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.     

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.     

Lycopene bioaccessibility and starch digestibility for extruded snacks enriched with tomato derivatives

To improve the nutritional value of energy-dense extruded snacks, corn grits were replaced with tomato paste and/or tomato skin powder at ratios of 5, 10, and 20% and extruded to make expanded snack foodlike products. Using a model digestion system, lycopene bioaccessibility and uptake from the snacks into Caco-2 cells were determined. The digestibility of the starch, the main nutrient component of the snacks, was also investigated. While extrusion cooking reduced the lycopene content of the snacks, the proportion of bioaccessible lycopene increased. Lycopene uptake by the Caco-2 cells from the extruded snacks exceeded that of the control in which the lycopene was not extruded, by 5% (p < 0.05). The digestibility of starch in the snacks varied depending on the type of tomato derivative and its concentration. Optimization of the extrusion cooking process and the ingredients can yield functional extruded snack products that contain bioavailable lycopene.     

Determination of zearalenone in cornflakes and other corn-based foods by thin layer chromatography, high pressure liquid chromatography, and gas-liquid chromatography/high resolution mass spectrometry

A simple cleanup procedure based on pH adjustments was used to obtain extracts of corn foods. The method gave good recoveries of zearalenone determined by thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). As little as 5 ng zearalenone was detected by TLC, using Fast Violet B Salt as the spray reagent; the lower limit of detection in cornflakes was about 20 microgram/kg. With HPLC on Spherisorb silica (5 micrometer) and detection by fluorescence at an excitation maximum of 310 nm as little as 5 microgram zearalenone/kg cornflakes could be determined. While the TLC method was also applicable to corn chips, cornmeal, popcorn, and frozen corn, an interference was observed in HPLC of the latter 3 products. This interference was separated from zearalenone by adding a second HPLC analytical column (Spherisorb ODS). Gas-liquid chromatography coupled with mass spectrometric single ion monitoring at high resolution, although of limited availability, was shown to be the most sensitive and selective method for determining zearalenone in corn foods. The natural occurrence of zearalenone in a sample of cornflakes (13-20 microgram/kg) was demonstrated by all 3 detection procedures.     

Effect of Separation and Grinding of Corn Dry-Milled Streams on Physical Properties of Single-Screw Low-Speed Extruded Products

Three streams of corn dry-milled products (corn grits, corn cones, and corn flour) were sieved and separated according to average diameter, and some segregated fractions were ground to produce nine streams. Corn grits were separated to produce grits with diameters of 1.19 and 0.841 mm, and selected fractions were ground into grits with average diameters of 0.297 and 0.210 mm. Corn cones were separated into average diameters of 0.595, 0.420, and 0.297 mm. Corn flour was separated into fractions with average diameters of 0.297 and 0.210 mm. The original and the additional streams were extruded at constant speed (50 rpm) and at three different processing extruder barrel temperature profiles: low (LTP, 100-110-120°C), medium (MTP, 110-120-130°C), and high (HTP, 120-130-140°C). The least significant difference (P < 0.01) test showed that additional grinding of corn grits affects the expansion ratio of extrudates processed at LTP and HTP. Additional separation of corn flour affects (P < 0.01) the bulk density and water solubility index of extrudates at HTP. At HTP, corn cones with diameters of 0.595 mm had significantly (P < 0.01) higher torque, specific mechanical energy, and die pressure than did the original corn cone extrudates without separation.     

