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Study on Aspergillus Species and Aflatoxin Levels in Sorghum (Sorghum bicolor L.) Stored for Different Period and Storage System in Kewet Districts, Northern Shewa, Ethiopia
Geremew Tassew weledesemayat1, Tilahun Bekele Gezmu2, Ashagrie Zewdu Woldegiorgis2 and Habtamu Fekadu Gemede2,3*
1Food, Medicine and Health Care Administration and Control Authority of Ethiopia, Addis Ababa, Ethiopia
2Center for Food Science & Nutrition, College of Natural Sciences, Addis Ababa University, Ethiopia
3Department of Food Technology and Process Engineering, Wollega University, Nekemte, Ethiopia

ABSTRACT
Sorghum serves as staple food for over 100 million people in Sub-Saharan African countries. It is the most important nutritional security crop and ranks third among major cereal crops in terms of area and production next to teff and maize in Ethiopia. However, Sorghum is susceptible to contamination by molds that produces aflatoxin that causes hepatotoxic and carcinogenic effects on humans and animals. This study was conducted to assess Aspergillus Species and aflatoxin level in Sorghum (Sorghum bicolor L.) stored under different storage system for different storage period. Thirty samples were analyzed for aflatoxin contamination using high performance liquid chromatography equipped with fluorescent detector and Aspergillus Species were isolated and identified using phenotypic features in a potato dextrose agar culture media. About 56.7%, 16.7%, and 23.3% of the Sorghum samples were found to be contaminated with Aspergillus flavus, Aspergillus niger and Aspergillus parasiticus, respectively. The level of total aflatoxin, aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 were in the range of 11.44 to 344.26µg/kg, 3.95 to 153.72µg/kg, 1.17 to 91.82µg/kg, 9.87 to 139.64µg/kg, and 3.22 to 52.02µg/kg, respectively. The concentration of aflatoxin in all Sorghum samples surpassed the maximum level set by the European commission and therefore, deserves attention to control them across the Sorghum value-chain.
KEYWORDS
Aflatoxin; Aspergillus Species; Sorghum; Storage Period; Storage System

Introduction
Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most important cereal crop that is grown for grain and fodder in the semi-arid tropics, mainly in Asian and African countries [1]. Sorghum is used as a major food and nutritional security crop for more than 100 million people in the Horn of Africa [2]. Ethiopia is one of the major centers of origin and diversity for Sorghum cultivation [3]. Sorghum ranks third among major cereal crops in terms of area and production next to teff (Eragrostis teff) and maize (Zea mays) throughout the country [4]. It is estimated more than 1.6 million hectares of the land covered with Sorghum production [5]. The lives of millions of Ethiopians depend on Sorghum as a staple food crop [1]. However, Sorghum crops have been affected by numerous mould contaminations [6].

Aflatoxins are naturally occurring toxic secondary metabolites of the storage fungi (Aspergillus flavus and Aspergillus parasiticus) which are produced in most agricultural products stored at inappropriate places, temperatures and moisture level. It is extremely persistent under most conditions of storage, handling and processing [7]. These two species are common and widely distributed in tropical and sub-tropical parts of the world. Aflatoxin has been found as contaminant in agricultural food products especially in cereals and animal feeds. Aspergillus flavus is common and widespread in nature and is most often found when certain grains are grown under stressful conditions such as drought. Additionally, mould occurs in soil, decaying vegetation, hay and grains undergoing microbiological deterioration and invades all types of organic substrates whenever and wherever the conditions are favorable for its growth [8,9].

Aflatoxin contamination of food causes hepatotoxicity, carcinogenicity, immunosuppression, mutagenic and teratogenic [10] and associated with childhood stunting, which cause severe economical losses to the country [11]. It is a serious health problem because factors which encourage the production of these toxins by mycotoxin (Aspergillus flavus and Aspergillus parasiticus) abound in Africa. Their presence in food interferes with micronutrients absorption and status in the body and as a consequence, they affect immunity and development [10]. Aflatoxin B1 is one of the most potent naturally occurring animals carcinogen; and immunosuppressive to immunosuppressant [12]. The extent to which mycotoxins affect human health is difficult to investigate in countries where health system lack capacity and resources are limited. For instance, factors such as immune suppression contributing to the overall burden of infectious diseases is difficult to quantify, but is undoubtedly significant. However, food safety remains an important opportunity for addressing current health problems in developing countries [13].

Previous reports have indicated aflatoxin contamination of cereals and pulses such as Sorghum, teff, wheat, maize, peanut, legumes collected from silos, warehouses, shops and market places in Ethiopia [14]. Many investigators have detected aflatoxin and other mycotoxins in many agricultural foods such as maize, teff, broad bean, Sorghum, beriberi, traditional spices (mitten shiro) and wheat in Ethiopia [15-20].

