1 Background

Aflatoxins are naturally occurring secondary metabolites of fungi produced by Aspergillus species, predominantly by Aspergillus flavus and Aspergillus parasiticus [1] There are four principal types of aflatoxins: aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2), where the B-types are produced by A. flavus and G-types are produced by A. parasiticus [2]. They predominantly occur in humid and hot regions of the world such as the Tropics and Sub-tropics. These aflatoxins contaminate the crops produced in the regions such as maize, groundnuts, millet, sesame seeds, wheat, rice, cocoa, fig, and spices due to fungal infection during pre-and post-harvest conditions [3]. Aflatoxins can cause several health-related effects including acute and chronic aflatoxicosis, liver cirrhosis, hepatic carcinoma, child stunting, and immune suppression. AFB1 is the most harmful and frequently found in contaminated samples [4]. A maximum permissible level of aflatoxin provided by the European Union for AFB1 is 2 µg/kg and is 4 µg/kg for total aflatoxins [5], 10 µg/kg for total aflatoxins by Codex Alimentarius Commission [1] in Cereals and processed foods and 2 µg/kg for AFB1 and 10 µg/kg for total aflatoxin according to Ethiopian Standard Agency [6] in maize and maize products.

Several studies conducted on mycotoxins revealed that about 25% of the world’s food crops are contaminated with aflatoxins [7]. The safety of food and beverages is influenced by several factors that can undermine the quality of the raw materials. Mycotoxicosis of the raw materials makes the consumption of the final product unsafe leading to adverse health effects [8, 9].

The presence of high concentrations of aflatoxins has been demonstrated in maize for consumption from many parts of the world. The study conducted in China on 44 pre- and post-nature drying maize kernels revealed contamination of only 1 sample with AFB1 level of 148.4 µg/Kg [10]. In a survey of 396 maize tortillas from Mexico, Average aflatoxin concentrations in 56 contaminated samples were: AFB1 (12.1 µg/Kg); AFB2 (2.7 µg/Kg); AFG1 (64.1 µg/Kg), AFG2 (3.7 µg/Kg), and total AF (20.3 µg/Kg) [11]. In another study held on 180 maize samples in Serbia, because of changes in weather conditions 68.5% (13) of samples showed a mean total aflatoxin content of 36.3 µg/kg (ranging from 1.01 to 86.1 µg /kg) [12].

Rural dwellers in Africa depend on homegrown crops such as maize, sorghum, millet, barley, and others for the production of food and beverages. The climate, poor agricultural practices, and storage conditions contribute to the occurrence of high contamination of cereals and cereal products in Africa. It is estimated that a 64% reduction in food quality in Africa is contributed by the aflatoxin contamination of the crops used for their food products [13].

High levels of several mycotoxins have been observed in maize from household stores and markets in West African countries. A mean level of 330 μg/kg with a maximum level of 1900 μg/kg of aflatoxins were found in farm-gate maize samples in Ghana and Nigeria [14]. In another study on 51 maize flour samples from Côte d'Ivoire, 107.9 μg/kg of mean total aflatoxin level was observed [15].

A study done on 19 maize samples from Burundi and the Eastern Democratic Republic of Congo (EDRG) showed the presence of aflatoxin in all samples, ranging from 2.5 to 350 μg/kg with a mean total aflatoxin level of 25.5 μg/kg [16]. In another study on maize flour samples from the markets, a mean total aflatoxin concentration of 7.6 ± 2.3 µg/kg was observed where about 20% of the samples showed a higher level of aflatoxin than the maximum acceptable level in East Africa (10 µg/kg) [17].

A study conducted on two popular cereal-based beverages (“Kunu-zaki” and “Pito”/) in Nigeria, showed that the fermentation process reduced at least 76% of mycotoxin contamination (in “Kunu-zaki” produced from sorghum) and reduced 59% (in “Pito” produced from maize) [18]. Another study conducted on sorghum malt obtained during the production of traditional beer “dolo” in Burkina Faso, revealed a mean concentration of 97.6 μg/kg AFB1, but after the beer was brewed and fermented detectable level of aflatoxin was not found in the beer [19].

