Occurrence and health hazard status of aflatoxin in human food and animal feed of wheat from Pakistan : A review paper

Aflatoxins (Afs) are the most important and naturally occurring health hazard mycotoxin which is carcinogenic and mutagenic found mainly in cereal grain food and feeds. Disease caused by ingestion of aflatoxin through food and feed are called aflatoxicosis. They cause hepatocellular carcinoma (HCC), or liver cancer mainly in humans and some animals. Considering its significant impact on human, animals and poultry health, the main aim of the present review is to provide a comprehensive overview on Afs affecting human food and animal feed of wheat from Pakistan. Afs are principal toxic secondary metabolites produced by toxigenic fungal plants (viz., Aspergillus flavus and A. parasiticus) on host crops either before harvest or post-harvest mainly during grain storage period. Research focuses that there are two main external factors e.g. humidity and temperature which affect the incidence of Afs producing Apergillus species. Warm and humid climatic condition prevails in most of the agricultural land of Pakistan. Humidity encourages Aspergillus to attack the wheat grains. Favorable condition for its growth is high temperature and high moisture content (>7%). The optimum temperature for Afs production is recorded as 30C. In Pakistan very little information regarding Afs status of cereal grains is available on toxicological data in general and wheat grains in particular. But few researchers highlighted the presence of Afs in wheat food and feeds. Based on their limited published work I found a mixed trend regarding the status of Afs in wheat food and feeds. Some analyzed samples were found above, and some were below the health hazard limit or permissible limit as proposed by European Union (EU) and FDA (US Food Drug Administration). Therefore, more research is needed to be carried out on the potential threats to human beings, animals and poultry after consumption of Afs contaminated wheat food and feeds.


Review Article
Occurrence and health hazard status of aflatoxin in human food and animal feed of wheat from Pakistan: A review paper Introduction Wheat (Triticum aestivum L.) is an important food crop grown throughout the globe.It is the third most produced cereal grain after maize and rice crops.It is a staple food of billions of people.China, India, USA, France, Russia, Australia, Canada, Pakistan, Germany and Turkey are the top ten leading countries producing about 125.6; 94.9; 61.8; 40.3; 37.7; 29.9; 27.0; 23.5; 22.4 and 20.1 million metric tones, respectively [1].In Pakistan, wheat is the topmost cereal crop cultivated on 8649.8 thousand hectares by producing 25979.4 [11].Among various mycotoxins, Af were assumed significant due to their deleterious effects on human beings, poultry and livestock.Approximately, 5-10% of agricultural commodities world wide are spoiled by fungi, to the extent that crops can not be consumed by human, animal and birds (poultry).This situation is more serious particularly in developing countries like Pakistan, where there are poor agricultural, harvesting, storage, transportation and marketing conditions, which ultimately lead to fungal growth and increase the risk of mycotoxin in general and aflatoxin in particular [12].According to reliable estimate, mycotoxin affect 1/4 th of the world's food crop which includes many basic food and feed stuffs of human and animals [13].Studies also showed that food contamination by Af may develop as a result of respective fungal attack before or during wheat harvest, or during storage [14].

Fungus Producing Aflatoxins (Afs)
Afs are mostly produced by filamentous fungi known as Aspergillus.This genus is distributed world wide and contains over 180 species.It is one of the most cosmopolitan and abundant of all groups of fungi, and also one of the most studied fungal groups [15,16,17].They can colonize and contaminate grain before harvest or during storage.This is also a type of phytopathogenic organisms that infect crops and fruits

Types of Aflatoxins
Aflatoxins (Afs) are the most potent carcinogens both in human and animal populations.In fact, the toxin-producing fungus was identified as Aspergillus flavus (1961) and the toxin was given the name Aflatoxin by virtue of its origin (A.flavis ------> Afla).There are more than 20 different types of aflatoxins (Af), but the most important and naturally occurring major members of Af in food and food stuffs are AfB1, AfB2, AfG1, AfG2, AfM1, AfM2.The last two are produced in milk and milk products.While among all these, AfB1 is the most toxic, abundantly occurring and well studied one is produced by both Aspergillus flavus and Aspergillus parasiticus [22,23].Letters 'B' and 'G' refer to its blue and green fluorescence colors produced by these compounds when exposed under UV light.

History of Aflatoxins (Afs)
The Afs represent a group of secondary fungal metabolites which were first discovered in Europe as contaminants of certain lots of animal feeds in year 1960.These toxic contaminants have a high order of acute toxicity both to human and many other animal species viz., chickens, swine, cattles, horses etc and also have shown to possess potent carcinogenic properties in several animals (including human).Their discovery in agriculture based animal feed and the subsequent demonstrations of various biological effects resulting from ingestion of contaminated diets by domesticated animals and birds have emphasized the potential public hazard which might arise from contamination of the food supply by mycotoxin in general and Af in particular [24].The first report of the toxicity syndrome in term of mortality of domestic animals were recorded in young turkeys [25], followed by the same sort of incidents in ducklings, and chickens [26], as well as in swines and calves [27, 28].

