Anti-inammatory Effect of Lactobacillus Plantarum IDCC 3501 and Its Safety Evaluation

This study investigated the anti-inammatory activity of L. plantarum IDCC 3501 isolated from kimchi (Korean fermented food) and its safety. When lipopolysaccharide (LPS)-induced RAW 264.7 macrophages were treated with cell-free supernatant from L. plantarum IDCC 3501, the mRNA expression level of inammatory markers (i.e., TNF-α, IL-1β, and IL-6) was signicantly reduced. The decreased cell viability by LPS was recovered and NO production in LPS-induced cell was also decreased. The genes responsible for antibiotic resistance and virulence were not detected from the genome analysis of this strain. Consistent with this, minimal inhibitory concentrations against various antibiotics, biogenic amines and D-lactate production, as well as enzymatic and hemolysis activities indicated that L. plantarum IDCC 3501 did not produce any harmful compounds during fermentation. Also, no acute toxicity and mortality were observed in a murine mouse model when feeding with L. plantarum IDCC 3501. Based on our ndings, L. plantarum IDCC 3501 is safe and benecial for human consumption. per kg body weight. Also, there were no signicant behavior changes, skin effects, impairment in feed intake, and body weight. Thus, these results show that is safe, both in vitro and in vivo.


Introduction
Lactobacillus plantarum is a major widespread species among the genus, Lactobacillus, due to its ecological niches, existing in dairy products, fermented foods, and host's mouth and intestinal tract [1]. As a facultatively heterofermentative species, L. plantarum has various bene cial health effects, such as antimicrobial activity [2], antiobesity [3], immune-boosting [4], and anti-in ammatory effects [5]. For this reason, L. plantarum has been used in various industrial food fermenters as a starter. As a probiotic, this species typically possess acid/bile tolerance and intestinal adhesion activity [6][7], and alleviating acute and chronic in ammation.
In ammation is an immunity-mediated response to viral or pathogenic infection, toxic compounds, or irradiation [8]. This response is typically regulated by proin ammatory mediators (e.g., iNOS and COX-2) and cytokines (e.g., TNF-α, IL-1β, and IL-6) [9], resulting in the recruitment of immune cells (e.g., macrophages) and systemic responses. Mainly, gut in ammation often causes acute symptoms, such as diarrhea, gastric bleeding, and abdominal pain and chronic symptoms, such as in ammatory bowel disease and colorectal cancer [10]. Therefore, the anti-in ammatory effect of probiotics has been intensively studied in the last decades to improve gut health. For example, L. plantarum has signi cant positive effects on Crohn's disease and ulcerative colitis by modulating the intestinal microbiota, suppressing pathogens, and boosting the immune system [11][12].
Previously, L. plantarum IDCC 3501 was reported to have the following characteristics as probiotics: coaggregation with pathogens, 25.0-66.1%; hydrophobicity, 39.2%; acid tolerance, and 84.9%; antibacterial effects against nine pathogens including E. coli O157:H7 and Salmonella Typhimurium [13]. However, the safety of L. plantarum IDCC 3501 needs to be carefully examined for human consumption on the strain basis.
In this study, the anti-in ammatory effects of L. plantarum IDCC 3501 and its safety were investigated. Here, cell viability, nitric oxide (NO) production, and the expression of in ammatory markers were investigated in LPS-stimulated RAW 264.7 macrophages. In vitro and in vivo studies, including hemolysis, minimal inhibitory concentration (MICs) tests, whole-genome analysis, and single-dose acute oral toxicity test in rats, were performed.

Material And Methods
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 Di co) 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 ltered using a 0.22 µm syringe lters (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 modi ed Eagle's medium (Thermo Fisher Scienti c, MA, USA), and supplemented with 10% (v/v) fetal bovine serum and 1% (w/v) penicillin/streptomycin at 37°C in a humidi ed atmosphere containing 5% CO 2 .
