Isolation of LAB strains and growth conditions. Four types of grains, rice, brown rice, black rice and hulled barley, were collected in the Republic of Korea. The grains were ground using blender and enriched for 7 days with distilled water under aerobic conditions. The grain samples were serially diluted with 0.85% (w/v) saline solution and spread on De Man-Rogosa-Sharpe (MRS; BD Difco, Sparks, MD, USA) agar. After incubation for 48 h at 30 ℃ or 37 ℃, LAB were isolated from the MRS agar plates and cultivated for 18 h at 30 ℃ or 37 °C in MRS agar. Lacticaseibacillus rhamnosus LGG (KCTC 5033), which was used as the experimental control strain for comparative analyses, was purchased from the Korean Collection for Type Cultures (Daejeon, Republic of Korea), and cultured at 37 ℃. All strains, including the experimental control strain, were stored at - 80 ℃ after suspension in 20% (w/v) glycerol solution (Georgiachem, GA, USA).
16S rRNA gene sequence analysis and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. Genomic DNA was extracted using a G-spin genomic extraction kit (iNtRON, Seongnam, Republic of Korea), according to the manufacturer’s protocol. Polymerase chain reaction (PCR) amplification, purification, and sequencing of 16S rRNA gene were performed as described previously20. Identification of the closest phylogenetic species based on 16S rRNA gene sequence was performed using the EzBioCloud server (https://www.ezbiocloud.net/)21.
Random amplified polymorphic DNA-PCR (RAPD-PCR), which was used to exclude replicates among LAB strains, was performed using two primers, ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) and ERIC1R (5’-ATGTAAGCTCCTGGGGATTCAC-3’) as described previously22. The PCR products were electrophoresed on 1.5% (w/v) agarose (LPS Solution, Daejeon, Republic of Korea) gel for 60 min, and after electrophoresis, the gel was stained with RedSafe (iNtRON, Seongnam, Republic of Korea).
Preparation of cell extract from LAB. Cell extracts of LAB were prepared as described previously23 with minor modifications. The cell mass was harvested using centrifugation and washed twice with phosphate-buffered saline (PBS, pH 7.2). The washed cells were resuspended in distilled water at a concentration of 100 mg/mL and sonicated using the method described previously16. The sonicated cell extracts were centrifuged at 13,000 rpm for 15 min at 4 °C and the supernatants were filtered using a 0.45 µM syringe filter (Sartorius Stedim Biotech GmbH, Göttingen, Germany) and lyophilized. The resulting powder was dissolved in sterile water to appropriate concentrations.
Cell culture, adipocyte differentiation and intracellular triglyceride content. C3H10T1/2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured following the method described previously24. A subculture of C3H10T1/2 cells was performed with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and antibiotics (penicillin and streptomycin, Hyclone). After seeding in 12-well plates, C3H10T1/2 cells were cultured in DMEM containing 10% FBS and antibiotics until confluency. Confluent cells were induced into adipocytes in DMEM supplemented with 10% FBS, antibiotics, 20 nM GW1929 (Sigma), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, MO, USA), 1 μM dexamethasone (Sigma-Aldrich), and 10 μg/mL insulin (Sigma-Aldrich). After 48 h, the differentiating cells were refreshed with media containing DMEM, 10% FBS, 20 nM GW1929, and 10 μg/mL insulin.
Cell extracts of LAB were adjusted by suspending to a concentration of 25,50, and100 μg/mL with sterile distilled water to create the same conditions. LAB cell extracts were treated during adipocyte differentiation of C3H10T1/2 cells, and sterile distilled water was treated as control. Then, the differentiated C3H10T1/2 cells were fixed with 4% formaldehyde (Sigma-Aldrich) in PBS (Hyclone) at room temperature overnight and stained with Oil Red O (Sigma-Aldrich). To quantify intracellular triglyceride content, stained cells from at least two independent experiments were resolved in isopropanol (Sigma-Aldrich) and measured with a spectrophotometer at 520 nm.
