Introduction

Lactic acid bacteria (LAB) have been used as probiotics and defined as a healthy diet as well as a way to provide health benefits such as cholesterol-lowering and immunomodulation effects. A reduction of 1% in serum cholesterol may alleviate the risk of coronary artery disease by 2 to 3% [1]. In addition, LAB immunomodulation is still of interest since it boosts immunity and is used to prevent and treat some immune diseases. Interleukin-12 (IL-12) is a pro-inflammatory cytokine involved in preventing infection and cancer [2]; IFN-γ plays a role in host defense against intracellular pathogens. Moreover, human beta defensin-2 (hBD2), human antimicrobial peptides, serve vital functions in the prevention of infection [3]. Nitric oxide (NO) also plays an important role in the prevention of infection and tumor cell proliferation [4]. There have been several studies of Lactobacillus and other LAB-inducing IL-12, IFN-γ, hBD2, and NO production [3, 5,6,7,8].

According to FAO/WHO [9], the probiotics must be isolated from safe sources, accurately identified, and characterized at the species level. Conventional methods and whole-genome sequencing (WGS) technology are required to accurately identify Lactobacillus species. Furthermore, WGS also plays an attractive and important role in assessing of pathogenicity and virulence, antibiotic-resistance genes, gastrointestinal tolerance genes, and health-promoting genes [10, 11].

In the investigation of LAB in Thai traditional fermented mussel (Hoi-dong), a darkish orange semi-solid with a sour (pH 3.1–6.1) and salty taste that was made of Perna viridis (Hoi-ma-laeng-poo) [12, 13], a strain LM14-2 was isolated and might be an attractive source for evaluating the possibility of discovering new potential probiotic isolates. Furthermore, no investigations have yet been published on the probiogenomic, cholesterol-lowering, and immunomodulation activities. Hence, this study aims to evaluate cholesterol-lowering and immunomodulation effects, probiotic properties, and the reliability of the omics approach for probiogenomic and safety assessment of Lactiplantibacillus sp. LM14-2.

Materials and Methods

Characterization of Strain

Strain LM14-2 was isolated from Hoi-dong (traditionally fermented mussel), which was collected from Chonburi province in Thailand. It was characterized based on the phenotypic and chemotaxonomic characteristics as previously described by E. Kingkaew et al. [14]. The genomic DNA was extracted using the Wizard Genomic DNA Purification kit (Promega Corporation, USA) according to the manufacturer’s protocol with some modifications. The modifications included the addition of 25 U/mL mutanolysin (Sigma-Aldrich, USA) in the 1 mg/mL lysozyme resuspending solution, incubation at 37 ℃ overnight for complete cell lysis, and additional centrifugation at 10,000 g for 3 min in the protein precipitation step. The DNA pellet was dissolved in DNase/RNase-free water. The concentration and quality of the DNA were measured using a NanoDrop ND-1000 spectrophotometer. At Omics Science and Bioinformatics Center, Chulalongkorn University, library preparation and genome sequencing of the strain LM14-2 were performed by Nextera XT DNA prep kit and Illumina Miseq sequencer. The quality of raw reads was checked using FASTQC software (Galaxy Version 1.1.5). Adaptors and poor-quality reads were removed using Trim Galore (Galaxy Version 0.6.3); filtered reads were used as an input for Unicycler, genome assembly program (Galaxy Version 0.4.8.0). The genomic quality and contamination were evaluated by CheckM [15]. The sequence similarity values between the strain LM14-2 and their related reference strains were computed using the EzBiocloud tool [16]. Then, the average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values were analyzed using the JSpeciesWS web server tool [17, 18] and the Genome-to-Genome Distance Calculator (GGDC 2.1) using the BLAST+ method with formular 2 [19]. The ANI of >95% and DDH of >70% are considered to be the same species [20]. A phylogenetic tree based on a whole-genome sequence was constructed by using the TYGS web server (https://tygs.dsmz.de/) [21]. Furthermore, a genomic circular map was constructed using CGView Server [22].

