1. Introduction
Benin, a developing country in West Africa, agriculture represents the basis of the Beninese economy. Although traditional, it provides populations with considerable income for their development. Each year, Government devotes a considerable part of its budget to improving productivity and limiting pre-and post-harvest losses. For that, Benin is divided into eight agroecological areas based on staple and feed commodity production [1] . During their growth, harvest, storage, transport, drying, selling, and transformation, raw materials are regularly contaminated by molds and their chemical hazards mycotoxins [2] [3] .
Mycotoxins are toxic substances generally produced by molds. These substances resist at oxidation, acidity, heat, and cold [4] . Among the 400 known mycotoxins, aflatoxins are the most dangerous and the most researched, due to their high toxicity [5] .
Aflatoxin is a poison produced by Aspergillus section Flavi. From this section, both A. flavus and A. parasiticus are the most dangerous species and are frequently found in crops, in food, and feed as they are plant pathogens that produce aflatoxins [6] . Among the more than 12 aflatoxins [5] , B1, B2, G1 and G2 are particularly dangerous for both humans and livestock, due to their consequences [7] . In fact, aflatoxin B1 has been classified as a carcinogen by the International Agency for Cancer Research since 2002 [8] .
Aflatoxin production can occur at any time. However, certain factors involving temperature, drought conditions, and heat are necessary [9] . High temperatures accentuate fungal and aflatoxic contamination [10] . Aflatoxins are resistant to heat destruction at temperatures that can be found in activities such as cooking, extrusion and even roasting [11] . In fact, aflatoxin B1 and B2 can resist destruction at temperatures as high as 289˚C [5] .
Aflatoxins have many consequences for human health. For example, they are involved in hepatic carcinogenesis and have immunosuppressive properties that can affect fetuses [12] . Other consequences are dependent on the quantity, nature, and duration of exposure to aflatoxins [13] . At high doses, they can cause immediate death, however, they generally, cause a chronic diseases such as cancer, cirrhosis of the liver, and stunted-growth in children [14] . Furthermore, molds and aflatoxins have caused 1.2 billion dollars in economic waste [15] and quart of crops gathering had been lost, according to data from the Food and Agriculture Organization.
Due to the damage, they inflict on health and the economy, we seek to learn about and characterize the several molds and corresponding aflatoxins that are present in the different agroecological areas of Benin. To achieve this, the objective of this current work is to construct a map of the geographical distribution of Aspergillus section Flavi found in food and feed in Benin. Specifically, this work aims to determine the morphological characteristics and metabolic and molecular profiles of aflatoxins, which would ultimately be represented in an aflatoxin distribution map in Benin.
2. Material and Methods
2.1. Origin of Raw Materials
The samples comprised 318 strains of Aspergillus section Flavi. The presence of these strains was isolated from several food and feed sources in the eight agroecological areas of Benin. These strains were previously isolated during food contamination surveys performed in Benin by strains of Aspergillus section Flavi. The food sources were corn (Zea mays), popcorn, smoked and fermented fish, dry spices, Manihot esculenta, Pennisetum glaucum, Sorghum bicolor, peanut (Arachis hypogaea) and bambara groundnut. Table A1 shows information regarding the origin of the strains.
2.2. Morphological Identification
Each strain was morphologically characterized on potato dextrose agar (PDA) and added to chloramphenicol. Macroscopic analysis was performed on malt extract agar (MEA) to isolate and characterize the strains of molds belonging to Aspergillus section Flavi. For microscopic analysis, an Olympus optic microscope, XSZ-107BN, No. 079931; at ×400, was used.
2.3. Molecular Identification
The DNA of the strain was extracted according the method of Aamir [16] with some modifications. Briefly, some quantity of mycelium was placed on the medium and crushed in a mortar with a lysis buffer (100 Mm Tris HCl; Ph = 8; 50 Mm EDTA; 3% SDS). Then, 1.5 mL was reground, placed in an Eppendorf tube and centrifuged at 12,000 rounds for 15 min. The supernatant was collected and mixed with 3 µl of RNase A (10 mg/ml), and incubated at 37˚C for 15 min. Then, an equal volume of phenol chloroform (50:50) was added and centrifuged at 12,000 rounds for 15 min. The chloroform phase described above was added to 1 mL of ethanol at 98˚ and incubated at −20˚C for 30 min.
DNA was quantified using a labelled Thermo Fisher Scientific spectrophotometer, model Evolution 60S, CAT 840-210100, SN 2R4326202, manufactured in China. Polymerization chain reaction (PCR) was used to amplify 1450 pb of the beta-tubulin sequence of the Aspergillus section Flavis gene according to the Go Taq protocol. An Eppendorf thermal cycler was used for the PCR analysis. The total volume of the reactional mix was 50 µl. For the reaction mix reactional, 5 µl of Taq buffer (10×) was added to 5 µl of MgCl2 (25 mM) with 5 µl of dNTP (2 mM) 1 µl of each primer (forward and reverse) (12.5 pM) and 1 µl of the DNA sample (10 ng). For RFLP, the PCR products were used with the BstYI restriction enzyme. The volume of the reaction mix was 20 µl. Then, 13 µl of the PCR product was added to 7 µl of the mix reaction mix from the total mix prepared as follows: 2 µl of BstYI, 1 µl of BstYI’ buffer, and 62 µl of PCR water. The sample were incubated at 60˚C for 10 min and 15 µl of the RFLP product was loaded on a 1% agarose gel and viewed under 360 nm illumination.
