Aflatoxin 4

Abstract
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1. INTRODUCTION
Nutritional security is effectively achieved when all people at all times consume food of
sufficient quantity and quality, in terms of variety, diversity, nutrient content, and safety, to meet
their dietary needs and food preferences for an active and a healthy life (FAO/AGN 2012).
Contaminated food is one of the major causes of undernutrition, morbidity, and mortality in sub‐
Saharan Africa, particularly among children, who are more vulnerable to diseases (Paudyal
et al., 2017). Ensuring food safety through the reduction of aflatoxin contamination can contribute
significantly to alleviating poverty, increasing food security, and improving nutrition. Also, this
has likely positive impacts on enhancing farm productivity, conserving natural resources, as well
as improving economic growth by meeting standards in domestic, regional, and international
trade.
Among the various mycotoxins, aflatoxins have garnered significant attention due to their
negative, and carcinogenic, effects on human and animal health (Klingelhöfer et al., 2018).
Although aflatoxins are produced by several Aspergillus species, the major causal agent of
contamination globally is A. flavus (Klich, 2007). There are four major aflatoxins, including B1, B2,
G1, and G2; however, aflatoxin‐B1 is the most toxic and prevalent and is classified as a Group 1A
carcinogen by the International Agency for Research on Cancer (IARC 2002). High‐dose exposure
to aflatoxins concentrations can cause acute health effects such as vomiting, abdominal pain, and
even possible death (Probst, Njapau, & Cotty, 2007; Sherif, Salama, & Abdel‐Wahhab, 2009), while
sublethal chronic exposure may lead to liver cancer, stunting in children, and immune system
suppression (Chan‐Hon‐Tong, Charles, Forhan, Heude, & Sirot, 2013; Wu & Khlangwiset, 2010). In
1981, for instance, the outbreak of aflatoxicosis as a result of ingestion of maize contaminated
with 3.2–12 mg/kg of aflatoxin‐B1 caused fatalities in Kenya (Obura, 2013). In another severe
aflatoxicosis outbreak, Azziz‐Baumgartner et al. (2005) also reported that aflatoxin contamination
was found to be the cause of over 125 deaths during 2004–2005 in Eastern province of Kenya.
Williams et al. (2004) estimated that over 5 billion people living in low‐income countries are at risk
of chronic exposure to aflatoxins.
The incidence of aflatoxin contamination in major food crops such as maize, groundnut,
sorghum, tree nuts, and dried fruits and spices as well as milk and meat products is widespread in
warm climates (CAST 2003; Chala et al. 2014; Mutegi, Ngugi, Hendriks, & Jones, 2009; Perrone
et al., 2014; Williams et al., 2004). In animals, aflatoxins may lower resistance to diseases, interrupt
vaccine‐induced immunity, and adversely affect growth and reproduction, causing serious
economic losses (CAST 2003; Fink‐Gremmels, 1999). When animal feeds are infected with aflatoxin‐
producing fungi, aflatoxins are introduced into animal source food chains and can be converted
to M‐type aflatoxins (De Ruyck, De Boevre, Huybrechts, & De Saeger, 2015; Iqbal, Jinap, Pirouz,
& Ahmad Faizal, 2015). Infection and production of aflatoxins by ubiquitous, air‐borne, and soil‐
inhabiting species of fungi begin at preharvest stages and may continue to increase until the grain
is consumed (Waliyar, Ntare, Diallo, Kodio, & Diarra, 2007; Waliyar et al., 2015).
The interplay between the safety of food and the adequacy of food is therefore crucial when
addressing the aflatoxins problem in low‐income countries. An earlier study by Brudzynski, Van
Pee, and Kornazewski (1977), for example, showed the presence of aflatoxins up to 1,000 μg/kg in
maize and groundnut from the DRC. Recently, Kamika and Takoy (2011) reported that 95% of
groundnut samples collected during the dry and the rainy seasons in Kinshasa contained
aflatoxin‐B1 over the maximum limit of 2 μg/kg prescribed by the EU as the standard for direct
human consumption. These studies were, however, limited to maize and groundnut conducted
only in the DRC. To expand insight, we conducted a comprehensive investigation on the
incidence of aflatoxin contamination in raw and processed materials from cassava, maize,
sorghum, beans, soybean, groundnut, and milk in the local markets of Burundi and Eastern DRC.
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2. MATERIALS AND METHODS
2.1. Sample collection

