Aflatoxins In Maize Research Articles

Foreword: aflatoxins in maize and other crops

Foreword: aflatoxins in maize and other crops

Total aflatoxin level and fungi contamination of maize and maize products

Samples of maize and maize products purchased from selected markets in parts of Kaduna State, Nigeria were analyzed for total aflatoxin and fungi contamination using Enzyme Linked Immunosorbent Assay (ELISA) and mycological procedures. The mean aflatoxin concentration of the maize and maize products obtained from this study shows that one hundred and fifty two (82.70%) of the maize products had aflatoxin contamination above the tolerance level of 20ppb stipulated by World Health Organization).The old maize had a mean aflatoxin level of 177ppb while the new maize had a mean aflatoxin level of 102ppb.The mean aflatoxin level in maize flour was 174ppb while the maize bran had a mean aflatoxin level of 213ppb. Significant differences exists between the mean aflatoxin contaminations of old maize, fresh maize, maize flour and maize bran. Five fungal genera namely: Aspergillus, Penicillium, Rhizopus,, Fusarium and Botrytis were isolated. Aspergillus was the most predominant genera isolated (62%).Among the Aspergillus, three species namely A. niger, A. tamari, and A. flavus were identified. The high level of aflatoxin in maize and maize products highlights the health problem associated with the consumption of these products with the toxin that could lead to adverse health effects such as liver, cervical, breast or oesophageal cancer.

Determination of Aflatoxin Levels in Maize Grain by High Performance Liquid Chromatography Using an Immunoaffinity Column Cleanup

The aim of this study was to determine aflatoxin contamination levels in maize grain produced in Samsun Province, Turkey. Forty samples of maize grain intended for animal feed in Samsun Province were analyzed. The amounts of the aflatoxins, B1, B2, G1 and G2, were quantified with a liquid chromatography (HPLC-FLD) and post-column derivatization. Recoveries ranged between 89 and 98%. The detection limits were 0.007 and 0.02 ng/g and the corresponding quantification limits were 0.022 and 0.063 ng/g. As a result, it has been shown that the accurate and sensitive HPLC-FLD method was confirmed as being appropriate for the detection of AFs in maize grain, besides, the incidence of aflatoxins in maize was very low (2.5%).

Discovery and confirmation of genes/proteins associated with maize aflatoxin resistance

Maize (Zea mays L.) is one of the major crops susceptible toAspergillus flavus infection and subsequent aflatoxin contamination. Many earlier studies indicated the roles of kernel proteins, especially constitutively expressed proteins, in maize resistance toA. flavus infection and aflatoxin production. In this review, we examined the past and current efforts in identifying maize genes and proteins from kernel, rachis, and silk tissues that may play an important role in resistance toA. flavus infection and aflatoxin contamination, as well as the efforts in determining the importance or involvement of them in maize resistance through biochemical, molecular and genetics studies. Through these studies, we gained a better understanding of host resistance mechanism: resistant lines appear to either express some stress-related and antifungal proteins at higher levels in endosperm, embryo, rachis and silk tissues beforeA. flavus infection or induce the expression of these proteins much faster compared to susceptible maize lines. In addition, we summarised several recent efforts in enhancing maize resistance to aflatoxin contamination using native genes from maize or heterologous and synthetic genes from other sources as well as fromA. flavus. These efforts to either suppressA. flavus growth or aflatoxin production, have all shown some promising preliminary success. For example, maize plants transformed with an ?-amylase inhibitor protein fromLablab purpurea showed reduced aflatoxin levels by 56% in kernel screening assays. The antifungal potentials of transgenic maize plants expressing synthetic lytic peptides, such as cecropin-based D4E1 or tachyplesin-based AGM peptides with demonstrated anti-flavus activity (IC50 = 2.5 to 10 ?M), are yet to be assayed. Further investigation in these areas may provide a more cost-effective alternative to biocontrol in managing aflatoxin contamination in maize and other susceptible crops.

