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. 2025 Jun 23;34(14):3331–3340. doi: 10.1007/s10068-025-01925-9

Safety assessment of potential probiotic lactic acid bacteria strains Pediococcus acidilactici SY21 and Pediococcus acidilactici SY22

Jaein Shin 1, Huijin Jeong 1, Na-Kyoung Lee 2, Dae‐Kyung Kang 3, Hyun‐Dong Paik 2, Young-Seo Park 1,✉
PMCID: PMC12408421  PMID: 40918487

Abstract

Pediococcus acidilactici SY21 and Pediococcus acidilactici SY22 exhibit anti-inflammatory activity; however, their safety has not been evaluated. The suitability as probiotic strains were evaluated by using phenotypic and genotypic analyses. Indole production, urease activity, mucin degradation, bile salt hydrolase activity, β-hemolysis, and gelatin liquefaction activity were not found. The minimum inhibitory concentrations of the nine antibiotics tested were below the cut-off values recommended by the European Food Safety Authority, except for kanamycin and clindamycin in P. acidilactici SY21 and kanamycin in P. acidilactici SY22. After confirming that the genetic basis of antibiotic resistance exceeded the cut-off value, all isolates showed intrinsic resistance. P. acidilactici SY21 and P. acidilactici SY22 did not produce biogenic amine, and β-glucuronidase activity. Neither strain was cytotoxic to Caco-2 cells. Antibiotic resistance or virulence genes were not detected with whole-genome sequencing. This study demonstrated the safety of the P. acidilactici SY21 and P. acidilactici SY22 strains as probiotics.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-025-01925-9.

Keywords: Safety assessment, Probiotics, Pediococcus acidilactici, Anti-inflammatory, Antibiotic resistance

Introduction

Probiotics are defined as “live microorganisms that when administered in adequate amounts, confer a health benefit to the host” (FAO/WHO, 2002). The use of probiotics is increasing because of their various health benefits, including antidiabetic, anti-inflammatory, antipathogenic, antiallergic, and anticancer effects (Kerry et al., 2018). Although many probiotic strains have been classified as Generally Recognized as Safe (GRAS) based on numerous studies, recent concerns have emerged regarding their safety, particularly with the increased use of strains isolated from potentially infectious sources (Zawistowska-Rojek and Tyski, 2018). To assess the safety of probiotics, the European Food Safety Authority (EFSA) evaluates the Qualified Presumption of Safety (QPS) status of probiotics, whereas the Food and Agriculture Organization (FAO)/World Health Organization (WHO) recommends safety assessments of new strains, including pathogenicity, toxicity, and antibiotic resistance (Jang et al., 2021).

Pediococcus, a genus of gram-positive lactic acid bacteria (LAB) in the Lactobacillaceae family, includes species, such as P. pentosaceus and P. acidilactici. These species are commonly used for producing pediocins, serving as starter cultures in fermentation processes, and probiotic supplements for humans and animals (Porto et al., 2017). The EFSA recognizes P. acidilactici as a QPS (EFSA, 2007), and certain strains have gained attention for their probiotic properties and beneficial effects on immune-related diseases.

Inflammation is an essential response of the body to chemical stimuli, microbial infections, and physical injury; however, if not properly regulated, it can lead to tissue damage and the development of chronic diseases (Wang et al., 2023). Probiotics have gained attention as potential therapeutic agents for the treatment of inflammation because of their immunomodulatory effects on the gut (Caruso et al., 2020).

Probiotics and their derived substances exhibit anti-inflammatory properties by suppressing the Toll-like receptor 4/myeloid differentiation primary response protein 88/nuclear factor (NF)-κB, mitogen-activated kinase (MAPK), and activator protein 1 (AP-1) signaling pathways (Kwon et al., 2020). In a previous study, P. acidilactici SY21 and P. acidilactici SY22 have shown similar effects in lipopolysaccharide-stimulated RAW 264.7 macrophages (Woo et al., 2024). The anti-inflammatory effects of P. acidilactici strains have been demonstrated; however, their safety has not been sufficiently evaluated (Woo et al., 2024). Therefore, the aim of this study is to evaluated the safety of P. acidilactici SY21 and P. acidilactici SY22 through genotypic and phenotypic analyses based on FAO/WHO international standards for their potential use as probiotics.