Kewet woreda is one of the Sorghum producing districts in the country and farmers use both underground pit and above ground storage systems. The storage system “Gotera” are made from different plants, clay, grass, ash, and cow dung. In the underground pit storage, they wash the pit with water and put the Sorghum grain until they are full, then the cover with grass, clay and flatted stone. Some farmers are using cleaning, insecticides and fumigants to prevent insect damage and adding the Sorghum grain in to the pits. The grain is stored for long periods; especially, this is the case during times of food scarcity. These storage systems are believed to protect against insect damage and theft, fire, domestic and wild animals and improve the quality of Sorghum as well. These Sorghum grains are stored under unhygienic conditions and very often spoiled by moulds and may develop mycotoxin contamination. Therefore, this particular research was endeavored to analyze the occurrence of Aspergillus Species and aflatoxin content in Sorghum grain stored at different time periods and different storage system.
Materials and Methods
Description of the study area
The sample was collected from North Showa Zone of Amhara region which is located at about 225 km to the north of Addis Ababa along the main road to Dessie. North Showa Zone consists of 26 districts and among these districts; Ataye, Kewet, Merahbete, Meda Oromo and Ensaro are high in Sorghum production area. Based on the storage system and periods and production rate, samples were collected from Kewet woreda of 5 Kebeles (Rassa, Ashgne yegeda, Gerenefara, and Teri and Yelen. From each of Kebeles, six role model farmers were proposed and selected on their production rate and one kilogram Sorghum sample was collected from each role model farmer. Kewet district is a lowland and semi-arid area that lies at an altitude ranging between 1280 and 2700 meters above sea level. It is located at longitude and latitude of 10˚00’N 39˚54’E and 10.000˚N 39.900˚E, respectively. The vast majority of people are rural small holding who depend on cultivation of 16,046 hectares of arable land. Kewet district has a population size of 100,760. The average annual rainfall is around 600-700mm and temperature of the area ranges from 17 to 30˚C.
Chemicals and reagents
The chemical and reagents used for aflatoxin analysis were HPLC grade. acetonitrile, methanol, n-hexane, MgSO4 anhydrous salt, NaCl, aflatoxin standard and deionized water. The AFG1, AFG2, AFB1 and AFB2 standards were obtained from Sigma-Aldrich. The pure reference standards were stored in dark place at 4˚C. The Aspergillus Species isolation and identification equipments and chemicals utilized were incubator, petridish and Potato Dextrose Agar (PDA), 10% sodium hypochlorite solution, and ethanol absolute (99.7%).
Survey questionnaire
The survey questionnaire was targeted on educational level, mould contamination, mould color, drying method, storage system, storage time, location of storage, cleaning and sanitation, critical problems and measure taken to control the problems, if any. The role model farmers were chosen purposively based on the information given by the DA (Agricultural Developmental Agents) workers. The farmers were interviewed by means of one to one correspondence.
Sampling and sample size
In this study, a total of 30 samples of Sorghum (15 samples from each storage type or 10 samples per each storage period) were collected purposively by taking the storage periods (< 12 month, 1-2 year and ≥ year) and storage system (above ground and underground pit storage systems) into consideration. About 1kg of Sorghum samples were collected from the aboveground storage and underground pit storage system from each role model farmers. The samples were collected in plastic bags by taking preventive measures to avoid adventitious contamination and were transported to the laboratory of food science and nutrition at Addis Ababa University. The Sorghum grains were separated from foreign matter and milled using miller and sieved to pass through 1 mm mesh size. The flour was packaged in tight polyethylene bags and stored in cool dry place. However, whole grains of Sorghum were utilized for Aspergillus Species isolation and identification. The sample collection and sampling were identified figure1 below.
Determination of moisture content
Moisture content was determined according to [21] using the official method 925.09. A crucible was dried in an oven at 105˚C for 1 hour and placed in desiccators to cool. The weight of the crucible (W1) was determined. 5 gm samples was weighed in the dry crucible (W2) and dried at 105˚C for 3 hours and after cooling to room temperature in desiccators it was again weighed (W3). The moisture content was determined by using Eq. (2).

Moisture content in (2)
Determination of aflatoxin
Method adaptation for analysis of aflatoxin
In order to perform the study on aflatoxin contamination level in the 30 Sorghum samples, methodology was performed as described by the QuEChERS method (Quick, Easy, Cheap, Effective, Rugged and Safe), by extraction with Milli Q water: methanol/acetonitrile, MgSO4 and NaCl, followed by centrifugation and filtration, and the quantification was carried out by high-performance liquid chromatography with florescence detector, without derivitization.