Optical detection of aflatoxins in maize using one- and two-photon induced fluorescence spectroscopy

The presence of aflatoxins in food and feed products is considered as one of the most important food safety problems in the world. Aflatoxins occur in a wide range of food products and can cause serious health risks. Moreover, they can nowadays only be detected by the use of destructive, time-consuming and expensive chemical analyses. We investigate the use of one- and two-photon induced fluorescence spectroscopy as nondestructive detection methods for the identification of aflatoxins. Particularly, as the samples under test, we consider the aflatoxin-contamination of different maize batches since maize is the staple food in many countries and cultivates in climates that show an extensive presence of the fungi. We first characterize the one- and two-photon induced fluorescence spectrum of pure aflatoxin B1, when excited with 365 nm and 730 nm laser light respectively. Subsequently, we experimentally investigate the fluorescence spectrum of various healthy and aflatoxin-contaminated maize samples, when excited with 365 nm, 405 nm, 730 nm, 750 nm, 780 nm and 810 nm laser light. For all excitation wavelengths, an intrinsic fluorescence signal of the maize grains is observed. However, for the contaminated maize grains, the present aflatoxin B1 significantly influences the intrinsic fluorescence. Depending on the excitation wavelength, we observe a different spectral contrast between the healthy and contaminated samples. The largest optical difference is observed for excitation with 365 nm and 780 nm, during the one- and two-photon induced fluorescence measurements respectively. The comparison of the measured fluorescence signals allows us to define a detection criterion for the optical identification of the contaminated maize samples. We can conclude that fluorescence spectroscopy can be a valuable tool for the measurement of aflatoxin-contents in maize, paving the way for real-time non-destructive industrial scanning-based detection.

An empirical evaluation of three vibrational spectroscopic methods for detection of aflatoxins in maize

Three commercially available vibrational spectroscopic techniques, including Raman, Fourier transform near infrared reflectance (FT-NIR), and Fourier transform infrared (FTIR) were evaluated to help users determine the spectroscopic method best suitable for aflatoxin analysis in maize (Zea mays L.) grain based on their relative efficiency and predictive ability. Spectral differences of Raman and FTIR spectra were more marked and pronounced among aflatoxin contamination groups than those of FT-NIR spectra. From the observations and findings in our current and previous studies, Raman and FTIR spectroscopic methods are superior to FT-NIR method in terms of predictive power and model performance for aflatoxin analysis and they are equally effective and accurate in predicting aflatoxin concentration in maize. The present study is considered as the first attempt to assess how spectroscopic techniques with different physical processes can influence and improve accuracy and reliability for rapid screening of aflatoxin contaminated maize samples.

Atoxigenic Aspergillus flavus endemic to Italy for biocontrol of aflatoxins in maize

Effective biological control of aflatoxin-producing Aspergillus flavus with atoxigenic members of that species requires suitable A. flavus well adapted to and resident in target agroecosystems. Eighteen atoxigenic isolates of A. flavus endemic in Italy were compared for ability to reduce aflatoxin contamination of maize in laboratory studies. Reduction in aflatoxin B1 concentrations ranged from 61 to 90 % with the most effective five atoxigenics, belonging to five different vegetative compatibility groups, causing reductions similar to, or greater than, those achieved by an atoxigenic that is the active ingredient in a product registered in the USA for aflatoxin management. The atoxigenic isolates were effective against each of the six Italian aflatoxin-producers tested and both with equal proportions of toxigenic and atoxigenic isolates and when atoxigenic isolates were only inoculated at 25 % the concentration of toxigenic. Equal proportions provided the highest percent reduction in contamination, but at the 4:1 ratio atoxigenics were the most efficient. The identified atoxigenic isolates will be of value as active ingredients in biocontrol products for reduction of aflatoxins in maize produced on the Italian Peninsula.

