Strains LC86 and LC89 were sourced from Wecare Probiotics Co., Ltd (Suzhou, China), cultivated under anaerobic conditions at 37°C using De Man-Rogosa-Sharpe medium (MRS, Qingdao Hi-Tech Industrial Park Hope Bio-Technology Co., Ltd, Qingdao, China). Two strains of Lactobacillus isolated from traditional yogurt and fermented foods were identified through a comprehensive analysis of cellular morphology, physiological and biochemical characteristics, 16S rRNA (Choudhary et al., 2019) gene sequences, dnaK gene sequences, and other experimental data, referring to the “Bergey's Manual of Systematic Bacteriology” and related research (2002). The two strains were identified as Lacticaseibacillus paracasei and Lacticaseibacillus casei, respectively. The strains were inoculated into a 25% (v/v) glycerol solution and stored at −80°C for preservation.

Employed a sophisticated nucleic acid extraction method to extract genomic DNA from LC86 and LC89, then sequenced them using the PacBio Sequel II and MGI G99 platform (Beijing Genomics Institute, Shenzhen, Guangdong, China). Predicted tRNA genes across the entire genome using tRNAscan-SE and predicted rRNA genes using Barrnap (http://www.vicbioinformatics.com/software.barrnap.shtml). The prediction of other non-coding RNAs was primarily obtained by comparison with the Rfam database; CRISPR elements were predicted using CRISPR finder, gene prediction for the whole genome sequence was performed using the GeneMarkS software. Performed average nucleotide identity (ANI) analysis using fastANI.

For an in-depth analysis of antibiotic resistance genes and virulence factors within the genomes, we aligned and screened all identified coding sequences (CDSs) using the following databases: the Comprehensive Antibiotic Resistance Database (CARD), ResFinder (on the foundation of the ARDB database), and AMRFinderPlus (NCBI Antimicrobial Resistance Gene Finder Plus). Additionally, we used ISEScan v.1.7.2.1 to identify the insertion sequence. In the screening for virulence factors, we conducted analysis using VirulenceFinder 2.0.5 (https://cge.food.dtu.dk/services/VirulenceFinder/) and the Virulence Factor Database (VFDB, http://www.mgc.ac.cn/VFs/). The VFDB, developed by the Chinese Academy of Medical Sciences, collects information on the composition, structure, function, pathogenesis, virulence islands, sequences and genomes of over 100 important pathogenic bacteria. Furthermore, we utilized the PathogenFinder 1.1 database (https://cge.food.dtu.dk/services/PathogenFinder/) to evaluate bacterial pathogenicity. We searched for biogenic amine synthesis genes on the LC86 and LC89 genome in the NR and KEGG databases, assessed genes/pathways related to the production of harmful metabolites in LC86 and LC89 through KEGG searches. Ultimately, to visually display the genome structure of LC86 and LC89, we generated circular genomic plots using cgview.

Conducted tolerance tests on strains LC86 and LC89 for temperature, acidity, artificial gastric fluid, artificial intestinal fluid and bile salts. The acid pH of the MRS liquid medium was adjusted to 2.5, 3.0 using HCl (Zhang et al., 2022). The artificial gastric juice was prepared by dissolving 0.1 g of pepsin (1:10,000, Sigma, St. Louis, MO, USA) in 10 ml of NaCl solution (0.5%, w/v), adjusted the pH to 2.5, 3.0 with HCl, filter sterilize. Prepared artificial intestinal juice by dissolving 0.1 g of trypsin (Sigma, St. Louis, MO, USA) in 10 ml of a 0.5% (w/v) NaCl solution, adjusted the pH to 8.0 with NaOH, filter sterilize. To prepare bile salt, MRS broth contained 0.1% pig bile salt (Sigma-Aldrich Corp., St. Louis, MO, USA) and then sterilized.

