Status on aflatoxin levels in groundnuts in Uganda

Status on aflatoxin levels in groundnuts in Uganda

Saphan Muzoora^1,&, Margaret Loy Khaitsa², Hartford Bailey², Peter Vuzi³

₁Department of Biomolecular Resources and Biolab Sciences, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda, ²Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Starkville, Mississippi, USA, ³Department of Biochemistry and Sports Sciences, College of Natural Sciences, Makerere University, Kampala, Uganda

^&Corresponding author
Saphan Muzoora, Department of Biomolecular Resources and Biolab Sciences, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda

Introduction    

Groundnuts possess a high nutritional and commercial value due to the presence of fatty acids, proteins, carbohydrates, minerals and vitamins [1]. Thus, groundnuts are widely consumed by humans, and are used as ingredients in formulation of animal feeds. Groundnuts are a popular crop worldwide because of their value as a plant protein source (23-25%) and oil (45-52%) [2]. In Uganda, the level of groundnut consumption is relatively high and are consumed either in a roasted or cooked form. Due to this demand, groundnuts rank second (0.7 metric tonnes/ha) after beans (1.5 metric tonnes/ha) in legumes grown in Uganda [3]. With increasing human population, the demand for groundnuts in Uganda is rapidly rising. The current annual human population growth rate of Uganda stands at 3.25%, and the rate is expected to double by the year 2030 [4]. This will have not only created the need for increased groundnut production, but also a need for prolonged storage periods. Improper storage conditions of foodstuffs can lead to growth of fungi that produce mycotoxins (patulin, ochratoxins, aflatoxins, citrinins and fumonisins). However, due to their high level of carcinogenicity, aflatoxins are mycotoxins of major public health significance worldwide [5, 6]. Aflatoxins are secondary metabolites produced primarily by Aspergilus flavus and Aspergilus parasiticus. These fungal species are major contaminants of main food crops. Presence of aflatoxins and their metabolites in animal products is documented. In Nigeria, aflatoxins were found in dried beef and fresh beef in concentrations of 0.003 ppb and 0.02 ppb, respectively. In milk, 0.15 ppb of aflatoxin M1 were found [7]. In Uganda, aflatoxins were present in food products in varying concentrations. For instance, 32 ppb in dried silver fish, 701 ppb in maize, 2000 ppb in groundnuts, 40 ppb in soybeans, 30 ppb in cassava chips and 25 ppb in sunflower seeds [8]. Of greatest concern, groundnuts provide a suitable substrate for fungal growth and aflatoxin production [9]. Aflatoxins are designated into 16 groups and those of public health interest are B₁, B₂, G₁ and G₂ and their metabolites M₁ and M₂. These cause cancer, suppress the immune system and retard growth in both animals and humans [10, 11]. Of those listed, aflatoxin B₁ is the most predominant, and is a known cause of hepatocellular carcinoma (HCC), especially in areas where Hepatitis B virus infection is endemic [12, 13]. It is estimated that more than 4.5 billion people in developing countries are at risk of developing chronic aflatoxicosis through consumption of aflatoxin contaminated foods [14-16]. In addition, chronic exposure to aflatoxins contributes to the development of between 4.6 and 26.2% of all human liver cancer cases worldwide. Of the 550,000 - 600,000 new human liver cancer cases recorded annually, approximately 25,200 - 155,000 are due to aflatoxin exposure. About 40% of these cases are found in sub-Saharan Africa [17], and approximately 1,000 human liver cancer cases are registered annually in Uganda [18]. It is further noted that besides the contribution from hepatitis viruses, the presence of aflatoxins in foods could be a major contributing factor in the development of hepatomas in Uganda. Although allowable concentrations of aflatoxins in foods vary from country to country, the Food and Drug Administration (FDA) in the United States of America (USA) fixed maximum levels for aflatoxins at 20 ppb in human food commodities and 0.5 ppb for M1 in milk [19]. However, Uganda National Bureau of Standards (UNBS) set the total aflatoxin limits in all foods and feeds at 10 ppb. Studies have reported increasing incidences in liver cancer cases in Uganda of which aflatoxins could be the major risk factor. The time lag between the previous study in aflatoxin levels in food samples in 2005 and the present situation, proved to be necessary for a study to determine the current aflatoxin levels in groundnuts in Uganda

