Effect of nixtamalization (Alkaline cooking) on fumonisin-contaminated corn for production of masa and tortillas

Studies were undertaken to determine the fate of the mycotoxins, fumonisins, during the process of alkaline cooking (nixtamalization), using normal-appearing corn that was naturally contaminated with fumonisin B(1) (FB(1)) at 8.79 ppm. Corn was processed into tortillas, starting with raw corn that was cooked with lime and allowed to steep overnight; the steeped corn (nixtamal) was washed and ground into masa, which was used to make tortillas. Calculations to determine how much of the original fumonisin remained in the finished products took into consideration that FB(1) will be converted to hydrolyzed fumonisin B(1) (HFB(1)) by the process of alkaline cooking. All fractions, including steeping and washing water, were weighed, and percent moisture and fumonisin content were determined. Tortillas contained approximately 0.50 ppm of FB(1), plus 0.36 ppm of HFB(1), which represented 18.5% of the initial FB(1) concentration. Three-fourths of the original amount of fumonisin was present in the liquid fractions, primarily as HFB(1). Nixtamalization significantly reduced the amount of fumonisin in maize.     

Liquid chromatographic determination of fumonisins B1, B2, and B3 in corn silage

Corn silage was dried, ground, and then extracted with 0.1 M ethylenediaminetetraacetic acid. The filtrate was applied to a FumoniTest immunoaffinity column. Fumonisins were derivatized with naphthalene-2,3-dicarboxaldehyde, separated on a C(18) liquid chromatographic column, and detected by fluorescence. The detection limits for fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 50, 25, and 25 ng/g of dried silage, respectively. Recoveries of fumonisin B(1), fumonisin B(2), and fumonisin B(3) from wet and dried corn silage spiked over the range of 100-5000 ng/g averaged 91-106%. The method was applied to corn silage samples collected from the midwestern area of the United States during 2001-2002. Of 89 corn silage samples, fumonisin B(1), fumonisin B(2), and fumonisin B(3) were found in 86 (97%), 64 (72%), and 51 (57%) of the samples. The mean positive levels of fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 615, 93, and 51 ng/g, respectively, in dried silage. This suggests that fumonisins may be frequent low level contaminants in corn silage.     

Characterization of fumonisin B(1)-glucose reaction kinetics and products

The reaction of fumonisin B(1) with the reducing sugar D-glucose can block the primary amine group of fumonisin B(1) and may detoxify this mycotoxin. A method to separate hundred milligram quantities of fumonisin B(1)-glucose reaction products from the excess D-glucose with a reversed-phase C(18) cartridge was developed. Mass spectrometry revealed that there were four primary products in this chain reaction when fumonisin B(1) was heated with D-glucose at 65 degrees C for 48 h: N-methyl-fumonisin B(1), N-carboxymethyl-fumonisin B(1), N-(3-hydroxyacetonyl)-fumonisin B(1), and N-(2-hydroxy, 2-carboxyethyl)-fumonisin B(1). The N-(1-deoxy-D-fructos-1-yl) fumonisin B(1) (fumonisin B(1)-glucose Schiff's base) was detected by mass spectrometry when fumonisin B(1) was heated with D-glucose at 60 degrees C. The nonenzymatic browning reaction of fumonisin B(1) with excess D-glucose followed apparent first-order kinetics. The activation energy, E(a), was 105.7 kJ/mol. Fumonisin B(1) in contaminated corn could precipitate the nonenzymatic browning reaction with 0.1 M D-glucose at 60 and 80 degrees C.     

Production of a single-chain variable fragment antibody against fumonisin B1

The selection of synthetic antibody fragments from large phage libraries has become a common method for the generation of specific antibodies. The technique is particularly valuable when antibodies against small, non-immunogenic molecules (haptens) or highly toxic substances have to be produced. In addition, haptens are usually coupled to protein carriers, bearing the risk that the free hapten is not detectable. Here, a single variable chain antibody (scFv) against the highly toxic mycotoxin fumonisin B1 has been produced. The hapten was coupled via a linker to biotin. Using this conjugate and a naive scFv library, it was possible to circumvent both the necessity of immunization and the risk of a disguised hapten. The scFv obtained after three panning rounds was found to bind specifically to both free fumonisin B1 and fumonisin-biotin conjugate. Also fumonisin B2 was bound by the scFv. Modeling of both scFv and fumonisin B1 molecule revealed a good fitting of structures. The antibody obtained can potentially be used for developing a rapid and affordable immunoassay for detection of food contamination and can be applied in immunoaffinity chromatography, usually carried out prior to HPLC analysis of mycotoxin-contaminated food and feed.     

