Fumonisin toxicity, bioaccessibility and control of fusarium spp. using essential oils.
Du Plesis, Belinda
Du Plesis, Belinda
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Abstract
Maize is a major staple food and the largest grain crop in South Africa. The mycotoxin, fumonisin B (FB), a Group 2B carcinogen produced by Fusarium spp., is a common contaminant in South African maize. The mode and mechanism whereby FB exerts toxicity is multifaceted and seems to vary in different cells and animal species. A major difficulty in the determination of FB in food is the occurrence of various forms of cryptic or masked FB (including modified and matrix-associated forms). These are not detected by routine analytical methods, which may result in an underestimation of the contamination levels and the associated health risk. The broad aim of the study was to gain a better understanding of the risks associated with fumonisins in South African maize. This was achieved by determining the toxicity of fumonisins, evaluating the interactions of fumonisins with the macrocomponents of maize, and determining their bioaccessibility from maize porridge. In addition, the in vitro potential of selected plant essential oils (EOs) as natural fungicides towards Fusarium spp., isolated from maize and soil, was investigated. In this study, the zebrafish embryo test (FET) was used to complement cell-based assays for the determination of the cytotoxicity, teratogenicity and acute toxicity of FB. The reaction between FB and starch, glucose, zein, amino acids and maize meal, respectively, were studied under different pH and temperature processing conditions. To evaluate the risk associated with the consumption of fumonisin-contaminated maize porridge, the bioaccessibility of FB from in stiff maize porridge was determined in the TNO Gastrointestinal Model of the stomach and small intestine (tiny-TIM). Soil and maize samples were collected from the two major maize-producing provinces in South Africa. The soil was characterised by determining the soil texture, moisture content, pH,
conductivity and trace element composition. Fungi in the soil and maize samples were isolated, characterised and identified. The potential of lemongrass, spearmint and thyme EOs for the biocontrol of Fusarium spp. isolated from soil and maize was determined using an in vitro toxic medium assay. Furthermore, the effect of the lemongrass EO on FUM1 and FUM21 gene expression and FB production was assessed using two fumonisin-producing F. verticillioides strains The results demonstrated that FB1 exhibited a very low cytotoxicity towards the RTL-W1 trout liver cell line when tested without pH adjustment (IC50 1 260 μg/mL), but no cytotoxicity when the pH was adjusted to physiological conditions. The mycotoxins FB1, FB2, FB3 and HFB1 were not genotoxic towards the RTL-W1 cell line. The zebrafish FET also yielded high LC50 values for FB1 (765 and 277 mg/L after 48 and 96 h exposure, respectively), indicating a low acute toxicity. After adjusting the pH of FB1 solutions to physiological conditions, the LC50 could only be obtained after 96 hpf using the concentrations tested. The LC50 for pH-adjusted FB1 (682 mg/L) was 2.5 times higher than the corresponding concentration without pH adjustment (277 mg/L). This indicates that the acute toxicity towards zebrafish embryos was reduced but not entirely eliminated when adjusting the pH. In the zebrafish FET assay, HFB1 proved to be less toxic than FB1. The LC50 values for HFB1 at 48 and 96 h were 1 090 and 471 mg/L, respectively. The teratogenicity index for FB1 (with and without pH adjustment) and for
HFB1 (without pH adjustment) were <2, indicating a lack of teratogenicity towards zebrafish embryos. The current study quantified the pH shifts caused by high
concentrations of FB1 and HFB1 and proved that the pH shifts contribute greatly to their toxicities. It also confirmed that the FET is a useful toxicity screening tool that may account for effects not observable in cell cultures. None of the processing treatments (pH 4, 7 and 10) or temperatures (30 and 90 °C)
resulted in a significant decrease in the recovery of FB1 from starch, glucose, zein or maize meal spiked with FB. These results confirm the stability of FB1 in maize porridge and the absence of formation of modified forms of FB. Although the recovery of FB1 from a mixture of amino acids and from glutamic acid alone was low, the ultraperformance liquid chromatography-quadrupole time-of-flight–high-definition mass spectrometry analysis demonstrated that no modified FB was formed in the presence of glutamic acid at ambient temperature. This highlights the challenges associated with the determination of FB in different food matrices, and the importance of validating the methods applied to each matrix to promote evidence-based decisions regarding the risk of FB to food safety.
Stiff maize porridge was prepared from maize meal naturally contaminated with fumonisins, as well as from fumonisin-free maize meal. To determine the
bioaccessibility of the mycotoxins, the porridge was digested using the tiny-TIM in vitro dynamic model with parameters simulating human digestion. The results proved that FB1, FB2 and FB3 are released rapidly from stiff maize porridge made from contaminated maize meal. After 360 min, 68%, 94% and 87% of FB1, FB2 and FB3, respectively, were bioaccessible, which confirms that the low bioavailability of FB is not due to strong association of the toxin with the intestinal content. The fact that the fumonisins in contaminated maize porridge become available rapidly for absorption during digestion, imply that it is likely that the intestine could be exposed to high concentrations of FB after consuming porridge prepared from contaminated maize meal. A total of 25 Fusarium cultures were isolated from soil and maize samples. The soil did not contain any fumonisin-producing fusaria, as only F. equiseti, F. solani and F. brachygibbosum were detected. Soil analysis confirmed that the samples represented a variety of textural classes, and that there were differences in the pH and the trace mineral composition of the soil samples collected from the different locations. Two species of Fusarium were isolated from maize, namely F. oxysporum and
F. verticillioides; with F. verticillioides being the most prevalent. Lemongrass EO was more efficient than spearmint and thyme in inhibiting the growth of the Fusarium spp. in vitro when tested in the toxic medium assay. At a concentration of 400 μL/L, lemongrass oil was able to prevent the growth of 18 Fusarium cultures isolated from maize and soil, as well as that of the reference culture MRC 826. The expression of FUM1 and FUM21 genes, which are involved in the biosynthesis of fumonisins, were also prevented at the minimum inhibitory concentration (MIC). Concentrations lower than the MIC (100 μL/L and 200 μL/L) resulted in an increase in FUM1 and FUM21 gene expression, and after 28 days, the highest fumonisin concentration (28.6 ng/mL) was detected in the
MRC 826 culture treated with 200 μL/L lemongrass EO. The results obtained are of concern, since they imply that EO treatments aimed at controlling pathogenic fungi may actually stimulate mycotoxin production in fungi that survive. This could pose health risks for consumers. This study was the first to use the RTL-W1 trout liver cell line and zebrafish FET to determine FB toxicity and has broadened the existing scientific knowledge concerning the toxic effects of FB. The stability of FB during the preparation of stiff maize porridge was confirmed and there was no evidence of the formation of modified FB under typical conditions used for the preparation of porridge. The current project presented new insights into the kinetics of FB release from porridge during simulated human digestion. The rapid release of FB from cooked porridge, thereby exposing the intestinal mucosa to FB, suggest that consumers using maize as a staple may be at risk. The inhibitory effects of lemongrass EO on FUM gene expression demonstrates potential for it to be used as a fungicide above the MIC. However, reported for the first time, is the potential risk of increased FB production by the fungus when exposed to concentrations below the MIC.
Description
Submitted in partial fulfilment of the requirements for the degree Doctor of Philosophy in Science in the Department of Biotechnology and Food Technology
Faculty of Science at the Tshwane University of Technology.
Date
2018-10-01
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Tshwane University of Technology