The USDA aflatoxin sampling program for shelled peanuts is an important component of broader industry efforts to minimize aflatoxin occurrence in the edible market. In this program, official samples are milled with either a traditional hammer/automatic sub-sampling mill, commonly called the Dickens Mill (DM) or with a vertical cutter mill (VCM). Particle size reduction and sample homogenization are the primary objectives of sample preparation (milling) to generate subsamples which best represent the parent sample composition for downstream analysis. DM particle size reduction is limited by the 3.2 mm round hole screens internal to the mill which prevent pasting of the sample. VCM grinding converts the sample to a paste while simultaneously homogenizing the sample. Experiments demonstrate that when testing aflatoxin contaminated peanuts for equivalent sized subsamples prepared from the two mill types, made into water slurries per USDA specifications and subsequently extracted and tested for total aflatoxin per USDA specifications, VCM subsamples are more normally distributed around the sample aflatoxin mean, whereas DM subsamples are more positively skewed (median lower than mean) around the sample aflatoxin mean. Accordingly, milling official samples with a DM compared to VCM promotes more lot misclassifications. It is also demonstrated that for a given subsample after extraction and immunoaffinity column (IAC) purification, the total aflatoxin measured by either high performance liquid chromatography (HPLC) or fluorometry (both USDA approved) are practically equivalent from an accuracy perspective. There are costs (time and resources) associated with decreasing natural variation due to sampling, sample preparation and analytical testing in an aflatoxin sampling/testing program. Sample preparation is a greater source of variation compared to that of the analytical testing. Resources would be better spent replacing DM with VCM mills than converting the final analytical step from IAC-fluorometry to IAC-HPLC in an effort to best classify peanut lots for the edible market.
Aflatoxins are toxic metabolites produced by a variety of
Aflatoxin was originally discovered in 1960 as the causative agent of "Turkey X" disease in which large numbers of turkey poults died in England after consuming contaminated peanut meal from Brazil (Lancaster
When present, aflatoxin contamination in a shelled lot typically affects only a small frequency of kernels. Early work in select lots demonstrated an approximate 0.1% to 2.5% frequency contamination (
Shelled peanut lot sizes in US commerce are typically 20 metric tons (MT) or ∼44,000 lbs but more rarely can be as large as 200,000 lbs. In the United States, the Federal State Inspection Service (FSIS) via USDA Agricultural Marketing Service (AMS) regulations, serves as the unbiased third party that collects official samples from positively identified shelled peanut lots. Sampling of shelled lots is best accomplished when the lot is being conveyed, typically just before final packaging, when multiple, regularly spaced incremental samples over the production of the lot can be automatically collected and subsequently aggregated (
The minimum sample considered for aflatoxin testing in the USDA AMS peanut program is 22 kg, which is commonly used to make a decision on a 20 MT lot. This represents about 0.11% of the lot in question. Ideally, the
Regardless of mill type, subsamples are collected for downstream extraction and analysis. In the official USDA extraction method, subsamples are first mixed with water (1.455 water to peanut solids ratio) and made into a slurry using a high speed blender. USDA AMS regulations allow anywhere from 900-1300 grams to be subsampled and slurried. This water slurry procedure was designed to provide further particle size reduction/mixing and to save on downstream organic solvent usage (and subsequent disposal of solvents) necessary for extraction (
After slurrying, a portion of the slurry is removed for downstream aflatoxin extraction and subsequent analytical measurement. Traditionally, thin layer chromatography (TLC) was the final analytical technique for aflatoxin testing (
The US Peanut industry follows a modified sequential plan where one, two or three samples are prepared and analysed for aflatoxin to either accept or reject the lot of raw peanuts being considered for the edible market (
Considering the infrequent kernel to kernel nature of aflatoxin contamination, the negative binomial distribution has been used to mathematically model aflatoxin contamination in actual shelled lots (
Given the heterogeneous nature of aflatoxin contamination in shelled peanuts, sampling is the largest source of variation in the sampling, sample preparation and analytical chain to determine a test result (
Disagreement exists within the US Peanut industry regarding the performance of the two mills types used to prepare samples for aflatoxin testing and their impact on sample results. Some in the peanut industry have seemingly observed that sample test results prepared from samples ground via a DM tend to skew lower than samples ground via a VCM (
While readily achievable, there are costs associated with reducing variation in the aflatoxin testing program that must be balanced against other factors. HPLC instrumentation is the preferred analytical measurement for aflatoxin detection (
A lot of commercial medium runner peanuts from the 2015 US crop that did not pass the official USDA aflatoxin program was identified in cold storage. Considering the official samples for this lot, the 1AB and 2AB measured a 37 and 45 ppb total aflatoxin, respectively, with an overall average of 41 ppb. From this lot, a 1 MT tote was randomly selected for these research purposes. This tote was subsequently repackaged using a scoop into 46 individual samples, each weighing about 22 kg (48-49 lb).
