) for 24 h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.2 Preparation of \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 samples\u003c/h2\u003e\n\u003cp\u003ePreparation of cell-free extracts (CFE): After incubating for 18 h at 37\u0026deg;C, LAB cells were harvested (6,000 g, 10 min, 4\u0026deg;C), washed twice with sterile water, resuspended to adjust the OD\u003csub\u003e600\u003c/sub\u003e to 0.5, and then sonicated in ice for 30 min (200 W, 5 s \u0026ldquo;on,\u0026rdquo; 8 s \u0026ldquo;off\u0026rdquo;). Subsequently, homogenized extracts were centrifuged for 15 min (8,000 g, 4\u0026deg;C), and then the supernatant was filtered with a 0.45 \u0026micro;L filter and lyophilized. For experiments, the powder was dissolved and adjusted to 30 \u0026micro;g/mL with sterile water [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003ePreparation of cell metabolites (CM): After incubating for 18 h at 37\u0026deg;C, LAB cells were harvested (6,000 g, 10 min, 4\u0026deg;C) and washed twice with sterile PBS buffer. Then, the cell concentration was adjusted to 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, serum-free DMEM was added, and the cells were incubated for 2 h at 37\u0026deg;C. After centrifuging for 10 min (6,000 g, 4\u0026deg;C), the supernatant was filtered through a 0.22 \u0026micro;L filter membrane to obtain the metabolite sample.\u003c/p\u003e\n\u003cp\u003ePreparation of heat-inactivated LAB (HK): After incubating for 18 h at 37\u0026deg;C, the LAB cells were harvested (6,000 g, 10 min, 4\u0026deg;C), washed twice with sterile water, and re-suspended to adjust the OD\u003csub\u003e600\u003c/sub\u003e to 0.5. The LAB cells were sterilized and heat-inactivated at 121\u0026deg;C for 15 min.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n\u003ch2\u003e2.4.3 Cell viability assay and Oil Red O Staining\u003c/h2\u003e\n\u003cp\u003eHepG2 cells were inoculated into a 96-well plate at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and incubated for 24 h at 37\u0026deg;C, and then cells were treated with OA at different levels (0, 200, 400, 800, 1000 nmol/L) for 24 h at 37\u0026deg;C. After drug treatment, 100 \u0026micro;L of culture medium with 10% CCK-8 (MCE, New Jersey, USA) was added into each well to replace the supernatant, and the culture was continued for 1 h at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eSubsequently, three components of \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 (CFE, CM, and HK) were added into each well and cultured for 24 h at 37\u0026deg;C. Cells were washed twice with PBS buffer, and then fixed with Oil Red O fixative for 30 min. The plate was treated with 60% isopropanol for 5 min, and the isopropanol was replaced with newly prepared Oil Red O staining solution. The plate was treated for 15 min and then washed five times with water. Afterwards, Mayer\u0026rsquo;s hematoxylin staining solution was added and mixed for 2 min to remove the excess dye-free solution, and Oil Red O buffer was added and mixed for 1 min. Finally, cells were covered with double-distilled water, and images of the stained lipid droplets were visualized with an inverted microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e2.5 Western blot\u003c/h2\u003e\n\u003cp\u003eAfter homogenizing in lysis buffer for 20 min, cell samples were centrifuged (12,000 g, 4 \u003csup\u003eo\u003c/sup\u003eC, 5 min) and harvested. The concentration of total protein was measured with a BCA Protein Assay Kit (Beyotime, Shanghai, China). The target proteins were separated using a 8% or 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes with 120 mA current for 1.5 h. The membranes were blocked with 5% nonfat milk for 1h at room temperature, and then the polyvinylidene difluoride membranes were treated with primary antibodies to PPAR\u0026gamma; (Boster, Wuhan, China), C/EBP\u0026alpha; (Boster, Wuhan, China), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR, BBI, Shanghai, China), cholesterol 7\u0026alpha;-hydroxylase (CYP7A1, BBI, Shanghai, China), sterol regulatory element binding protein-1c (SREBP-1c, BBI, Shanghai, China), or glyceraldehyde-3-phosphate dehydrogenase (GADPH, BBI, Shanghai, China), in 5% nonfat milk in TBST buffer overnight at 4\u0026deg;C. Secondary antibodies were incubated with the membranes at room temperature for 1 h at 25\u0026deg;C 5% nonfat milk in TBST buffer. The immunoreactive proteins were detected using enhanced chemiluminescence. The expression level of target proteins was determined by comparison to GADPH protein levels.