Aflatoxin contamination in groundnut and maize food products in Eastern and Northern Uganda

Abstract Groundnut and maize are among the economically important crops grown widely in Uganda for household food and income security. However, those crops and their products are vulnerable to aflatoxigenic fungi and aflatoxin contamination. The present study sought to establish the levels of aflatoxin in the different products of groundnut and maize in local markets and roadside retail shops. A cross-sectional survey was conducted in ten districts in the eastern and northern regions of Uganda. Groundnut products: paste, unshelled, shelled/grain, and flour/powder were collected; and maize products: cobs (covered and uncovered), grain, flour, bran, mixed feed, flour reject, and roasted/boiled maize were also collected. A total of 241 samples (133 groundnut and 108 maize samples) were collected from the community and analysed for aflatoxin contamination using ELISA test. Regardless of the source, 41.8% of groundnuts had aflatoxin levels higher than 20 parts per billion (ppb), with groundnut paste (196.52 ± 437.24 ppb) and flour (187.90 ± 289.95 ppb) being the most contaminated. Aflatoxin levels in 62.8% of maize products were higher than 20 ppb, with maize cobs having the highest levels (126.4 ppb). Groundnut and maize products from the eastern region were the most contaminated. These results indicate that most of the maize and groundnut products in the markets away from major urban centres are also highly contaminated and unsafe for food and feed. This calls for immediate action to develop mitigation measures to curb the impact of aflatoxin contamination on the health and income of households, particularly through sensitization and training on appropriate pre-and post-harvest handling practices of the products.


