Design and analysis of sampling plans to estimate aflatoxin concentrations in shelled peanuts

Methodology for use in the design and evaluation of sampling plans to estimate aflatoxin concentrations in lots of shelled peanuts is presented. Use of the operating characteristic curve for comparing and evaluating processor and consumer risks related to various sampling plans and application of the negative binomial distribution to estimate probabilities associated with sampling lots of shelled peanuts for aflatoxin concentration are discussed. Operating characteristic curves are developed for two different single-sample plans, an attribute multiple sample plan, and the plan presently used by the peanut industry to estimate aflatoxin concentrations in commercial lots of shelled peanuts. An estimated prior distribution of lots according to aflatoxin concentration is used to predict, among others, such values as the per cent of all lots tested that will be accepted by the sampling plans and the average aflatoxin concentration in the accepted lots. All four of the sampling plans described in the paper are compared on the basis of values such as these. Other factors to be considered in the critical evaluation and selection of sampling plans for estimating aflatoxin concentrations in commercial lots of shelled peanuts are discussed. Paper number 3197 of the Journal of Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C.     

Variability associated with testing corn for aflatoxin

The sampling, subsampling (both coarse and fine ground meal), and analytical variances associated with testing shelled corn for aflatoxin were estimated by the use of 500 g samples, 50 g subsamples, and the CB method of analysis. The magnitudes of the variance components increased with an increase in the aflatoxin concentration. Functional relationships were developed to predict the variance for a given aflatoxin concentration and any size sample, subsample, and number of analyses. At 20 ppb total aflatoxin, the coefficient of variantion associated with a 4.54 kg sample, 1 kg subsample of coarsely ground meal (passes a #14 screen), a 50 g subsample of finely ground meal (passes a #20 screen) and one analysis were 21, 8, 11, and 26%, respectively.     

Fluorescence in pistachio nuts contaminated with aflatoxin

Bright greenish-yellow fluorescence under long wave ultraviolet light was observed on the shells of 7% of the nuts in samples from 46 aflatoxin contaminated commercial lots of Iranian pistachio nuts. Kernels from the fluorescent nuts contained 50% of the aflatoxin in the samples. No aflatoxin was found in any of the shells. When kernels and shells were cultured, toxicogenic fungi grew from only 4% of the shells and 21% of the kernels from fluorescent nuts and from 9% of the shells and 15% of the kernels from nonfluorescent nuts.     

Aflatoxin distribution in fines and meats from decorticated cottonseed

Nine samples of fuzzy cottonseed potentially high in aflatoxin were separated into hulls, fines and meats. Assays for aflatoxin on triplicate 50-g portions from fines (small, dry particles of kernels) and meats from each of the nine samples indicated a marked concentration of toxin in the fines. On average, there was a 17-fold difference between the aflatoxin content of the fines and that of the meats; the average toxin level in fines was 4024 μg/kg and that in the meat samples was 232 μg/kg. These results indicate a potential for marked reduction in aflatoxin content of processed cottonseed meal by physical removal of fines from meats after dehulling and before processing of meats into oil and meal.     

Bright greenish-yellow fluorescence and aflatoxin in recently harvested yellow corn marketed in north carolina

Corn kernels that exhibited bright greenish-yellow fluorescence (BGYF) under long-wave ultraviolet light were hand-picked from samples of yellow corn produced in eastern North Carolina. The BGYF kernels from 113 4-kg samples contained an average of 8665 parts per billion (ppb) aflatoxin compared to an average of 46 ppb in the non-BGYF kernels. A regression analysis between the ppb aflatoxin concentration and the wt % BGYF kernels in 2,304 4.5-kg samples produced the regression equation: ppb in sample =197 (wt % BGYF). The correlation coefficient for the analysis was 0.90. Testing programs to reduce aflatoxin concentrations in purchased lots of corn based on either the BGYF method or the AOAC chemical assay method were compared. The average aflatoxin concentration in lots accepted by the AOAC method was 4 ppb, 10 ppb or 18 ppb when an acceptance level of < 20 ppb, < 50 ppb or < 100 ppb, respectively, was used. For the BGYF method, the average aflatoxin concentration in accepted lots was 10 ppb, 16 ppb or 22 ppb when an acceptance level of < 0.10% BGYF, < 0.25% BGYF or < 0.50% BGYF, respectively, was used. Approximately the same percentage of lots were accepted by both methods when either the low, medium or high acceptance level was used. Paper no. 6930 of the Journal Series of the North Carolina Agriculture Research Service (NCARS), Raleigh, NC.     