A total of 244 food samples intended for human consumption, including cassava, maize,
sorghum, beans, soybean, groundnut, milk, and their products, were randomly collected from
local markets in Burundi and Eastern DRC during May–July in 2016. During the time of survey,
an average temperature and relative humidity in Burundi were 24.6°C and 70.7%, respectively,
while Eastern DRC had an average temperature of 20.5°C and relative humidity of 73.6%. To
collect a representative data set, we first obtained the list of villages in each province/district
from Provincial Inspection Office. From each province/district, the villages were then randomly
selected. In Burundi, the samples were collected from five villages in Gitega province (Gishora,
Ruhanza, Tuba‐Murayi, Kobero, and Nyamuhogoti) and eleven villages in Cibitoke province
(Rukana, Munyika, Mparambo I, Mparambo II, Rusigabangazi, Kanazi, Murambi, Gasenyi rural,
Ruhagarika, Kansege, and Kaburantwa). The samples from the DRC were collected from five
villages in Kabare district (Murhesa, Mudaka, Miti Centre, Kavumu, and Katana), Bukavu town
and five villages in Uvira district (Kamanyola centre, Katogota, Luberizi, Sange, and Bwegera)
of Eastern DRC. These areas were contrasted by altitude, average temperature, and rainfall.
Gitega province of Burundi and Kabare district of Eastern DRC is considered as high altitude
area, while Chibitoke province of Burundi and Uvira district of Eastern DRC represents low
altitude area. The farm households in these areas are mainly subsistence farmers and grow
rainfed crops. The main food crops in Gitega province and Kabare district include cereal crops
such as maize and legumes, while in Chibitoke province and Uvira district the main crops are
cassava and legumes.
For each sample, 1 kg of the commodity was bought and collected from different parts of the
seller's container and thoroughly mixed, while 1 L of milk and yoghurt was purchased as sellers
prepared in plastic bottle. The sellers were randomly selected from each market, and the type of
samples was collected depending on available samples from each seller. After collection,
samples were labeled with the name of village and collection date and then subdivided into three
portions. The first portion was kept as a backup, while the second was directly used for moisture
analysis. The third was examined to determine the level of aflatoxin contamination. All samples
were sealed in polyethylene plastic bags under normal atmospheric conditions, whereas the milk
and dairy products were kept in plastic bottles. The package seal was carefully inspected to avoid
any possibility of leakage. Subsequently, the sealed packages were stored at a temperature of 4°C
for dried samples and −4°C for milk and dairy products about 2 weeks, without direct sunlight
until further analysis.
2.2. Chemical analysis
2.2.1. Moisture content

Moisture content (MC) of each sample was determined by drying the samples in the hot air oven
at 105°C for 12 hr, following technique 950.46 (AOAC 2006). The tests were conducted in
triplicates, and the moisture content was calculated using the following formula: [(original
weight of sample – weight of sample after drying)/original weight of sample] *100.
2.2.2. Analysis of aflatoxins in grains

For each sample except flour, 200 g was ground into fine powder using a laboratory blender
(model 37BL85; Dynamics Corporation of America, USA). Approximately 10 g ground sample
was added to 50 ml 65% ethanol (v/v) in a 100 ml media bottle. The resulting suspension was
shaken (model HS 501 D Shaker; IKA, Germany) at 200 rpm for 3 min to extract aflatoxins. The
suspension was allowed to settle, filtered through Whatman No. 1 paper, and filtrate collected.
To analyze the aflatoxins concentration, a Reveal Q+ test kit (Neogen Corporation, USA) was
used as a single step lateral flow immunochromatographic assay based on a competitive
immunoassay format. A total of 500 μl diluent was mixed with 100 μl of the sample filtrate and
then carefully mixed by pipetting up and down five times in a dilution cup. A 100 μl portion of
the mixture was transferred to a new clear sample cup. Subsequently, a Reveal Q+ for aflatoxin
test strip was placed into the sample cup for 6 min; the strip was removed and inserted to the
AccuScan® reader (AccuScan Pro, model AX‐2; Neogen Corporation, Australia). Aflatoxin
concentration was displayed in parts per billion (ppb). All samples were analyzed in duplicate
from a separate 10 g measure.
2.2.3. Analysis of aflatoxins in milk and dairy products

To determine aflatoxin‐M1 in milk and yogurt, the method developed by Gizachew, Szonyi,
Tegegne, Hanson, and Grace (2016) was adapted, while the method of Škrbić, Antić, and Živančev
(2015) was modified to determine aflatoxin‐M1 in cheese products. One hundred milliliter of milk
and yogurt samples was warmed to 37°C in a water bath and then centrifuged at 10°C with
3500 g for 10 min (model TDL‐5‐A; Lab companion, Korea). After discarding the upper cream
layer, the remaining skimmed milk was filtered through Whatman No. 4 filter paper before
aflatoxin‐M1 analysis. For the cheese products, 2 g of homogenized samples were weighed and
blended with 40 ml dichloromethane for 15 min. The filtrates were evaporated via rotary
evaporator (model R‐II; Büchi, Postfach, Switzerland) at 60°C. A solution of 0.5 ml methanol,
0.5 ml phosphate buffer saline (PBS), and 1 ml hexane was added to the residue and then
centrifuged at 15°C with 2,700 g for 15 min. The lower methanolic phase was collected. Prior
aflatoxin‐M1 determination, 100 μl of this methanolic phase was diluted with PBS to achieve a
dilution of 1:5.
Assay procedure was followed according to the protocol provided by
RIDASCREEBN® Aflatoxin‐M1 (R‐Biopharm AG, Darmstadt, Germany). Briefly, 100 μl
solutions from the mixing wells were transferred to the assay wells coated with aflatoxin‐
M1 antibodies and incubated for 15 min at room temperature in the dark. After adding 100 μl
horseradish peroxidase as a conjugate to aflatoxin‐M1, incubation continued for further 30 min.
Then the liquid was poured out of the wells, and the wells were washed with PBS‐Tween 20
buffer solution three times. The wells were tapped face down on a layer of absorbent to remove
the residual wash buffer. Subsequently, 100 μl of tetramethylbenzidine (TMB), as enzyme
substrate, was added into each well, incubated for 15 min at room temperature in the dark, and
then 100 μl of the stop solution was added to the microplate wells, which changes the color from
blue to yellow. The optical density (OD) was measured at 450 nm using the enzyme‐linked
immune‐sorbent assay (ELISA) plate reader (model BDSL, Immunoscan plus; Lab systems,
Finland). All analyses were run in duplicates.
Two sets of standard solutions were prepared for aflatoxin‐M1 calibration curves. The lower
concentrations were in the range of 0.05–0.1 mg/L, whereas the higher concentrations were in
the range of 0.1–2.0 mg/L. Samples that were beyond the range of the highest standard
concentration were diluted, and the ELISA experiments were repeated.
2.2.4. Total aflatoxin and aflatoxin‐M1 validation

