Survey of aflatoxin B1 in helva,a traditional Turkish food,by TLC

A total of 102 helva samples consisting of 34 plain helva, 34 helva containing cacao, and 34 helva containing pistachio nuts purchased from helva-factories and supermarkets in Adana of Turkey were analysed for aflatoxin B₁ (AFB₁) by thin-layer chromatography. The detection limit of AFB₁ was 1 μg kg⁻¹. Recovery experiments were carried out with spiked samples in the range 2–10 μg kg⁻¹ of AFB₁. No AFB₁ was found in any plain helva and helva containing cacao samples. On the other hand, of 34 helva containing pistachio nuts AFB₁ was determined in eight samples. AFB₁ was found in excess of Turkish legal limit of 5 μg kg⁻¹ in 4 of 102 helva samples. This paper reports the data of the first survey for the presence of AFB₁ in helva in Turkey.     

Aflatoxin contamination level in Iran's pistachio nut during years 2009–2011

An efficient monitoring system for sampling, analyzing and issuing the export certificates for pistachio consignments has been established in Iran in recent years. Accordingly, 3181 commercial raw pistachio nut lots were supplied for testing for European export certification since January 2009 till December 2011. Aflatoxin analysis was carried out by high-performance liquid chromatography with fluorescence detection after immunoaffinity column clean up with recoveries ranging from 77 to 99%. Amongst 8203 sub-samples analyzed, aflatoxin B₁ (AFB₁) was detected in 1921 cases (23.4%) with the mean and median values of 2.18 ± 13.1 ng/g and <LOD, respectively. Total aflatoxin (AFT) was detected in 1927 sub-samples (23.5%) with the mean and median values of 2.42 ± 14.7 ng/g and <LOD, respectively. AFB₁ level in 556 (6.78%) and 428 (5.22%) sub-samples was above the maximum tolerable levels set for AFB₁ in Iran (5 ng/g) and European Union (EU) (8 ng/g). The mean contamination levels of AFB₁ (2.18 ng/g) and AFT (2.42 ng/g) were lower than the maximum tolerable levels set in Iran and EU. The contamination levels of pistachio nut for export to EU were ∼50% of those found in 2002–2003 indicating a satisfying improvement in hygienic conditions of pistachio cultivation, harvesting and post-harvesting practices in Iran.     

Aflatoxins in bulk and pre-packed pistachios sold in Spain and effect of roasting

The occurrence of aflatoxins in national and imported pistachios available in Aragón (NE Spain), purchased from commercial outlets during 2007, was surveyed. Thirty-two samples of roasted pistachios were analyzed for aflatoxins by immunoaffinity cleanup with liquid chromatography and fluorescence detection using post-column photochemical derivatization. Results showed that the incidence of aflatoxin B₁ and B₂ in pre-packed pistachios was 19% and 6%, while in bulk pistachios was 50% and 12.5%, respectively. All positive samples originated from Iran, while pistachios from USA, Turkey and Spain tested negative for aflatoxins. The low degree of contamination ranged from 0.12 to 0.29 μg/kg, and no sample exceeded the maximum permitted level for aflatoxins in pistachio nuts. Naturally occurring aflatoxin B₁ was not noticeably reduced by commercial roasting at 120 °C for 20 min.     

Natural occurrence of aflatoxin B1 and aflatoxin M1 in “halva” and its ingredients

A total 431 samples including halva (56), pistachio (71), almond (63), semolina (69), cardamom (34), raisins (46), halva puri (39) and wheat powder (53) were analyzed using HPLC equipped with florescence detector. The results have shown that 32 (57%) samples of halva, 45 (63%) pistachio, 43 (68%) almond, 46 (67%) semolina, 21 (62%) cardamom, 19 (41%) raisins, 21 (54%) halva puri and 22 (42%) of wheat powder samples were found contaminated with AFB₁, and 11 (20%), 23 (32%), 34 (54%), 12 (17%), 11 (32%), 7 (15%), 9 (23%) and 11 (21%) samples, respectively were above the European Union permissible limit (2 μg/kg). The results have shown that 20 (59%) samples of halva that contained milk were found contaminated with AFM₁ and 3 (9%) samples were found above the recommended limit for AFM₁ i.e. 0.05 μg/kg. Limit of detection (LOD) and Limit of quantification (LOQ) for AFB₁ and AFM₁ were 0.04 μg/kg, 0.12 μg/kg, and 0.004 μg/L, 0.012 μg/L, respectively.     