Materials and methods

Bacterial strains, media, and growth conditions

P. acidilactici SY21 and P. acidilactici SY22, and Lactiplantibacillus plantarum KU15122 strains were sourced from Konkuk University (Seoul, Korea) and grown in de Man, Rogosa, and Sharpe (MRS) medium (Oxoid, Hampshire, UK). The medium consisted of 10 g protease peptone No. 3, 10 g beef extract, 5 g yeast extract, 20 g dextrose, 1 g polysorbate 80, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g magnesium sulfate, 0.05 g manganese sulfate, and 2 g dipotassium hydrogen phosphate per liter. The cells were cultured at 37 °C for 24 h.

Brevibacillus parabrevis KCCM 41421, Streptococcus pyogenes KCCM 11873, Proteus vulgaris KCCM 40211, and Klebsiella pneumoniae subsp. pneumoniae KCCM 41433 were purchased from the Korean Culture Center of Microorganisms (Seoul, Korea).

B. parabrevis KCCM 41421 was cultured in tryptic soy broth (TSB, BD Biosciences, Heidelberg, Germany) containing 17 g tryptone (pancreatic digest of casein), 3 g soytone (peptic digest of soybean), 2.5 g glucose, 5 g sodium chloride, and 2.5 g dipotassium phosphate per liter, adjusted to pH 7.3, at 30 °C for 24 h. S. pyogenes KCCM 11873 was cultured anaerobically in TSB supplemented with 5% sheep blood (MB cell, Seoul, Korea) at 37 °C for 24 h. P. vulgaris KCCM 40211 was cultured in nutrient broth composed of 3 g beef extract and 5 g peptone per liter at 30 °C for 24 h. Escherichia coli ATCC 10536 was cultured in TSB at 30 °C for 24 h. K. pneumoniae subsp. pneumoniae KCCM 41433 was cultured in nutrient broth at 30 °C for 24 h.

Cell culture conditions

Caco-2 cells (ATCC® HTB-37™) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 1% (v/v) penicillin–streptomycin (Gibco) and 10% (v/v) fetal bovine serum (FBS; Corning Inc., Corning, New York, NY, USA). The cells were cultured at 37 °C in a 5% CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA). To assess the cytotoxicity, the viability of Caco-2 cells was measured before and after treatment with the probiotic strains.

Measurement of the minimum inhibitory concentration

The bacterial colonies were suspended in 0.88% NaCl to achieve a concentration equivalent to the 0.5 McFarland standard solution (0.5 mL of 0.048 M BaCl₂, 99.5 mL of 0.18 M H₂SO₄), corresponding to a turbidity of approximately 1.5 × 108 cells/mL. The suspension was then spread onto LAB susceptibility test medium, which consisted of 90% Oxoid Iso-Sensitest Broth (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 10% MRS agar, using a sterile swab. Nine antibiotic E-test strips (ETEST®, bioMérieux, Marcy-l’Étoile, France) were placed on the surface of the agar plates using sterile forceps, and the plates were incubated at 37 °C for 24 h. The minimum inhibitory concentration (MIC) of each antibiotic was determined and compared against the cut-off values established by the EFSA (2012) for each antibiotic. The cut-off value refers to the highest MIC value for a given antibiotic at which a bacterial strain is still considered susceptible. Strains with MIC values above the cut-off are considered resistant.

Genomic analysis of the antibiotic resistance and virulence genes in P. acidilactici SY21 and P. acidilactici SY22

The complete genome sequences of P. acidilactici SY21 and SY22, provided by Dankook University (Cheonan, South Korea), have been deposited in the NCBI database under BioProject accession numbers PRJNA1258167 and PRJNA1258165, and BioSample accession numbers SAMN48290200 and SAMN48290199, respectively. Antibiotic resistance genes were analyzed using ResFinder v4.6.0 (http://genepi.food.dtu.dk/resfinder) based on the ResFinder database, RGI v6.0.3 based on the Comprehensive Antibiotic Resistance Database (CARD) v3.3.0 (https://card.mcmaster.ca/analyze/rgi), and HGTree2 (http://hgtree2.snu.ac.kr/). To determine horizontal gene transfer, the plasmid conjugation was analyzed using PlasmidFinder 2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/), the bacteriophage transduction was evaluated with PHAge Search Tool with Enhanced Sequence Translation (PHASTEST) (http://phastest.ca), and transposon translocation was analyzed using MobileElementFinder (https://cge.food.dtu.dk/services/MobileElementFinder/). Virulence genes were identified using VirulenceFinder 2.0 (https://cge.food.dtu.dk/services/VirulenceFinder).