The presence of aflatoxigenic fungi and aflatoxin contamination are detected in many food commodities in Ethiopia. AFB1 is the most common aflatoxin detected in several crops in Ethiopia. A mean aflatoxin B1 level of 29.5 μg/kg was analyzed in sorghum collected from farmers’ stores in Ethiopia which is higher than the permissible level by the European Union for food cereals (2 μg/kg) [20]. A study conducted on 150 maize samples from Dilla revealed that all the maize samples showed aflatoxin toxicity higher than the limits set by the European Union (EU) and Food and Drug Administration (FDA), in the case revealed 53 ppb mean aflatoxin concentration for all samples analyzed [21]. The maize collected from Ambo, Adama, and Diredawa showed a total aflatoxin content ‹ 5 µg/kg [22]. Another study conducted, on maize grains from west Gojjam indicated a mean total aflatoxin levels of 18.38 µg/kg and 43.43 µg/kg for pre-harvest and post-harvest maize samples respectively [23].

In Ethiopia, there are many types of traditional beverages produced from various cereal crops in different localities by indigenous processing methods under uncontrollable natural conditions. Among these “Tej”, “Tella”, “Borde”, “Shamita”, “Cheka” and “Araki” are studied traditional beverages in Ethiopia [20, 24]. The studies conducted on these traditional beverages were mainly focused on processing operations, and microbiological and physicochemical parameters [24,25,26] instead of their mycotoxin contamination level. “Cheka” is a cereal‐based traditional fermented beverage that is consumed in Southwestern parts of Ethiopia mainly in Dirashe, Konso, Alle, and their neighboring localities. It is often prepared from cereal ingredients such as maize, sorghum, finger millet, barley, wheat, and some vegetable additives. In particular, brewers of “Cheka” in Konso utilize maize as the main ingredient to prepare “Cheka” for both home consumption and market. All age groups, sexes, and socio-economic classes consume “Cheka” as a routine recip. On average an adult man can consume up to 8 L per day [26]. The traditional processing methods, ingredients utilized, and the uncontrollable involvement of different microorganisms in the spontaneous fermentation of “Cheka” are, the possible sources of contamination of “Cheka” with aflatoxin.

Studies showed that exposure to aflatoxin contamination contributes to between 4.6 and 28.2% of the entire cases of liver cancer in the world and; those people with positive results of hepatitis B virus (HBV) would have 30 times the risk of getting hepatic carcinoma than those who are HBV negative when exposed to aflatoxin contamination [27]. Several recent studies showed that many individuals die from liver diseases which is increasing unprecedentedly in Ethiopia [25]. It is suspected that consumption of “Cheka”, is a common risk factor for liver disease. Besides this, there is a lack of awareness about the effects of aflatoxin contamination which causes huge economic and health damage [28]. Therefore, our study was conducted to evaluate aflatoxin contamination of “Cheka” and its major ingredient (maize).

2 Methods

2.1 Study design and period

This study followed a completely randomized experimental design to evaluate the level of aflatoxins in traditionally brewed “Cheka” (from “Cheka” vending houses) and its major ingredient (maize seeds from farms and “Cheka” vending houses) samples in the Konso zone, located in southwestern Ethiopia from November 2019 to June 2020.

2.2 Description of study area

The study was conducted in the Konso zone, located in the South Nations Nationalities and Peoples Region (SNNPR) of south Ethiopia, and the experiments were carried out in the Bless Agri-Food laboratory located 20 km east of Addis Ababa at Legetafo town. Konso is located 595 km from Addis Ababa, the capital city of Ethiopia. It has 4 weredas and 1 city administration with a total population size of about 359,000. It is located at 5° 15′ N 37° 29′ E Latitude and 5.250° N 37.483 ° E Longitude and is elevated 1650 m above sea level. Its average annual temperature and rainfall are 23.95 °C and 507 mm respectively [29]. (Fig. 1 and Table 1)Samples and Sampling.

