**Background:** Aflatoxin B1 (AFB1), a Class I carcinogen produced by Aspergillus flavus and Aspergillus parasiticus, is a ubiquitous contaminant in poultry feed that causes hepatotoxicity, immunotoxicity, and impaired performance. Biological detoxification using probiotic microorganisms offers a promising approach due to its specificity and safety. This study aimed to evaluate two novel bacterial-based detoxifiers and compare them with a commercial mycotoxin deactivator in laying hens.
**Methods:** AFB1 was produced by fermenting Aspergillus parasiticus PTCC-5286 on rice, yielding a powder that was added to basal diets to achieve 500 ppb AFB1. Eighty-four 32-week-old Bovans white laying hens (initial BW 1,530 ± 50 g) were allocated to 5 treatment groups with 6 replicate cages (2 birds/cage) for a 12-week feeding trial: negative control (NC, no AFB1, no detoxifier), positive control (PC, 500 ppb AFB1, no detoxifier), MF (500 ppb AFB1 + 2 g/kg Mycofix Plus MTV INSIDE containing Trichosporon mycotoxinivorans), MTA (500 ppb AFB1 + 2 g/kg Magnotox-alphaA, without detoxifying bacteria), and MTB (500 ppb AFB1 + 2 g/kg Magnotox-alphaB, containing Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans at 10^12, 10^11, 10^9 cfu/kg respectively, and Bifidobacterium bifidum at 10^9 cfu/kg). Performance parameters, egg quality, liver fat content, ileum microbiota, serum biochemistry, β-carotene, vitamin A, and malondialdehyde (MDA) concentrations were measured.
**Key Results:** In vitro, the bacterial consortium reduced zearalenone by 98.8% and patulin by 94.5% within 1 hour, and achieved 80.1% reduction of AFB1 after 24 hours. In vivo, the PC group showed significantly impaired performance: lowest egg weight (53.80 g vs. 60.01 g in NC, P<0.05), lowest average daily feed intake (77.67 vs. 91.11 g/d, P<0.05), lowest egg production (68.83% vs. 90.66%, P<0.05), lowest egg mass (39.64 vs. 54.33 g, P<0.05), and poorest feed conversion ratio (1.98 vs. 1.68, P<0.05). The MTB group exhibited the best performance among AFB1-treated groups, with egg production of 95.74% (P<0.05), egg mass of 57.55 g (P<0.05), and FCR of 1.62 (P<0.05), comparable to or exceeding the NC group. Eggshell strength was significantly reduced in PC (3.84 kg) and MTA (3.94 kg) compared to NC, MF, and MTB (4.39, 4.38, and 4.37 kg respectively, P<0.05). Haugh unit was lowest in PC (67.9, P<0.05) and highest in MTB (83.95). Liver fat content was highest in MF (48.19%) and PC (42.21%), while MTB (35.39%) approached NC levels (30.64%, P<0.05). Serum β-carotene was significantly lower in NC, PC, and MTB (12.79, 12.98, and 12.02 μg/dL) compared to MF and MTA (20.16 and 21.90 μg/dL, P<0.05). Serum vitamin A was lowest in PC (19.39 μg/dL) and highest in MTA (40.81 μg/dL) and MF (33.83 μg/dL, P<0.05). MDA concentrations were significantly elevated in PC across all samples (P<0.05), while MF, MTA, and MTB groups showed reduced levels. Ileum microbiota analysis revealed that MF and MTB significantly reduced E. coli populations (4.35 and 4.48 log10 CFU/g, P<0.05) compared to MTA (5.84 log10 CFU/g). Serum glucose was significantly depressed in PC (137.49 mg/dL) compared to all other groups (210.06–242.50 mg/dL, P<0.05), and triglycerides were also lowest in PC (383.47 mg/dL vs. 778.54–1,099.53 mg/dL in other groups, P<0.05).
**Clinical Implications:** The bacterial consortium in MTB effectively mitigated AFB1-induced toxicosis in laying hens, restoring performance, egg quality, and liver health to levels comparable to uncontaminated controls. The multi-species approach (Bacillus spp. and Bifidobacterium bifidum) appeared more effective than the commercial product or the detoxifier without bacteria, particularly for egg production, egg mass, and liver fat reduction. These findings support the use of carefully formulated probiotic consortia as a practical, safe strategy for mycotoxin mitigation in poultry production, though further research is needed to elucidate the specific enzymatic mechanisms and identify optimal bacterial combinations.