ResFinder analysis was conducted with a sequence similarity threshold of over 90% and a minimum length of 60%. RGI analysis considered sequence similarity of over 95% as a strict hit and determined that sequence similarity of over 70% indicated the presence of antibiotic resistance genes. MobileElementFinder analysis was performed with a minimum alignment coverage of 95%, a minimum sequence identity of 90%, and a maximum truncation length of 30 bases (nt).

Hemolytic activity test

P. acidilactici SY21 and P. acidilactici SY22 were cultured on blood agar plates prepared with 42.5 g blood agar base No. 2 (Sigma-Aldrich, St. Louis, MO, USA; 15.0 g of agar, 2.50 g of liver extract, 15.0 g of proteose peptone, 5.0 g of sodium chloride, and 5.0 g of yeast extract per liter, adjusted to pH 7) and 50 mL defibrinated sheep blood (KisanBio, Seoul, Korea), per liter. A single colony of each strain was cultured on blood agar plates and the plates were incubated anaerobically at 37 °C for 48 h. S. pyogenes KCCM 11873 was used as a positive control. The formation of a clear zone surrounding the bacterial colonies was identified as an indicator of β-hemolysis.

Mucin degradation property

Mucin degradation test for P. acidilactici SY21 and P. acidilactici SY22 was performed as described by Prakash et al. (2011). P. acidilactici SY21 and P. acidilactici SY22 were cultured in a 30 mL MRS basal medium with 0.3% (w/v) mucin (type III, Sigma-Aldrich), with or without 1% (w/v) glucose, at 37 °C for 24 h. The non-MRS medium was used as the control. Bacterial growth was assessed by measuring the change in turbidity at 600 nm using a spectrophotometer (BioSpec-mini; Shimadzu Co., Kyoto, Japan) and monitoring the pH of the culture.

Cell cytotoxicity on Caco-2 cells

Caco-2 cells were seeded at a concentration of 5 × 104 cells per well in a 96-well plate with DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. The cells were incubated at 37 °C with 5% CO2 for 20 h. Pediococcus acidilactici SY21 and P. acidilactici SY22 were cultured in MRS broth at 37℃ for 18 h. For the positive control, K. pneumoniae subsp. pneumoniae KCCM 41433 was cultured in nutrient broth at 37 °C for 18 h. Overnight cultures were inoculated at 1% (v/v) into 10 mL MRS medium and incubated at 37 °C for 3 h. Bacterial cells were collected using centrifugation at 20,781×g for 1 min, washed thrice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene, Gyeongsan, Korea), and resuspended in 1 mL of DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. The bacterial suspension was added to Caco-2 cells at multiplicities of infection (MOI; ratio of bacterial cell number to Caco-2 cell number) of 125, 250, 500, and 1000, followed by incubation at 37 °C in a 5% CO2 incubator for 24 h. After 24 h, the cells were washed twice with DPBS and treated with 200 μL of DMEM containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin, along with 20 μL of EZ-CYTOX (DoGenBio Co., Ltd., Seoul, Korea), and incubated at 37 °C in a 5% CO2 incubator for 30 min. The absorbance was measured at 450 nm using a Take3 Micro-Volume Plate (Epoch microplate reader, Biotek Instruments, Inc., Winooski, VT, USA).

Bile salt deconjugation

The deconjugation of bile salts was evaluated using MRS agar medium supplemented with 0.5% (w/v) taurodeoxycholic acid (TDCA; bile acid; Sigma-Aldrich). P. acidilactici SY21 and P. acidilactici SY22 were cultured on MRS agar medium with 0.5% (w/v) TDCA by streaking and incubated at 37 °C for 48 h. Strains showing sedimentation around the colonies were identified as strains producing bile salt hydrolases. L. plantarum KU15122 was used as the positive control.