Fig. 1
figure 1

Map of the study area (konso) (Gelanew et al., 2010)

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Table 1 Geographical locations of the origins of the sample
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A total of 12 samples; 6 “Cheka” samples from “Cheka” vending houses and 6 maize samples (3 from pitfall, 3 from upper ground storage (“Gotera”), were taken and analyzed in duplicate. Crude “Cheka” samples (1 L each) were collected within clean plastic containers with tight seals. Maize seed samples (1 kg each) were collected within sealed polyethylene plastic bags purposively by considering the processing methods and fermentation time for “Cheka”. All maize samples were collected from the harvest of 2019. “Cheka” samples prepared from respective maize samples were collected on the same day of fermentation. All samples were coded with the first letter of the site followed by the first letter of storage type (or sample type) and transported to the Bless Agri-Food laboratory located 20 km east of Addis Ababa at Legetafo town using an icebox. Subsequently, the sealed packages were stored at a temperature of 4 °C for maize seed samples and − 18 °C for “Cheka” samples to prevent further fermentation until the analysis of aflatoxin was carried out. (Figure 2).

Fig. 2
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Traditional maize storage types (A) Upper ground (“Gotera”) (B) underground storage (Pitfall)

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2.3 Determination of aflatoxins in maize and “Cheka” samples

Analysis of total aflatoxins (B1, B2, G1, and G2) in the “Cheka” samples and maize seed samples was carried out by using the AOAC (Association of Official Agricultural Chemists) official method (2005. 08) and LCTech sample preparation and analysis manual with some modifications and method validations.

2.4 Materials and accessories

The materials used in the experiment of aflatoxin evaluation were; grand laboratory mills, spoon, homogenizers, mechanical shaker for extraction, Beaker (50 ml, 1000 ml), magnetic stirrer, Whatman filter paper, funnel, single-use syringes(10 ml pp), millipore filter, filter disks (0.2 μm), ImmunoAffinity column, laboratorystand with clump, plastic bags, icebox, vials with screw cap and sealing ring, Graduated pipettes ( 10 ml, 25 ml, and 50 ml), micropipettes, micropipette tips, Sample label marker, volumetric flask (10 ml, 25 ml, 50 ml, 100 ml, 500 ml, and 1000 ml), vacuum pump, graduated cylinder (100 ml), conical flask (250 and 500 ml), pH meter, Electronic balance, AflaCLEAN™ (LCTech P/N 10514), HPLC system consisting of auto-sampler with the injector, pump, column oven, Link, Degasser, fluorescence detector, post-column derivatizer and computer with chromatography software, UVE™ Photochemical Reactor, and C-18 reversed-phase HPLC column.

2.5 Chemicals and reagents

HPLC-grade Acetonitrile, methanol, distilled water, phosphate-buffered saline (PBS), and n-hexane in different concentrations were used.

Extraction solution: Methanol–water (4:1 v/v).

PBS stock solution (PH 7.2): Sodium Chloride (NaCl: 40.48 g/l), Sodium Phosphate Dibasic (Na2HPO4.12H2O: 27.6 g/l), Sodium Dihydrogen Phosphate (NaH2PO4.H2O: 71.63 g/l).

HPLC Mobile Phase: Water–Methanol-Acetonitrile (60:25:15, v/v/v).

Aflatoxin standards: Aflatoxins (B1, B2, G1, G2, and Mixed) purchased from Sigma Aldrich (St. Louis, MO, USA) were used. All the chemicals and reagents were obtained from Bless laboratory.

2.6 HPLC method validation

Evaluation of aflatoxin content in “Cheka” and its ingredients was carried out with the Shimadzu HPLC system after its analytical performance and validation were evaluated by primarily preparing aflatoxins standard solutions and injecting them into the machine, identifying injected aflatoxins by their retention times, determining the limit of detection (LOD) and Quantification (LOQ), Accuracy, Precision, Standard curve & Linearity before running the sample for analysis. The replicate analyses of the standard reference material and proficiency testing materials were used during HPLC method validation.

