Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG.

The surface of Lactobacillus rhamnosus strain GG (LGG) has previously been shown to bind aflatoxin B(1) (AFB(1)) effectively, it being a food-borne carcinogen produced by certain species of Aspergillus fungi. To establish which components of the cell envelope are involved in the AFB(1) binding process, exopolysaccharides and a cell wall isolate containing peptidoglycan were extracted from LGG and its AFB(1) binding properties were tested. LGG was also subjected to various enzymatic and chemical treatments and their effects on the binding of AFB(1) by LGG were examined. No evidence was found for exopolysaccharides, cell wall proteins, Ca(2+) or Mg(2+) being involved in AFB(1) binding. The AFB(1) binding activity of the cell wall isolate indicates that AFB(1) binds to the cell wall peptidoglycan of LGG or compounds tightly associated with the peptidoglycan.

Kinetics of adsorption and desorption of aflatoxin B1 by viable and nonviable bacteria.

The reactions involved in the binding (adsorption) and release (desorption) of aflatoxin B1 (AFB1) to and from the surface of bacteria were investigated. Viable and heat-killed Lactobacillus rhamnosus GG, L. rhamnosus LC-705, and Propionibacterium freudenreichii subsp. shermanii JS were incubated in phosphate-buffered saline containing variable concentrations (0.0017 to 13.3 microg/ml) of AFB1. The relationship between the bacterial surface hydrophobicity and the AFB1 adsorption affinity was also investigated. A linear relationship was observed between the specific rate of AFB1 adsorption and the AFB1 concentration for all bacteria. The nature of desorption of adsorbed AFB1 was investigated by repetitive aqueous washes. A linear relationship was observed between the natural log value of the concentration of AFB1 adsorbed and the number of washes for all bacteria studied. The desorption constants were strain-dependent and were lower for heat-killed bacteria than for viable bacteria. Heat treatment appears to alter the surface properties of the bacteria rather than expose new adsorption sites. No correlation was found between the hydrophobicity and the AFB1 adsorption affinity.

Selective in vitro binding of dietary mutagens, individually or in combination, by lactic acid bacteria.

Specific strains of lactic acid bacteria possessing antimutagenic properties are suggested to remove mutagenic contaminants of foods through binding and an investigation of their substrate specificity is required. The ability of Lactobacillus rhamnosus strains GG and LC-705 in viable and non-viable (heat- and acid-treated) forms to remove both dietary mutagens and other aromatic dietary substrates from solution was studied using HPLC. Overall, removal increased in the order: caffeine = vitamin B12 =folic acid < ochratoxin A < aflatoxin B1 = PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) < Trp-P-1 (3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole) (p < 0.05). Aflatoxin B1, Trp-P-1 and PhIP were removed in high amounts (77-95%) and ochratoxin A was removed in moderate amounts (36-76%). By contrast, only minimal amounts of caffeine, vitamin B12 andfolic acid were removed (9-28%). The significant removal of selected mutagens, but not other substrates, suggests these strains may be useful for dietary detoxification. Since exposure to multiple mutagens is likely, the removal of aflatoxin B1 and Trp-P-1 from a mixture of these substrates was also investigated. Removal of AFB1 significantly increased (p < 0.05) in the presence of Trp-P-1, while removal of Trp-P-1 significantly decreased (p < 0.05) in the presence of AFB1. Overall, no significant differences in removal were found between bacterial strains or between viable, heat- and acid-treated bacteria.

Surface binding of aflatoxin B(1) by lactic acid bacteria.

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B(1) complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B(1) remained bound. Nonviable bacteria retained the highest amount of aflatoxin B(1). Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B(1) from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B(1) to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B(1). Variation in temperature (4 to 37 degrees C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B(1) released. Binding of aflatoxin B(1) appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.