SASA

Action Against Aflatoxins

ICRISAT’s Approach to Protecting Human Health

August 2009 (Issue No.3)

The global challenge – Growing demand for food safety

Today, more than 800 million people, many of them children, are hungry and malnourished with serious impact on growth, learning capacity and the ability to lead fully productive lives. Most of these people are found in the developing world where food is often contaminated or adulterated, thus reducing its nutritional quality and inflicting severe harm to human nutrition.

Food safety - a serious concern, especially for these tender tots.

Concern for food safety has become widespread all over the world. Food is a basic human need, and people are normally concerned about whether their food is tasty, nutritious, and safe. However, about 1.5 billion cases of food-borne disease outbreaks are reported annually, resulting in 3 million deaths globally.

Nevertheless, the foregoing global challenge can be surmounted. Scientific innovations, especially by international agricultural research, have enhanced food quality and allowed a better understanding of the nutritional qualities of food and its health implications.

These innovations have led consumers to become more discriminating in food, demanding protection from inferior quality and unsafe foods. Today, consumers expect that domestic and imported foods will meet basic quality and safety standards and requirements related to food safety.

Mycotoxins – A major health threat

Agricultural products are often at risk of fungal invasion that can produce toxic metabolites called “mycotoxins”. Among these, aflatoxins have assumed economic importance because of their influence on the health of human beings and livestock, and on the marketability of agricultural products. Over 4.5 billion people in developing countries are chronically exposed to high levels of mycotoxin contamination from food crops.

The (magnified) Aspergillus fungus that produces aflatoxin.

Aflatoxins – a group of toxic, carcinogenic, teratogenic and immuno-suppressive substances are produced when toxigenic strains of the fungi Aspergillus flavus and A. parasiticus contaminate agricultural commodities such as groundnut, maize, cotton and chillies. Aflatoxicoses in humans has been reported in many countries, such as India, China, Thailand, and several African countries. In African and Asian countries, where environmental conditions favor the aflatoxin contamination, threat to human health from aflatoxins is quiet high.

Such exposure to toxins compromises immune systems and interferes with the metabolism of various proteins and micronutrients in the human body, leading to reduced growth rate and higher probability of liver cancer.

Aflatoxin contaminated groundnuts.

It is essential to analyze food products to ensure their safety. In most developing countries limited or no facilities exist for monitoring these toxins in food and feed. The monitoring systems are based on physicochemical methods such as thin layer chromatography (TLC), and high performance liquid chromatography (HPLC). However, these are very expensive and may not be available every where.

Facing the challenge

The management of the mycotoxin contamination of food and feed has proven to be a difficult challenge due to lack of awareness, inadequate food safety regulations, pre-disposing environmental conditions and farming practices, and, last but not least, lack of easily available, cost-effective diagnostic tools.

On a global level, access by developing countries to food export markets in general, and of the industrialized world in particular, depends on their capacity to meet the regulatory requirements of importing countries. For most developing countries, agriculture lies at the center of their economies, and food exports are a major source of foreign exchange and means livelihood for the rural population. The long-term solution for these countries to sustain a demand for their agricultural products in world markets, lies in building up the trust and confidence of importers in the quality and safety of their food supply systems. This requires cutting edge scientific innovations that could help farmers improve crop production and post-harvest operations and food industries for food quality and safety. Such efforts will greatly help in increasing the relatively small share of developing countries in the international food trade.

Principal Scientist Farid Waliyar displaying aflatoxin quantification test using the ELISA reader.

In this context, the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) and its partners are doing research on aflatoxin contamination. ICRISAT’s strategy is to develop mycotoxin-tolerant cultivars of ICRISAT’s mandate crops, particularly groundnuts, through conventional plant breeding, biotechnology and pre-and post-harvest technologies. Related to this, ICRISAT also develops and shares improved agronomic and cultural practices, biological control and the development of simple and low-cost mycotoxin diagnostic tools for monitoring purposes. This is shared with farmers and other stakeholders through the mass media, information and communication technology (ICT) and innovative technology commercialization approaches such as those promoted through its Agri-Science Park.

The already existing inexpensive aflotoxin-detection kit ELISA allows profiling of the extent and intensity of mycotoxin contamination. Hence, it will help farmers select aflatoxin-free groundnuts to meet international standards.

The Testing Tool

To test food for the presence of aflatoxins, immunological methods are preferred over analytical methods because of their simplicity and cost-effectiveness. ICRISAT and the Scottish Crop Research Institute (SCRI) produced high quality monoclonal antibodies for aflatoxins, and developed methodologies to use antibodies for aflatoxin estimation by the enzyme-linked immunosorbent assay (ELISA) in different agricultural commodities. The results were comparable with those of HPLC. Costs for performing this test procedure were compared with those of TLC and HPLC and was found to be the least expensive.

ELISA kit for aflatoxin detection and food products that are susceptible to infection.

The method developed is therefore simple, robust, versatile and cost-effective. Constant monitoring of food and feed will contribute to improvement of human health and livestock, and will also enhance export potential leading to increased income for poor farmers in developing countries. In recent years ICRISAT’s efforts helped to further simplify the technique and has produced both polyclonal and monoclonal antibodies for several mycotoxins.