Cell viability assay. Cell viability was determined using methyl thiazolyl tetrazolium salt (MTS) colorimetric assays (ab197010, Abcam). C3H10T1/2 cells were seeded at 1.5 × 104 cells per well in 96-well plates, then treated with various strain doses (1, 12.5, 25, 50, 100, and 200 μg/mL) and sterile distilled water as control in triplicate. After 24 h, MTS (20 μL/100 μL in medium) was added into the media and cells incubated for 4 h at 37 ℃. The absorbance of formazan dye was measured at 490 nm using a microplate reader (BioTek, Winooski, VT, USA).
Quantitative real-time polymerase chain reaction (RT PCR) analysis. Total RNA was extracted from C3H10T1/2 cells using QIAzol lysis reagent (QIAGEN, Germantown, MD, USA). First-strand complementary DNA was synthesized from 0.5 μg of total RNA using ReverTra Ace Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. Quantitative RT PCR was performed in 25 μL final reaction volume containing Power SYBR Premix ExTaq (RP041A; Takara, Shiga, Japan), primers, and cDNA using thermal cycler machine (Takara). The primer sequences used for the PCR were described previously24.
Tolerance assays against acid and bile salts. Tolerance to acid was measured as described previously25 with minor modifications. LAB strains were cultured overnight (18 h) at 30 °C (for RP21 and K28) or 37 °C (for RP12 and LGG), harvested for 10 min at 7,000 rpm at 4 °C, and washed twice with PBS buffer (pH 7.2). Bacterial cells (approximately 109 CFU/mL) were resuspended in liquid MRS medium (pre-adjusted to pH 1.0, 2.0, 2.5, and 3.0) and incubated for 3 h at optimal temperatures. Viability was determined in triplicate in terms of viable colony counts using the plate count method. Tolerance to bile salts was measured as described previously26 with minor modifications. LAB strains were suspended in liquid MRS medium containing 0.3, 0.5, 1.0, and 2.0% oxgall (Sigma-Aldrich) at a concentration of approximately 109 CFU/mL. After incubation for 6 h at 30 °C or 37 °C, suspension was poured into MRS agar plates and incubated at optimum growth temperatures for 48 h. Tolerance assays against acid and bile salts were performed in triplicate and LGG was used as the comparative strain.
In vitro adhesion assays. The adherence assay was performed according to the method described previously27 with minor modifications. Caco-2 cells used for the adherence assay were purchased from the Korean Cell Line Bank (Seoul, Korea). The Caco-2 cells were cultured in high glucose DMEM supplemented with 10% (v/v) FBS (Hyclone) and 1% (v/v) penicillin-streptomycin at 37 °C in 5% CO2 atmosphere. The Caco-2 cells were seeded at 2 × 105 cells/well in 6-well tissue culture plates. The adherence assay was performed at post-confluence. The monolayer was washed with sterile PBS (Hyclone) twice. LAB cells were diluted with DMEM to approximately 109 CFU/mL and added to the wells. Plates were incubated for 90 min at 37 °C in 5% CO2 atmosphere. The Caco-2 monolayers were washed three times with sterile PBS (Hyclone) and treated with EDTA-trypsin solution for 3 min. The cell suspensions were serially diluted and spread on MRS agar plates. Cell viability was counted after incubation for 48 h. The adhesion ability of LAB was calculated as the percentage between remaining bacteria and initial bacteria per well. The same passage Caco-2 cells were used in adhesion assays and assays were repeated in triplicate.
Antibiotic susceptibility. Susceptibility to antibiotics was examined using the disc-diffusion method with application of modified agar diffusion method described previously28,29. LAB inoculated in MRS agar were adjusted to approximately 108 CFU/mL and paper discs (Advantec, Tokyo, Japan) were dispensed. Each disc was treated with 10 μL of specific antibiotic. The concentrations of antibiotics tested are listed in Supplementary Table 1. The inhibition zone diameters were measured and evaluated in terms of sensitive, intermediate sensitive, and resistant according to the interpretative standard table (Supplementary Table 1). The 2013 Clinical and Laboratory Standards Institute criteria30 were used for interpretation.
Enzyme activity test. Enzyme activity of the LAB was investigated as described previously16 using the API ZYM kit (BioMérieux, Marcy l’Etoile, France).
Statistical analysis. Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. Significance differences between groups in triglyceride content were determined using Duncan's multi-range test. Significance differences in gene expression and adhesion ability were determined by comparison with control using two-tailed unpaired Student’s t-test. A p-value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS Inc. software (version 19.0).