2.7 Identification

Aflatoxin identification was performed by running individual and mixed standard aflatoxins (AFG2, AFG1, AFB2, and AFB1) injected at the same condition and recording their retention time or elution order of the specific Aflatoxin peaks.

2.8 Limit of detection (LOD) and limit of quantification (LOQ)

The limit of detection is a minimum quantity of aflatoxin substance giving a signal-to-noise ratio of at least 3 (S/N ≥ 3). The LOD was measured for all 4 aflatoxins by injecting standard solutions of different concentrations and measuring for each substance the amount injected on the column and the S/N values. Approximate LOD values range from 0.23 to 0.94 µg/Kg. LOQ is the lowest concentration of aflatoxin that can be determined with acceptable precision and accuracy. This also corresponds to the lowest concentration of the calibration curve, which was set at 0.5 μg/L for AFG2 and AFB2, and 2 μg/L for AFG1 and AFB1, corresponding to back-calculated concentrations of 0.7 and 2.8 μg/kg for the samples (Table 2).

Table 2 Limit of Detection and Limit of quantification of Aflatoxins
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2.9 Accuracy and precision

Analysis of samples containing known aflatoxin amounts was used to determine the method's accuracy and precision. Test standards which are prepared separately from the calibration standards, the standard reference material, and the proficiency test sample were used for this purpose. During accuracy checking of the method for the determination of aflatoxins in test samples, the recovery acceptance criterion for the concentration range was between 60 and 115%. The calculated recovery was within the acceptable range (from 94.2 to 108.3%). The precision acceptance criterion for the concentration range was around 21%. The calculated precision was within the acceptable range (from 2.1 to 9.4%).

2.10 Standard curve and linearity

For all four aflatoxins, standard curves were plotted by using the standard solutions covering the concentration ranges from 0.5 to 5 μg/l for Aflatoxin B2 and G2 and 2 to 20 μg/l for Aflatoxin B1 and G1 respectively. Peak areas against the concentrations of the different aflatoxins were plotted and linear regression analysis was used to calculate the equation and the correlation coefficient of the standard curves. The results of the standard curves are summarized in Table 3 and Fig. 3. The acceptance criterion for correlation is > 0.99. All correlation curves were ≥ 0.9985, demonstrating the linearity over the concentration ranges studied.

Table 3 Aflatoxins concentration and peak area of the calibration solutions
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Fig. 3
figure 3

Calibration Curve for Linearity test at different concentration of Aflatoxins

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2.11 Procedures for sample preparation and clean up

50 g of each sample of maize seed was taken and finely ground by the laboratory mills and mixed thoroughly. 20 g of each ground maize sample was taken and added to a conical flask with 2 g of NaCl, mixed with 100 ml extraction solvent containing methanol (80%) and water (20%) and 50 ml n-hexane followed by homogenizing at high speed for 45 min on a shaker. The solution was filtered through Whatman filter paper and the collected filtrate was allowed for phase separation in which a lower phase was used for the next steps. 7 ml of the lower filtered extract was taken and diluted with 43 ml of phosphate buffer saline solution (PBS) adjusted to pH 7.2. The filtrate diluted with PBS to 50 ml solution was allowed for cleaning-up of aflatoxin from other impurities by Reversed-Phase ImmunoAffinity column at a flow rate of 2 ml/min using a gentle vacuum until all samples drained through the column and no more samples remained in the column. The column was then washed with 10 ml distilled water and the residual water was removed by a gentle vacuum pumping. Aflatoxin trapped inside the column was eluted into a graduated glass vial by 2 consecutive additions of 1 ml methanol to break the aflatoxin-antibody bond to aid total leaving from the column for injection into the HPLC system. Except for milling, all sample preparation, extraction, and clean-up processes, steps, and amounts are similar to that of maize samples for “Cheka” samples.