Gene Prediction and Functional Annotation

The draft genome was annotated by using the DFAST sever [23], Rapid Annotation Server Technology (RAST) [24], PATRIC [25], and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [26]. Plasmid was detected by PlasmidFinder [27]. The PATRIC, CheckM, RAST, DFAST, NCBI, and PlasmidFinder tools were used for the determination of genomic features (Table 1). Antibiotic resistance genes were determined using the Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca) [28] and ResFinder web-based tool [29]. The pathogenicity was predicted by PathogenFinder web-based tool [30], and the PHAge Search Tool Enhanced Release (PHASTER) was used to identify and annotate putative prophage sequences [31]. Identification of carbohydrate-active enzymes was performed using the dbCAN meta server (https://bcb.unl.edu/dbCAN2/blast.php) with HMMER: biosequence analysis with profile hidden Markov models (version: 3.3.2), and all data generated in dbCAN were based on the family classification from the CAZy database (http://www.cazy.org/) [32, 33]. The biosynthesis of antimicrobial peptides gene clusters was detected and visualized by BAGEL4 (BActeriocin GEnome mining tool; http://bagel4.molgenrug.nl) [34]. Genes responsible for virulence and undesirable characteristics may be identified using public databases and manually inspected to confirm its identity and function. Precautions are required in the interpretation of the findings since genes involved in survival and adaptation should not be treated as virulence genes for non-pathogenic bacteria. The search uses the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.kegg.jp) [35] for the pathways and genes.

Table 1 Genomic features of Lactiplantibacillus plantarum LM14-2, L. plantarum 299V, and Lacticaseibacillus rhamnosus GG (ATCC 53103)
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In Vitro Probiotic Assays

Preparation of LAB Cell Suspension

The cell suspension was prepared for studying probiotic characteristics. The strain LM14-2 was cultured twice in MRS broth for 24 h at 30 ℃. Then, the cells were harvested by centrifugation at 14,000 rpm for 10 min at 4 ℃, washed twice with phosphate buffer (0.1 M, pH 7.2, containing 0.85% (w/v) NaCl), and resuspended in phosphate buffer (0.1 M, pH 7) to get a cell suspension with A600 = 1 and 109 CFU/mL.

Bile Salt Hydrolases Activity

The BSH activity was evaluated as described by Shehata et al. [36]. On MRS agar supplemented with 0.037% (w/v) calcium chloride (CaCl2) and 0.5% (w/v) taurodeoxycholic acid sodium salt (TDCA), 20 µl of cell suspension was spotted. Plates were incubated anaerobically for 72 h at 37 ℃. The BSH activity was indicated by the formation of halos around colonies or white opaque colonies. The non-modified MRS served as the control.

Cholesterol Assimilation

The cholesterol assimilation ability of strain LM14-2 was determined in MRS broth supplemented with cholesterol-polyethylene glycol (PEG) 600 (Sigma, India) at a final concentration of 100 µg/mL; 1% of cell suspension was inoculated into MRS-cholesterol-PEG 600 and incubated anaerobically for 24 h at 37 ℃. The cholesterol in MRS broth was extracted using the method described in [37]. The quantity of residual cholesterol was measured using the modified technique of L. L. Rudel and M. Morris [38]. The following cholesterol concentrations were used to construct a standard absorbance curve with cholesterol concentrations: 0 μg/mL, 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL, and 125 μg/mL in MRS. The cholesterol concentration was compared to the standard curve constructed using the cholesterol stock solution. The assimilate cholesterol ability was presented as the cholesterol assimilated (%) at each incubation as follows:

$$\begin{array}{l}\mathrm{Cholesterol\;assimilated\;(\mu g/ml)}=\mathrm{{[Cholesterol\;(\mu g/ml)]}}_\mathrm{0h}-\mathrm{[Cholesterol\;(\mu g/ml)]}_\mathrm{24h}\\ \mathrm{\%\;Cholesterol\;assimilated}=\left[\frac{\mathrm{Cholesterol\;assimilated(\mu g/ml)}}{\mathrm{Cholesterol\;{(\mu g/ml)}}_\mathrm{0h}}\right]\times 100\end{array}$$

