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Production of ethanol and xylitol from corn cobs by yeasts.

Saccharomyces cerevisiae and Candida tropicalis were used separately and as co-culture for simultaneous saccharification and fermentation (SSF) of 5-20% (w/v) dry corn cobs. A maximal ethanol concentration of 27, 23, 21 g/l (w/v) from 200 g/l (w/v) dry corn cobs was obtained by S. cerevisiae, C. tropicalis and the co-culture, respectively, after 96 h of fermentation. However, theoretical yields of 82%, 71% and 63% were observed from 50 g/l dry corn cobs for the above cultures, respectively. Maximal xylitol concentration of 21, 20 and 15 g/l from 200 g/l (w/v) dry corn cobs was obtained by C. tropicalis, co-culture, and S. cerevisiae, respectively. Maximum theoretical yields of 79.0%, 77.0% and 58% were observed from 50 g/l of corn cobs, respectively. The volumetric productivities for ethanol and xylitol increased with the increase in substrate concentration, whereas, yield decreased. Glycerol and acetic acid were formed as minor by-products. S. cerevisiae and C. tropicalis resulted in better product yields (0.42 and 0.36 g/g) for ethanol and (0.52 and 0.71 g/g) for xylitol, respectively, whereas, the co-culture showed moderate level of ethanol (0.32 g/g) and almost maximal levels of xylitol (0.69 g/g).

Acetic Acid↗

Process and economic evaluation of the extraction and purification of recombinant beta-glucuronidase from transgenic corn

A process model for the recovery and purification of recombinant beta-glucuronidase (rGUS) from transgenic corn was developed, and the process economics were estimated. The base-case bioprocessing plant operates 7500 h/year processing 1.74 million (MM) kg of transgenic corn containing 0.015% (db) rGUS. The process consists of milling the corn into flour, extraction of protein by using 50 mM sodium phosphate buffer, and rGUS purification by ion exchange and hydrophobic interaction chromatography. About 137 kg of rGUS of 83% (db) purity can be produced annually. The production cost amounted to $43 000/kg of rGUS. The cost of milling, protein extraction, and rGUS purification accounted for 6, 40, and 48% of annual operating cost, respectively. The cost of transgenic corn was 31% of the raw material costs or 6% of the annual operating cost. About 78% of the cost of buffer and water were incurred in the protein extraction section, while 88% of other consumables were from the purification section. The sensitivity analysis indicated that rGUS can be produced profitably from corn even at the 0.015% (db) expression level, assuming a selling price of $100 000/kg GUS. An increase in rGUS expression levels up to 0.08% significantly improves the process economics.

Journal Article↗

Mycoflora and fumonisin contamination in Brazilian corn from sowing to harvest.

The present study aimed to analyze the mycoflora and potential mycotoxin contamination of soil and corn samples collected at different plant maturity stages in Capão Bonito and Ribeirão Preto, two regions of the State of São Paulo, Brazil. In addition, the data obtained were correlated with the occurrence of wind-dispersed fungi and the predominant climatic conditions of the two regions studied. Corn mycoflora profiles showed that Fusarium verticillioides prevailed in 35% of the samples from Capão Bonito and in 49% of the samples from Ribeirão Preto. Examination of wind-dispersed fungi also revealed a high incidence of F. verticillioides. Soil mycoflora analyses showed that Penicilliumwas the most prevalent genus, although F. verticillioides was present in 55.5% of Capão Bonito's samples and in 26.7% of Ribeirão Preto's samples. With respect to water activity, the corn kernels most contaminated with F. verticillioides had water activity levels of 0.70-0.80. HPLC analysis of fumonisins revealed that 88.5% of Capão Bonito's kernels were contaminated with fumonisin B(1) (FB(1)) (0.09-10.87 microg/g) and 53.8% with fumonisin B(2) (FB(2)) (0.05-0.52 microg/g); Ribeirão Preto's kernels presented contamination levels of 93.5% for FB(1) (0.11-17.69 microg/g) and 61.3% for FB(2) (0.05-5.24 microg/g). No aflatoxins were detected by thin-layer chromatography in corn grains of either region. The concomitant occurrence of F. verticillioides and fumonisins in most of the field corn assayed demonstrates the importance of an effective control of cultivation throughout the plant maturity stages.