Distribution of ammonia-related aflatoxin reaction products in cottonseed meal

The fate of aflatoxin during ammoniation of contaminated cottonseed meal was studied under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to 27.7 kg meal (14% moisture) that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with 4% ammonia at 40 psi, 100 C for 30 min. This treatment reduced the chemically detected aflatoxin B₁ to less than 4 μg/kg. In control nonammoniated meals, 90% of the radiolabeled material was accounted for in the methylene chloride extract. Duplicate 2-kg samples of the ammoniated meal were fractionated and the distribution of radioactivity was determined. Ca. 86% of the radioactivity was detected in the meal after initial air-drying. Ca. 25% of the added radioactivity was extracted from the air-dried meal with methylene chloride and another ca. 5% was extracted from this residue with methanol. Weak acid released 3% of the added radioactivity from the residue after methanol extraction, bicarbonate released 1% and Pronase digestion, including methylene chloride extraction of the residue, accounted for nearly 19% of the total added radioactivity. Only 37% of the added radioactivity remained in the meal matrix following solvent extractions and chemical and enzymic treatments.     

Variability associated with testing cottonseed for aflatoxin

The sampling, subsampling, and analytical variance associated with testing cottonseed for aflatoxin were estimated by use of 4.54 kg samples, 100 g subsamples, and the Velasco method of analysis. Regression analysis indicated that each of the above variance components is a function of the concentration of aflatoxin in the populations tested. Functional relationships are presented to determine the sampling, subsampling, and analytical variance for any size sample, subsample, and number of analyses. Paper 4821 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.     

Survey of aflatoxins in California tree nuts

Surveys of the California walnut and almond crops to determine incidence of aflatoxins are reported. Average proportions of contaminated nuts from the field were one in 28,250 for walnuts and one in 26,500 for almonds. It was shown that there is a high correlation of contamination with damaged nuts which are removed by standard sorting procedures. Statistical treatment of data from the surveys indicates some of the problems in sampling tree nuts for analysis.     

Comparison of the observed distribution of aflatoxin in shelled peanuts to the negative binomial distribution

Suitability of the negative binomial distribution for use in estimating the probabilities associated with sampling lots of shelled peanuts for aflatoxin analysis has been studied. Large samples, called “minilots,” were drawn from 29 lots of shelled peanuts contaminated with aflatoxin. These minilots were subdivided into ca. 12 lb samples which were analyzed for aflatoxin. The mean and variance of these aflatoxin determinations for each minilot were determined. The shape parameterk and the mean aflatoxin concentrationm were estimated for each minilot. A regression analysis indicated the functional relationship betweenk andm to be:k=(2.0866+2.3898m) × 10⁻⁶. The observed distribution of sample concentrations from each of the 29 minilots was compared to the negative binomial distribution by means of the Kolmogorov-Smirnov test. The null hypothesis that each of the true unknown distribution functions was negative binomial was not rejected at the 5% significance level for all 29 comparisons. Journal Series Paper of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C.     

Sample preparation for aflatoxin assay: The nature of the problem and approaches to a solution

Cases have been reported of individual peanuts, cottonseeds or Brazil nuts so highly contaminated with aflatoxin that, for a 50 g portion to be representative of the whole, the sample preparation procedures should grind each unit to a large number of particles and distribute them uniformly throughout the sample. Assuming uniform contamination of the individual kernel, each 50 g sample should contain 1/100 of that kernel. Even though these extreme cases may be encountered only infrequently, the more usual situation still presents difficulties because of great variability in individual kernel contamination. However, if the extreme can be handled, one can expect to handle the more usual situation. Equipment and procedures to achieve this distribution goal are described. The equipment studied includes a food chopper (Hobart), a nut mill (Thomas Mills), a disc mill (Bauer), a hammer mill (Fitzpatrick Model D comminuting machine), a hammer mill designed specifically for peanut samples (Dicken’s subsampling mill), a Polytron homogenizer (Bronwill Scientific), a vertical cutter-mixer (Hobart), and a sample splitter (Jones riffle). Commodities examined were shelled peanuts and in-shell Brazil nuts, walnuts, pecans and almonds. Comminution and mixing effectiveness were determined by particle size analysis, by distribution of kernels made radioactive by neutron activation and by aflatoxin analysis of naturally contaminated products. From the results we conclude that the ultimate in sample uniformity can be achieved with a disc mill, solvent addition to obtain a fluid system and mixing and grinding of the fluid with a dispersion mixer-grinder. A practical uniformity can be achieved in a vertical cutter-mixer with less expenditure of time and effort for the commodities studied. Presented in part at the AOCS-AACC Joint Meeting, Washington, D.C., April 1968.     