Gastrointestinal Transit Tolerance

An in vitro assay that simulated the gastrointestinal fluids and conditions was prepared and conducted following the procedure of M. Minekus et al. [39]. Briefly, the cell suspension was mixed with simulated gastric fluid (SGF) containing pepsin 2,000 U/mL and incubated anaerobically for 3 h at pH 3 and 37 ℃. The samples were collected at 0 h (initial time) and 3 h (gastric-emptying time) of incubation time for viably bacterial enumeration. After the incubated time of the gastric phase, the gastric chyme was transferred, mixed with simulated intestinal fluid (SIF) containing pancreatin (based on trypsin activity at 100 U/mL) and 10 mM of bile, and incubated anaerobically for 5 h at pH 7 and 37 ℃. The samples were collected at 0 h (initial time) and 5 h (small intestinal-emptying time) of incubation time for viably bacterial enumeration. The number of viable LAB was quantified using a serial 10-fold dilution and spot plate technique. The Lacticaseibacillus rhamnosus GG was used as control. The viable bacteria were reported as logarithms of colony-forming units per milliliter (log CFU/mL).

Adhesion Ability

The strain LM14-2 was selected to investigate the adhesion ability, with slight modifications procedure of Q. Han et al. [40]. Caco-2 cells were routinely grown at 37 ℃ in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS) in a humidified condition of 95% air and 5% CO2. Caco-2 cells were seeded into 24-well tissue culture plates at a concentration of 5×105 cells/mL for the adhesion test. The tissue plates were incubated at 37 ℃ in 5% CO2 until the Caco-2 cells developed a confluent-differentiated monolayer. Caco-2 cells were washed twice in PBS, and the bacterial cells were collected by centrifugation at 14,000 rpm for 10 min at 4 ℃, and bacterial cells were resuspended in DMEM without antibiotics. The bacterial cell suspension was inoculated into a well and incubated for 90 min at 37 ℃ in a 5% CO2. After the incubation period, Caco-2 cells were washed three times with PBS to eliminate unbounded bacterial cells. Then, the Caco-2 cells were lysed with 0.05% Triton-X100 solution. The number of adherent bacteria was enumerated by the spot plate technique on the MRS agar and then incubated at 37 ℃ on MRS agar for 48 h. The L. rhamnosus GG was used as a control. The adherent ability was calculated according to the following equation:

$$\mathrm{Adhesion\; percentage}\ (\%)= \frac{\mathrm{N_t}}{\mathrm{N_0}} \times 100$$

where Nt is the number of adherent LAB cells to the Caco-2 cells, and N0 is the total number of LAB cells inoculated.

The Immunomodulation Effects

The immunomodulation effect of strain LM14-2 was determined by the procedure of Y. Hosaka et al. [41].

Preparation of Sterilized Lactic Acid Bacteria Powder

The strain LM14-2 was cultivated in MRS broth and incubated at 30 ℃ for 24 h, according to the procedure of Y. Hosaka et al. [41]. The cultures were heated at 100 ℃ for 20 min, and the heat-killed cells were collected by centrifugation at 1,000 rpm for 10 min. After that, the heat-killed cells were washed with sterile distilled water and the heat-killed cells were collected by centrifugation at 1,000 rpm for 10 min. Next, the heat-killed cells were lyophilized to prepare powder. The heat-killed cell powders were suspended in PBS to 200 µg/mL to prepare the test sample.

Cell Culture and Cell Differentiation

RAW264.7 cells were cultured in DMEM supplemented with 5% FBS and 0.25% PS at 37 ℃ in 5% CO2. Professor Shinichi Yokota of Sapporo Medical University School of Medicine provided the Caco-2 cells. Caco-2 cells were grown in DMEM supplemented with 5% FBS and 0.25% PS in a 5% CO2 at 37 ℃. THP-1 cells were grown in RPMI 1640 media supplemented with 10% FBS and 0.20% PS in a 5% CO2 incubator at 37 ℃.

Caco-2 cells (1.5×105 cells) were cultivated for 72 h in cell culture inserts (Falcon, 24-Well Hanging Inserts 0.4 m). After 72 h, the solution containing 5 mM sodium butyrate was replaced, and the cells were cultured for 96 h to promote differentiation. Differentiated cells were evaluated by transepithelial electrical resistance (TEER) using Millicell-ERS (Merk), and differentiated cells (TEER values >400 Ωxcm2) were used. THP-1 cells were seeded on a multi-well plate (24 well, Falcon) and incubated for 72 h in media supplementing 100 ng/mL cholecalciferol (vitamin D3) and 10 nM phorbol12-myristate13-acetate (PMA) to differentiate into macrophage-like cells. Following differentiation, Caco-2 and THP-1 cells were co-cultured in Transwell.