Samples were milled with one of two mill types: 1) DM or 2) VCM. Two DM's were used for these experiments, and the units were used alternately and equally throughout the experiments. Each DM had two spouts where subsamples are automatically discharged during milling. This is the standard design for a DM in the US peanut industry. As the DM blades rotate, they are surrounded by a cylindrical screen with 3.2 mm round hole openings and all ground material is forced through this screen. The two sup-sampling spouts correspond to two channels surrounding this screen, and these two channels each collect about 1100 g of the material passing through the screen at those locations. The remainder of the milled peanuts passes through the screen, but is not collected in the two subsample channels. Instead, this milled material falls out the bottom of the mill; however, this 'fall thru' is equivalent in particle size reduction and comminution to that collected via the subsampling spouts. When milling a sample with a DM, samples were slowly metered into the DM's and subsamples collected from the two spouts built into the mill. Additionally, for some experiments, the 'fall thru' was also collected for subsequent testing.
Two Stephan (Hameln, Germany) VCM's were used in this research, a model 44 and model 60, with 44 L and 60 L stainless bowl capacities, respectively. Sharpened, dual serrated blades were used for both VCM's and each had 220V, 33A motors with 3600 RPM in addition to motorized, scrape surface baffles. The two VCM's were used alternately and equally throughout the experiments, and all samples were milled for 6 minutes.
After VCM milling, paste was manually sub-sampled by randomly collecting milled sample around the bowl with a spatula. For a given subsample, roughly equal portions of paste are taken from each of four quadrants within the bowl and aggregated to provide an 1100 g subsample.
After milling (VCM or DM) all subsamples were slurried with water per USDA guidelines for the domestic, edible, shelled peanut aflatoxin sampling and testing program. While USDA guidelines for preparing water slurries allow milled subsamples weights to range from 900-1300 grams, all starting peanut material weights were 1100 grams and corresponding water weight was 1600 grams. Slurries were blended on high for 3 min. Immediately after slurry preparation, a 122.8 gram portion (50 gram peanut equivalent) was weighed into a tared blender jar, and 10.0 g of NaCl were added along with 177 ml of 85/15 methanol/water. This slurry portion with added methanol/water and NaCl was subsequently blended on high for an additional 2 min. Methanol and NaCl were both American Chemical Society grade. After blending, the material was gravity filtered with a P8 filter paper from Fisher Scientific (Fair Lawn, NJ). 20 ml of filtered extract was mixed 1:1 with deionized water and then gravity filtered through a G6 glass fiber filter from Fisher Scientific (Fair Lawn, NJ). Post filtration, 10 ml of the extract was then passed through disposable IAC's (Pi Biologigue; Seattle WA) at 1-2 drops/second using a vacuum manifold. Columns were then washed twice with 10 ml of deionized water before elution with 1 ml of methanol. If total aflatoxin measured above 60 ppb at the final analytical stage, the extract was diluted 1:10 and rerun thru a new IAC.
Post IAC, final eluates were analyzed for total aflatoxin via 1) fluorometry or 2) HPLC based on AOAC Method 991.31. For fluorometer measurements, the 1 ml eluates were diluted 1:1 with AflaTest® developer (VICAM; Watertown, MA) and total fluorescence measured via a VICAM (Watertown, MA) Series-4EX Fluorometer, and measured fluorescence was converted to ppb based on known calibration standards measured daily.
