Maize, rice, wheat and barley are the world’s four major cereal crops. In Africa, maize is the
major staple cereal crop for the majority of the population and most of the traditional
fermented products are maize-based (e.g. ogi, kenkey, mawe and mahewu). Similar to other
cereals, maize is also at risk of infestation by mycotoxigenic fungi. International studies have
indicated that fumonisins (64%) take the lead in mycotoxins contamination worldwide
followed by deoxynivalenol (59%), zearalenone (45%), aflatoxin (33%) and ochratoxin A
(28%). Various African studies have indicated fumonisins occur in maize at levels that can
exceed 10 μg/g. Fumonisins are most widely known to cause leucoencephalomalacia in
horses and high levels of fumonisin B1 (FB1) has been linked to increased incidence of
oesophageal cancer in Eastern Cape formerly known as Transkei. Further research has shown
the consumption of FB1 by pregnant women can cause birth defects. Due to their structural
similarity to sphingonine and sphingosine, fumonisins interfere with sphingolipid metabolism
and this result in liver disease and tumours in the liver and kidneys. Many methods have
been investigated for the detoxification of mycotoxins. A new method of detoxification has
recently been developed wherein some mycotoxins have a natural affinity to bind to
microorganisms. Recent studies have indicated that probiotics, specifically lactic acid
bacteria (LAB), have a natural ability to bind to fumonisins and introducing a detoxifying
effect on the fermented product. In this project, we investigated the ability of the following
dominant probiotic LAB strains isolated from traditional fermented maize-based products
(ogi and mahewu) namely, Lactobacillus plantarum FS2, L. delbrueckii subsp. delbrueckii
CIP 57.8T and Pediococcus pentosaceus D39, against a positive control strain, L. plantarum
R1096, to reduce the toxicity of fumonisins (B1 and B2).
In completion of the main objective, each LAB strain was evaluated for its binding ability.
Visualization of the binding interaction was achieved by derivatization of the fumonisins and
interaction with the non-fluorescent LAB cells (viable and non-viable) under confocal laser
scanning microscopy (CLSM) with the appropriate controls in place. Results provided
physical evidence that the fluorescent fumonisins (B1 and B2) bound to the LAB cells
externally, as both the viable and non-viable cells fluoresced green under CLSM. To the best
of our knowledge, this is the first study to visualize the interaction between LAB and
fumonisins. Each LAB strain was evaluated to quantify the binding of fumonisin molecules
to a certain number of bacterial cells, proof of adsorption as well as reproducibility and repeatability. The high binding percentages obtained indicated successful binding strength
being achieved across all LAB strains for both FB1 and FB2. For viable cells, FB1 bound the
most to L. plantarum FS2 but the least to P. pentosaceus D39, whereas, the highest binding
for FB2 was with L. plantarum R1096 but the least with L. delbrueckii CIP 57.8 T. Similarly,
non-viable cells of L. plantarum FS2 indicated the highest binding to FB1 and FB2, while P.
pentosaceus D39 and L. delbrueckii CIP 57.8 T bound the least to FB1 and FB2,
respectively. Overall, non-viable cells bound more to FB1 and FB2 in comparison to viable
cells due to the heat shock causing the cell wall to disrupt exposing more favourable binding
sites to FB1 and FB2. Binding occurred with viable and non-viable cells which confirmed the
results seen with the visualization of binding. The binding strength was repeated in two
independent trials to establish repeatability and reproducibility where the statistical analyses
indicated no significant differences.
To test the stability of the LAB – fumonisin complex, the main parameters were based on the
fermentation process of ogi: incubation temperature of 30 oC and decreasing pH from 6 to 4
that occurs in a natural ogi fermentation process; same decreasing pH values and optimal
growth temperature of the LAB strains which is 37 oC; simulated gastric condition of a lower
pH of 2 and temperature of 37 oC and storage period of 7 days at pH 4 at 30 oC were selected.
Results indicated that whether ogi fermentation process occurs at 30oC or 37 oC, the binding
strength for both fumonisins was still very high, in addition, as the pH decreases it can be
seen that the binding for both the fumonisins increased. Even at the lower pH of 2 and
incubation temperature of 37 oC, binding was quite high and the complex was still stable. For
the storage conditions (7 days at pH 4 and 30 oC), contrary to previous binding strength
results in this project, L. plantarum R1096 was the only strain that increased in binding
strength whereas the other strains decreased in binding. Generally, as pH decreased the
binding increased, but across all strains, FB2 was bound more than FB1.
Due to the liver being the main site of toxicity, the bound complex was evaluated on HepG2
cell line with controls and the LAB strains in order to give a comparative analysis and have a
clear understanding of its toxicity in vitro for the objective of reducing the toxicity of
fumonisins by use of LAB strains. HepG2 cells were observed in the presence of fumonisin
only, LAB cells only and then with a combination of fumonisin and LAB cells to assess
toxicity using the sulforhodamine B (SRB) assay. The results indicated that the LAB cells did
not attach, aggregate or disrupt the HepG2 cells as the absorbance readings remained constant throughout the 24-48 h incubation period. Observation of the HepG2 cells exposed to the
fumonisins alone indicated the IC50 value of 308.6 μg/ml. The comparative observation of the
HepG2 cells exposed to a combination of the fumonisins and LAB cells indicated that the
LAB cells “protected” the HepG2 cells by binding to the fumonisins and increasing the IC50
values. L. plantarum R1096 “protected’ the HepG2 cells the most by increasing the IC50
value to 903.1 μg/ml followed by L. delbrueckii CIP57.8T at 857.3 μg/ml, similarly with L.
plantarum FS2 at 856.5 μg/ml and the least by P. pentosaceus D39 at 701.4 μg/ml. All LAB
strains had more than doubled the IC50 value by being present and binding to the fumonisins.
In conclusion, L. plantarum FS2, L. delbrueckii CIP 57.8T and P. pentosaceus D39, test
strains isolated from African traditional fermented foods, have been successfully tested
against the positive control strain L. plantarum R1096 and can be used as detoxifying agents
in reducing the toxicity of fumonisin B1 and B2 in fermented cereal based products.