Measurement of NO Production

The nitric oxide (NO) production was measured as described by Y. Yang et al. [42]. RAW264.7 cells were suspended in DMEM medium (5% FBS + 0.2% PS) at a concentration of 3×105 cells/mL, seeded in each 24-well multi-well plate, and incubated for 24 h at 37 ℃ in a 5% CO2. The sample was examined at a final concentration of 20 µg/mL to stimulate the cells. PBS was used as a negative control, while LPS (10 µg/mL) (Fujifilm Wako) was used as a positive control. After 24 h of stimulation, the supernatant was collected and centrifuged at 12,000 rpm for 20 min and determined by Griess reaction as reported by K.-S. Baek et al. [43]; 100 µl of each Griess reagent, medium supernatant sample, and 1.56 to 100 µM of sodium nitrite standard solution were added to 96-well microplates and incubated for 20 min at room temperature. The absorbance at 550 nm was measured in a microplate reader. The amount of nitrite in the medium supernatant was enumerated using a calibration curve generated from the sodium nitrite standard solution.

Intestinal Immunity Model

An in vitro intestinal immune model was simulated by co-culture of cell culture inserts (apical side) and multi-well plates (basal side). The samples were suspended in RPMI 1640 medium that was added to the apical side (final concentration 20 µg/mL), and the cells were stimulated in a 5% CO2 incubator at 37 ℃ for 2 days. After incubation, the basal side of the medium was collected and centrifuged at 12,000 rpm for 20 min. For determination of IL-12 and IFN-γ concentration, proteins were precipitated by adding a 25% volume of 100% trichloroacetic acid (TCA) to the culture medium supernatant sample. The precipitated proteins were washed with acetone to eliminate TCA and dissolved in 1X sample buffer for protein enrichment after heat treatment at 100 ℃ for 2 min. SDS-PAGE was used to separate the proteins performed according to U. K. Laemmli [44], and the target proteins were detected by Western blot according to the method reported by H. Towbin et al. [45]. Calibration curves were prepared with IL-12 and IFN-γ standard to calculate the production of IL-12 and IFN-γ. The IL-12 and IFN-γ concentration were corrected by measuring β-actin as an endogenous control. The PVDF membrane was reacted with an IL-12p35 antibody [46] (diluted 500 times) with an anti-rabbit AP IgG (BioRad, diluted 10,000 times) or an anti-rabbit HRP IgG (Biorad, diluted 15,000 times). Samples were treated with a BCIP-NBT solution (Nacalai Tesque) or an ECL Plus Western Blotting Detection System (Cytiva). The immunoblotting bands obtained were analyzed by ImageJ. For hBD2, unenriched medium supernatant was measured by the Dot blot, and the amount of produced hBD2 was corrected from the amount of total protein by CBB staining. The values were evaluated relative to the no stimulation test section with PBS.

Statistical Analysis

All experiments were done in triplicates, and the results were described as the mean ± standard deviation. All data were analyzed by independent t-test using SPSS 22.0 software. For the comparison between initial time and gastric/small intestinal-emptying time, paired t-test was used for analysis. The significant differences were accepted at P-value ≤0.05

Results

Characterization of Strain

Strain LM14-2 was Gram-stain-positive and facultatively anaerobic rods. Growth occurred at 15–45 ℃, pH 3.0–8.0, and in the presence of 1–8% (w/v) NaCl. It did not produce gas from glucose. LM14-2 produced DL-lactic acid from glucose. Negative for catalase, arginine hydrolysis, and nitrate reduction. Strain LM14-2 contained meso-DAP in the cell wall peptidoglycan. In API 50 CH tests, acid formation is described in Supplementary Table 1. Based on the full 16S rRNA gene sequence (1,567 bp), strain LM14-2 was closely related to L. plantarum ATCC 14917T, L. argentoratensis DSM 16365T, L. pentosus DSM 20314T, and L. paraplantarum DSM 10667T with 100%, 100%, 99.93%, and 99.73% similarity, respectively.