Selection of non-toxigenic strains of Aspergillus flavus for biocontrol of aflatoxins in maize in Thailand

Aflatoxin production in maize and peanuts remains a major public health problem, especially in developing countries. One promising method for combating aflatoxin formation is biocontrol using competitive exclusion, a management strategy currently being studied in maize crops in Thailand. It is important that the strains of Aspergillus flavus used in biocontrol be non-toxigenic and be incapable of reversion to toxigenicity. In the current study, 80 non-toxigenic strains of A. flavus, randomly selected from commercially produced dried maize samples from several sources in Thailand, were screened for their potential as biocontrol strains by examining the 24 aflatoxin biosynthesis genes, using a PCR assay. Assessment of the presence or absence of PCR products provides an indication of the function of pathway genes. Of the 80 strains, 78 showed no PCR products from one or more genes in the aflatoxin biosynthesis pathway. Twenty-three isolates showed only one failure, in the aflD (nor-1) gene, but most isolates failed to produce a PCR product for two or more genes. Nineteen isolates (24%) failed to show a PCR product in 10 or more genes. Altogether, 45 PCR product patterns were observed, usually common to only one or two isolates, indicating great diversity in the aflatoxin biosynthesis pathway in A. flavus isolates taken from dried Thai maize. Although the absence of a particular PCR product is not conclusive evidence that the particular gene is non-functional, the absence of several such PCR products provides reasonable evidence that the isolate in question will not revert to toxigenicity in the field.

A rapid quantitative lateral flow test for the detection of total aflatoxins in maize

IntroductionAflatoxins (B1, B2, G1 and G2) are toxic secondary metabolites of, primarily, the fungi Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are potent carcinogens which cause liver diseases in livestock, domestic animals and humans throughout the world. Regulations for aflatoxins in commodities and food exist in more than 100 countries. A rapid quantitative lateral flow test, namely “Agrastrip”, was developed to measure total aflatoxin levels in maize. The test is developed based on the competitive immuno-chromatography principle. The total assay time for a sample extract is approximately 5 minutes. The results from the test are comparable to the reference HPLC or LC-MS/MS methods. ObjectiveTo validate the quantitative AgraStrip total aflatoxin test in maize samples. MethodsAflatoxins were extracted from ground maize sample using 70% methanol. To start an assay, buffer was added into a microwell to dissolve the pre-coated antibody-colloidal gold conjugate particle in the microwell. This was followed by mixing the dissolved conjugate with sample extract. A test strip was then placed into the mixture in microwell and incubated in a heat block incubator for 3 minutes. The test line zone in the test strip is able to capture the conjugate and form a visible line. The concentration of aflatoxins present in the sample is inversely proportional to the colour intensity of the test line. A line is always visible in positive control zone regardless of the presence of aflatoxins. The test strip is analyzed by the AgraVision® Reader and concentration of aflatoxins is determined. Results & DiscussionThe test was able to measure aflatoxins in the range of 5–100μg/kg. Limit of detection for the test kit on corn sample was 3.6μg/kg. Accuracy and precision of the test on maize sample naturally contaminated with aflatoxins were comparable to the results form reference HPLC or LC-MS/MS methods. The shelf-life of the test kit was 12 months. ConclusionAgrastrip total aflatoxin test is able to screen total aflatoxins in maize samples in its quantitation range.

Evaluation of the bright greenish yellow fluorescence test as a screening technique for aflatoxin-contaminated maize in Malawi

The bright greenish yellow fluorescence (BGYF) test has been used with varying success in screening for aflatoxins in maize. This test was applied to 180 maize samples collected from different markets within 12 districts of Malawi in order to evaluate its performance against high performance liquid chromatographic analysis. The number of BGYF grains in 2.5 kg unground samples ranged from 0 to 35 and about 49% of all tested samples had aflatoxin concentrations ranging from 1 to 382 μg/kg. A total of 65 (36%) of the examined unground samples showed no BGYF. The European Commission recommends a false negative rate of less than 5% for a screening technique to be acceptable. In this study, four BGYF grains per 2.5 kg unground maize sample successfully indicated an aflatoxin contamination of >10 μg/kg (10 μg/kg being the maxium tolerable level proposed by the Common Market for Eastern and Southern Africa), with a 4.4% false negative rate. In this case, the amount of confirmatory analyses would be reduced by 63%, if the BGYF test was employed as a screening method. The screening technique therefore offers a practical tool for Malawi and possibly for the Sub-Saharan region.