The HPLC was previously calibrated with 5-point standard aflatoxin and conditioned for mobile phase containing Acetonitrile: Methanol: Water (15: 25: 60, v/v) at a flow rate of 1.2 ml/min and Column Oven temperature adjusted to 39 °C. A fluorescence detector was set at excitation and emission wavelengths of 365 and 440 nm, respectively for detection purposes. The injected sample was run for 11 min. The print-out results per sample were reported as the sum of the detected aflatoxins (sum of B1, B2, G1, and G2) at parts per billion (μg/kg). The levels of total aflatoxins in both “Cheka’ and maize samples determined by IAC cleanup and HPLC with fluorescence detection were calculated by using the following equation.

$${\text{Aflatoxin}}\,\left( {{{{\mu g}} \mathord{\left/ {\vphantom {{{\mu g}} {{\text{kg}}}}} \right. \kern-0pt} {{\text{kg}}}}} \right)\, = \,A\, \times \,\left( \frac{T}{I} \right)\, \times \,\left( \frac{1}{W} \right)$$

where;-

A = amount of aflatoxin as elute injected in μg/kg.

T = final test solution elute volume (ml).

I = volume of elute injected into LC (ml).

W = mass of commodity represented by final extract (g).

Each sample was analyzed in duplicate and the average total concentration of aflatoxin was determined as a mean of these two average values which were finally reported in µg/kg.

2.12 Statistical data analysis

The print-out data was imported into IBM SPSS version 25. Independent student T-test was used to compare the mean values of total aflatoxins between maize of different storage sites, between maize and the respective “Cheka” samples, and between “Cheka” prepared from maize of different storage types. P-value less than 0.05 (P < 0.05) was considered an indicator of the presence of statistical significance between the mean values.

2.13 Data quality assurance

To keep the quality of data, each step of this project starting from the collection of samples, transportation, laboratory experiment, data processing, and analysis was strictly followed. To prevent cross-contamination, maize and “Cheka” samples were taken neatly and cleanly. The laboratory instruments and reagents used for the determination of aflatoxin were of standard quality. The internal quality control had been designated and the obtained results were checked for completeness daily. Standard reference material was checked whenever big changes were observed in the test method or instrument.

3 Results

3.1 Aflatoxin concentration of “Cheka” major ingredients (maize Seeds)

A duplicate run was performed to analyze the total aflatoxin of both maize and “Cheka” samples. The level of mean total aflatoxin analyzed in major ingredients of “Cheka” (maize samples) was 6.78 ± 8.28 μg/kg. The maize taken from above-ground storage (“Gotera”) showed a mean total aflatoxin level of 10.23 ± 10.12 μg/kg, ranging from (‹ LOQ—20.25 μg/kg). Maize taken from underground storage (pitfall) showed a mean total aflatoxin of 3.35 ± 5.79 μg/kg, ranging from (‹ LOQ 10.23 ± 10.12 μg/kg). The Aflatoxin G family was not detected in maize samples taken from both storage types. Aflatoxin B family, predominantly aflatoxin B1 contributed to the total Aflatoxin level of both storage types (Table 4).

Table 4 Aflatoxin concentration of “Cheka” major ingredients (maize Seeds)
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3.2 Levels of aflatoxin in “Cheka” Samples

Aflatoxin concentration of “Cheka” samples from maize of different storage types was determined. The mean total aflatoxin level analyzed in whole “Cheka” samples was 10.76 ± 4.50 μg/kg. The “Cheka” samples prepared from respective maize of upper ground storage (“Gotera”) showed a mean total aflatoxin level of 10.19 ± 6.35 μg/kg, ranging from (3.39–15.9 μg/kg) whereas the “Cheka” samples prepared from respective pitfall maize showed a mean total aflatoxin level of 11.34 ± 3.06 μg/kg, ranging from (8.73–14.71 μg/kg). Similar to the maize samples, the aflatoxin G family was not detected in “Cheka” samples while aflatoxin B1 and B2 contributed to the total aflatoxin. Aflatoxin B1 was predominantly detected (Table 5).

Table 5 Aflatoxin concentration of “Cheka” samples from maize of different storage types
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3.3 Comparison of mean total aflatoxin in maize samples from different storage types

The independent sample t-test was determined to compare the total aflatoxin level in maize samples from different storage types. The Independent sample t-test showed that there is no significant difference between the mean total aflatoxin among the maize samples from upper-ground storage and pitfall storage (P = 0.379) (Table 6).