Introduction
Groundnut (Arachis hypogaea L.) and maize (Zea mays L.) are some of the economically important crops grown widely in Uganda.The crops are a major source of food and income for both smallholder and commercial farmers in the country, contributing significantly to food and nutrition security and poverty alleviation Sserumaga et al., 2020Sserumaga et al., , 2021)).Specifically, groundnut seeds are rich in oil (48-50%) and protein (25-28%) and are the source of several vitamins, minerals, and other nutritional factors such as antioxidants, flavonoids, polyphenols, and isoflavones (Bonku & Yu, 2020).Groundnut is majorly cultivated in eastern and northern parts, and consumed widely in the country (Baluka et al., 2017;Sserumaga et al., 2021).The raw, roasted, blanched, raw grains pounded into flour (ebinyebwa); roasted grains ground into peanut butter, and may be applied on bread or eaten as an accompaniment to cassava and sweet potato; and pastes or flour mixed with traditional dishes as a sauce are the different forms of groundnut products eaten in the country On the other hand, maize grains are a major source of macronutrients like starch (70%), fiber, protein (8-11%), and fat; micronutrients like vitamin B complex, ß-carotene; and essential minerals like magnesium, zinc, phosphorus, and copper, among others (Bathla et al., 2019).Maize also contains a booster of antioxidants like polyphenols and phytosterols that protect against various degenerative diseases.In Uganda, maize is cultivated and consumed country-wide; produced by over 3 million farmers (Daly et al., 2016); and is one of the staple foods depended on by many households (Epule et al., 2021).Maize grains are eaten roasted, boiled, or ground into flour for making bread (Posho), porridge, and other value-added products, as well as bran for feed (Ahmed and Ojangole, 2014).
Despite their importance, groundnuts and maize are among the main sources of human exposure to risks associated with mycotoxin contamination, such as aflatoxin and fumonisin (Jallow et al., 2021).This is because groundnut and maize crops are infected by mould fungi both in the field and at post-harvest stages, which could possibly produce aflatoxin in groundnut and maize products.Several studies have reported on the susceptibility of the crops to fungal attacks and subsequent mycotoxin contamination (Kimanya, 2015;Mutiga et al., 2015;Omara et al., 2020;Sasamalo et al., 2018).This is largely influenced by prevailing suitable conditions such as optimum temperatures, high humidity, and high rainfall that facilitate fungal growth and mycotoxin biosynthesis (Sujayasree et al., 2022).According to Ankwasa et al. (2021), the high susceptibility of these crops to mycotoxin contamination, including aflatoxin, does not only pose significant food safety concerns but greatly lowers their economic value, thus affecting the earnings of commercial farmers.Aflatoxin is a group of mycotoxins known to be produced by Aspergillus species, mainly section Flavi such as, A. flavus and A. parasiticus.These fungi are capable of contaminating diverse agricultural crops including groundnut and maize, whenever there is a favourable condition for mould development.According to Omara et al. (2020), at least 18 different types of aflatoxin have been documented, but the six main types are aflatoxin B1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), aflatoxin G2 (AFG 2 ), aflatoxin M 1 (AFM 1 ), and aflatoxin M 2 (AFM 2 ).Aflatoxin B 1 , B 2 , G 1 , and G 2 , with aflatoxin B 1 being the most toxic (Sujayasree et al., 2022) Aflatoxin M 1 and M 2 are commonly found in the milk of animals that have consumed contaminated feed (Nguyen et al., 2020).The potency of toxicity, carcinogenicity, and mutagenicity of the different aflatoxin types varies in decreasing order from B (Omara et al., 2020;Ülger et al., 2020).
Thus, aflatoxin contamination is becoming a major challenge to the economy, food and nutritional security, and human and animal health (Baluka et al., 2017;Kang et al., 2015;Lauer et al., 2019;Natamba et al., 2015;Okonji, 2019;Omara et al., 2020;Osuret et al., 2016).It is estimated that aflatoxin contamination of crops in Africa causes a yearly loss of US $750 million (Gbashi et al., 2018).Huge economic losses due to aflatoxin contamination have been reported in Uganda (Lukwago et al., 2019) and the neighbouring countries (Kamala et al., 2018;Kamika et al., 2016).Aflatoxins have carcinogenic, haemorrhagic, mutagenic, tremorgenic, genotoxic, immunesuppressing, and growth-retarding effects in both humans and animals (Sujayasree et al., 2022.The Association of Official Analytical Chemists (AOAC) has published a number of official methodologies to identify aflatoxin contamination in food samples (P.Kumar et al., 2017).Enzyme-Linked Immunosorbent Assay (ELISA) is the most commonly used method owing to its rapid detection; followed by a few chromatographic techniques like High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectroscopy (LCMS), and Thin Layer Chromatography (TLC) (A.Kumar et al., 2021;Norlia et al., 2019).Rapid methods for detecting aflatoxins are emerging, such as; Spectroscopic techniques for detecting quality and safety (Kaavya et al., 2020); Fourier transform-infrared (FT-IR), Near-infrared (NIR) and Raman spectroscopic techniques for evaluation of grain and grain-based products (Pandiselvam et al. (2023); Hyperspectral imaging (HSI) technique for detection of fungal contamination in maize kernels (Mansuri et al., 2022).
Strategies for aflatoxin contamination control and elimination include the use of genetic engineering, physical methods, chemical methods, good agricultural practices, biological control, postharvest precautions, and breeding for resistance (A.Kumar et al., 2021;Jallow et al., 2021;Mahato et al., 2019).Despite numerous recommendations on aflatoxin management that are abundant in the literature, aflatoxin contamination of important crops such as groundnuts and maize by the toxins remains high in Sub-Saharan regions.
In Uganda, several studies on Aspergillus species and aflatoxin contamination in different commodities, especially in groundnuts and maize, were presented (Ankwasa et al., 2021;Omara et al., 2020).Additional information, particularly on groundnuts and maize, is available (Acur et al., 2020;Sserumaga et al., 2020Sserumaga et al., , 2021)).It is known that the distribution and the levels of contamination of foods and feeds with aflatoxin vary with the status of post-harvest management (mainly poor pre-, peri-, and post-harvest activities), poor government legislation, lack of awareness, and low levels of education among farmers, entrepreneurs, and consumers (Omara et al., 2020).The level of marketing of food products (PACA, 2017), where higher aflatoxin contamination was reported among the retailers than the wholesalers (Omara et al., 2020), prevailing weather conditions (Sserumaga et al., 2021), the type of crop affected (Muzoora et al., 2017), and fungal population (Acur et al., 2020) are equally important.According to Omara et al. (2020), the other factors that could influence aflatoxin production are water activity, alkalinity and acidity, the concentration of oxygen and carbon dioxide, microbial competition, mould lineage, plant stress, and use of pesticides and fertilizers.
Several studies have been conducted in Uganda on the levels of aflatoxin contamination in different commodities, including maize and groundnuts.According to Omara et al. (2020) and Ankwasa et al. (2021), most of these studies focused on food safety risks associated with commodities for export, but no deliberate effort has been made to assess the aflatoxin contamination in the different forms of products of groundnut and maize The study specifically sought to: 1) establish the levels of aflatoxin contamination in the different forms of products of groundnut and maize; and 2) determine how the levels of aflatoxin contamination vary with the point of access of the various products.It is envisaged that the information generated by our study ameliorates and significantly contributes to the design and approach of implementing an integrated mycotoxin management system at the household and community levels.

