Detection of aflatoxins and zearalenone contamination in wheat derived products

In present study the natural occurrence of aflatoxins (AFs) and zearalenone (ZEN) has determined in 147 samples of wheat derived products i.e. spaghetti (25), noodles (34), macaroni (29), lasagne (37), and bucatini (22), collected from major districts of Punjab, Pakistan. The mean of AFs was found 9.12 in spaghetti, 7.35 in noodles, 5.91 in macaroni, 8.39 in lasagne, and 9.61 μg/kg in bucatini and 28, 18, 17, 22 and 27% of samples were found above the European Union's legal limit (i.e. 4 μg/kg) for total AFs, respectively. However, mean value of ZEN was found 7.36 in spaghetti, 6.80 in noodles, 4.98 in macaroni, 6.90 in lasagne, and 8.89 μg/kg in bucatini samples and 16, 15, 21, 19 and 36% of samples were found to be above the recommended limit. The study urged the need to focus more comprehensive survey for these toxins in wheat derived products from Punjab, Pakistan.     

Distribution of deoxynivalenol and zearalenone in milled germ during wheat milling and analysis of toxin levels in wheat germ and wheat germ oil

A method consisting of solvent extraction using hexane for defatting, multifunctional cleanup column, and HPLC determination was validated for the analysis of deoxynivalenol (DON) and zearalenone (ZEA) in wheat germ and wheat germ oil. A total of 36 batches of grain wheat were subjected to industrial milling and the distribution factors in milled germ were 47% for DON and 71% for ZEA. A survey of 50 samples of germ-based dietary supplements revealed that 60% of wheat germ and 40% of wheat germ oils contained DON at mean values of 111 and 41 μg/kg, respectively, while none of germ samples and 16% oils contained ZEA (mean 6 μg/kg). Contamination levels lead to a daily intake of 1.3 μg DON and 0.03 μg ZEA, representing 1.9% and 0.23% of their respective tolerable daily intakes (TDI).     

Effect of milling on the content of deoxynivalenol,nivalenol, and zearalenone in Japanese wheat

The effect of milling on the content of zearalenone (ZEA), a Fusarium mycotoxin, in a semi-processed wheat product was investigated and compared with that of deoxynivalenol (DON) or nivalenol (NIV). Two grain samples of Japanese soft wheat varieties, norin 61 and chikugoizumi, were milled to obtain three breaking flours (1B, 2B, 3B), three middling flours (1M, 2M, 3M) and two outer-layer fractions (bran and shorts). Patent flour for human consumption was made from 1B, 1M, 2B and 2M flours, while low-grade flour for animal feed was made from 3B to 3M flours. The contents of ZEA in patent flour, low-grade flour, bran and shorts were analyzed by an in-house validated analytical method using solvent extraction, multifunctional cartridge cleanup, and HPLC-fluorescence detection. A greater than 50% reduction in ZEA was observed in the patent flour of both samples, while 4–74% reduction in DON and NIV in the patent flour of the same two samples was observed. The results of this study revealed that the transfer ratios of ZEA had different features from those of deoxynivalenol and nivalenol.     

Natural occurrence of ochratoxin A in Tunisian cereals

Ochratoxin A (OTA) is a mycotoxin produced by several fungal species from Aspergillus and Penicillium genera. It is widespread in food and feed and its occurrence has been reported in cereals, cereal-derived products, dried fruits and spices. This mycotoxin was implicated in several human and animal pathologies such as the Balkan Endemic Nephropathy (BEN) and the Tunisian Chronic Interstitial Nephropathy (CIN) of unknown cause. In Tunisia, a clear correlation has been established between the consumption of OTA contaminated food and the induction of specific pathologies. Thereby, OTA was detected in human blood and tissues. The aim of our study was to investigate the presence of OTA in widely consumed cereals commercialized in Tunisia. The analytical methods used in our study involved the extraction of OTA by acidified toluene, immunoaffinity (IAC) clean-up and HPLC quantification with fluorescence detection. Levels and percentages of OTA contamination in different types of cereals, 110 wheat, 103 barley, 113 sorghum and 96 rice samples, were evaluated with incidences of 38%, 40%, 38% and 28%, respectively. The average of contamination by OTA found were 55, 96, 44 and 117 μg/kg, respectively, for wheat, barley, rice and sorghum. Our results showed that contamination percentages and levels in the period from 2004 to 2005 were higher then usual norms (5.0 μg OTA/kg) established by the European commission in 2002. The present report is the first one ever carried out on the natural occurrence of OTA in cereals, largely consumed by the Tunisian population.     