To test the sensitivity of the method, the total aflatoxin standard solution at two different
concentrations was added to the all samples. The extraction and the recovery of the spiked
samples were performed as previously described, in duplicate. The validation of Reveal Q+ and
ELISA methods was carried out with the determination of the recoveries and the coefficient of
variation (%CV) as presented in Table 1.
Table 1
Validation data of methods for total aflatoxins in dried food, milk, and dairy products samples
Category Total aflatoxin level added % Coefficient of variation

(μg/kg) Recovery (%CV)
Cassava
Dried root 2.0 80.2 3.0
10.0 82.3 3.7

Flour 2.0 85.7 2.9
10.0 87.6 5.1
Ubuswage a 2.0 83.5 6.0
10.0 81.2 2.9
Bakery products (bread, 2.0 77.8 4.0

cookies)
10.0 83.1 1.2
Maize
Grain 2.0 93.6 2.1
10.0 90.8 3.6
Flour 2.0 84.3 4.3

10.0 90.1 2.4
Sorghum
Grain 2.0 83.4 1.7
10.0 85.2 2.5
Flour 2.0 80.5 3.3
10.0 87.7 4.7
Germé b 2.0 78.9 2.6
10.0 77.6 4.1
Beans
Grains 2.0 82.8 3.9
10.0 88.1 1.6
Soybean
Grains 2.0 83.2 3.2
10.0 84.6 5.5
Flour 2.0 85.0 4.1
10.0 88.5 3.6
Groundnut
Grain 2.0 90.4 1.3

10.0 92.6 3.2
Roasted 2.0 88.5 2.4
10.0 83.9 1.8
Flour 2.0 87.2 1.1
10.0 86.1 2.4
Milk
Fresh milk 0.5 91.5 1.7
1.0 101.2 1.1
Yogurt 0.5 95.4 2.8

1.0 98.5 4.2
Cheese 0.5 91.7 1.9
1.0 101.5 2.1
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a
Ubuswage is the traditional cassava product in Central African region.
b
Germe is the germinated sorghum for beer processing.
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3. RESULTS AND DISCUSSION
3.1. Moisture content of samples

The MC of grain samples collected from local markets in Burundi and Eastern DRC is shown in
Table 2. There was no significant difference in mean MC of the samples from the two countries.
In the market, grain samples were mostly kept in open containers, while processed samples were
stored in plastic closed containers or paper bag. Only traditionally fermented cassava foods
(ubuswage) were wrapped in plantain leaves. Overall, the MC ranged between 6.7% and 15.0%
for grain samples and between 5.5% and 13.6% for flour, with the lowest being recorded in
groundnut flour and the highest in cassava flour. Much higher MC content was recorded in the
cassava prepared for ubuswage, with an average MC of 59.8%. In addition, the MC of fresh
milk, yogurt, and cheese ranged between 79.3% and 89.3%.
Table 2
Presence of moisture content in food samples collected from local markets in Burundi and
Eastern DRC
Category Moisture content (g 100/g w.b.)
Burundi Eastern DRC
Cassava
Dried root 14.95 ± 1.23 –
Flour 13.58 ± 2.41 13.14 ± 1.52
Ubuswage a 59.76 ± 4.41 –
Bakery products (bread, – 13.07 ± 1.18

cookies)
Maize
Grain 11.21 ± 1.49 11.92 ± 1.26
Flour 10.36 ± 1.34 10.58 ± 1.28
Sorghum
Burundi Eastern DRC
Grain 12.23 ± 0.52 12.52 ± 1.44
Flour 10.76 ± 1.91 10.71 ± 0.76
Germé b 10.39 ± 0.65 –
Beans
Grain 11.52 ± 1.03 11.85 ± 1.30
Soybean
Dried 9.54 ± 0.30 8.74 ± 1.15
Flour 6.76 ± 2.35 7.60 ± 1.29

Burundi Eastern DRC
Groundnut
Grain 7.00 ± 1.00 6.65 ± 1.67
Roasted 4.56 ± 1.05 5.21 ± 1.26
Flour 6.52 ± 0.63 5.52 ± 1.04
Milk
Fresh milk 89.25 ± 1.41 89.12 ± 1.35
Yogurt 88.02 ± 1.25 87.72 ± 1.47
Cheese – 79.34 ± 1.08

Notes
Value is the mean ± SD.
a
b
3.2. Occurrence of aflatoxins in crop samples

The occurrence and concentration of total aflatoxins in crop samples collected from Burundi and
Eastern DRC are summarized in Tables 3 and and4.4. All the 218 samples were contaminated
with aflatoxins, which ranged from 1.3 to 2,410.0 μg/kg. Nowadays, the EU has set the strictest
standards, such that any products for direct human consumption can only be marketed with
concentrations of aflatoxin‐B1 and total aflatoxins not >2 and 4 mg/kg, respectively (EC, 2007, 2010).
Likewise, US regulations have specified the maximum acceptable limit for total aflatoxins at
20 mg/kg (Wu, 2006). In India, a tolerance limit of 30 mg/kg for aflatoxins in all foods has been
defined. Kenya adopted a maximum allowed level of 10 mg/kg of aflatoxin‐B1 in groundnuts
and several grain foods. Brazil has fixed the limit of total aflatoxins in nuts at 30 mg/kg (Freitas‐
Silva & Venâncio, 2011). As Burundi and DRC do not have regulations for aflatoxins, in this study,
we applied the EU standard as the strictest standards to compare for all crop samples.
Table 3
Distribution of total aflatoxins in dried food samples found on local markets in Burundi and
Eastern DRC
Category Burundi Eastern DRC Overall
Incid Ave Me Ra Incid Ave Me Ra Incid Ave Median( Ran

