Western honeybees (Apis mellifera L.) face elevated colony mortality from pathogens (Paenibacillus larvae, Nosema spp., Melissococcus plutonius, Aspergillus spp.), varroa mites, viruses, and insecticides including coumaphos, chlorpyrifos, and neonicotinoids. Antibiotic use in beekeeping (e.g., tylosin against American foulbrood) can induce dysbiosis and harm pollinators. Lactic acid bacteria (LAB) are Generally Recognized as Safe (GRAS) probiotic microorganisms that strengthen immunity, modulate microbiota, inhibit pathogens via organic acids and bacteriocins, and can detoxify pesticides. The study aimed to characterize 10 previously selected LAB strains for honeybee probiotic development.
Ten LAB isolates—Pediococcus acidilactici (4/1, 11/3, 18/1, 21/1), Pediococcus pentosaceus (5/2, 7/1, 14/1, 25/1, OK-S), and Lacticaseibacillus casei 12AN—were sourced from honeybee-related environments (flowers, honey, fermented cucumbers, human feces). Apilactobacillus kunkeei DSM 12361 served as the honeybee-derived reference strain. Assays included: (1) organic acid profiling by HPLC; (2) survival in simulated gastric (pepsin, α-amylase, pH 3.0) and intestinal (bile salts, pancreatin, pH 7.5) juices; (3) survival in three sugar syrups (A, B, C) at 20 °C for 24–48 h; (4) cytotoxicity of cell-free supernatants (CFS) at 1–20% on Caco-2 cells via Neutral Red Uptake; (5) protective activity of P. pentosaceus 14/1 CFS (0.1 and 1 mg/mL) against chlorpyrifos (25, 50 µg/mL) and coumaphos (12.5, 25 µg/mL) in Caco-2 cells; (6) antibiotic susceptibility by disc diffusion against 15 antibiotics; (7) hemolysis on blood agar; (8) mucin degradation; (9) bile salt hydrolase (BSH) activity; (10) hydrogen peroxide production; and (11) mutual antagonism via agar slab method.
ORGANIC ACID PROFILES
All 10 LAB strains produced lactic, acetic, and malonic acids. The highest combined acid production (>300 µg/mL) was by A. kunkeei DSM 12361, P. pentosaceus 14/1, and P. acidilactici 18/1. P. acidilactici 18/1 produced the highest lactic acid concentration. Butyric acid was not detected in P. acidilactici 18/1 and P. pentosaceus 25/1. Propionic acid was not produced by A. kunkeei DSM 12361. P. pentosaceus 14/1 yielded the highest propionic and butyric acids.
GASTROINTESTINAL SURVIVAL
All isolates maintained viable counts above 10^4 CFU/mL after sequential gastric and intestinal exposure. P. acidilactici 4/1, 18/1, and P. pentosaceus 5/2 showed significantly decreased gastric survival vs. reference (p = 0.0008, p = 0.0007, p = 0.0006, Independent Samples t-tests), while P. acidilactici 11/3 was significantly increased (p = 0.049). No statistically significant differences were observed in intestinal juice viability (p > 0.05).
SUGAR SYRUP SURVIVAL
After 48 h, all strains maintained >10^6 CFU/mL. Viability in Syrup A ranged from 6.19 log CFU/mL (P. acidilactici 18/1) to 8.72 log CFU/mL (L. casei 12AN); P. pentosaceus 5/2, P. acidilactici 18/1, and 21/1 showed significant decreases (p = 0.017, p = 0.026, p = 0.008). P. pentosaceus 7/1 reached 7.91 log CFU/mL in Syrup B after 48 h.
CYTOTOXICITY
At 20% CFS concentration, Caco-2 viability was highest for P. pentosaceus 5/2 (89.81% ± 8.74%) and 25/1 (75.65% ± 15.2%), comparable to the reference strain A. kunkeei DSM 12361 (78.72% ± 6.66%).
INSECTICIDE CYTOPROTECTION
CFS from P. pentosaceus 14/1 at 1 mg/mL reduced coumaphos cytotoxicity by 19.32% (at 12.5 µg/mL) and 17.23% (at 25 µg/mL), with the 25 µg/mL reduction reaching statistical significance (p = 0.003, Kruskal–Wallis test). Chlorpyrifos cytotoxicity was only slightly reduced at 0.1 mg/mL CFS.
SAFETY PROFILE
No strain exhibited β-hemolytic activity, mucin degradation, hydrogen peroxide production, BSH activity, or mutual antagonism. Antibiotic resistance was strain-dependent; greatest resistance was shown by P. acidilactici 4/1 (also resistant to lincomycin), while chloramphenicol produced the strongest growth inhibition.
Four strains—P. acidilactici 21/1, P. pentosaceus 5/2, P. pentosaceus 14/1, and L. casei 12AN—demonstrated the most favorable combination of probiotic properties for honeybee application. Their tolerance to sugar syrups supports practical delivery via beekeeper supplementation. The cytoprotective activity of P. pentosaceus 14/1 CFS against coumaphos highlights a novel mechanism for mitigating insecticide toxicity relevant to honeybee and potentially human health through contaminated hive products. In vivo confirmation is required before field deployment.