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 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.     

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.     

Predicting the positional distribution of docosahexaenoic and docosapentaenoic acids in aquatic animal triglycerides

The positional distribution of 22∶6 and 22∶5 in aquatic animal triglycerides can be predicted by simple proportionality equations of the typey=kx. The mole % 22∶6 at the 1, 2, or 3 position (y) is obtained by multiplying the proportionality constant for that position (k ₁,k ₂, ork ₃) times the mole % 22∶6 in the total triglycerides (x). For fish and invertebrate triglycerides,k ₁=0.28,k ₂=2.06, andk ₃=0.66. For marine mammal blubber triglycerides,k ₁=0.94,k ₂=0.22, andk ₃=1.84. The same equations apply to both 22∶6 and 22∶5.     

Minicolumn chromatography: State of the art

It was recognized in the early 1960s that a rapid screening method for aflatoxin was needed. Holaday first proposed the minicolumn chromatography method as a rapid screening method in 1968. Since that time, many improvements have been made in this method. The latest minicolumn method has a limit of detection of 5μg/kg and can be completed in 5-7 min. The minicolumn technique has been expanded to include screening commodities other than peanuts for aflatoxin, as well as for other mycotoxins including ochratoxin, zearalenone and aflatoxin M 1.     

Determination of the length of polymethylene chains in salts of saturated and unsaturated fatty acids by infrared spectroscopy

The length of polymethylene chains is determined by counting the number of, or measuring the position of, methylene vibration peaks in the 1070–710 cm⁻¹ and/or the 1380–1170cm⁻¹ regions of the IR spectrum of salts of fatty acids. Plotting this peak position against the phase relationship of the vibration in adjacent methylenes gives a curve which is independent of the chain length. (Thephase relationship, Φ/π=k/(m+1); where φ is the phase difference in radians between adjacent methylenes in a chain;m is the number of methylenes in the chain;k=1,2,3,…m, withk=1 generally assigned to the in-phase vibration.) Separate curves are obtained for methylene wagging and for two arrays of coupled twisting-rocking vibrations. Coupled twisting-rocking vibrations give as many as one peak per methylene group in the 1070–710 cm⁻¹ region with silver, sodium, potassium and barium salts of saturated acids. Lead salt peaks split. These peaks show the total length of salts of both saturated andtrans-unsaturated acids, but only the length of the carboxylate segment in salts ofcis-unsaturated acids. (The carboxylate segment comprises the carbons from the carboxylate carbon to the first unsaturated carbon, inclusive.) Wagging vibrations in the 1380–1170 cm⁻¹ region show the total chain length of saturated salts and the length of the carboxylate segment in unsaturated salts, bothcis andtrans. This region also has peaks for twisting-rocking vibrations, and they are most conspicuous in the spectra of silver and barium salts. Presented in part at the AOCS meeting in Toronto, Canada, 1962.     

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.     

Reaction between Hydrosulfide and Iron/cerium (hydr)oxide: Hydrosulfide Oxidation and Iron Dissolution Kinetics

Hydrosulfide oxidation and iron dissolution kinetics were studied at normal pressure, under inert (N₂) atmosphere, in a liquid–solid mechanically-stirred slurry reactor. The kinetic variables undergoing variations were: hydrosulfide initial concentration (0.90–3.30 mmol/L), oxide initial surface area (16–143 m²/L) and pH (8.0–11.0). The hydrosulfide consumption and products (thiosulfate and polysulfide) formation were quantified by means of capillary electrophoresis, while iron dissolution was monitored through atomic absorption spectroscopy. Most of Fe(II) produced at pH = 9.5 remained associated with the oxide surface in the time-scale of the experiments. The hydrosulfide oxidation by the iron/cerium (hydr)oxide was found to be surface-controlled, with rates (R_i) of both sulfide oxidation and Fe(II) dissolution expressed in terms of an empirical rate equation: R_i = k_i[HS⁻]_t=0^−0.5[A]_t=0[H⁺]_t=0^−0.5 , where k_i represents the apparent rate constants for the oxidation of HS⁻ (k_HS) or the dissolution of Fe(II) (k_Fe), [HS⁻]_{t = 0} is the initial hydrosulfide concentration, [A]_{t = 0} is the initial Fe/Ce (hydr)oxide surface area and [H⁺]_{t = 0} is the initial proton concentration. The rate constant, k_HS, for the oxidation of hydrosulfide at pH = 9.5 was (3.4219 ± 0.65) × 10⁻⁴ mol² L⁻¹ m⁻² min⁻¹, with the rate of hydrosulfide oxidation being ca. 10 times faster than the rate of Fe(II) dissolution (assuming a 1:2 stoichiometric ratio between HS⁻ oxidized and Fe(II) produced; k_Fe = (3.9116 ± 0.41) × 10⁻⁵ mol² L⁻¹ m⁻² min⁻¹).     