Biogenic amine production

The biogenic amines produced by P. acidilactici SY21 and P. acidilactici SY22 were analyzed using HPLC. LAB seed cultures were inoculated into MRS medium supplemented with 400 ppm each of arginine, histidine, lysine, ornithine, phenylalanine, and tryptophan (Sigma-Aldrich), and 100 ppm of tyrosine. Thereafter, the cultures were incubated at 37 °C for 24 h. Approximately, 0.5 mL of a saturated sodium carbonate solution was added to the 1 mL of culture supernatant, followed by 0.8 mL of 1% (w/v) dansyl chloride acetone solution. The mixture was then allowed to react at 45 °C for 1 h. After the reaction, 0.5 mL of 10% (w/v) proline and 5 mL and diethyl ether were added to the derivatized mixture, vortexed for 10 min, and evaporated under nitrogen gas. The resulting solution was diluted with 1 mL of acetonitrile, passed through a 0.22-μm Polyvinylidene Fluoride filter, and analyzed using HPLC. The derivatized biogenic amines were examined using HPLC on a DIONEX UltiMate 3000 system (Thermo Fisher Scientific) equipped with a UV detector set at 254 nm and an Agilent 5 TC-C18 column (250 × 4.6 mm, 5 μm, Agilent Technologies, Amstelven, Netherlands). The column oven temperature was maintained at 40 °C. After a 30-min equilibration with acetonitrile/water (55/45), 20 μL of the dansylated biogenic amines were introduced into the column. To achieve optimal separation, a flow rate of 1 mL/min was used with a gradient elution. The standard biogenic amines analyzed in this study were agmatine, β-phenylethylamine, histamine, putrescine, serotonin, spermidine, tryptamine, and tyramine (Sigma-Aldrich).

d-/l-Lactate formation

The LAB culture was centrifuged at 16,000×g for 10 min. The supernatant was analyzed using a d-lactate assay kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Absorbance was measured at 340 nm using a BioSpec-mini spectrophotometer (Shimadzu).

Enzymatic activity using API ZYM

The activities of 19 enzymes were measured using an API ZYM Kit (API Identification Systems, bioMérieux, France). After culturing the samples in MRS medium at 37 °C for 24 h, the cells were centrifuged and washed three times with saline. The samples were then resuspended in saline and adjusted to a turbidity of 5–6 McFarland. A volume of 65 μL of the suspension was dispensed into each cupule of the ZYM strip, followed by incubation at 37 °C for 4 h. To enhance enzyme activity, ZYM A was added, followed by the addition of one drop of ZYM B reagent to each cupule and allowed to react for 5 min. Enzyme activity was assessed by observing the color changes.

Toxic metabolite production test

Urease activity test

Urease activity was evaluated by measuring the change in pH in urea agar inoculated with P. acidilactici SY21 and P. acidilactici SY22. Next, P. vulgaris KCCM 40211 was used as a positive control, and the inoculated tubes were anaerobically incubated at 37 °C for 24 h. A strain was identified as positive for ammonia production when the color of the medium changed from pink to red.

Gelatin liquefaction test

The gelatin liquefaction assay was performed by inoculating P. acidilactici SY21 and P. acidilactici SY22 into MRS medium containing 12% (w/v) gelatin in test tubes. P. acidilactici SY21, P. acidilactici SY22, and the positive control B. parabrevis KCCM 41421, were inoculated into tubes and incubated at 37 °C for 72 h. Following incubation, the tubes were cooled in an ice bath for 30 min. If the gelatin liquefied, it was considered a positive result for gelatin liquefaction.

Indole production test

Indole production was determined using tryptophan medium (10.0 g casein enzyme hydrolysate, 5.0 g NaCl, 1.0 g dl-tryptophan per liter). E. coli ATCC 10536 was used as a positive control. After 18 h of incubation, five drops of Kovac’s reagent (Sigma-Aldrich) were added. A change in the color of the medium from red was considered positive.

Statistical analysis

The results are expressed as the mean ± standard deviation from three independent experiments. To assess the statistical significance of the results, one-way analysis of variance (ANOVA) was conducted using IBM SPSS Statistics version 28.0 (SPSS Inc., Chicago, IL, USA). Duncan’s test was used to assess significant differences between the groups, with a significance level of p < 0.05.