In phylogenomic tree analysis (Supplementary Fig. 1), strain LM14-2 was formed in the same cluster within several L. plantarum strains. Strain LM14-2 was closely related to L. plantarum DSM 20174T or ATCC 14917T with a digital DNA–DNA hybridization (dDDH) value of 93.7%, which was the highest dDDH value observed with closely related species (Supplementary Table 2). The values of ANIb and ANIm between strain LM14-2 and L. plantarum DSM 20174T or ATCC 14917T were 98.2% and 99.27%, respectively (Supplementary Table 2). The strain LM14-2 was identified as L. plantarum. The genomic features of L. plantarum LM14-2 (accession no. JALPQH000000000), L. plantarum 299V (accession no. LEAV00000000), and L. rhamnosus GG (accession no. FM179322) (Table 1) lead to a circular genome map construction (Fig. 1). The draft genome sequence of strain LM14-2 was 3,311,812 bp, with a genomic DNA G + C content of 44.3%, N50 of 331,723, L50 of 4, and genome coverage of 250×. CheckM showed 99.35% genome completeness. The Prokaryotic Genome Annotation Pipeline annotation identified 3,115 genes (total), 2,999 are genes (coding), 41 pseudo genes (total), 76 genes (RNA), 70 tRNAs, and 4 ncRNAs; while the Rapid Annotation of microbial genomes using Subsystems Technology (RAST) showed 3,254 (no. of CDSs) and 67 RNA genes. Furthermore, DDBJ Fast Annotation and Submission Tool (DFAST) did not detect CRISPRS in strain LM14-2, and the subsystem is shown in Supplementary Fig. 2.

Fig. 1
figure 1

Circular genomic map of L. plantarum LM14-2. The information is indicated as follows: open reading frames (ORFs) (blue), GC skew (+) (green), GC skew (−) (yellow), and GC content (pink)

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Safety Assessment

These genomic findings demonstrate the probiotic potential of strain LM14-2. The L. plantarum LM14-2 was predicted as a non-human pathogen by PathogenFinder web-based tool (Table 2). From the DFAST annotation database, the virulence-associated genes were detected, such as exopolysaccharides biosynthesis protein (cps2B and cps4B) and capsular polysaccharide biosynthesis protein (cpsE). Although these genes were identified as virulence elements, these genes play a key role in stress defense. In addition, the hemolysin III gene (hlyIII) was found in the genome of the strain LM14-2. The hemolysis test using sheep-blood agar demonstrated a hazy zone of hemolysis around the growth of bacteria, similar to the zone surrounding the probiotic L. rhamnosus GG, indicating that the two strains had comparable hemolysis activity. From the Comprehensive Antibiotic Resistance Database (CARD), PlasmidFinder and PHASTER web-based tools, the strain LM14-2 did not contain AR genes and plasmid. Three prophage regions were found (see Supplementary information (PHASTER.xlsx) for details).

Table 2 Pathogenicity prediction, prophage detection, and antibiotic resistance genes (ARGs) analysis from PathogenFinder of CGE and PHASTER (default program settings applied)
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Carbohydrate-active Enzyme Analysis

The genome of strain LM14-2 comprised 101 carbohydrate-active enzyme genes (Supplementary information, Cazy_LM14-2.xlsx), including 39 glycosyltransferase (GT) genes, 51 glycoside hydrolase (GH) genes, five carbohydrates esterase (CE) genes, three carbohydrate-binding molecules (CBMs), and three auxiliary activities (AA).

Identification of Genes Associated Stress Response, Microbe-Host Interactions, and Bacteriocin Biosynthesis

The genomic data of L. plantarum LM14-2 discovered subsystems and genes coding for stress response, adhesion, nutritional synthesis, cholesterol-lowering effect, lactate synthesis, transcriptional regulators, and metabolic rearrangement, all of which contribute to probiotic characteristics, survival in acidic and bile salt conditions, and prevention of pathogen colonization in a gut environment (Table 3). Furthermore, this study discovered that strain LM14-2 possesses six genes that are crucial for the production of the class IIb bacteriocin: plantaricin NC8-α, F, E, J, NC8-β, and K with 98.60 bit-score, 107.07 bit-score, 112.46 bit-score, 112.46 bit-score, 112.85 bit-score, 114.01 bit-score, respectively (Fig. 2); however, this did not contain the other genes for bacteriocin production, including plnA, plnB, plnC, plnL, plnN, plnO, plnQ, plnR, plnT, plnU, plnV, plnW, and plnX genes, which are also essential for bacteriocin production. In addition, the no antimicrobial activity of this strain was confirmed by in vitro study. The plantaricin genes of strain LM14-2 and reference strains are shown in Table 4.

