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.     

Aflatoxins in hull and meats of cottonseed

We have found that when aflatoxins are a contaminant of cottonseed, they may be distributed both in the hulls and in the meats. The concentrations in hulls and meats do not appear to be correlated. Aflatoxins were found in hulls and not in meats of some seed samples, and the reverse situation also was observed. The amounts of toxins were generally much greater in meats, which contained up to 10,200 ppb, than in hulls, which contained up to 390 ppb aflatoxins. Hulls as well as meats fromAspergillus flavus-damaged seed represent a potential source of aflatoxin contamination, and both should be analyzed in order to accurately assess the total aflatoxins in seeds. Crops Research Division, ARS, USDA. So. Utiliz. Res. Dev. Div., ARS, USDA.     

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.     

Relationship betweenAspergillus flavus growth fat acidity,and aflatoxin content in peanuts

The influence of fungal growth, under standardized conditions, on fat acidity in large-seeded Virginia-type peanuts inoculated withAspergillus flavus and relationships between fat acidity and aflatoxin, a toxic metabolite produced byA. flavus were studied. Fat acidity increased quadratically and was highly correlated with visible fungal growth. A lag in aflatoxin production in relation to fat acidity increase was noted; fat acidity reached 60 mg KOH per 100 g kernels before aflatoxin became detectable. This relationship suggests that a rapid method of determining fat acidity might be used to sereen peanut samples for the possible presence of aflatoxin. A rapid method of determining fat acidity is cited and compared with the official A.O.A.C. method.     

Destruction of aflatoxins in peanut protein isolates by sodium hypochlorite

Sodium hypochlorite has been tested for destruction of aflatoxins during the preparation of peanut protein isolates from raw peanuts and defatted peanut meal. The treatments were evaluated by determination of the aflatoxins in the products by thin layer chromatography. Effects of sodium hypochlorite concentration, reaction pH, temperature, and time were studied. Results show that both the sodium hypochlorite concentration and pH are important factors in reducing the concentration of aflatoxins in the protein isolates to nondetectable levels. The treatment with 0.4% sodium hypochlorite at pH 8 produced protein isolates with trace amounts of aflatoxins B₁ and B₂ from ground raw peanuts containing 725 ppb aflatoxin B₁ and 148 ppb aflatoxin B₂, whereas untreated protein isolates contained 384 ppb aflatoxin B₁ and 76 ppb aflatoxin B₂. At pH 9, 0.3% sodium hypochlorite reduced the aflatoxin B₁ content in the protein isolates from 300 ppb to below detectable quantities and the aflatoxin B₂ content from 52 ppb to 2 ppb. Similar results were obtained at pH 10 for 0.3% sodium hypochlorite concentration. In the case of defatted peanut meal which contained 136 ppb aflatoxin B₁ and 36 ppb aflatoxin B₂, 0.25% sodium hypochlorite concentration at pH 8 (0.20% at pH 9; 0.15% at pH 10) reduced both the aflatoxin B₁ and B₂ contents to below detectable quantities in protein isolates as compared to aflatoxin levels of ca. 75 ppb B₁ and 17 ppb B₂ in the untreated protein isolates. Reaction temperature and time did not affect the destruction of aflatoxins significantly.     

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.     

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.     

Effect of carbon dioxide,temperature, and relative humidity on production of aflatoxin in peanuts

Effects of carbon dioxide (CO₂) in combination with reduced relative humidities (RH) and temperatures on growth and aflatoxin production byAspergillus flavus in peanuts were investigated. Sound mature kernels of Early Runner peanuts were surface disinfested, inoculated withA. flavus, and incubated at various temperatures, RH, and CO₂ concentrations. Visible growth, aflatoxin production, and free fatty acid (FFA) formation byA. flavus was inhibited at approximately 86% RH by 20% CO₂ at 17C and by 60 and 40% CO₂ at 25C. Aflatoxin and FFA levels decreased as RH decreased from approximately 99% to 92% to 86%. At a constant temperature, an increase in CO₂ concentration caused a decrease in aflatoxin and percentage FFA; and, at a given CO₂ concentration, lowering the temperature resulted in a decrease in aflatoxin and percentage FFA.     

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.     