High performance liquid chromatography equipped with fluorescence detector was used for analyzing the aflatoxin level of the Sorghum samples. The column size was 250mm×4.6mm and the Rheodyne injector size was 20µL. Milli Q water and mixture of acetonitrile and methanol (in the ratio of 71.5/28.5) were used as a mobile phase. The Wavelength florescence detector was set at 440nm. The flow rate was limited to 1.0ml/mm. The parameters selected for method adaptation were linearity, specificity, and accuracy, limit of detection and quantification and precision.
Sample extraction and clean-up
Extraction of aflatoxin from the Sorghum samples were performed according to the method that was validated by Ghent University faculty of Pharmaceutical science, Laboratory of Food Analysis, Belgium, Europe [22]. 1.0g of Sorghum flour sample was accurately weighed, 5ml of water was added, vortexed briefly and allowed to stand for 30 minutes. 5ml of extraction solvent (100% ACN) was added and mixed by using a vortex mixer and shacked for 30 minute at using shaker. Weigh 2.0 ± 0.05 g of anhydrous salt MgSO4 and 0.5 ± 0.01g of NaCl and added to the test tube. Shacked immediately to the tubes briefly to prevent agglomeration of the salts and vortex for 2 min, centrifuge at 4000g for 15 min. then transferred 4ml of the top organic layer to a new tube and evaporate under Rotta Vapor and 200μL of injection solvent (A: B 50/50) and 200μL of n-hexane were added to the residue and vortexed immediately to dissolve the residue. Then, dissolved residue was transfer to a centrifuge filter and centrifuge at 10000g for 10 min.

The final extracts were filtered through a 0.45μm PTFE membrane, which is 150μl of the lower phase added in to vials and 20μL were injected into a high performance liquid chromatography column, using a Shimadzu high pressure liquid chromatography (Kyoto, Japan) with fluorescence detector (excitation at 365 nm and emission above 440 nm).
Isolation and identification of fungi
Fifteen Sorghum seeds per sample were surface sterilized with 10% sodium hypochlorite solution for 1 min, followed by immersion in sterile distilled water for 1 min. Surface sterilized seeds were then placed on freshly prepared Potato Dextrose Agar (PDA) plates (ten seeds per plate) and incubated for three days at 25˚C. Pure cultures of different out growing fungi were obtained by transferring fungal colonies to new PDA plates using sterile toothpicks, and incubating the plates for 5-7 days at 25˚C. Pure cultures of each isolate were then stored at 4˚C in test tube containing 2.5ml of sterile distilled water for further use.