Methods     

Study area

Uganda is a landlocked country in Eastern Africa with an area of 241,038 km². Kampala is its largest city (population 1.5 million), as well as its capital. Uganda is divided into four regions; Northern, Central, Eastern and Western, with a total of 112 districts [20]. The eight study districts were: Kampala, Mubende, Gulu, Pader, Mbarara, Masindi, Soroti, and Kaberamaido and are located in the four regions of Uganda. The urban districts were Kampala, Gulu, Soroti and Mbarara, while Mubende, Pader, Masindi and Kaberamaido were the rural districts. These districts were chosen to take care of possible variations in fungal contamination levels of the foodstuff along the value chain. Additionally, the two districts selected from each region dominate in groundnut production and/or consumption in Uganda [20]. As of Uganda´s 2014 National Population and Housing Census, the total population was 34,634,650; Central region contained 27% of the country´s population, Western region contained 26%, Eastern region contained 25%, and the Northern region contained 22%. The population of the study districts was Kampala at 1,507,080; Mubende at 684,337; Gulu at 436,345; Pader at 178,004; Mbarara at 472,629; Masindi at 291,113; Soroti at 296,833 and Kaberamaido at 215,026 persons [20].

Sample collection

One hundred twenty (120) groundnut samples were conveniently purchased from traders. From each region, 30 samples were collected. They were categorized as; milled and seeds (Figure 1). In total, 74 samples were milled and 46 samples were seeds. For each sample, 250g were weighed, placed in a sterile lockable and waterproof sample bag and labeled with the sampling area code and sample number (e.g. KN 02). The samples were then placed in a cold box with icepacks (-8°C), and transported to a Biotechnology/Microbiology laboratory at Uganda Industrial Research Institute, located in Kampala, for immediate extraction. Samples that were not immediately extracted were kept at -20°C to prevent microbial growth and were processed within one month.

Sample size determination

According to the Central Limit Theorem [21], 30 groundnut samples were collected by convenience from each of the four regions of Uganda.

Analysis of Aflatoxins in Groundnuts

Sample extraction for Thin Layer Chromatography (TLC)

Extraction was done according to the method described by Schuller et al., (1983) with minor modifications customized to our laboratory settings [22]. One hundred seventy (117) samples were screened as three samples were found contaminated. Briefly, prior to extraction, groundnut seeds were ground using a laboratory blender. Fifty grams (50g) of each sample were placed into a sterile stomacher bag and 4g of sodium chloride added to remove oil. Then 150mLs of a mixture of methanol-water (8 + 2v/v) were added into each sample and homogenized for 2 minutes using a bag mixer. The homogenized samples were transferred into conical flasks and allowed to stand for the emulsion to separate. After separation, the supernatant was decanted and placed into a measuring cylinder, into which 10mLs of a mixture of distilled water-chloroform (50% + 50%v/v) were added. The resultant mixture was then transferred into clean conical flasks and shaken for 10 minutes on a rotator shaker. Thoroughly mixed emulsions were transferred into separating funnels and chloroform extract collected into labeled bijou sample bottles.

Aflatoxin screening by TLC

To detect different groups of aflatoxins, the TLC technique was used. The B group (B1 & B2) exhibits a blue fluorescence, whereas the G group (G1 & G2) exhibits a yellow-green fluorescence when exposed to the ultra-violet (UV) light. Briefly, 5μl of each of the chloroform extracts and the aflatoxin standard extracts were spotted on a marked TLC silica gel plate, and then the plate was allowed to dry. Initially, the dried TLC plate was placed in diethyl ether until the mobile phase moved a distance approximately equal to half of its length to remove the remaining oils. It was then removed and allowed to dry before it was viewed under a UV lamp. The sample spotted TLC plate was subjected to a second treatment in which it was placed into a mobile phase composed of acetone- chloroform mixture (50% + 50%v/v). After drying, the TLC plate was observed under a UV lamp. The results from the two treatments were recorded as either positive or negative.

Aflatoxin quantification by total aflatoxin MaxSignal ELISA kit

The remainder of TLC positive samples were subjected to the second extraction procedure for aflatoxin quantification using c-ELISA technique.

Sample extraction for ELISA technique

This was done according to the manufacturer´s manual (Total Aflatoxin ELISA Test Kit Manual; Catalog #: 1030-07) with minor modifications. In brief, 0.2g of sodium chloride and 25mLs of 70% methanol were added in 5g of milled groundnut sample. The mixture was vortexed for 10 minutes using a multi- vortexer. After thorough mixing, the mixture was then centrifuged for 20 minutes at 2500rpm. The filtrate was collected into labeled Eppendorf tubes and kept at 25°C until use.

