Evaluation of reduced toxicity of zearalenone by extrusion processing as measured by the MTT cell proliferation assay

The objective of this study was to determine loss of toxicity of zearalenone in extruded cereal-based products by the MTT (tetrazolium salt) cell proliferation assay using a sensitive MCF-7 human breast cancer cell line and to compare the results to chemical (high-performance liquid chromatography, HPLC) and biochemical (enzyme-linked immunosorbent assay, ELISA) methods of analysis. A split-split plot design was used for the extrusion process experiments at temperatures of 150, 175, and 200 degrees C and screw speeds of 70 and 140 rpm. The initial zearalenone concentration in the artificially contaminated corn grits with Fusarium graminearum was found at a mean concentration of 37.88 microg/g as measured by HPLC. The percent reductions of zearalenone in the contaminated corn grits upon extrusion processing were in the ranges of 67-81, 60-72, and 66-78% as measured by HPLC, ELISA, and the MTT cell proliferation assay, respectively. The MTT cell proliferation assay results were more closely correlated with HPLC results (r = 0.96) than ELISA results (r = 0.83). The MTT cell proliferation assay was demonstrated to be a useful method for quantification of zearalenone as well as a potential toxicity screening method for contaminated extruded cereal-based products.     

Liquid chromatographic determination and stability of the Fusarium mycotoxin moniliformin in cereal grains

Moniliformin is a mycotoxin produced by Fusarium subglutinans and other Fusarium species. A rapid, liquid chromatographic method for its determination in corn and wheat is described. Samples are extracted in acetonitrile-water (95 + 5); following defatting with n-hexane, an aliquot of the extract is evaporated and cleaned up on small C18 and neutral alumina columns successively. Reverse-phase liquid chromatography (LC) is conducted on a C18 column with 10 or 15% methanol or acetonitrile in aqueous ion-pair reagent as mobile phase, with detection by ultraviolet absorption at 229 and 254 nm. Average recoveries of moniliformin (potassium salt) added to ground corn and wheat at levels of 0.05-1.0 micrograms/g were 80% (n = 20) and 85% (n = 12), respectively, and the limit of detection was ca 0.01-0.18 micrograms/g, depending on LC conditions. Analysis of 24 samples of wheat, 4 samples of rye, and 12 samples of corn showed moniliformin in only 2 corn samples (0.06 and 0.2 micrograms/g). Moniliformin was also detected in a sample of artificially damaged (slashed) corn (0.2 micrograms/g) and selected kernels of corn that were field-inoculated with F. subglutinans and F. moniliforme (50 micrograms/g and 0.5 micrograms/g, respectively). In stability studies, moniliformin (potassium salt, 1 microgram/g) in ground corn and ground wheat heated at 50, 100, and 150 degrees C for 0.5-2 h decomposed moderately, e.g., 55% remained in corn after 0.5 h at 100 degrees C.     

Effect of extrusion temperature on the solubility and molecular weight of lentil bean flour proteins containing low cysteine residues

Lentil flour was extruded at die temperatures of 135, 160, and 175 degrees C. The soluble protein content in the extrudates decreased by 40.1% in the extracting buffer (1% sodium dodecyl sulfate in 50 mM sodium phosphate buffer, pH 6.9) as the extrusion die temperature was increased to 175 degrees C. The most insoluble proteins in the extrudates extruded at die temperatures of up to 175 degrees C could be resolubilized by using sonication. The total disulfide content and sulfhydryl content in the extrudates decreased. The SDS-PAGEs showed that the molecular weight distribution of proteins in the lentil flour changed little before and after extrusion as well as during reduction. The results from this study show that the extrusion temperature had less effect on the solubility and molecular weight of the lentil proteins, which contain a lower level of cysteine residues than wheat proteins.     

Susceptibility of wheat and Aspergillus niger phytases to inactivation by gastrointestinal enzymes

The activity of wheat and Aspergillus niger phytases was determined following preincubation for 60 min at 37 degrees C alone or in the presence of pepsin or pancreatin to examine their ability to survive in the gastrointestinal tract. At pH 3.5 both phytases were stable, but at pH 2.5 wheat phytase rapidly lost activity. Following preincubation at pH 3.5 in the presence of 5 mg of pepsin/mL, A. niger phytase retained 95% of its original activity, whereas only 70% of the wheat phytase activity was recovered. The stability of A. niger phytase in the presence of pepsin was the same at pH 2.5 as at pH 3.5. Results similar to those with pepsin at pH 3.5 were obtained following preincubation of the phytases in the presence of pancreatin at pH 6.0.     