Isolates were identified to a species level based on morphological (phenotypic) features as described by [23]. For this purpose: isolates representing each pure culture were grown on PDA Agar at 25˚C for 5-7 days. Fungal colonies that grow rapidly and produce colors of white, yellow, yellow-brown, brown to black or shades of green, mostly consisting of a dense felt of erect conidiophores were broadly classified as Aspergillus Species. while those that produce blue spores were considered as Pencillium species. Isolates with dark green colonies and rough conidia were considered as Aspergillus parasiticus. The major distinction currently separating Aspergillus niger from the other species of Aspergillus is the production of carbon black or very dark brown spores from Biseriate phialides [24].
Data analysis
Sorghum samples were analyzed in duplicates and the data obtained were analyzed statistically to calculate the level of significance of various parameters using Analysis of Variance (one way-ANOVA) for storage period and paired comparison for storage system, using the SPSS software version 20.0. The results were reported as mean ± SD and percentages. Least Significant Difference (LSD) was utilized for mean separation and P-value < 0.05 was considered to be significant.
Results and Discussion
Information on farmer’s awareness of mould contamination
A total of 30 farmers (4 female and 26 male) were interviewed for their knowledge regarding aflatoxin contamination in their locality. About 14 farmers (46.7%) respondents reported they were aware of mould contamination and the remaining 16 farmers (53.33%) were not. On the other hand, the 26.6% of respondents answered that the color of mould is Black and 6 farmers (20%) responded it as white. The major critical problems in Sorghum production and storage identified by the farmers included insects (33.33%), rodent (33.33%) and mould contamination (16.7%). About 75% of the farmers reported they used insecticides to control insect damage to Sorghum grains and about 5 (16.7%) of the farmers described they use other control mechanisms such as fire wood smoking. All farmers in the study area used natural drying method (sun drying) to adjust moisture of the grains prior to storage. With respect to location of the Sorghum storage system, 50% of the farmers stored their grains in the field, 16.7% stored inside house and 33.3% stored out in the courtyard. Nine farmers (30%) used above ground storage system and about 10 farmers (33.3%) used underground pit storage and 11 farmers (36.7%) used both aboveground (Gotera) and pit storage system. According to the informants, Sorghum can be stored for up to four years (Table 1).
Aflatoxin level in Sorghum stored at different storage periods and storage system
All 30 Sorghum samples were collected and assayed in duplicate for total aflatoxin contents and the average value for each sample was calculated. The total moisture contents of Sorghum samples in the present study were in the range of 8.8-11.86 % for Sorghum grain stored for different storage periods (< 12 month, 1-2 year and ≥ 2 year) and storage system (aboveground and underground pit storage). An indication of the moisture content and aflatoxin detected in Sorghum were shown in table 2.
Aflatoxin B1 and total aflatoxin content in Sorghum stored for less than 12 month
The moisture content of Sorghum samples stored for less than 12 months in the study was 9.53- 11.4% and 8.77-11.5% for aboveground and underground pit storage, respectively. Aflatoxin contamination levels detected in Sorghum samples stored above ground (Gotera) storage system for less than 12 months were in the range of 2.15-27.12µg/kg for AFB2, 3.1-41.96µg/kg for AFG2 and 16.62-139.64µg/kg for AFG1. Sorghum samples stored underground were detected in the range of 14.17-20.38µg/kg for AFB2, 7.46-26.16µg/kg for AFG2 and 16.98-56.39µg/kg for AFG1 (Table 2). The mean aflatoxin B1 content of Sorghum stored for less than 12 months above ground (Gotera) was in the range of 3.96-69.79µg/kg which was relatively lower than Sorghum stored underground (5.3-82.34µg/kg). The mean total aflatoxin content of Sorghum stored for < 12 month above ground was in the range of 16.59-183.16µg/kg and was slightly greater than Sorghum stored underground (46.48-177.33µg/kg). Therefore, the study showed that both Sorghum grains stored underground pit and aboveground (Gotera) were highly contaminated with aflatoxin. The reason for high aflatoxin content in samples stored aboveground could be because most farmer in the study are use improper construction materials such as clay, grass, caw dung, stone, plane water, ash and Sorghum stalk which encourages mould growth. Moreover, farmers repeatedly use the same storage system without properly cleaning it and without drying the Sorghum grains.
Aflatoxin B1 and total aflatoxin content in Sorghum stored for between one and two year
The moisture content of Sorghum samples stored for between one and two year in the current study was 9.33- 11.86% and 9.4-12.5% for aboveground (Gotera) and underground pit storage, respectively. Aflatoxin contamination levels detected Sorghum samples stored above ground (Gotera) storage system between one and two years were in the range of 2.76-52.05µg/kg for AFB2, 4.65-51.01µg/kg for AFG2 and 18.96-101.79µg/kg for AFG1. Sorghum samples stored underground pit were detected in the range of 3.06-35.01µg/kg for AFB2, 2.04-52.84µg/kg for AFG2 and 42.30-116.13µg/kg for AFG1 (Table 2). The mean aflatoxin B1 content of Sorghum stored between one and two year above ground storage (Gotera) was in the range of 21.75-88.90µg/kg which was relatively lower than Sorghum stored for underground (3.95-153.72µg/kg). The mean total aflatoxin content of Sorghum stored for between one and two year above ground storage was in the range of 44.27-210.53µg/kg and was relatively lower than Sorghum stored underground (11.44-344.26µg/kg). Therefore, the study showed that the Sorghum grain stored underground was highly infected by mould and hence contained higher aflatoxin concentration. The reason for high aflatoxin contamination in Sorghum stored underground could be because most farmers in the study area had misunderstanding about mould color and Sorghum handling.
Aflatoxin B1 and total aflatoxin content in Sorghum stored for two or more than year
The moisture content of Sorghum samples stored for two or more than years in the present study were 8.9- 11.3% and 9.4-11.75% for aboveground and underground pit storage, respectively. Aflatoxin contamination levels detected Sorghum samples stored aboveground for two or more years were in the range of 1.17-91.82µg/kg for AFB2, 16.95-72.65µg/kg for AFG2 and 12.92-114.10µg/kg for AFG1. Sorghum samples stored in underground pit were detected in the range of 6.89-21.35µg/kg for AFB2, 3.22-12.58µg/kg for AFG2 and9.87-129.26µg/kg for AFG1 (Table 2). The mean aflatoxin B1 content of Sorghum stored above ground for two or more years was in the range of 12.95-105.96µg/kg which was relatively higher than Sorghum stored underground pit storage (4.59-15.57µg/kg). The mean total aflatoxin content of Sorghum stored for two or more years aboveground (Gotera) was in the range of 65.17-247.92µg/kg and was relatively higher than Sorghum stored underground (17.68-164.3µg/kg). Therefore, the study showed that Sorghum grain stored aboveground (Gotera) storage was highly susceptible to aflatoxin contamination. The reasons for high aflatoxin contamination levels in the Sorghum sample stored aboveground (Gotera) could be because of most farmers in the study area use poor cultivation and harvesting, inadequate handling for example drying, insect control, cleaning, and storage practice.