Dehulling cottonseed and separating kernels and hulls: Comparison of several varieties of seed

The proteinaceous components of cottonseed can be converted into several different forms for use in human food. All of them require nearly complete separation of kernels and hulls. In research on improving separation processes, eight multiton lots of cottonseed were processed through pilot size commercial-type dehulling and kernel-hull separating machinery. The machinery was operated to produce the cleanest separations possible. Each lot of seed was from a different variety of cotton. For six of the lots, seed were delinted to 2 levels of ca. 7.0 and 2.5% residual linters; and separate dehulling runs were made on seed of each level. Weak hulled seeds were the only lots showing any important differences in dehulling characteristics. They produced higher yields of coarse kernels than the other lots. In terms of nearly pure kernels, good results were obtained with all lots. Yields of kernels averaging 90% of the total kernels from each variety were concentrated into a product which contained less than 1.0% hulls. These products could be converted into meals of more than 55% protein and less than 3% crude fiber. With the addition of a specific gravity separator to the process, loose hulls in coarse kernels can be reduced to nearly zero. One of seven papers presented at the symposium, “Processing Methods for Oilseeds,” AOCS Spring Meeting, New Orleans, April 1973.     

Rapid method for detecting aflatoxins in peanuts

The thin-layer chromatographic procedure for detecting and quantifying aflatoxin in peanuts is too time-consuming, costly, and difficult to be practical for use by untrained personnel. A method based on millicolumn chromatography was tested and found to be both rapid and simple. Columns are prepared by filling a length of 4-mm glass tubing with silica gel to a depth of about 4.5 cm. The millicolumn is developed in a chloro-form-methanol extract of a peanut sample. If aflatoxin is present, a blue fluorescent band at the lower end of the column is observed when the column is exposed to long-wave ultraviolet radiation. Sensitivity is in the order of 5 ppb and an assay can be completed in 15–25 min. Some degree of quantification is possible by comparison with columns developed in extracts with known aflatoxin contents.     

Aflatoxin control program for Peanuts

Under provisions of a USDA Marketing Agreement, an aflatoxin control program for peanuts produced in the United States is administered by the Peanut Administrative Committee composed of peanut growers and shellers. Regulations of this committee contain provisions about the quality of peanuts acquired from farmers, storage of unshelled peanuts, aflatoxin testing, quality and disposition of processed lots, and indemnification of handlers for losses caused by lots which test over 25 parts-perbillion aflatoxin. Effects of the control program on aflatoxin concentrations in peanut products are discussed. Paper number 4978 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.     

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.     

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.     

Aflatoxin effects in livestock

Feeding trials were conducted with swine, beef cattle, dairy cattle and poultry to determine adverse effects, if any, of graded levels of aflatoxins in rations. In addition, samples of meat, eggs and milk from these animals were analyzed chemically to determine if aflatoxin was transmitted into these products. In growing-fattening swine, no evidence of toxic effects was observed when the aflatoxin level fed was 233 ppb or less. In a swine reproduction experiment, no adverse effects were detected in pigs produced from sows fed 450 ppb aflatoxin. No toxic effects were observed at levels of 300 ppb or lower in cross-bred beef steers fed aflatoxin rations for 4.5 months. Using recognized chemical methods, we detected no aflatoxin in meat from swine and cattle fed rations containing 800 and 1000 ppb of aflatoxin, respectively. In dairy cows, weekly intakes of 67 to 200 mg of aflatoxin B₁ per cow produced 70 to 154 ppb aflatoxin M₁ in lyophilized milk. Rapid disappearance of aflatoxin M₁ in the milk took place after withdrawal of aflatoxin from the ration. No adverse effects were discernible in broilers fed from one day to eight weeks of age a ration containing 400 ppb aflatoxin. Lyophilized meat from broilers fed 1600 ppb aflatoxin for eight weeks contained no detectable aflatoxin. Striking differences in aflatoxin susceptibility were observed in 17 different breeds and strains of poultry and game birds fed from two to six weeks of age a ration of 800 ppb aflatoxin B₁. New Hampshire chicks and turkey poults were highly susceptible to aflatoxin in contrast to the resistance of Barred Rock and Australop chickens and guinea fowl. Hybrid chicks from a New Hampshire-White Leghorn cross were highly resistant to aflatoxin. Eggs and meat from White Leghorn hens fed a ration containing 2700 ppb aflatoxin contained no detectable aflatoxin. One of 21 papers presented at the Symposium, “Oilseed Processors Challenged by World Protein Need,” ISF-AOCS World Congress, Chicago, September 1970. W. Utiliz. Res. Dev. Div., ARS, USDA.     