Natural incidence of aflatoxins,ochratoxin A and zearalenone in chicken meat and eggs

Aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEN) were analyzed in 115 chicken meat and 80 eggs samples, collected from central areas of Punjab, Pakistan. The study was carried out using reverse phase HPLC, equipped with fluorescence detector. The results revealed that 35% samples of chicken and 28% samples of eggs were found contaminated with AFs, and maximum level of AFB₁ and total AFs was found in the liver part of chicken (layer) 7.86 and 8.01 μg/kg, respectively. Furthermore, 41% samples of chicken and 35% sample of eggs were found contaminated with OTA and maximum level 4.70 μg/kg was found in the liver part of chicken meat. However, 52% samples of meat and 32% samples of eggs were found contaminated with ZEN and maximum level 5.10 μg/kg was found in the liver part of chicken meat. The occurrence and incidence of AFs, OTA and ZEN in chicken meat and eggs are alarming and it may produce health hazards and urged the need of continuous monitoring for these toxins in chicken meat and eggs.     

Occurrence and co-occurrence of Fusarium mycotoxins in wheat grains and wheat flour from Romania

In this study, the presence of fourteen Fusarium mycotoxins, legislated by the European Union – deoxynivalenol, zearalenone, HT-2 and T-2 toxins (EC/1881/2006; 2013/165/EU), or non-legislated (five trichothecens and five “emerging” mycotoxins), was evaluated in 31 whole unprocessed wheat samples and 35 white wheat flour samples from different areas of Romania. For this purpose, a validated multi-mycotoxins liquid chromatography tandem mass spectrometry method was applied. Seventy three percent of the analyzed samples contained at least one mycotoxin. The highest occurrence was for enniatin B, 71% of the analyzed samples being positive (21–407 μg kg⁻¹). Regarding the legislated mycotoxins, deoxynivalenol was detected in 14% (111–1787 μg kg⁻¹) of the samples, while zearalenone was detected in 9% (51–1135 μg kg⁻¹). Only one sample was positive for neosolaniol. Concerning the co-occurrence, 42% of the samples were contaminated with two to five mycotoxins, the most frequent being the binary or tertiary combinations of enniatins. This is the first study applied to Romanian wheat grains and flour samples using a high sensitive multi-mycotoxins method, and which included also “emerging” mycotoxins.     

Effect of deoxynivalenol detoxification by ozone treatment in wheat grains

Considering the wide existence of mycotoxin deoxynivalenol (DON) in wheat and derived products, finding ways to detoxify DON in wheat grains as original resources for food-chain contamination is very important to protect human health and reduce economic losses. This study aimed to evaluate the effectiveness of destroying DON in wheat via ozonation under different conditions, such as moisture content (MC), ozone concentration, exposure time, and form of raw-material. The achieved data showed that DON reduction significantly improved with increased ozone concentration and exposure time. The whole wheat flour (WWF) was easier to degrade than the wheat kernels. Higher MC decreased larger amounts of DON. The maximum reduction of DON was 20.10% for the MC group, compared with the 11.79% and 16.29% for the other MC groups. DON concentration decreased from 3.89 mg/kg to 0.83 mg/kg under the generally recognized Maximum Residue Limit (MRL) level of 1 mg/kg, when WWF was treated with 100 mg/L of ozone treatment for 60 min. The first-order kinetic model established in this work showed a good R-squared value (R² > 0.877) and was consistent with the results. Therefore, ozonation is an effective and rapid way to degrade DON in wheat, especially in whole wheat flour.     

Incidence of aflatoxins, ochratoxin A and zearalenone in tunisian foods

A total of 209 samples of different groups of foods widely consumed by the Tunisian population were collected during 2004–2005 years. Samples were analyzed for contamination with aflatoxins, ochratoxin A and zearalenone, using competitive enzyme-linked immunosorbent assay (ELISA). The predominant mycotoxin was ochratoxin A with a mean level of 3.5 ± 5.3 ng g⁻¹ in 59.8% of studied samples. Furthermore, Aflatoxins were detected in all analyzed commodities with a contamination frequency of 50.5%. In addition, aflatoxin B1 was found in 37% of the samples. The zearalenone was detected around 15% with a mean level of 10.4 ± 11.8 ng g⁻¹. Species, dried fruits and sorghum were the most contaminated samples by aflatoxin and ochratoxin mycotoxins, whereas Rice was the least contaminated commodity. The most frequent mycotoxins co-occurrence included aflatoxins and ochratoxin A, which have been detected in 33.8% of analyzed samples. Furthermore, the simultaneous contamination by aflatoxins, ochratoxin A and zearalenone was observed in 7.2% of studied samples.     