Aspergillus flavus↗

Fumonisin contamination and fusarium incidence in corn from Santa Catarina, Brazil.

In Brazil, the southern region has the highest incidence of esophageal cancer and also the highest production and consumption of corn (Zea mays) products. Corn samples intended for human consumption from the western, northern, and southern regions of the state of Santa Catarina, southern Brazil, had mean total fumonisin B (B(1), B(2), and B(3)) levels of 3.2, 3.4, and 1.7 mg/kg, respectively. Fusarium verticillioides, the predominant fungus in the corn samples, had mean incidences (percent of kernels infected) of 14, 11, and 18% for the three regions, respectively. Additional corn samples intended for animal feed from the southern region had a mean total fumonisin level of 1.5 mg/kg and a mean F. verticillioides incidence of 10%. The fumonisin levels in corn from the state of Santa Catarina, Brazil, were similar to the high levels determined in other high esophageal cancer incidence regions of the world.

Animal Feed↗

Evaluation of Bt (Bacillus thuringiensis) corn on mouse testicular development by dual parameter flow cytometry.

The health safety of Bt (Bacillus thuringiensis) corn (Zea mays L.) was studied using mouse testes as a sensitive biomonitor of potential toxic effects. Pregnant mice were fed a Bt corn or a nontransgenic (conventional) diet during gestation and lactation. After they were weaned, young male mice were maintained on the respective diets. At 8, 16, 26, 32, 63, and 87 days after birth, three male mice and an adult reference mouse were killed, the testes were surgically removed, and the percentage of germ cell populations was measured by flow cytometry. Multigenerational studies were conducted in the same manner. There were no apparent differences in percentages of testicular cell populations (haploid, diploid, and tetraploid) between the mice fed the Bt corn diet and those fed the conventional diet. Because of the high rate of cell proliferation and extensive differentiation that makes testicular germ cells highly susceptible to some toxic agents, it was concluded that the Bt corn diet had no measurable or observable effect on fetal, postnatal, pubertal, or adult testicular development. If data from this study were extrapolated to humans, Bt corn is not harmful to human reproductive development.

Animals↗

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.

Chromatography, High Pressure Liquid↗

Influence of drying temperature, water content, and heating rate on gelatinization of corn starches.

The gelatinization properties of starch extracted from corn and waxy corn dried at different temperatures were determined at various water contents and heating rates by differential scanning calorimetry. All gelatinization transition temperatures increased with drying temperature and heating rate. Onset and peak temperatures remained relatively constant, whereas end temperature decreased in the presence of excess water. The gelatinization enthalpy (deltaH(g)) of corn starch decreased with drying temperature at 50% water; however, it remained constant for waxy corn starch. The effects of water content and heating rate on deltaH(g) were dependent on each other. The minimum water levels required for gelatinization of starch extracted from corn dried at 20 and 100 degrees C are 21 and 29%, respectively. The activation energy (E(a)) was calculated using an Arrhenius-type equation and two first-order models; the degree of conversion (alpha) was predicted using a newly proposed model that produced good results for both E(a) and alpha.

Calorimetry, Differential Scanning↗

Corn silk induces nitric oxide synthase in murine macrophages.

