Inspection, sampling and analysis of maize and groundnuts for aflatoxin
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by George M. Ware
The effect of environmental and cultural factors on aflatoxin contamination in maize have been studied and have identified the 5 major factors that affect the development of toxin-producing fungi; inoculum level, moisture, temperature, time, and damaged kernels, Each of these variables play a complex role in toxinproducing capability of fungi. From the farmer prospective the following practices are major contributors to the overall high aflatoxin level in maize.
1) Harvesting before maturity where the moisture of the grain is high (greater than 25%).
2) Shelling grain with high moisture content causes severe hernel damage.
3) Temporary storage of moist grain (very common) (Known as bulking up in the maize trade)
4) Storage of grain in mould infested bin (last year mould spores will infect this year crop). A bottle of bleach and water will reduce the inoculum level in many storage bins and it is inexpensive.
In most cases this will require more than one sample and one analysis. Because of the variability associated with test methods for aflatoxin in maize, analyses of samples from good lots may indicate that the lots are bad (low level of aflatoxin); (exporters' risk) and analyses of samples from bad lots (high level of aflatoxin) may indicate that the lots are good (importer's risk). Testing program for aflatoxin has been developed for maize in the United States. This testing program involves taking a 4.5 kg. sample from a lot, grinding the entire sample and subdividing into five 50 9. samples. The aflatoxin is solvent-extracted from the maize meal by the AOAC CB method. If the analysis of one sample is less than 20 PPM, the lot is accepted. Otherwise, two more samples are analyzed. If the difference between each of the 3 analyses and their average (A) is less than 0.3 A. The lot is accepted when a is less than 25 PPB and the lot is rejected when the average is greater than 25 PPB. When any one of the 3 analyses is not within the range of (A ± 0.3 A) PPB. the fourth and fifth samples are analyzed. The lot is accepted when the average of the 5 anlyses is greater than 25 PPB.
Even with this elaborate test program, the exporter still has a 1% probability of making a wrong decision based on the results of 5 analyses, and the importer has a 7.3% probability of making a wrong decision.
Decisions based on mini-column method, that is not qualitative, will expose the exporter to a great risk and likelihood of being wrong with respect to the amount of aflatoxin in particular lot of maize being exported. Also, the importer has the same increase risk of being wrong using a non-quantitative method.
Special Precaution and Decontamination Procedures
Handle aflatoxins as very toxic substance, use gloves when handling standards. Perform manipulations under hood whenever possible. Take special precautions with toxins in dry form because of electrostatic nature and tendency to disperse in work area. Decontaminate all volumetric flasks used in making standard solutions and all vials containing concentrated sample extract or reference standard solutions by the following procedure. Prepare decontamination solution by adding 3 ml of sodium hypochlorite solution (household bleach) per microgram of each aflatoxin. After mixture is left for several hours or overnight, an equal volume of 5% aqueous acetone is added. After 30 minutes, the mixture may be safely discarded. Oils and solids extracts should be shaken well with the sodium hypochlorite solution. Large volume of organic solvent should be evaporated and then add the sodium hypochlorite to residue. It is very important to add the acetone to sodium hypochlorite solution. One of the products of treatment of waste containing aflatoxin b1, -2, 3 dichloride, which is also carcinogenic. Rinsings of glassware and TLC plates should be done in sink in washer room to minimize bleach use in area where aflatoxin analysis is ongoing.
Use dust mask when grinding grains, feeds or any other products generating dust into atmosphere during preparation. Sample preparation area should be specially cleaned after preparation of sample containing high levels of mycotoxin.
Extraction and clean-up steps should be performed under hood whenever possible. Since aflatoxin is destroyed by alkali and strong acids, do not handle samples in same area where vapors of strong acid/base may be present. Spot TLC plates under hood and avoid exposure of spotted plates to reactive vapors such as O3, SO2 and HC1, which effect stability of aflatoxins on plates and which can also effect the absorbents. Dry plates thoroughly before exposure to UV light since presence of solvent enhances degradation of aflatoxins expased to UV light including fivorescent Light
Samples may be taken from crops growing in the field, during handling, during storage, and at other points in the production, marketing, and processing system.
The most effective sampling method is to take small portions from a moving stream at periodic intervals and combine the portions into a sample. The stream should be sampled frequently but the amount taken at each interval should be small to avoid accumulating too large a sample. The samples must be taken from the stream throughout the time the lot is moved.
Probe sampling is probably adequate for lots which have relently been blended by harvesting lots which have relently been blended by harvesting or handling operations. Recommended methods for taking probe samples are published by the American Oil Chemists Society Official Methods of Analysis.
Diagram each field and divide the field into approximately N equal sections. If the field is rectangular, a count of rows for width and a count of paces for length can be helpful in dividing and identifying N equal field sections. Devise a method to randomly (table of random number) select one ear from each section. Shell and composite the N ears in a mesh bag as a single sample.