2.4. Metabolites Characterization
The strains isolated and characterized on MEA were cultured on coconut cream agar medium to determine the production of aflatoxin under UV light and on Aspergillus flavus parasiticus agar to determine the production of aspergillic acid under sunlight by Aspergillus section Flavi after five days of incubation at 25˚C. Furthermore, secondary metabolites were extracted directly from the food and feed by the Bruce and al. method with some modifications. Secondary metabolites were also isolated from the strains cultured on PDA medium by the Azzoune [17] method. In this method, 5 mL of chloroform is added to a medium in which molds are grown and is vortexed for 2 min. The chloroform is 1.5 ml is placed in the Eppendorf tube and dried. Finally, 40 µl of methanol 90˚ added to the tube. The isolated mycotoxins were analyzed by chromatography plaque tests (10 cm × 10 cm). Chromatography plaques with aflatoxin standards (B1, B2, G1, G2) and samples were observed with 360 nm light.
3. Results
3.1. Morphological Characterisation
Based on the macroscopic characterization from Pitt and Hocking macroscopic characterization [18] , all tested strains showed a color between yellow and green on the MEA medium, depending on the production of sclerotes such as Aspergillus section Flavi spp. ( Table A2). Some strains showed a powdery form whereas others contained aerial mycelium. Several strains produced structures such as exudates and sclerotia. These observations are similar to those of Frisvad [19] , who observed the same characteristics regarding Aspergillus section Flavi on MEA. This demonstrates that strains tested belonged to Aspergillus section Flavi. (Figure 1 and Figure 2; Table A3).
The morphological analysis of secondary metabolites permitted the classification of Aspergillus section Flavi strains. Of these strains, 42% were identified as A. parasiticus, 21% as A. flavus, 19% as A. coremiiformis, and 7% as A. tamarii. It is important to remember that according to the morphological analysis, some strains contained two or three different Flavi subsections. It is because the strains numbers in the previous table are large over the initial strains numbers. In agroecological sites 1, 3, and 5, the strains were identified as mainly A. nomius, whereas in site 2, A. flavus was more present. In sites 5 and 8, A. parasiticus was
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Figure 1. Visual aspect of two Aspergillus section Flavi strains on MEA J+7.
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Figure 2. Visual aspect of two other Aspergillus section Flavi strains on MEA J+7.
abundant ( Table A4).
3.2. PCR-RFLP
After beta-tubulin 2 gene amplification, 1450 pb of the DNA fragments were obtained (Figure 3). RFLP revealed two groups of the Flavi section, one group comprising three fragments of A. flavus at 336 pb, 438 pb, and 581 pb, and the other group comprising four fragments of A. parasiticus at 176 pb, 263 pb, 334 pb, and 576 pb (Figure 4).
3.3. Secondary Metabolites Characterization
3.3.1. Aspergillic Acid Identification
Of all the strains tested, 191 (60%) produced aspergillic acid: ( Table A5).
3.3.2. Mycotoxins Analysis
Nearly half; of the A. section Flavi strains (41.82%) produced both aflatoxins B and G, whereas 28.93% produced only aflatoxins B. Furthermore, agroecological
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Figure 3. Agarose’s gel (1%), result of beta tubulin 2 gene amplication by PCR migration.
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Figure 4. Agarose’ gel (1%) result of RFLP migration (weight of DNA fragment: 1450 pb; weight of each DNA fragment: first line fragment from top: 576 pb; second line: 438 pb; last line: 336 pb).
sites 1, 3, and 4 were mainly contaminated by aflatoxins B, sites 5, 6, 7, and 8 contained both aflatoxins B and G, and in site 2, similar quantities of aflatoxins B and G were observed (Table 5A, Table 6A, and Table 7A).
4. Discussions
All food and feed samples were contaminated by several molds, mostly Aspergillus-flavus. Strains of A. parasiticus, A. nomius, A. parvisclerotigenus, A. novoparasiticus, A. flavus, and A. togoensis were identified from their morphological and metabolites characteristics. They presented a color between green and yellow on MEA after the seventh day of growth [19] . Similar results were found by [20] and [21] .
Indeed, Benin is a West African country located between 1˚ and 30˚N-S of latitude, with an average temperature varying between 26˚C and 32˚C [22] . These environmental conditions are ripe for molds development, most Aspergillus species grow primarily between 26˚C and 35˚C [23] with optimal growth occurring at a temperature of 33˚C [18] . The behavior of A. section Flavi in West Africa has been demonstrated in previous research. In addition, [24] showed the occurrence of Aspergillus flavus in peanut (Arachis hypogaea) in Burkina Faso. Furthermore, [25] demonstrated the same results as well as the existence of other molds such as A. parasiticus and A. tamarii in agroecological site 8.