Enzyme-assisted extraction of moniliformin from extruded corn grits

Water has been known to be the ideal solvent for moniliformin but is not suitable to extract this toxin from cooked matrices due to instant swelling upon addition of the solvent. In this study, an improved method to extract moniliformin from extruded corn grits using alpha-amylase was developed. In an effort to optimize the method, the efficacy of using a protease was also studied. Treatment with alpha-amylase resulted in a clear solution with decreased suspended solid content as measured by transmittance (%T), which improved from 0 to 96% in 10 min. The detected level of moniliformin from extruded corn grits was increased to 4.02 mug/g when extracted with 1% tetrabutylammonium hydrogen sulfate following alpha-amylase treatment compared to 2.56 microg/g when it was extracted with 90% acetonitrile without enzyme treatment. The average recovery of moniliformin from extruded corn grits was 96% when alpha-amylase was used in the extraction procedure. Overall, the amounts of moniliformin detected in extruded corn grits increased significantly by using enzyme hydrolysis. Chromatographic separation was also benefited by lesser interference and improved peaks.     

Physical and biochemical properties of maize hardness and extrudates of selected hybrids

Protein and starch determinants of maize kernel hardness and extruded products were characterized to better define the role of endosperm texture during extrusion. Maize physical properties were correlated with total proteins and zein subclasses (p < 0.01). The extrusion process significantly altered protein solubility and increased protein fragmentation as measured by RP-HPLC and size exclusion chromatography. Harder grits and extrudates demonstrated higher amylose content, lower degree of starch damage, and fragmentation at different screw speeds than softer grits and extrudates. Differences in extrudate expansion ratio, water absorption index, water solubility index, oil absorption capacity, and breaking stress between harder and softer hybrids were related to protein aggregation and fragmentation as well as starch damage and fragmentation.     

Extrusion conditions affect chemical composition and in vitro digestion of select food ingredients

An experiment was conducted to determine the effects of extrusion conditions on chemical composition and in vitro hydrolytic and fermentative digestion of barley grits, cornmeal, oat bran, soybean flour, soybean hulls, and wheat bran. Extrusion conditions altered crude protein, fiber, and starch concentrations of ingredients. Organic matter disappearance (OMD) increased for extruded versus unprocessed samples of barley grits, cornmeal, and soybean flour that had been hydrolytically digested. After 8 h of fermentative digestion, OMD decreased as extrusion conditions intensified for barley grits and cornmeal but increased for oat bran, soybean hulls, and wheat bran. Total short-chain fatty acid production decreased as extrusion conditions intensified for barley grits, soybean hulls, and soybean flour. These data suggest that the effects of extrusion conditions on ingredient composition and digestion are influenced by the unique chemical characteristics of individual substrates.     

Effect of Temperature and Screw Speed on Stability of Fumonisin B1 in Extrusion-Cooked Corn Grits

Corn grits spiked with fumonisin B₁ (FB₁) at a level of 5 μg/g were extrusion cooked in a corotating twin-screw extruder at different temperatures (140, 160, 180, and 200°C) and screw speeds (40, 80, 120, and 160 rpm). Good recoveries of FB₁ were obtained from the nonextruded as well as the extruded grits by using high-performance liquid chromatography. Both the barrel temperature and the screw speed significantly (P ≤ 0.05) affected the extent of fumonisin reduction in extruded grits. As expected, the FB₁ recovered decreased with an increase in temperature and a decrease in screw speed. The amount of FB₁ lost from cooking grits at the different extrusion parameters used in this study ranged from 34 to 95%. About 46–76% of the spiked FB₁ was lost when the grits were cooked at temperatures and screw speeds that resulted in acceptable product expansion and color.     

Effect of Some Extrusion Variables on Residual Quantity of Cyanogenic Compounds in an Organic Breakfast Cereal Containing Passion Fruit Fiber