encea rage dian nge encea rage dian nge encea rage μg/kg) ge
(μg/ (μg/ (μg/ (μg/ (μg/ (μg/ (μg/ (μg/
kg) kg) kg) kg) kg) kg) kg) kg)
Cassava
Dried 8/8 3.7 3.6 2.5– – – – – 8/8 3.7 3.6 2.5–

root 5.4 5.4
Flour 10/10 2.8 2.6 1.9– 18/18 2.7 2.7 1.3– 28/28 2.7 2.6 1.3–

4.6 5.0 5.0
Ubus 2/2 3.8 4.0 3.3– – – – – 2/2 3.8 4.0 3.3–

wage b 4.0 4.0
Baker – – – – 3/3 3.4 3.3 2.3– 3/3 3.4 3.3 2.3–

y 5.6 5.6
produc
ts
(bread
,
cookie
s)
Maize

Grain 10/10 38.7 4.3 2.7– 9/9 10.7 3.2 2.2– 19/19 25.5 3.9 2.2–
330. 73.2 330.
0 0
Flour 10/10 41.9 5.9 3.2– 9/9 47.9 7.5 2.5– 19/19 44.7 6.9 2.5–
350. 320. 350.
0 0 0
Sorghum
Grain 12/12 7.1 6.4 5.6– 11/11 4.1 4.3 2.5– 23/23 23.3 4.8 2.5–
490. 5.5 490.
0 0
Flour 5/5 6.1 5.9 4.0– 7/7 4.9 4.9 3.1– 12/12 5.4 5.2 3.1–
8.5 6.5 8.5
Germ 3/3 6.2 6.3 5.2– – – – – 3/3 6.2 6.3 5.2–


é c 6.9 6.9
Beans
Grains 21/21 3.9 3.7 2.5– 10/10 3.5 3.4 1.9– 31/31 3.7 3.7 1.9–
6.6 6.4 6.6
Soybean
Grains 8/8 3.4 3.5 2.3– 3/3 3.7 3.8 2.8– 11/11 3.5 3.6 2.3–
4.1 4.2 4.2
Flour 5/5 6.9 4.8 3.5– 4/4 4.1 4.4 2.3– 9/9 5.6 5.7 2.3–
12.3 5.5 12.3
Groundnut

Grain 7/7 7.1 4.6 3.9– 9/9 3.4 3.4 2.2– 16/16 5.0 3.9 2.2–
29.3 5.4 29.3
Roaste 10/10 220. 34.0 4.3– 11/11 4.0 3.9 2.9– 21/21 107. 5.3 2.9–
d 3 1,08 5.7 0 1,08
0.0 0.0
Flour 10/10 824. 550. 310. 2/2 1027 101 470. 12/12 857. 550.0 310.
0 0 0– .5 0.0 0– 9 0–
2,41 1,62 2,41
0.0 0.0 0.0
Total 121/1 99.6 4.5 1.9– 97/97 29.3 3.7 1.3– 218/2 68.1 4.0 1.3–
21 2,41 1,62 18 2,41
0.0 0.0 0.0

a
Incidence number is represented by the number of samples with aflatoxins above the detectable level/total sample in
a particular category.
b
c
Table 4
Levela of total aflatoxins contamination in dried foods marketed in Burundi and Eastern DRC
<4 μg/ 4–10 μ >10 μg/ <4 μg/ 4–10 μ >10 μg/ <4 μg/ 4–10 μ >10 μg/

kg g/kg kg kg g/kg kg kg g/kg kg
Cassava
Dried 7 1 0 – – – 7 (87.5) 1 0
root (87.5)b (12.5) (12.5)
Flour 9 (90) 1 (10) 0 16 2 0 25 3 0

(88.9) (11.1) (89.3) (10.7)
Ubuswag 1 (50) 1 (50) 0 – – – 1 (50) 1 (50) 0

e c
Bakery – – – 3 (100) 0 0 3 (100) 0 0

products
(bread,
cookies)
Maize

Grain 4 (40) 4 (40) 2 (20) 7 (77.8) 1 1 (11.1) 11 5 3 (15.8)

(11.1) (57.9) (26.3)
Flour 3 (30) 2 (20) 5 (50) 2 (22.2) 3 4 (44.5) 5 (26.3) 5 9 (47.4)

(33.3) (26.3)
Sorghum
Grain 0 9 (75) 3 (25) 4 (36.4) 7 0 4 (17.4) 16 3 (13)

(63.6) (69.6)
Flour 0 5 (100) 0 2 (25.0) 6 0 2 (15.4) 11 0

(75.0) (84.6)
Germé d 0 3 (100) 0 – – – 0 3 (100) 0
Beans

Grain 14 7 0 8 (80) 2 (20) 0 22 (71) 9 (29) 0

(66.7) (33.3)
Soybean
Grain 7 (87.5) 1 0 2 (66.7) 1 0 9 (81.8) 2 0

(12.5) (33.3) (18.2)
Flour 1 (20) 3 (60) 1 (20) 2 (50) 2 (50) 0 3 (33.3) 5 1 (11.1)

(55.6)
Groundnut
Dried 1 (14.3) 5 1 (14.3) 7 (77.8) 2 0 8 (50.0) 7 1 (6.2)

(71.4) (22.2) (43.8)
Roasted 0 3 (30) 7 (70) 7 (63.6) 4 0 7 (33.3) 7 7 (33.3)

