Biodegradation of aflatoxin by Flavobacterium aurantiacum (Exiguobacterium aurantiacum)

Author

Randall Kent Phebus

Date of Award

12-1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Food Science and Technology

Major Professor

F.A. Draughon

Committee Members

R.D. Linnabary, J.L. Collins, G.E.Christen

Abstract

Flavobacterium aurantiacum (Exiguobacterium aurantiacum) has been shown to remove aflatoxins from solution, although the mechanism for this elimination is not known in detail. This investigation was undertaken to determine substrate specificity and inducibility of aflatoxin degrading activity in F. aurantiacum cultures. Finally, the cellular fraction exhibiting aflatoxin degrading activity was isolated.

Cells were preincubated for 24 h in 5.0 and 0.5 μg/ml of aflatoxin B₁ (AB₁) to serve as an induced inoculum. The induced inoculum was compared to noninduced cells grown in tryptic soy broth only. AB₁ removal of initial concentrations of 4.0 and 2.0 μg/ml was determined over 212 and 53 h of incubation, respectively, by high performance liquid chromatography. When F. aurantiacum was induced with 5.0 μg AB₁/ml and tested against 4.0 μg/ml, the non-induced population increased more rapidly during the initial 12 h of incubation. However, the induced population removed 23% more AB₁ during this period. Decreasing the preincubation AB₁ concentration tenfold (0.5 μg/ml) followed by testing at 2.0 μg/ml resulted in both a slower population growth and 58% less AB₁ removal during the initial 12 h of incubation. Chromatograms indicated that AB₁ removal by F. aurantiacum was a stepwise process producing two or more intermediates. Since non-induced cultures metabolized AB₁ without a lag period, a constitutive enzyme(s) seems to be involved. Preincubation in the higher AB₁ concentration probably resulted in selection of an AB₁ tolerant population explaining the rapid removal of toxin by the induced cells.

Substrate specificity of F. aurantiacum to eight forms of aflatoxin (B₁, G₁, B₂, G₂, M₁, M₂, Q₁ and R₀ was determined. All forms except R₀ were irreversibly removed from broth by the organism, although at varying degrees of efficiency. When AB₁ and AG₁ (2.0-8.0 μMg/ml each) were tested in combination, AG₁ was preferentially removed. This was, again, seen when AB₂ and AG₂ (1.0- 4.0 μg/ml each) were tested simultaneously. The bacterium removed AB₁ and AB₂ more efficiently when the toxins were tested individually. Degradation of AB₁ and AG₁ resulted in the production of at least one intermediate compound for each toxin as observed in chromatograms.

A F. aurantiacum culture was cultivated for 24 h followed by separation into cell-free extract (extracellular components), cytosol and membrane cellular fractions. Each of these components was tested for AB₁ degrading activity during 48 h of incubation at 30 °C. The cell-free extract removed only 16% of AB₁ (initial concentration of 2.0 μg/ml). From an initial AB₁ level of 5.0 μg/ml, the cytosol fraction effectively removed 99% of the toxin, whereas, only 27% was removed by the membrane component. It was concluded that the cytosol fraction of F. aurantiacum cells may be a valuable source of an aflatoxin degrading enzyme(s) which is constitutively produced by the organism.

Recommended Citation

Phebus, Randall Kent, "Biodegradation of aflatoxin by Flavobacterium aurantiacum (Exiguobacterium aurantiacum). " PhD diss., University of Tennessee, 1992.
https://trace.tennessee.edu/utk_graddiss/7575