Rapid thin layer chromatographic determination of zearalenone in corn, sorghum, and wheat

A rapid method is described for determining zearalenone in corn, sorghum, and wheat. The mycotoxin is extracted with a mixture of acetonitrile and 4% KCl in HCl. The extract is cleaned up with isooctane, evaporated, and redissolved in chloroform. Zearalenone is separated by thin layer chromatography; identity is confirmed with various developing solvents and spray reagents. Zearalenone is then quantitated by the limit detection method. The minimum detectable concentration is 140-160 micrograms/kg when aluminum chloride solution is used as spray reagent, and 85-110 micrograms/kg when Fast Violet B salt is used as spray reagent.     

Gelling properties of heat-denatured beta-lactoglobulin aggregates in a high-salt buffer

Thermal denaturation, rheological, and microstructural properties of gels prepared from native beta-lactoglobulin (beta-LG) and preheated or heat-denatured beta-LG (HDLG) aggregates were compared. The HDLG was prepared by heating solutions of 4% beta-LG in deionized water, pH 7.0, at 80 degrees C for 30 min and then diluted to the desired concentration in 0.6 M NaCl and 0.05 M phosphate buffer at pH 6.0, 6.5, and 7.0. When reheated to 71 degrees C, HDLG formed a gel at a concentration of 2% protein. At pH 7.0, 3% HDLG gelled at 52.5 degrees C and had a storage modulus (G') of 2200 Pa after cooling. beta-LG (3%) in 0.6 M NaCl and 0.05 M phosphate buffer, pH 7.0, did not gel when heated to 71 degrees C. The gel point of 3% HDLG decreased by 10.5 degrees C and the G' did not change when the pH was decreased to 6.0. The HDLG gel microstructure was composed of strands and clumps of small globular aggregates in contrast to beta-LG gels, which contained a particulate network of compacted globules. The HDLG formed a gel at a lower concentration and lower temperature than beta-LG in the high-salt buffer, suggesting an application in meat systems or other food products prepared with salt and processed at temperatures of < or =71 degrees C.     

Occurrence of zearalenone-4-beta-D-glucopyranoside in wheat

An LC-MS method was developed for the analysis of zearalenone-4-beta-D-glucopyranoside and zearalenone in wheat (Triticum aestivum). The limit of determination for zearalenone-4-beta-D-glucopyranoside and zearalenone was 10 microg/kg. The recovery rates were calculated to be 69% and 89% at a concentration of 100 microg/kg for zearalenone-4-beta-D-glucopyranoside and zearalenone, respectively. Twenty-four Bavarian wheat samples from a 1999 harvest were analyzed. Zearalenone was present in 22 of 24 field samples, the levels ranged from 11 to 860 microg/kg. Zearalenone-4-beta-D-glucopyranoside was found in 10 of the zearalenone positive samples (42%) at levels ranging from 17 to 104 microg/kg. The amounts of zearalenone-4-beta-D-glucopyranoside were correlated to those of zearalenone (r2 = 0.86, b = 0.10). After gastrointestinal hydrolyzation, zearalenone-4-beta-glucopyranoside might be implicated in the development of a zearalenone-syndrome. Therefore, more attention should be focused on conjugated mycotoxins in food and feed.     

Formation of peptide-bound Heyns compounds

The reaction of the Nalpha-hippuryllysine (BzGK) with fructose was investigated in two model systems to obtain an insight in fructose-induced modification of lysine in bakery products. After BzGK and fructose had been heated in a buffered low-moisture model system (80 degrees C, 60 min, aW = 0.86, pH 7.4), formation of epimeric Heyns compounds Nalpha-hippuryl-Nepsilon-glucosyl-lysine (BzGGlcK) and Nalpha-hippuryl-Nepsilon-mannosyl-lysine (BzGManK) was clearly demonstrated using RP-HPLC with UV as well as MS detection. The Amadori compound Nalpha-hippuryl-Nepsilon-fructosyl-lysine (BzGFruK) was formed in glucose-containing samples. When BzGK was added to the dough of fructose-containing biscuits, the Heyns compounds were detectable after baking at 175 degrees C for 7 min. The yields of the Heyns compounds in the fructose-containing biscuits and the yield of the Amadori compound in the glucose-containing biscuits were determined to 33 and 63%, pointing to the fact that formation of substantial amounts of Heyns products is very likely in fructose-containing bakery products.     

