Determination of aflatoxins in individual peanuts and peanut sections

Subsamples of a given lot of peanuts may vary greatly in aflatoxin content due to extreme variability in the degree of contamination of individual kernels. A micro method, adapted from the aqueous acetone procedure recently proposed by Pons and Goldblatt for the determination of aflatoxins in cottonseed products, was developed to permit accurate determination of aflatoxins in individual kernels and kernel sections. Use of this procedure permitted the topographic distribution of aflatoxins within single kernels to be mapped and indicated that the toxins are not uniformly distributed within contaminated kernels, even when the kernel contains a high level of aflatoxins. Although wrinkling or discoloration sometimes indicated that a kernel was contaminated, this type of physical damage was not found to be a reliable indication of aflatoxin content. Also it was noted that a few apparently sound and mature kernels contained high levels of aflatoxins. Presented at the AOCS Meeting, Houston, April 1965. Honorable Mention Bond Award Competition. So. Utiliz. Res. Dev. Div., ARS, USDA.     

Unavoidable low level aflatoxin contamination of peanuts

A major portion of aflatoxin contamination of peanuts probably occurs when decayed or discolored peanuts are incompletely removed by sorting. Quality control measures have been instituted in the United States to insure that unavoidable aflatoxins in consumer peanuts and peanut products do not exceed 20 μg/kg. However, low level aflatoxin contamination, from trace amounts to about 50 μg/kg in sound mature unblemished peanuts, can occur before peanuts are dug. This low level contamination is not related to high levels of Aspergillus flavus infection or to current production practices. Low level aflatoxin contamination of peanuts may be endemic, and current sorting procedures may not be effective in removing unblemished contaminated peanuts.     

Theoretical investigations into the accuracy of sampling shelled peanuts for aflatoxin

Within a population of shelled peanuts, aflatoxin may be concentrated in less than 0.5% of the peanuts. Those peanuts containing aflatoxin might have concentrations up to 1,000,000 μg of aflatoxin per kilogram of peanuts. Because of the distribution pattern, sample means vary widely, and the true average level of aflatoxin in the population is difficult to estimate. The objective of this study was to determine the effect of sample size, N, on sampling accuracy. The negative binomial distribution of aflatoxin since it allowed for a high probability of zero counts along with small probabilities of large counts. Using both the Monte Carlo technique and a direct computation method, the effect of sample size on sampling accuracy was quantitatively described. Journal Paper No. 2775, North Carolina State University Agricultural Experiment Station, Raleigh, N.C.     

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.     

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.     

A method for determining kernel moisture content and aflatoxin concentrations in peanuts

A method was developed to determine kernel moisture content (KMC) and aflatoxin concentration in discrete peanut samples. Shelled peanuts were weighed to the nearest 0.01 g, and a water slurry was made by blending the peanuts for 2 min with 2.2 ml of water per g of peanuts. The slurry (10 g) was withdrawn and dried at 130°C for 3 h to determine KMC. Methanol was added to the remaining slurry and blended for an additional 1 min, and aflatoxins were quantitated with high-performance liquid chromatography. Comparison of the slurry method with an official peanut moisture method showed good agreement between the two over a range of moisture levels. Recovery of aflatoxin B₁ from spiked samples averaged 97% with an average coefficient of variation of 3.6%. The method enables determination of both KMC and aflatoxin content in peanut samples without degradation of aflatoxin that would occur when using the official moisture method.     

An improved rapid physicochemical assay method for aflatoxin in peanuts and peanut products

A practical, short cut, sensitive method for more rapidly determining aflatoxin in peanuts and peanut products has been developed. This was in response to the need to reduce the time required for analyses of peanut products in process. Through reductions in solvent volumes, utilization of pressure filtration for clarification, and substitution of liquid:liquid extraction for a lengthy column clean up, equivalent results are possible in less than one half the time required for the current official procedures. Sensitivity, precision and accuracy are comparable to the current methods for raw nuts and peanut butter. It is now possible to analyze a given ground sample of peanuts within a period of less than 90 min and one analyst can assay more than 16 samples within an 8 hr working day. Presented at the AOCS-AACC Joint Meeting, Washington, D.C., April 1968.     