Table 6 Comparison of mean total aflatoxin level between maize samples from different storage types
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3.4 Comparison of total mean aflatoxin content among brewed “Cheka” samples

The independent sample t-test was determined to compare the total aflatoxin level among brewed “Cheka” samples. There is no significant difference between the mean total aflatoxin among the “Cheka” samples brewed from upper-ground storage (“Gotera”) maize and “Cheka” prepared from pitfall maize (P = 0.796). (Table 7).

Table 7 Comparison of total mean aflatoxin levels among a "Cheka” samples from maize of different storages
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3.5 Comparison of total aflatoxin content in maize samples with total aflatoxin content in brewed “Cheka” samples

The independent sample t-test was determined to compare the total aflatoxin level in maize samples with brewed “Cheka” samples. There is no significant difference between the mean total aflatoxin content in maize samples and brewed Cheka samples (P = 0.332). (Table 8).

Table 8 Comparison of mean aflatoxin levels between maize ingredients and brewed “Cheka” samples
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3.6 Comparison of aflatoxin content of maize samples based on storage types with their respective brewed “Cheka” samples

The independent sample t-test was determined to compare the total aflatoxin content in maize samples based on storage types with their respective brewed “Cheka” samples. The mean total aflatoxin level between “Gotera” maize samples and samples of “Cheka” prepared from “Gotera” maize did not show a significant difference (P = 0.997)(Table 9). The mean AFB1 content between the Pitfall maize sample and samples of “Cheka” prepared from Pitfall maize showed a significant difference (P = 0.033) but the mean total aflatoxin level between Pitfall maize samples and samples of “Cheka” prepared from Pitfall did not show a significant difference (P = 0.124) (Table 10).

Table 9 Comparison of aflatoxin content in maize sample from "Gotera” with corresponding “Cheka” samples
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Table 10 Comparison of aflatoxin content in pitfall maize samples with corresponding “Cheka” samples
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4 Discussion

We evaluated aflatoxins content among 12 samples comprising, 6 “Cheka” and 6 maize samples collected from “Cheka” vending houses in Konso. The samples were analyzed in duplicate for total aflatoxin (sum of AFB1, AFB2, AFG1, and AFG2) contamination by HPLC. In this study, out of 6 maize samples, 3 samples showed a mean total aflatoxin less than the limit of quantification. In the remaining 3 samples, the level of both AFB1 and mean total aflatoxin showed results above the maximum allowable level of aflatoxin set by the European Union ( 2 µg/kg and 4 µg/kg) respectively [5]. Similarly, these 3 samples also showed mean total aflatoxin levels exceeding the maximum permissible levels (10 µg/kg) set by the Codex Alimentarius Commission (CODEX) and Ethiopian Standard Agency (ESA) [1, 30]. Specifically, the mean total aflatoxin level of “Gotera” maize (10.23 µg/kg) is above the permissible limits of EU, ESA, and CODEX, while the mean total aflatoxin level of pitfall maize (3.35 µg/kg) was found below the maximum limit of the above authorities. However, AFB1 which is the most toxic type was found above the permissible limit (2 µg/kg) set by EU in the pitfall maze samples (2.18 µg/kg).In the present study, all samples of maize obtained from “Gotera” showed a quantifiable level of aflatoxin except one while in 2 maize samples taken from the pit, aflatoxins were detected but not quantified. AFB1 is the most predominantly detected aflatoxin in both samples followed by the small amount of AFB2. Aflatoxin G family was not detected in both sample types. There was no difference in the level of contamination between the mean total aflatoxin of “Gotera” maize and that of pitfall maize samples (P = 0.379).

