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QUANTIFICATION OF AFLATOXINS IN MAIZE SAMPLES COLLECTED FROM VARIOUS PARTS OF THE PUNJAB, PAKISTAN.

Byline: M. Manzoor, Z. Farooq, S. Iqbal, H. Mukhtar and M. Nawaz

ABSTRACT

Maize, one of the main cash crops of Pakistan is cultivated under various ecological conditions. Ma ize quality deteriorates due to many factors like rainy season, adverse temperature, and traditional practices of harvesting and insufficient storage facilities that stimulate fungal infection (aflatoxins). The present study was designed to quantify the aflatoxins (AFB1, AFB2, AFG1 and AFG2) in maize grains collected from various districts of the Punjab. Seventy two samples of maize grains were collected from 12 districts of Punjab. Toxins were extracted by MycoSep columns (product code 226) and analyzed by High-Performance Liquid Chromatography (HPLC) for the quantification of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). The study showed that AFB1 and AFB2 are the primary contaminants. Out of the analyzed samples, 77.7% of yellow maize grains were contaminated with AFB1, 61.1% AFB2 and 38.8% with high levels of AFG2.

The positive samples showed higher than the legal limits as set by the European Union (EU), United States Food and Drug Administration (USFDA) Department and Pakistan Standards and Quality Control Authority (PSQCA). Study further found out that aflatoxin production was mainly caused due to inappropriate storage conditions. Variation in agro-ecological conditions and inappropriate storage cause aflatoxin contamination in maize grains which can be controlled by improvement in the storage conditions and use of proper detoxifying technologies.

Keywords: Aflatoxins, Maize grains, HPLC, Food safety.

INTRODUCTION

Maize (Zea mays) is the vital cereal crop of Pakistan after wheat and rice and generally used as a source of food and feed. The average production of maize crop in Pakistan is 2850 kg ha- 1 (Tariq and Iqbal 2010) out of which about 60% is grown by irrigation and 36% in rain fed areas (Abuzar et al. 2011). It has big contribution as a food in the form of cob, parched, boiled, fried, roasted, ground and fermented maize used for the manufacturing of breads, porridges, cakes, alcoholic beverages, corn protein, corn sugar, corn oil and syrup (Akmal et al. 2013; Ahmad et al. 2007). It provides 15 to 56% of the total calories in the diets of people where protein from animal source is deficient and costly and not available to majority of the population (Prasanna et al. 2001). Cereal grains especially maize grains get easily contaminated by fungus in the field and during storage condition. Usually these fungi are capable of producing mycotoxins which are stable compounds.

These mycotoxins are not destroyed during processing of food, which may lead to contamination of cereal based foods. Maize crops are easily attacked by the mycotoxins producing fungi in field as well as during poor storage conditions. Generally, these mycotoxins are produced by the filamentous fungi more specifically, Aspergillus, Fusarium and Penicillium. Contamination of maize with Aspergillus flavus and Aspergillus parasiticus and production of aflatoxins during storage is one of the serious problems all over the world (Kaaya and Kyamuhangire. 2006). These two types of fungi (Aspergillus flavus and Aspergillus parasiticus) are responsible for spoilage of stored grains (Paster. 1995). Long-term storage under un-hygienic, hot and humid condition is responsible for fungal growth (Williams and McDonald. 1983; Hell et al. 2000). The main aflatoxins naturally occurring in foods are designated as AFB1, AFB2, AFG1, and AFG2. Aflatoxin B1 is the most toxic and usually predominant (FAO. 1997).

These fungi survive in a broad range of environments and can be found in soil, in plant and animal remains, and in grains and seeds such as maize, peanuts, and tree nuts (Pitt. 2000). Aspergillus flavus is the main fungus that causes pre harvest aflatoxins contamination in field crops. Aflatoxins have been identified in the early 1960s. They are secondary metabolites of Aspergillus flavus and Aspergillus parasiticus and target a variety of staple foods, especially maize and groundnuts, in low income countries (Wild and Gong. 2010). Williams et al. (2004) have reported a probability that 4.5 billion of the world's population is exposed to aflatoxins. The Food and Agriculture Organization of the United Nations (FAO) estimated that up to 25% of the world's cereal grains are contaminated by aflatoxins (Liu et al. 2006).