Strains LC86 and LC89 were cultured in MRS medium and centrifuged at 4°C (10,000 × g, 10 min). The pellet was resuspended in MRS liquid medium and incubated at temperatures of 30, 37, 42, 45°C for 24 h. After incubation, the turbidity of the culture broth was observed. The supernatant was discarded, the pellet was resuspended in 10 ml of either acidic MRS liquid medium, artificial gastric juice, artificial intestinal juice, or bile salt solution. The mixtures were thoroughly vortexed, and the viable bacterial count was assessed by plating at time zero (0 h), establishing the initial count (N0) as the control. Subsequently, the mixtures were incubated at 37°C for 2 h (Chen et al., 2024), after which the viable bacterial count was determined again using the same plating method. The survival rate was calculated using the formula: Survival rate/% = lgNi/lgN0 × 100, where Ni represents the viable bacterial count after “I” h of treatment. This procedure was conducted in triplicate to ensure reliability of the results.

The fermentation broth cytotoxicity and bacterial cell toxicity for LC86 and LC89 were assessed (Hardy et al., 2018). The bacterial cell cytotoxicity was assessed using the Lactate Dehydrogenase Cytotoxicity Assay Kit (Promega, USA), while the fermentation broth cytotoxicity was evaluated with the CCK-8 Assay Kit (TargetMol, USA). Seeded Caco-2 (Lee et al., 2024) cells at a density of 1.5 × 10⁴ cells per well in a 96-well plate to assess fermentation broth cytotoxicity. Centrifuged the bacterial fermentation liquid (5,000 × g, 10 min), then collected the supernatant, adjust the pH to 7.4 using 5 M NaOH, filtered it through a 0.22 μm filter. Performed the same procedure with fresh MRS medium. Prepared Minimum Essential Medium [MEM, Thermo Fisher Scientific (China) Co., Ltd, Shanghai, China] with 20% Fetal Bovine Serum [FBS, Thermo Fisher Scientific (China) Co., Ltd, Shanghai, China], supplemented with either 10% or 20% of the bacterial supernatant and also prepared MEM with 20% FBS containing 10% or 20% of the fresh MRS medium as a control. Replaced the Caco-2 cell culture medium with the prepared supernatant media and incubated for 24 h. After incubation, added the CCK-8 solution and incubated for an additional 3 h. Measured the optical density at 450 nm (OD₄₅₀) to determine the cell viability. To evaluate Bacterial Cell Toxicity, began by seeding Caco-2 cells at a rate of 1.5 × 10⁴ cells per well in a 96-well plate. Next, centrifuged the bacterial suspension (5,000 × g, 10 min), then pelleted the bacterial cells and resuspend them in 1 ml of MEM medium containing 20% FBS. Created a series of dilutions to achieve bacterial suspensions with concentrations of 10⁷, 10⁸, 10⁹ CFU/ml. Prior to the assay, washed the Caco-2 cells with PBS to clear away the culture medium and any dead cells, repeating the wash twice. Subsequently, added 200 μl of each bacterial suspension to the wells containing the Caco-2 cells, with quadruplicate wells for each concentration, incubated under conditions of 37°C and 5% CO₂. At intervals of 4 and 8 h post incubation, collected 50 μl of the culture supernatant from each group and assessed cell toxicity employing the LDH-Glo cytotoxicity assay kit, which utilizes a chemiluminescence detection method.

Determined the adhesive properties of LC86 and LC89 to Caco-2 cells (Lee et al., 2024). To assess the adhesive properties of LC86 and LC89 to Caco-2 cells, seeded the cells at a density of 3 × 10⁵ cells per well in a six-well plate. After centrifuging (5,000 × g, 10 min) the bacterial suspension, resuspended the pellet in 10 ml of MEM supplemented with 20% FBS (without antibiotics) and adjusted the concentration to 10⁸ CFU/ml. Washed the Caco-2 cells with PBS to remove the MEM medium, repeating the wash twice. Added 1 ml of the bacterial suspension to each well, with duplicates for each group and incubated at 37°C for 4 h. Use MEM with 20% FBS (without antibiotics) as a control. After incubation, washed the wells with Phosphate-Buffered Saline [PBS, Thermo Fisher Scientific (China) Co., Ltd, Shanghai, China] five times to remove non-adherent bacteria. Add 1 ml of 0.3% Triton-X100 lysis solution to each well, incubated at room temperature for 15 min, then added 1 ml of MRS liquid medium and mixed thoroughly. Performed serial dilutions of the lysate (1:100, 1:1,000) and spread 400 μl onto MRS agar plates. After anaerobic incubation at 37°C for 24 h, counted the digested Caco-2 cells in the control wells to determine the total cell count (N_c). Counted the colonies on the MRS plates to calculate the total number of adhering bacteria (N_b) using the formula: N_b = (average number of colonies) × (2 ml/0.4 ml) × (dilution factor). Finally, calculated the average number of probiotic bacteria adhering to every 100 Caco-2 cells as N_b/N_c × 100.