Designing of multiple deferred state sampling plan for generalized inverted exponential distribution

In this article, we propose a sampling plan called a multiple deferred state sampling plan for sentencing a lot based on the information of current and successive lot samples. Based on this sampling plan, the median life of the product is assured based on a time-truncated life test where the lifetime of the product follows the generalized inverted exponential distribution. The quality of the product is measured by its median life. The optimal parameters of the proposed plan are obtained by using the approach of two points on the operating characteristic curve. Tables are also constructed for determining the optimal parameters with various shape parameters. The implementation of the proposed plan is illustrated with examples. The performance of the proposed plan is compared with the performance of existing sampling plans under the generalized inverted exponential distribution.     

Preparation of lot samples of nut meats for mycotoxin assay

A procedure has been devised for preparing lot samples of mycotoxin-contaminated nut meats so that a representative analytical sample may be removed. The sample is rapidly reduced to coarse size. A relatively large portion (about 1/10 of total sample) of subsample is then split out and further comminuted to a fine particle size with the aid of a fat solvent (meat-solvent, w/v, 3:2). The analytical sample is removed from this mixture. The procedure was tested with shelled almonds and shelled walnuts using radioactive nuts to simulate the mycotoxin contamination and provide a simple, precise measure of the contaminated nut meat distribution. The pooled coefficient of variation was 18% for the subsamples and 4.4% for the analytical samples. Considering the dilution factors used (1.50 and 2.14 contaminated nuts/10⁴ nuts) and the low degree of reliability of the lot sample, the sample preparation methods tested appear to be practical and reliable.     

Aflatoxin content of peanut hulls

The degree of aflatoxin contamination in peanut hulls was determined by analyzing inoculated hand-shelled hulls and hulls from peanuts known to contain aflatoxin. Hulls adjusted to 20% moisture, inoculated withAspergillus flavus, and incubated 7 days at 25 C supported growth ofA. flavus but not aflatoxin production. Peanuts from 20 selected Segregation III (visibleA. flavus) lots contained 13–353 ppb of aflatoxin. The machine-shelled hulls from these lots were analyzed and 3 lots contained no detectable aflatoxin, 13 lots contained 4–88 ppb and 4 lots contained >116 ppb. Aflatoxin concentrations of 53–87 ppb were detected in hulls when peanuts containing relatively high levels of aflatoxin (up to 26.8 ppm in damaged kernels) were carefully machine-shelled. Hulls from the same samples obtained by hand-shelling contained no detectable aflatoxin. When machine-shelled hulls were screened through successively smaller screens, the aflatoxin concentration of the smallest fraction (<3.18 mm) was always highest and indicated that small peanut kernels and peanut parts in the hulls actually contained the aflatoxin. Separating hulls over a 4.76 mm round-hole screen appeared to provide a means of removal of most aflatoxin in peanut hulls. No aflatoxin was found in hulls from uncontaminated peanuts.     

Effects of ammoniation on aflatoxins in rations fed lactating cows

A multifaceted cooperative research program involving industry, government and universities was initiated to determine the effects of feeding lactating dairy cows rations containing various levels of cotton-seed and cottonseed meal that had been naturally contaminated with aflatoxins. Evidence is presented that ammoniation of aflatoxin-contaminated cottonseed and cottonseed meal eliminates the aflatoxins, producing a product safe for feeding to ruminants. The aflatoxin M₁ content of milk samples of individual cows receiving rations containing (a) prime cottonseed meal, (b) aflatoxin contaminated meal, and (c) aflatoxin contaminated meal that had ammoniation treatment is reported. Data comparing results with (d) prime cottonseed, (e) aflatoxin contaminated seed, and (f) aflatoxin-contaminated seed that had ammoniation treatment are also reported. None of the milk samples from cows fed ammoniated rations contained any detectable M₁ by the modified Jacobson et al. methodology used. The sensitibity of the method in this laboratory is 0.1 μg M₁/liter of milk. Under the conditions of this study, aflatoxin M₁ levels are related to the levels of aflatoxin B₁ consumed in the diet of the cows. Conversion ratios are reported. Aflatoxin M₁ levels in the milk, relative to the time of the cows’ initial ingestion of aflatoxin B₁, the persistence of M₁ in the milk after discontinuing ingestion of B₁, and disappearance of M₁ under the conditions of the analytical methodology used relative to storage time and temperatures, are reported for liquid milk and for frozen milk. Milk containing the highest level of aflatoxin M₁ was treated with rennet. An 80:20 partion of aflatoxin M₁ was observed between curd and whey, respectively.     