Corn silk has been purified as an anticoagulant previously and the active component is a polysaccharide with a molecular mass of 135 kDa. It activates murine macrophages to induce nitric oxide synthase (NOS) and generate substantial amounts of NO in time and dose-dependent manners. It was detectable first at 15 h after stimulation by corn silk, peaked at 24 h, and undetectable by 48 h. Induction of NOS is inhibited by pyrolidine dithiocarbamate (PDTC) and genistein, an inhibitor of nuclear factor kappa B (NF-kappaB) and tyrosine kinase, respectively, indicating that iNOS stimulated by corn silk is associated with tyrosine kinase and NF-kappaB signaling pathways. IkappaB-alpha degradation was detectible at 10 min, and the level was restored at 120 min after treatment of corn silk. Corn silk induced nuclear translocation of NF-kappaB by phosphorylation and degradation of IkappaB-alpha.

Animals↗

ABE production from corn: a recent economic evaluation.

This article details an economic assessment of butanol production from corn using the newly developed hyper-butanol-producing strain of Clostridium beijerinckii BA101. Butanol is produced in batch reactors and recovered by distillation. For a plant with 153,000 metric tons of acetone, butanol, and ethanol (ABE) production capacity, the production equipment cost and total working capital cost is US$33.47x10(6) and US$110.46x10(6), respectively. Based on a corn price (C(p)) of US$79.23 x ton(-1) (US$2.01 x bushel(-1)), an ABE yield of 0.42 (g ABE/g glucose) butanol price is projected to be US$0.34 x kg(-1). An improved yield of 0.50 will reduce this price to US$0.29 x kg(-1). Assumptions, such as by-product credit for gases and complete conversion of corn steep liquor (CSL) to fermentation by-products, have been taken into consideration. An increased price of corn to US$197.10 x ton(-1) would result in a butanol price of US$0.47 x kg(-1). A grass-rooted plant would result in a butanol price of US$0.73 x kg(-1) (C(p) US$79.23 x ton(-1)). In a worst case scenario, the price of butanol would increase to US$1.07 x kg(-1) (C(p) 197.10 x ton(-1) for a grass-rooted plant and assuming no credit for gases). This is based on the assumption that corn price would not increase to more than US$197.10 x ton(-1).

Acetone↗

A quantitative risk assessment for fumonisins B1 and B2 in US corn.

Quantitative risk analysis permits modifying risk estimates with changes in variables such as exposure. This analysis for exposure to the mycotoxin fumonism describes the magnitude of adverse effects, variability in the population and uncertainty of models as a range of possible outcomes. The most sensitive adverse response in rats, nephrotoxic lesions, was used for the dose-response analysis. Dietary intake of corn products was estimated from a 3-day consumption survey. Levels of corn in each product were estimated by standard methods. Fumonisin levels in corn products were estimated from Food and Drug Administration (FDA) surveillance data and distributions of fumonisin consumption were modelled for each eater in the survey population. Uncertainty for predictions made from each model and uncertainty resulting from model selection were described. Results of the dose-response and exposure analyses were assimilated in a two-dimensional Monte-Carlo simulation. Distributions representing variability and uncertainty were iteratively selected to form an array of estimates of the risk. On the basis of this analysis, current dietary levels of fumonisin would not result in renal lesions even at upper levels of exposure. To avoid toxicity at much higher doses, limiting corn intake would be more effective than would limiting the level of fumonisin in corn.

Animals↗

Comparison of extraction and clean-up procedures for analysis of zearalenone in corn, rice and wheat grains by high-performance liquid chromatography with photodiode array and fluorescence detection.