In a survey of 396 maize tortillas from Mexico, average aflatoxin concentrations in 56 contaminated samples were: AFB1 (12.1 µg/Kg); AFB2 (2.7 µg/Kg); AFG1 (64.1 µg/Kg), AFG2 (3.7 µg/Kg), and total AFT (20.3 µg/Kg) [11]. When compared with our findings, the AFB1 level of our finding is consistent with the AFB1 level in this study but AFB2 and the mean total aflatoxin level of this study is higher than AFB2 and the mean total aflatoxin level of our findings. Additionally, AFG1 and AFG2 were not detected in our samples. The difference may be due to differences in weather conditions and storage types.

The study conducted in China on 44 pre- and post-nature drying maize kernels revealed contamination of only 1 sample with an AFB1 level of 148.4 µg/Kg [10]], which is much higher than the AFB1 of our findings.

In Contrast to our finding, aflatoxins were not detected in the research done in Serbia on 180 maize samples analyzed in the 2009- 2011 period [12]. This might be due to the difference in environmental weather conditions as well as the type of storage. In the study by Kos et a, due to the change of weather conditions in Serbia to hot and dry with a prolonged drought during spring and summer in 2012, mean total aflatoxin the level of 36.3 µg/Kg was observed which is higher than our finding.

When it was compared with studies conducted in African countries, the mean total aflatoxin of maize samples (6.78 ± 8.28 μg/kg) was relatively lower than the mean total aflatoxin levels observed in farm-gate maize samples from Ghana and Nigeria (West Africa) with mean total aflatoxin level of 330 μg/kg [14]. On the other hand, the mean total aflatoxin observed in 51 maize flour samples from Côte d'Ivoire with 107.9 μg/kg of mean total aflatoxin level was higher than our finding [15]. The possible reason for a lower level of mean total aflatoxin in our maize samples may be due to the difference in the period of harvest and storage, environmental conditions, and analytical method used [21, 30].

Our finding is in line with previous research conducted in Ethiopia, that AFB1 is the predominant type of aflatoxin contaminating most crops in the country [20]. The result of our study showed lower AFT content than the finding on 150 maize samples collected from the Gedeo zone, southern Ethiopia with a mean total aflatoxin level of 50 ppb on samples analyzed with immune chromatographic assay and 52.1 ppb on those analyzed with thin layer chromatography [21]. This may be due to differences in analytical methods, weather conditions, and storage types.

Our finding is consistent with a study done on maize collected from Ambo, Adama, and Diredawa, which showed a total aflatoxin content of < 5 µg/kg [22]. Another study conducted on maize grains from west Gojjam reported mean total aflatoxin levels of 18.38 µg/kg and 43.43 µg/kg for pre-harvest and post-harvest maize samples respectively [23]; which are relatively higher than our finding.

All six samples of “Cheka” showed a quantifiable level of AFB1 above the maximum permissible level of aflatoxin (2 μg/kg) and five samples contained a level of mean total aflatoxin level exceeding the maximum limit (4 μg/kg) set by EU. Four samples of “Cheka” showed a mean total aflatoxin content exceeding the maximum limit (10 µg/kg) set by the Codex Alimentarius Commission (CODEX) and Ethiopian Standard Agency (ESA) [1, 6]]. “Cheka” prepared from “Gotera” and pitfall maize showed a mean total aflatoxin of 10.19 µg/kg and 11.34 µg/kg respectively, which is above the maximum permissible mean total aflatoxin level of EU, CODEX, and ESA. This indicates that “Cheka” was contaminated by aflatoxin level which can lead to negative health effects such as liver diseases, stunting in children and others when frequently consumed.

In a comparison of the mean total aflatoxin level of Cheka samples with the mean total aflatoxin of maize samples, there was no statistically significant difference observed between the means (P = 0.332). This indicates that the contamination of maize with AFT also contaminates the respective “Cheka” prepared from it [23, 31, 32].

Comparing the mean aflatoxin level of “Cheka” prepared from “Gotera” maize with “Cheka” prepared from pitfall maize, there was no statistically significant difference between their mean total aflatoxin level (p = 0.796). This indicated that both types of cheka samples were contaminated by aflatoxin which they got from respective maize samples.