Kinetic study of the reaction between tocopheroxyl radical and unsaturated fatty acid esters in benzene

A kinetic study of the reaction between a tocopheroxyl radical and unsaturated fatty acid esters has been undertaken. The rates of allylic hydrogen abstraction from various unsaturated fatty acid esters (ethyl oleate2, ethyl linoleate3, ethyl linolenate4, and ethyl arachidonate5) by the tocopheroxyl radical (5,7-diisopropyltocopheroxyl6) in benzene have been determined spectrophotometrically. The second-order rate constants, k₃, obtained are 1.04×10⁻⁵ M⁻¹s⁻¹ for2, 1.82×10⁻² M⁻¹s⁻¹ for3, 3.84×10⁻² M⁻¹s⁻¹ for4, and 4.83×10⁻² M⁻¹s⁻¹ for5 at 25.0°C. Thus, the rate constants, k_abstr/H, given on an available hydrogen basis are k₃/4=2.60×10⁻⁶ M⁻¹s⁻¹ for2, k₃/2=9.10×10⁻³ M⁻¹s⁻¹ for3, k₃/4=9.60×10⁻³ M⁻¹s⁻¹ for4, and k₃/6=8.05×10⁻³ M⁻¹s⁻¹ for5. The k_abstr/H values obtained for the polyunsaturated fatty acid esters3,4, and5 containing H-atoms activated by two π-electron systems are similar to each other, and are about three orders of magnitude higher than that for the ethyl oleate2 containing H-atoms activated by a single π-system. From these results, it is suggested that the prooxidant effect of α-tocopherol in edible oils and fats may be induced by the above hydrogen abstraction reaction.     

Comparative characterization of the distribution of biomarker hydrocarbons in the chemical transformation products of oxygen-containing precursors of petroleum

The distribution of biomarker hydrocarbons in the thermolysis and thermocatalysis products of oxygen-containing compounds (possible petroleum hydrocarbon precursors) was studied by gas chromatography-mass spectrometry. It was found that n-alkylcyclohexanes and steranes were generated in the course of thermolysis along with expected hydrocarbons—n-alkanes and isoprenes, which resulted from the loss of a functional group. In this case, the molecular-weight distributions of n-alkanes and n-alkylcyclohexanes correlated well with each other. In the course of thermocatalysis, the homologous series of n-alkanes and n-alkylcyclohexanes were also generated; however, an n-alkylcyclohexane distribution maximum had an even number of carbon atoms in the molecule (C₁₈). It was experimentally found that the sterane fragments of oxygen-containing compounds formed upon thermolysis were thermodynamically unstable; this was supported by the formation of biological C₂₇–C₂₉ 5α, 14α, 17α, 20S, and 20R and 5β, 14α, 17α, and 20R (coprostane) steranes as a result of thermolysis. Unlike thermolysis, all of the four epimers of regular C₂₇–C₂₉ (5α, 14α, 17α, 20S, and 20R and 5α, 14β, 17β, 20S, and 20R) steranes were formed as a result of thermocatalysis. It was found that the relative distribution of biomarker hydrocarbons in the thermolysis products of various oxygen-containing compounds largely reflected the structure of the parent organic substance, as compared with that upon thermocatalysis in the presence of active aluminosilicate.     

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.     

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.     

Comparison of the oxidizability of various glycerophospholipids in bilayers by the oxygen uptake method