Inhibition of Aspergillus flavus and Aflatoxin in Maize by Acorus calamus (Bojo)

This study was carried out to reveal the effect of Bojo powder on Aflatoxin production and growth of Aspergillus flavus 'ITCC 5192' in maize grain stored at four different moisture levels of 13, 17, 21 and 24%. For the inhibition study, Bojo powder was used in the concentration of 0.0, 0.5, 0.75, 1.0, 1.25 and 1.5% (w/w) in maize samples. After inoculation of Aspergillus flavus .ITCC 5192. and incubation for 15 days, Aflatoxins were extracted and quantified by using Thin Layer Chromatography (TLC). Maize sample with moisture content 17% and Bojo powder concentration at 1.0% prevented the growth of Aspergillus flavus and production of Aflatoxin. Aflatoxins as high as 1416ppb were detected in control sample (i.e. no Bojo added) and stored at 17 and 21% respectively. Formation of Aflatoxins and growth of Aspergillus flavus 'ITCC 5192' was not completely inhibited in maize grains at high moisture levels (i.e. 21 and 24%). J. Food Sci. Technol. Nepal, Vol. 6 (59-64), 2010 DOI: http://dx.doi.org/10.3126/jfstn.v6i0.8261

Global Risk Assessment of Aflatoxins in Maize and Peanuts: Are Regulatory Standards Adequately Protective?

The aflatoxins are a group of fungal metabolites that contaminate a variety of staple crops, including maize and peanuts, and cause an array of acute and chronic human health effects. Aflatoxin B1 in particular is a potent liver carcinogen, and hepatocellular carcinoma (HCC) risk is multiplicatively higher for individuals exposed to both aflatoxin and chronic infection with hepatitis B virus (HBV). In this work, we sought to answer the question: do current aflatoxin regulatory standards around the world adequately protect human health? Depending upon the level of protection desired, the answer to this question varies. Currently, most nations have a maximum tolerable level of total aflatoxins in maize and peanuts ranging from 4 to 20ng/g. If the level of protection desired is that aflatoxin exposures would not increase lifetime HCC risk by more than 1 in 100,000 cases in the population, then most current regulatory standards are not adequately protective even if enforced, especially in low-income countries where large amounts of maize and peanuts are consumed and HBV prevalence is high. At the protection level of 1 in 10,000 lifetime HCC cases in the population, however, almost all aflatoxin regulations worldwide are adequately protective, with the exception of several nations in Africa and Latin America.

Natural occurrence of aflatoxins in maize harvested in Serbia during 2009–2012

The aim of the present study was to determine concentration of aflatoxins (AFs) B1, B2, G1 and G2 in 380 maize samples collected from different regions of Serbia during four years period and to analyze the influence of weather conditions on the AFs production.Aflatoxins content was determined by direct competitive Enzyme Linked Immunosorbent Assay (ELISA) method.In 180 maize samples analyzed in 2009–2011 period AFs were not detected. However weather condition changes in 2012 caused AFs presence in 137 (68.5%) of samples in the concentration range from 1.01 to 86.1 μg/kg with the mean level of 36.3 μg/kg.The obtained results indicate great influence of hot and dry weather with prolonged drought during spring and summer 2012 on a contamination of maize with AFs.Since the obtained results present the first national report of AFs maize contamination this study could be of a great interest for establishing monitoring programs of AFs in Serbia.

Maize aflatoxin accumulation segregates with early maturing selections from an S2 breeding cross population.

Maize breeders continue to seek new sources of aflatoxin resistance, but most lines identified as resistance sources are late maturing. The vast difference in flowering time makes it hard to cross these lines with proprietary commercial lines that mature much earlier and often subjects the reproductive phase of these resistant lines to the hottest and driest portion of the summer, making silking, pollination and grain fill challenging. Two hundred crosses from the GEM Project were screened for aflatoxin accumulation at Mississippi State in 2008, and a subset of these lines were screened again in 2009. The breeding cross UR13085:S99g99u was identified as a potential source of aflatoxin resistance, and maturity-based selections were made from an S2 breeding population from this same germplasm source: UR13085:S99g99u-B-B. The earliest maturing selections performed poorly for aflatoxin accumulation, but later maturing selections were identified with favorable levels of aflatoxin accumulation. These selections, while designated as “late” within this study, matured earlier than most aflatoxin resistant lines presently available to breeders. Two selections from this study, designated S5_L7 and S5_L8, are potential sources of aflatoxin resistance and will be advanced for line development and additional aflatoxin screening over more site years and environments.