Organic passion fruit fiber is obtained from organic passion fruit rind and is an interesting source of dietary fiber with potential for use in food products such as breakfast cereals. However, various researchers have confirmed the presence of cyanogenic compounds in passion fruit. The objective of this study was to evaluate the effect of the thermoplastic extrusion process on the residual quantity of total cyanogenic compounds (TCC) in extruded organic breakfast cereal produced with corn flour and different levels of passion fruit fiber added to the formulation. For the production of the extrudates, a 2³ complete factorial design was followed, that permitted the analysis of the results by response surface methodology. The effects of the quantity of passion fruit fiber (0–30%), feed moisture content (18–28%) and barrel temperature (120–160°C) on the residual quantity of TCC were studied. The raw passion fruit fiber presented 748.3 mg/kg of TCC. The extruded products presented TCC contents of 0–254.1 mg/kg, increasing significantly with the increase of the quantity of passion fruit fiber. The residual quantity of TCC was influenced by feed moisture, while temperature had no significant effect on this response. Nevertheless, only a small reduction of cyanogenic compounds was verified in the breakfast cereals produced by thermoplastic extrusion. Thus, it was concluded that the toxicity of the cereal blends was not improved by the extrusion process.     

Development of a fluorescence polarization assay for the determination of aflatoxins in grains

Aflatoxins produced by Aspergillus flavus are commonly found in human and animal foods including grains, cereals, peanut products, sorghum, and soy seeds. Exposure to aflatoxins has been associated with carcinogenicity. This paper reports a simple, portable, and rapid fluorescence polarization (FP) assay for aflatoxin determination in grains. This immunoassay is field portable, homogeneous, and without any washing and cleaning steps. The assay is based upon the competition between free aflatoxin and an aflatoxin-fluorescein tracer for an aflatoxin-specific monoclonal antibody in solution. A series of naturally contaminated corn, sorghum, peanut butter, and peanut paste samples were analyzed by FP and compared with HPLC results. Similarly, spiked popcorn samples were analyzed by FP. FP results of naturally contaminated samples correlated well with HPLC (r (2) = 0.97). FP analysis of spiked popcorn samples (with a mixture of B(1)/B(2)/G(1)/G(2), 7/1/3/1, w/w) gave a good correlation with spiked values (r (2) = 0.99). However, FP consistently underestimated the aflatoxin contents. This was perhaps due to low cross-reactivity of the antibody used toward B(2), G(1), and G(2) aflatoxins. These results combined with the portability and simplicity of the assay suggest that the assay can be used for screening total aflatoxin in grains.     

Effect of industrial processing on the distribution of aflatoxins and zearalenone in corn-milling fractions

The aim of this study was to investigate the distribution of aflatoxins and zearalenone levels in various corn-milling fractions. Corn kernels and six derived milling fractions (germ, bran, large and small grits, flour, and animal feed flour) were sampled in an industrial plant; both conventional and organic corns were sampled. To evaluate the effect of cooking, samples of polenta were prepared starting from naturally contaminated flour. Conventional and organic lots showed mycotoxin contamination. For both lots, germ, bran, and animal feed flour showed a marked concentration factor from 239 to 911% accounting for both the low yields of the derived products and the distribution of aflatoxins and zearalenone contamination in the outer parts of the kernels. Conversely, a reduction factor of at least four times from raw material to finished products was observed. Polenta samples were unaffected by the cooking process, with levels of contamination similar to those of starting flour.     

Effect of Additional Separation and Grinding on the Chemical and Physical Properties of Selected Corn Dry-Milled Streams

Three streams of corn dry-milled products (corn grits, corn cones, and corn flour) from three different commercial corn dry-millers were further separated by particle size according to the major portion of each stream. They were separated into corn grits (1.190 and 0.841 mm), corn cones (0.595, 0.420, and 0.297 mm), and corn flour (0.297 and 0.210 mm). Besides separation, corn grits were also ground and then separated into ground corn grits (0.297 and 0.210 mm). The original streams, streams with additional separation, and streams with additional grinding were analyzed for protein content, ash content, crude fat content, and color properties. Duncan's significant difference tests (P < 0.01) showed that additional separation and grinding of the commercial corn grits, corn cones, and corn flour affected protein, crude fat content, and color parameter (L, a, and b) distribution of the products. The tristimulus parameters (L, a, and b) were good indicators of the protein content of the corn dry-milled streams studied.     