Table 3 Potential genes associated with various probiotic characteristics from L. plantarum LM14-2 genome
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Fig. 2
figure 2

Genetic organization of pln locus of L. plantarum LM14-2; L. plantarum DHCU70; L. plantarum DKP1; L. plantarum NC8; and L. plantarum WCFS1; Indicators: α – NC8α, β – NC8β, J – plnJ, K – plnK, E – plnE, F – plnF; red color indicated immunity protein. Data generated from the BAGEL4 tool

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Table 4 Comparison of pln genes of L. plantarum LM14-2, DHCU70, DKP1, NC8, and WCFS1
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Cholesterol-lowering Effects

Bile Salt Hydrolase Activity

L. plantarum LM14-2 exhibited bile salt hydrolase activity by the formation of white opaque colonies and contained the choloylglycine hydrolase (also known as bile salt hydrolase; bsh) gene. This study demonstrated that BSH-producing LAB could be found from non-human origin.

Cholesterol Assimilation

In this study, strain LM14-2 showed a great cholesterol assimilation ability at 86.07 ± 5.03% with no statistically significant difference compared to L. rhamnosus GG (81.40 ± 4.00%) (Table 5).

Table 5 Cholesterol-lowering effects, gastrointestinal tolerance and its adhesion ability of L. plantarum LM14-2 and L. rhamnosus GG
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The Gastrointestinal Transit Tolerance

The strain LM14-2 was incubated in simulated gastric condition supplemented with pepsin (2,000 U/mL) at pH 3 for 3 h, and the viability was reduced from 9.44 ± 0.03 to 7.93 ± 0.08 log CFU, while the viability of L. rhamnosus GG was reduced from 9.38 ± 0.04 to 7.78 ± 0.18 log CFU (Table 5). After that, strain LM14-2 was transited and incubated in an intestinal condition supplemented with pancreatin (based on trypsin activity 100 U/mL) at pH 7 for 5 h. The viability of strain LM14-2 showed a log reduction of 1.79 compared with the log CFU in intestinal condition at 0 h. While L. rhamnosus GG showed a log reduction of 1.85 compared with the log CFU in intestinal condition at 0 h.

The Adhesion Ability

L. plantarum LM14-2 and L. rhamnosus GG showed adhesion ability at 1.91 ± 0.88% and 1.26 ± 0.22%, respectively (Table 5). The adhesion ability of strain LM14-2 was not statistically significantly different from the adhesion ability of L. rhamnosus GG.

Immunomodulation Effects

The immunomodulatory effects of the L. plantarum LM14-2 are shown in Fig. 3. The L. plantarum LM14-2 stimulated IL-12, IFN-γ, and NO production at 35.49 ± 6.85 ng/mL, 44.89 ± 14.61 ng/mL, and 19.98 ± 0.28 µM, respectively. Furthermore, the hBD2 expression was also enhanced by L. plantarum LM14-2 (2.15 ± 0.07 of relative value). These results indicated that the dead cell of L. plantarum LM14-2 still had immunomodulatory effects.

Fig. 3
figure 3

The immunomodulation effects of L. plantarum LM14-2; a the level of IL-12 and IFN-γ production and the relative value of hBD2; b NO production. Results are expressed as means ± SD. *P < 0.05, compared to PBS (no stimulation) within each column; **P < 0.05, compared to LPS (positive control)

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Discussion

This study highlighted the characterization, probiogenomic, cholesterol-lowering, and immunomodulation effects of L. plantarum LM14-2 isolated from Thai fermented mussel (Hoi-dong). The strain LM14-2 was identified as L. plantarum based on the dDDH and ANI values [47] that were above the species boundary value (ANI > 95–96%) [48]. For these reasons, the characteristics and whole-genome analysis supported strain LM14-2 as representing a member of Lactiplantibacillus plantarum.