For HPLC measurements, the 1 ml eluates were diluted 1:1 with 1% acetic acid and loaded into HPLC vials. An Agilent HPLC 1100, equipped with a fluorescence detector, and a post-column photochemical reactor for enhanced detection (PHRED) (Aura Industries, NY, NY) was used for aflatoxin measurements. Fifteen μl of solution was injected onto a 4.6 x 150mm, Waters Nova-Pak C18 4μm analytical column held at 30°C. An isocratic method using 55:45 water: methanol [HPLC Grade] at a flow rate of 1.0 ml/min was used to separate individual aflatoxins at a pressure of approximately 200 bar. PHRED-enhanced peaks were detected by fluorescence with an excitation wavelength of 360 nm and emission wavelength of 440 nm. Aflatoxin B1, B2, G1, and G2 were quantified using a purchased 4-component aflatoxin mix from Supelco (Bellefonte, PA)
Sampling, sample preparation and analytical variation were systematically investigated by measuring aflatoxin distributions within a 1 MT tote of medium runners. As detailed in the Materials section, this tote was isolated from a shelled lot that failed the official USDA aflatoxin sampling program. In the first experiment, three sets (66 kg/set) of peanuts were prepared by selecting three samples (22 kg) randomly from the repackaged tote, thoroughly mixing these samples using a 3-way splitter and then dividing equally into three new samples (22 kg) per set. This generated three sets, three samples each, for a total of 9 samples. Three samples of this size are equivalent to the total sample available in the official USDA sampling and testing program for aflatoxin. All samples were first milled with a DM and the two spout subsamples collected. Additionally, the corresponding 'fall thru' from each sample was collected, milled in a VCM, and four subsamples collected after the VCM grind to determine the average aflatoxin in the 'fall thru'. This VCM milled 'fall thru' was assumed to have a particle size reduction similar to a typical VCM grind.
All subsamples, regardless of the mill used in preparation, were made into water slurries per USDA AMS specifications and extracted for aflatoxin per USDA AMS specifications using IAC's coupled with fluorometry or HPLC to measure total aflatoxin. Both fluorometry and HPLC coupled with IAC clean-up have been recognized as official AOAC International methods since 1991 for aflatoxin detection in peanuts (
Comparison of aflatoxin measurements for subsamples after DM and VCM sample preparation. Three sets, 3 samples each, were prepared by thoroughly mixing ∼66 kg of medium runners which were then randomly split into 3 samples (22 kg). All samples were randomly selected from a 1 MT tote of medium runners that was in turn randomly selected from a 20 MT lot that had failed aflatoxin (1AB, 2AB average = 41 ppb). Each of the 9 samples was first milled via a DM and the two automatic subsamples (spouts) collected. Additionally, the 'fall thru' after DM milling was collected, milled in a VCM and then 4 subsamples collected. All subsamples, regardless of mill type, were extracted and analyzed equivalently, including water slurry preparation, extraction, IAC column cleanup and measurement of total aflatoxin via fluorometry.
USDA AMS regulations require that all subsample extracts be split and analysed in duplicate, and this protocol was followed for data collected in
Comparison of fluorometer total aflatoxin measurements prepared from equivalent subsample extracts and two immunoaffinity columns, A and B.
HPLC is the most accurate and precise analytical measurement
Comparison of total aflatoxin measured by fluorometry vs total aflatoxin measured by HPLC for subsample extracts split prior to immunoaffinity cleanup. See manuscript for details.
Subsample variation for the paired DM subsamples is directly observed and CV's ranged from 4.0 to 132.7%, and average 60.1% across the nine samples (
To further understand sample preparation performance, the 'fall thru' (see Methods) from the various DM preparations was collected, milled in a VCM and then 4 subsamples (1100 gram) removed, slurried, extracted, passed thru IAC's and total aflatoxin measured (
When present in peanuts, aflatoxin contamination is highly positively skewed (median lower than mean), on a kernel to kernel basis, and sampling is the largest source of variation in the final test result determining lot acceptability (
Given the large differences observed in aflatoxin measured for subsamples prepped from the DM versus a VCM, further experiments were pursued to understand implications of these differences. From the repackaged tote, 11 additional samples (representing approximately 24% of the tote) were pulled at random, prepared via VCM, and an additional 11 samples were pulled at random and prepared via DM. From each milled sample, a minimum of 12 individual 1100 gram subsamples were selected, slurried, extracted, and analysed post IAC for total aflatoxin via a fluorometer. Note that for DM subsamples, the two subsample spouts were collected and the 'fall thru' was riffle divided extensively before manually collecting additional subsamples. This riffle dividing, if anything, provided additional mixing for the 'fall thru' which is equivalent in particle size reduction and mixing for that collected in the spouts. The median, mean, standard deviation and CV among subsamples from each of the 22 samples are summarized in
Summary of total aflatoxin measured for multiple subsamples (1100 gram) prepared after milling samples (48-49 lb) with either a DM or VCM. 11 samples were milled using a VCM and 11 samples were milled using a DM. All samples were randomly selected from a 1 MT tote of medium runners that was in turn randomly selected from a 20 MT lot that had failed aflatoxin (1AB, 2AB average = 41 ppb). After milling each sample and collecting multiple 1100 gram subsamples (minimum of 12 per sample), all subsamples were extracted and analyzed equivalently, including water slurry preparation, extraction, IAC cleanup, and total aflatoxin measurement via fluorometry. Medians, means, standard deviations and CV among subsamples for all samples were calculated.