Strains LC86 and LC89 were inoculated on Columbia blood agar plates (Qingdao Hi-Tech Industrial Park Hope Bio-Technology Co., Ltd, Qingdao, China) containing 5% (w/v) fresh sheep blood and incubated at 37°C for 48 h. Subsequently, the plates were examined to determine if hemolysis rings were present. A viridans hemolysis ring indicated that an organism is α-hemolytic, whereas a transparent hemolysis ring indicated that it was β-hemolytic. The assay was conducted in triplicate on three separate occasions (Chen et al., 2024; Arellano et al., 2020).

Performed the catalase test by taking a colony from the cultured strain and spreading it onto a microscope slide. Applied a drop of 3% hydrogen peroxide (H₂O₂, Sinopharm Chemical Regent Co., Ltd., Shanghai, China) to the colony. Observed for the production of bubbles. The presence of bubbles signified a positive catalase reaction, while their absence indicated a negative result.

Prepared the MRS agar medium by incorporating 0.5% sodium deoxycholate, 0.2% sodium mercaptoacetate, 0.2% calcium chloride. After inoculating the test strain onto the plates, incubated them at 37°C under anaerobic conditions for 72 h to observe bile salt precipitation. The presence of precipitated bile salts indicated that the test strain possessed bile salt hydrolysis activity.

Inoculated the test strain into a gelatin biochemical tube and incubated it at 37°C under anaerobic conditions for 24 h. Following incubation, chill both the inoculated test tube and an uninoculated control tube at 4°C for 30 min. Then, assessed for gelatin liquefaction to determine the strain's activity.

Subcultured the strain through two successive generations and streak-inoculated it onto Columbia blood agar plates. Transferred bacterial colonies to sterile saline to achieve a turbidity of ~3.75, which corresponds to a concentration of around 10⁸ CFU/ml, using a McFarland turbidity meter (Zhengzhou Bioda Instrument Co., Ltd., Henan, China) for calibration. Pipetted 2 drops (~0.06 ml) of the bacterial suspension into both arginine hydrolysis biochemical tubes and amino acid control biochemical tubes. Overlayed the medium's surface with 5–8 drops of sterilized liquid paraffin to prevent evaporation and oxidation. Incubated the tubes at 37°C for 72 h. Interpreted the results as follows: a blue-green color in the test tube and a yellow color in the control tube indicated a positive reaction. If both tubes turned yellow, the result was negative.

Prepared two batches of 50 CHL culture medium: one with 0.3% mucin (Sigma–Aldrich, Inc., MO, USA) and another without mucin. Inoculated each batch with a 1% bacterial suspension and incubated at 37°C under anaerobic conditions for 48 h. For analysis, took 75 μl of the culture liquid, mixed it with 25 μl of 4 × sample buffer, heated the mixture in a 100°C water bath for 5 min. Subsequently, performed SDS–PAGE electrophoresis and visualized the proteins by staining with Coomassie Brilliant Blue (Abe et al., 2010).

Prepared a series of standard solutions containing a mixture of biogenic amines (spermine, spermidine, cadaverine, putrescine, tryptamine, histamine) at concentrations ranging from 0 to 50.0 mg/L in increments of 5.0 mg/L, using 0.1 mol/L hydrochloric acid as the diluent. For the sample preparation, extracted the LC86/LC89 fermentation liquid with an equal volume of 10% (M/V) trichloroacetic acid for 1 h. Filter the mixture through double-layer filter paper and transferred 2 ml of the filtrate to a derivatization vial. To the vial, added 1 ml of 2 mol/L NaOH to adjust the pH to alkaline conditions. Then, added 10 μl of benzoyl chloride and derivatized the sample in a 30°C water bath for 20 min. To halt the derivatization, added 2 ml of saturated NaCl and heated the mixture at 60°C for 5 min. After cooling, added 3 ml of ether, shook well to mix, allowed the layers to separate. Transferred the upper organic phase to a clean test tube. Evaporated the ether under a stream of nitrogen to dryness and then dissolved the residue in 1 ml of methanol. Finally, injected 20 μl of this solution into the HPLC system to determine the biogenic amine content.

