Bacterial strain, culture conditions, and preparation of cell-free supernatants
Lactobacillus plantarum IDCC 3501 (ATCC BAA-2838), isolated from kimchi (Korean fermented food), has been manufactured in Ildong Bioscience (Pyeongtaek, Korea) since 2015. L. plantarum IDCC 3501 and Lactobacillus rhamnosus GG were anaerobically cultured in De Man, Rogosa, and Sharpe (MRS; BD Difco, Franklin Lakes, NJ, USA) medium at 37°C in a static incubator. Staphylococcus aureus ATCC 25923 was used as a positive strain for hemolysis assay, and it was cultured in brain heart infusion (BHI; BD Difico) medium at 37°C with shaking at 200 rpm. Supernatants of 16 h-cultured L. plantarum IDCC 3501 and L. rhamnosus GG (positive control) were prepared by centrifugation at 8000 rpm and filtered using a 0.22 µm syringe filters (Merck Millipore, Burlington, MA, USA).
Cell culture
RAW 264.7 cells were purchased from Korean Cell Line Bank (Seoul, Korea) and were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, MA, USA), and supplemented with 10% (v/v) fetal bovine serum and 1% (w/v) penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2.
Macrophage viability assay
The viability of RAW 264.7 cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, the cells (1 × 105 cells·well− 1) were seeded in a 96-well plate for 24 h and treated with L. plantarum IDCC 3501 cell-free supernatants for 2 h. Then, MTT was added to each well and incubated for 4 h at 37°C. After removal of media and MTT, dimethyl sulfoxide (DMSO) was added to the well to dissolve the formazan. Finally, the developed color was measured at 540 nm using a microplate reader (BioTek, Winooski, VT, USA).
Measurement of nitric oxide production
Nitric oxide contents were determined by the Griess reaction (Promega, Madison, WI, USA). Briefly, 50 µL of cell supernatant was collected and mixed with 50 µL of Griess A (1% sulfanilamide) and 50 µL of Griess B (0.1% N-1-naphthylethylenediamine dihydrochloride). The mixture was then incubated at room temperature for 10 min, and nitric oxide contents were determined at 540 nm according to the calibration curve.
Quantitative reverse transcription-polymerase chain reaction (qRT- PCR)
Total RNA from RAW 264.7 cells treated with LPS and supernatant of either L. plantarum IDCC 3501 or L. rhamnosus GG, was isolated using TRIzol reagent (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription (Superscript IV First-Strand Synthesis System; ThermoFisher Scientific, MA, USA) was then performed to synthesize cDNA with 2 µg of total RNA and random hexamers. Finally, PCR was performed using the primers listed in Table S1, and the bands were analyzed and quantified using Image Lab (Bio-Rad, Hercules, CA, USA).
Gene search for antibiotic resistance and virulence
The complete genome sequence of L. plantarum IDCC 3501 was previously reported, consisting of a circular 3,242,587 bp chromosome with a GC content of 44.52% [14] (GenBank accession no. CP031702). The assembled sequence was compared with the reference sequences in the ResFinder database, using ResFinder v.3.2 (https://cge.cbs.dtu.dk/servies/ResFinder). The search parameters were sequence identity greater than 80% and a 60% coverage. Virulence genes were searched using the BLASTn algorithm with VFDB database [15]. The identification thresholds were identity greater than 70%, coverage greater than 70%, and E-value less than 1E-5.
Determination of MIC
The susceptibility of L. plantarum IDCC 3501 to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol was evaluated. A single colony was inoculated into MRS broth and cultured for 16–18 h. The cultured cells and antibiotic solution were mixed in a 96-well plate to obtain an initial cell density of 5 × 105 CFU mL− 1 and antibiotic concentration of 0.125–1,024 µg mL− 1. The plate was then anaerobically incubated at 37°C for 20 h. Finally, optical density was measured using a microplate reader (BioTek, Winooski, VT, USA), and MICs for each antibiotic were determined as the lowest concentration that completely inhibited cell growth.
Biogenic amine and lactate concentration determination
The supernatants from L. plantarum IDCC 3501 were collected by centrifugation at 6,000 rpm for 5 min at 4°C and were filtered using a 0.2-µm pore-size membrane. Then, 1 mL of supernatant was mixed with 200 µL of saturated NaHCO3, 20 µL of 2 M NaOH, and 0.5 mL of dansyl chloride (10 mg mL− 1 acetone), and the mixture was incubated at 70°C for 10 min for derivatization. The derivatized sample was added with 200 µL of proline (100 mg mL− 1 H2O) and incubated in a dark room for 15 min. The sample was made up to 5 mL with acetonitrile and filtered with a 0.45 µm membrane. Finally, biogenic amines were analyzed using high-performance liquid chromatography (HPLC; LC-NETI/ADC, Jasco, Macclesfield, UK) equipped with an Athena C18 column (4.6 mm × 250 mm, ANPEL Laboratory analysis, Shanghai, China). Aqueous acetonitrile solution (67:33 of H2O) was used as the mobile phase at 0.8 mL min− 1. Peaks were detected at 254 nm using a UV detector (UV-2075 Plus, Jasco, Macclesfield, UK) and quantified according to the calibration curves of each biogenic amine.
The quantities of L- and D-lactate in L. plantarum IDCC 3501 supernatant were measured using an assay kit (Megazyme, Bray, Ireland) according to the manufacturer’s protocol.
Hemolytic and extracellular enzyme activities
L. plantarum IDCC 3501 and Staphylococcus aureus ATCC 25923 were streaked on sheep blood agar plates (BBL Microbiology Systems, Cockeysville, MD) and incubated at 37°C overnight. Then, β-hemolytic activity was determined by observing clear zones around colonies.
Enzyme activities of L. plantarum IDCC 3501 were assessed using the API-ZYM kit (BIOMÉRIUX, Marcy-l'Étoile, France), which can evaluate 19 hydrolytic enzyme activities according to the manufacturer’s protocol.
Acute oral toxicity test
Acute oral toxicity test was performed by Korea Testing and Research Institute (Hwasun-gun, Jeollanam-do, Korea) according to the OECD guidelines (Test No. 425) [16]. Briefly, 12 Crl:CD(SD) female rats aged 9–10 weeks were divided into 4 groups of 3 rats each. Each group's rats were orally administered at 300 mg kg− 1 or 2000 mg kg− 1, corresponding to 3.4 × 1011–3.6 × 1011 CFU and 2.3 × 1012–3.4 × 1012 CFU, respectively. Then, toxicity, morality, and body changes were observed for 14 d. Finally, 100-mL isoflurane was treated to euthanize the rats, and an autopsy for the examination of organs was performed.