Utilization of Indigenous Plant-Based Treatments and Nixtamalization for Mitigating Mycotoxin Contamination in Maize in Eastern Democratic Republic of Congo

Abstract

Food security considerations have shifted in recent years with the recognition that available food should also be nutritious and safe. Mycotoxins, which are secondary metabolites produced by a number of mycotoxigenic molds are a major food safety concern, especially aflatoxin and fumonisin contamination of maize, a major staple food crop in the Democratic Republic of Congo. To ensure food safety regarding maize, mitigation measures to prevent mold contamination and subsequent mycotoxin contamination need to be considered and put in place. The purpose of this study was to assess the levels of contamination of maize with aflatoxin and fumonisin and to determine the potential use of plant extract and nixtamalization techniques to mitigate the production of these secondary fungal metabolites along the supply chain in South Kivu. A total of 335 maize samples (grains and flour) were randomly collected for mycotoxin analysis. Total aflatoxins and total fumonisins were determined by a single-step lateral flow immunochromatographic assay. A survey of 335 stakeholders (farmers, processors, and traders) in two counties (Ruzizi Plain and Kabare) and one town (Bukavu) was carried out using a structured questionnaire. Focus group discussion, face-to-face interviews, and observation were used to assess the knowledge and awareness of mycotoxin contamination among different actors along the value chain. AFB1 and FB1 were determined by enzyme-linked immunosorbent assay method. Mycotoxigenic fungi were characterized according to gene sequencing data. Standard techniques were used to determine the phytochemical composition of the moringa, cloves, and ginger extracts; while the antimicrobial activity of extracts of moringa, cloves, and ginger was investigated using the agar-well diffusion method. Atomic absorption spectrophotometry determined the concentrations of minerals in composite flour while high-performance liquid chromatography determined phytate, and UV-VIS spectrophotometry determined tannins in composite flour. Aflatoxin was found in all maize samples, with freshly harvested dry maize having the lowest content with a mean and range of 3.2±0.3 µg/kg, and 0.3-18.5 µg/kg respectively. Aflatoxin levels were high in the market, with a range of 1 - 823.2 µg/kg of flour. At the market level, nearly all maize flour collected from the three areas had a high contamination level that exceeded the maximum tolerable limit of 10 µg/kg. At the processor level, there were significant differences (p<0.05) in aflatoxin contamination between flour and grain. The flour had the highest aflatoxin contamination with a mean of 148.9±164.5 μg/kg. The overall maximum concentration of aflatoxin in maize grain at the processor level was 97.9±182 μg/kg with levels ranging from 1.16 to 841.5 μg/kg. The high value for aflatoxin contamination was found in samples collected in Bukavu town. Fumonisin was found in all samples at varying concentrations. Almost all maize samples (80%) collected from the three areas had a degree of contamination that did not exceed the maximum tolerable limit of 4 µg/g. According to the interview, the majority of respondents were unaware of the mycotoxin contamination of maize. According to the interview, most of the respondents were aware of the mycotoxin contamination in maize: 63.7% and 69.3% of the farmers and processors, respectively. Most of the traders were unaware of the mycotoxin contamination. Contamination of both FB1 and AFB1 in flour was higher at the market level than at processor and farmer levels. At the market level, AFB 1 in flour was 12.3 ± 02.9 μg/kg, while in grain it was 9.2 ± 4.2 μg/kg. The farmer levels showed the least contamination, with concentrations of 0.15 ± 0.12 μg/g and 6.64 ± 3.79 μg/kg, respectively. Microscopic and molecular characterization was investigated on thirty isolates of fungi producing aflatoxin and thirty isolates producing fumonisin. Results have shown that 90% of Aspergillus isolates were categorized as yellow-dark green colonies, and all the isolates had conidia that were rough, while Fusarium isolates had whitish to purplish colonies on Potato Dextrose Agar. Molecular characterization was used to confirm their identities using a PCR-sequence analysis of the internal transcribed spacer (ITS) region of rDNA, which revealed that all the isolates from South Kivu that produce aflatoxin were A. flavus and F. verticillioides that produce fumonisin. Based on the seven target genes (aflR, aflM, aflP, aflD, aflQ, aflS, and aflO) producing aflatoxin, A. flavus was classified into five different groups: group I (9 isolates), characterized by the presence of the seven genes; group II (3 isolates), with the presence of aflR, aflM, aflP, and aflD; group III (three isolates), showing three (AflM, aflS, and aflQ); group IV (two isolates), with only two amplicons (aflR and aflD); and finally, group V (13 isolates), with none target gene. For Fusarium isolates, the target gene was Fumarate hydratase 1, mitochondrial (FUM1); only two groups were found: 15 isolates that amplify for the gene FUM1 and 15 that didn’t amplify. Different plants, namely moringa, clove, and ginger, were screened to see their potential to be used as natural products in the mitigation of aflatoxin and fumonisin. After screening the different phytochemicals, the clove methanolic extract had all three types of phytochemicals namely saponins, alkaloids, and terpenoids. The cloves extracts made with methanol and ethanol had a lot of flavonoids (78.3 ± 6.9 mg QE/g and 72.7 ± 1.25 mg QE/g), as well as total phenol (360.4 ± 3.6 mg GAE /g and 470.6 ± 2.4 mg GAE /g). The moringa extract had a high content of tannins (35.4±4.1 mg/g for ethanol and 31.2±2.3 mg/g for methanol). Ginger, clove, and moringa extracts in water and organic solvents had different antimicrobial effects that depended on the concentration. Each microorganism had a very different inhibition zone at 25 mg/mL. It had inhibition zones that were 20.9 ±1.3 mm for Fusarium verticillioides and 18.8 ±1.2 mm for A. flavus when the clove was extracted with ethanol. The minimum inhibitory concentration ranged from 2.5–10 mg/mL in clove extracts. Maize was subjected to nixtamalization and the flour was used to prepare some composite flour with soybean. Results show that the pH value varied with the nixtamalization process, from 6.8 ± 0.7 to 9.3 ± 0.4. The energy content of the composite flour was in the range of 354.77 ± 3.2 to 429.5 ± 4 kcal, meeting the minimum standard set for a cereal-based composite flour which is 400 Kcal. The protein values of the CFs (9.4 ± 0.3 to 13.9 ± 1.9%) on a dry weight basis were slightly below 15% of the recommended levels set by Codex. In conclusion, the composite flour made with 25% hydrothermal treated soybean and 75% nixtamalized maize can be considered the best composite flour concerning its nutritional properties, mineral, antinutrient, and mycotoxin content. Aflatoxin and fumonisin contamination in maize, a staple food in South Kivu, presents a significant public health risk due to its potential adverse effects on consumers. To address this issue, there is an urgent need to implement targeted awareness programs and capacity-building initiatives for key stakeholders along the maize value chain, including farmers, traders, and consumers. Furthermore, this study identifies plant extracts and the nixtamalization process as effective mitigation strategies for reducing contamination levels, highlighting their potential for improving food safety and protecting public health.