Control and removal of aflatoxin

The best approach to contain the problem of aflatoxin is prevention and enough is now known about prevention to reduce contamination drastically. Guidelines for preventing mycotoxins in farm commodities have been suggested by the U.S. Department of Agriculture. Moisture is the single most important parameter and prompt drying to safe levels is essential for control of toxigenic molds. Foreign matter and damaged seed should be removed. Provision of clean, dry, adequately cooled and ventilated storage is important and good sanitation is essential to minimize mold contamination during storage and processing: Genetic approaches which may result in resistance to elaboration of aflatoxins are under investigation. When aflatoxin is found in a sample of oilseeds the contamination generally resides in only a small proportion of the kernels, commonly less than 1%. Sorting or separation can concentrate the vast majority of aflatoxin-contaminated kernels into relatively small fractions and only a small loss is incurred as a result of their removal. Aflatoxin is frequently found deeply imbedded within individual kernels so removal by simple washing does not seem feasible. However, extraction with polar solvents such as alcohols and ketones to achieve essentially complete removal of aflatoxins appears technically feasible. Heat is relatively ineffective for destruction of aflatoxin although normal roasting, as of peanuts for the preparation of peanut butter, results in considerable reduction in aflatoxin content. Treatment withFlavobacterium aurantiacum removes aflatoxin and may be useful for beverages. Oxidizing agents readily destroy aflatoxin, and treatment with hydrogen peroxide may be useful. Treatment of defatted oilseed meals with ammonia can reduce aflatoxin content to very low or undetectable levels with only moderate damage to protein quality. One of 21 papers presented at the Symposium, “Oilseed Processors Challenged by World Protein Need,” ISF-AOCS World Congress, Chicago, September 1970. So. Market. Nutr. Res. Div., ARS, USDA.     

Absence of aflatoxin from refined vegetable oils

The present investigation is the first definitive study of the fate of the aflatoxins in vegetable oils undergoing processing. Crude oils, obtained by solvent extraction or by hydraulic pressing of ground moldy peanuts (not suitable for human consumption), contained only small fractions of the aflatoxin originally present in the peanuts; the meals retained the bulk of the aflatoxin. Conventional alkali refining and washing of the oils reduced aflatoxin content to a range of 10 to 14 ppb. The subsequent bleaching operation essentially eliminated aflatoxin from the oils; the concentrations were now less than 1 ppb. The above results were confirmed using corn oils obtained from corn germ deliberately contaminated in the laboratory withAspergillus flavus. The nonfluorescing forms of aflatoxins, capable of being produced during the alkali refining operations, are also absent from the refined vegetable oils; these aflatoxin derivatives are readily converted to their original form on acidification and thereby measurable by fluorescence, if present. Presented in part at the AOCS Meeting, Los Angeles, April 1966.     

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.     

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.     

Limiting temperature and relative humidity for growth and production of aflatoxin and free fatty acids byAspergillus flavus in sterile peanuts

Sound mature kernels, broken mature kernels, immature kernels, and unshelled Early Runner peanuts were heat-treated in controlled environment cabinets and inoculated with spores ofAspergillus flavus. Treatments were incubated at 97-99% relative humidity at different temperatures ranging from 5 to 55C and also at 30C with relative humidities ranging from 55 to 99%. Samples were removed after 7 and 21 days and assayed for aflatoxin, free fatty acids, and peanut kernel moisture. The limiting relative humidity for aflatoxin production byA. flavus was 85ŷ1% relative humidity for 21 days at 30C. The limiting low temperature for visible growth and aflatoxin production by the fungus was 13ŷ1C for 21 days at 97-99% relative humidity. Damaged kernels, however, developed some afllatoxin in 21 days at 12C. The maximum temperature for aflatoxin production was 41.5ŷ1.5C for 21 days at 97-99% relative humidity. Fungus growth and sporulation at 43C were equal to that at 40C, but no aflatoxin was produced. Moisture content of immature kernels was higher at equilibrium with the same relative humidity than the moisture content of sound mature kernels, damaged kernels, or kernels from unshelled peanuts. There appeared to be no proportional quantitative correlation between synthesis of aflatoxin and production of free fatty acids in nonliving peanuts, but no aflatoxin was produced without a simultaneous increase in free fatty acids.     