Structural characterization of alpha-zein

A variety of published physical measurements, computational algorithms, and structural modeling methods have been used to create a molecular model of 19 kDa alpha-zein (Z19). Zetaeins are water-insoluble storage proteins found in corn protein bodies. Analyses of the protein sequence using probability algorithms, structural studies by circular dichroism, infrared spectroscopy, small-angle X-ray scattering (SAXS), light scattering, proton exchange, NMR, and optical rotatory dispersion experiments suggest that Z19 has approximately 35-60% helical character, made up of nine helical segments of about 20 amino acids with glutamine-rich "turns" or "loops". SAXS and light-scattering experiments suggest that in alcohol/water mixtures alpha-zein exists as an oblong structure with an axial ratio of approximately 6:1. Furthermore, ultracentifugation, birefringence, dielectric, and viscosity studies indicate that alpha-zein behaves as an asymmetric particle with an axial ratio of from 7:1 to 28:1. Published models of alpha-zein to date have not been consistent with the experimental data, and for this reason the structure was re-examined using molecular mechanics and dynamics simulations creating a new three-dimensional (3D) structure for Z19. From the amino acid sequence and probability algorithms this analysis suggested that alpha-zein has coiled-coil tendencies resulting in alpha-helices with about four residues per turn in the central helical sections with the nonpolar residue side chains forming a hydrophobic face inside a triple superhelix. The nine helical segments of the 19 kDa protein were modeled into three sets of three interacting coiled-coil helices with segments positioned end to end. The resulting structure lengthens with the addition of the N- and C-terminal sections, to give an axial ratio of approximately 6 or 7:1 in agreement with recent experiments. The natural carotenoid, lutein, is found to fit into the core of the triple-helical segments and help stabilize the configuration. Molecular dynamics simulations with explicit methanol/water molecules as solvent have been carried out to refine the 3D structure.     

Influence of the chemical environment on metolachlor conformations.

Metolachlor exists in multiple, different stable conformations in solution. Assignment of the NMR frequencies to chemical structure is a prerequisite to understanding the behavior of individual conformations. (1)H NMR experiments of metolachlor in different chemical environments identified the labile sites of metolachlor and environments that influence conformational/configurational changes. Within very specific chemical environments, metolachlor atropisomers aS,12S (aR,12R) and aR,12S (aS,12R) freely interchange, and consequently, the multiple conformations also interchange. The changes in chemical environments, which most alter the conformations and molecular dynamics of metolachlor, identify the most critical components affecting its environmental fate. These results enable a structural interpretation of conformational changes that can influence the environmental fate of metolachlor.     

N-(1-deoxy-D-fructos-1-yl) fumonisin B(1), the initial reaction product of fumonisin B(1) and D-glucose

Incubation of fumonisin B(1) and D-glucose in aqueous solutions resulted in the formation of N-(1-deoxy-D-fructos-1-yl) fumonisin B(1) in addition to the previously reported N-(carboxymethyl) fumonisin B(1). N-(1-Deoxy-D-fructos-1-yl) fumonisin B(1) is the first stable product formed after the Amadori rearrangement of the Schiff base formed by the reaction of the primary amine of fumonisin B(1) and the aldehyde group of D-glucose. N-(1-Deoxy-D-fructos-1-yl) fumonisin B(1) was synthesized by reacting fumonisin B(1) with an excess of D-glucose in methanol and heating for 6 h at 64 degrees C. It was purified using C(18) and strong cation exchange solid-phase extraction cartridges and characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry. Subsequently, N,N-dimethylformamide was found to be a better reaction solvent, requiring reaction for only 2-3 h at 64 degrees C and eliminating the formation of methyl esters. Alkaline hydrolysis of N-(1-deoxy-D-fructos-1-yl) fumonisin B(1) gave a mixture of hydrolyzed fumonisin B(1) and hydrolyzed N-(carboxymethyl) fumonisin B(1).     