Studies of long term administration of aflatoxin to rats as a natural food contaminant

The effect of feeding aflatoxin, as a natural food contaminant, to rats over long periods of time was studied using multigeneration and longevity tests. The test animals in the multigeneration study consisted of three groups of rats fed diets containing 0, 1 and 10 ppb of aflatoxin (predominantly B₁) continued over four generations, with animals of the first and fourth generation fed the diets for 104 weeks. These diets were in proper nutritional balance and included 35% ground roasted peanut products; the ration with 0 ppb aflatoxin excluded the peanuts usually discarded; the one with 1 ppb had the roasted discards returned, while the ration with 10 ppb included the discards in amount 10 times that which had been initially removed. Another longevity study was also performed in which rats were fed diets containing aflatoxin at a level of 80 ppb. In this case, the test peanuts, also fed as a simulated peanut butter at 35% concentration, consisted entirely of roasted peanut discards. Control diets provided no peanut components. Animals fed the low levels of aflatoxin grew as well and actually had a higher percentage survival at 104 weeks than did the animals on the control, aflatoxinfree diets. Organ weights, liver total lipid and cholesterol levels were comparable in all groups. Pathological abnormalities, e.g., hemorrhagic and opaque spots and mottling in some of the livers, were attributed to the aging process since the abnormalities appeared in the control as well as the experimental groups. In the animals fed the aflatoxin at 80 ppb, which has been reported by several investigators to produce well-defined hepatomas in rats, there was liver involvement and some biochemical changes occurred that were not noted in the controls. However, no hepatomas were observed in these animals even after 21 months on this diet. The liver lesions, indicative of a toxic effect, have not been associated with the development of hepatomas. It is possible that some components of the diet used in these experiments may have protected the animal against hepatoma formation. Our studies indicate that there may be a tolerance for aflatoxin as judged by results in one species of rats when whole ground roasted peanuts provide the natural contaminant. Presented in part at the AOCS Meeting, New York, October 1968.     

A fluorometric rapid screen method for aflatoxin in peanuts

Peanuts were screened for aflatoxin using a rapid, inexpensive fluorometric method. Peanuts were ground and extracted with methanol, and the extract was treated with acidified zinc-acetate-sodium chloride solution, filtered, and diluted with water. Fluorescence of the extracts was compared with that from aflatoxin-free control peanuts. Test samples (160) of several varieties and grades of sources and were screened for the presence of aflatoxin. One hundred thirty-five samples (84%) were identified by this method as aflatoxin positive (15 ppb+) or aflatoxin negative (<15 ppb). Although 22 samples (13.6%) were incorrectly labeled as aflatoxin positive, most of these showed evidence of the presence of mold metabolites other than aflatoxin. Three samples (1.8%) were incorrectly labeled as aflatoxin negative when they actually contained 20, 33, and 34 ppb aflatoxin.     

Elaboration of aflatoxin on cottonseed products byAspergillus flavus

In controlled laboratory experiments heat sterilized and unautoclaved glanded and glandless whole cottonseed or decorticated kernels and sterilized cottonseed meals were found to be utilized as substrates by an aflatoxin elaborating strain ofA. flavus with the production of high levels of aflatoxins B₁, B₂, G₁ and G₂. Gossypol pigments in cottonseed products are apparently not a barrier to either mold invasion or aflatoxin production. Cottonseed hulls, lint cotton, and cottonseed linters were found to be poorly utilized as substrates for either mold growth or aflatoxin production. So. Utiliz. Res. Dev. Div., ARS. USDA.     

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.     

Aflatoxin in Arizona cottonseed: Lack of toxin formation following aspergillus flavus inoculation at sutures

Aflatoxin assays were conducted on seeds from cotton bolls inoculated withAspergillus flavus in commercial fields in Arizona. Inoculations were at sutures at the initiation of boll opening either in the morning or evening in August over a three-year period. One morning inoculation was followed by a water treatment that simulated rain. Fully fluffed bolls were harvested after two or four weeks, and lint and seed linters were examined for bright-green-yellow-fluorescence (BGYF). Ginned seed were assayed for aflatoxin. While BGYF of lint was detected, no seed linters exhibited BGYF and no seeds from the 140 bolls examined contained aflatoxin. Results imply that boll infection byA. flavus occurs before the initiation of boll opening, an observation that is in agreement with recent reports ofA. flavus infection but contrary to conclusions made in earlier reports.