The aims of this study were twofold: develop a convenient and rapid procedure for assessing the oxidizability of small quantities of glycerophospholipids in bilayers by the oxygen uptake method, and determine and compare the oxidizability of various glycerophospholipids in bilayers. Our purpose was to educidate phospholipid oxidation characteristics in membranes. The quantitative autoxidation kinetics of dilinoleoyl phosphatidylcholine (DLPC) (18∶2/18∶2) was studied in large unilamellar vesicles (LUV) in aqueous dispersions with watersoluble initiator—2,2′-azobis(2-amidinopropane) dihydrochloride—and inhibitor 2-carboxy-2,5,7,8-tetramethyl-6-chromanol. The kinetic data indicated a high efficiency of free radical production, resulting in shortening of measuring time; the very low kinetic chain length, particularly in the induction period, suggested the possibility of including large errors in thekinetics data. Nevertheless, the autoxidation of DLPC obeyed the classic rate law: R _p =k _p [LH]R _i ^1/2 /(2k _t )^1/2 (where R _p -rate of oxygen consumption, k _p =rate constant for chain propagation, [LH]-substrate concentration; R _i ^1/2 -square root of rate of chain initiation, and 2k _t =rate constant for chain termination) in a mixed bilayer system with saturated dimyristoyl PC (14∶0/14∶0), which provided precise and reproducible data. Therefore, the system was used to assess the relative oxidizability of each glycerophospholipid DLPC (18∶2/18∶2), dilinolenoyl PC (18∶3/18∶3), 1-palmitoyl-2-linoleoyl PC (16∶0/18∶2), 1-palmitoyl-2-arachidonoyl PC (16∶0/20∶4), 1-palmitoyl-2-docosahexaenoyl PC (16∶0/22∶6), and dilinoleoyl PE (18∶2/18∶2) in bilayers. The results suggested that the oxidizability of glycerophospholipid in bilayers is substantially influenced by the number of intramolecular oxidizable acyl chains and the content of bis-allylic hydrogen in a structured environment, and showed deviation of the rate law for autoxidation in PC and PE mixed LUV, which possibly was due to nonhomogeneous phospholipid distribution in vesicles.     

CFD evaluation of internal manifold effects on mass transport distribution in a laboratory filter-press flow cell

The internal manifold geometry strongly influences the flow distribution inside an electrochemical reactor. The mass transport coefficient is a function of the flow pattern and is a key parameter in successful electrochemical reactor design and scale-up. In this work, a commercial computational flow dynamics (CFD) package was used to describe the flow pattern in the FM01-LC reactor at controlled volumetric flow rates (corresponding to mean linear flow velocities past the electrode surface between 0.024 and 0.11 m s^?1). Numerical Re numbers were obtained for each local flow velocity at different positions in the reactor channel. From a known mass transport correlation (based on dimensionless groups, i.e. Sh, Re, Sc), numerical k _m values were obtained (in the range 200 < Re < 1,000) at different positions in the reactor channel. Computed k _m numbers are compared against experimental values. This computational approach could be useful in reactor design or selection since it facilitates a fast, preliminary reactor flow and mass transport characterisation without experimental electrochemical measurements.     

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.     

Rate constants for quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and α-tocopherol in liposomes

The ¹O₂ quenching rate constants (k _Q ) of α-tocopherol (α-Toc) and carotenoids such as β-carotene, astaxanthin, canthaxanthin, and lycopene in liposomes were determined in light of the localization of their active sites in membranes and the micropolarity of the membrane regions, and compared with those in ethanol solution. The activities of α-Toc and carotenoids in inhibiting ¹O₂-dependent lipid peroxidation (reciprocal of the concentration required for 50% inhibition of lipid peroxidation: [IC₅₀]⁻¹) were also measured in liposomes and ethanol solution and compared with their k _Q values. The k _Q and [IC₅₀]⁻¹ values were also compared in two photosensitizing systems containing Rose bengal (RB) and pyrenedodecanoic acid (PDA), respectively, which generate ¹O₂ at different sites in membranes. The k _Q values of α-Toc were 2.9×10⁸M⁻¹s⁻¹ in ethanol solution and 1.4×10⁷ M⁻¹s⁻¹ (RB system) or 2.5×10⁶ M⁻¹s⁻¹ (PDA system) in liposomes. The relative [IC₅₀]⁻¹ value of α-Toc in liposomes was also five times higher in the RB system than in the PDA-system. In consideration of the local concentration of the OH-group of α-Toc in membranes, the k _Q value of α-Toc in liposomes was recalculated as 3.3×10⁶ M⁻¹s⁻¹ in both the RB and PDA systems. The k _Q values of all the carotenoids tested in two photosensitizing systems were almost the same. The k _Q value of α-Toc in liposomes was 88 times less than in ethanol solution, but those of carotenoids in liposomes were 600–1200 times less than those in ethanol solution. The [IC₅₀]⁻¹ value of α-Toc in liposomes was 19 times less than that in ethanol solution, whereas those of carotenoids in liposomes were 60–170 times less those in ethanol solution. There were no great differences (less than twice) in the k _q and [IC₅₀]⁻¹ values of any carotenoids. The k _Q values of all carotenoids were 40–80 times higher than that of α-Toc in ethanol solution but only six times higher that of α-Toc in liposomes. The [IC₅₀]⁻¹ values of carotenoid were also higher than that of α-Toc in ethanol solution than in liposomes, and these correlated well with the k _Q values.     