Brie y, the cells (1 × 10 5 cells·well − 1 ) were seeded in a 96-well plate for 24 h and treated with L. plantarum IDCC 3501 cellfree 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). Brie y, 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-1naphthylethylenediamine 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 Scienti c, Waltham, MA, USA). Reverse transcription (Superscript IV First-Strand Synthesis System; ThermoFisher Scienti c, 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 quanti ed 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 identi cation 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 × 10 5 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 ltered using a 0.2-µm pore-size membrane. Then, 1 mL of supernatant was mixed with 200 µL of saturated NaHCO 3 , 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

Results And Discussion
Effects of L. plantarum IDCC 3501 on cell viability The cytotoxicity of the supernatants of L. plantarum IDCC 3501 on RAW 264.7 cells, which in ammation was induced by 1 µg per mL of lipopolysaccharide (LPS), was determined using MTT assay (Fig. 1A). The supernatants of L. rhamnosus GG were also applied as a control. After treatment using the supernatants of L. plantarum IDCC 3501, the decreased cell viability by LPS was recovered, showing no negative effect on cell viability. Like cell viability results, LPS addition showed a remarkable increase in NO production, which shows early stimulation exerted on activated macrophages (Fig. 1B). However, the supernatants of L. plantarum IDCC 3501 considerably inhibited NO production in a level of more than 60%. Thus, both cell viability and NO production results suggested that L. plantarum IDCC 3501 supernatants positively affected the relief of oxidative stress in cells.
The effect of L. plantarum IDCC 3501 on pro-in ammatory cytokines Activated macrophages which were regulated by LPS-induced pro-in ammatory cytokine mediators could release various cytokines to enhance immune defense mechanisms, such as TNF-α, IL-1β, and IL-6 in RAW 264.7 cells. To demonstrate that L. plantarum IDCC 3501 inhibits LPS-induced nitrite and PEG 2 production, iNOS and COX-2 expression were investigated using semi-RT-PCR ( Fig. 2A and B). The expression levels of both iNOS and COX-2 were signi cantly increased in LPS-induced cells. L. plantarum IDCC 3501 decreased iNOS and COX-2 expression by 26.5% and 55.5% than the values of LPSinduced cells, respectively. Although, TNF-α, IL-1β, and IL-6 were inhibited by 38.2%, 37.9%, and 73.8%, respectively (Fig. 2C-E), IL-6 was the mostly affected among these cytokines.
There are many reports about whole cells (e.g., alive or heat-killed cells) or cell wall components of lactic acid bacteria.
Recently, much attention has been paid to cell-free supernatants containing biologically active metabolites which are secreted by live bacteria [17]. Previous studies have shown anti-in ammatory response on intestinal epithelial cells and macrophages by reducing proin ammatory mediators or cytokines [18]. Among them, the deregulation of IL-6 has been implicated in the pathogenesis of many diseases, especially Crohn's disease and colon cancer [19]. According to our ndings, the supernatants of L. plantarum IDCC 3501 exert anti-in ammatory effects because it leads to iNOS, COX-2, TNF-α, IL-1β, and IL-6 inhibition under viable conditions (Fig. 2). Thus, cell-free bacterial supernatants are promising antiin ammatory agents for treating gut in ammation. The administration of our strains to infected mice can clarify the effects for further study.