The results of genomic sequences are necessary for comprehensive safety assessments and proposal of probiotic application of strain. The prediction of pathogenicity found that strain LM14-2 was predicted as a non-human pathogen. Exo- and capsular-polysaccharide genes related to virulence factors were detected. However, exo- and capsular-polysaccharides protein (cps2B, cps4B, and cpsE) and polysaccharides are involved in stress tolerance. Exopolysaccharide help bacteria survive osmotic, desiccation, and oxidative stress conditions [11, 49,50,51] and play a role in cryoprotection [52]. In addition, exopolysaccharides play a role in cell adhesion [53]. Besides, capsular polysaccharides are involved in discovering the strain-specific properties important for probiotic action, such as stress resistance, adhesion, and the defense mechanism of the host [11, 49]. Capsular polysaccharides have been discovered in bacterial colonization in the digestive tract, and they play a role in modulating the immune system [54]. Notably, virulence factors (i.e., adhesions) were also encoded in the genomes of commensal bacterial [49]. Additionally, the hemolysin III gene (hlyIII) was also observed in various commercial probiotics, including an accepted Generally Recognized as Safe (GRAS) probiotic strain L. plantarum 299V, L. rhamnosus GG, and numerous other Lactobacillus strains in the GenBank [55]. Generally, the strains containing the hlyIII gene have been proven safe and are commercially available worldwide. The bacterium containing this gene should not pose a safety risk, provided that no other pathogenesis genes are observable in the genome. There are numerous reports on hemolysin III and its safety in lactobacilli; hence, this toxin protein is not considered of significant concern [56,57,58]. Nevertheless, these virulence genes could be deemed advantageous to the bacterium without pathogenesis mechanisms. These genes were identified as virulence factors in the virulence factor database as they were also involved in pathogenic bacterial adaptation, survival, or attachment in the hostile/host environment. These genes could be regarded as beneficial to the bacterium since they increase bacterial fitness and may be desirable where live cells are needed. Not only the enhancing role of bacterial endurance but also these genes were observed in the genomes of several commensal bacteria [50, 55]. The concern of AR genes in beneficial bacteria is their transfer possibility to other pathogens, which may cause serious problems. This study focused on mobile elements, plasmids and bacteriophages, because they are the most plausible vehicles for inter-cellular genetic exchange. The strain LM14-2 did not contain AR genes and plasmid; consequently, no oriT was found in any of the plasmids, indicating that LM14-2 is incapable of self-transmission through conjugative transfer. No ARGs (antibiotic resistance genes) were located in the prophage regions. As a result of these findings, it was established that the absence of AR genes in LM14-2 does not present a risk of transfer to other bacteria; therefore, the strain does not provide a safety issue for the functional and transferrable ARG characteristic.

The presence of genes involved in carbohydrate metabolism in strain LM14-2 is essential for its potential adaptability to the environment of the gut environment and its interaction with the human host, hence enhancing its survival, competitiveness, and longevity. The strain LM14-2 comprised several carbohydrate-active enzyme genes. Consequently, strain LM14-2 could use several mono- and polysaccharides as energy sources and produce complex compounds. Oligosaccharides are the source of prebiotics, which are related to human gut health [59]. The presence of GH13 and GH32 are key oligosaccharide-degrading enzymes. Furthermore, GH families play essential roles in synthesizing oligosaccharides that may be preferentially utilized as prebiotics by strain LM14-2 and other probiotic bacteria [60]. In addition, glycosyltransferases catalyze the conversion of sugars, which is necessary for constructing surface structures recognized by host immune systems [61]. Thus, strain LM14-2 containing the numerous GT genes could be a potential probiotic, especially for immune stimulation and pathogen prevention.