Description of study sites
The study was carried out in Uganda's eastern and northern regions, with Serere, Soroti, Budaka, Iganga, Namutumba, and Tororo districts chosen from the eastern region and Arua, Zombo, Nwoya, Pader, and Dokolo districts chosen from the northern region.The districts were classified into sub-regions, namely Busoga, Bukedi, and Teso in the eastern region; and Lango, Acholi, and West Nile sub-regions in the northern region.These sub-regions and the districts therein are not only predominantly cereals and legumes producers and consumers, but also differ in agroecological conditions and the nature of maize and groundnut products utilized, thus putting the population at a high risk of aflatoxin contamination.The unique characteristics of these subregions and the agro-ecological zones (AEZs) to which they belong have been described by Sserumaga et al. (2020), and other information in this regard is available at the FAO website (http://www.fao.org/agriculture/seed/cropcalendar/searchbycountry.do).On average, the sampling points were located at an altitude of between 900 and 1600 meters above sea level (masl).The products were accessed from households, roadside selling points, retail shops, mini-supermarkets, small-scale millers, animal feed shops, and markets.

Sample collection
Samples of groundnut and maize products were collected from the various points along the value chain during the month of September, 2019.All the samples were collected from seven different sampling points, including: farmer's home/store/crib, drying area/yard, farmer's field (after harvest), markets (roadside and central market), grinding/milling areas, feed stores, and shops/minisupermarkets.Different food products of groundnut: paste (processed from half and full-roasted groundnuts), unshelled (raw, roasted and boiled), shelled/grain (raw and roasted), and flour/ powder were collected.While maize cobs (covered and uncovered), grain, flour, bran, mixed feed, flour reject, and roasted/boiled maize were also collected.Additional data was collected on the farmer's details (bio-data), location (region, district, sub-county, and parish), and GPS coordinates (altitude, latitude, longitude, and accuracy).
In total, 241 samples (133 groundnuts and 108 maize, each weighing 300 grams) from the two regions were obtained (Table 1).In each district, 10 to 20 maize and 11 to 26 groundnut samples were collected.The product samples were collected and packaged in well-labeled zip-lock bags; the labels included the sample number, the name of the commodity, the product type, the form of the product type, the name (optional) and contact information of the farmer.Then all collected samples were transported to the laboratory at the National Semi-Arid Resources Research Institute (NaSARRI), Uganda, where they were stored at −4 °C until analysis.

Sample compositing and processing
Further processing such as sorting out inert materials and compositing was done to the samples under sterile condition.Composite samples were formed from the samples collected using the batch method (Fabrizio et al., 1995), where similar samples (type and form of products) from the same sampling point are bulked and homogenized together to form a bulk sample from which smaller samples were taken to constitute samples for analysis.This was done to reduce the sample number and cut costs of analysis.As a result, a total of 43 out of 108 maize and 67 out of 133 groundnut composite products were reconstituted for aflatoxin analysis.
At the mycotoxin laboratory, all the samples were oven-dried at 48℃ for 48 hours to approximately 11% moisture content.Groundnut samples in pods and maize in cobs were hand-shelled to obtain the grains prior to drying.All samples were ground into a fine powder and homogenized using Bunn Coffee Mill.The samples were then divided into two sub-samples-one for aflatoxin contamination, and the second batch was stored in the freezer at −70℃ as a backup.

