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.     

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.     

Application of TLC chemical confirmatory tests to minicolumn chromatography of aflatoxins

The minicolumn (MC) proposed by Holaday and Lansden was developed with standard aflatoxin solution and also with the extracts of corn, rice, wheat, cottonseed, peanut cake and black pepper; each having different levels of aflatoxins. One-half mL each of 2,4-dinitrophenylhydrazine,p-anisaldehyde, 20% H₂SO₄, 20% HCl and trifluoroacetic acid (TFA) with 25% HNO₃, which were used for confirming aflatoxins on TLC, were applied to the developed column. Among these, all the 3 acid reagents changed the blue fluorescence of aflatoxins to yellow and thus were found to be satisfactory confirmatory tests. The TFA with 25% HNO₃ had the lowest detection limit-5 ppb.     

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.     

Membrane degumming of crude rice bran oil: Pilot plant study

The procedure for the classical chemical refining of vegetable oils consists of degumming, alkali neutralization, bleaching, and deodorization. Conventional refining of rice bran oil using alkali gives oil of acceptable quality, but the refining losses are very high. A critical work has been carried out to study the application of membrane technology in the pretreatment of crude rice bran oil. Oils intended for physical refining should have a low phosphorus content, and this is not readily achievable by the conventional acid/water degumming process. The application of membrane technology for the pretreatment of rice bran oil has been investigated. The process has already been successfully applied to other vegetable oils. Ceramic membranes, which are important from the commercial point of view, were examined for this purpose. The results showed that the membrane‐filtered oils met the requirements of physical refining, with a substantial reduction in color. It was observed that most of the waxy material was also rejected. Experiments were carried out to establish the relationship between permeate flux and rejection with membrane pore size, trans‐membrane pressure and micellar solute concentration.     

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.     

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.     

Fatty Acid,Phytosterol, and Polyamine Conjugate Profiles of Edible Oils Extracted from Corn Germ,Corn Fiber,and Corn Kernels

This study compared the profiles of fatty acids, phytosterols, and polyamine conjugates in conventional commercial corn oil extracted from corn germ and in two “new-generation” corn oils: hexane-extracted corn fiber oil and ethanol-extracted corn kernel oil. The fatty acid compositions of all three corn oils were very similar and were unaffected by degumming, refining, bleaching, and deodorization. The levels of total phytosterols in crude corn fiber oil were about tenfold higher than those in commercial corn oil, and their levels in crude corn kernel oil were more than twofold higher than in conventional corn oil. When corn kernel oil was subjected to conventional degumming, refining, bleaching, and deodorization, about half of the phytosterols was removed, whereas when corn fiber oil was subjected to a gentle form of degumming, refining, bleaching, and deodorization, only about 10% of the phytosterols was removed. Finally, when the levels of polyamine conjugates (diferuloylputrescine and p-coumaroyl feruloylputrescine) were examined in these corn oils, they were only detected in the ethanol-extracted crude corn kernel oil, confirming earlier reports that they were not extracted by hexane, and providing new information that they could be removed from ethanol-extracted corn kernel oil by conventional degumming, refining, bleaching, and deodorizing.     

Effects of plant‐scale alkali refining and physical refining on the quality of rapeseed oil

The physical refining of soybean oil was introduced as an energy saving and environmentally friendly procedure alternative to the traditional alkali refining, and the process was successfully applied to other vegetable oils. We had compared the two procedures in industrial refining under conditions, which enable a clear comparison. In nine plant‐scale experiments, crude rapeseed oil, taken from the same tank of crude oil, was processed on the same day both by alkali refining and by physical refining. Quality changes (free fatty acids, peroxide value, conjugated fatty acids, polar lipids, minor constituents) were determined, and also their stability against oxidation (Rancimat and Schaal Oven Test), and the fatty acid composition. In refined oils, the sensory acceptabilities and the sensory profiles were assayed. Finally deodorized oils, produced by the two methods, did not appreciably differ in their sensory characteristics and chemical composition, excepting slightly higher concentration of isomeric polyunsaturated fatty acids in physically refined oils. During storage for one year in commercial packagings at 15 °C, oxidative and sensory changes were negligible.     

Minor constituents of vegetable oils during industrial processing

We report the effects of individual steps of industrial refining, carried out in Brazil, on the alteration of selected minor constituents of oils, such as corn, soybean, and rapeseed oils. Total sterols, determined by capillary gas chromatography (GC), decreased by 18–36% in the fully refined oils, compared with the crude oils. The total steradienes, dehydration products of sterols, were determinedvia a simple clean-up on a short silica gel column, followed by high-performance liquid chromatography (HPLC) with ultraviolet detection. The level of steradienes, normally not present in crude oils, increased after each refining step, especially after deodorization. Thus, the content of steradienes increased after deodorization by about 15- to 20-fold in corn and soybean oils, and by about 2-fold in rapeseed oil. The total steryl esters were also determinedvia clean-up on a short silica gel column, followed by HPLC with evaporative light scattering mass detection. A minor decrease in the level of steryl esters was observed after complete refining. The individual tocopherols and tocotrienols were determined by HPLC with a fluorescence detector. The level of total tocopherols and tocotrienols decreased by about 2-fold after complete refining of corn oil and by about 1.5-fold in soybean and rapeseed oils. In all three cases, maximum reduction of tocopherols was observed after the deodorization step. The level of polymeric glycerides, determinedvia clean-up on a short silica gel column followed by size-exclusion HPLC, increased to some extent (0.4–1%) during refining. The level oftrans fatty acids, determined by capillary GC, also increased to a substantial extent (1–4%) after refining. Part of doctoral thesis of Roseli Ap. Ferrari to be submitted to Faculdade de Engenharia de Alimentos, Universidade de Campinas, Campinas, Brazil.     