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e2.6 Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e\n\u003cp\u003eCells were gently washed twice with cold PBS and lysed in RIPA buffer. Total RNA was isolated using an RNA extraction kit (Takara-Bio, China). cDNA was synthesized using a PrimeScript\u0026trade; RT Reagent Kit with a gDNA Eraser (Takara-Bio, Shuzo, Japan). Real-time PCR was carried out using SYBR\u0026reg; Premix Ex Taq\u0026trade; (Takara-Bio, Shuzo, Japan) on a LightCycler\u0026reg; 96 (Roche, Basel, Switzerland). Sequences used in this study are listed in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e\n\u003cp\u003eAll experimental results were based on three replicates. Statistical analysis was conducted using SPSS software (version 19.0; IBM Corporation, Armonk, NY, USA). Results are expressed as mean\u0026thinsp;\u003cem\u003e\u0026plusmn;\u003c/em\u003e\u0026thinsp;standard deviation from three measurements.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Screening of lipid-lowering LAB\u003c/h2\u003e\n \u003cp\u003eIn this study, 39 colonies were screened with the MRS plates. They were opaque, white or light yellow, round, rod-shaped, catalase-negative, and Gram-positive. Each of the 39 strains was also taxonomically identified according to its 16S rDNA sequence. Based on the physiological and phylogenetic experiments, these strains belonged to \u003cem\u003eL. plantarum\u003c/em\u003e (22), \u003cem\u003eL. pentosus\u003c/em\u003e (8), \u003cem\u003eL. paraplantarum\u003c/em\u003e (5), and \u003cem\u003eL. fermentum\u003c/em\u003e (4) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Meanwhile, the cholesterol-lowering ability of the 39 LAB was also evaluated. We found that different strains exhibited different physiological properties for degrading cholesterol, with degradation from 4.47\u0026ndash;55.15% (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Among of them, there were ten strains that exhibited higher cholesterol degradation, including SS02 (44.12%), SS05 (51.96%), SS08 (52.10%), SS13 (49.76%), SDJ01 (46.70%), SDJ07 (55.15%), SDJ09 (48.16%), SDJ13 (53.30%), SC04 (42.75%), and SC20 (45.26%). These ten strains were further tested for their inhibition of pancreatic lipase, and five strains had higher inhibition: SDJ07 (34.24%), SDJ09 (33.73%), SS02 (32.45%), SC20 (32.01%), and SS05 (31.43%) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLactic acid bacteria (n\u0026thinsp;=\u0026thinsp;39) isolated from the ten fermented foods used herein\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eClosest species\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolation source\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of strain\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSS02 SS03 SS05 SS08 SS09\u003c/p\u003e\n \u003cp\u003eSS10 SS11 SS13\u003c/p\u003e\n \u003cp\u003eSDJ01 SDJ02 SDJ07 SDJ08 SDJ09 SDJ10 SDJ12 SDJ13\u003c/p\u003e\n \u003cp\u003eSC01 SC03 SC11 SC12\u003c/p\u003e\n \u003cp\u003eSC13 SC17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esour bamboo shoots\u003c/p\u003e\n \u003cp\u003emilk fans\u003c/p\u003e\n \u003cp\u003esour bean\u003c/p\u003e\n \u003cp\u003esauerkraut\u003c/p\u003e\n \u003cp\u003epickles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSS01 SS04 SS06 SS07\u003c/p\u003e\n \u003cp\u003eSDJ14 SDJ15\u003c/p\u003e\n \u003cp\u003eSC19 SC20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus pentosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esour bamboo shoots\u003c/p\u003e\n \u003cp\u003esour bean\u003c/p\u003e\n \u003cp\u003esauerkraut\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSC04 SC06 SC07 SC09\u003c/p\u003e\n \u003cp\u003eSC16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLactiplantibacillus paraplantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esauerkraut\u003c/p\u003e\n \u003cp\u003epickles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSDJ03 SDJ04 SDJ06 SDJ11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esour bean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Investigating the probiotic properties of LAB\u003c/h2\u003e\n \u003cp\u003eAfter a 3-h exposure to various conditions, including acid, bile, simulated gastric digestion, and intestinal fluid, the survival rates of the five strains were investigated in detail (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). When cells were incubated at pH 2.0, the survival rate of the five strains ranged from 29.41\u0026ndash;98.50%, in which strains SDJ07 and SDJ09 exhibited the highest survival rates, reaching 98.50% and 62.96%, respectively. Similarly, after incubation with 3 g/L of bile salt, the survival rates of the five strains were higher than that of the control strain LGG, and strains SDJ09 and SS05 had the highest survival rates of 61.9% and 42.35%, respectively. In artificial gastric fluid, the survival rates of the five strains were also higher than that of LGG, ranging from 62.75\u0026ndash;96.61%. After incubation in simulated intestinal fluid, the survival rate of the five strains ranged from 36.44\u0026ndash;67.77%, in which strains SC20 and SDJ09 exhibited the highest survival rates, reaching 67.77% and 61.38%, respectively.