Total aflatoxin quantification by c-ELISA

The detection and quantification of total aflatoxins was performed by c-ELISA technique according to the manufacturer´s instructions (Total Aflatoxin ELISA Test Kit Manual Catalog # 1020-07). The extracts were diluted to different concentrations (1:10; 1:1000 & 1:10,000) based on the results obtained after optimization of c-ELISA method with TLC positive samples. Briefly, 50μl of the filtrate were added into different wells of a pre-coated plate. This was followed by adding 100μl of aflatoxin B1-HRP conjugate. The plate was then rocked for 1 minute to obtain a uniform mixture and incubated at room temperature (25°C) for 30 minutes. After 30 minutes of incubation, the wells were washed three times by adding 250μl of a 20-fold concentrated wash solution into each well and the plate blotted on a clean paper after each wash. After proper washing, 100μl of the tetramethylbenzidine (TMB) were added into each well and plate incubated at 25°C for 15 minutes. The reaction was stopped by adding 100μl of 4M H2SO4 into each well. The absorbance readings were taken within 15 minutes using a microplate reader at 450nm. A standard curve was constructed using ELISA absorbance readings of the total aflatoxin standards (0, 0.02, 0.06, 0.2, 0.6 and 1.5 ng/mL) to determine aflatoxin concentrations (Figure 2).

Data analysis

The mean absorbance readings and concentrations of the standard solutions were entered into a Microsoft Excel 2013 spreadsheet, and the standard curve was generated. A Cochran-Armitage Test was performed to establish contamination trends among the sample types (fresh seeds and milled groundnuts). Proportions of samples that contained aflatoxins were compared using the chi- square test and/or fisher's exact test. Odds ratios and their associated 95% confidence intervals were estimated to quantify the magnitude and direction. Analysis of differences in mean aflatoxin levels in groundnut seed and milled samples was done using student´s t-test. All tests were performed at a 5% level of significance.

Results     

Out of 117 samples analyzed, 72 (62%) samples were TLC positive for aflatoxin, of which 19 (26%) had B₁, B₂, G₁ and G₂ all present, and 53 (74%) samples had only B₁ and G₁. Among the 72 total positive samples, 20 (28.2%) had Type G, while 22 (31%) had Type B. The state of groundnuts (fresh seeds, seeds, and milled) increased the probability of the thin layer chromatography (TLC). Among the positive TLC, an increasing trend in the proportion was noted with increasing levels of the state of groundnuts from 4.2% to 78.9% (Figure 3). The proportion of milled groundnuts (56/75, 78.9%) that tested positive for aflatoxins was higher than that of seeds (12/30, 16.9%) and/or fresh seeds (3/13; 4.2%). Positive TLC was 5.5 times more likely to be associated with milled groundnuts, compared to groundnut seeds (p value = < 0.0001; OR=5.5; 95% CI (2.44 - 12.43)). Excluding the milled groundnuts, positive TLC was 2.2 times more likely to be associated with groundnuts seeds compared to fresh groundnut seeds (p value = < 0.4871; OR= 2.2; 95% CI (0.5 - 9.8)). The proportion of samples that tested positive for aflatoxin (in ascending order) by region were: Northern (24.6), Central (25.4%), Eastern (25.4%), Western (26.8%); and by district: Masindi (5.6%), Pader (7.0%), Mubende (9.9%), Kaberamaido (9.9), Soroti (12.7%), Gulu (14.1%), Kampala (19.7%), and Mbarara (21.1%). However, there were no significant differences in aflatoxin contamination levels in groundnut samples among regions (p value = 0.391) and districts p value= 0.2249). Urban samples had a higher proportion of positive samples (51/76; 67.1%) compared to rural (20/42; 47.6%); p= 0.0384; OR=2.24 95% CI (1.04 - 4.86). The c-ELISA results indicate that out of 72 TLC positive samples, 58 (81%) were aflatoxin positive. Total aflatoxin concentrations in groundnuts varied greatly and ranged from 0.31 to 11,732 ppb (1,277.5 + 382.2 ppb) in milled samples and 1.6 to 516 ppb (84.7 + 43.8 ppb) in seed samples (Table 1). However, differences in mean total aflatoxin levels between two sample types were not statistically significant (t (56) = 1.67, p= 0.051). Total aflatoxin concentrations of 30 (52%) samples exceeded the 20 ppb and 10 ppb FDA/WHO and Uganda National Bureau of Standards (UNBS) regulatory limits, respectively.