Reduction of fumonisin B1 in corn grits by single-screw extrusion

This study was designed to determine the efficacy of extrusion in reducing fumonisin B1 in corn flaking grits in the presence and absence of glucose. In addition, degradation products of fumonisin B1 during extrusion were identified and quantitated with a mass balance approach. Uncontaminated clean corn grits, grits spiked with 30 microg/g fumonisin B1, and grits fermented with Fusarium verticillioides M-2552 (40-50 microg/g fumonisin B1) were extruded in the presence and absence of glucose (10%, w/w) using a single-screw extruder. Extrusion decreased fumonisin B1 by 21-37%, whereas the same process with added glucose further decreased fumonisin B1 by 77-87%. LC-fluorescence and LC-MS showed that most fumonisin in the extruded samples without added glucose was the fumonisin B1 form, whereas the main degradation product in grits extruded with glucose was N-(deoxy- d-fructos-1-yl)fumonisin B1. The formation of hydrolyzed fumonisin B1 was not significant during extrusion. Results suggest that extrusion in the presence of glucose may reduce fumonisin B1 in corn grits significantly.     

Bound fumonisin B1: analysis of fumonisin-B1 glyco and amino acid conjugates by liquid chromatography-electrospray ionization-tandem mass spectrometry

To study the formation of fumonisin artifacts and the binding of fumonisins to matrix components (e.g., saccharides and proteins) in thermal-treated food, model experiments were performed. Fumonisin B(1) and hydrolyzed fumonisin B(1) were incubated with alpha-d-glucose and sucrose (mono- and disaccharide models), with methyl alpha-d-glucopyranoside (starch model), and with the amino acid derivatives N-alpha-acetyl-l-lysine methyl ester and BOC-l-cysteine methyl ester (protein models). The reaction products formed were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of d-glucose with fumonisin B(1) or hydrolyzed fumonisin B(1) resulted in the formation of Amadori rearrangement products. Whereas conjugates were found following the reaction of sucrose, methyl alpha-d-glucopyranoside, and the amino acid derivatives with fumonisin B(1), the heating with hydrolyzed fumonisin B(1) yielded no artifacts. For structural determination, the stable reaction product formed by heating of methyl alpha-d-glucopyranoside (as starch model) with fumonisin B(1) was purified and identified by nuclear magnetic resonance spectroscopy as the diester of the fumonisin tricarballylic acid side chains with methyl alpha-d-glucopyranoside. These model experiments demonstrate that fumonisins are able to bind to polysaccharides and proteins via their two tricarballylic acid side chains.     

Fate of fumonisin B(1) in corn kernel steeping water containing SO(2)

Six 100 ppm fumonisin B(1) (FB(1)) solutions were prepared by dissolving pure standard in six different solvents containing SO(2). Two of the solvents contained 0.2 or 0.4% SO(2) in distilled water. The other four solvents were obtained by steeping corn kernels at 60 degrees C in a 0.2% SO(2) aqueous solution for 6, 12, 24, or 48 h. After the addition of FB(1), all solutions were maintained at 60 degrees C for 7 days. Fumonisin B(1) content in each solution was determined in triplicate by HPLC. Steeping corn kernels in 0.2% solution at 60 degrees C for 6 h seems to be the most effective treatment to decrease the amount of FB(1).     

Structure and natural occurrence of stereoisomers of the fumonisin B series mycotoxins

1H and 13C NMR spectroscopy of both fumonisin B3 and B4, as well as high-performance liquid chromatography (HPLC) analysis of samples of fumonisin B3 used as standards, showed in each case the presence of two stereoisomers, which could not be separated by preparative chromatography. The 2,3-anti relative configuration for the two minor stereoisomers of fumonisin B3 and B4 was deduced from the NMR data, and their 2S,3R absolute configurations were established by application of Mosher's method using the fumonisin B3 sample. Samples of fumonisin B3 and B4 can contain between 10 and 40% of fumonisin B compounds of the 3-epi series. The 3-epi-FB3, determined by HPLC with fluorescence detection of the o-phthaldialdehyde derivative and confirmed by liquid chromatography-tandem mass spectrometry, was found to occur naturally in a range of maize samples at levels much lower than FB3 (< 20%). The identification of members of the 3-epi-fumonisin B series provides insight into the order and selectivity of steps in fumonisin biosynthesis.     