Detoxification of zearalenone by three strains of lactobacillus plantarum from fermented food in vitro

Zearalenone (ZEA) occurs naturally in many important crops worldwide with high concentrations. Lactic acid bacteria (LAB) are a promising solution to reduce exposure to dietary mycotoxins. Twenty-seven Lactobacillus plantarum strains isolated from traditional fermented food were evaluated using HPLC-MS-MS for the ability of these strains to remove ZEA from a MRS medium. Furthermore, the detoxification dynamics of the strains with high degrading abilities as well as the impact of a variety of factors on the abilities of the high degrading strains to remove ZEA from the MRS medium or PBS were carefully studied. The results indicated that strains Lp22, Lp39, and Lp4 cleared 47.80%, 38.06%, and 39.50% of ZEA from the solution, respectively, providing the highest ZEA degrading abilities of the strains studied. Apart from the bacterial cell density, the removal of ZEA also depended on the concentration of toxins, the viability of bacteria and the incubation temperature in the MRS medium. Removal of ZEA by Lactobacillus plantarum was a slow and continuous process, and approximately 45% of ZEA was removed in 48 h.     

Detoxification of zearalenone by Lactobacillus pentosus strains

Zearalenone (ZEA) contamination in food samples plays a critical role in food safety, since it causes serious health problems. Usage of microorganisms, such as lactic acid bacteria (LAB), is a promising new approach for detoxification. Eight Lactobacillus pentosus strains were evaluated for their ability to remove ZEA from a sodium acetate buffer solution with initial ZEA concentrations of 5.51–74.70 μg/mL. The adsorption capacity increased with increasing ZEA concentrations. The strain JM0812 showed the highest adsorption capability, at 83.17%, in solution containing 74.70 μg/mL ZEA, followed by UM054 (82.78%) and UM055 (81.69%), respectively. Three adsorption isotherms were applied to predict the removal efficiency of ZEA and the Freundlich isotherm appeared to have the best-fit for ZEA sorption onto bacterial cells. Our results indicate that Lb. pentosus strains are novel promising strains to reduce mycotoxin contamination in food products.     

Esterase activity inspired selection and characterization of zearalenone degrading bacteria Bacillus pumilus ES-21

Zearalenone (ZEN), mainly produced by Fusarium species, is an estrogenic mycotoxin which causes reproductive disorders in livestock. In this study, we described a simple and rapid method for screening of ZEN-degrading bacteria by esterase activity assay. Soil bacteria strains were first tested for their esterase activities, then active strains were further evaluated for their ZEN-degrading potentials. A bacterial strain named Bacillus pumilus ES-21 was detected to be able to eliminate ZEN in the culture medium. ZEN degradation conditions were optimized through response surface methodology and the result showed that the degradation rate of ZEN by Bacillus pumilus ES-21 was up to 95.7% at the ZEN concentration of 17.9 μg/ml within 24 h. One of the degradation product was proposed to be 1-(3,5-dihydroxyphenyl)-6′-hydroxy-l′-undecen-l0′-one according to LC-TOF-MS/MS analysis. This study provided a strategy for the isolation of ZEN degrading microbes and a promising degrading strain.     

Development of a rapid magnetic bead-based immunoassay for sensitive detection of zearalenone

Here we demonstrate a novel magnetic bead-based enzyme-linked immunosorbent assay (MB-ELISA) for zearalenone (ZEN) detection. Firstly, an anti-ZEN monoclonal antibody (mAb) was prepared by hybridoma technique, and immobilized on carboxyl modified MBs to obtain mAb-MBs. In addition, the biotinylated ZEN-BSA was labelled by streptavidin-HRP for use as competitor. Based on the mAb-MBs and streptavidin-HRP labelled ZEN-BSA, a MB-ELISA which contains only one 20 min antigen-antibody reaction step and takes no more than 45 min for dozens of samples analysis was developed. The half maximal inhibitory concentration (IC₅₀) and limit of detection (LOD) of the MB-ELISA are 1.78 ng/mL and 0.13 ng/mL, respectively. And the MB-ELISA working range for corn samples analysis is from 5.0 μg/kg to 255.2 μg/kg. The recoveries for ZEN spiked corn samples ranged from 82.3 to 110.5% with coefficient of variation (CV) under 8.9%. For natural corn samples analysis, the results of MB-ELISA showed good agreement with the results of conventional direct competitive ELISA (R² = 0.9742).     