(36.4) (33.3)

Flour 0 0 10 (100) 0 0 2 (100) 0 0 12 (100)
Total 47 45 29 60 30 7 (7.2) 107 75 36

(38.8) (37.2) (24.0) (61.9) (30.9) (49.1) (34.4) (16.5)

a
The EU permissible level and the WHO advisory level for total AFs are 4 and 10 μg/kg, respectively, for foods
intended for direct human consumption.
b
The first integer is the number, and the integer in parenthesis is the percent of samples containing a specified level
of aflatoxins.
c
d
About 60% of these samples contained aflatoxins above the EU maximum permissible limit
(4 μg/kg) for total aflatoxins in maize intended for human consumption (EC 2007; EC 2010). As other
countries found within the tropics, aflatoxin contamination in food commodities from Burundi
and Eastern DRC can be attributed to high temperatures and drought conditions driven by
climate change, resulting in crop stress which favors A. flavus infection in the production field
and proliferation during postharvest period (Bandyopadhyay et al., 2016; Kamika, Koto‐te‐Nyiwa,
& Tekere, 2016; Kamika & Takoy, 2011; Paterson & Lima, 2010; Schmidt‐Heydt, Abdel‐Hadi, Magan,
& Geisen, 2009). In addition, high aflatoxin contamination levels can be compounded by other
farm practice factors, including poor weeding, infertile soils particularly in Burundi, poor crop
rotation, high planting densities, and delayed time of harvesting. The poor storage of agricultural
produce can also lead to accelerated aflatoxin contamination as a result of proliferation of
aflatoxin‐producing fungi. This has been demonstrated by many authors (Azziz‐Baumgartner
et al., 2005; Mwalwayo & Thole, 2016). Some socioeconomic factors may also contribute to
aflatoxins contamination, including informal marketing systems, inadequate transportation
modes, unavailability of needed materials, tools, and equipment, lack of information and
knowledge on appropriate pre‐ and postharvest managements, and poor governmental
regulations and legislations. Moreover, some of these countries have experienced conflicts,
resulting in poor outcomes in health, education, and living standards. Food insecurity and
malnutrition, especially among children, in resource‐poor households are also common
occurrence. It is therefore not surprising that aflatoxin contamination was detected in all of the
samples collected in this study, especially in processed products which have lower MC. The
average aflatoxin contamination was high in the samples from Burundi (99.6 μg/kg) when
compared to those of Eastern DRC (29.3 μg/kg). This result can be explained by the fact that
Burundi is relatively hotter and drier, a situation that favors the growth of mycotoxin‐producing
fungi. Further details of the incidence of aflatoxin contamination in specific crops are also
presented below.
3.2.1. Cassava
Aflatoxin levels in cassava samples ranged from 1.3 to 5.6 μg/kg. More than 88% of the samples
met the EU regulatory threshold for aflatoxin of 4.0 μg/kg. All the samples met the proposed
East Africa regulatory threshold of 10 μg/kg. Similar observations regarding the low
contamination by aflatoxins in cassava are reported in Ghana (Wareing, Westby, Gibbs, Allotey,
& Halm, 2001), Republic of Benin (Adjovi et al., 2014; Gnonlonfin et al., 2012) and Tanzania (Sulyok
et al., 2015) The occurrence of aflatoxins in cassava chips from Cameroon was only detected after
4 weeks’ storage (Essono et al., 2009). These results suggested that fresh cassava is safe regarding
aflatoxin contamination; however, processing methods such as heat treatment, sun drying, or
freezing may alter the ability of cassava to block toxinogenesis, leading to secondary
contamination. Another possible explanation associated with this observation is that the effect of
fermentation process generally employed in the processing of cassava into dried cassava, cassava
flour, and ubuswage favors the growth of lactic acid bacteria (LAB) or some microorganisms
like Saccharomyces cerevisiae strains. The ability of these microorganisms to bind or degrade
aflatoxins, especially aflatoxin‐B1 and aflatoxin‐M1, in foods and feeds has been reported
(Ahlberg, Joutsjoki, & Korhonen, 2015; El‐Nezami & Gratz, 2011; Peltonen, El‐Nezami, Haskard,
Ahokas, & Salminen, 2001). Aflatoxin binding seems to be strongly related to several factors such
as LAB strains, matrix, temperature, pH, and incubation time (El‐Nezami & Gratz, 2011; Shetty,
Hald, & Jespersen, 2007). Moreover, the MC of cassava has been shown to influence the shelf life
of samples rather than aflatoxin occurrence.
3.2.2. Maize
Among the grain samples, the high concentrations of total aflatoxins were obviously detected in
maize, followed by groundnut, sorghum, beans, and soybean, respectively (Table 3). Notably,
aflatoxin levels in maize flour ranged from 2.5 to 350.0 μg/kg. Kamika et al. (2016) also reported
that aflatoxin contamination in the DRC along the maize supply chain. They showed that
contamination increased of up to 500 times from preharvest (3.1–103.9 μg/kg) to city stores
(2,070.5 μg/kg) and to distribution markets (2,806.5 μg/kg). They attributed this trend to
inappropriate storage practices as well as a lack of drying facilities in the country. Similar studies
in SSA countries have reported high levels of aflatoxin contamination in maize. Kaaya and
Kyamuhangire (2006), for instance, reported more than 20 μg/kg of aflatoxins in maize kernels
from Uganda after 6 months of storage, while very high content of aflatoxins in homegrown
maize was found in Kenya when compared to purchased or relief maize (Daniel et al., 2011). Lewis
et al. (2005) indicated that the contaminated homegrown maize may represent a source of aflatoxin