Liquid chromatographic determination of alpha-zearalenol and zearalenone in corn: collaborative study

The liquid chromatographic determination of alpha-zearalenol and zearalenone in corn was collaboratively studied. Each of 13 collaborators received 7 corn samples; 2 were blanks and 5 were spiked to contain 50, 100, and 200 ng alpha-zearalenol/g and 50, 100, 500, 1000, and 4000 ng zearalenone/g. Four sets (including blanks) of blind duplicates were included in the study. Five naturally contaminated corn samples (one in duplicate) were also provided. All collaborators detected both mycotoxins at 50 ng/g. Average recoveries reported by all collaborators ranged from 81.9% at 200 ng/g to 100.3% at 50 ng/g for alpha-zearalenol and from 77.8% at 1000 ng/g to 123% at 50 ng/g for zearalenone. Three collaborators reported false positives for both alpha-zearalenol and zearalenone. The within-laboratory CV values based on blind duplicates were 22.6% for alpha-zearalenol and 31.4% for zearalenone. The CV values based on laboratory-sample interaction were 25.6 and 33.8% for alpha-zearalenol and zearalenone, respectively. The CV values for naturally contaminated samples (including duplicates) were 47.0% for alpha-zearalenol and 37.7% for zearalenone. The method has been adopted official first action.     

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.     

Factors Affecting Expansion of Corn Meals with Poor and Good Expansion Properties

Two corn meals, one with good and one with poor expansion properties, were used to study the critical factors responsible for poor expansion during corn curl extrusion. Screening tests revealed that the corn meal with poor expansion had a slightly larger particle size. This sample also had a larger proportion of opaque particles compared to the corn meal with good expansion. Extrusion of coarse corn grits showed that larger particle size alone could cause poor expansion. Water diffusion tests showed that the sample containing more opaque particles was more competitive for water. As a result, in corn that contained both opaque and vitreous particles less water was available to the vitreous particles. The underplastisized (dry) vitreous particles remained glassy (unmelted) during extrusion, resulting in reduced expansion of the extrudates. The results suggest that addition of water to the conditioning cylinder of the extruder would overcome poor expansion.     

Determination of zearalenone in corn: collaborative study.

Corn samples spiked at levels of 100, 300, 1000, and 2000 mug zearalenone/kg were sent to 22 collaborators for analysis by the Eppley method. All samples were yellow corn except one white corn sample spiked at 2000 mug/kg. Results from 16 collaborators were statistically analyzed. Only 4 of 16 collaborators detected zearalenone in the sample containing 100 mu/kg, but 11 detected the toxin in the sample containing 300 mug/kg. Average recoveries from all samples were 129% at 300 mug/kg, 101% at 1000 mug/kg, and 88% at 2000 mug/kg. The between-laboratory coefficients of variation were 53.0% at 300 mug/kg, 38.2% at 1000 mug/kg, and 27.0% at 2000 mug/kg. Five naturally contaminated corn samples, one in triplicate, were also provided. The mean level of zearalenone in the naturally contaminated samples ranged from 431 to 7622 mug/kg. The mean coefficient of variation for all samples was 40.5%. Two collaborators measured quantities of zearalenone on thin layer chromatographic plates densitometrically. Their results were not included in the statistical analysis, but the results indicated that densitometric measurement, given proper dilutions of solutions, could be used. The method has been adopted as official first action.     