The aim of this work was the optimization of some procedures usually used in the analysis of zearalenone (ZEA) in corn and other cereals by high-performance liquid chromatography (HPLC) with photodiode array and/or fluorescence detection. The comparison of five extraction solvents is presented. Three solid-phase extraction cartridges (C-18, silica, Florisil) and immuno-affinity columns were also compared to obtain the best recovery of the mycotoxin with the minimal presence of co-extractives in the chromatograms. Mixtures of methanol-1% aqueous NaCl (80.20 or 60:40 v/v) were the best extraction solvents. Florisil provided higher recovery of ZEA than C-18, and silica proved unsuitable. The immuno-affinity column was very efficient in cleaning the extracts, but its sample capacity was lower than that of SPE columns due to saturation. The mobile phase (methanol-water 80:20 v/v) gave a low retention time for ZEA (approximately 5 min), high sensitivity and acceptable separation between this mycotoxin and alpha-zearalenol. The optimized protocol is straightforward, provides high ZEA recoveries in spiked corn (mean 102.4%), has an acceptable sensitivity and has a lack of interference with fluorescence detection (detection limit 4 ng ZEA g(-1) corn). The photodiode array detector was useful, except at very low ZEA levels, to confirm the identity of the mycotoxin. The method was applied to search for ZEA accumulation in corn, wheat and rice grains inoculated with selected strains of Fusarium graminearum, F. oxysporum and method was applied to search for ZEA accumulation in corn, wheat and rice grains inoculated with selected strains of Fusarium graminearum, F. oxysporum and F. culmorum.

Chromatography, High Pressure Liquid↗

Anti-mutagenic compounds from corn.

In previous studies with aflatoxin-contaminated corn an uncharacteristic response for AFB1 in the Salmonella/microsomal mutagenicity assay (Ames test) was observed and the presence of anti-aflatoxin factors in the corn was suggested. In the current study, corn was extracted and fractionated using thin layer chromatography (TLC) using different developing solvent systems and the Ames test was used to monitor for anti-mutagenic activity in the corn fractions. Both Salmonella tester strains TA98 and TA100 with metabolic activation (S9) were used. Several corn fractions, at different stages in the isolation and purification process, showed anti-mutagenic dose-responses when exposed to pure AFB1. Corti extracts were non-toxic to the tester strains and TLC fractions that showed the best anti-mutagenic dose-responses were selected for further partial characterization analyses.

Aflatoxin B1↗

Post-harvest storage of corn: effect of beginning moisture content on mycoflora and fumonisin contamination.

The effect of storage on mycoflora profile was monitored bimonthly in 36 corn (Zea mays L.) samples, dividing the same sample into groups dried to 11 and 14% moisture content (1008 analysis). These groups were further subdivided based on the initial total count (moulds and yeasts) up to 10(4) CFU g(-1) (12 samples, range 1.6 x 10(4) to 9.0 x 10(4), mean 3.8 x 10(4) CFU g(-1)) and up to 10(5) CFU g(-1) (24 samples, range 1.0 x 10(5) to 5.0 x 10(5), mean 2.7 x 10(5) CFU g(-1)). In the corn group dried to 11%, the fumonisin content was analysed at the initial stage (freshly harvested) and at the end of 12-month storage. Fusarium spp. and Penicillium spp. prevailed at the freshly harvested stage (100%), maintaining this profile throughout 12 months, in corn dried to both 11 and 14%. Cladosporium spp., Aspergillus spp. and Phoma spp. were also detected at lower frequencies during the storage. Fusarium spp. and the total fungal colony count during 12-month storage carried out with samples dried to 11 or 14% moisture content were statistically evaluated using ANOVA for randomized complete block design. The correlation between storage time and Fusarium spp. and total fungal colony count data was analysed by Pearson's correlation test. There was no difference in Fusarium spp. and total counts in the 10(4) CFU g(-1) initial total count group throughout the storage time (p < 0.05). There was a negative correlation between fungal population and storage time (p < 0.05) in the 10(5) CFU g(-1) initial total count group. Fumonisins were detected in all freshly harvested corn, at a mean concentration of 9.9 +/- 6.0 micro g g(-1) (range 0.74-22.6 micro g g(-)1). These values did not change in the 12-month stored corn (mean of 9.9 +/- 5.8 micro g g(-1), range 0.81-23.7 micro g g(-1)). These post harvest data indicated the importance of moisture content at the crop harvesting/predrying stage to control fungal growth and further fumonisin production.

Agriculture↗

Distribution of ammonia/aflatoxin reaction products in corn following exposure to ammonia decontamination procedure.