contamination in market maize, especially when local farmers sold a portion of their farm
household stores to market vendors. In Tanzania, Kamala et al. (2015) also reported that 87% of
maize samples were co‐contaminated with aflatoxins and fumonisins.
3.2.3. Sorghum
In this study, all sorghum samples in grain, flour, and germé forms contained detectable
concentrations of aflatoxins, ranging between 2.5 and 490.0 μg/kg. Additionally, total aflatoxins
exceeded the regulatory levels for direct human consumption as set by the EU in 84.6% of the
sorghum samples. The levels of aflatoxin contaminations may also be associated with the poor
pre‐ and postharvest practices as well as processing methods. Sorghum, in particular, is used as a
malted grain (germé) in beer production in Burundi. The traditional processing technique, which
involves the use of Enterobacteruaceae and molds, may cause aflatoxin contamination
in germé (Bationo et al., 2015). Although zearalenone is reported as the most common mycotoxin
found in sorghum (Chala et al., 2014), high levels of aflatoxins, ranging 340–476 μg/kg, were also
found in malted sorghum (Matumba, Monjerezi, Khonga, & Lakudzala, 2011). Another study by
Ayalew, Fehmann, Lepschy, Beck, and Abate (2006) reported that about 6% of field samples of
sorghum in Ethiopia are contaminated with aflatoxin‐B1 up to 26 μg/kg, whereas
Bandyopadhyay, Kumar, and Leslie (2007) found that 5% of sorghum grain samples exceeded the
Nigerian safety threshold of 20 μg/kg.
3.2.4. Beans
Aflatoxin was present in 100% of bean samples from Burundi and Eastern DRC and ranged from
1.9 to 6.6 μg/kg. This low level of aflatoxin contamination in the bean samples is perhaps due to
the ability of phenolic compounds, particularly gallic and chlorogenic acids, to inhibit fungal
amylase activities (Telles, Kupski, & Furlong, 2017). Pagnussatt, Bretanha, Sílvia, Garda‐Buffon,
and Badiale‐Furlong (2013) also mentioned that the synergistic effect of different compounds in
beans can contribute to a defense barrier against development of toxigenic species. Literature
reports a few instances of aflatoxins in red kidney beans, split peas, chickpea, and cowpea such
as in Pakistan (Lutfullah & Hussain, 2012).
3.2.5. Soybean
All soybean samples analyzed were positive for total aflatoxins with 40.0% of these samples
exceeding 4 μg/kg. The highest concentration of aflatoxins was found in flour than in dried
grains. It has been reported that aflatoxin contaminations in soybean are relatively low, but there
are conflicting explanations to the possible cause of low aflatoxin contamination in soybean. One
of the initial studies associated this phenomenon to the zinc binding ability of phytate in soybean,
as it is an important intermediate substrate of aflatoxin biosynthesis (Gupta &
Venkitasubramanian, 1975). However, Ehrlich and Ciegler (1985) showed that phytate level does not
influence aflatoxin biosynthesis. Burow, Nesbitt, Dunlap, and Keller (1997) hypothesized that
lipoxygenase in soybean can produce hydroxyl fatty acids which are capable of inhibiting
aflatoxin production in A. parasiticus. With regard to aflatoxin inhibition, Mellon and Cotty (2002)
reported that soybean grains with lipoxygenase might not deter increased seed pathogen
susceptibility, but seed coat integrity and seed viability may play more determinant role in seed
resistance to aflatoxin contamination. There is hence the need for further understanding of the
possible cause of low aflatoxin contamination in soybean.
3.2.6. Groundnut
In this study, total aflatoxins concentration in groundnut products from the local markets in
Burundi and Eastern DRC ranged from 2.2 to 2,410.0 μg/kg. The highest contamination level
was found in groundnut flour (2,410 μg/kg), followed with roasted groundnut (1,080 μg/kg) and
dried kernels (29.3 μg/kg), respectively. About 69.4% of the groundnut samples exceeded the EU
aflatoxin regulatory limits. None of the groundnut flour samples were fit for human consumption
according to any existing regulation globally, with some samples surpassing the EU maximum
permissible limit of 4 μg/kg by 600‐fold. Aflatoxins were found more in processed groundnut
than in unprocessed dried grains (Tables 2 and and3).3). Processed groundnut, often prepared
from low quality groundnut, can be exposed to a wide range of environmental conditions, such
as high temperature and humidity as well as to oxygen and mold, which can trigger further
increase in aflatoxin contamination. Nonetheless, other factors including biological, nutritional,
and climatic factors can be responsible for aflatoxins contamination, especially in groundnut and
maize, some of which are either difficult or impracticable to control. Groundnut is a preferred
substrate for aflatoxin‐producing fungi (Bankole, Schollenberger, & Drochner, 2006; Ezekiel
et al., 2013; Monyo et al., 2012). The range of aflatoxin contamination in groundnut samples in this
study was comparable to those reported from local vendors, markets, and retail shops in Nigeria
where aflatoxin‐B1 detected in 64.2% of dry roasted groundnut (Bankole, Ogunsanwo, &
Eseigbe, 2005). In Kenya, about 87.0% of groundnut were contaminated with <4 μg/kg of
aflatoxin‐B1, while 7.5% exceeded national regulatory limited of 20 μg/kg (Mutegi et al., 2009).
Similarly, 70% of groundnut samples from the DRC were found to contain higher than 5 μg/kg
aflatoxins (Kamika & Takoy, 2011). Matumba, Van Poucke, Monjerezi, Ediage, and De Saeger
(2015) also revealed that groundnut samples from informal markets in Malawi contained aflatoxins
up to 47 times as compared with samples destined as export goods.
3.3. Occurrence of aflatoxin‐M1 in milk and dairy products