Aflatoxin analysis in groundnut and maize sample products
Aflatoxin analysis was done at the Regional Mycotoxin Research Laboratory-Machakos, Nairobi, Kenya.Accuscan Pro reader was used to analyze the aflatoxin levels in the samples following the manufacturer's instructions (Neogen Corp., Lansing, Michigan).Each sample was homogenized by shaking for 1 minute, and later 10 g of the sample (in duplicates) was mixed with 50 ml of 65% ethanol for three minutes using an orbital shaker (HS501, IKA-WERKE, Germany).The mixture was filtered through Whatman No. 4 filter paper, and the filtrate was obtained in a Tripod beaker.
Red sample dilution cups and clear sample dilution cups were placed in the sample cup rack and labeled appropriately.To each red sample dilution cup, 500 µL of sample diluent was added.A total of 100 µL of sample extract was added to the red dilution cup with sample diluents and mixed to ensure uniformity by pipetting up and down seven times.Then, 100 µL of diluted sample extract was transferred into a new sterile, clear sample cup.A reveal Q+ strip (GIPSA-approved Neogen Reveal ® Q+ for Aflatoxin kit) was placed into the clear sample cup and incubated for six minutes, and the test strip was read within one minute.Aflatoxin contamination levels were read in parts per billion with a lower detection limit of 2 ppb and an upper detection limit of 150 ppb.Samples with more than 150 ppb were serially diluted in 65% ethanol, re-analyzed, and the new dilution factor was considered during interpretation of the results.

Data analysis
The aflatoxin contamination levels in groundnut and maize product samples were grouped into three categories-i) ≤ 4 ppb, ii) >4 ≤ 20 ppb, iii) > 20 ppb, corresponding to the total aflatoxin limits established by the European Union (EU; 4 ppb), the combined limits for the Uganda National Bureau of Standards (UNBoS; 10 ppb), and the United States Food and Drug Administration (FDA; 20 ppb) and those beyond the limits.The analysis of variance (ANOVA) for the values for aflatoxin contamination levels was carried out using the general linear model (GLM) for unbalanced data where crop type was used as a compounding factor.All statistical tests were performed with Genstat 15 th Edition (Payne et al., 2012).
Means were compared using one-way tests and means were separated using Fisher's protected least significant difference (LSD) tests at a 5% significance level (α = 0.05).Aflatoxin contamination levels values were first log-transformed before analysis to normalize the variance.In the creation of the table, means of untransformed data generated using excel statistical were used for interpretation.Regions, sub-regions, districts, sampling points, altitude range, crop type, and product forms were the main independent variables.

Aflatoxin contamination in groundnut and maize products
Higher aflatoxin contamination was recorded in the Eastern than Northern region with the highest aflatoxin contamination levels in groundnuts and maize recorded in the Busoga and Lango subregions (Table 2).Among the districts, Iganga, Namutumba, and Dokolo exhibited higher contamination in groundnuts.While Namutumba, Dokolo, and Pader districts showed higher aflatoxin levels in maize.Serere District had the lowest levels of aflatoxin contamination in both groundnuts and maize.
With regard to the product types, the aflatoxin contamination levels in groundnut were higher than that in maize products.Groundnut paste and flour recorded the highest contamination, followed by unshelled groundnuts.The lowest aflatoxin contamination level was recorded in maize flour and roasted groundnuts.Among the maize products, maize on cob and grains were the most contaminated, while maize flour was the least contaminated (Table 3).

Categories of aflatoxin contamination levels
The proportions of samples per the different categories of aflatoxin contamination levels in the different crop products and the regions are shown in Table 4. Higher proportions of groundnuts in the eastern region (46.2%)compared to the northern (26%) were contaminated with aflatoxin above 20 ppb.Also, a higher proportion of maize from the eastern region was contaminated with aflatoxin at levels above 20 ppb (40% and 27%) respectively.Regardless of the regions, 41.8% of groundnut samples had aflatoxin contamination level above 4 ppb compared to 62.84% of the maize samples.
In terms of the product samples, in groundnuts, unlike groundnut flour and paste, a large proportion of the other products, roasted groundnuts, shelled and unshelled, have aflatoxin levels of less than 20 ppb.On the other hand, most maize grains (63.6%) and flour (88.2%) showed aflatoxin levels lower than 20 ppb (Table 5).
There was a highly significant (p < 0.001) difference in the level of aflatoxin contamination in the groundnut and maize products (Table 6).Though insignificant, higher aflatoxin contamination was recorded in the eastern than in the northern.Aflatoxin contamination also varied significantly (p < 0.009) with the form of processing that the product had undergone.