Results and discussion

Measurement of the minimum inhibitory concentration

The antibiotic susceptibilities of P. acidilactici SY21 and P. acidilactici SY22 were evaluated and compared with the cut-off values established by EFSA (2012). The MIC of the antibiotics was below the cut-off value recommended by the EFSA, except for kanamycin and clindamycin in P. acidilactici SY21 and kanamycin in P. acidilactici SY22 (Fig. 1, Tables S1 and S2). Lee et al. (2014) showed that all Pediococcus strains were sensitive to antibiotics commonly used to treat bacterial infections, such as ampicillin, chloramphenicol, erythromycin, kanamycin, streptomycin, and tetracycline.

Fig. 1.

Fig. 1

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E-test for the assessment of antibiotic resistance. A Pediococcus acidilactici SY21, B P. acidilactici SY22

The EFSA recommends avoiding the use of bacterial strains with transferable antibiotic resistance genes in probiotics and fermented foods (EFSA, 2007). This precaution is due to the risk of spreading antibiotic resistance among the gut microbiota, highlighting the need to select strains that lack transferable resistance elements or the capability to facilitate plasmid transfer (Salminen et al., 2000). Although Pediococci are typically regarded as safe regarding antibiotic susceptibility, an antibiotic susceptibility evaluation must be performed of all newly isolated strains before their use in food products. In Lactobacillus plantarum, Lactobacillus casei, Pediococcus, and other Lactobacillus species, the C-terminal D-Ala in the cell wall is substituted with D-lactate, providing these bacteria with intrinsic resistance to vancomycin (Martínez et al., 2020). According to EFSA guidelines, testing for vancomycin susceptibility in P. acidilactici is not required.

Genomic analysis of the antibiotic resistance and virulence genes in P. acidilactici SY21 and P. acidilactici SY22

As a result of the HGTree2 analysis, a total of 30 horizontal gene transfer (HGT) genes were predicted in P. acidilactici SY21 and P. acidilactici SY22, of which 17 were identified as received from external sources and 13 as donated genes (Table S3). Among the total genomes, HGT genes accounted for 1.47%, with recipient genes comprising 0.83% and donor genes 0.64%. Antibiotic resistance gene analysis using HGTree2 and RGI revealed no antibiotic resistance genes within the HGT genes.

Plasmids are known as genetic elements that enable bacteria to inactivate or evade the action of specific antibiotics, often by carrying antibiotic resistance genes. Plasmid transfer occurs through conjugation, a horizontal gene transfer method in which the pilus of a donor strain connects to a recipient strain to transfer a copy of the plasmid. If the plasmid includes both the tra gene and an antibiotic resistance gene, it is considered acquired resistance (Bennett, 2008). PlasmidFinder 2.0 analysis detected no plasmids in either P. acidilactici SY21 or P. acidilactici SY22.

Transduction is divided into a generalized type and a specialized type. The generalized type can randomly deliver host DNA, whereas the specialized type can deliver specific genes. Similar to plasmid conjugation, transduction may promote the spread of antibiotic resistance. Bacteria that acquire antibiotic resistance genes may transmit them vertically to their offspring or spread them to surrounding bacteria through transduction and other horizontal gene transfer mechanisms (Bennett, 2008). As a result of PHASTEST analysis, two intact prophage regions were identified in both P. acidilactici SY21 and P. acidilactici SY22 (Table S4). In the Resfinder analysis, no antibiotic resistance genes were found in the intact prophage regions. In the RGI analysis, the HelR gene associated with rifamycin resistance was detected in P. acidilactici SY21 with 28.83% homology (Table S5). However, due to low homology, it was determined that antibiotic resistance genes were not present in the intact prophage regions.

A transposon is a mobile DNA segment involved in DNA rearrangement within cells, distinct from plasmids or bacteriophages. However, when carrying an antibiotic resistance gene and inserted into a plasmid, it may spread to other bacteria through conjugation (Bennett, 2008). In the MobileElementFinder analysis of P. acidilactici SY21, seven mobile genetic elements were identified, and no antibiotic resistance genes were found in the ResFinder analysis. In the RGI analysis, the tetracycline-associated tetA(58) gene was detected with 33.64% homology, and the nalD gene was detected with 30.17% homology (Table S6). However, due to low homology, it was determined that no antibiotic resistance genes were present in the intact prophage regions. Similarly, seven mobile genetic elements were identified in P. acidilactici SY22, and no antibiotic resistance genes were detected in the ResFinder or RGI analyses.