Milk and dairy products are important for growth and development as well as maintenance of
good health in humans, especially babies and children. The occurrence of aflatoxin‐M1 in milk
and its products collected in Burundi and Eastern DRC is presented in Tables 5 and and6.6.
According to the EU regulations, the maximum residue level of aflatoxin‐M1 in raw milk and
dairy products is 50 ng/L, while this level based on USA regulations was adjusted to 500 ng/kg
(Campagnollo et al., 2016; Iqbal et al., 2015; Mulunda & Mike, 2014). Aflatoxin‐M1 was detected in all
samples collected for this study, with concentrations ranging between 4.8 and 261.1 ng/kg.
Among the 13 fresh milk samples analyzed, 4 (30.8%) contained aflatoxin‐M1 above the
maximum permissible limit of 50 ng/kg, as set by the EU for raw milk, heat‐treated milk, and
milk for the manufacture of milk‐based products (EC 2006). Of the eight yogurt samples, only two
samples (25%) were contaminated with aflatoxin‐M1 above the limit of 50 ng/kg, with the
concentration ranging between 4.8 and 63.2 ng/kg. Brackett and Marth (1982) explained that the
changes in casein structure due to fermentation process may cause adsorption or occlusion of
toxins, including aflatoxin‐M1, in the precipitate. Montaseri et al. (2014) also referred to this
behavior as the possible reason why LAB is capable of removing aflatoxin‐M1 from yogurt.
Furthermore, the low concentration of aflatoxin‐M1 in yogurt might be associated with
processing variables such as pH, formation of organic acids, or other fermented by‐products
(Govaris, Roussi, Koidis, & Botsoglou, 2002).
Table 5
Incidence and concentration of aflatoxin‐M1 in milk and dairy products sampled from local
markets in Burundi and Eastern DRC
Incide Aver Med Ran Incide Aver Med Ran Incide Aver Med Ran
ncea age ian ge ncea age ian ge ncea age ian ge
(μg/k (μg/ (μg/ (μg/k (μg/ (μg/ (μg/ (μg/
g) kg) kg) g) kg) kg) kg) kg)
Milk
Fres 10/10 31.4 43.6 8.4– 3/3 37.3 37.5 25.6 13/13 42.6 40.5 8.4–
h 82.8 – 82.8
milk 49.8
Yog 6/6 32.5 33.5 8.2– 2/3 18.0 16.1 4.8– 8/8 27.7 25.0 4.8–
urt 63.2 26.0 63.2
Che – – – – 5/5 170.0 142. 18.5 5/5 170.0 142. 18.5

ese 0 – 0 –
261. 261.
1 1
Total 16/16 32.5 39.8 8.2– 10/10 37.3 83.3 4.8– 26/26 56.6 32.5 4.8–
82.8 261. 261.
Incide Aver Med Ran Incide Aver Med Ran Incide Aver Med Ran
ncea age ian ge ncea age ian ge ncea age ian ge
(μg/k (μg/ (μg/ (μg/k (μg/ (μg/ (μg/ (μg/
g) kg) kg) g) kg) kg) kg) kg)
1 1

Incidence number is represented by the number of samples with aflatoxins above the detectable level/total sample in
a
a particular category.
Table 6
Number of samples with ≤50 ng/kg or more of aflatoxin‐M1 concentration in milk and dairy
products marketed in Burundi and Eastern DRC
≤50 ng/k >50 ng/kg ≤50 ng/k >50 ng/kg ≤50 ng/k >50 ng/kg

g g g
Milk
Fresh milk 6 (60)a 4 (40) 3 (100) 0 9 (69.2) 4 (30.8)
Yogurt 4 (66.7) 2 (33.3) 2 (100) 0 6 (75) 2 (25)

≤50 ng/k >50 ng/kg ≤50 ng/k >50 ng/kg ≤50 ng/k >50 ng/kg

g g g
Cheese – – 1 (20) 4 (80) 1 (20) 4 (80)
Total 10 (62.5) 6 (37.5) 6 (60) 4 (40) 16 (61.5) 10 (38.5)