The present survey demonstrated that food and feed from all 8-eight agroecological sites in Benin were contaminated by aflatoxins. Indeed, food and feed from the northern zones (1, 3, and 4) were shown to be strongly contaminated by aflatoxin B, while those from the southern zones (5, 6, 7, and 8) were contaminated by both aflatoxins B and G. The production of aflatoxins B is associated with the presence of A. flavus and A. togoensis, while both aflatoxins B and G are associated with the presence of A. parasiticus, A. nomius, A. parvisclérotigenus, and A. novoparasiticus in food and feedstuffs.
Generally, northern Benin is the primary agricultural zone. The majority of raw materials and staple foods are produced there. To increase production, farmers regularly use products such as fertilizer and; pesticides. Moreover, feedstuffs and staples are no longer kept in place. In addition, the northern zone is characterized by rough environmental conditions, demonstrating why a smaller population of Aspergillus and just one type of aflatoxins B were produced there. However, in the south, agricultural products are kept for a long time. In fact, southern Benin has the largest market in the country and even the West African zone. Indeed, agricultural products from several agroecological zones of the country and other countries such as Nigeria, and Togo are maintained in the international market of Dantokpa. The long duration of feedstuff and staple food storage facilitates the development and growth of many types of molds such as Aspergillus, and even the production of large quantities of the two types of aflatoxins (B and G). These results confirmed the previous survey regarding the link between the development of molds, aflatoxin production, and storage duration [26] [27] . The high production of aflatoxins in the south can be explained by the better environmental conditions for mold development, and environmental stresses such as drought, and temperature further aflatoxin production. In the south, as opposed to the North, there are four seasons that alternate throughout the year. The relative humidity of the environment encourages molds development and environment stress as the drought, temperature further aflatoxins production. In addition, the presence of aflatoxins B and G particularly in fish demonstrates cross-contamination among staples foods, as several types of fishes (dry, smoked and fermented) were generally sold with species that are strongly contaminated by Aspergillus and aflatoxins [28] . The transfer of fungal strains was responsible for the production of aflatoxin in fish [25] . In particular, A. parvisclerotigenus and A. novoparasiticus; were identified in cassava in the transition zone between the north and south [29] .
The map in (Figure 5) presents the distribution of Aspergillus section Flavi and their aflatoxins in Benin.
5. Conclusion
In summary, the present survey shows that staples foods, raw materials, and feedstuffs, including cooked food, are contaminated by aflatoxins in Benin. The north is contaminated by A. flavus and A. togoensis, producers of aflatoxins B (AFB1 and B2). The south, however, is mainly contaminated by A. parasiticus and A. nomius, producers of both aflatoxins B and G. Exposure to aflatoxins by
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Figure 5. Repartition risk map linked of aflatoxins production in 8 agroecologic areas in Benin.
the population is inevitable owing to the consumption of contaminated food through mold development and the presence of mycotoxins. Further economic damage, which can increase poverty, molds contamination, and aflatoxins contamination, is very dangerous for population health. High exposure can lead to death. So, molds and aflatoxins food contamination could be avoided by observation of the good practices of harvest and good storage conditions.
Acknowledgments
Warm thanks to Dr. Yann ADJOVI who leds the present survey and supplied all reagents, dishes, culture mediums. We thank all members of the Laboratoire de Biochimie et de Biologie Moléculaire, which hosted the tests and manipulations.
Appendix
All Tables: https://docs.google.com/document/d/1361T6YVrK92Tts_3nWWd_8t-e1PQrm_i/edit#heading=h.gjdgxs
Table A1. List of strains from Aspergillus section Flavi collected within agroecologic area in Benin.
https://docs.google.com/document/d/130Cn8CNW_Jhz_oM78XAH1mzOabusaKSPovdDCoKE2U8/edit
Table A2. References section Flavi’s clades.
https://docs.google.com/document/d/1TbER0QnrgYJVfYXQum-mBSbsKkOq1_lxr8nhmV-Nxgs/edit?usp=sharing
Table A3. Mascroscopic’s characterization of strains.
https://docs.google.com/document/d/1GVvZ8wr4CAq4W2f7tUberr2TfDY09wDij4FCYq3WefU/edit?usp=sharing
Table A4. Global metabolic profile of strains.
https://docs.google.com/document/d/1-5s_Wl2aopaB0hRK8OKW3Iik_W3M6yNKMDIZhPB7owQ/edit?usp=sharing
Table A5. Morphological identification results.
https://docs.google.com/document/d/1xrY-wEI_nGz96rzZ6goeGk0ykW9lKiMNTl_nYAcxaPc/edit?usp=sharing
Table A6. Aflatoxins production distribution.
https://docs.google.com/document/d/1Vqtev8UgEj8IEVL_O5ZdTe9sTSpXhNsF-dG-gkgxwIk/edit?usp=sharing
Table A7. Synthesis of strains identification.
https://docs.google.com/document/d/1PZ6n8Mfxu566dLB1p3Aj9bJ6bTGry45NMFmtFsjaVaM/edit?usp=sharing