The influence of drought on the occurrence of aflatoxins in maize

In this study, a total of 78 maize samples harvested during September and October 2012 in Vojvodina were analyzed. Presence of aflatoxins (AFs) was deter?mined by enzyme-linked immunosorbent assay (ELISA) method. Among the 78 analyzed maize samples, even 44 (56.4%) samples were contaminated with AFs. Concentration interval between 1-10 ?g/kg, 10-50 ?g/kg and 50-80 ?g/kg were found in 23.1%, 17.9% and 15.4% of analyzed maize samples, respectively. It was supposed that prolonged drought during spring and summer of 2012 had a great influence on high contamination frequency and concentration of AFs.

Comprehensive assessment of maize aflatoxin levels in Eastern Kenya, 2005-2007.

Background: Aflatoxin, a potent fungal toxin, contaminates 25% of crops worldwide. Since 2004, 477 aflatoxin poisonings associated with eating contaminated maize have been documented in Eastern Kenya, with a case-fatality rate of 40%.Objective: We characterized maize aflatoxin contamination during the high-risk season (April–June) after the major harvests in 2005, 2006 (aflatoxicosis outbreak years), and 2007 (a non-outbreak year).Methods: Households were randomly selected each year from the region in Kenya where outbreaks have consistently occurred. At each household, we obtained at least one maize sample (n = 716) for aflatoxin analysis using immunoaffinity methods and administered a questionnaire to determine the source (i.e., homegrown, purchased, or relief) and amount of maize in the household.Results: During the years of outbreaks in 2005 and 2006, 41% and 51% of maize samples, respectively, had aflatoxin levels above the Kenyan regulatory limit of 20 ppb in grains that were for human consumption. In 2007 (non-outbreak year), 16% of samples were above the 20-ppb limit. In addition, geometric mean (GM) aflatoxin levels were significantly higher in 2005 (GM = 12.92, maximum = 48,000 ppb) and 2006 (GM = 26.03, maximum = 24,400 ppb) compared with 2007 (GM = 1.95, maximum = 2,500 ppb) (p-value < 0.001). In all 3 years combined, maize aflatoxin levels were significantly higher in homegrown maize (GM = 17.96) when compared with purchased maize (GM = 3.64) or relief maize (GM = 0.73) (p-value < 0.0001).Conclusions: Aflatoxin contamination is extreme within this region, and homegrown maize is the primary source of contamination. Prevention measures should focus on reducing homegrown maize contamination at the household level to avert future outbreaks.

Developing resistance to aflatoxin in maize and cottonseed.

At this time, no “magic bullet” for solving the aflatoxin contamination problem in maize and cottonseed has been identified, so several strategies must be utilized simultaneously to ensure a healthy crop, free of aflatoxins. The most widely explored strategy for the control of aflatoxin contamination is the development of preharvest host resistance. This is because A. flavus infects and produces aflatoxins in susceptible crops prior to harvest. In maize production, the host resistance strategy has gained prominence because of advances in the identification of natural resistance traits. However, native resistance in maize to aflatoxin contamination is polygenic and complex and, therefore, markers need to be identified to facilitate the transfer of resistance traits into agronomically viable genetic backgrounds while limiting the transfer of undesirable traits. Unlike maize, there are no known cotton varieties that demonstrate enhanced resistance to A. flavus infection and aflatoxin contamination. For this reason, transgenic approaches are being undertaken in cotton that utilize genes encoding antifungal/anti-aflatoxin factors from maize and other sources to counter fungal infection and toxin production. This review will present information on preharvest control strategies that utilize both breeding and native resistance identification approaches in maize as well as transgenic approaches in cotton.