Discussion     

Results of this study indicate that aflatoxin B1 was the predominant aflatoxin, and total aflatoxin concentrations varied greatly among groundnut samples. Higher total aflatoxin levels were found in milled groundnut samples than in seeds, but there was no significant difference between the mean aflatoxin levels in two sample types (t (56) =1.67, p=0.051). The largest proportion (52%) of positive samples had aflatoxin concentrations exceeding 20 ppb (FDA/WHO regulatory limit). In addition, samples from urban areas were more contaminated than those from rural areas. This study was able to detect the aflatoxin groups in groundnut samples using a sensitive TLC screening technique and quantify total aflatoxins using c- ELISA. The c-ELISA is a sensitive technique and a choice for many researchers in the measurement of aflatoxins and their metabolites in different types of samples. Precautions were taken to prevent further fungal growth in samples by immediately processing them, and those not handled were stored at -20°C until extraction. However, during sampling, samples were collected from the top layer in the packaging material where conditions could not have favored fungal growth as it would have been the case with the bottom layer.

Presence of aflatoxin B₁ in all c-ELISA positive samples was in agreement with results of previous studies [12, 13]. Conversely, relative differences in contamination levels in the two sample types observed in this study may be attributed to the practice of traders in Uganda in which fungal contaminated groundnuts are milled to shield evidence of spoilage from consumers. In addition, it may be attributed to the non-homogenous nature of aflatoxin contamination in groundnut seeds than milled products. Milling allows homogenous contamination and creates a conducive environment for the growth of Aspergillus Spp. The higher contamination levels of urban groundnut samples than rural ones may be attributed to longer storage periods of agricultural produce in urban areas. Studies have demonstrated that significant contamination of agricultural produce occurs during storage [5, 6].

The presence of higher levels of aflatoxins in groundnut samples analyzed in this study compared to results of previous studies in Uganda indicates an increasing trend in contamination of foodstuffs with aflatoxins. This may be associated with increasing rural-urban migrations that have led to the increasing need for storage of food by traders for relatively longer periods compared to the past. With the increasing annual growth rate in this country, the situation is expected to worsen in the near future. Although there are no recent reports on acute aflatoxicosis in Uganda, higher aflatoxin levels obtained in this study may be responsible for the increasing hepatoma frequency in Uganda. This observation corroborates with findings of other studies. For instance, the numbers of acute aflatoxicosis are not large relative to the populations at risk because humans are usually an aflatoxin- tolerant species [16]. Consumption of aflatoxin-contaminated products is detrimental to human health since aflatoxins are teratogenic, immunosuppressive, carcinogenic and cause growth retardation in both humans and animals [12, 13]. Therefore, based on the findings of this study, there is an urgent need to quantify aflatoxin levels in other foodstuffs countrywide to establish aflatoxin contamination trends, conduct a dietary assessment to ascertain human aflatoxin exposure levels and establish the likely contribution of aflatoxins to liver damage particularly in the development of hepatocellular carcinoma.

Conclusion     

This study has demonstrated that Uganda´s groundnuts are highly contaminated with aflatoxins in levels that are likely to cause acute aflatoxicosis if consumed in relatively large quantities, and can cause liver damage, immunosuppression and growth retardation on chronic exposure. Results further indicated that urban groundnut samples were more contaminated than rural ones. In addition, milled groundnuts were more contaminated than seeds. Thus, good storage practices are needed to prevent aflatoxin contamination of groundnuts, as well as, other foodstuffs. We recommend that an epidemiological study be conducted in Uganda to fully understand the dangers associated with high aflatoxin presence in groundnuts (the second staple plant protein source after beans) in Uganda.

Competing interests     

The authors declare no competing interest.

Authors´ contributions     

The primary author (Dr. Muzoora), Dr. Khaitsa and Dr. Bailey developed the research concept. Dr. Vuzi assisted with analysis of groundnut samples. All authors participated in writing the article, reviewed several drafts, and approved the version to be published.

Acknowledgments     

The authors wish to acknowledge the United States Agency for International Development´s (USAID) support through the “Capacity building in Integrated Management of Transboundary Animal Diseases and Zoonoses (CIMTRADZ)” project that enabled the primary author (Dr. Muzoora) to visit Mississippi State University, USA for three months under Makerere University junior faculty exchange. The International Institute, Mississippi State University, USA, financially supported this study. The authors also acknowledge the contribution of Uganda Industrial Research Institute (UIRI) that provided laboratory space and equipment. We particularly acknowledge Mr. Wacoo Alex of UIRI for the technical input into this study. Finally, we thank all traders who gave us groundnut samples analyzed in this study.

Table and figures     

Table 1: mean total aflatoxin levels (ppb) in positive groundnut samples (n=58)

Figure 1: distribution of 120 groundnut samples by Region and District of Uganda

Figure 2: aflatoxin standard curve

Figure 3: aflatoxin results of thin layer chromatography in groundnut sample types

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