Incidence of mycotoxigenic fungi during processing and storage of bambara groundnut (Vigna subterranea) composite flour

Abstract

Fungal contamination of food commodities is a global food security challenge that impacts negatively on the health of consumers. Mycotoxins are produced as secondary metabolites by some pathogenic fungi and may contaminate agricultural products while on the field or during harvesting and storage. Processing operations and storage conditions of temperature and relative humidity have marked effect on the ability of fungal pathogens to grow and produce mycotoxins in agricultural food commodities. The consumption of mycotoxin- contaminated foods, even at low doses over a prolonged period of time, may have deleterious effects on health of consumers.

Bambara groundnut (Vigna subterranea (L.) Verdc) is an African legume gaining wide acceptance in various food applications due to its favourable nutritional composition, especially the high protein content. In several parts of Africa, it is used as a supplement in cereal-based foods, especially in weaning food for infants and young children. Bambara groundnut grows near or under the soil, which serves as inoculum of pathogenic fungi. Very little information is presently available on fungal and mycotoxin contamination of Bambara groundnut from Southern Africa. Hence, its safety for consumption from a mycological standpoint requires further studies.

To establish the profiling of fungal contaminants in food commodities consumed in Durban, South Africa, 110 samples of regularly consumed food samples which included rice (23), spices (38), maize and maize-derived products (32), and Bambara groundnut (17) were randomly collected over a period of five months from retail stores and open markets. The food samples were screened for fungal contamination using conventional and molecular methods. Fungal isolates were characterized following DNA extraction, polymerase chain reaction and sequencing. Using a modified QuEChERS method, the detection and quantification of mycotoxins in Bambara groundnut was performed via Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), and isolation and detection of the causative pathogen was carried out. The effect of processing operations of milling, a combination of roasting and milling, and spontaneous fermentation on the survival of the natural fungal population of Bambara groundnut, and aflatoxin production under simulated tropical conditions of storage was further studied. Processed Bambara groundnut flour samples were stored at temperature of 30±1 °C and 85±2% relative humidity for 30 days,

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and samples withdrawn at 5-day intervals for analyses, i.e., fungal counts, aflatoxin accumulation and changes in water activity during storage.

Following the detection of aflatoxins in Bambara groundnut flour and the isolation of aflatoxigenic Aspergillus flavus in the seed, the effect of milling, roasting and milling, or lactic acid bacteria fermentation on the survival, growth and aflatoxin production of A. flavus in Bambara groundnut flour was studied. Irradiated seeds of Bambara groundnut were artificially inoculated with a 3-strain cocktail of A. flavus (2 x 106 spores/mL) and processed by milling, roasting at 140 °C for 20 min and milling. Slurries of irradiated Bambara groundnut flour were also inoculated with A. flavus spores and 1 x 108 CFU/mL inoculum of Lactobacillus fermentum or Lactobacillus plantarum. All inoculated samples were incubated at 25 °C for 96 h, samples withdrawn every 24 h were analyzed for viable A. flavus counts, changes in water activity during incubation, and aflatoxin production using Enzyme- linked Immunosorbent Assay (ELISA). Bambara groundnut flour samples fermented with lactic acid bacteria were further analyzed for pH, total titratable acidity, and viable lactic acid bacteria counts over the incubation period. The degradation of aflatoxin (AF) B1 by both lactic acid bacteria was also studied. Slurries of irradiated Bambara groundnut flour were spiked with 5 µg/kg of aflatoxin B1 (AFB1) and the percentage reduction over the incubation period was determined using HPLC.

The survival, growth and aflatoxin production of A. flavus in Bambara groundnut and maize- composite flours as affected by milling, roasting and milling or lactic acid bacteria fermentation during storage was also studied. Processed and irradiated Bambara groundnut flour, maize flour and maize-bambara composite flour (70:30) were inoculated with 2 x 107 spores/ml of A. flavus and stored for up to 10 weeks at a temperature of 25±2 °C and relative humidity of 75±2%. Samples were withdrawn weekly and analyzed for viable populations of A. flavus, concentrations of aflatoxins B1, B2, G1 and G2, changes in pH and water activity over the storage period. The colonization of Bambara groundnut by A. flavus and the effects of fungal infection on the seed coat, storage cells and tissue structures were also studied. Irradiated Bambara groundnut seeds were artificially inoculated with spore suspension of aflatoxigenic A. flavus (2 x 106 spores/mL) and stored at a temperature of 25±2 °C and relative humidity of 75±2% for 14 days. Samples were withdrawn at 24 h intervals for 4 days, then at 7 and 14 days and examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