Sampling must yield a minimum of one 4.54 kg. sample per unit sample. If more than 4.54 kg. is taken, reduce by dividing or by blending and quartering.
Note on Sampling Methods
Stream sampling is preferable to probe sampling which in turn is preferable to field sampling of ears of corn. For example, the estimated coefficients of variation (%) among aflatoxin test when the indicated number of ears of maize is randomly selected from a field with an average of 100 ppm aflatoxin a 133% for 36 ears harvested and shelled, 83% for 100 ears, 62% for 200 ears, 48% for 400 ears, and 39% for 800 ears. However, if the same entire field is harvested, shell and a stream sample is taken the estimated coefficient of variation is only 29%
Sample Collection Report
A sample collection report should have a sample number and give the date sample was collected, where collected, observation at collection site (insects, rodents), description of sampling method used to collect sample, size of lot sample was drawn from, where sample is stored in laboratory and a description of method used to prevent aflatoxin accumulation during period of sample collection and sample analysis.
Handling and Safe Storage
Each sample should be handled in a manner that minimizes the post-collection production of aflatoxin. Moist samples should be held in cloth or paper bags, cooled if feasible and transported to drying facilities as soon as possible. Samples should not be placed in plastic bags unless seeds are dead and dry, or any other confined area where humidity and temperature can increase around them.
Significant mould growth and aflatoxin production can occur in just a few hours and this must be prevented. As soon as possible after collection, the samples should be dried at approximately 80-90 °C for 3 hours or more to reduce the grain moisture to about 12-13% (where moulds are to be studied, 60°C for a longer time is recommended). If samples were refrigerated before drying, they should be dried immediately and kept dry until analysed.
The entire 4.54 kg. sample should be ground to pass a No. 14 sieve, thoroughly blended, and properly sub-divided to a 1 kg. sample. The entire 1 kg. sample should be ground to pass a No. 20 sieve, thoroughly blended, and properly subdivided to 50 9 analytical samples. When feasible, the samples may be ground and sub-sampled immediately after collection. The subsamples can be analyzed immediately, stored under refrigeration, or dried for storage.
Methods of Analysis
The method for analysis should be selected in advance and used uniformly throughout the survey. Official methods that have been collaboratively studied should be used as the method of analysis for aflatoxin. Unofficial methods or methods reported in literature should be validated with respect to the method accuracy and variability before survey is started. The selected method should be retested during the survey by periodic analyses of blank, standards and "spiked" samples. Analytical procedures once set must be followed to the letter. No alternative procedures are permitted unless expressly authorised in operations manual or by project leader.
A standard format for reporting data should be adopted and used throughout the survery. Laboratory report form should include sample number, date analysed, method used, commodity, analyst's name, all calculations of results, source of standards and concentration of standard.
Preparation and Storage of Aflatoxin Standards
Aflatoxin standards should be prepared and stored as per AOAC Official Book of Method 14 edition.
A survey summary should be used to capture data generated by the survey. This form should be kept by the project leader of the survey and can be used to summarize the progress of the survey. The form should include sample number, date collected, date analysed, analyst's name, analytical result, and space for reporting miscellaneous observations, such as recovery from spiked sample and/or duplicate samples analyzed at different times by different analysts.
Prevention and Control
The development of reliable aflatoxin prevention programs requires considerable scientific knowledge about when and where in the marketing system that aflatoxin contamination occurs. Guidelines based on lesser scientific knowledge most often are costly and ineffective. The environmental conditions that Thai maize is subjected to during post-harvest handling will determine the contamination level at the exporter's silo. Reducing moisture content of maize to below the safe level (13-14%) before any mould growth occurs is at present the safest and most reliable method of preventing aflatoxin contamination. However, reducing moisture content of very large volume of maize in a short period of time in high humidity environment is costly. Other methods should be investigated (particularly aeration, on cob drying on plant, effects of insects, and inoculum levels in bin) to determine if control of these environmental conditions will retard the accumulation of aflatoxin during post-harvest processing. Optimization of these parameters may increase the time moist maize may be safely stored before final drying and final storage.
Guildelines for Mould Control and Aflatoxin Prevention in High-Moisture Maize
The project dedication and very gracious hostpitality of Mrs. Dara Buangsuwon and Mr. Prawat Tanboon-ek is gratefully acknowledged. Many thanks are also extended to Mrs. Kanjana Bhudasam for assistance in many ways. Special thanks go to Dr. Donald DeVivo, UNDP. The friendly cooperation of all the laboratory personnel was much appreciated. Also highly appreciated was the efficient assistance of Mr. Prachop Milindachinta, UNDP. Special thanks also go to Mr. Prachop's secretary for her efficient typing of this report.
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