Survey of mycotoxins in dates and dried fruits from Tunisian and Spanish markets

In an effort to enforce the knowledge on mycotoxin occurrence and co-occurrence in dates and dried fruits, 228 samples purchased from Tunisian and Spanish markets were subjected to multi-mycotoxin liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of 16 mycotoxins. At least one mycotoxin was detected in 160 samples (70%). The frequency of contaminated samples was 83%, 80%, 64%, 59% and 26% for dates, dried vine fruits, figs, apricots and plums, respectively but none of the analyzed samples contained detectable levels of fumonisins (FB₁, FB₂, FB₃) or T-2 toxin (T2).The incidence and levels of mycotoxins varied in samples. The most prevalent mycotoxin was enniatin B (EnnB) (54%), followed by enniatinA₁ (EnnA₁) (36%); aflatoxins (AFs) (23%) and ochratoxin A (OTA) (22%). Thirteen samples contained AFs in levels that exceed the maximum limits established in EU legislation. The simultaneous contamination with 2–6 mycotoxins was observed in 51% of the analyzed samples. Lastly, intakes of the detected mycotoxins were calculated for average adult consumers and compared with the tolerable daily intakes (TDI). Data obtained were used to estimate the potential exposure levels.     

Fumonisin B1 and its co-occurrence with other fusariotoxins in naturally-contaminated wheat grain

The natural occurrence of fumonisin B₁ (FB₁) and its co-occurrence with zearalenone (ZEA), T-2 toxin and deoxynivalenol (DON) were surveyed in 103 winter wheat samples collected after four to six-month storage in family barns from different locations in Serbia. All 103 samples were mycotoxin positive. The mean concentrations of all mycotoxins except ZEA were greater in 2005 than in 2007. FB₁ was detected in 82.1% and 92.0% of all samples with ranges of 750-5400 μg kg⁻¹ (mean, 2079.45 μg kg⁻¹) and 750-4900 μg kg⁻¹ (mean 918.76 μg kg⁻¹) in 2005 and 2007, respectively. Moderate positive correlations were found between FB₁ and DON concentrations (r = 0.56 in 2005 and r = 0.54 in 2007) and between FB₁ and ZEA concentrations (r = 0.48 in 2005 and r = 0.60 in 2007), while a moderate negative correlation was detected between the production of FB₁ and T-2 toxin in 2007 (r = −0.33). This is the first report of FB₁ occurrence in naturally-contaminated wheat grain and its simultaneous occurrence with ZEA, DON and T-2 toxin in Serbia. Moreover, this is one of the rare reports presenting the occurrence of FB₁ on wheat in the world.     

Plasma inactivation of bacterial endospores on wheat grains and polymeric model substrates in a dielectric barrier discharge

Motivated by the frequent microbial contamination of granular food products like cereal grains or sprout seeds, an atmospheric pressure dielectric barrier discharge (DBD) was developed to study plasma inactivation of microorganisms on granular materials. Wheat grains as well as polypropylene model substrates were artificially contaminated with endospores of Geobacillus stearothermophilus as a model organism and treated in a pulsed argon plasma discharge applying different combinations of treatment time, pulse voltage and frequency. While the treatment of polypropylene substrates resulted in an efficient reduction of microbes, wheat grains, having a rough surface and a deep ventral furrow, turned out to be more challenging to decontaminate. However, an improvement in treatment efficiency could be achieved by applying longer treatment, faster pulse frequency or higher pulse voltage. Furthermore, experiments demonstrated that endospore reduction was not caused by thermal, mechanical or electrical stress factors, but a direct effect of plasma-generated species, and chemical sputtering is supposed to be the predominant inactivation mechanism. Finally, it could be shown that functional wheat grain properties (Falling number, gluten content) are not negatively affected by our plasma treatment.     