The distribution of aflatoxin decontamination reaction products in corn following ammonia decontamination treatment was determined. The parameters of the ammoniation procedure used to decontaminate aflatoxin contaminated corn were 2% NH3, 16% moisture, 55 psi, 40-45 degrees C, and 60 min duration. Uniformly ring-labelled 14C-aflatoxin B1 was added to corn (1.0 microCi/kg) containing 7500 micrograms naturally-incurred aflatoxin B1 (AFB1)/kg. Aflatoxin levels were reduced by ca 93% after ammonia treatment. Distribution of radiolabelled AFB1 was used to follow the modification of AFB1 and the ammonia/aflatoxin reaction products were separated and isolated through a series of chemical extraction/partition procedures. Samples of the ammoniated product were fractionated through sequential extraction with methylene chloride and methanol, then either treated with acetic acid and sodium hydroxide or exposed to proteolytic enzyme digestion followed by methylene chloride extraction. Approximately 88% of the added radioactivity was detected in the corn after treatment (i.e. 12% of aflatoxin reaction products were volatile), ca 20% was extracted with methylene chloride and ca 13% was extracted with methanol. Treatment with acid and base released 18.8% of the added radioactivity. Similar amounts (19.1%) of aflatoxin-related compounds were liberated after enzymatic digestion with Pronase E. The remaining corn matrix after acid-base treatment or Pronase digestion contained ca 37.0% of the original radioactivity. A fluorescent spot on the TLC plate represented 6.1% of the CH2Cl2-extractable compounds and contained a compound which reacts chromatographically similarly to AFB2a.

Aflatoxins↗

Aspergillus flavus-induced chitosanase in germinating corn and peanut seeds: A. flavus mechanism for growth dominance over associated fungi and concomitant aflatoxin production.

Chitosanase, a new class of enzymes with antifungal properties was induced by toxigenic Aspergillus flavus in both germinating corn and peanut seeds. The enzyme was partially purified and fractioned by SDS-Polyacrylamide Gel Electrophoresis activity and copolymerized with chitosan or glycolchitosan as substrate, then quantified by scanning densitometry and a 2-dimensional analysis software program. Chitosanase enzyme was markedly induced by toxigenic A. flavus growing in germinating corn and peanut seeds, as compared to control (water) which showed the lowest activity (almost nil in corn). However, chitosanase induction was higher in seeds treated with chitosan from crustacea. Overall, enzyme activity was higher in peanut than in corn seeds. However, electrophoresed gels from peanut treated with A. flavus or water showed more polypeptides (three and one, respectively) than gels from corn seeds, which only showed one polypeptide for both A. flavus and water treatment. The enzyme molecular weight was estimated to be between 36,000 and 45,000.

Aflatoxins↗

Determination of hydrolysed fumonisin B1 (HFB1) in corn by competitive direct enzyme-linked immunosorbent assay.

Fumonisin B1, a mycotoxin produced by certain Fusarium moulds, consists of two tricarballyic acid groups esterified to a 20-carbon backbone. Under alkaline conditions, or through metabolism, the aminopentol backbone, also known as hydrolysed Fumonisin B1 (HFB1) can be formed and is itself cytotoxic. Although the occurrence of HFB1 in corn-based foods is suspected, because of the ubiquitous nature of FB1 in corn, analytical methods for its detection are difficult. In the present report we describe a monoclonal antibody-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for the rapid analysis of HFB1 in corn. The concentration required to inhibit enzyme conjugate binding by 50% (IC50) was 36 ng/ml. The limit of detection of HFB, by the CD-ELISA was 2ng/ml, when HFB1 was added in bovine serum albumin-phosphate buffered saline. The antibody also cross-reacted with the hydrolysis products of FB2, FB3, and FB4, having IC50 values of 331, 174, and 1700 ng/ml respectively. The antibody did not react with the intact fumonisins, sphingosine, sphinganine, or tricarballylic acid. Samples of corn spiked with HFB1 over the range of 5-1000 ng/g indicated the CD-ELISA has a limit of detection of 5 ng/g and an IC50 of 41 ng/g in the matrix. The CD-ELISA provides a sensitive and rapid tool for the analysis of corn-based foods for HFB1.