Both P. acidilactici SY21 and P. acidilactici SY22 confirmed intrinsic resistance through the above analysis. Intrinsic resistance is not caused by prior exposure to antibiotics, and has a low potential for transfer. Intrinsic antibiotic resistance in probiotic strains is beneficial because it allows survival in the gastrointestinal tract during antibiotic treatment (Das et al., 2020). Additionally, no virulence genes were detected in the genome sequence compared with those in the sequences of the pathogens.

Hemolytic activity test

When the hemolytic activity was assessed using sheep blood agar, P. acidilactici SY21 and P. acidilactici SY22 did not display a clear zone around the colonies, whereas the positive control S. pyogenes KCCM 11873 showed a distinct clear zone surrounding the colony (Fig. 2). This indicated that both P. acidilactici SY21 and P. acidilactici SY22 were γ-hemolytic (non-hemolytic). This result is consistent with previous studies showing that Pediococcus species do not exhibit β-hemolysis (Shin et al., 2020).

Fig. 2.

Fig. 2

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Hemolysis test. A Streptococcus pyogenes KCCM 11873 displayed a clear zone surrounding the bacterial colony; B Pediococcus acidilactici SY21 displayed a non-clear zone surrounding the bacterial colony; C P. acidilactici SY22 displayed a non-clear zone surrounding the bacterial colony

Hemolysis refers to the destruction of the red blood cell membrane, causing the release of intracellular contents into the surrounding plasma. Hemolytic activity is indicated by a clear zone around the colony (β-hemolysis), whereas green discoloration (α-hemolysis) or no zone (γ-hemolysis) is considered non-hemolytic (De Vuyst et al., 2003).

Mucin degradation

The mucin layer of the gastrointestinal tract is composed of glycoproteins, which protect epithelial cells from digestive enzymes and external factors and act as a barrier to prevent potential damage caused by pathogens. Mucin-degrading enzymes can contribute to the toxicity of pathogenic microorganisms; when the mucous membrane is damaged, it may become more vulnerable to harmful substances and pathogens (Mangia et al., 2019). According to Daliri et al. (2022), the addition of mucin to MRS did not enhance the growth of Weissella cibaria SCCB2306, P. acidilactici SDL1402, Lactobacillus rhamnosus JDFM6, or P. acidilactici SDL1406 compared to the positive control, E. coli ATCC 35150, for which mucin was added to TSB.

The assessment of mucin degradation by P. acidilactici SY21 and P. acidilactici SY22 using the four modified media showed no decrease in pH or increase in absorbance values in the mucin-supplemented media (Fig. 3). This confirms that these strains do not use mucin as a carbon source.

Fig. 3.

Fig. 3

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Mucin degradation property of Pediococcus acidilactici SY21 and P. acidilactici SY22. A Left panel, pH of the P. acidilactici SY21 culture medium; right panel, growth of P. acidilactici SY21 measured by optical density at 600 nm. B Left panel, pH of the P. acidilactici SY22 culture medium; right panel, growth of P. acidilactici SY22 measured by optical density at 600 nm. Non-MRS, MRS broth without glucose; Mucin, MRS broth containing 0.3% mucin; Glucose, MRS broth containing 1% glucose; Mucin+Glucose, MRS broth containing 0.3% mucin and 1% glucose. Different letters represent significant differences between groups at p < 0.05

Cell cytotoxicity

Evaluation of the cytotoxicity of P. acidilactici SY21 and P. acidilactici SY22 on Caco-2 cells, a positive control, K. pneumoniae subsp. pneumoniae KCCM 41433, showed cell viabilities of 71.6% at MOI 125, 73.8% at MOI 250, 92.9% at MOI 500, and 56.8% at MOI 1000. No cytotoxicity was observed for P. acidilactici SY21 and P. acidilactici SY22 at any MOI (Fig. 4).

Fig. 4.

Fig. 4

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The effect of Pediococcus acidilactici SY21 and P. acidilactici SY22 on Caco-2 cell viability. Left bar, Caco-2 cells were incubated with P. acidilactici SY21; Center bar, Caco-2 cells were incubated with P. acidilactici SY22; Right bar, Caco-2 cells were incubated with Klebsiella pneumoniae subsp. pneumoniae KCCM 41433. Different letters represent significant differences between groups at p < 0.05

Lee et al. (2025) confirmed that L. brevis KU15006 exhibited no cytotoxicity at any MOI compared to K. pneumoniae subsp. pneumoniae KCCM 41433, which was used as the positive control. The Caco-2 and HT-29 cell lines are well-established models for simulating intestinal ecosystems in vitro. These epithelial cell monolayer cultures serve as useful tools for studying the colonization, cytotoxicity, and antibacterial effects of probiotics against enteric pathogens (Dunne et al., 2001).