Whole-genome analysis and antibiotic susceptibility of L. plantarum IDCC 3501 In lactic acid bacteria, antibiotic resistance and virulence factors are easily acquired from an exogenous source. Gene transfer commonly involves bacterial conjugative plasmids, transposable elements, and integron systems [20][21]. Thus, whole-genome analysis is becoming an essential procedure for safety assessment in the probiotic industry. Notably, L. plantarum IDCC 3501 has no antibiotic resistance genes and virulence factors [15]. Meanwhile, L. plantarum IDCC 3501 showed susceptibility to all antibiotics, except kanamycin (Table 1). Lactobacillus strains are susceptible to antibiotics related to protein synthesis (e.g., chloramphenicol, erythromycin, clindamycin, and tetracycline) but are aminoglycosidesresistant (neomycin, kanamycin, streptomycin, and gentamicin) [22]. For example, approximately 79% of isolated probiotic strains showed kanamycin resistance [23]. Thus, L. Plantarum IDCC 3501 resistance to kanamycin might be considered aminoglycoside intrinsic because of membrane impermeability by potential e ux mechanisms [24]. According to hemolytic activity results identi ed as a virulence factor, L. plantarum IDCC 3501 showed no hemolysis zones on the sheep blood agar plates, unlike Staphylococcus aureus ATCC 25923 which was used as a positive control showing a distinct hemolytic zone (Fig. S1). In conclusion, L. plantarum IDCC 3501 was safe regarding antibiotic resistance and virulence factors.
In vitro safety evaluation of L. plantarum IDCC 3501 In this study, observations of biogenic amines (BAs), D-lactate, and potential toxin-producing enzymes in L. plantarum IDCC 3501 were evaluated. BAs are low molecular weight compounds derived by the decarboxylation of amino acids. BAs are present in various foods and beverages, such as meat, sh, cheese, and vegetables [25]. However, BAs are precursors for forming N-nitroso compounds known as cancer-causing agents [26]. Also, high BA consumption may cause symptoms, such as headache, heart palpitations, vomiting, diarrhea, and hypertensive crises in humans and animals [27]. In this study, BAs' production including tyramine, histamine, putrescine, 2-phenethyamine, and cadaverine, was not observed during L. plantarum IDCC 3501 fermentation (data not shown).
Lactate, a key molecule produced during the fermentation of lactic acid bacteria, exists in two forms, D-lactate and L-lactate. In humans, more than 99% of lactate found in the blood is L-lactate. Even though D-lactate appears in human tissue, it possesses D-2-hydroxy acid dehydrogenase that converts D-lactate to pyruvate, resulting in a decrease in acidosis risk [28].
In this study, L. plantarum IDCC 3501 produced 20.1 g L − 1 of L-lactate (99.85%) and 0.03 g L − 1 of D-lactate (0.15%) ( Table 2). D-lactate production in L. plantarum IDCC 3501 was much lower than in other lactic acid bacteria. Next, an essential criterion in safety assessment is the absence of harmful enzymatic activity. For example, α-chymotrypsin has been associated with virulence and local tissue injury during infective endocarditis [29]. β-glucuronidase can cleave glucuronic acid-conjugated carcinogens related to toxic compounds [30]. Also, β-glucosidase may have damaging effects on the colon [31]. In this study, L. plantarum IDCC 3501 had no α-chymotrypsin and β-glucuronidase (Table 3 and Fig. S2). Although a low level of β-glucosidase activity was shown, it was relatively low compared with other lactic acid bacteria. In contrast, βgalactosidase expressed in probiotics has contributed to the relief of lactose maldigestion symptoms [32]. Also, aminopeptidases, such as leucine arylamidase, valine arylamidase, and cystine arylamidase, are involved in cheese ripening by supplying free amino acids that further affect metabolism of cheese avor [33]. Conclusively, L. plantarum IDCC 3501 retained these bene cial enzymes (Table 3 and Fig. S2).  In vivo safety evaluation of L. plantarum IDCC 3501 by oral administration A 14-d oral acute toxicity study in female rats aged 9-10 weeks was performed to investigate the oral consumption safety of L. plantarum IDCC 3501 (Table 4). L. plantarum IDCC 3501 did not cause any toxic symptoms or mortality, and the rats lived up to 14 d after administering 300 mg or 2000 mg single dose per kg body weight. Also, there were no signi cant behavior changes, skin effects, impairment in feed intake, and body weight. Thus, these results show that L. plantarum IDCC 3501 is safe, both in vitro and in vivo. Jung conceived of the study and participated in its design and coordination, and J Yang managed this project and helped to nalize the manuscript. All authors read and approved the nal manuscript.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.