Animals↗

The natural occurrence of fumonisins in Brazilian corn kernels.

Since animal intoxication related to corn-based feed is frequently observed in the State of Paraná, Brazil, natural contamination by fumonisins in 48 corn samples (39 from the State of Paraná, and 9 from the Brazilian tropical states, Mato Grosso do Sul and Goias) harvested in 1990-1991 was investigated, along with fungal flora. The total mould count ranged from 6.3 x 10(2) to 5.5 x 10(7) cfu/g, and Fusarium moniliforme and Aspergillus species belonging to section Flavi were detected in 41 and 33 samples, respectively. Regarding the samples from the State of Paraná, F. moniliforme was present in 33 samples at a count of 1.0 x 10(2) to 1.6 x 10(7) cfu/g and Aspergillus spp. in section Flavi in 27 samples at 1.0 x 10(2) to 1.0 x 10(6) cfu/g. HPLC analysis of fumonisins in the corn showed that fumonisins B1 (FB1) and B2 (FB2) were positive for 97.4% and 94.8% of samples respectively. All the corn from North Paraná was positive for fumonisins, with average FB1 levels of 4.79 micrograms/g and average FB2 levels of 3.95 micrograms/g: the Central-West region had average levels of 3.30 and 2.52 micrograms/g, and the Central-East had average of 3.25 and 2.34 micrograms/g, respectively. Except for one negative sample all the corn samples from the Central Region were positive for fumonisins, averaging FB1 levels being 5.45 micrograms/g and FB2 levels being 5.09 micrograms/g. Out of eight samples from the tropical state of Mato Grosso do Sul, F. moniliforme was detected in seven and Aspergillus spp. in section Flavi in five samples with average FB1 levels of 10.59 micrograms/g and average for FB2 levels of 10.31 micrograms/g. The samples from Goias were also contaminated with these two fungi, with the FB1 contamination being 5.83 and the FB2 contamination 3.62 micrograms/g.

Aspergillus↗

Natural co-occurrence of aflatoxins and Fusarium mycotoxins (fumonisins, deoxynivalenol, nivalenol and zearalenone) in corn from Indonesia.

Sixteen corn samples collected from Indonesia were analysed for aflatoxins (AF), fumonisins (FM), trichothecenes and zearalenone (ZEA) using high-performance liquid chromatography and gas chromatography-mass spectrometry. AF were detected in 11 (69%) samples at a mean level of 119 ng/g (maximum 487 ng/g) and FM in all of the samples at a mean level of 895 ng/g (maximum 2970 ng/g). Deoxynivalenol (DON), nivalenol (NIV) and ZEA were each detected in two (12%) samples; 21 and 32 ng/g, 49 and 169 ng/g, and 11 and 12 ng/g, respectively. All of the AF-contaminated samples were co-contaminated with FM. Mycological study showed all of the AF-contaminated samples were infected with A. flavus/A. parasiticus, and the FM-contaminated samples were either infected with F. moniliforme (50%), F. proliferatum (12%), F. nygamai (6%) or F. decemcellulare (38%). Supportive mycological studies showed that Fusarium species isolated from Indonesian corn were capable of producing a mean level of 10,000 micrograms/g FM. Based on these results, the correlation between FM-producers and AF-producers on kernel infection and contamination of these mycotoxins in corn was discussed. This is the first report on the natural co-occurrence of AF and various Fusarium mycotoxins, including DON and NIV in corn from Indonesia, and also the first report on the natural occurrence of DON in corn from hot areas of Southeast Asia.

Aflatoxins↗