Procedures

Sampling is the most critical step in the process of estimating the presence of aflatoxin in food and feed. It is highly recommended by FAO and the EU as a standard procedure.

Groundnut seed extracts: Powder 100 g of groundnut kernels using a blender. Take 20 g of this powder and add 100 ml 70% methanol (v/v-70 ml absolute methanol in 30 ml distilled water) containing 0.5% KCl in a blender, until the seed powder is thoroughly pulverized. Transfer the extract to a conical flask, seal it with parafilm and shake it for 30 min at 300 rpm in a mechanical shaker. Filter the extract through Whatman No. 4 filter paper, and store at 4^oC till needed for analysis. In the same way prepare a toxin-free sample extract, which will be used for dilution of standards as well as a negative control.

ELISA test: ICRISAT developed two types of ELISAs for the analysis of aflatoxins (i) indirect competitive ELISA and (ii) direct competitive ELISA. Both types are heterogeneous competitive assays that involve the separation of free (un-reacted) toxin in liquid phase from the bound toxin in solid-phase. The basic principle of ELISA lies in trapping the antigen on a solid surface, or capturing the antigen by specific antibodies, and probing with specific immunoglobulins carrying an enzyme label. The enzyme, retained in the case of a positive reaction is detected by adding a suitable substrate. The enzyme converts the substrate to a product that can be easily recognized by its color. Moreover, both ELISAs were developed using alkaline phosphatase, pencillinase, horseradish peroxidase enzyme systems separately, and they do not significantly differ from each other.

Equipment: ELISA reader, incubator, orbital shaker, ELISA plate shaker, a set of micro-pipettes including 12 channel one, balance, pH meter, fume hood, vortex mix, waring blender with mini jars, ELISA plates (Nunc, Maxisorp).

Preparation of reagents

Aflatoxin B1-BSA conjugate (AFB1-BSA): Dissolve 1 mg AFB1-BSA (Sigma 6655) in 1 ml sterile distilled water.

Carbonate buffer (coating buffer) Na₂CO₃ 1.59 g, NaHCO₃ 2.93 g, distilled water 1.0 L, pH of buffer should be 9.6 (No need to adjust the pH).

Phosphate buffer (PBS): Na₂HP0₄ 2.38 g, KH₂P0₄ 0.4 g, KCl 0.4 g, NaCl 16.0 g, dist. water 2 L

Phosphate buffered saline with Tween (PBS T): PBS: l.0 L, Tween 20: 0.5 mL

PBST-BSA: Dissolve 200 mg bovine serum albumin (BSA) (Sigma A 6793) in 100 ml PBS-T

ELISA plate design with end color development.

Substrate buffer for alkaline phosphatase system: It is preferable to use the p-nitrophenyl phosphate (pNPP) chemical in tablet form (available in 5, 15 or 20 mg tablets). Prepare 10% diethanolamine (v/v) in distilled water, adjust pH to 9.8 with conc. HCl. Prepare 0.5 mg ml 1 pNPP in 10% diethanolamine,
pH 9.80 (for each 15 mg tablet 30 ml solution is required).

Sample analysis design: Aflatoxin standards and samples should be placed in the plate as shown in the diagram. Standards and samples should be tested in two wells. Occasionally the border wells give non-specific backgrounds, so it advisable to avoid the border rows. In both ELISAs, the plate should be incubated at 37^oC preferably on an ELISA plate shaker.

Allied support systems

Open Markets: Lack of incentives is one of the main reasons that discourage farmers from producing toxin-free products. Linking them to markets more effectively, creating demand for improved quality produce and conducting proactive consumer education helps overcome this obstacle. One example is contract farming, which ensures predictable and reliable returns. ICRISAT is well placed to undertake these activities involving public-private-people partnerships through its Agri-Science Park including the Agri-Business Incubator.

Learning to build up a stack for curing groundnuts.

Capacity Building: Training farmers and other stakeholders in pre- and post-harvest technologies, and also supply chain on production technology, quality issues, value-addition and risk reduction helps reduce aflatoxin contamination.

Economic Risk Reduction: Aflatoxin contamina¬tion of groundnuts can be very high in rainfed areas due to recurrent drought. Measures have to be applied to reduce involved economic risk, eg, crop insurance.

Cost Effective Technologies: Locally adapted and affordable technologies for different farming systems need to be developed and demonstrated. Again, the involvement of Agri-Science Park@ICRISAT will strengthen this.

In conclusion

ICRISAT joins forces in research and implementation to minimize health threats occurring from mycotoxin contamination. The scale of intervention includes several areas: multi-disciplinary research and technology dissemination, public awareness and knowledge sharing, policy facilitation and capacity building.

Vimala Feeds, one of the large poultry manufacturers in Andhra Pradesh, benefits from use of the ELISA kits.

The development and dissemination of high quality and low cost diagnostic tools for estimating the risk of human exposure to aflatoxins and the quantity of mycotoxins in crops will monumentally assist in ensuring food health and security in the developing world.

Reference

To learn more about ICRISAT’s work on aflatoxin research go to http://www.icrisat.org/aflatoxin