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Polyphasic Assessment of Aflatoxin Production Potential in Selected Aspergilli
Stephen Abiola Akinola1, Collins Njie Ateba1, Mulunda Mwanza2
1Bacteriophage Therapy and Phage Bio-control Laboratory, Department of Microbiology, Faculty of Natural and Agricultural Sciences, North-West University, Private Bag X2046, Mmabatho 2745, South Africa.
Abstract:
This study investigated the aflatoxin production potentials of selected fungi using a polyphasic approach. Internally transcribed spacer region of the fungi was amplified using the polymerase chain reaction. Forty-five Aspergillus strains were further assessed for aflatoxin production using the conventional methods such as growth on yeast extract sucrose, β-cyclodextrin neutral red desiccated coconut agar (β-CNRDCA); expression of the aflatoxin regulatory genes and the use of both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A large proportion (82.22%) of the isolates harbored the Nor-1 gene while 55.56%, 68.89%, and 80% possessed the ver-1, omt-A, and aflR genes, respectively. All 100% the isolates harbored the aflJ gene. Twenty-three isolates were positive for aflatoxin production based on the yeast extract sucrose medium (YES) test; ammonium vapor test (51%), yellow pigment production (75.5%), and β-CNRDCA tests; and blue/green fluorescence (57.7%). Based on TLC detection 42.2% produced aflatoxins while in the HPLC, total aflatoxin (AFTOT) production concentrations ranged from 6.77-71,453 µg/g. Detectable aflatoxin B1 (AFB1) concentrations obtained from the HPLC ranged between 3.76 and 70,288 µg/g; 6.77 and 242.50 µg/g for aflatoxin B2 (AFB2); 1.87 and 745.30 µg/g for aflatoxin G1 (AFG1); and 1.67 and 768.52 µg/g for aflatoxin G2 (AFG2). AFTOT contamination levels were higher than European Union tolerable limits (4 µg/kg). The regression coefficient was one (R = 1) while significant differences exist in the aflatoxin concentrations of Aspergillus (p ≤ 0.05). This study reports the potentials of Aspergillus oryzae previously known as a non-aflatoxin producer to produce AFG1, AFG2, AFB1, and AFB2 toxins. Aspergillus species in feedlots of animals reared for food are capable of producing aflatoxins which could pose hazards to health.
Insights
This study found that several Aspergillus strains, including Aspergillus oryzae, can produce aflatoxins at levels exceeding EU limits. These findings highlight potential health risks from contaminated animal feed.
Area of Science:
- Mycology
- Food Safety
- Molecular Biology
Background:
- Aflatoxins are toxic secondary metabolites produced by certain fungi, posing significant risks to human and animal health.
- Understanding the production potential of various Aspergillus species is crucial for effective risk assessment and management in the food chain.
Purpose of the Study:
- To investigate the aflatoxin production capabilities of selected Aspergillus strains using a polyphasic approach.
- To assess the presence of key aflatoxin biosynthesis genes and correlate them with actual toxin production.
Main Methods:
- Polymerase chain reaction (PCR) amplification of the internally transcribed spacer (ITS) region for fungal identification.
- Conventional methods including yeast extract sucrose (YES) agar, ammonium vapor test, and pigment production assays.
- Gene expression analysis of aflatoxin regulatory genes (Nor-1, ver-1, omt-A, aflR, aflJ).
- Detection and quantification of aflatoxins using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC).
Main Results:
- A high percentage of isolates harbored aflatoxin biosynthesis genes, with aflJ present in 100% of strains.
- Conventional tests indicated aflatoxin production in a significant proportion of isolates.
- HPLC analysis confirmed total aflatoxin (AFTOT) concentrations ranging from 6.77-71,453 µg/g, exceeding EU limits.
- Aspergillus oryzae, previously considered non-aflatoxigenic, demonstrated the ability to produce AFB1, AFB2, AFG1, and AFG2.
Conclusions:
- Selected Aspergillus strains, including Aspergillus oryzae, possess the genetic machinery and capability for aflatoxin production.
- Aflatoxin contamination in animal feedlots presents a potential health hazard due to levels exceeding regulatory limits.
- The study underscores the need for continuous monitoring and risk assessment of aflatoxigenic fungi in food production systems.
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