Ability of Lactobacillus plantarum MON03 to mitigate aflatoxins (B₁ and M₁) immunotoxicities in mice

Figures & data

Table 1. Gastric juice tolerance and Caco-2 adherence of LAB strains (log cfu/ml).

		Resistance to gastric juice
Lactobacillus strain	Initial mean counts	pH 2	pH 3	Caco-2 adhesion^a
L. plantarum MON03	9.77 ± 0.19	9.23 ± 0.46	9.16 ± 0.56	466.3 ± 109.1
L. rhamnosus GG^b	9.92 ± 0.18	9.21 ± 0.47	9.13 ± 0.58	493.2 ± 97.3

^aNumber of lactobacilli strains adhered to Caco-2 cells in 20 random microscopic fields.

^bLactobacillus rhamnosus GG: used as control.

Each value shown is the mean ± SD from three trials.

Table 2. Characterization of bacterial surface—adhesion to solvent (microbial adhesion to solvents method).

	% adhesion to solvent
	L. plantarum MON03		E. coli ATCC 10536
Solvent	Live biomass	Dead biomass	Live biomass
Hexadecane	7.63 ± 0.35	38.52 ± 0.05	21.13 ± 0.09
Chloroform	59.30 ± 0.24	42.91 ± 0.32	8.78 ± 0.06
Ethyl acetate	12.51 ± 0.05	23.43 ± 0.41	30.85 ± 0.28

Table 3. Percentage AFB₁ and AFM₁ removal from PBS by LP alone.

	AFB1 (50 µg/ml)			AFM1 (50 µg/ml)
Treatment	0 h	12 h	24 h	0 h	12 h	24 h
PBS only	2.3 ± 0.1	2.1 ± 0.2	2.4 ± 0.1	3.0 ± 0.2	2.4 ± 0.2	2.3 ± 0.2
Live LP	20.1 ± 4.1*	54.3 ± 7.3*	82.3 ± 8.3*	25.9 ± 5.8*	64.5 ± 5.2*	89.1 ± 6.2*
Killed LP	18.1 ± 0.4	32.6 ± 0.7	39.8 ± 0.4	23.1 ± 0.3	42.5 ± 0.6	52.2 ± 0.5

To determine the effect of bacteria viability on the binding affinity to AFB₁ and AFM₁, ‘killed’ bacteria (10⁸ CFU/ml) were heated at 90°C for 15 min before being used in the assay.

Values shown are mean ± SD from three determinations per experimental scenario tested.

*All values shown for bacterium-containing systems are significantly different from both control (PBS only and heated bacteria) at p ≤ 0.05.

Figure 1. Effect of treatments on feed consumption by Balb/c mice. Results shown are mean ± SD. In each histogram, values (bars) with superscripts bearing different letters differ significantly (p < 0.05).

Table 4. Body weight gain and spleen and thymus indexes in the mice.

	Body weight (g)			Organ indexes
Treatment	Initial	Final	Net weight gain (g)	Spleen	Thymus
Control	25.90 ± 1.21	34.01 ± 1.60	8.93 ± 0.39	0.697 ± 0.028	0.053 ± 0.005
LP alone	30.30 ± 2.13	41.94 ± 3.33	11.64 ± 1.02	0.677 ± 0.035	0.057 ± 0.003
AFB1 alone	22.41 ± 1.84	23.64 ± 2.62*	1.23 ± 0.78*	0.858 ± 0.071*	0.077 ± 0.001*
AFM1 alone	23.35 ± 1.82	25.38 ± 2.37*	2.02 ± 0.56*	0.895 ± 0.055*	0.075 ± 0.004*
AFB1 + LP	32.30 ± 2.16	46.74 ± 4.32	14.44 ± 2.16	0.675 ± 0.034	0.050 ± 0.001
AFM1 + LP	32.12 ± 2.20	47.11 ± 4.67	14.99 ± 2.48	0.664 ± 0.021	0.052 ± 0.001

Balb/c mice were orally exposed daily (for 15 days) to saline, LP (2 × 10⁹ cfu/kg) alone, AFB₁ (0.25 mg/kg) alone, AFM₁ (0.27 mg/kg) alone, or to co-treatments with LP + AFB₁ or LP + AFM₁.

Values shown are mean ± SD from three determinations per experimental scenario tested.

*Value significantly differs from control, LP alone, or corresponding co-treatment values (p < 0.05).

