INCIDENCE OFMYCOTOXIGENICFUNGIDURING PROCESSING AND STORAGE OF BAMBARA GROUNDNUT (Vigna subterranea)COMPOSITE FLOUR

Olagunju, Omotola Folake (2019) INCIDENCE OFMYCOTOXIGENICFUNGIDURING PROCESSING AND STORAGE OF BAMBARA GROUNDNUT (Vigna subterranea)COMPOSITE FLOUR. Doctoral thesis, Durban University of Technology, Durban, South Africa.

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Abstract

Fungal contamination of food commodities is a global food security challenge that impacts negatively on thehealth of consumers. Mycotoxinsare produced as secondary metabolitesby some pathogenic fungi andmay contaminate agricultural products while on the field or during harvesting and storage. Processing operations andstorage 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 prolongedperiod 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 highprotein content. In several parts of Africa, it is used as a supplement in cereal-based foods, especially inweaning food for infants and young children. Bambara groundnut grows near or under the soil,whichservesas 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 establishthe profilingof fungal contaminantsin 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 reactionand sequencing. Using a modified QuEChERS method, thedetection and quantificationof mycotoxinsin Bambara groundnutwas performedvia LiquidChromatography Tandem Mass Spectrometry (LC-MS/MS),and isolation and detection of the causative pathogenwas carried out. The effect of processing operations of milling, a combination of roasting and milling, andspontaneous fermentation on thesurvival of the natural fungal population of Bambara groundnut,and aflatoxin production under simulated tropical conditions of storagewas further studied. Processed Bambara groundnut flour samples were stored at temperature of 30±1°Cand 85±2% relative humidityfor 30 days, viiand sampleswithdrawn at 5-day intervals for analyses, i.e., fungal counts, aflatoxin accumulationand changes in water activity during storage.Following the detection of aflatoxins in Bambara groundnut flour and the isolation of aflatoxigenic Aspergillus flavusin the seed, the effect of milling, roasting and milling, or lactic acid bacteria fermentationon the survival, growth and aflatoxin production of A. flavusin Bambara groundnutflour was studied.Irradiated seeds of Bambara groundnutwere artificially inoculated with a 3-strain cocktail of A. flavus(2 x 106spores/mL) and processed by milling, roasting at 140°C for 20 minand milling. Slurries of irradiated Bambara groundnut flour were also inoculated with A. flavusspores and 1 x 108CFU/mL inoculum of Lactobacillus fermentumor Lactobacillus plantarum. All inoculated samples were incubated at 25°C for 96 h,samples withdrawn every 24 h wereanalyzed for viable A.flavuscounts, changesin water activityduring incubation,and aflatoxin productionusing 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 countsover the incubation period. The degradation of aflatoxin (AF) B1by both lactic acid bacteriawas also studied. Slurries of irradiated Bambara groundnut flour were spiked with 5 μg/kgof aflatoxin B1(AFB1) and the percentage reduction over the incubation period was determinedusing HPLC.The survival, growth and aflatoxin production ofA.flavusin 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 107spores/ml of A.flavusand 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, G1and G2, changes in pH andwater activityover the storage period.Thecolonization of Bambara groundnut by A. flavusand the effects of fungal infection on the seed coat, storage cells and tissue structureswere also studied. Irradiated Bambara groundnut seeds were artificially inoculated with spore suspension of aflatoxigenic A.flavus(2 x 106spores/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 scanningelectron microscopy (SEM)and transmission electron microscopy(TEM). viiiVarious fungal genera were isolated from the food samples under studywith 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. flavusand other Aspergilli, Penicilliumcitrinumand Fusarium oxysporumwere isolated from Bambara groundnut seeds. AflatoxigenicA.flavuswas isolated from Bambara groundnut seed, with a co-occurrence of Aflatoxin (AF) B1 (0.13–6.90μg/kg),AFB2 (0.14–2.90μg/kg), AFG1(1.38–4.60μg/kg), and AFG2 (0.15–1.00μg/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 Log10CFU/g in Bambara groundnut flour to 6.55 Log10CFU/g in fermented Bambara groundnut flour.Aflatoxin concentrationwas affected markedly by the processing methodsinBambara groundnut flour(0.13 μg/kg) and fermented Bambara groundnut flour(0.43μg/kg), aflatoxin was not detected in roasted Bambara groundnut flour. The survival and growth of A.flavuswas also markedly affected by lactic acid bacteria fermentation and roasting during incubation. Within 24 hof fermentation with L.fermentum, significant (p≤0.05) changes were recorded inviable population of A. flavus (6.30‒5.59 Log10 CFU/mL),lactic acid bacteria count(8.54‒13.03 Log10CFU/mL), pH (6.19‒4.12), total titratable acidity (0.77‒1.87%) and a reduction by 89.2% in aflatoxin B1concentration.Similar significantchanges were recorded in Bambara groundnut flour fermented with L.plantarum.Aspergillusflavusin the artificially contaminated seeds were completely eliminated by roasting.Aflatoxin production was not detected in Bambara groundnut flour samplesover the incubation period.During storagefor 10 weeks, the population of A.flavussignificantly (p≤0.05) decreasedin roasted Bambara groundnut flour from 7.18to 2.00 Log10 CFU/g. Similar significant(p≤0.05)decreasein A. flavusviable countswas recorded in fermented Bambara groundnut flour from 6.72to 2.67 Log10CFU/g, howeverafter 7 weeks of storage and beyond,A. flavus was not detected. Significant (p≤0.05) decreasein aflatoxin B1(0.36‒0.26 μg/kg) and aflatoxin G1(0.15‒0.07 μg/kg) accumulationwasalso recorded in roasted Bambara groundnut flour. While A. flavusviable population significantly(p≤0.05)decreased in maize-Bambara composite flourfrom 6.90to 6.72 Log10CFU/g, aflatoxin B1accumulationsignificantly (p≤0.05) increasedfrom 1.17to 2.05 μg/kg.Microscopy studies showed that the seed coat of Bambara groundnut was rapidly colonized by A.flavuswithin 24 h of inoculation. The infection of internal tissues of the cotyledon was through the ruptured seed ixcoat, resulting in a disruption of the cellular architecture. Cell wall collapse,developmentof cavities in parenchymatous cells and ruptured storage cells resulted from A.flavus infection of the seed.This studyreportsa high prevalence of fungal contaminationin some food commoditiesconsumed in Durban, South Africa. The isolation of live mycotoxin-producing fungi from the food commoditiesnecessitates the need for regular routine checks to ensure the mycological safety of agricultural products offered for sale to consumers. The detection of aflatoxigenic A.flavusand aflatoxins in Bambara groundnut flour at levels above the maximum tolerablelimitsraises 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. fermentumorL.plantarumiseffective in eliminating A. flavusand degrading AFB1in Bambara groundnut flour.Compositing Bambara groundnut with maize increased aflatoxin production in the flour.It is therefore necessary to implement legislationforaflatoxinsin Bambara groundnut,and develop effective management practicesduring planting, harvesting and storagethat willmitigate A.flavus infection in Bambara groundnut

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