Bile salt deconjugation

When deconjugation of bile salts was assessed using MRS agar containing TDCA, P. acidilactici SY21 and P. acidilactici SY22 did not precipitate around the colonies or form white colonies. However, L. plantarum KU15122, which precipitated around the colonies and formed white colonies, served as a positive control (Fig. 5). These findings suggest that P. acidilactici SY21 and P. acidilactici SY22 could not deconjugate taurodeoxycholate sodium or transform bile salts into secondary bile acids.

Fig. 5.

Fig. 5

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Bile salt deconjugation of Pediococcus acidilactici SY21 and P. acidilactici SY22. A Lactiplantibacillus plantarum KU15122 produced a white precipitate around its colonies. B P. acidilactici SY21 does not produce a white precipitate around its colonies. C P. acidilactici SY22 did not produce a white precipitate around its colonies

Tanaka et al. (2022) confirmed that L. rhamnosus YM2-1 and YM2-3 showed no precipitation around their colonies in media containing six types of bile acids (taurocholic acid, taurochenodeoxycholic acid, TDCA, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid), confirming the absence of bile salt hydrolase activity.

Bile acids emulsify fat, which promotes the action of lipases and helps to break down fatty acids. When a probiotic strain exhibits bile acid hydrolase activity, bile acids are decoupled, which protects the probiotic and increases intestinal settlement (Dashkevicz and Feighner, 1989). However, decoupling bile acid reduces its emulsifying function, making it difficult to break down fatty acids and posing a potential risk of producing toxic secondary metabolites by metabolizing other intestinal microorganisms (Dashkevicz and Feighner, 1989).

Biogenic amine production

The analysis of biogenic amine (BA) production revealed that both P. acidilactici SY21 and P. acidilactici SY22 did not produce agmatine, histamine, β-phenylethylamine, putrescine, serotonin, spermidine, tryptamine, and tyramine (Table S5). According to Ruiz‐Moyano et al. (2010), all 12 strains of P. acidilactici were found to have low biogenic amine production capacity (less than 100 mg/L).

Biogenic amines (BAs) are organic compounds with low molecular weights that are produced during fermentation by the microbial decarboxylation of amino acids (Erdag et al., 2018). These amines play essential roles in various physiological functions, including the regulation of blood pressure and cell growth. Although small amounts of BAs are important for maintaining health, excessive levels can trigger various physiological issues, such as nausea, respiratory problems, headaches, sweating, palpitations, and abnormal blood pressure (either hypertension or hypotension) (Erdag et al., 2018).

l-/d-Lactate formation

The evaluation of lactate production showed that P. acidilactici SY21 produced 96.0 mM of l-lactic acid and 63.7 mM of d-lactic acid, while P. acidilactici SY22 produced 91.5 mM of l-lactic acid and 61.4 mM of d-lactic acid (Tables S6 and S7).

According to Ruiz‐Moyano et al. (2010), Pediococci strains produced total lactic acid in the range of 28.0–34.0 g/L (311.1–377.8 mM) in the culture medium. Strains PS955, PS194, and PP54 produced less than 10% d-lactic acid, whereas strains PS171 and PS200 produced > 30% lactic acid. Compared to the result of Ruiz‐Moyano et al. (2010), it can be confirmed that P. acidilactici SY21 and P. acidilactici SY22 produce lower levels of D-lactic acid, confirming that there are no safety concerns.

The human body can metabolize l-lactate, but not d-lactate, which is an isomer. Some probiotic strains contain an enzyme (dl-lactate racemase) that converts l-lactate into d-lactate, leading to the accumulation of d-lactate, which can cause metabolic acidosis in newborns, children, and patients with congenital bowel syndrome. Therefore, precautions should be taken during its ingestion (Kang et al., 2019).

Enzymatic activity using API ZYM

The API ZYM kit was used to assess the inhibition of harmful enzymes. β-glucuronidase activity hydrolyzes p-nitrophenyl-β-d-glucuronide to form nitrophenol (Thomas and Sobin, 1995). β-glucuronidase may be associated with carcinogenic compounds and increase the risk of colorectal cancer (Kim and Jin, 2001).

