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Aflatoxins are toxic metabolites produced by certain fungi in/on foods and feeds. They are probably the best known and intensively found mycotoxins associated with various diseases, such as aflatoxicosis in livestock, hepatocellular carcinoma (HCC) in human and domestic animals throughout the world. The incidence of these toxins in agricultural commodities has a negative impact on the economies of the affected regions, where harvest and post-harvest techniques for the prevention of mold growth are seldom practiced. Keeping in view the potential toxicity of aflatoxins the current project has been designed for the qualitative and quantitative estimation of aflatoxins by HPLC coupled with fluorescent detector in processed foods available in market mostly taken by adults and infants. The data thus obtained will be analyzed using analysis of variance (ANOVA).
Food is one of the most intimate and important components of our chemical environments. Whether we accept or reject food depending mainly on its flavour. Recently, the concept of good food has changed considerably. Good food is not only expected to look fresh and tasty but it must be free from all contaminants. The presence of contaminant residues in food products is a clear violation of this simple reasonable expectation.
Mycotoxins are natural food and feed contaminants, mainly produced by moulds of genera Aspergillus, Penicillum and Fusarium. The number of mycotoxins known to exert toxic effect on human and animal health is constantly increasing as well as the legislative provisions taken to control their presence in food and feed. Extensively considered mycotoxins are aflatoxins (AFs), ochratoxin A (OTA), Fusarium toxins and patulin (Miraglia and Brera, 2000). Epidemiological, clinical, and experimental studies reveal that exposure to AFB1 may develope liver cancer, especially persons having hepatitis B antigens. World Health Organization (WHO) classifies AFB1 as a human carcinogen (Chen et al., 2005). So, the US Food and Drug Administration (FDA) has established specific guidelines for acceptable levels, less than 20 and less than 100 ppb for humans and animals, respectively (Ambrose et al., 2009). The aflatoxin AFM1 is major metabolite of AFB1, which can be detected in the blood, urine, milk, and internal organs of animals which ingest AFB1 containing feed (Chen et al. 2005).
Consumption of aflatoxtion-contaminated foods has been associated with several cases of human poisoning, or aflatoxtion, sometimes resulting in death (Bathnagar and Garcia, 2001).
Despite lots of studies on aflatoxins in corn, maize, chillies and peanuts only a few are concerned with the amount of aflatoxins in processed food products that are mostly part of our daily diet. A variety of the processed food products containing large amounts of corn, maize, chillies, peanuts or other cereals are available in the market and is taken by adults and infants as a part of their diet.
The aim of this study is, therefore to provide information on AF levels in processed foods marketed in different regions of Pakistan. The results of this study will contribute to the evaluation of processed products, consumed by people of Pakistan from the point of view of food safety.
AIMS AND OBJECTIVES:
The present study has been designed with the following objectives.
To investigate Aflatoxins contents in different commercially available processed food products.
Comparison with reported studies.
REVIEW OF LITERATURE
Edmond et al. (2002) studied the most frequent toxigenic fungi in Europe which are Aspergillus, Penicillium and Fusarium species. They produce aflatoxin B1 transformed into aflatoxin M1 found in the milk, as well as Ochratoxins and Zearalenone, Fumonisin B1, T-2 toxin, HT-2 toxin and deoxynivalenol (vomitoxin), which are of increasing concern in human health. These mycotoxins are under continuous survey in Europe, but the regulatory aspects still need to be set up and/or harmonized at European level. They are found in foodstuffs and are not destroyed by normal industrial processing or cooking since they are heat-stable. Some of their metabolites are still toxic and may be involved in human diseases. Their toxic effects (liver, kidney and hematopoetic toxicity, immune toxicity, reproduction toxicity, foetal toxicity and teratogenicity, and mainly carcinogenicity) are mostly known in experimental models, the extrapolation to humans being always inaccurate. The inaccuracy of extrapolation to humans may be explained by the lack of adequate food consumption data, lack of knowledge about relative health risks associated with specifically proposed limits and by the possibility of synergism with other mycotoxins present in the same food commodities. Other pathological causes are viral hepatitis, immune or hormonal deficiencies or organ dysfunction. The acceptable daily intake limits are mostly based on animal data and may be too high, due to the differences in the sensitivity of different animal species.
Erdogan (2004) investigated aflatoxins contamination using TLC in 44 red-scaled pepper and 26 red powder pepper samples obtained from various spice retailers in Erzurum, and 20 isot (red pepper produced in Sanliurfa). Aflatoxin (B+G) was found in 8 red pepper samples (18.2%), and 3 red powder peppers (10.7%), and 1 isot sample (5%). The aflatoxin amount ranged from 1.1 to 97.5 ppb in all samples investigated. The highest amount of aflatoxin was found in red-scaled pepper. Species of Aspergillus, Penicillium and Rhizopus were generally contaminants of all samples.