Kinetic model of asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CGMCC No.2266

The kinetic model of asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester using Saccharomyces cerevisiae CGMCC No.2266 with 10% glucose as co-substrate to realize the regeneration of NADPH was established. The effect factors on reduction, the type and the content of co-substrate and coenzyme, and the changes of the substrate and product content vs. time during the reaction process were investigated. The results indicate that 10% glucose can increase the reaction conversion from 23.0% to 98.4% and NADPH is reducer. The reduction process conforms with sequence mechanisms. The model parameters are as follows: v _m =5.0×10⁻⁴ mol·L⁻¹·h⁻¹, k₁=1.5×10⁻⁶ mol·L⁻¹·h⁻¹, k₂=3.0×10⁻³ mol·L⁻¹·h⁻¹. The kinetic model is in good agreement with the experimental data.     

Preparation of14C-labeled aflatoxins and incorporation of unlabeled aflatoxins in a blocked versicolorin-A-accumulating mutant ofaspergillus parasiticus

We have compared 2 methods for preparing radiolabeled aflatoxins from [¹⁴C] acetate for use in biosynthetic studies at the end of the aflatoxin pathway. The Salhab-Edwards method (SE) used a 3-day-old mycelium and a defined medium containing MnCl₂ with incubation at 28 C. The Lee-Bennett method (LB) used a 2-day-old mycelium and a defined medium containing low levels of Mn with incubation at 30 C. Generally, the LB method produced lower quantities of aflatoxin but the product had higher specific activities (sp act). The SE method produced 0.157μmol of aflatoxin B₁ and 0.028μmol of G₁ compared to the LB method with 0.058μmol of aflatoxin B₁ and 0.001μmol of G₁. The sp act (inμCi/μmol) for aflatoxin produced by the LB method were: B₁ = 1.379; B₂ = 0.130; G₁ = 5.0 and G₂ - 0.063. The sp act of aflatoxin produced by the SE method were: B₁ = 0.267; B₂ = 0.014; G₁ = 0.178; and G₂ =0.133. Unlabeled aflatoxins were presented to resting cell cultures of the versicolorin-A-accumulating mutant ofAspergillus parasiticus. No conversion of aflatoxin B, was noted. However, when aflatoxins B₂ or G₁ were presented low levels of aflatoxins B₁ and G₂ were recovered. Aflatoxins B₂ and G₁ were recovered when aflatoxin G₂ was presented. Similar low levels of recovery were obtained in experiments using autoclaved mycelia. Thus we conclude that these minor quantities of aflatoxins recovered were not produced enzymatically.     

Kinetic study of the prooxidant effect of tocopherol. Hydrogen abstraction from lipid hydroperoxides by tocopheroxyls in solution

A kinetic study of the prooxidant effect of vitamin E (tocopherol, TocH) has been carried out. The rates of hydrogen abstraction (k₋₁) from methyl linoleate hydroperoxide (ML-OOH) by α-tocopheroxyl (α-Toc^.) (1) and eight types of alkyl substituted Toc^. radicals, (2–9) in benzene solution have been determined spectrophotometrically. The results show that the rate constants decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of Toc^. increases. The k₋₁ value (5.0×10⁻¹M⁻¹s⁻¹) obtained for α-Toc^. (1) was found to be about seven orders of magnitude lower than the k₁ value (3.2×10⁶M⁻¹s⁻¹) for the reaction of α-TocH with peroxyl radical, which is well known as the usual radical-scavenging reaction of α-TocH. The above reaction rates (k₋₁) obtained were compared with those (k₃) of methyl linoleate with Toc^. (1–9) in benzene solution. The rates (k₋₁) were found to be about six times larger than those (k₃) of the corresponding Toc^.. The results suggest that both reactions may relate, to the prooxidant effect of α-TocH at high concentrations in foods and oils. The effect of the phytyl side chain on the reaction rate, of Toc^. in micellar dispersions has also been studied. We have measured the rate constant, k₋₁, for the reaction of phosphatidylcholine hydroperoxide with a Toc^. radical in benzene,tert-butanol and in Triton X-100 micellar dispersions, and compared the observed k₋₁ values with the corresponding values for ML-OOH.