In this study, β-glucuronidase activity was not detected in P. acidilactici SY21 or P. acidilactici SY22 (Table S8). Similarly, Abbasiliasi et al. (2017) measured the enzyme activity of P. acidilactici Kp10 and did not detect β-glucuronidase activity.

Toxic metabolites production

P. acidilactici SY21 and P. acidilactici SY22 showed no color change in urease activity evaluations, confirming the absence of ammonia or urease activity (Fig. 6A). When evaluating gelatin liquefaction, B. parabrevis KCCM 41421, which possesses enzymes capable of breaking down proteins, liquefied gelatin, whereas P. acidilactici SY21 and P. acidilactici SY22 maintained the gelatin in its solid form without liquefaction (Fig. 6B). When evaluating indole production, P. acidilactici SY21 and P. acidilactici SY22 showed no color change, whereas the positive control, E. coli ATCC 10536, exhibited a red ring at the top of the tube, indicating indole production. Therefore, P. acidilactici SY21 and P. acidilactici SY22 did not produce indoles (Fig. 6C).

Fig. 6.

Fig. 6

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Toxic metabolite production. A Urease activity; B gelatin liquefaction assay; C indole production. 1. positive control (A Proteus vulgaris KCCM 40211; B Brevibacillus parabrevis KCCM 41421; C Escherichia coli ATCC 10536), 2. Pediococcus acidilactici SY21 and 3. P. acidilactici SY22

According to Cho et al. (2024), none of 12 P. pentosaceus strains produced gelatinases or ureases. Gelatinases and ureases are enzymes associated with probiotic pathogenicity. Gelatinases degrade gelatin and collagen, and urease increases the pH by producing ammonia, contributing to bacterial virulence (Collins and D’Orazio, 1993). Urease-related ammonia production can lead to serious health issues, such as kidney and liver failure, as well as nephrotic syndrome (Collins and D’Orazio, 1993). When tryptophan is broken down by tryptophanase, pyruvate, ammonia, and indole are produced (Watanabe and Snell, 1972). Indole plays an important role in microbial community signaling and affects the human digestive and immune systems. However, at high concentrations, indole can act as a toxic substance in the gut, causing inflammation or disrupting intestinal cell function. Gupta and Sharma (2017) reported that none of their isolates, including Lactobacillus and Pediococcus, exhibited urease activity or indole production.

Although P. acidilactici is less commonly used as a commercial probiotic than Lactobacillus or Bifidobacterium species, certain strains have demonstrated promising health-promoting effects, including immunomodulation and pathogen inhibition. In this study, P. acidilactici SY21 and P. acidilactici SY22, which were previously confirmed to exert anti-inflammatory effects, were selected to investigate their safety. While MIC tests confirmed resistance to kanamycin and clindamycin in P. acidilactici SY21 and resistance to kanamycin in P. acidilactici SY22, genomic analysis showed that both strains possessed intrinsic resistance without transferable antibiotic resistance genes and lacked virulence genes associated with pathogenic bacteria, confirming their safety. P. acidilactici SY21 and P. acidilactici SY22 showed negative results for hemolysis and mucin degradation, and no cytotoxicity to Caco-2 cells, confirming their non-pathogenic nature. Toxic reactions, such as ammonia and indole production and gelatin liquefaction, were not observed. In addition, negative responses were observed for bile salt deconjugation, biogenic amine production, and β-glucuronidase activity. Based on these results, P. acidilactici SY21 and P. acidilactici SY22 have been proposed as safe probiotics for human consumption, with demonstrated anti-inflammatory activity and favorable safety profiles. These findings support their potential application as probiotic candidates with the possibility of attaining GRAS status and contribute novel insights into the probiotic potential of underexplored P. acidilactici strains through comprehensive phenotypic and genomic evaluation.

Supplementary Information

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Supplementary file1 (PDF 228 KB) (228.4KB, pdf)

Acknowledgements

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry through the High Value-Added Food Technology Development Program funded by the Ministry of Agriculture, Food, and Rural Affairs (grant number: 321035052HD020).

Funding

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (Grant No. 321035052HD020).

Declarations

Conflict of interest

The authors declare no conflict of interest.

Footnotes

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