A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples

This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 μg/kg to 106 μg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 μg/kg) and ochratoxin A (相似文献   

Mycotoxin contamination of dietary and medicinal wild plants in the Eastern Cape Province of South Africa

Nineteen dietary and 30 medicinal wild plants used by residents of the Eastern Cape Province of South Africa were investigated for the presence of fumonisin B1 and aflatoxin B1. The plants were extracted in water, and cleanup was undertaken on immunoaffinity cartridges; analysis was by HPLC using fluorescence detection. None of the plant extracts contained detectable levels of aflatoxin B1; however, eight plants, four dietary and four medicinal, were positive for fumonisin B1 at levels ranging from 34 to 524 microg/kg and from 8 to 1553 microg/kg, respectively. The presence of fumonisin B1 was confirmed by LC-MS/MS using positive ion electrospray ionization. Fumonisin B1 provided characteristic fragment ions at m/z 704, 686, 546, 528, 370, and 352 corresponding to sequential loss of H2O and tricarboxylic acid moieties from the alkyl backbone. These results indicate that exposure to fumonisin B1 is much more widespread than initially thought and is the first report of mycotoxin contamination in South African medicinal and dietary wild plants.     

Effect of solutes and matrix structure on water mobility in glycerol-agar-water gel systems: a nuclear magnetic resonance approach

Nuclear magnetic resonance spectroscopy (NMR) has been widely used to determine water molecular mobility in food systems. This study aimed to examine the effects of matrix structure and solutes on the dynamics of water molecules in model mixed systems, glycerol-agar-water gels, using low- and high-resolution NMR. Simple models to explain water relaxation rates and self-diffusion coefficients in mixed systems were developed using the experimental values obtained for the individual binary systems (glycerol-water solutions and agar-water gels). The spin-lattice relaxation of mixed systems was influenced by interactions of both glycerol and agar with water, while the spin-spin relaxation of mixed systems was dominated by the interaction of agar with water. Water diffusion was influenced by not only molecular interactions between all components but also the gel matrix structure. These models are able to differentiate the effect of solutes from that of matrix structure on water molecular dynamics.     

Fumonisin production by Fusarium verticillioides strains isolated from maize in Mexico and development of a polymerase chain reaction to detect potential toxigenic strains in grains

Fumonisins are mycotoxins produced by Fusarium verticillioides (Sacc. Nirenberg) in maize (Zea mays L.), a staple crop in Mexico. In this study, we report the isolation and identification of 67 Fusarium strains isolated from maize kernels collected in Northwest and Central Mexico. The strains were characterized regarding fumonisin B(1) production and the presence of the FUM1 gene. F. verticillioides was the predominant species isolated in both geographic regions, but the isolates from Northwest Mexico produced higher levels of fumonisin. A polymerase chain reaction (PCR)-based method, to detect a region of the FUM1 gene involved in fumonisin biosynthesis, was developed and employed to detect mycotoxigenic fungi in pure culture and in contaminated maize. The presence of the FUM1 gene was associated with fumonisin production in most isolates, except seven that did not synthesize fumonisin but contained the gene in their genome. The PCR method allowed the direct detection of fungal contamination in ground corn and could be employed to screen for the presence of potential mycotoxigenic fusaria.     

Evaluation of conventional and organic italian foodstuffs for deoxynivalenol and fumonisins B(1) and B(2)

Two lots of human foodstuffs from conventional and organic brand foods were purchased from supermarkets and analyzed for three Fusarium toxins, deoxynivalenol, by GC-ECD, and fumonisins B(1) and B(2) (FB(1)-FB(2)), by LC-MS. The occurrence of deoxynivalenol contamination was higher than 80% in both organic and conventional foods; fumonisin B(1) was found in 20% of organic foods and in 31% of conventional ones and fumonisin B(2) in more than the 32% of the food samples from both the agricultural practices. The highest median concentration of deoxynivalenol occurred in conventional rice-based foodstuffs (207 microg/kg): that of fumonisin B(1) in conventional maize-based foods (345 microg/kg) and that of fumonisin B(2) in organic wheat-based foods (210 microg/kg).     