Figure 2. Thymocyte and splenocyte cell numbers for mice after the 15 days of treatment. Mice were orally exposed to LP (2 × 10⁹ CFU/kg), AFB₁ (0.25 mg/kg), AFM₁ (0.27 mg/kg) or to a co-treatment with LP + AFB₁ or LP + AFM₁. Data shown are as mean ± SE. In each histogram, values (bars) for each given type of cellularity with superscripts bearing different letters differ significantly (p < 0.05).

Table 5. Effect of treatments on hematological parameters.

Parameter	Control	LP	AFB1	AFM1	LP + AFB1	LP + AFM1
WBC (10^3/µl)	9.80 ± 0.11	9.39 ± 0.36	16.26 ± 0.07*	18.35 ± 0.08*	10.16 ± 0.34	9.49 ± 0.82
RBC (10^6/µl)	8.79 ± 0.21	8.46 ± 0.18	7.78 ± 0.11*	7.44 ± 0.23*	8.87 ± 0.09	8.63 ± 0.19
Hb (g/dl)	14.51 ± 0.12	14.69 ± 0.23	13.10 ± 0.05*	13.73 ± 0.20*	14.65 ± 0.40	14.20 ± 0.26
Ht (%)	43.14 ± 0.47	44.07 ± 0.31	38.25 ± 0.05*	39.50 ± 0.69*	43.61 ± 0.42	43.00 ± 0.53
Platelets (10^3/µl)	393.03 ± 20.22	442.83 ± 23.10	602.00 ± 2.10*	454.80 ± 4.28*	400.00 ± 14.72	392.90 ± 29.59

Balb/c mice were orally exposed daily (for 15 days) to saline, LP (2 × 10⁹ cfu/kg) alone, AFB₁ (0.25 mg/kg) alone, AFM₁ (0.27 mg/kg) alone, or to co-treatments with LP + AFB₁ or LP + AFM₁.

Values shown are mean ± SD from six determinations per experimental scenario tested.

*Value significantly differs from control, LP alone, or corresponding co-treatment value (p < 0.05).

RBC, red blood cells; WBC, white blood cells; Hb, hemoglobin; Ht, Hematocrit.

Figure 3. Representative photomicrographs of H&E-strained spleen of mice after the 15 days of treatment. (A and B) Control mice and mice treated daily with LP (2 × 10⁹ CFU/kg). (C and D) Mice treated daily with AFB₁ (0.25 mg/kg) or AFM₁ (0.27 mg/kg). (E and F) Mice co-treated daily with LP + AFB₁ or LP + AFM₁. Magnification = 40×. WP, white pulp; RP, Red pulp; MZ, Marginal zone; Mg, Megakaryocyte; L, Lymphocyte; M, Macrophage; N, Neutrophil.

Figure 4. Representative photomicrographs of H&E-strained thymus of mice after the 15 days of treatment. (A and B) Control mice and mice treated daily with LP (2 × 10⁹ CFU/kg bw). (C and D) Mice treated daily with AFB₁ (0.25 mg/kg) or AFM₁ (0.27 mg/kg). (E and F) Mice co-treated daily with LP + AFB₁ or LP + AFM₁. Magnification = 40×. C, Cortex; M, medulla.

Table 6. Effect of treatments on splenic mRNA expression of select inflammatory cytokines.

Parameter	Control	LP	AFB1	AFM1	LP + AFB1	LP + AFM1
IFNγ	1.38 ± 0.08	1.39 ± 0.06	0.96 ± 0.07*	0.91 ± 0.07*	1.26 ± 0.04	1.30 ± 0.02
TNFα	1.32 ± 0.08	1.33 ± 0.08	1.00 ± 0.01*	1.00 ± 0.03*	1.27 ± 0.09	1.23 ± 0.09
IL-4	0.90 ± 0.02	0.86 ± 0.02	1.08 ± 0.04*	1.06 ± 0.03*	0.90 ± 0.06	0.90 ± 0.06

Balb/c mice were orally exposed daily (for 15 days) to saline, LP (2 × 10⁹ cfu/kg) alone, AFB₁ (0.25 mg/kg) alone, AFM₁ (0.27 mg/kg) alone, or to co-treatments with LP + AFB₁ or LP + AFM₁.

Values shown are mean ± SD ratios of the cytokine-specific RT-PCR product vs the corresponding cyclophilin band intensity (expressed in arbitrary units [AU]) from six determinations per experimental scenario tested.

*Value significantly differs from control, LP alone, or corresponding co-treatment value (p < 0.05).