Ability of Lactobacillus rhamnosus GAF01 to remove AFM₁in vitro and to counteract AFM₁ immunotoxicity in vivo

Figures & data

Table 1.  Percentage of AFM1 removal from PBS and reconstituted milk by Lactobacillus plantarum MON03 and Lactobacillus rhamnosus GAF01 strains.

Times	PBS
	0.05 µg AFM1/ml			0.10 µg AFM1/ml			0.20 µg AFM1/ml
	0 hr	6 hr	24 hr	0 hr	6 hr	24 hr	0 hr	6 hr	24 hr
L. plantarum MON03	16.1 ± 1.4	51.1 ± 4.2	71.6 ± 6.1	16.0 ± 2.4	56.3 ± 5.2	74.9 ± 8.2	15.9 ± 1.6	67.4 ± 5.8	78.6 ± 7.1
L. rhamnosus GAF01	26.2 ± 2.0	61.5 ± 5.2	84.9 ± 7.1	25.9 ± 2.5	66.6 ± 6.2	91.3 ± 7.2	26.0 ± 2.0	86.6 ± 7.1	94.7 ± 8.2
PBS only	5.1 ± 1.1	4.6 ± 0.9	4.2 ± 0.7	3.9 ± 0.8	5.0 ± 0.5	4.1 ± 0.6	4.4 ± 0.7	4.2 ± 0.1	4.2 ± 0.5
Reconstituted milk
L. plantarum MON03	15.3 ± 1.5	48.2 ± 4.7	72.3 ± 5.9	15.8 ± 2.2	57.1 ± 5.4	72.9 ± 8.9	15.2 ± 1.3	69.1 ± 5.3	76.9 ± 8.0
L. rhamnosus GAF01	25.1 ± 2.0	60.3 ± 5.1	85.8 ± 7.5	26.1 ± 2.7	65.3 ± 6.5	90.7 ± 7.5	25.3 ± 2.2	85.9 ± 7.4	95.1 ± 8.3
Reconstituted milk	5.3 ± 0.9	4.3 ± 0.6	4.1 ± 0.6	4.1 ± 0.7	5.2 ± 0.6	4.4 ± 0.3	4.6 ± 0.6	4.0 ± 0.3	4.4 ± 0.2

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

All values shown for the bacteria-containing systems are significantly different from control at p ≤ 0.05.

Figure 1.  High performance liquid chromatography (HPLC) chromatograms. (a) Untreated (positive control) aflatoxin M₁ (at 0.15 µg AFM₁/ml)-phosphate buffered saline (PBS) solution; (b) AFM₁-free PBS solution; or (c) AFM₁ (0.15 µg/ml)-PBS solution after 6 h treatment with Lactobacillus rhamnosus GAF01 at 10⁸ CFU/ml.

Figure 2.  Effect of washing on bacteria:AFM₁ complex. Initial binding was determined after bacteria (10⁸ CFU/ml) and AFM₁ (0.05 µg/ml) were incubated together for 6 h at 37°C. Bacteria/AFM₁ complexes formed were subjected to centrifugation and then washing with 5 ml PBS, before the amount of AFM₁ released was determined as outlined in the Methods. Results shown are the mean (± SD) from triplicate samples.

Figure 3.  Influence of AFM₁ and Lactobacillus rhamnosus GAF01 (alone or in combination) on lymphocyte sub-types. Balb/c mice received daily (for 14 days) by gavage distilled water (open bar), AFM₁ (100 µg/kg BW) (solid bar), L. rhamnosus GAF01 (1 mg/kg BW) (shaded bold bar), or AFM₁ + L. rhamnosus GAF01 (shaded fine bar). Lymphocyte counts were then performed on blood samples collected on Day 15 of the experiment (i.e., 1 day after final exposure in each regimen). Results are expressed as mean [± SE] from n = 12 mice/group. A Student’s t-test was used to compare differences between groups: ^aValue significantly/^bnot significantly different from control at p ≤ 0.05.

Figure 4.  Influence of AFM₁ and Lactobacillus rhamnosus GAF01—alone or in combination—on blood cell counts. Balb/c mice received daily (for 14 days) by gavage distilled water (open bar), AFM₁ (100 µg/kg BW) (solid bar), L. rhamnosus GAF01 (1 mg/kg BW) (shaded bold bar), or AFM₁ + L. rhamnosus GAF01 (shaded fine bar). Erythrocyte and leukocyte counts were then performed on blood samples collected on Day 15 of the experiment (i.e., 1 day after final exposure in each regimen). Results are expressed as mean [± SE] from n = 12 mice/group. A Student’s t-test was used to compare differences between groups: ^aValue significantly/^bnot significantly different from control at p ≤ 0.05.