Subsample variation as measured by the standard deviation for both the VCM and DM increased as sample means increased (
Subsample standard deviation versus sample mean for VCM and DM preparations representing eleven ∼22 kg samples for each mill. Samples were randomly selected from a 1 MT of medium runners that was in turn randomly selected from a 20 MT lot that had failed aflatoxin (1AB, 2AB average = 41 ppb).
For the current study, in addition to improved CV values for VCM preparations versus DM, overall averages of the mean and median values among subsamples were more equivalent after VCM preparation: mean = 20.4 and median = 21.0, compared to DM preparation: mean = 23.9 and median = 16.2 (
Aflatoxin histograms for multiple subsamples (1100 gram) prepared after milling multiple samples (22 kg) with either a DM or VCM. Eleven samples were milled using a VCM (top panel) and 11 samples were milled using a DM (bottom panel). Samples were randomly selected from a 1 MT of medium runners that was in turn randomly selected from a 20 MT lot that had failed aflatoxin (1AB, 2AB average = 41 ppb). After milling each sample and collecting multiple 1100 gram subsamples (minimum of 12 per sample), all subsamples were extracted and analyzed equivalently, including water slurry preparation, extraction, IAC cleanup, and total aflatoxin measurement via fluorometry. A reference line is provided at 15 ppb, which is the USDA accept/reject limit.
Subsample variation is inversely proportional to subsample size, and directly proportional to particle size reduction and degree of homogenization during milling (
There are opportunities to reduce variation in an aflatoxin sampling/testing program, but the benefits of implementing such opportunities must be balanced against their costs (
Given sample preparation, this data provides some practical examples of how DM sample preparation can increase the chances of good lots being rejected and bad lots being accepted. The price of a DM is estimated to be $5000 whereas a VCM equivalent to those used in this research, which are sized to effectively process 22 kg of shelled peanuts, is estimated to be closer to $40,000. The VCM must be appropriately sized and built, as it has been reported that the conversion of oilseeds to a paste can clog mills and prevent mixing during sample preparation (
Given technological advances in analytical procedures over the past approximate 20 years, the analytical variation in the sampling, sample preparation and analytical chain to generate a test result is very low in modern aflatoxin sampling/testing programs. As previously mentioned, an exception is TLC, which while inexpensive to purchase instrumentation, has inherently high analytical variability, increased operational inefficiencies and requires the use of solvents which pose environmental and health risks. TLC is not advised for a modern aflatoxin sampling/testing program. However, while HPLC is the gold standard of
These experiments demonstrate the increased potential to misclassify shelled lots of peanuts in an aflatoxin testing program when preparing samples with a DM versus a VCM. A VCM is more expensive, but the clear benefits in better classifying aflatoxin contamination, operational efficiency and operator safety justify this expense. Furthermore, assuming an appropriate IAC is used upstream during extraction, a fluorometer can provide near equivalent performance in accuracy versus an HPLC for detecting total aflatoxin.
The authors are Jack P. Davis, Ph.D., Director of Technical Services, JLA International, Albany, GA, 31707 and Adjunct Faculty, Dept. of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695; James M. Leek, Chairman of the Board, JLA International; Mike Jackson, President, JLA International; and Mansour Samadpour, Ph.D., President and CEO of IEH Laboratories, Lake Forest Park, WA 98155.