From the total 30 samples, 96.66%, 93.33%, 96.7%, and 90% were contaminated by aflatoxin B1, B2, G2 and G1, respectively. These showed total aflatoxin contamination by the level ranged from 11.44µg/kg to 344.26µg/kg and the mean total aflatoxin value of 123.85µg/kg. Aflatoxin contamination levels were detected Sorghum samples in range of 1.17-91.82µg/kg for AFB2, 3.22-139.64µg/kg for AFG2 and 9.87-139.64µg/kg for AFG1 (Table 2). Enormous variation of aflatoxin contamination was observed between 23 Sorghum samples and 7 samples had aflatoxin levels below detection limits (3 for AFG1, 2 for AFB2, 1 for AFB1 and AFG2). This may be due to the variation in fungal colonization, especially Aspergillus flavus, and spore density during the grain development stage. It had comparable to the variation of this fungus in developing Sorghum grain was studied earlier by Ratnavathi et al. [25].

About 96.66% of the total aflatoxin in the Sorghum samples was attributed to AFB1 which ranged from 3.95- 153.72µg/kg. Generally speaking, AFB1 is the most toxic aflatoxin among all types of aflatoxin and is considered to be hepatocarcinogenic and immunosuppressive as well [12]. Therefore, the high concentration of the AFB1 in Sorghum samples indicates that there could be a serious problem of aflatoxin contaminations in the study area.

The incidence of aflatoxin contamination in the examined Sorghum grain samples appear to be lower than those reported for Sorghum and maize [16] in which the aflatoxin contamination reached up to 1000µg/kg for Sorghum and 1388µg/kg for wheat, respectively. However, this study result showed a very high aflatoxin levels above the permissible limits. The maximum aflatoxin B1 content found in this research (153.72µg/kg) was below the highest amount reported by Habtam and Kelbesa (692µg/kg) [17]. On other hand, the presence of aflatoxin contamination (96.6%) reported in the present study was relatively higher than the previous study reported by Ayalew [26] aflatoxin infection (88%) in maize from Ethiopia.

The presence of aflatoxin B1 detected in the present study (153.72µg/kg) was generally higher than from the previous study by Chala et al., [15] on Sorghum in Ethiopia (29.5µg/kg). However, the amount of aflatoxin B1 (153.72µg/kg) in the Sorghum samples in this research was much relatively lower than the finding reported by Alpert et al., [27] which was as high as 1000µg/kg. The concentrations of aflatoxin G1 obtained in the present study were larger (139.64µg/kg) when compared with previous reports for Sorghum grain (29.65µg/kg) by Chala et al. [15].

On other hand, the percent of AFB1 level (96.66%) reported in the present study was relatively higher than the previous study reported in Brazil which was 39% for pre-fermented and 32% for post-fermented Sorghum samples [28] with maximum values of 5.10µg/kg and 30.05µg/kg, respectively. Therefore, the current study also showed aflatoxin contamination in Sorghum stored under different storage system and period. This indicates that there is high problem of aflatoxin contamination in the country.
Effects of storage periods on the level of aflatoxin contamination in Sorghum
Sorghum stored for two or more years had high level of aflatoxin B1 (52.19µg/kg) followed by Sorghum stored for less than 12 months (38.24µg/kg). Although storage period had no significant (P>0.05) effect on the level of most of the aflatoxin, it resulted in significant difference in the level of aflatoxin B1 in Sorghum. Significant difference (P<0.05) in the aflatoxin B1 was observed between Sorghum grains stored for up to two years (52.19 µg/kg) and stored for more than two years (27.02µg/kg). This indicates that storing Sorghum for a period between one and two years result in higher contamination by aflatoxin B1. However, Sorghum stored for over two years had high level of aflatoxin B2 (21.42µg/kg) and Sorghum stored for less than 12 months had high concentration of the aflatoxin G1 (57.7µg/kg). Sorghum grains stored for above two year had high level of total aflatoxin than the remaining storage periods but with no significant difference (P>0.05) (Table 3). Based on the result of the present study, aflatoxin contamination can occur in grains stored for any storage period given no proper Sorghum cultivation, harvesting, transporting, trashing, cleaning, insect prevention and storage practices.

The level of aflatoxin B1 contamination (52.19µg/kg) in Sorghum storage period (< 12 month, 1-2 year, and ≤ 2 year) were lower than AFB1 in ground pepper, shiro, legumes and spices (incidences of AFB1 contamination occur 13.33% and 8.33%, and the levels of contamination range 250 to 525µg/kg and 100 to 500µg/kg, respectively) previously stated by Habtamu and Kelbessa [29].