Highly sensitive toxin microarray assay for aflatoxin B1 detection in cereals

We report a rapid, highly sensitive microarray method for quantitative aflatoxin B₁ (AFB₁) detection in cereal samples. Following optimisation using an indirect competitive immunoassay, optimised amounts of AFB₁-bovine serum albumin (AFB₁-BSA)-conjugate were contact-printed onto 16 isolated sub-arrays on multi-pad nitrocellulose coated slides subsequently used in competitive binding assays.The toxin microarray working range for AFB₁ was established in the range of 15 pg g⁻¹ to 3.04 ng g⁻¹, with a detection limit of 1 pg g⁻¹. To determine assay sensitivity in contaminated food models, wheat flour and barley grains samples were spiked with AFB₁ standard dilutions. Following extraction, the working ranges of 0.11–4.15 and 0.18–4.31 ng g⁻¹ were determined, with detection limits of 30 and 90 pg g⁻¹, respectively. The sensitivity of the developed assay is below the European commission limit set for AFB₁ detection and the assay procedure was completed in 3 h time. Good recoveries (98% ± 11%) obtained demonstrate the suitability of the proposed method for rapid and sensitive quantification of AFB₁ in contaminated cereal samples.     

Effect of ozone treatment on aflatoxin B1 and safety evaluation of ozonized corn

This paper studies the ozone treatment effect on degradation of aflatoxin B₁ (AFB₁) in corn with different moisture content (MC). The toxicity of the degradation products (DPs) of the ozone-treated AFB₁-Contaminated Corn (ACC) was also evaluated using the human hepatocellular carcinoma cell line (HepG2) as model cells. The degradation rate of AFB₁ in corn increases with ozone concentration and treatment time. The results showed that ACC with 13.47% MC was easier to be degraded by ozone than with 20.37% MC. Treated with 90 mg L⁻¹ ozone for 20 min and 40 min, AFB₁ in corn with 13.47% MC decreased from 83 μg kg⁻¹ to 18.12 μg kg⁻¹ and 9.9 μg kg⁻¹, respectively, well meeting the China National Standard of AFB₁ in corn (20 μg kg⁻¹). In order to evaluate the safety of ozone used on ACC, the impacts of AFB₁ as well as untreated and ozone-treated ACC with the same level of AFB₁ content on HepG2's survival rate, morphology, and apoptosis were studied. The results showed that ACC had high cell toxicity while the toxicity of ozone-treated ACC had no significant difference with that of the AFB₁-free culture solution. It is concluded that ozonation can quickly and effectively degrade AFB₁ in corn and diminish ACC's toxicity, and therefore, ozonation is expected to be an effective, fast, and safe method for AFB₁ degradation in ACC.     

Degradation and detoxification of AFB1 by Staphylocococcus warneri,Sporosarcina sp. and Lysinibacillus fusiformis