Various fungal genera were isolated from the food samples under study with Aspergillus (52.5%) and Penicillium (31.8%) as the dominant genera. All the 110 food samples were contaminated with more than one fungal species. A. flavus and other Aspergilli, Penicillium citrinum and Fusarium oxysporum were isolated from Bambara groundnut seeds. Aflatoxigenic A. flavus was isolated from Bambara groundnut seed, with a co-occurrence of Aflatoxin (AF) B1 (0.13–6.90 µg/kg), AFB2 (0.14–2.90 µg/kg), AFG1 (1.38–4.60 µg/kg), and AFG2 (0.15–1.00 µg/kg) in the flour. The fungal counts of the samples during storage significantly (p≤0.05) increased, irrespective of the processing method from 6.3 Log10 CFU/g in Bambara groundnut flour to 6.55 Log10 CFU/g in fermented Bambara groundnut flour. Aflatoxin concentration was affected markedly by the processing methods in Bambara groundnut flour (0.13 µg/kg) and fermented Bambara groundnut flour (0.43 µg/kg), aflatoxin was not detected in roasted Bambara groundnut flour.

The survival and growth of A. flavus was also markedly affected by lactic acid bacteria fermentation and roasting during incubation. Within 24 h of fermentation with L. fermentum, significant (p≤0.05) changes were recorded in viable population of A. flavus (6.30‒5.59 Log10 CFU/mL), lactic acid bacteria count (8.54‒13.03 Log10 CFU/mL), pH (6.19‒4.12), total titratable acidity (0.77‒1.87%) and a reduction by 89.2% in aflatoxin B1 concentration. Similar significant changes were recorded in Bambara groundnut flour fermented with L. plantarum. Aspergillus flavus in the artificially contaminated seeds were completely eliminated by roasting. Aflatoxin production was not detected in Bambara groundnut flour samples over the incubation period.

During storage for 10 weeks, the population of A. flavus significantly (p≤0.05) decreased in roasted Bambara groundnut flour from 7.18 to 2.00 Log10 CFU/g. Similar significant (p≤0.05) decrease in A. flavus viable counts was recorded in fermented Bambara groundnut flour from 6.72 to 2.67 Log10 CFU/g, however after 7 weeks of storage and beyond, A. flavus was not detected. Significant (p≤0.05) decrease in aflatoxin B1 (0.36‒0.26 µg/kg) and aflatoxin G1 (0.15‒0.07 µg/kg) accumulation was also recorded in roasted Bambara groundnut flour. While A. flavus viable population significantly (p≤0.05) decreased in maize-Bambara composite flour from 6.90 to 6.72 Log10 CFU/g, aflatoxin B1 accumulation significantly (p≤0.05) increased from 1.17 to 2.05 µg/kg. Microscopy studies showed that the seed coat of Bambara groundnut was rapidly colonized by A. flavus within 24 h of inoculation. The infection of internal tissues of the cotyledon was through the ruptured seed

coat, resulting in a disruption of the cellular architecture. Cell wall collapse, development of cavities in parenchymatous cells and ruptured storage cells resulted from A. flavus infection of the seed.

This study reports a high prevalence of fungal contamination in some food commodities consumed in Durban, South Africa. The isolation of live mycotoxin-producing fungi from the food commodities necessitates the need for regular routine checks to ensure the mycological safety of agricultural products offered for sale to consumers. The detection of aflatoxigenic A. flavus and aflatoxins in Bambara groundnut flour at levels above the maximum tolerable limits raises health concerns on its utilization in food applications, and in supplementary feeding for infants and young children. Although roasting was effective in degradation of aflatoxins in Bambara groundnut seeds, elimination of fungal contaminants was not achievable which resulted in continued production of aflatoxin during storage. Fermentation using L. fermentum or L. plantarum is effective in eliminating A. flavus and degrading AFB1 in Bambara groundnut flour. Compositing Bambara groundnut with maize increased aflatoxin production in the flour. It is therefore necessary to implement legislation for aflatoxins in Bambara groundnut, and develop effective management practices during planting, harvesting and storage that will mitigate A. flavus infection in Bambara groundnut.