Discussion
The present study builds on recent field survey studies on maize (Sserumaga et al., 2015(Sserumaga et al., , 2020) ) and groundnuts (Sserumaga et al., 2021) that reported high aflatoxin contamination levels in the two crops.The current study focused on the assessment of the aflatoxin contamination levels in different forms of products of groundnuts and maize in selected ten districts in eastern and northern Uganda.The levels of aflatoxin contamination in groundnut products were high, exceeding all applicable limits, and they were consistent with the overall levels of aflatoxin contamination reported by earlier studies (Baluka et al., 2017;Muzoora et al., 2017;Osuret et al., 2016;Sserumaga et al., 2021).Some of the drivers of the high aflatoxin contamination levels in groundnut have been reported (;Ankwasa et al., 2021;Waliyar, Osiru, et al., 2015;Agbetiameh et al., 2018).Groundnut flour and groundnut pastes (peanut butter) were the most unsafe with regard to aflatoxin contamination.The conditions of processing the groundnut flour and how they influence the aflatoxin contamination level have been described (Ankwasa et al., 2021;Omara et al., 2020).Groundnuts kept in pods had a relatively lower aflatoxin contamination level, incidence, and density of Aspergillus section Flavi species, which is similar to earlier study reports in Ghana (Agbetiameh et al., 2017).This is because the pod walls of the groundnuts stored in pods provide a physical barrier for the entry of fungal species.
Unlike an earlier report by Baluka et al. (2017) that reported high aflatoxin contamination levels in groundnut samples from the market and no contamination in homemade samples, our study showed no significant difference in aflatoxin contamination levels across the different sampling points.But for groundnuts, most of the contaminated products were from the community and the roadside markets, rather than from homes.This is because most of the products at the homes were kept in pods and those in flour or pastes are prepared instantly for making sauces, while those in the markets were flours and pastes that stay up to two to three weeks on the shelves, as observed by the sellers.This prolonged stay on the shelves predisposes the products to aflatoxin-producing fungal growth and aflatoxin contamination.It should be noted that groundnut ranks second after beans in importance as a source of cheap protein in the study region (eastern and northern Uganda) and contributes significantly to the livelihood of the population (Sserumaga et al., 2021).The seeds are consumed severally as confectionary or traditional sauce, ebinyebwa Therefore, efforts to curtail the levels of contamination, especially in food products, should be made quickly to reduce the cases of illness associated with high aflatoxin levels.
The majority of the products of maize were unsafe under the European Union (EU; 4 ppb), the Uganda National Bureau of Standards (UNBoS; 10 ppb), and the United States Food and Drug Administration (FDA; 20 ppb).In contrast, maize products had a higher proportion of aflatoxin contamination levels than the international, regional, and national limits as reported in previous studies (Paul Wacoo et al., 2018).Specifically, the maize cobs, grain, and maize-based livestock or poultry feed products were the most contaminated maize products.The high level of aflatoxin contamination in cobs is very dangerous since the storage of maize in cobs is a common practice among smallholder farmers in Uganda.These high levels could be attributed to high incidences of aflatoxin-causing Aspergillus section Flavi species in the field (Sserumaga et al., 2020) and poor storage conditions that favour Aspergillus section Flavi fungal growth and aflatoxin contamination (Acur et al., 2020;Muzoora et al., 2017;Omara et al., 2020).High levels of aflatoxin contamination above acceptable limits in poultry and related feed ingredients have been reported recently, where limited knowledge about aflatoxins, aflatoxin contamination predisposing factors, dangers of aflatoxin to animals and humans, and mitigation strategies; and poor feed and feed ingredient handling and storage practices are major contributing factors (Nakavuma et al., 2020).