However, the aflatoxin content and mycotoxin development in Sorghum grain stored for different period may increase due to moisture migration from the surrounding and storage condition (temperature and humidity) reported by Mohamed et al., Mashilla et al., and Habtamu and Kelbessa [30,31,17]. The level of aflatoxin increment stated by Shephard [13], flood damage to grain (mainly Sorghum) in underground storage areas resulted in visible fungal contamination and these harsh realities; it is not surprising that fungal contamination of staple food. The current study showed that the study area has a suitable condition for mycotoxin development and farmers may not used sufficiently good handling, harvesting and storage practice. This practice may lead to elevated aflatoxin production.
Effects of storage system on the level of aflatoxin contamination in Sorghum
Storage of grains under poor storage system and conditions often result in aflatoxin contaminations. However, in this study, storage system did not result in significant variation between the mean aflatoxin contents of Sorghum grains. Grains stored above ground had high aflatoxin level of 49.67µg/kg (G2), 63.91µg/kg (G1), 21.57µg/kg (B2) and 44.0µg/kg (B1) than grains stored in the underground pit which in that order contained 30.05µg/kg, 44.92µg/kg, 14.56µg/kg and 34.30µg/kg. Storage type also did not significantly affect the level of total aflatoxin (p>0.05), but the level of aflatoxin in storage type still larger than the maximum level stated by EU for aflatoxin B1 (5μg/kg) and for total aflatoxin (10μg/kg) and FDA established maximum acceptable level of 20μg/kg for aflatoxin in maize, and Sorghum (Table 4).

The reason for aflatoxin level increment in the current study may be factors, including soil quality, crop yields, and the biological environment of crops such as the abundance of pests and plant pathogens determine the mould growth of stored Sorghum [30]. These factors encourage mycotoxin invasion of grains and thereby impart a food-borne risks. Basically, the ability of fungi to produce mycotoxins is largely influenced by temperature, relative humidity, insect attack, and stress conditions of the plants.
HPLC chromatogram of the aflatoxin in Sorghum sample
A typical HPLC chromatogram showing the clear separation of 1 ppb standard mixture of four aflatoxins with respect to retention time G2 (19 min), G1 (21 min), B2 (23.5min) and B1 (25.8min) and the standard curve, black sample, mould infected sample and Sorghum sample was described in figure 2 and figure 3.
Isolation and identification of Aspergillus Species from Sorghum sample
Aspergillus Species. isolation representing each pure culture was grown on PDA Agar at 25˚C for 5-7 days. The first species isolated from the Sorghum samples was Aspergillus niger. The major distinction of separating Aspergillus niger from the other species of Aspergillus is the production of carbon black or dark brown spores of Biseriate phialides were indicated as plate 7. Aspergillus flavus was the second species identified in this study. Colonies of this fungus were characterized by yellow to dark, yellowish-green pigments, consisting of a dense felt of conidiophores or mature vesicles bearing phialides over their entire surface. In general, Aspergillus flavus was known as a velvety, yellow to green or the old colony was brown mould with a goldish to red-brown on the reverse. Aspergillus parasiticus was the third species identified from Sorghum samples tested in the current study. Colonies representing this species produced dark green and rough conidia on PDA at 25 and 37˚C after 5-7 days of incubation. The three identified Aspergillus Species were indicated in figure 4 below.

In this study all Sorghum samples come from regions with temperatures ranging from 17 to 30˚C which supports the growth of Aspergillus Species. Under tropical condition, stored products are more susceptible to Aspergillus Species than other fungi as many Aspergillus Species are favored by the combination of low water activity (aw) and relatively high storage temperature [17]. Although, cereal grains belong to corn, rice, barley; wheat and Sorghum are found susceptible to aflatoxin accumulation by aflatoxogenic fungus.

In table 5 indicated that the occurrence of Aspergillus spp. in Sorghum grain stored at different storage period and storage system; 56.7%, 16.7% and 23.33% of Aspergillus flavus, Aspergillus parasiticus and Aspergillus niger was found, respectively from the total 30 samples.

The incidence of Aspergillus flavus (56.7%) in the present study was bigger than the occurrences of Aspergillus flavus in maize samples from Ethiopia were indicated by Ayalew [26]. However, the invention of Aspergillus fulvous (56.7%) in Sorghum sample was lower than the occurrence of Aspergillus flavus (72.7%) in Sorghum grain reported from India [32]. Therefore the occurrence of aflatoxin content in Sorghum sample is associated with Aspergillus flavus and Aspergillus parasiticus. These indicate aflatoxin contaminations in Sorghum sample collected from the study area is highly significant.

However, the current results showed that Sorghum was more profoundly colonized by aflatoxin producing Aspergillus Species, with overall aflatoxin levels being correspondingly higher. The Sorghum grain contamination by Aspergillus Species. and the production of aflatoxin are highly influenced by the weather conditions prevailing during the grain development stage, i.e., seed set to physiological maturity stage [25]. In addition, this may be caused by the variations in cultivars, storage periods, and storage system and over all handling practices used.