a
Figures in parenthesis indicate proportion (%) of samples in a particular category.
Four out of five (80.0%) cheese samples had concentration of aflatoxin‐M1 below the EU
maximum limit of 250 ng/kg. The contamination of aflatoxin‐M1 in these samples can be
attributed to the intake of aflatoxigenic mold contaminated feeds by milk‐producing animals.
Variability of aflatoxin‐M1 in milk and dairy products is influenced by several factors such as
geographical region, seasons, type and quality of feed, feed storage conditions, and processing
methods and conditions (Gizachew et al., 2016; Škrbić et al., 2015).
Several studies have reported the occurrence of aflatoxin‐M1 in milk and dairy products. Milk
samples from urban centers in Kenya contained aflatoxin‐M1 up to 6,800 ng/L (Kang'ethe &
Lang'a, 2009). In Sudan, 95% of milk was contaminated with aflatoxin‐M1 ranging between 220
and 6,800 ng/L (Elzupir & Elhussein, 2010), whereas 6–527 ng/L of aflatoxin‐M1 was detected in
15% of cow milk samples from Cameroon (Tchana, Moundipa, & Tchouanguep, 2010). The
concentration of aflatoxin‐M1 varied between 150 and 170 ng/L in commercial and rural milk in
South Africa (Mulunda & Mike, 2014), while 8.0% of milk samples in Ethiopia contained
aflatoxin‐M1 <5 ng/L (Gizachew et al., 2016). In Iran, Feta cheese samples contained aflatoxin‐
M1 with concentration ranging from 150 to 2,410 ng/kg (Kamkar, Karim, Aliabadi, &
Khaksar, 2008), whereas white cheese was contaminated with 52 to 745 ng/kg of aflatoxin‐
M1 (Fallah, Jafari, Fallah, & Rahnama, 2009). In Serbia, Tomašević et al. (2015) identified that 56.3%
of raw milk, 32.6% of heat‐treated milk, and 37.8% of milk product samples contaminated
aflatoxin‐M1 above the EU maximum residue permitted amount.
Go to:
4. CONCLUSIONS
This first report on the incidence of aflatoxin contamination in agricultural products from local
markets in Burundi and Eastern DRC showed that of the 244 crops, milk, and their processed
products sampled, the percentage of aflatoxin positive samples was 100%. In addition, 50.9% of
crop, 28.6% of milk and yogurt, and 20.0% of cheese samples had aflatoxin concentrations
higher than the regulatory limits set by the EU. The processed samples presented higher aflatoxin
contamination when compared to unprocessed samples. Therefore, the presence of aflatoxin in
local food products from Burundi and Eastern DRC is a problem in the context of food
sufficiency, public health, and economic benefits. Appropriate pre‐ and postharvest management
strategies need to be promoted among actors along the food value chains, especially farmers and
processors, to achieve significant reduction in aflatoxin contamination in agricultural
commodities. This can increase food availability, accessibility, utilization, and stability, as well
as economic sustainability in the two countries. At the subsistence farm and processing levels,
application of biocontrol tools, in conjunction with other aflatoxin‐management practices such as
drying and storage technologies, as well as the proper and effective regulatory standards are
required as part of efforts to reduce the risk of aflatoxin contamination. Mitigation measures
must, however, be backed up by further insights on the causes of contamination and possible
variations in contamination levels across regions as well as crop commodities. Further work, for
example, on the microbiology, especially on etiology, on‐farm, and postharvest as well as
marketing structures need to be studied further. To further strengthen the county's efforts in
abating contamination, risk assessments are proposed in order to establish country regulatory
thresholds that the local consumer population can depend on and which can be used to monitor
safety across the country. These thresholds can also be used to monitor safety of food
commodities across the county's boarders.
Go to:
CONFLICT OF INTEREST
The authors have no conflict of interests.
Go to:
ETHICAL STATEMENT
This study does not involve any human or animal testing.
Go to:
ACKNOWLEDGMENTS
The authors gratefully acknowledge the “ILRI/IITA Crop Livestock Integration Project (PJ‐
002057)” for giving the opportunity to prepare this article. The authors also acknowledge the
support of the project “Aflatoxin contamination prevention and control in grain” which is funded
by the Bill & Melinda Gates Foundation (OPP1007117).
Go to:
Notes
Udomkun P, Mutegi C, Wossen T, et al. Occurrence of aflatoxin in agricultural produce from
local markets in Burundi and Eastern Democratic Republic of Congo. Food Sci Nutr.
2018;6:2227–2238. 10.1002/fsn3.787 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Go to:
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Aflatoxin 4

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Abstract

2. MATERIALS AND METHODS

2.1. Sample collection

2.2. Chemical analysis

2.2.1. Moisture content

2.2.2. Analysis of aflatoxins in grains

2.2.3. Analysis of aflatoxins in milk and dairy products

2.2.4. Total aflatoxin and aflatoxin‐M1 validation

Category Total aflatoxin level added % Coefficient of variation

Dried root 2.0 80.2 3.0

10.0 82.3 3.7

Flour 2.0 85.7 2.9

10.0 87.6 5.1

Ubuswage a 2.0 83.5 6.0

10.0 81.2 2.9

Bakery products (bread, 2.0 77.8 4.0

10.0 83.1 1.2

Grain 2.0 93.6 2.1

10.0 90.8 3.6

Flour 2.0 84.3 4.3

10.0 90.1 2.4

Grain 2.0 83.4 1.7

10.0 85.2 2.5

Flour 2.0 80.5 3.3

10.0 87.7 4.7

Germé b 2.0 78.9 2.6

10.0 77.6 4.1

Grains 2.0 82.8 3.9

10.0 88.1 1.6

Grains 2.0 83.2 3.2

10.0 84.6 5.5

Flour 2.0 85.0 4.1

10.0 88.5 3.6

Grain 2.0 90.4 1.3

10.0 92.6 3.2

Roasted 2.0 88.5 2.4

10.0 83.9 1.8

Flour 2.0 87.2 1.1

10.0 86.1 2.4

Fresh milk 0.5 91.5 1.7

1.0 101.2 1.1

Yogurt 0.5 95.4 2.8

1.0 98.5 4.2

Cheese 0.5 91.7 1.9

1.0 101.5 2.1

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3. RESULTS AND DISCUSSION

3.1. Moisture content of samples

Burundi Eastern DRC

Dried root 14.95 ± 1.23 –

Flour 13.58 ± 2.41 13.14 ± 1.52

Bakery products (bread, – 13.07 ± 1.18

Grain 11.21 ± 1.49 11.92 ± 1.26

Flour 10.36 ± 1.34 10.58 ± 1.28

Burundi Eastern DRC

Grain 12.23 ± 0.52 12.52 ± 1.44

Flour 10.76 ± 1.91 10.71 ± 0.76

Grain 11.52 ± 1.03 11.85 ± 1.30

Dried 9.54 ± 0.30 8.74 ± 1.15