\u003c/p\u003e\n \u003cp\u003eFurthermore, the bacteriostatic abilities of the five strains were also investigated and shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. These showed antibacterial properties against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e, and their inhibition zone diameters were greater than 10 mm. In addition, the surface hydrophobicity of the five strains was also evaluated based on the adhesion to the hydrocarbon phase in solutions containing xylene. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, strains SDJ07 and SDJ09 had better hydrophobicity towards xylene, and the hydrophobicity value reached 89.50% and 91.57%, respectively. More interestingly, strain SDJ09 exhibited the highest auto-aggregation ability, 67.01% at 2 h, suggesting that strain SDJ09 had higher adhesion than that of other LAB strains.\u003c/p\u003e\n \u003cp\u003eIn addition, we tested the five strains for their susceptibility to 10 antibiotics. As shown in Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e, the five strains were resistant to vancomycin and resistant or intermediate to polymyxin but were susceptible or intermediate to eight or more antibiotics. More importantly, when the five strains were grown on amniotic blood plates, all strains exhibited \u0026gamma;-hemolysis on plates without green rings, clear rings, or hemolytic phenomena (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating that the five strains were safe at the cellular level.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of acid tolerance, bile tolerance, simulated gastric digestion tolerance, simulated intestinal fluid tolerance, and bacteriostatic abilities of the five strains\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eStrains\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eAcid Tolerance (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eBile tolerance (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eSimulated gastric digestion tolerance (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eSimulated intestinal fluid tolerance (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eDiameter of inhibition zone (mm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSS02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.35\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSS05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSDJ07\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSDJ09\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.96\u0026thinsp;\u0026plusmn;\u0026thinsp;5.70b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.90\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSC20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.56\u0026thinsp;\u0026plusmn;\u0026thinsp;8.77ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.77\u0026thinsp;\u0026plusmn;\u0026thinsp;3.37a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e* Results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD tested in triplicate. Different letters represent statistically significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between groups.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Effect of \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 on lipid accumulation in 3T3-L1 adipocytes\u003c/h2\u003e\n \u003cp\u003eTo assess the potential of SDJ09 to inhibit lipid accumulation in adipocytes, mature 3T3-L1 adipocytes were used as an in vitro model. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI, we found that the lipid area (red) of the 3T3-L1 mature adipocytes was effectively reduced by CFE, CM, and HK from SDJ09 according to the result of Oil Red O staining. Similarly, triglyceride (TG) content in mature 3T3-L1 cells also significantly decreased after treatment with CFE, CM, and HK from SDJ09 from 0.33 mg/mg protein to 0.13, 0.08, and 0.12 mg/mg protein, respectively (Fig.\u0026nbsp;3II). Meanwhile, to further investigate the physiological mechanism of inhibiting lipid accumulation, we assessed the effect of CFE, CM, and HK from \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 on the expression of PPAR\u0026gamma; and C/EBP\u0026alpha; in 3T3-L1 cells. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, CFE and CM decreased PPAR\u0026gamma; expression by 12% and 11%, respectively, while HK decreased C/EBP\u0026alpha; expression by 70%. Therefore, strain SDJ09 probably had its lipid-lowering effect through inhibiting the expression of PPAR\u0026gamma; and C/EBP\u0026alpha; in mature 3T3-L1 cells.\u003c/p\u003e\n \u003cp\u003eCorrespondingly, the qRT-PCR results also showed that these three components of SDJ09 influenced the expression of PPAR\u0026gamma;, C/EBP\u0026alpha;, LPL, and aP2 genes to different degrees. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, CFE significantly decreased the expression of PPAR\u0026gamma; (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), C/EBP\u0026alpha; (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and aP2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by 17%, 24%, and 15%, respectively, but did not affect the expression of LPL. However, CM did not affect the expression of C/EBP\u0026alpha; but significantly inhibited the expression of PPAR\u0026gamma;, LPL, and aP2 (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by 47%, 22%, and 42%, respectively. HK significantly decreased the expression of PPAR\u0026gamma; and C/EBP\u0026alpha; by 27% and 32%, respectively (both P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), but did not affect the expression of LPL or aP2.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Effect of \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 on lipid accumulation in HepG2 nonalcoholic steatosis cells\u003c/h2\u003e\n \u003cp\u003eOil Red O staining results showed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI) that normal cells grew in a long rhomboid shape, with a complete nuclear membrane and a clear edge, and only a few red lipid droplets were seen inside the cells (a), while in the treated group (b\u0026ndash;e), many red lipid droplets surrounded the nucleus. With an increase in oleic acid concentration, the red lipid droplets also increased. High doses of oleic acid had toxic effects on the cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eII). At oleic acid concentrations lower than 400 \u0026micro;mol/L, there was no significant effect on the cell survival rate. The TG content in the liver steatosis model resulting from 400 nmol/L OA treatment was 2.4 times that of the normal cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eIII), indicating that the liver steatosis cell model was successfully established. We found that 24-h treatment with CFE, CM, and HK from SDJ09 decreased the TG accumulation by 56%, 51%, and 61%, respectively, in 400 nmol/L OA-treated HepG2 cells.\u003c/p\u003e\n \u003cp\u003eFurthermore, to investigate the effect of strain SDJ109 on the inhibition of fat accumulation and cholesterol pathway mechanisms in HepG2 cells, the expression levels of several enzymes (including SREBP-1c, FAS, ACC, HMGCR, and CYP7A1) were analyzed by western blot and RT-PCR. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, CFE, CM, and HK from SDJ09 significantly decreased the expression of HMGCR: CFE (60%), CM (59%), and HK (50%). Similarly, the CFE and HK slightly decreased the expression of SREBP-1c by 16% and 17%, respectively, but the CM did not affect the expression of SREBP-1c. However, CFE, CM, and HK significantly enhanced the expression of CYP7A1 with increases of 42%, 46%, and 56%, respectively. Meanwhile, qRT-PCR was used to verify the results of western blot at the mRNA level, in which both CFE and CM effectively decreased the expression of SREBP-1c (71% and 82%), HMGCR (59% and 79%), ACC (6% and 7%), and FAS (50% and 61%) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significantly increased the expression of CYP7A1 (100% and 30%) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). Correspondingly, HK significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) decreased the expression of SREBP-1c (59%), HMGCR (57%), and ACC (16%) but did not affect the expression of CYP7A1 or FAS. Therefore, \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 effectively decreased the hepatic lipid accumulation by inhibiting the expression of SREBP-1c and HMGCR and promoting the expression of CYP7A1.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAt present, excessive cholesterol and triglycerides in the daily diet and blood have been shown to be the main risk factors for obesity and metabolic diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Probiotics can effectively reduce serum triglycerides and cholesterol, and thus are promising for attenuating obesity and related metabolic diseases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, although probiotics have been widely screened for anti-obesity treatment, it is still necessary to screen strains with good therapeutic effects and strong probiotic properties.\u003c/p\u003e \u003cp\u003eFor probiotics, strong resistance to gastrointestinal stress conditions (including acidity, bile, pepsin, and trypsin), antimicrobial activity against enteric pathogenic microorganisms, good adhesion, and safety are the initial requirements. In this study, \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 was selected from several fermented foods and exhibited high tolerance to the gastrointestinal environment with a survival rate of 62.96% at pH 2.0 for 3 h, 42.35% after a 3-h exposure to 3 g/L bile salt, 86.98% after a 3-h exposure to 3 g/L pepsin (pH\u0026thinsp;=\u0026thinsp;3.0), and 61.38% after an 8-h exposure to 1 g/L trypsin (pH\u0026thinsp;=\u0026thinsp;8.0). More importantly, \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 had strong cholesterol-lowering and pancreatic lipase inhibition activity, showing a 48.16% reduction in cholesterol and 33.73% pancreatic lipase inhibition, which was higher than that of \u003cem\u003eL. plantarum\u003c/em\u003e SMFM2017-NK2 (10.95\u0026thinsp;\u0026plusmn;\u0026thinsp;5.87%), \u003cem\u003eL. fermentum\u003c/em\u003e SMFM2017-NK3 (14.43%), and \u003cem\u003eP. pentosaceus\u003c/em\u003e SMFM2017-GK1 (18.92%) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs one of the important indicators of adhesion to the intestine, hydrophobicity is considered to be a key factor for adhesion to intestinal epithelial cells. Generally, a strain with a hydrophobicity greater than 60% is considered to be highly hydrophobic [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, strains with strong auto-aggregation ability can achieve high cell density in the gastrointestinal tract, which is conducive to adhering to the intestine and avoiding colonization by pathogenic microorganisms [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 showed higher hydrophobicity (91.57%) than that of \u003cem\u003eL. plantarum\u003c/em\u003e Lb37 (57.40% \u0026plusmn; 1.19%) and \u003cem\u003eL. plantarum\u003c/em\u003e S57 (67.4% \u0026plusmn; 1.3%) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and had a significant auto-aggregation ability at 2 h, with a self-aggregation ability of 67.01%.\u003c/p\u003e \u003cp\u003eLipid accumulation occurs when preadipocytes differentiate into mature adipocytes, which is regulated by several transcription factors. Among them, \u003cem\u003ePPARγ\u003c/em\u003e and \u003cem\u003eC/EBPα\u003c/em\u003e work synergistically to drive the terminal differentiation of preadipocytes and then control the transcriptional activation of mature adipocyte markers [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For example, lipoprotein lipase (LPL) is a key enzyme involved in TG clearance and attaches to the luminal surfaces of capillary endothelial cells in muscle and adipocytes to hydrolyze TG [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and aP2 is a soluble fatty acid carrier and promotes the removal of fatty acids from adipocytes. Both LPL and aP2 are regulated by \u003cem\u003ePPARγ\u003c/em\u003e and \u003cem\u003eC/EBPα\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, three fractions of \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 effectively decreased the accumulation of TG in differentiated 3T3-L1 cells and impacted expression of PPARγ, C/EBPα, LPL, and aP2. CEF significantly upregulated PPARγ and C/EBPα and downregulated aP2 expression but did not significantly affect LPL. CM did not significantly affect C/EBPα but significantly downregulated LPL and aP2 gene expression. Therefore, \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 has a multipronged effect on lipid accumulation through multiple mechanisms, including the downregulation of the expression of PPARγ, C/EBPα, LPL, and aP2, providing an effective way to combat obesity and a potential candidate for an anti-obesity drug [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, antilipidemic drugs include statins, PCSK9 inhibitors, bile acid chelators, and cholesterol absorption inhibitors, in which statins are the most effective lipid-lowering drugs through inhibiting cholesterol synthase HMGCR [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and bile acid chelators can promote bile acid synthesis and deplete cholesterol in the body through increasing the activity of CYP7A1 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. As a new drug target, acetyl CoA carboxylase (ACC) can reduce liver triglyceride content by simultaneously reducing fatty acid synthesis and stimulating fatty acid oxidation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. ACC and FAS are important lipogenic enzymes for fatty acid synthesis and are typically regulated by SREBP-1c [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. More interestingly, the three fractions of \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 significantly decreased the expression of HMGCR protein and increased the expression of CYP7A1 protein, indicating that \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 effectively reduced hepatic lipid accumulation by inhibiting the expression of SREBP-1c and HMGCR and promoting the expression of CYP7A1. \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 downregulated the expression of lipid synthesis genes (e.g., ACC and FAS) by reducing the expression of SREBP-1c, inhibiting the expression of cholesterol synthase HMGCR, and promoting bile acid synthesis through upregulating the expression of CYP7A1, thereby decreasing lipid accumulation in steatotic HepG2 cells.