Table 6 showed that the Aspergillus flavus (13.33% for < 12 months 23.33% for 1-2year and 20% for ≥ 2 year) and Aspergillus parasiticus (6.67% for < 12 months 6.67% for 1-2year and 3.33 % for ≥ 2 year) were occurred Sorghum stored at different periods. Aspergillus flavus and Aspergillus parasiticus stored at 1-2year was slightly larger than Sorghum grains stored with ≥ 2 year and < 12 month. This indicated that aflatoxin contamination in Sorghum grain was highly disposed from storage periods. The presence of Aspergillus niger (3.3%) in Sorghum stored between one and two year had lower than both 10 % of Aspergillus flavus and Aspergillus parasiticus. Sorghum stored with two or more years had relatively lower Aspergillus parasiticus (3.33%) than both 6.67% of Aspergillus parasiticus at Sorghum stored in less than 12 month and between one and two year.
Evaluation of aflatoxin results against different international standards
The maximum tolerable limits of aflatoxins in foodstuffs are laid by European Union legislation are specified for aflatoxin B1 (5μg/kg) and maximum total aflatoxin (10μg/kg) [33]. Another regulation, the US Food and Drug Administration (FDA) have established maximum acceptable level of 20 ppb for aflatoxin in maize, Sorghum and other cereals for human consumption, with the level in milk being even lower (0.5ppb) [34]. Likewise, FAO underline the maximum tolerated levels limit of 20µg/kg for mycotoxins in foodstuffs, Maize, kidney beans, rice, Sorghum, groundnuts and groundnut butter. Based on the result of the current study, the mean average of aflatoxin concentration in range of AFB1 (3.95-153.72µg/kg) and total aflatoxin (11.44-344.26μg/kg) is higher than the EU regulation limit and FDA maximum tolerable limit.

The aflatoxin content measured in all the Sorghum samples showed that natural aflatoxin was higher and 96.66 % of samples were contaminated by toxin as compared to highly susceptible crops like maize and groundnut [35]. However, the result also above the safety limit (20µg/kg) recommended by the Codex Alimentarius Committee.

In East African standard specification (CD-ARS 462:2012 (E) for Sorghum, it is stated that the Sorghum grains shall comply with those maximum mycotoxin limits established by the Codex Alimentarius Commission. In particular, total aflatoxin levels in Sorghum grains for human consumption shall not exceed 10µg/kg with AFB1 not exceeding 5µg/kg when tested according to ISO 16050. However, the present study showed that the total aflatoxin concentration and AFB1 were surpassed the specified tolerable levels by the above mentioned regulations.
Conclusion
The level of total aflatoxin in Sorghum samples was above tolerable limits set by different organizations. This can be more hazardous to individuals who are more sensitive and prone to toxic effects of such highly carcinogenic food contaminants. Therefore, this situation clearly demands wider national or international programs for the control of aflatoxin contamination in Sorghum. In conclusion, aflatoxin control programs should focus on addressing all the factors that contribute to fungal growth across the value chain (i.e., pre-harvest to household practices). Hence, the concepts like HACCP “Farm to Fork” should be applied.
Acknowledgment
We are grateful to Center for Food Science and Nutrition of Addis Ababa University, Food, Medicine and Healthcare Administration and Control Authority of Ethiopia (EFMHACA) and Micronutrient Initiatives for funding this research.

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Figures


Figure 1: About 1kg of Sorghum samples were collected from the aboveground storage and underground pit storage system from each selected role model farmer.



Figure 2: HPLC Chromatogram of 1 ppb standard mixture of four aflatoxin.



Figure 3: Chromatogram of blank sample (A), standard calibration curve (B) and naturally contaminated fungal sample (peanut) (C), Sorghum sample (D), (elution order AFG2, AFG1, AFB2 and AFB1).



Figure 4: Morphological (phenotypic) feature of Aspergillus Species, (Photo by-Author).

Tables
Characteristics Response Frequency Percent (%)
Age 19-38 14 46.7
38-55 13 33.33
> 55 3 10
Education Illiterate 19 63.33
Primary school 9 30
Secondary school 2 6.7
Sex Female 4 13.3
Male 26 86.7
Yes Yes 14 46.7
No 16 53.3
Color Black 8 26.6
White 6 20
Problem Yes 0 0
No 30 100
Critical problem Insect 15 50
Rodent 10 33.33
Mould 5 16.7
Other 0 0
Measure taken to control Insect sides 25 75
Fire wood smoke 5 25
Drying technology Sun drying 30 100
Other technology 0 0
Cleaning and sanitation Yes 30 100
No 0 0
Storage Location Field 15 50
Inside house 5 16.7
Courtyard 10 33.3
Mixing Yes 0 0
No 30 100
Storage system Above ground (Gotera) 9 30
Underground (pit) 10 33.3
Both (Pit and Gotera) 11 36.7
Duration of storage time periods Six month 11 36.7
One year 15 50
Two year and above 4 13.3
More than four year All 100
Table 1: Socio demographic data and farmers’ awareness about mould contamination.