Extrusion of Cross‐Linked Hydroxypropylated Corn Starches II. Morphological and Molecular Characterization

A series of cross‐linked hydroxypropylated corn starches were extruded with a Leistritz micro‐18 co‐rotating extruder. Extrusion process variables including moisture (30, 35, and 40%), barrel temperature (60, 80, and 100°C), and screw design (low, medium, and high shear) were investigated. Scanning electron microscopy (SEM) of extruded starches showed a gel phase with distorted granules and granule fragments after extrusion at 60°C. After extrusion at 100°C only a gel phase was observed with no granular structures remaining. High performance size exclusion chromatography (HPSEC) equipped with multiangle laser light‐scattering (MALLS) and refractive index (RI) detectors showed extruded starches degraded to different extents, depending on extrusion conditions. The average molecular weight of the amylopectin of unextruded native corn starch was 7.7 × 10⁸. Extrusion at 30% moisture, 100°C, and high shear reduced the molecular weight of amylopectin to 1.0 × 10⁸. Hydroxypropylated normal corn starch extruded at identical conditions showed greater decreases in amylopectin molecular weight. With the addition of cross‐linking, the amylopectin fractions of the extruded starches were less degraded than those of their native and hydroxypropylated corn starch counterparts. Similarly, increasing moisture content during extrusion lowered amylopectin degradation in the extruded starches. Increasing temperature during extrusion of cross‐linked hydroxypropylated starches at high moisture content (e.g., 40%) lowered amylopectin molecular weights of the extruded starches, whereas increasing extrusion temperature at low moisture content (30%) resulted in less degraded molecules. This difference was attributed to the higher glass transition temperatures of the cross‐linked starches.     

Determination of N-(carboxymethyl)fumonisin B(1) in corn products by liquid chromatography/electrospray ionization--mass spectrometry

It is well-known that fumonisin B(1) (FB(1)) in corn meal decreases during baking, frying, and cooking, but it is still not exactly clear how heating affects the formation of N-(carboxymethyl)fumonisin B(1) (NCM-FB(1)), the reaction product of FB(1) and reducing sugars. In model experiments corn grits were spiked with FB(1) (2 mg/kg) and D-glucose (50 g/kg) or sucrose (50 g/kg) and manufactured into extrusion products at various temperatures (160--180 degrees C) and moisture levels (16--20%). A liquid chromatography/electrospray ionization-mass spectrometry method using isotopically labeled fumonisin FB(1)-d(6) as an internal standard was developed for the determination of NCM-FB(1). For sample cleanup solid-phase C18 cartridges were used. The detection limit achieved with this method was 10 ng/g (signal-noise ratio = 3:1) using the protonated molecule [M + H](+) signal of NCM-FB(1) (m/z 780) in the selected ion monitoring mode. Low concentrations of NCM-FB(1) (29-97 ng/g) were detected in all samples spiked with D-glucose and FB(1), whereas those spiked with FB(1) and sucrose showed only NCM-FB(1) in samples produced at 180 degrees C (NCM-FB(1) = 27 ng/g). Various corn-containing food samples from the German market were analyzed for the presence of NCM-FB(1), FB(1), and hydrolyzed fumonisin B(1) (HFB(1)). All samples were contaminated with FB(1) (22--194 ng/g) and HFB(1) (5--247 ng/g). Six of nine samples contained NCM-FB(1) in low concentrations ranging from 10 to 76 ng/g. From these data and the low toxicity of NCM-FB(1) it can be concluded that the significance of NCM-FB(1) in food seems to be a minor one.     

Functionality Behavior of Raw and Extruded Corn Starch Mixtures

Relationships between the structural properties of raw and extruded corn starches and their functionalities were investigated using mixtures of these starch types. Extruded starch had higher water absorption and water solubility indices, and produced lower RVA viscosity profiles when compared with raw starch. It also had no differential scanning calorimetry (DSC) endotherm. Gel cohesiveness and adhesiveness of both starch types were similar, while extruded starch gels were softer. Extruded starch produced lower Rapid Visco Analyser (RVA) viscosity profiles than raw starch due to starch degradation during extrusion. The raw and extruded starch components had negative interaction coefficients, thus RVA viscosity parameters were lowered as the fraction of extruded starch in the mixture increased. Starch degradation in the extruded starch was a likely significant factor associated with low viscosity profiles. Mixtures of raw and extruded starches could be commercially prepared to obtain finished starch products with a range of functional attributes.