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.     

A water slurry method of extracting aflatoxin from peanuts

A water slurry method in which 1100 g of comminuted peanuts was blended with 1500 ml of tap water for 3 min in a blender and the aflatoxin in a 130-g portion of the water slurry was extracted by solvent according to methods similar to those used in Method II of AOAC was compared to the method presently used by the Food Safety and Quality Service, USDA. The proposed water slurry method requires only 180 and 60 ml per sample, respectively, of methanol and hexane compared to the 1650 and 1000 ml, respectively, required by the FSQS method. Blending comminuted peanuts with water reduced the average particle size and distributed the contaminated particles throughout the slurry. Ninety-four percent of the blended particles passed a sieve with 149-μ openings compared to only 66% of the unblended product. Variance among analyses with the FSQS method did not differ significantly from the variance among analyses with the slurry method. However, analyses with the slurry method averaged 16% more aflatoxin than with the FSQS method. Paper No. 6189 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC.     

Relationship of physical appearance of individual mold-damaged cottonseed to aflatoxin content

Over 700 individual aflatoxin-suspect cottonseed were hand-selected from a heterogenous stockpile of ginned seed. The seed were categorized on the basis of (a) bright greenish-yellow, fluorescence termed cateye, on the linter fibers under ultraviolet light; (b) partially bald seed with part of the linter fibers removed by ginning; (c) a combination of cateye and balding; (d) thin and discolored lint; and (e) bluish, not cateye, fluorescence. Aflatoxin assays on each of the 771 selected seed showed that 142 out of 771 (18%) were contaminated by aflatoxin (B₁+B₂) in the range of 150 ppb—5.75 million ppb. Some 93% of the aflatoxin-contaminated seed was concentrated in categories (a), (b), and (c), with the highest concentration, 61%, in category (b). Eight seed in these three categories contained over 1 million ppb of aflatoxins. The data suggest that removal of cateye and partially bald seed from contaminated lots of cottonseed should be more effective for controlling aflatoxin contamination in cottonseed than removal of cateye seed alone.     

Limiting temperature and relative humidity for aflatoxin production byAspergillus flavus in stored peanuts

Sound mature kernels, broken mature kernels, immature kernels and unshelled cured Early Runner peanuts were inoculated with spores ofAspergillus flavus and incubated up to 84 days in controlled environment cabinets. In a series of experiments temperatures ranged from 8 to 49 C in combination with 98±1% relative humidity (RH); in others RH was varied from 70% to 99% at 30±1/2 C and from 83% to 99% at 20±1/2 C. Samples were removed after 7, 21, 42 and 84 days of incubation and assayed for aflatoxin, free fatty acids and peanut kernel moisture. Aflatoxin was formed in sound mature kernels at 40 C and 14 C and in broken mature kernels at 13 C, but none was formed at 41 C after 21 days or at 12 C after 84 days in 98±1% RH. The limiting temperatures for aflatoxin formation in peanut kernels with intact shells were 41 C for 21 days and 16 C for 84 days of incubation. The limiting RH at 30 C for aflatoxin production in sound mature kernels was 84%, whereas in broken mature and immature kernels it was 83% and in kernels from unshelled peanuts the limiting RH was 86% for 84 days of incubation. The limiting RH at 20 C for sound and broken mature kernels was 83%, whereas it was 86% RH for immature kernels and 92% for kernels from unshelled peanuts. Free fatty acid formation was correlated with visible growth of fungi rather than with aflatoxin production. Aflatoxin formation was generally correlated with kernel moisture contents of 10% or higher.     