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.     

Distribution of aflatoxins in various fractions separated from raw peanuts and defatted peanut meal

The present investigation is the first definitive study of the distribution of aflatoxins in a wet-milling process of raw peanuts. The results show that the majority of the aflatoxins originally present in the peanuts remained in the solid fractions, particularly the protein fraction, during wet-milling. In the protein concentrate preparation, the concentrates carried 81–89% of the total toxin; crude oil, 5–8%; and whey fraction, 3–14%. In the case of protein isolate preparation, 51–56% of the total toxin remained with the isolates, 22–26% with the residue, 11–17% with the whey, and 7–8% with the crude oil. Distribution of aflatoxins in the preparation of protein isolates from defatted peanut meal showed that 55–65% of the total toxin originally present in the meal remained with the protein isolates, 20–28% with the residue, and 10–20% with the whey fraction. Changes in extraction pHs for the preparation of protein isolates either from raw peanuts or defatted meal did not alter the distribution pattern mentioned above. A new approach based upon charge-transfer (electron acceptor-donor) complex formation is suggested to shift this aflatoxin distribution from protein products to disposable whey or residue fraction during the processing of raw peanuts and defatted meal for protein products.     

Reduction of aflatoxin levels in cottonseed and peanut meals by ozonization

Cottonseed and peanut meals were treated with ozone to destroy or eliminate aflatoxins. High meal moistures (cottonseed 22%, peanut 30%), high temperature (100C), and longer treatment times favored inactivation as measured by thin-layer chromatography. Aflatoxins B₁ and G₁ were readily destroyed by the ozone processes whereas aflatoxin B₂ appeared relatively resistant. In cottonseed meal, 91% of the total aflatoxins was destroyed in 2 hr, a decrease from 214 to 20 ppb; in peanut meal, 78% was destroyed in 1 hr, a decrease from 82 to 18 ppb. In both meals, aflatoxin B₁ was totally inactivated within the times specified. E. Util. Res. Dev. Div., ARS, USDA.     

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.     

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.     

Experience of Prerefining of Vegetable Oils with Acids

Prerefining of vegetable oils with acids serves general purposes. One is to remove impurities like phosphatides etc. from the crude oils to such a degree that the oil can then be physically refined. Another purpose is to facilitate subsequent alkali refining and to reduce pollution in effluent from alkali refining. After a review of some earlier work results from tests in laboratory scale on soya oil, rapeseed oil and linseed oil will be presented and discussed. Finally, an industrial procedure for acid refining, called “special degumming” will be described.     

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.     

Effects of dietary peanut oil on serum lipoprotein patterns of rats

Oils prepared from two varieties of peanuts and from a hybrid corn having linoleic acid concentrations substantially different from the respective commercial oils were compared with commercial oils for their effects on serum lipids of weanling female rats. In the first experiment, serum lipid patterns appeared to reflect linoleic acid content of the dietary oil. However, with a longer feeding period in the second experiment, serum lipid patterns were determined by the plant source of the dietary oil rather than its linoleic acid content; all peanut oils differed from both corn oils in their physiological effects. Diets containing triglyceride, hydrocarbon and sterol fractions obtained by liquid chromatography of peanut and corn oils were fed to female rats. The data provide no evidence that the hydrocarbon or sterol fractions of peanut oil are responsible for its unusual atherogenicity when fed as the sole fat source or that similar fractions from corn oil are protective against the effects of peanut oil.     

A new analytical procedure to differentiate virgin or non‐refined from refined vegetable fats and oils

The effects of individual steps of industrial refining on the alteration of triacylglycerides (TAG) are reported. The level of dimer triglycerides, normally not present in crude oils, increased after each refining step, especially after steam‐washing and desodorisation. A good correlation between the applied temperatures and dimer triglycerides content was found. The forming of dimer triglycerides starts at 90 °C and increases corresponding to the extension of thermal treatment like normal heating or desodorisation. The data for various types of vegetable oils demonstrate that there is no clear‐cut different tendency to form dimers. Heated oils with different contents of linoleic acid produced nearly the same amount of dimers. Other criteria, like the determination of trans fatty acids, steradienes, or the UV‐absorption, were found not to be appropriate to detect a thermal treatment at temperatures below 150—170 °C. The formation of steradienes mainly depends on the total sterol contents, the percentage of added bleaching earth, and its acidity and moisture. Over 160 commercial vegetable oil samples were analysed to obtain a data range on the content of dimerised triglycerides. Mostly, vegetable oils Iabelled as non‐refined (which may be steam‐washed) did not exceed dimer contents of 0.1%. Virgin vegetable oils did not contain dimers (< 0.05%). The content of dimer triglycerides in vegetable oils was determined by a new method via clean‐up on a short silica gel column, followed by size‐exclusion HPLC with refractive index detection.     

Inactivation of aflatoxins in peanut and cottonseed meals by ammoniation

Aflatoxins in peanut and cottonseed meal can be inactivated by treatment with gaseous ammonia. In pilot plant runs, contaminated peanut meal was ammoniated at two levels each of moisture content, reaction time, temperature and ammonia pressure. Thin layer chromatography indicated that ammoniation inactivated the aflatoxins (121 ppb) in the meal to a nondetectable level. With a similar treatment, total aflatoxins (350 ppb) in cottonseed meal were reduced to 4 ppb. A series of runs was made with large scale equipment using cottonseed meal containing an average of 519 ppb total aflatoxins. Under optimum processing conditions, aflatoxin content of this meal was reduced to below 5 ppb and non-detectable levels. So. Utiliz. Res. Dev. Div., ARS, USDA.