Aflatoxins occur usually in tropical regions with high temperature and humidity and they mount up to post-harvest stage when food commodities are stored under conditions that promote fungal growth. Tropical conditions such as high temperatures and moisture, monsoons, unseasonal rains during harvest and flash floods lead to fungal proliferation and production of mycotoxins. Poor harvesting techniques, inappropriate storage and less optimal conditions during transportation, marketing and processing can also contribute to fungal growth and increase the risk of aflatoxins production (Wagacha and Muthomi. 2008). The variations in climatic conditions as well as the food cultivation and production chains are characteristic in most parts of Pakistan. Consequently, the threat of aflatoxin contamination of foods and feeds resulting in human and livestock poisoning is real and of major concern (Wagacha and Muthomi. 2008).

Aflatoxins have carcinogenic and hepatotoxic actions, depending upon the extent and level of exposure. Chronic exposure of aflatoxins in dietary practices is a major risk factor for hepatocellular carcinoma, predominantly in the areas where hepatitis B virus infection is endemic. Ingestion of higher quantity of aflatoxins can produce acute aflatoxicosis, which is manifested as hepatotoxicity and in severe cases, fulminant liver failure (Fung and Clark 2004). Contamination in varieties of foods by these naturally occurring toxins is a great concern in rural communities of developing countries (Lewis et al. 2005). Aflatoxin causes illness and even death when consumed in large quantities. Low-level, chronic exposure is carcinogenic and has been linked to growth retardation in children. The present study was designed to evaluate the occurrence of aflatoxins in maize, grown in different regions of Punjab, Pakistan and stored under local storage conditions.

MATERIALS AND METHODS

Twelve districts from the three regions of Punjab province were selected for sampling purpose according to climatic conditions. 72 samples of yellow and white variety of stored maize grains were procured from the whole sale markets of Southern Punjab (Multan, Vehari, Bahawalpur, Rahim Yar Khan) Central Punjab (Lahore, Faisalabad, Sargodha, Gojar Khan) and Northern Punjab (Rawalpindi, Chakwal, Khushab, Attock) of Pakistan. Samples of maize grains (yellow maize, white maize) were randomly collected from each district in triplicates and stored at 4AdegC in polythene bags with proper codes and identification marks for analysis.

Chemicals and Regents: Standard solutions of Aflatoxins (AFB1, AFB2, AFG1 and AFG2) were prepared in 50 ug mL-1 in acetonitrile. Aflatoxin standards were purchased from Sigma-Aldrich (St. Louis, Mo., USA). MycoSepA(r) column 226 (AflaZone) was purchased from Romer Labs (Union, Mo., USA). HPLC grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany) and Trifluoroacetic Acid (TFA) was obtained from Sigma-Aldrich (St. Louis, Mo., USA).

Extraction and purification: Extraction and purification of samples were carried out by using a slightly modified method of Iqbal et al. (2012). The maize samples were ground to uniform consistency by laboratory grinding mill (ZM 200. Germany). A portion from each homogenous ground samples (25g) was used for extraction with 100 ml of acetonitrile/water (80:20 v/v) and 5 gram of sodium chloride (NaCl) by shaking in water bath for 60 min at 50 rpm at room temperature in 250 ml Erlenmeyer flask fitted with a stopper. The solutions were filtered through Whatman No.5 papers. Then 9 ml of the filtrates were acidified with 70ul acetic acid and the mixture was transferred to MycoSep columns (product code 226) and passed through at a flow rate of 2 ml/min. The aflatoxins were passed through the column and 2 ml portion of each eluate was taken and evaporated to dryness by nitrogen evaporator in a centrifuge glass tube.

After drying the mixture 200ul n-hexane was added and vortex mixed the sample for 1-2 min. Then 50ul Trifluoroacetic Acid (TFA) was added and mixed by vortex mixer. Then 1.95 ml of water and acetonitrile (1:0.95) were added and mixed again. The 1 ml of sample filtrate obtained from syringe filter was collected in HPLC vials. A 20ul portion of each sample was subjected to HPLC analysis.

Mobile phase: For aflatoxins analysis, mobile phase of acetonitrile/methanol/water (20:20:60 v/v/v) was applied which was degassed by sonication. The HPLC (Shimadzu, Kyoto, Japan) was fitted with a Supelco C18 Column (Discovery HS) with a fluorescence detector (RF-530). Aflatoxins were identified at excitation and emission wavelengths of 360 nm and 440 nm, respectively. The flow rate was 1 ml/min and the column was maintained at 40AdegC. The injection volume was 20ul. Resultant Chromatographs showed quality resolutions of peaks. For the validation of this method, calibration curves were drawn using a series of calibration solutions of aflatoxins in methanol. Standard solutions were chromatographed in duplicate.