Natural Occurrence of Aflatoxins from Maize in Iran

Fifty-one maize samples, intended for animal feed and human consumption, were collected from the four main maize production provinces in Iran and analyzed by high-performance liquid chromatography (HPLC) for contamination by four naturally occurring aflatoxin analogues (AFB1, AFB2, AFG1, and AFG2). AFB1 was detected in 58.3, and 80% of the maize samples obtained from Kermanshah and Mazandaran provinces, respectively. The maximum AFB1 (276.3 μg/kg) and highest level of total aflatoxins (AFT) (316.9 μg/kg) were detected in a maize sample collected from Kermanshah province. The mean aflatoxin level from contaminated samples (52.60 μg/kg) from Kermanshah was significantly higher (P < 0.0001) than those in maize from the other three provinces and exceeded all the maximum tolerated levels (MTLs) set for AFT in maize. The level of AFB1 in 15.68% of the total samples was above the MTL (5 μg/kg) for AFB1 in maize in Iran. The mean contamination level of AFT (23.86 μg/kg) in the positive samples was higher than MTL for maize in Iran (20 μg/kg) intended for animal feed. The levels of AFB1, AFB2, AFG1, and AFG2 ranged between not detected (<0.1 μg/kg) and 276.3, 30.4, 9.1, and 1.1 μg/kg in maize grain, respectively.

Effect of Microwave Heating during Alkaline‐Cooking of Aflatoxin Contaminated Maize

To evaluate the effectiveness of maize detoxification achieved with a modified tortilla-making process (MTMP), maize contaminated with aflatoxin B(1) (AFB(1)) and aflatoxin B(2) (AFB(2)) at levels of 22.46, 69.62, and 141.48 ng/g (AFB(1)+ AFB(2)) was processed into tortillas. Aflatoxin content was determined according to the 991.31 AOAC official method. Based on the results obtained with spiked samples (0.78 to 25 ng/g), the mean recovery was 92%, with a standard error of 1.2, and a coefficient variation value of 4.4%. The MTMP caused 68, 80, and an 84% decrease in aflatoxin content, respectively. Extract acidification (as occurs during digestion) prior to mycotoxin quantification caused some reformation of the aflatoxin structure in tortillas (up to 3%). According to these results, the MTMP seems to be safe for decontamination since a low percentage of the initial aflatoxin concentration can be reverted to the original fluorescent form upon acidification. The potential presence of aflatoxins in maize destined for human consumption is a serious problem to the Mexican food supply, as these toxic compounds may persist during the traditional alkaline-process for tortilla elaboration. Consequently, new detoxification procedures are needed that eliminate or at least minimize the aflatoxin risk, through lowering aflatoxin concentration in maize-based products. Under these considerations, the use of MTMP is recommended, since it has definite advantages including non-production of wastewater and reduced energy/time consumption.

Development of a quantitative lateral flow immunoassay for the detection of aflatoxins in maize

An immunoassay-based lateral flow device for the quantitative determination of four major aflatoxins in maize has been developed. The one-step assay has performance comparably with that of other screening methods, as confirmed by the intra- and the inter-day precision of the data (RSD 10–22%), and can be completed in 10 min. Quantification was obtained by acquiring images of the strip and correlating intensities of the coloured lines with analyte concentration by means of a stored calibration curve carried out by diluting aflatoxins in the extract from a blank maize sample. Limit of detection (1 µg kg–1) and dynamic range (2–40 µg kg−1) allows the direct assessment of aflatoxin contamination in maize at all levels of regulatory relevance. All reagents are immobilized on the lateral flow device. In addition, very simple sample preparation, using an aqueous buffered solution, has been demonstrated to allow the quantitative extraction of aflatoxins. Twenty-five maize samples were extracted with the aqueous medium and analyzed by the developed assay. A good correlation was observed (y = 0.97x + 0.07, r 2 = 0.980) when data was compared with that obtained through an official method. The developed method is reliable, rapid and allows for application outside the laboratory as a point-of-use test for screening purposes.