Rapid screening method for deoxynivalenol in agricultural commodities by fluorescent minicolumn

A rapid screening procedure based on the selective adsorption of deoxynivalenol (DON) from extracts of wheat and corn has been developed. DON is extracted from the sample with acetonitrile-water (85 + 15) and partially purified on a preparative minicolumn. Solvent is evaporated and the residue is dissolved in toluene-acetone (95 + 5) and chromatographed on a novel detector minicolumn which selectively adsorbs DON. A blue fluorescence is produced when the column is heated 5 min at 100 degrees C. The procedure is capable of detecting DON at greater than or equal to 500 ng/g. Forty-three wheat samples, contaminated with DON at 60-6300 ng/g, were assayed by gas chromatography-mass spectroscopy (GC-MS) of the heptafluorobutyryl derivative of DON and by the selective adsorption procedure. Comparison of results showed 91% agreement between data from the 2 methods. Selective adsorption assays were positive for all samples that were greater than or equal to 500 ng/g by GC-MS (no false negatives) and were negative for 85% of samples less than 500 ng/g (4/27 false positives). These four samples contained greater than 200 ng/g by GC-MS. Samples of wheat (64), corn (23), soybeans (8), and sorghum (6) were extracted and extracts were assayed by thin-layer chromatography and the selective adsorption procedure. Selective adsorption assays agreed with TLC results.     

Sephadex LH-20 cleanup, high pressure liquid chromatographic assay, and fluorescence detection of zearalenone in animal feeds

A high pressure liquid chromatographic (HPLC) method is described to determine zearalenone in animal feeds at levels as low as 0.01 ppm. Samples are extracted with chloroform-ethanol and initially purified using a SEP-PAK silica cartridge, followed by column chromatography using Sephadex LH-20. Separation by normal phase HPLC is followed by fluorescence detection. Recoveries at levels of 1.0-0.01 ppm averaged greater than 90%. Confirmation included HPLC analysis of the sample and a zearalenone standard, using 3 different excitation wavelengths, and comparison of fluorescence responses obtained. The method was successfully applied to the analysis of 1 corn and 3 cornmeal samples. Zearalenone was detected in all 4 samples at levels of 0.379-19.2 ppm.     

Survey of 1975 wheat and soybeans for aflatoxin, zearalenone, and ochratoxin

Wheat samples (102 lots) were collected from Virginia, North Carolina, southeastern Missouri, southern Illinois, and Kentucky. Soybean samples (180 lots) were collected from Virginia, Illinois, Iowa, Minnesota, Nebraska, Alabama, Arkansas, and Texas. Samples of both commodities were analyzed for zearalenone, aflatoxin, and ochratoxin by the Eppley method. None of the 3 mycotoxins was detected in soybeans. Aflatoxins and ochratoxin A were not detected in wheat, but zearalenone was detected in 19 of 42 samples collected in Virginia. Half of the Virginia samples were collected because they were mold-damaged. Zearalenone levels ranged from 0.36 to 11.05 ppm; the identity of the zearalenone was confirmed by gas-liquid chromatography and mass spectroscopy. Gibberella zea infection (6-60%) was detected in all of the zearalenone-positive samples; 6-60% of the kernels in the samples tested contained G. zea.     

Reduction in fusarium toxin levels in corn silage with low dry matter and storage time

Under unfavorable climatic conditions, Fusarium spp. can contaminate corn plants in the field and produce toxins that are present at the time of ensiling. The stability of deoxynivalenol, fumonisins B1 and B2, and zearalenone in corn silage was tested over two consecutive years. Variables studied were corn dry matter (DM) and storage length and temperature. The concentration of all Fusarium toxins decreased upon ensiling ( P < 0.001). Increasing the length of storage and ensiling with low DM resulted in a higher rate of toxin disappearance, particularly for the water soluble toxins deoxynivalenol and fumonisin B1. Toxin disappearance ranged from 50% for zearalenone to 100% for deoxynivalenol. In contrast, temperature did not have any effect on stability ( P > 0.05). These results indicate that low DM at ensiling as well as a prolonged storage could be a practical way to reduce or eliminate some Fusarium toxins in contaminated silages.     