Effects associated with aflatoxins (AFs), principally aflatoxin B₁ (AFB₁) have necessitated strategies to eliminate their occurrence in commodities along the food chain. This study therefore, investigated the AFB₁ biodegradation ability of Staphylococcus warneri, Sporosarcina sp. and Lysinibacillus fusiformis liquid cultures and cell lysates (disrupted in the presence or absence of protease inhibitors to obtain lysates). These were incubated with AFB₁ (2.5 μg/mL) for 3, 6, 12, 24 and 48 h. AFB₁ degradation was subsequently monitored on high performance liquid chromatography (HPLC) and results indicated that after 48 h, % AFB₁ degradation by the liquid cultures of Lysinibacillus fusisormis, S. warneri and Sporosarcina sp. were 61.3, 47.7 and 46.9%, respectively. After 12 h of incubation, a 100% AFB₁ degradation was observed for all protease inhibited lysates tested. To establish toxicity of the AFB₁ biotransformed products, results from a cytotoxicity study against human lymphocytes demonstrated that the products exhibited significantly (p ≤ 0.05) lower cytotoxic effect compared to the parent AFB₁. From this study, it can be deduced that the mechanism of AFB₁ degradation was enzymatic and that protease inhibition of cells before disruption, could increase this enzymatic activity. Conclusively, the potential of these lysates as a biotechnological approach towards decontaminating AFB₁ is promising.     

Inactivation of aflatoxigenic fungi and the reduction of aflatoxin B1 in vitro and in situ using gamma irradiation

Detailed investigation on the effect of gamma (γ) irradiation on germination, sporulation, and growth of aflatoxigenic moulds (Aspergillus parasiticus 2999, Aspergillus flavus 305, and Aspergillus niger 388), as well as on the reduction of aflatoxin B₁ (AFB₁) level in artificially and naturally contaminated maize/feed samples was performed. The results of in vitro and in situ experiments with aflatoxigenic moulds demonstrated that 5 kGy-γ irradiation manages to prevent sporulation, germination and growth of the tested moulds both when in form of a pure and when in form of a mixed culture. In the feed samples artificially contaminated with AFB₁ (50 μg kg⁻¹) 5 kGy-γ irradiation reduced AFB₁ level by around 60%, while 10 kGy-dose reduce it for around 85%. Similarly, in feed samples spiked with AFB₁ in the concentrations of 100 μg kg⁻¹ 5 kGy-dose reduced the AFB₁ level by approximately 70%, while the dose of 10 kGy reduced it by approximately 90%. The experiments on naturally contaminated maize samples (n = 30) confirmed these observations; following a 5 kGy-irradiation, the overall mean AFB₁ reduction equalled to 69.8%, while the irradiation with a 10 kGy-dose achieved the overall mean toxin reduction of 94.5%. The obtained results indicate that γ irradiation can be used to prevent the growth of aflatoxigenic moulds and to reduce the AFB₁ levels in various goods intended for animal and human consumption, thus minimizing the animal and human exposure to this carcinogenic mycotoxin.     

Degradation of AFB1 in aqueous medium by electron beam irradiation: Kinetics,pathway and toxicology

The degradation study of aflatoxin B₁ (AFB₁) in aqueous medium was performed under electron beam irradiation (EBI) at various AFB₁ initial concentrations. It has been proven that the degradation of AFB₁ in the selected ranges of concentrations follows pseudo first-order reaction kinetics well (R² > 0.95). Five degradation products of AFB₁ in aqueous solution were identified by UPLC-Q-TOF/MS, and the possible degradation pathway was proposed. The Ames and cytotoxicity tests were employed to evaluate the toxicity of the AFB₁ degradation products in aqueous solution, and the results indicated that the mutagenicity and cytotoxicity of EBI treated samples decreased significantly compared with that of untreated samples, but were not completely disappeared. The study provided clues involving the application of EBI methods in AFB₁ decontamination.     