Chen et al. (2005) studied that animal feed is contaminated with aflatoxin and even pasteurized milk contains the AFM1. To confirm its presence, the solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) were used. In this study 200 mL of milk was extracted using a C18 disk at a flow rate of 100 mL/min. The AFM1 was quantified by HPLC tandem mass spectrometry with negative electrospray ionization. The efficacy of Mycosep multifunctional cleanup columns (MFC) was compared with that of immunoaffinity columns (IAC). Average recovery and detection limits of whole milk and low-fat milk cleaned up by IAC were significantly more than obtained with the MFC (78-87% and 0.59-0.66 ng/L, respectively). The new procedure improves extraction speed, sensitivity, and specificity.
Zinedine et al. (2005) studied sixty samples of cereals (20 of corn, 20 of barley, and 20 of wheat) and 55 samples of spices (14 of paprika, 12 of ginger, 14 of cumin, and 15 of pepper) purchased from popular markets of Rabat and Sale in Morocco, for mycotoxins. Cereals samples were all analyzed for ochratoxin A (OTA). The average levels of contamination were 1.08, 0.42 and 0.17 µg/kg for corn, wheat and barley respectively. Samples of corn were also analyzed for zearalenone (ZEA) and fumonisin B1 (FB1), the average contaminations were 14 and 1930 µg/kg, respectively. Co-occurrence of OTA, FB1, and ZEA was also checked. Spices samples were analyzed only for aflatoxins (AFs) and the average contaminations found for aflatoxin B1 (AFB1) were 0.09, 0.63, 2.88 and 0.03 µg/kg for black pepper, ginger, red paprika and cumin, respectively. It was found that higher level of contamination was found in red paprika (9.68µg/kg).
Giorni et al. (2007) studied on Aspergillus section Flavi and isolated from maize in northern Italy. Significant problems arose with colonization and contamination of maize destined for animal feed with Aspergillus section Flavi and aflatoxins (AFs). This resulted in milk and derived products being contaminated with AFM1 at levels above the legislative limit. There was little knowledge and experience of this problem in Italy. The objectives of this research were thus to study the populations of Aspergillus section Flavi in six northern Italian regions and obtain information on the relative role of the key species, ability to produce sclerotia, production of the main toxic secondary metabolites, aflatoxins and cyclopiazonic acid, and tolerance of key environmental parameters. A total of 70 strains were isolated and they included the toxigenic species A. flavus and A. parasiticus. A. flavus was dominant in the populations studied, representing 93% of the strains. Seventy percent of strains of Aspergillus section Flavi produced AFs, with 50% of strains also producing cyclopiazonic acid. Sixty-two percent of A. flavus strains and 80% of A. parasiticus were able to produce sclerotia at 30 °C. Using 5/2 agar, only 1 strain developed S sclerotia and 19 L sclerotia. With regard to ecological studies, growth of Aspergillus section Flavi was optimal at between 25 and 30 °C, while AFB1 production was optimal at 25 °C. Regarding water availability (water activity, aw), 0.99 aw was optimal for both growth and AFs production, while the only aflatoxin produced in the driest condition tested (0.83 aw) was AFB1. This information will be very useful in identifying regions at risk in northern Italy by linking climatic regional information to levels of fungal contamination present and potential for aflatoxin production in maize destined for animal feed. This would be beneficial as part of a prevention strategy for minimising AFs in this product.
Ozdemir et al. (2007) studied that milk and dairy products contain aflatoxin M1 as a result by the eating of feedstuffs tainted with aflatoxin B1 by dairy goats. This study was carried out to find out the levels of AFM1 in goat milk usually consumed in the city of Kilis. For this purpose, one hundred and ten samples of milk were collected at random. The analysis was carried out by ELISA to know the level of AFM1. AFM1 was not present in 17 samples (15.46%), whereas 93 samples (84.54%) had AFM1 at different levels. The range of AFM1 was between 5.16 and 116.78 ng/l in 70 of the 110 samples. AFM1 level in 7 (6.36%) samples was more than the maximum acceptable limit (50 ng/l) accepted by the Turkish Food Codex. Ozdemir determined that AFM1 84.54% of the goat milk samples was taken by the people in the Kilis province. Moreover 6.36% of samples had AFM1 at risky levels for human health.