Transmission and distribution of aflatoxin in contaminated beef liver and other tissues

A 160-kg Holstein steer was fed 52 mg₁, equivalents/day (orally) for 5 consecutive days and then slaughtered. The liver, kidneys, spleen, heart and skeletal muscle (round) were retained and assayed for aflatoxin. The liver was cut into 14 samples and each was analyzed to determine the distribution of aflatoxin in liver. Afla-toxin levels in samples from the edges of the liver were 44% lower than averages of all the remaining samples, which were 25.0 ng B,, 15.4 ng M₁ and 47.1 ng total aflatoxin/g; however, cross-sectional samples would give representative assay results. Lower total aflatoxin concentrations (16.0 ng, 18.5 ng and 12.9 ng/g) were found for heart, spleen and muscle, respectively. The kidneys had the highest level of aflatoxin (145.9 ng/g) with M₁ levels (105.5 ng/g) 3.6 times the B, concentration (29.3 ng/g). In the other tissues, B₁ concentrations were 1.6-2.9 times greater than aflatoxin M₁.     

An investigation of oven methods for determining the moisture content of shelled peanuts

Air-oven methods, using ground, sliced, and whele shelled peanuts, were investigated. The use of ground samples was found to be unsatisfactory. Slightly more accurate results were obtained with whole nuts than with sliced nuts. The method of heating 50-g. samples of whole shelled peanuts for 3 hrs. at 130°C. in a foreed-draft oven gave results agreeing closely with those obtained by the Karl Fischer method for samples of low moisture content.     

Characteristics of Iranian almond nuts and oils

Almonds of nine different iranian cultivars were studied. The characteristics of almond nuts and almond oils from these cultivars were determined. They also were studied for possible deterioration of their oils during long storage time. Almond nuts from some cultivars showed significant differences. The variations found in the oil characteristics of different cultivars are not definitely due to varietal differences. The majority of these data does not agree with that found in literature. Acid and peroxide values are very low in both 3 and 12 month old samples, proving the good keeping quality of almonds with regard to their oil content.     

Raffinose content may influence cottonseed susceptibility to aflatoxin contamination

The effect of the cotton storage trisaccharide raffinose and cottonseed storage protein (CSP) in combination on aflatoxin production by Aspergillus flavus was investigated. The ability of ground whole cottonseed and water-extracted cottonseed meal to support fungal biosynthesis of aflatoxin also was assessed in vitro. Dose response data showed that utilization of raffinose as a sole carbon source supported growth and aflatoxin production by A. flavus. When raffinose was a carbon source and CSP was the sole nitrogen source, aflatoxin levels were stimulated up to fourfold above those in raffinose reference media. Results with ground whole cottonseed as a sole carbon/nitrogen source demonstrated the capacity for aflatoxin production in A. flavus cultures. Lipid extraction of ground whole seed severely reduced aflatoxigenesis potential; however, lipid extraction followed by water extraction of ground whole seed restored much of the aflatoxin biosynthetic potential, suggesting the presence of a water soluble inhibitory factor. Accessible carbon appeared to be the limiting resource in water-extracted meal, as a raffinose supplement stimulated aflatoxin production. Either cottonseed storage lipid or raffinose was capable of providing accessible carbon to A. flavus. Raffinose and CSP in developing and mature cottonseed may predispose seed to potentially high levels of aflatoxin contamination by A. flavus upon seed infection.     

Changes in the tocopherol content of almond,pecan and macadamia kernels during storage

The individual tocopherols present in almond, pecan and macadamia nuts were determined by HPLC. Changes in tocopherol content during 16-months' storage at 30°C and 55% relative humidity were related to keeping quality. The large amount of total tocopherols, mainly α-tocopherol, in almonds accounted for their good storage ability. Pecan nuts with less total tocopherols and mainly γ-tocopherol became rancid after 4 months' storage and macadamia nuts, with practically no tocopherols, were rancid after 2 months' storage. Total tocopherol content decreased in all nuts during storage, possibly as a result of its inhibitory function during auto-oxidation.     

Sampling cottonseed lots for aflatoxin contamination

Large samples called “sublots” were drawn from 41 commercial lots of contaminated cottonseed. Each sublot was subdivided into twenty 5 lb samples which were analyzed for aflatoxin. The mean, median, variance, coefficient of variation, and the estimated range among the sample concentrations were computed. The results indicated that: (A) the variance among sample concentrations was large and was found to be a function of sample concentration and (B) the distribution of sample concentrations was skewed; the density of sample values was greater below the sublot concentration.