Used the D-Lactate Enzyme Assay Kit (r-biopharm, Germany) and the D/L-Lactate Enzyme Assay Kit (r-biopharm, Germany) to test the ability of LC86 and LC89 to produce D-lactic acid and L-lactic acid, respectively. Performed the assays using the fermentation broth of LC86 and LC89. D-Lactic Acid: Added 100 μl of distilled water to 2,000 μl of reagent 1 to serve as the reagent blank. Added 100 μl of bacterial liquid to 2,000 μl of reagent 1 as the sample solution. Thoroughly mixed and incubated at 37°C for 3 min. Then measured the absorbance (A1). Subsequently, added 500 μl of reagent 2, mixed well again, incubated at 37°C for 10 min before measuring the absorbance (A2). D-/L-Lactic Acid: added 100 μl of distilled water to 2,000 μl of reagent 1 as the reagent blank. Added 100 μl of bacterial liquid to 2,000 μl of reagent 1 as the sample solution. After mixing well, incubated at 37°C for 1 min and measured the absorbance (A1). Then, added 500 μl of reagent 2, mixed thoroughly, incubated at 37°C for 10 min to measure the absorbance (A2). The experiment was conducted in triplicate and the average value was taken.

The antibiotic susceptibility of strains LC86 and LC89 was assessed in accordance with the European Food Safety Authority (EFSA) guidance (European Food Safety Authority, 2018), which encompass testing for eight antibiotics: ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol (Sigma-Aldrich Corp., St. Louis, MO, USA). As per the EFSA guidelines, testing for vancomycin sensitivity was not required. The bacterial solution was adjusted to a concentration of 3 × 10⁵ CFU/ml using saline solution. Antibiotics were then added at varying concentrations through a serial dilution process. The minimum inhibitory concentration (MIC) was identified as the lowest antibiotic concentration that completely inhibits bacterial growth, with no visible increase in cell numbers observed.

The evaluation of the acute oral toxicity of Lacticaseibacillus paracasei LC86 and Lacticaseibacillus casei LC89 was conducted in accordance with the GB 15193.3-2014 [“Guobiao” (Chinese National Standard), National Food Safety Standard—Acute Oral Toxicity Test]. Selected 6-week-old SPF-grade ICR mice from Shanghai Shenchang Biotechnology Co., Ltd. (SCXK (Shanghai) 2021-0002, Shanghai, China), with a body weight of 18–22 g. The feed was provided by Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd. [Su Feed Approval (2019) 01008, Nanjing, China], in compliance with the relevant provisions of GB/T 14924.1-2001 [“Guobiao” (Chinese National Standard), Laboratory animals—General quality standard for formula feeds]. This experiment was approved by the Animal Ethics Committee of Technical Center for Animal Plant and Food Inspection and Quarantine of Shanghai Customs [permit number: SYXK (Shanghai) 2019-0033]. All experimental mice were housed in an SPF animal room with free access to food, under a temperature of 20–22°C and a relative humidity of 45%−65%. Twenty mice (half male and half female, unmated and non-pregnant female mice) were all subjected to a 5-day period of environmental adaptation and quarantine observation in the experimental setting. The mice were fasted (with unrestricted access to water) for 6 h before the experiment. A single-limit method was used and the test substance (LC86, LC89) was administered orally at a dose of 2 × 10¹⁰ CFU/kg body weight (BW), with a maximum gavage volume of 20 ml/kg BW. The general condition, behavioral changes, signs of poisoning, mortality of the animals were observed twice daily. The animals were weighed once every 7 days. Gross necropsy and observation were conducted on animals that died from poisoning and on those that survived for 14 days post-exposure.