Fu et al. (2008) determined aflatoxins in corn and peanuts collected from China. A rapid and simple method using ultra-high-pressure liquid chromatography with UV detection for the determination of aflatoxins B1, B2, G1 and G2 in corn and peanuts has been developed. In this method, aflatoxins were extracted with a mixture of acetonitrile and water (86:14) and then purified by solid-phase clean-up with a MycoSep#226 AflaZon+ column. The toxins were determined by UPLC-UV without derivatizing aflatoxins in real samples, which has not been used in other studies. The mean recoveries of aflatoxins from non-infected peanut and corn samples spiked with aflatoxins B1, B2, G1 and G2 at concentrations from 0.22 to 5μg/kg were between 83.4% and 94.7%. The detection limits (S/N = 3) for B1, B2, G1 and G2 were 0.32, 0.19, 0.32 and 0.19 μg/kg, and the corresponding quantification limits (S/N = 10) were 1.07, 0.63, 1.07 and 0.63 μg/kg, respectively. We also applied this method on real samples. Among 16 peanut samples, 2 (12.5% incidence) were contaminated with aflatoxin; among 18 corn samples, 4 (22% incidence) were contaminated. The proposed method is rapid, simple and accurate for monitoring aflatoxins in corn and peanuts
Juan et al. (2008) studied the occurrence of aflatoxins (AFs) in dried fruits and nuts available in Morocco. One hundred samples of dried fruits and nuts, purchased from retail shops and local markets from January to October 2006, were analyzed for AFs content by immunoaffinity (IAC) clean-up with liquid chromatography using fluorescence detection. Results showed that the incidences of total aflatoxins (AFT) and aflatoxin B1 (AFB1) in peanut, dried raisins, dried figs, walnut, and pistachio were 5%, 20%, 30%, 30% and 45% and 5%, 20%, 5%, 30% and 45%, respectively. The highest contamination levels of AFB1 were found in one walnut sample (2500 µg/kg) and one pistachio sample (1430 µg/kg).The samples of pistachio, walnut and dried raisins, 5%, 20% and 20% respectively exceeded the maximum tolerable limit (2µg/kg) set for AFB1 by EU regulations. While 15% of dried figs samples showed the maximum limit (4µg/kg) set by EU regulations for AFT.
Oliveira et al. (2009) studied peanut products traded in the Northeast region of Sao Paulo, Brazil to determine aflatoxin levels. For this purpose, 240 samples of peanut products were collected. The analysis of samples was carried out for aflatoxins (AF) B1, B2, G1 and G2 by high performance liquid chromatography. 44.2% samples positive for AF at levels of 0.5 to 103.8 μg·kg-1. The daily intake (PDIM) of aflatoxins from peanut products in the Northeast region of Sao Paulo was estimated to be 0.23 ng kg b.w. day-1. The PDIM value was relatively low, but when we consider the highly susceptible consumers. For example, it should be noticed that children are potentially exposed to aflatoxins, since they consume peanut candies in large quantity, and these products had the highest number of samples positive for AFB1.
Viktoria et al. (2010) analysed eighty-one rice samples from different markets in Vienna for their aflatoxin content. The samples were extracted using methanol in water (80/20 v/v) followed by immunoaffinity clean up. The determination was carried out by HPLC-FLD coupled to a Kobracell. Different samples including basmati rice, whole grain rice, long grain rice, short grain rice as well as puffed rice were investigated. Moreover, conventionally and organically produced rice were compared. The results revealed that 24 out of 81 samples contained detectable amounts of aflatoxins. Aflatoxin B1 could be quantified in 15 samples and aflatoxin B2 in one sample. The contamination range was noted to be between 0.45 μg kg−1 and 9.86 μg kg−1 for aflatoxin B1 and 1.5 μg kg−1 for aflatoxin B2. Aflatoxins G1 and G2 were not detected in any sample. Three samples exceeded the maximum levels set in the European Union; having AFB1 concentrations of 2.16, 2.85 and 9.86 μg kg−1. In the three organic produced rice samples only traces of aflatoxins were found
Beltran et al. (2011) developed method for the ultrasensitive and selective determination of various regulated mycotoxins ( aflatoxins G1, G2, B1, B2, M1, and ochratoxin A) in baby food commodities and milk, using ultra high pressure liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS/MS). The high sensitivity required for these compounds made necessary the application of a pre-concentration step based on solid phase extraction with immunoaffinity columns, after sample extraction with acetonitrile:water (80:20). Thanks to the fast high-resolution of UHPLC and the enhanced selectivity obtained with the triple quadrupole mass analyser in SRM mode, the chromatographic separation was achieved in only 4 min. Validation of the method was carried out in four different matrices (cereals infant formula, powdered milk for babies, milk with cereals for infants, and raw milk) by recovery experiments, using samples spiked at 0.025 and 0.1 μg kg−1 in quintuplicate. Satisfactory recoveries, between 80% and 110%, with RSDs lower than 15%, were obtained in all food matrices tested.
MATERIAL AND METHODS
COLLECTION OF SAMPLES:
The samples of processed food products will be collected from local market of Pakistan.
EXTRACTION OF AFLATOXINS
Aflatoxins will be extracted by shaking for 60 minutes using 100ml of mixture of acetonitrile-water (84 : 16v/v). Mycosep # 226 Aflazon + Column will be used for clean up.
Aflatoxins will be analyzed by HPLC coupled with Fluorescent detector (Fu et al., 2008)
The data thus obtained will be analyzed using analysis of variance (ANOVA). (Steel et al. 1996).