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, based on the investigation of probiotic properties, we found that \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 exhibits exceptional tolerance to treatment that simulates the GI tract, inhibitory effects against pathogens, strong adhesion, and safety. More importantly, \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 effectively reduced lipid synthesis by inhibiting the expression of transcription factors and genes associated with lipid synthesis. Furthermore, \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 also promoted the expression of lipolysis-related genes, facilitating fat metabolism and decreasing TG content, suggesting that \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 has the potential to serve as a probiotic candidate strain in functional foods targeted towards anti-obesity and NAFLD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work was funded by the National Natural Science Foundation of China (grant number: grant number: 32160558), Key Projects in Guangxi (grant number: 2019GXNSFDA245008), and the \u0026ldquo;Bagui Young Scholars\u0026rdquo; Special Project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe first draft of the manuscript was written by Baoxin Yang and all authors commented on previous versions of the manuscript. Data collection was performed by Wenxuan Wang,Cuiwen Jian and Beibei Lv. Formal analysis was performed by Hailin He and Miao Wang. Funding acquisition and material preparation were performed by Shubo Li. The manuscript was revised by Yuan Guo. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Yuan Guo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors will make availability of data and materials on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSuk, L. E., Ji, C. S., Don, K. H., Hye, K. M., YunJeong, J., GeumSoog, K., \u0026amp; Yeong, J. G. (2022). 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AMPK Phosphorylates and Inhibits SREBP Activity to Attenuate Hepatic Steatosis and Atherosclerosis in Diet-Induced Insulin-Resistant Mice. \u003cem\u003eCell Metabolism\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(4), 376\u0026ndash;388.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lactiplantibacillus plantarum, probiotic, lipid-lowering, lipid metabolism, anti-obesity","lastPublishedDoi":"10.21203/rs.3.rs-4115321/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4115321/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, 39 strains of lactic acid bacteria were screened from several fermented foods. Based on the evaluation of functional and prebiotic properties, \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e SDJ09 was selected as a promising candidate. It gave a 48.16% cholesterol reduction and 33.73% pancreatic lipase inhibition in cells; exhibited high resistance to acid, bile salts, and gastrointestinal fluid; and had strong antibacterial activity and high adhesion capabilities. More importantly, the lipid-lowering effect of \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 was also investigated using 3T3-L1 mature adipocytes and HepG2 nonalcoholic fatty liver disease models. \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 effectively decreased triglyceride accumulation by more than 50% in both cell models, in which the expression of PPARγ, C/EBPα, aP2, and LPL in 3T3-L1 cells was significantly downregulated by \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09. \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 also improved lipid metabolism by downregulating the expression of HMGCR, SREBP-1c, ACC, and FAS and upregulating the expression of CYP7A1 in HepG2 nonalcoholic steatohepatitis cells. Therefore, \u003cem\u003eL. plantarum\u003c/em\u003e SDJ09 has the potential to effectively decrease obesity and non-alcoholic fatty liver disease (NAFLD) by inhibiting lipid accumulation, providing a prospective probiotic agent for anti-obesity.\u003c/p\u003e","manuscriptTitle":"Screening of the lipid-lowering probiotic Lactiplantibacillus plantarum SDJ09 and its anti-obesity mechanism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-02 05:05:08","doi":"10.21203/rs.3.rs-4115321/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-03-29T00:43:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-29T00:38:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2024-03-22T13:39:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2024-03-22T08:23:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"04fcc2c9-c867-424d-b1a7-3ccad0fc9bb1","owner":[],"postedDate":"April 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T16:08:46+00:00","versionOfRecord":{"articleIdentity":"rs-4115321","link":"https://doi.org/10.1007/s12010-024-05034-x","journal":{"identity":"applied-biochemistry-and-biotechnology","isVorOnly":false,"title":"Applied Biochemistry and Biotechnology"},"publishedOn":"2024-08-02 15:57:23","publishedOnDateReadable":"August 2nd, 2024"},"versionCreatedAt":"2024-04-02 05:05:08","video":"","vorDoi":"10.1007/s12010-024-05034-x","vorDoiUrl":"https://doi.org/10.1007/s12010-024-05034-x","workflowStages":[]},"version":"v1","identity":"rs-4115321","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4115321","identity":"rs-4115321","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}