Salisu, Baha’uddeen
(2022)
Characterisation of aspergillus section flavi and development of aflatoxins level detection methods in food grains and poultry feeds from Malaysia and Nigeria.
Masters thesis, Universiti Sains Malaysia.
Abstract
Aspergillus section Flavi (ASF) and their aflatoxins are among the most critical food
and feed contaminants with deleterious economic and public health impacts. It
contributes to about 20% of global cancer-related deaths annually. However, research
data is lacking from most states in Malaysia and Nigeria, where ASF contamination is
usually high. This study determines the bioburden and distribution of mycoflora in 660
Malaysian and Nigerian food grains and poultry feeds by utilising microbiological
dilution plating techniques; in identifying, classifying, and screening the isolated ASF
for aflatoxigenicity by phenotypic, biochemical, molecular, and phylogenetics
methods. In addition, simplified chromatographic (Thin Layer Chromatography (TLC)
and High Performance Liquid Chromatography (HPLC)) and spectroscopic
(Attenuated Total Reflectance – Fourier Transformed Infrared Spectroscopy (ATRFTIR))
aflatoxin quantification methods were developed and validated; hence applied
to determine the aflatoxins level in the samples. The average dietary exposure risks to
aflatoxins (DERA) and attributable liver cancer (HCC) risks were also determined. A
total of 142 and 185 filamentous fungal isolates with average bioburden of 8.9 x 104 ±
1.6 x 105 to 1.0 x 106 ± 2.5 × 105 CFU/g and 1.2 x 105 ± 1.7 x 105 to 4.0 x 105 ± 5.8 x
104 CFU/g were obtained from the food grains and poultry feeds from Malaysia and
Nigeria, respectively. Based on the phenotype, extrolites and gene sequence data (β –
tubulin gene and nuclear ribosomal internal transcribed spacer (ITS) gene), 74 isolates
(Malaysia = 27, Nigeria = 47) were identified as ASF (60 A. flavus and 14 A. oryzae),
of which 47 (Malaysia = 13, Nigeria=34) produced aflatoxins on solid media and
possessed the aflatoxin biosynthesis genes (aflR, aflP, aflD and aflM). On the other
hand, the developed chromatographic and spectroscopic methods showed high
accuracy and sensitivity in quantifying aflatoxins in the order HPLC (R2 > 99.9%) >
FTIR (R2 = 99.59%) > TLC (R2 > 99%). The HPLC showed that the levels of aflatoxins
(8.68 to 77.40 ng/g) in samples from Malaysia signified low DERA (3.27 to 35.88 ng
of aflatoxins/KgBw/day) and HCC risks (1.67 to 18.31% incidence of HCC/100,000
peoples/year) compared the levels of aflatoxins (0.21 to 114.41 ng/g) in samples from
Nigeria (mean DERA = 23.04 to 50.08 ng/KgBw/day, HCC risk = 26.61 to 57.94%
incidence of HCC/100,000 peoples/year). Results showed that (i) improved aflatoxin
extraction and quantification methods (FTIR, TLC and HPLC) in food grains and
poultry feeds were developed and validated in this study with accuracy values of 90%
to 103%; (ii) A. flavus and A. oryzae are the main ASF species identified in the
samples, with aflatoxigenic chemotypes being significantly higher than the nonaflatoxigenic
groups and (iii) significant number of the samples analysed have fungal
bioburden and aflatoxins above the international regulatory limit which could lead to
above than 10% HCC cases in the study regions. Hence, fungal/aflatoxin control and
prevention strategies should be strengthened in the study regions.
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