High pressure liquid chromatographic determination of zearalenone in chicken tissues

A method is reported for the extraction and analysis of zearalenone in chicken fat, heart muscle, and kidney tissue by using high pressure liquid chromatography (HPLC). Zearalenone is extracted with acetonitrile, cleaned up with hexane, and extracted further with ethyl acetate. Zearalenone is determined by HPLC using a reverse phase radial compression separation system, an ultraviolet absorbance detector, and a mobile phase of acetonitrile-water (60 + 40) (v/v). Recoveries of zearalenone added at levels from 50 to 200 ng/g are in the range 82.6-95.1%.     

Impact of Fusarium culmorum on the polysaccharides of wheat flour

To assess the effects of Fusarium infection on the polysaccharides of winter wheat grain (Triticum aestivum L.), grain samples obtained from plants artificially inoculated with Fusarium culmorum were analyzed. Microscopy revealed obvious damage to the starch granules in the seriously infected samples. The Fusarium infection had no analytically detectable influence on the starch and total insoluble dietary fiber content of the wheat grain. There were significantly positive relationships between alpha-amylase activity, cellulase activity, total soluble dietary fiber content, pentosan content, and degree of infection quantified by an enzyme-linked immunosorbant assay, which would indicate the importance of fungal enzymes. A distinct higher Hagberg falling number (FN) was determined in the seriously infected samples, while the viscosity and sucrose content of the flour decreased. However, the addition of a liquid medium contaminated with F. culmorum led to a significant decrease in the FN. Depending on the type of buffer used, the alpha-amylase of F. culmorum demonstrated its maximum activity between pH 5.5 and pH 7.0 at 30-50 degrees C. Remarkably, this fungal alpha-amylase showed a thermostable characteristic and was active over a wide range of temperatures, from 10 to 100 degrees C. This type of thermostability suggests that the alpha-amylase of F. culmorum may damage starch granules throughout the processing of wheat flour, thereby inducing weak dough properties and unsatisfactory bread quality.     

Confirmation of reduced toxicity of deoxynivalenol in extrusion-processed corn grits by the MTT bioassay

The objective of this study was to determine the loss of toxicity of deoxynivalenol in extruded cereal-based products by the tetrazolium salt (MTT) bioassay using a sensitive Chinese hamster ovary (CHO-K1) cell line and to compare the results to chemical (high-performance liquid chromatography, HPLC) and biochemical (enzyme-linked immunosorbant assay, ELISA) methods of analysis. A split-split plot design was used for the extrusion process experiments at temperatures of 150, 175, and 200 degrees C and screw speeds of 70 and 140 rpm. The initial mean deoxynivalenol concentration in the corn grits artificially contaminated with Fusarium graminearum was found to be 23.5 mug/g as measured by HPLC. The percent reductions of deoxynivalenol in the contaminated corn grits upon extrusion processing ranged from 22 to 35%, from 21 to 34%, and from 21 to 37% as measured by HPLC, ELISA, and MTT bioassay, respectively. The MTT bioassay results were more closely correlated with HPLC (r = 0.90) results than with ELISA results (r = 0.78). The MTT bioassay, using a sensitive mammalian cell line, was demonstrated to be a useful method for quantification of deoxynivalenol as well as a potential toxicity screening method for contaminated extruded cereal-based products.     

Screening method for the detection of aflatoxin, ochratoxin, zearalenone, penicillic acid, and citrinin.

A modification of the official method for ochratoxins and a screening method for zearalenone, aflatoxin, and ochratoxin is described and expanded to include citrinin and penicillic acid. The method uses 0.5N phosphoric acidchloroform (1+10) in the initial extraction; the extract is divided and eluted from 2 columns to provide a quantitative thin layer chromatographic (TLC) method for aflatoxin and ochratoxin in corn and dried beans. Aflatoxin and zearalenone are eluted from one column and ochratoxin, penicillic acid, and citrinin from the other. Ochratoxin A recoveries are low (50%) in peanuts. Zearalenone, penicillic acid, and citrinin were qualitatively recovered from corn and beans; zearalenone and penicillic acid were recovered from peanuts but citrinin was not. Several TLC solvents were used to separate interferences.     

Liquid chromatographic determination of zearalenone and zearalenol in animal feeds and grains, using fluorescence detection

A liquid chromatographic (LC) method was developed for the determination of zearalenone and zearalenol in grains and mixed animal feeds. Samples are extracted with chloroform and purified by a base-acid liquid-liquid partition. Zearalenone and zearalenol are separated by reverse phase LC and determined by fluorescence detection, excitation wavelength 236 nm with a 418 nm cutoff filter. The method was applied to the determination of zearalenone and zearalenol in 395 survey samples of corn, oats, barley, sorghum, silage, and finished feeds. The limit of detection is 10 ng/g for both toxins. The range of naturally occurring toxins found was 10-4,000 ng/g. Average recoveries were 84% for zearlenone and 69% for zearalenol. Coefficients of variation were 24.6% for zearalenone and 30.8% for zearalenol for crop year 1980, and 28.3% for zearalenone and 22.0% for zearalenol for crop year 1981.     