Chemistry of Aflatoxins (Afs)
They are secondary metabolites produced by the moulds on food and feed stuffs.Though there are more than 20 naturally occurring Afs, but the most important one are AfB1, AfB2, AfG1 and AfG2.Af B2 and AfG2 are the dihydro derivatives of the parent compounds (Fig. 1).They all exist as colorless to pale-yellow crystals at room temperature [29].They are oxygenated hydrocarbons and are slightly soluble in water, soluble in organic solvents like methanol, acetone, and chloroform, and insoluble in non-polar solvents.Dichloromethane has good solubility for aflatoxins and has been used in analytical procedures to extract and purify aflatoxin for their assay.Aflatoxins are relatively unstable in light and air, particularly in polar solvents or when exposed to oxidizing agents, ultraviolet light, or solutions with a pH below 3 or above 10.There are various isoforms of Af viz.B1, B2, G1, G1.Afs decompose at their melting points, which are between 268-289°C (B1 & B2) and 237-246°C (G1 & G2), but are not destroyed under normal cooking conditions (Table 1).They can be completely destroyed by autoclaving in the presence of ammonia or by treatment with bleach [30].

b) Chronic aflatoxicosis:
It is produced when there is a low to moderate level of Afs are ingested through contaminated food and feed.The effects are usually subclinical and difficult to recognize.Few of the common symptoms are impaired food conversion and slow rates of growth with or without having an overt Af syndrome [44].In addition to carcinogenic effects, it also causes an immune system problem both in human and animals.

Status of Aflatoxin in Food and Feed of Wheat
There are about five billion peoples in developing countries of world which are at the risk of chronic exposure to aflatoxins through contaminated food.As compare to develop nations, there have been very few scientific enquiries for Af in wheat food and feed in Pakistan though wheat is being consumed as the topmost staple food by the general public of the country [45,46].A very little attention has been paid to study the mycotoxin in general and Af in particular in wheat food and feed of Pakistan.However, based on limited published literature, the quantity of Af in 56 analyzed wheat samples from Sindh province were not detected within the detectable limits [3].The studies of [47] indicated an average mean value of 1.42 µg kg -1 (Table 2).In another study about 20% of wheat samples were contaminated by total Afs and the highest level was found in one sample.Among all the contaminated samples, two wheat samples were above the suggested limit (4 µg kg -1 ), and their average mean value of 6.6 µg kg -1 was also above the safe limit set by EU regulations (Lutfullah & Hussain, 2012) [21].Similarly wheat bran and wheat bread were found to be higher than safe limit recommended by FDA [48].[49] also stated that out of 19 wheat bran samples, 6 samples were found positive by producing average mean values of AfB1 i.e. 19.0 µg kg -1 .However, a maximum level of 39 µg kg -1 was also recorded by them.More than eight species of Aspergillus is recorded from stored wheat grains of Pakistan.None of the stored wheat grain samples of three provinces (viz.

Conclusions
Keeping in view the above mentioned pivotal role of fungi in Afs production in wheat food and feed, it is strongly recommended that seed lots before and after harvest should regularly be monitored through certain modern technologies in order to determine the health hazard status of seeds prior to be locally consumed or exported.
The wheat crop seeds contaminated by Apergillus spores should be treated at pre harvest level as a control measure of spread of disease caused by Aspergillus.Present review also suggests that Aspergillus growth needs to be controlled both in fields prior to harvest and post harvest at storage stage before to be consumed by human and animals.The chances of Afs contamination in wheat grains increase on account of high moisture content, and inadequate storage temperature.Therefore, wheat grains must be maintained at proper storage conditions to stop the Aspergillus proliferation.
Post-harvest agricultural practices also need to be properly improved by using the modern technologies in threshing, drying, storage, and transportation of wheat grains.Detoxification and decontamination and cleaning are few other remedies which could be adopted in order to minimize or reduce the rate of Afs contamination.To ensure the health safety of national and international consumers, the concerned regulatory authorities of Pakistan are suggested to take into consideration this serious issue of wheat food and feed contamination caused by fungal growth, and controlling strategies should be practiced and quality control system of food should also be adopted and needs to be improved.

Fig 1 .
Fig 1. Common aflatoxins (Afs) found in wheat food and feeds.

Table 2 . Average mean values of aflatoxins (Afs) in wheat food and feed samples reported from Pakistan.
[54] and humid climatic condition prevails in most of the agricultural land of Pakistan.Humidity encourages Aspergillus to attack the wheat grains or crop.Favorable condition for its growth is high temperature and high moisture content (>7%).The optimum temperature for Afs production is recorded as 30 0 C, while no toxin is produced at 10 0 C [53].The positive control in all the batches of Aspertgillus flavus and Aspergillus parasiticus isolates produced maximum Afs in grains at 16% moisture, and 25 0 C temperature in a laboratory experiment[54].