Food uses of peanut protein

Approximately 19 million metric tons of peanuts (Arachis lypogae L.) are harvested annually, and contribute over 3.5 million tons to the world’s protein pool for food and feed uses. Peanut is the world’s fourth most important source of edible vegetable oil and the third most important source of vegetable protein feed meal. About 70% of the U.S. Crop is consumed domestically or exported as peanut kernels, peanut butter, and confections. Crushing is limited primarily to culls and kernels containing aflatoxin; and to stabilize the market. However, in countries such as India, Senegal, Brazil and Argentina, 75 to nearly 100% of the crop is crushed or exported for use as oil and livestock meal. The peanut is perhaps the world’s most widely researched food protein oilseed. Advantages over other oilseeds include relatively bland flavor, minor color problems, and minimal preparation requirements. Products in use throughout the world include boiled peanuts, roasted full-fat or partially defatted peanuts, peanut butters, grits and flours (full-fat or defatted), defatted peanuts, protein concentrates, and protein isolates. Compounded food applications include fortified breads and bakery products, snacks, meat products, extended milks, cheese and curd type products, and various mass-feeding foods in developing countries. Challenges encountered in peanut utilization include improvement of flavor levels and stability, identification of nutritional adequacy and fortification requirements, elimination of antinutritional factors, development of new products and improved processes, and elimination of aflatoxin problems.     

Sampling diced almonds for aflatoxin

To ensure that diced almonds meet the current FDA guideline limit for total aflatoxin, it is necessary to have a sampling plant that will allow representative sampling with defined precision-i.e., with confidence limits on the average aflatoxin found. A sequential sampling plan using 4.54-kg samples of diced almonds or 150-g samples of meal by-product (fines screened from diced nuts during production) was constructed with data from a study of aflatoxin distribution among samples of 2 selected lots of almonds. These 2 lots of whole nuts, estimated to have 400 and 25 ppb aflatoxin, were diced and boxed with normal processing equipment and procedures to approximate the distribution of aflatoxin in the product during commercial production. With a square root trans-formation of the data from 4.54-kg samples of diced nuts, the aflatoxin in samples of both lots approximated a normal distribu-tion and the within-lot variances were not significantly different, which allowed the statistical plan described. A supplemental study was made of aflatoxin distribution in the meal by-product. The lack of a significant difference between the results for diced nuts and those for the corresponding meal suggests that diced almonds can be monitored for aflatoxin indirectly by sampling the meal, which will allow the use of fewer analyses of 150-g samples of less expen-sive product to reach a decision.     

Measurements of High Oleic Purity in Peanut Lots Using Rapid,Single Kernel Near-Infrared Reflectance Spectroscopy

High oleic peanuts have improved shelf life vs. conventional peanuts. Purity (percentage of high oleic peanuts within a lot) is critical to ingredient performance and final lot value. Contamination can result from unintentional mix-ups at the breeder/seed level, improper production handling, or due to physiologically immature high oleic kernels. Therefore, industry groups have established unofficial sampling plans to monitor purity. Assuming equivalent measurement performance and simple random sampling, increasing the sample size decreases variance among replicated sample test results and increases the precision of estimated lot purity. A novel instrument (QSorter Explorer by QualySense AG) using near-infrared reflectance spectroscopy was evaluated for high speed (20 kernels per second) high oleic purity measurements. The study objectives were to assess instrument performance in: (1) measuring oleic acid (%) in runner peanuts and (2) estimating the true high oleic purity of artificially mixed peanut lots. Three grades (Jumbo, Medium, and No 1) of US Runner mini-lots each at seven different contamination levels (0, 5, 10, 20, 30, 50, and 100%) were prepared. Oleic acid (%) of individual kernels was measured by scanning replicated samples of 10, 50, 100, and 500 kernels using the QSorter Explorer. The variance associated with each sample size and lot contamination level on returned purity values is discussed in the context of binomial sampling. Overall, the demonstrated measurement performance and capacity of the QSorter Explorer to process much larger sample sizes suggest this instrument can better identify true high oleic peanut lot purity vs. other currently available technologies.     

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.