Generation of anti-zearalenone scFv and its incorporation into surface plasmon resonance-based assay for the detection of zearalenone in sorghum

Recombinant scFvs to zearalenone were isolated by phage display, expressed in Escherichia coli and the analytical sensitivity of selected scFvs determined by competitive inhibition enzyme-linked immunoassay (CI-ELISA) and surface plasmon resonance (SPR)-based inhibition assay. The results demonstrated higher sensitivity for the SPR-based assay compared to CI-ELISA. The applicability of the SPR-based assay for the detection of zearalenone in food samples was evaluated using sorghum.     

Biofilm forming Salmonella strains exhibit enhanced thermal resistance in wheat flour

The biofilm lifestyle of bacteria confers a remarkably increased tolerance to antimicrobial interventions and environmental stresses, however little is known about influence of biofilms on thermal resistance of Salmonella in low-moisture foods. This study was aimed to assess the correlation between the ability of biofilm formation of Salmonella Enteritidis (S. Enteritidis) strains and their capacity to survive desiccation and thermal treatment in wheat flour as a model for low-moisture food. The production of the biofilm in S. Enteritidis strains was analyzed qualitatively and quantitatively using calcoflour fluorescence, congo-red binding, pellicle formation and microtiter-plate test. Subsequently, three biofilm-forming and four non-forming S. Enteritidis strains were selected. Survival after desiccation was evaluated by population counts before and after equilibration for 4–5 days at 45% RH. Thermal resistance (D_{80 °C, 0.45 aw}) of S. Enteritidis in wheat flour was evaluated by fitting the thermal inactivation kinetic data with the first order kinetics model. The biofilm forming ability was not associated with resistance to desiccation. However, thermal resistance (D_{80 °C, 0.45 aw}) and pre-formed biofilm amount (OD_492 nm) showed a linear correlation (Spearmen correlation ρ = 0.8, p < 0.05), indicating more biofilm production confers more thermal resistance. Average thermal resistance (D_{80 °C, 0.45 aw}) was significantly (p < 0.05) higher among biofilm formers (14.1 ± 0.6 min) when compared with non-formers (6.0 ± 0.2 min). This study shows that the amount of biofilm produced by Salmonella on congo red-calcofluor media is linearly correlated with the thermal resistance of Salmonella in wheat flour. The findings reinforce the necessity of appropriate management in sanitation and biofilm removal in plants that process low-moisture foods.     

Fumonisin occurrence in naturally contaminated wheat grain harvested in Argentina

A survey was carried out to determine fumonisin contamination in 135 common and 40 durum wheat samples collected during 2011 harvest season (non-FHB epidemic year) in the main wheat production area of Argentina using LC–MS/MS. A 93% of total samples showed fumonisin contamination, with levels ranging from 0.16 to 680.44 ng/g in common and from 0.15 to 1304.39 ng/g in durum wheat samples, respectively. FB₁ was the fumonisin most frequently found during the evaluated year. Twenty five wheat samples (15 common and 10 durum) were selected for a deoxynivalenol (DON) analysis among all the samples analyzed for fumonisin content using different contamination levels as selection criteria. DON contamination was present in 24 out of 25 wheat samples, the levels ranging from 50.60 to 28650 ng/g. Nine out of 25 wheat samples reached values higher than 1000 ng/g. However there was no correlation between fumonisin and DON contamination. This is the first report of natural fumonisin presence in common wheat grains in Argentina, as well as of DON co-occurrence in both types of wheat.     

Natural occurrence of deoxynivalenol in soy sauces consumed in China

A total of 40 samples of domestic and imported soy sauces with the greatest diversity were investigated for the presence and contents of deoxynivalenol (DON) by using GC–MS after pre-column derivatisation. Results showed that 97.5% of samples investigated were contaminated with DON, and there was a remarkable variation in the DON contents across soy sauce samples. The average incidence rate of DON in domestic soy sauces was 97.1% (range 4.5–1245.6 μg/l, average value 141.5 μg/l), while for those imported from Japan was 100% (range 30.5–238.3 μg/l, average value 113.7 μg/l). Manufacturing process and quality grade were found to have significant influences on the levels of DON in soy sauces. Generally, domestic soy sauces produced with low salt solid state fermentation and with higher quality grade exhibited higher DON contents. The present results also indicated that the exposure to DON from the consumption of soy sauces is at a very low risk level.