Comparing the mean total aflatoxin level between pitfall maizes and their respective cheka samples showed that, although there was no significant difference between the mean total aflatoxin content of pitfall maize and respective cheka samples (p = 0.124), a significant difference was observed between the AFB1 mean of Gato pitfall maize and their respective cheka samples (P = 0.033). This finding was in agreement with the study conducted by Castillo-Urueta et al. [11], where there was no significant difference observed between total aflatoxin levels for comparison made with ‘‘type of stores’’, but a significant difference was observed only for AFB 1 (p = 0.0320).

Our finding was not in agreement with the study conducted by Bationo et al. [19], where the mean concentration of AFB1 was 97.6 μg/kg in sorghum malt during the production of traditional beer (dolo) but not detected after the beer was brewed and fermented. It was reasoned out that processing by milling and fermentation causes a drastic reduction in aflatoxin levels [32]. This was supported by the evidence given in the study conducted in Nigeria, where the fermentation process reduced at least 76% of mycotoxin contamination in “kunu-zaki” (a beverage produced from sorghum) and 59% in pito (a beverage produced from maize)[18].

Compared to studies conducted on traditional beverages across some African countries, “Cheka” showed a low level of mean total aflatoxin. A study done on traditional maize opaque beer from Malawi showed a mean total aflatoxin level of 90 μg/kg [33], which is much higher than the result of our finding. This might be due to differences in contamination of ingredients (maize), processing, malting, and other factors that brought deviations.

According to our findings, “Cheka” had higher aflatoxin content than “Thobwa” (sorghum opaque and sweet traditional beverage) of Malawi which showed a mean total aflatoxin level of 4.5 µg/kg [32]. This might be due to the difference in ingredients used for brewing the beverages (maize is more prone to aflatoxins than sorghum [34], processing, environmental conditions, and others.

In our study result, maizes from both storage types (pitfall & “Gotera”) and their respective cheka samples showed a positive result for aflatoxin contamination where most of them showed a level above a permissible limit set by different authorities. This indicates both of these traditional storage types enhance the growth of aflatoxigenic fungi and promote the production of aflatoxin [21]. Consumption of foods contaminated with aflatoxin causes liver diseases like liver cancer which is a high risk in carrier people with Hepatitis B and Hepatitis C positive [4, 35, 36].

It might be better to use recent technologies like polyethylene bags and gunny bags for storing maize in comparison to traditional storage in light of controlling aflatoxin contamination [37].

5 Conclusions

As per the objective of this study, aflatoxin contamination was detected in all maize and “Cheka” samples. Three maize samples were contaminated with a non-quantifiable (< LOQ) level of total aflatoxin, while all “Cheka” samples showed a quantifiable level of total aflatoxin (> LOQ). AFB1 was determined as the predominant contributor to the total aflatoxin content found in all samples. In contrast, the aflatoxin G family was neither detected nor quantified in all samples evaluated in this study.

Three maize samples showed a level of AFB1 above the maximum advisable level set by the European Union and Ethiopian Standard Agency and a mean total aflatoxin level exceeding the maximum permissible level set by the European Union, Codex Alimentarius Commission, and Ethiopian Standard Agency.

All samples of “Cheka” showed a quantifiable level of both AFB1 and total aflatoxin levels. All samples showed AFB1 levels exceeding the maximum limit set by the European Union while five samples contained a level of mean total aflatoxin level exceeding the maximum limit set by the European Union. Moreover, four samples of “Cheka” showed a mean total aflatoxin content exceeding a maximum limit set by the Codex Alimentarius Commission and the Ethiopian Standard Agency.

There was no statistically significant difference in the mean total aflatoxin levels between maize samples from different storage types, between “Cheka” samples prepared from these respective maize samples, and between “Cheka” and their corresponding maize samples. Although there was no significant difference between the mean total aflatoxin levels of pitfall maize and respective “Cheka” samples, a significant difference (P = 0.033) was observed between AFB1 levels of Gato pitfall maize and respective “Cheka” samples. In both cases, the AFB1 level was found above the maximum limit set by the European Union and the Ethiopian Standard Agency.