All data in this article were analyzed using SPSS 26.0 software and were represented as mean ± standard deviation (SD). Group differences were assessed utilizing one-way Analysis of Variance (ANOVA) to determine the presence of statistically significant variations across the study cohorts. If the ANOVA revealed significant effects, Tukey's Honestly Significant Difference (HSD) test was employed for pairwise comparisons between groups, allowing for the identification of specific inter-group disparities. The threshold for statistical significance was set at a p-value of < 0.05, which denoted that observed differences between the experimental and control groups were unlikely to occur by chance and are thus considered to be meaningful.

The chromosome length of LC86 was 3,102,337 base pairs (bp) and the plasmid length was 48,752 bp (Figures 1A, B). The complete genome GC content of LC86 was 46.34%, which was essentially consistent with the 46.5% GC content of the L. paracasei strains SMN-LBK, CBA3611, TCI727 and JCM 8130, as completed and sequenced in National Center for Biotechnology Information (NCBI). According to the analysis results of Average Nucleotide Identity (ANI), the ANI value between LC86 and L. paracasei SMN-LBK (whole genome accession number: GCF_024498315.1) was 99.99%, confirming this strain as Lacticaseibacillus paracasei, which was consistent with the results of bacterial identification. Using the AMRFinder, CARD and ResFinder databases for gene searches, no potential antibiotic resistance genes were detected in either the chromosome or the plasmid. Potential virulence-related genes were searched using the VirulenceFinder and VFDB databases, with no virulence-related genes identified in the chromosome or the plasmid. Pathogenicity gene analysis was conducted with the PathogenFinder database, confirming that this strain was non-pathogenic to humans. By searching the LC86 genome for biogenic amine synthesis genes using the KEGG and NR databases, the gene for ornithine decarboxylase was identified, located at positions 1,817,943–1,820,033 bp on the genome. Additionally, KEGG assessment of LC86 for genes related to the production of metabolic products indicates that LC86 did not contain bacterial toxins. LC86 did contain genes associated with the metabolic products of pyruvate, such as D-lactate dehydrogenase, and with the metabolic products of ornithine, such as ornithine decarboxylase. However, LC86 lacked genes related to the metabolic products of primary and secondary bile acids.

The chromosome length of LC89 was 2,940,964 bp, and the plasmid length was 28,032 bp (Figures 1C, D). The complete genome GC content of LC89 was 47.86%, which was essentially consistent with the 48% GC content of the L. casei strains ATCC393 and LC130, as completed and sequenced in NCBI. According to the analysis results of ANI, the ANI value between LC89 and L. casei ATCC393 [whole genome accession number: AP012544.1 (Chromosome), AP012545.1 (Plasmid 1), AP012546.1 (Plasmid 2)] was 99.95%, confirming this strain as Lacticaseibacillus casei, which was consistent with the results of bacterial identification. No antibiotic resistance or virulence genes were found in its genome. The strain was non-pathogenic to humans. LC89 had genes for ornithine decarboxylase and D-lactate dehydrogenase but lacked genes for primary and secondary bile acid metabolism. The gene for ornithine decarboxylase was identified, located at positions 1,600,679–1,602,769 bp on the genome.

Strains LC86 and LC89 demonstrated survival rates higher than 95% after being cultured for 2 h in acidic environments (pH 2.5, 3.0) and in artificial gastric juice (pH 2.5, 3.0). Furthermore, the survival rate of LC86 in artificial intestinal juice and bile salts were 99.93% and 96.89%, respectively, while the survival rates for LC89 in artificial intestinal juice and bile salts were 98.42% and 99.90% (Table 1). These findings indicated that both LC86 and LC89 possessed strong tolerance to artificial gastric juice, artificial intestinal juice, and bile salts. The ability of strains LC86 and LC89 to grow at temperatures of 30, 37, 42, 45°C indicated that these two strains had a broad temperature adaptation range. Their temperature tolerance should have been taken into consideration during production and storage processes to ensure product quality and safety (Table 2).