Aflatoxin B1 degradation by culture and lysate of a Pontibacter specie

The presence of aflatoxin B₁ (AFB₁) along the food chain poses a significant threat, thus propelling the need for an effective approach to control it. This study was therefore, aimed at investigating AFB₁ degradation of liquid cultures and lysates of an isolated Pontibacter sp. (VGF1). Liquid cultures, lysed bacterial cells in the absence (uninhibited lysates) and presence of protease inhibitors (protease inhibited lysates) were respectively incubated with AFB₁ for 3, 6, 12, 24 and 48 h. AFB₁ degradation was monitored during this period on high performance liquid chromatography (HPLC) and results obtained revealed that after 6 h of incubation, the protease inhibited (PI) lysates yielded a 65% AFB₁ degradation, whereas after 12 h, no residual AFB₁ was detected. Conversely, after 48 h of incubation, a significantly (p≤0.05) lower AFB₁ degradation of 50 and 36% by the liquid culture and uninhibited lysate, respectively, were noted. It was further confirmed that the degradation mechanism was enzymatic. Data from cytotoxicity studies against human lymphocytes further demonstrated that extracts of biotransformed AFB₁ were less toxic when compared to that of AFB₁. Findings from this study have demonstrated an alternative approach for the decontamination and biocontrol of AFB₁ in various agricultural commodities.     

Isolation and characterization of a Bacillus subtilis strain with aflatoxin B1 biodegradation capability

Aflatoxins are type of mycotoxins mainly produced by Aspergillus flavus and a common contaminant of food and grain, posing a serious economic and health problem worldwide. In order to find efficient bacteria to remove or detoxify these mycotoxins, a bacterial strain capable of degrading aflatoxin B₁ (AFB₁) was isolated from soil samples using a culture medium containing coumarin as the sole carbon source. Based on 16S rRNA gene sequence analysis, this isolate was identified as Bacillus subtilis JSW-1; its further characterization showed that it could inhibit the growth of A. flavus with an inhibition ratio of 58.3% and could degrade AFB₁ by 67.2% after incubation at 30 °C for 72 h. The aflatoxin B₁-degrading activity of isolate JSW-1 was predominantly attributed to the cell-free supernatant and this activity was found to be heat stable but sensitive to proteinase K treatment, indicating that the extracellular proteins or enzymes are responsible for the AFB₁ degradation. In addition, no degradation products of AFB₁ could be detected by liquid chromatography-mass spectrometry (LC-MS) analysis, indicating that the parent AFB₁ might be biotransformed to compounds with chemical properties different from that of AFB₁.     

Aflatoxin B1 and ochratoxin A in breakfast cereals from athens market: Occurrence and risk assessment

A method for aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) determination in breakfast cereals is described using a simultaneous methanolic-aqueous extraction followed by immunoaffinity columns clean-up step and High Pressure Liquid Chromatography (HPLC) with Fluorescence Detector (FD). Recoveries were found to be 78% and 83% for AFB₁ and OTA, respectively, while the detection limit (DL) was 0.02 ng g^?1 for both mycotoxins. Both determinations were applied in fifty five samples of breakfast cereals purchased from Athens market. Results revealed the presence of AFB₁ in 56.3% of the samples examined (mean 1.42 ng AFB₁ g^?1). Seven samples (median 3.5 ng AFB₁ g^?1) were found to be contaminated at levels higher than the EU limit (2 g g^?1). OTA was detected in 60% of the samples (mean 0.18 ng g^?1). Nineteen samples were found to be contaminated by both mycotoxins. In addition in the present study the daily exposure to AFB₁ and OTA is discussed.     

Amperometric biosensor for aflatoxin B1 based on aflatoxin-oxidase immobilized on multiwalled carbon nanotubes

An amperometric aflatoxin biosensor developed by aflatoxin-oxidase (AFO), embedded in sol-gel, linked to multiwalled carbon nanotubes (MWCNTs)-modified Pt electrode was reported for the first time. The covalent linkage between AFO and MWCNTs retained enzyme activity and responsed to the oxidation of afltoxin B₁ (AFB₁). Its apparent Michaelis-Menten constant for AFB₁ was 7.03 μmol·L^?1, showing a good affinity. The sensor exhibited a linear range from 3.2 nmol·L^?1 to 721 nmol·L^?1 (1 ng/ml to 225 ng/ml) with limits of detection of 1.6 nmol·L^?1 (signal-to-noise ratio = 3), an average response time of 44 s (less than 30 s when AFB₁ Conc. is bigger than 45 ng/ml), and a high sensitivity of 0.33 × 10² A mol^?1·L cm^?2. The active energy was 18.8 kJ mol^?1, demonstrating the significant catalyzation of AFO for oxidation of AFB₁ in this biosensor.     