Statistical Analysis: Data was analyzed by using SPSS software version 16. Independent sample T-test was applied to analyze data. P-value 20 ug/kg. A total of 61.11% (22) yellow maize grains were contaminated with aflatoxin B2, out of which 36.11% (13) were at higher limits then acceptable levels and 25% (09) showed up to the acceptable levels (Table 3). Ghiasian et al. (2011) reported the incidence of AFB1 80% and AFB2 60% recovered from maize grain in Mazandaran which was also similar to those reported by Yazadanpanah et al. (2001). Pearson et al. (2011) also observed almost same type of results in his study.

The highest level of AFB1 was 134 ug/kg observed in the samples of Chakwal and Rawalpindi and the highest of AFB2 was 33.13 ug/kg from the samples of Chakwal. Ghiasian et al. (2011) studied that in Iran maize grains collected from different districts AFB1 and AFB2 were in the range from not detected to 276.3ug/kg and not detected to 30.4ug/kg respectively. 33.3% of the yellow maize grains showed the results of higher levels of both B1 and B2. Lutfullah and Hussain (2012) observed 40% of maize grains were contaminated with high level of AFB1 and AFB2 in Khyber Pakhtun Khan (KPK), Pakistan. AFG1 was not detected in the present study. Yazadanpanah et al. (2001) reported no contamination of AFG1 and AFG2 from maize grains of high incidence area of Iran. Out of total 36 samples of yellow maize grains, 14 (38.8%) samples were found to be contaminated with high levels of AFG2 (Table No. 3). Samples of yellow maize collected from Lahore were found to have highest levels (20 ug/kg) of AFG2.

Results of the presence of AFB1 and AFG2 were further confirmed through HPLC chromatogram (Figure No. 3). Ghiasian et al. (2011) observed two samples of maize grains from Kermanshah province, contained detectable concentrations ranged from not detected to 9.1 ug/kg of AFG1and AFG2 in Iran. Out of 36 samples of white maize grains 55.5% (20) were contaminated with aflatoxins B1, out of which 19.4% (07) were at higher limits than acceptable levels and 36.1% (13) of white maize grains showed under the acceptable levels. 36.11% (13) out of total samples of white maize grains were contaminated with aflatoxins AFB2, out of which 16.6% (06) were at higher limits than acceptable levels and 5.5% (02) of white maize grains showed up to the acceptable levels and 19.4% (07) were contaminated by AFG2 and 16.6% (06) showed upper limits (Table No. 3).

Lewis et al. (2005) observed similar results of aflatoxins contamination in Eastern and Central Kenya during an outbreak of Acute Aflatoxicosis where high proportion (55%) of maize samples from the local markets in all four study districts had aflatoxin levels greater than the regulatory standards. 16.7% of the white maize grains showed the results of higher levels of both AFB1 and AFB2. Hell et al. (2003) observed similar results of aflatoxins contaminations of the maize grains which were improperly stored more than six months. Hell et al. (2003) observed that aflatoxins were produced by the unhygienic and dissimilar conditions of storage of maize grains for prolong time period. Overall, aflatoxins were detected with variable levels of infestations in 28 and 22 samples of yellow and white maize respectively, (Table No. 4). Data related to aflatoxins quantification were statistically analyzed by using independent t-test (Shah et al. 2010).

For aflatoxins quantification AFB1 in two different varieties of maize (yellow and white), the independent sample "t" test was applied to confirm the mean difference between samples. Independent "t" test was also applied to check the significance levels of total aflatoxins in maize grains. P < 0.05 showed the significant results (Table 5).

Conclusion: The discussion concluded that the maize grains of yellow and white variety contains good nutritional value but the main concern is the contamination of AFB1, AFB2, AFG1 and AFG2 presence in the maize grains. Variation in agro-ecological conditions and poor storage condition directly influence the aflatoxins contamination so there is need to control it and properly monitor the storage conditions and storage time period and use of proper detoxifying technologies to prevent the maize crop from the fungal attack which could help in reduction of aflatoxins contamination.

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Publication:Journal of Animal and Plant Sciences
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Date:Dec 31, 2018
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