As reported by Omara et al. (2020) and PACA, (2020), there was no significant variation in aflatoxin contamination levels at regional (eastern vs. northern), sub-regional, and district levels.However, comparatively, the eastern region and specifically the Busoga sub-region recorded the highest aflatoxin contamination levels.Iganga District, as reported by Sserumaga et al. (2021), recorded the highest aflatoxin contamination level in groundnuts, in addition to Namutumba and Dokolo districts.The latter two districts and the Pader District in the northern part of Uganda also showed higher aflatoxin contamination in maize products.This could be associated with the differences in weather conditions, cropping systems, and postharvest practices employed by the farmers which end up  favoring the multiplication of the aflatoxin producing fungi.In addition, the most dominant groundnut product in the Busoga sub-region was groundnut flour, which also recorded the highest level of contamination.
Generally, our results are similar to earlier studies that showed high levels of aflatoxin contamination in groundnuts and maize from the field to the market as reported by Ankwasa et al. (2021).Aflatoxin contamination of groundnuts and maize products may be facilitated by prevailing humid conditions, high temperatures, and damage caused by birds or insects (Ankwasa et al., 2021) that predispose the crops to fungal attack and aflatoxin biosynthesis.These high levels of contamination pose a great risk to both the health of the population through the development of aflatoxin-related illness as illustrated by Sujayasree et al. (2022), and the economy of the country through rejection in international markets (Lukwago et al., 2019), since the majority of the resource-poor Ugandan population's livelihood depends to a large extent on the two crops.
In addition, there was a significant difference in aflatoxin contamination in processed and unprocessed maize and groundnut products.This could be explained by the fact that different processing techniques may reduce the quantity but not completely remove or destroy aflatoxin and other mycotoxins in food products (Jallow et al., 2021).However, physical removal of contaminated or damaged kernels by sorting and cleaning may lower mycotoxin concentrations without altering the product.In a previous study, hand sorting of contaminated peanuts resulted in to a 96.7% reduction in aflatoxin levels (Xu et al., 2017).According to Jallow et al. (2021), grain and nut sorting has evolved from handpicking through air-floating, automated sorting based on grain size and color, to sensorbased optical sorting for practical reasons such as higher production volumes.It was also noted that milling processes do not destroy or completely remove all mycotoxins but rather redistribute the toxins into different milled fractions.According to Zhong et al. (2015), 60 seconds of milling totally eliminated all aflatoxin B 2 and fivefold decreased the quantity of aflatoxin B1 in rice, but significantly higher aflatoxin levels were detected in the bran.
Previous research have identified a number of aflatoxin control measures, including the use of cutting-edge agricultural technologies, sound agricultural practices, and the use of appropriate storage technologies (Mahato et al., 2019).Ozone technology is emerging for microbial decontamination and mycotoxin degradation in cereals (Sivaranjani et al., 2021).Ozone has a high rate of oxidation and produces more free radicals, which can attack the functional groups of different mycotoxin functional groups by altering their molecular structures resulting in products with lower molecular weights, fewer double bonds, and less toxicity (Khanashyam et al., 2022;Sujayasree et al., 2022).On the other hand, Taha et al. (2022) has reported on the use of ultrasound technology for deactivation of microbes, degradation of toxins and inactivation of some enzymes.

Conclusion
Overall, results from the current study showed that most of the maize and groundnut products in the market are highly contaminated with aflatoxin and aflatoxin-producing fungal organisms that render the products unsafe for food and feed.However, the level of aflatoxin contamination varied significantly with the different forms of products in the market.Unlike maize cobs, most of the highly contaminated products were processed.This calls for the need to investigate how and the conditions under which these products are processed and whether due diligence is taken when preparing the raw materials for processing.The results obtained in this study and preceding studies, as suggested, may serve to develop aflatoxin mitigation strategies from household to market level against the high aflatoxin contamination levels in Uganda.

Table 1 .
Number of samples collected per region per district for the quantification of aflatoxin contamination levels in maize and groundnut