Storage system Sample Code B1 (µg/kg) B2 (µg/kg) G2 (µg/kg) G1 (µg/kg) Total Afs (µg/kg) Moisture content (%)
< 12 month stored Sorghum
Aboveground storage S01 28.82 11.17 3.1 16.62 75.57 11.4
S02 3.96 4.29 50.2 111.25 16.59 10.4
S03 31.76 2.15 7.65 24.94 60.41 9.73
S04 71.52 22.81 26.25 139.64 176.49 9.53
S05 69.79 27.12 41.96 113.36 183.16 10.2
Underground pit storage S06 82.34 14.77 8.44 16.98 177.33 11.3
S07 31.49 20.38 7.46 56.39 100.97 10.33
S08 28.64 18.55 26.16 31.11 91.85 10.43
S09 28.8 19.11 23.07 50.31 93.4 8.77
SO10 5.3 16.54 8.47 17.24 46.48 11.53
1-2 year stored Sorghum
Aboveground storage SO11 21.75 2.76 21.02 101.79 44.27 11.86
SO12 46.16 3.48 15.73 68.3 88.61 11.43
SO13 88.9 24.43 51.01 ND 210.53 10.78
SO14 33.17 52.05 26.28 18.96 175.16 9.82
SO15 31.06 40.56 4.65 23.87 145.61 9.33
Underground pit storage SO16 8.86 35.01 12.65 116.13 94.28 11.05
SO17 3.95 2.01 2.04 80.7 11.44 12.53
SO18 64.68 17.33 18.74 42.3 152.19 9.47
SO19 69.69 3.06 52.84 61.82 128.86 10.8
SO20 153.72 33.45 37.89 ND 344.26 9.4
≥ 2 year stored Sorghum
Aboveground storage SO21 26.27 6.58 19.4 12.92 65.17 9.4
SO22 51.28 1.17 32.91 114.1 199.46 9.4
SO23 12.95 30.26 16.95 78.17 138.33 8.9
SO24 105.96 91.82 24.61 25.53 247.92 9.19
SO25 36.75 2.85 72.65 109.2 221.45 11.13
Underground pit storage SO26 15.57 6.89 12.58 129.26 164.3 9.6
SO27 9.6 21.35 15.62 51.79 98.36 8.8
SO28 4.59 10.08 3.22 9.87 17.68 11.75
SO29 7.25 ND 4.28 9.94 21.47 10.72
SO30 ND ND ND ND ND 9.4
% of contamination level 96.66 93.33 96.66 90 ----- -----
Table 2: Level of aflatoxin content in Sorghum sample stored for different period and storage system.
ND -means not detected.
Aflatoxin content (µg/kg) Aflatoxin content (µg/kg)
AFG2 AFG1 AFB2 AFB1 Total aflatoxin
< 12 months 20.28a 57.78a 15.69a 38.24ab 64.31a
1-2 years 24.28a 51.39a 17.09a 52.19a 59.31a
> 2 years 22.22a 54.08a 21.42a 27.02b 81.64a
Table 3: Effect of storage period (< 12 month, 1-2 year, and ≤ 2 year) on the level of aflatoxin (µg/kg) in Sorghum.
Means within the same column followed by the same letter superscript indicate no significant difference (p > 0.05).

Storage type Average aflatoxin content (µg/kg)
AFG2 AFG1 AFB2 AFB1
Above ground 49.67a 63.91a 21.57a 44.1a
Underground pit 30.05a 44.92a 14.56a 34.3a
Table 4: Effect of storage system (above ground and underground pit) on the level of aflatoxin.
Means within the same column followed by the same letter superscript indicate no significant difference (p > 0.05).

Aspergillus species Number of sample % of Aspergillus species found
Aspergillus flavus 17 56.66
Aspergillus parasiticus 5 16.66
Aspergillus niger 7 23.33
Total 29 96.65
Table 5: Aspergillus Species identified from Sorghum sample using PDA agar.

Duration of Storage period Aspergillus species(%)
Aspergillus flavus Aspergillus parasiticus Aspergillus niger
< 12 months 13.33 6.67 10
1-2year 23.33 6.67 3.33
≥ 2 year 20 3.33 10
Table 6: The occurrence of Aspergillus Species in different storage periods.

Citation: weledesemayat GT, Gezmu TB, Woldegiorgis AZ, Gemede HF (2016) Study on Aspergillus Species and Aflatoxin Levels in Sorghum (Sorghum bicolor L.) Stored for Different Period and Storage System in Kewet Districts, Northern Shewa, Ethiopia. J Food Sci Nutr 3: 010.