Aflatoxin B1 degradation by Bacillus subtilis UTBSP1 isolated from pistachio nuts of Iran

Pistachio nuts are among the commodities with the highest risk of aflatoxin contamination in Iran. Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for humans and livestock. In nature, there are microorganisms which are capable of reducing aflatoxins contamination in food and feed products. In this study, Bacillus subtilis strain UTBSP1 was isolated from pistachio nuts and studied for the degradation of AFB1. The AFB1 contents were determined by the use of HPTLC and HPLC as well as multiple reactions monitoring (MRM) method in LC-MS/MS. The results indicated B. subtilis UTBSP1 could considerably remediate AFB1 from nutrient broth culture and pistachio nut by 85.66% and 95%, respectively. Cell free supernatant fluid caused an apparent 78.39% decrease in AFB1 content. The optimal conditions for AFB1 degradation by cell free supernatant appeared at 35-40 °C, during 24 h. Furthermore, the results indicated that AFB1 degradation is enzymatic and responsible enzymes are extracellular and constitutively produced. The destructive AFB1 differed from standard AFB1 chemically, and lost a fluorescence property.     

Aflatoxin B1 decontamination by UV-mutated live and immobilized Aspergillus niger

The decontamination of Aflatoxin B₁ (AFB₁) by immobilized cells of a new mutant strain, prepared on a base of HSCAS (hydrated sodium calcium aluminosilicate), was studied. Novel strains were induced by UV irradiation, from which 50 were screened according to their degradation efficacy on AFB_1, compared with the wild strain (FS-Z1). The FS-UV1 strain exhibited highest degradation efficacy, which was confirmed by 18SrDNA to be Aspergillus niger. The results indicate that both immobilized cells and this mutant strain which are incubated for 48 h at 30 °C, would considerably remediate AFB₁ in nutrient broth culture, by 95.32% and 82.43%, respectively. By the application of samples of contaminated cottonseed meal, with results of 93.46%∼96.82%, the degradation rate was also validated. The results of Ames test indicate the mutagenic activity of treated AFB₁ is greatly abated, with treated controls. The Application of LC-q-TOFMS (liquid-chromatography, quadrupole, time-of-flight mass spectrometry) deduces the structure and molecular formulas of the degradation products. In the vivo study, the damages of photomicrographic evidence are decreased in kidney and liver and the serum biochemical parameters is improved, in response to preventative treatment with immobilized cells. This is the application of HSCAS-prepared, immobilized A. niger cells to degrade AFB₁ of contaminated samples. The investigation in this paper offers a novel path for economical, time-saving biodegradation of AFB₁ in foods and feeds.     

Annual and regional variations of aflatoxin B1 levels seen in grains and feed coming from Croatian dairy farms over a 5-year period

The aim of the study was to investigate annual and regional differences in the level of aflatoxin B₁ (AFB₁) in grains and dairy cattle feed. Maize (n = 972), wheat (n = 201), barley (n = 147), oat (n = 136), grain mixtures (n = 168), and dairy cattle feed (n = 325) were sampled from 2009 to 2013 on different farms and in different farm factories situated in four Croatian regions. The samples were analysed for AFB₁ using the validated ELISA immunoassay. AFB₁ was determined in 16.4% of all investigated samples, among which maize was proven to be the most contaminated, with 21.7% of the samples recovered during 2013 harbouring AFB₁ in concentrations over the permissible ones. Levels higher than permitted were observed in 17.9% and 12.3% of grain mixtures and dairy cattle feed, respectively, whereas concentrations of AFB₁ determined in other crops throughout the investigated period met the stipulated requirements. The results revealed the AFB₁ occurrence to be significantly (p < 0.05) dependent on the cultivation region, with the highest levels generally found in maize harvested in 2013 and consequently in grain mixtures and cattle feed that can most likely be associated with climatic conditions as the most critical factor for mould formation, and thus also AFB₁ production.     