Effect of pH, temperature, and moisture on the formation of volatile compounds in glycine/glucose model systems

Mixtures of glycine, glucose, and starch were extrusion cooked using sodium hydroxide at 0, 3, and 6 g/L of extruder water feed, 18% moisture, and 120, 150, and 180 degrees C target die temperatures, giving extrudates with pH values of 5.6, 6.8, and 7.4. Freeze-dried equimolar solutions of glucose and glycine were heated either dry or after equilibration to approximately 13% moisture at 180 degrees C in a reaction-tube system designed to mimic the heating profile in an extruder. Volatile compounds were isolated onto Tenax and analyzed by gas chromatography-mass spectrometry. For the extrudates, total yields of volatiles increased with decreasing pH at 180 degrees C, reached a maximum at pH 6.8 at 150 degrees C, and increased with increasing pH at 120 degrees C. Amounts increased with temperature at all pH values. Pyrazines were the most abundant class for all sets of conditions (54-79% of total volatiles). Pyrroles, ketones, furans, oxazoles, and pyridines were also identified. Yields of volatiles from the reaction-tube samples increased by >60% in the moist system. Levels of individual classes also increased in the presence of moisture, except pyrazines, which decreased approximately 3.5-fold. Twenty-one of the compounds were common to the reaction-tube samples and the extrudates.     

Survey for aflatoxins and zearalenone in 1973 crop corn stored on farms and in country elevators.

A total of 315 marketable and 57 obviously damaged corn samples were collected at 116 different farms and country elevators located in the United States in countries selected from among those producing more than 1 million bushels of corn in 1972. The samples were analyzed for aflatoxins and zearalenone. The most striking correlations observed were between geographical area and mycotoxin contamination. Aflatoxin contamination was most frequently encountered in the Southeast-Appalachia areas with a 44% incidence of marketable corn with detectable aflatoxins. Zearalenone was most frequently encountered in the Corn Belt with 10% incidence in marketable corn from that region. When mycotoxin contamination was found in an establishment, most of the samples from that establishment were contaminated. There was no correlation between mycotoxin contamination and storage practices nor could the observed contamination of marketable corn be related to the contamination of the obviously damaged grain. These observations plus correlations with the geographic incidence and aflatoxin level distribution of published field contamination data suggest the possibility of a common contamination mode.     

Effect of heat treatment on the antioxidant activity of extracts from citrus peels

The effect of heat treatment on the antioxidant activity of extracts from Citrus unshiu peels was evaluated. Citrus peels (CP) (5 g) were placed in Pyrex Petri dishes (8.0 cm diameter) and heat-treated at 50, 100, or 150 degrees C for 10, 20, 30, 40, 50, and 60 min in an electric muffle furnace. After heat treatment, 70% ethanol extract (EE) and water extract (WE) (0.1 g/10 mL) of CP were prepared, and total phenol contents (TPC), radical scavenging activity (RSA), and reducing power of the extracts were determined. The antioxidant activities of CP extracts increased as heating temperature increased. For example, heat treatment of CP at 150 degrees C for 60 min increased the TPC, RSA, and reducing power of EE from 71.8 to 171.0 microM, from 29.64 to 64.25%, and from 0.45 to 0.82, respectively, compared to non-heat-treated control. In the case of WE from CP heat-treated at the same conditions (150 degrees C for 60 min), the TPC, RSA, and reducing power also increased from 84.4 to 204.9 microM, from 15.81 to 58.26%, and from 0.27 to 0.96, respectively. Several low molecular weight phenolic compounds such as 2,3-diacetyl-1-phenylnaphthalene, ferulic acid, p-hydroxybenzaldoxime, 5-hydroxyvaleric acid, 2,3-diacetyl-1-phenylnaphthalene, and vanillic acid were newly formed in the CP heated at 150 degrees C for 30 min. These results indicated that the antioxidant activity of CP extracts was significantly affected by heating temperature and duration of treatment on CP and that the heating process can be used as a tool for increasing the antioxidant activity of CP.