The experimental data indicated that Caco-2 cells cultured in 10% or 20% fermentation broth of the LC86 and LC89 bacterial strains for 3 h showed good survival, when Caco-2 cells were co-cultured with the bacterial cells of LC86 and LC89 at three different concentrations (10⁷, 10⁸, 10⁹ CFU/ml) for 4 and 8 h, there was no significant difference compared to the control group cultured in the medium alone (P > 0.05); this suggested that the bacterial cells and fermentation broth of LC86 and LC89 were non-toxic to Caco-2 cells (Figure 2).

The cell adhesion assay results showed that the total cell count was N_c = 6.365 × 10⁴ CFU. For the adhered bacteria, the count was N_b = 38,250 CFU for LC86 and N_b = 19,750 CFU for LC89. The amount of LC86 bacteria adhering to every 100 cells was N_b/N_c × 100 CFU = 60.09 CFU. Similarly, the amount of LC89 bacteria adhering to every 100 cells was N_b/N_c × 100 CFU = 31.03 CFU. The study above demonstrated that LC86 and LC89 had good adhesive properties to Caco-2 cells. LC89 exhibited slightly lower adhesion ability to Caco-2 cells compared to LC86.

No hemolytic zones were observed around the LC86 and LC89 colonies grown on Columbia blood agar plates, indicating hemolytic negativity (Figure 3).

Under the conditions of this test, catalase activity was negative in LC86 and LC89.

The precipitation of white bile salts around the bacteria growing on the medium indicated that strains LC86 and LC89 possess bile salt hydrolysis capability (Figure 4).

Under the conditions of this test, LC86 and LC 89 were negative for gelatin hydrolysis.

After the experiment, it was observed that both the test tube and the control tube turned yellow, indicating that LC86 and LC89 showed a negative result for arginine hydrolysis.

The experimental results showed that, compared to the uninoculated medium, LC86 and LC89 did not reduce the mucin zone. This indicated that LC86 and LC89 did not have mucin degradation activity.

The concentrations of tryptamine, putrescine, cadaverine and spermidine in the LC86 fermentation liquid were 2.73, 1.59, 1.98, 2.30 mg/L, respectively. Histamine and spermine were not detected, with concentrations below the detection limit of the method (1 mg/L). In the LC89 fermentation liquid, the concentrations of putrescine, cadaverine, and spermidine were measured at 1.38, 1.85, 2.37 mg/L, respectively. The absence of tryptamine, histamine, spermine suggested that their levels were below the detection limit of the method (1 mg/L).

The L-lactic acid content in the fermentation broth of LC86 and LC89 was 0.96 and 0.891 g/L, respectively, while the D-lactic acid content was 0.023 and 0.035 g/L, respectively. The D-lactic acid content of both strains was significantly lower than that of L-lactic acid (Supplementary Table S1).

The minimum inhibitory concentrations (MICs) of various antibiotics against LC86 were found to be 0.5 μg/ml for ampicillin, 8 μg/ml for gentamicin, 32 μg/ml for kanamycin, 32 μg/ml for streptomycin, 0.125 μg/ml for erythromycin, 0.125 μg/ml for clindamycin, 0.5 μg/ml for tetracycline, 2 μg/ml for chloramphenicol. The MIC of LC89-Ampicillin was 0.032 μg/ml, LC89-Kanamycin was 32 μg/ml, LC89-Gentamicin was 8 μg/ml, LC89-Streptomycin was 16 μg/ml, LC89-Erythromycin was 0.032 μg/ml, LC89-Clindamycin was 16 μg/ml, LC89-Tetracycline was 1 μg/ml, LC89-Chloramphenicol was 2 μg/ml (Supplementary Table S2). The determined MICs for LC86 and LC89 were at or below the species-specific breakpoints defined by the EFSA guidance, demonstrated that the strain is susceptible to pertinent antibiotics.

During the 14-day acute oral toxicity study in mice, no signs of poisoning or mortality were observed in the test animals. There were no abnormal changes in weight gain for either male or female animals. At the end of the observation period, a gross pathological dissection was performed on the test animals, and no pathological changes were found in any organs. The oral LD₅₀ of both LC86 and LC89 in mice were >2 × 10¹⁰ CFU/kg (Table 3).