For potential genes associated with various probiotic characteristics, sortase class A (srtA) is involved for the LPXTG proteins covalently to the cell wall and plays a role in adherence to host surfaces. Mucus-binding proteins contribute to the adherence of bacteria to the intestinal mucosa [62]. Stain LM14-2 harbors gene coding for mucus-binding protein mub. Thirty-two genes responsible for acid and bile salt stress were identified (Table 3). The F1F0-ATPase is encoded by the atp operon, which comprises the following genes: atpC, atpD, atpG, atpH, atpF, atpB, atpE, and atpA. The atp genes are vital for the survival or tolerance of acidic environments. The “atp” operon is primarily related to the pumping of protons [63]. S-Ribosylhomocysteinase (luxS) plays an essential role in the adhesion ability and the autoinducer-2 synthesis that promotes stress resistance [64]. Additionally, moonlighting protein genes or multifunctional protein genes, such as elongation factor Tu and chaperonin GroEL, has been associated with adhesion to epithelial cells and immunomodulation [60]. Furthermore, probiotics perform an essential function in the host gut by synthesizing micronutrients and factors such as amino acids, fatty acids, oligosaccharides, vitamins, and enzymes. The strain LM14-2 comprised subsystems and genes responsible for synthesizing and metabolizing of amino acids and derivatives, carbohydrates, fatty acids, lipids, cofactors, prosthetic groups, and vitamins (Table 3). The seven subsystems are responsible for vitamin biosynthesis, including biotin, folate, niacin, pantothenate, pyridoxine, thiamin, and riboflavin. Overall, the draft genome information of L. plantarum LM14-2 will help us properly comprehend the molecular basis for its probiotic effects. The absence of antimicrobial activity was explained by the lack of several genes, including plnA, plnB, plnC, plnL, plnN, plnO, plnQ, plnR, plnT, plnU, plnV, plnW, and plnX genes, which are also essential for bacteriocin production, transcriptional regulation, and bacteriocin secretion [65, 66]. The incomplete plantaricin gene cluster, in agreement with in vitro findings, where no bacteriocin-like activity was detected. This finding is consistent with the previous study of Tegopoulos et al. [67]. Thus, the desirable probiotic characteristics of strain LM14-2 were also supported by the in silico findings.

For the cholesterol-lowering effects, the presence of the bsh gene and BSH activity indicated the cholesterol-lowering effects (lower total and low-density lipoprotein; LDL cholesterol). In the Guidelines for the Evaluation of Probiotics in Food issued by FAO/WHO, bile salt hydrolase activity is recognized as an additional criterion for selecting probiotics and safety assurance [9, 68]. Conversely, a high level of deconjugated bile may impair lipid digestion, disrupt normal intestinal conditions, induce gallstone formation, and may be further modified to carcinogenic secondary bile salts. After considering all the advantages and risks using the scientific evidence revealed above, this study suggested that the bile salt deconjugation property could be desirable when the strain could not modify the deconjugated bile into harmful secondary bile products. Aside from the choloylglycine hydrolase, no genes associated with the secondary bile salts biosynthesis were discovered in LM14-2. Regarding its inability to produce harmful secondary bile products, this study considers LM14-2 to pose no safety issues from this property. Moreover, the strain LM14-2 showed cholesterol assimilation ability. The BSH and cholesterol assimilation activities are the cholesterol removal mechanisms and desirable characteristics of probiotics [69]. Because probiotics can consume cholesterol for their metabolism, as a result, luminal cholesterol levels available for absorption are reduced.

For the immunomodulatory effects, the advantages of dead/dormant cells of probiotics include a reduced risk of probiotic sepsis and drug resistance, as well as a longer shelf-life because there is no need to preserve the storage system to preserve the viability of the probiotics [70, 71]. The immunomodulation ability of heat-killed LM14-2 cells is consistent with the previous studies [41, 72, 73]. In addition, the defensins activation may be an appealing novel treatment method for strengthening innate immunity. As a result, strain LM14-2 could enhance immunity [74, 75].

Conclusion

This study is the first to investigate the characterization, probiogenomic analysis, cholesterol-lowering, and immunomodulation effects of L. plantarum LM14-2 isolated from Hoi-dong. The strain LM14-2 exhibited BSH activity by forming an opaque white colony and potentially assimilated cholesterol by more than 75%. It could tolerate gastrointestinal conditions and greatly adhered to Caco-2 cells. Additionally, the L. plantarum LM14-2 provided immunostimulatory effects. L. plantarum LM14-2 could stimulate the production of IL-12, IFN-γ, and NO and promote the hBD2 secretion.

Furthermore, the in silico analysis of L. plantarum LM14-2 emphasized the attractiveness of this strain as a potential probiotic. This strain was considered safe, and it did not contain antibiotic resistance genes, plasmid, and virulence elements. In addition, it is composed of various genes that play roles in acid and bile salt tolerance, adhesion, and other positive benefits. Therefore, the combination of in vitro and in silico analysis suggested that L. plantarum LM14-2 is considered a potential probiotic because it exhibited health-promoting effects and probiotic characteristics. Also, the genomic data of this strain supported the desirable features.