Mycoflora and fumonisin mycotoxins associated with cowpea [Vigna unguiculata (L.) Walp] seeds

Cowpea seed samples from South Africa and Benin were analyzed for seed mycoflora. Fusariumspecies detected were F. equiseti, F. chlamydosporum, F. graminearum, F. proliferatum, F. sambucinum, F. semitectum, and F. subglutinans. Cowpea seed from South Africa and Benin and F. proliferatum isolates from Benin, inoculated onto maize patty medium, were analyzed for fumonisin production. Samples were extracted with methanol/water and cleaned up on strong anion exchange solid phase extraction cartridges. HPLC with precolumn derivatization using o-phthaldialdehyde was used for the detection and quantification of fumonisins. Cowpea cultivars from South Africa showed the presence of fumonisin B(1) at concentrations ranging between 0.12 and 0.61 microg/g, whereas those from Benin showed no fumonisins. This is believed to be the first report of the natural occurrence of FB(1) on cowpea seed. Fumonisin B(1), B(2), and B(3) were produced by all F. proliferatum isolates. Total fumonisin concentrations were between 0.8 and 25.30 microg/g, and the highest level of FB(1) detected was 16.86 microg/g.     

Efficacy of a mycotoxin binder against dietary fumonisin, deoxynivalenol, and zearalenone in rats

It was hypothesized that a mycotoxin binder, Grainsure E, would inhibit adverse effects of a mixture of fumonisin B1, deoxynivalenol, and zearalenone in rats. For 14 and 28 days, 8-10 Sprague-Dawley rats were fed control diet, Grainsure E (0.5%), toxins (7 μg fumonisin B1/g, 8 μg of deoxynivalenol/g and 0.2 μg of zearalenone/g), toxins (12 μg of fumonisin B1/g, 9 μg of deoxynivalenol/g, and 0.2 μg of zearalenone/g + Grainsure E), or pair-fed to control for food intake of toxin-fed rats. After 28 days, decreased body weight gain was prevented by Grainsure E in toxin-fed female rats, indicating partial protection against deoxynivalenol and fumonisin B1. Two effects of fumonisin B1 were partly prevented by Grainsure E in toxin-fed rats, increased plasma alanine transaminase (ALT) and urinary sphinganine/sphingosine, but sphinganine/sphingosine increase was not prevented in females at the latter time point. Grainsure E prevented some effects of fumonisin B1 and deoxynivalenol in rats.     

Occurrence of fumonisins B(2) and B(4) in retail raisins

Concerns that raisins may be contaminated by fumonisins stem from the persistent occurrence of Aspergillus niger spores on raisins and the recent discovery of fumonisin production by A. niger on grapes, which leads to the widespread occurrence of fumonisin B(2) in wine. This study presents an LC-MS/MS survey of fumonisins in retail raisins. In 10 of 21 brands collected in Denmark, Germany, and The Netherlands, fumonisins B(2) and B(4) were detected at levels up to 13 and 1.3 μg/kg, respectively. Only fumonisin B(2) has been detected in wine, so the presence of fumonisin B(4) in raisins suggests that the fumonisins are produced mainly during the drying process concomitant with the decreasing water activity. Analysis of multiple packages from one manufacturer showed a 3-fold package-to-package variation, suggesting that a few raisins per package are contaminated.     

SA-IP-SPS型保水剂及其对土壤物理性能的影响

以未经预处理的工业级丙烯酸、聚乙烯醇为原料，首先通过氯磺酸磺化法在聚乙烯醇分子中引入强亲水性离子基团，然后采用静态水溶液聚合法合成了吸盐水率较高、凝胶机械强度较高的的聚丙烯酸钠-聚乙烯醇硫酸钠互穿网络(SA-IP-SPS）型保水剂。研究了SA-IP-SPS型保水剂含量、颗粒大小对南方赤红壤的水分抑蒸发性能和团粒结构的影响，比较研究结果表明，SA-IP-SPS型保水剂对赤红壤的水分抑蒸发性能和团粒结构改良性能与丙烯酸-丙烯酰胺交联共聚型保水剂效果相当，但明显优于传统的交联聚丙烯酸钠保水剂。