Survival and inhibition of Staphylococcus aureus in commercial and hydrated tahini using acetic and citric acids

Tahini (sesame paste) is a low-moisture ready-to-eat food that has been linked to foodborne outbreaks and recalls. The objectives of this study were to investigate the behavior of Staphylococcus aureus in commercial and hydrated tahini at 10, 21 and 37 °C and to inhibit S. aureus in these products by 0.1, 0.3 and 0.5% acetic or citric acid. S. aureus was able to survive in commercial tahini with reductions of 3.3, 1.6 and 0.7 log₁₀ CFU/g at 37, 21 and 10 °C, respectively; while it grew in hydrated tahini with an increase of 3.9, 3.0 and 1.8 log₁₀ CFU/ml at 37, 21 and 10 °C, respectively, by 28d. Citric or acetic acid at ≤ 0.5% reduced S. aureus in commercial tahini by ≤ 2.3 log₁₀ CFU/ml by 28d compared to control at all of the tested temperatures. However, acetic and citric acid were more inhibitory at 37 and 10 °C, respectively. In hydrated tahini, viable S. aureus cells were not detected in the presence of 0.5 or 0.3% acetic acid after 7 and 14d, respectively, at both 21 and 37 °C; and after 14 and 28d, respectively at 10 °C. Acetic acid at 0.1% also reduced S. aureus numbers to undetectable levels after 14 and 28d at 21 and 37 °C, respectively. S. aureus cells were also not detected in the presence of 0.5% citric acid by 21d at all of the tested temperatures, or 0.1 and 0.3% citric acid by 28 and 21d, respectively at 21 °C. Acetic and citric acids could be used in tahini or tahini-based products to reduce the potential risk associated with S. aureus.     

Survey of aflatoxins and ochratoxin A in retail market chilies and chili sauce samples

The aim of this study was to determine the levels of the aflatoxins (AFs) and ochratoxin A (OTA) in 312 samples of whole chili, chili powder, crushed chili and chili sauce samples from suburbs, open market and food restaurants of Punjab, Pakistan. The analysis was carried out using HPLC with fluorescence detector, after immunoaffinity column clean-up. The results have shown that 176 out of 312 (56.4%) samples were to be positive with AFs and 126 out of 312 (40.4%) sample of chilies were found to be contaminated with OTA. Total mean level of AFB₁ and total AFs in chilies were 12.50 ± 1.91, 15.16 ± 2.22 μg/kg, respectively. The total mean level of OTA in chilies was found 16.68 ± 2.58 μg/kg, ranged from LOD to 120.9 μg/kg. Sample 39.5, 26.3 and 32.7% of chilies were found containing level of AFB₁, total AFs and OTA, higher than the recommended limits for EU, respectively. The dietary levels of 3.26, 3.52, and 3.84 μg/kg were determined for AFB₁, total AFs and OTA in chilies. The incidence and levels of AFs and OTA in chilies are higher and could pose serious health hazards for consumers.     

Kinetics of ochratoxin A destruction during coffee roasting

In the present study, Coffea arabica was artificially contaminated with spores of toxigenic Aspergillus westerdijkiae. The contaminated coffee was roasted in a vertical spouted bed roaster at four different temperatures (180 °C, 200 °C, 220 °C and 240 °C) and three different time periods (5, 8 and 12 min), in order to obtain more accurate results for the development of the kinetic model for ochratoxin A (OTA). Chlorogenic acids (CGA) content during coffee roasting was also evaluated to investigate the effect of the heat employed to destroy OTA in these health promoting compounds. Coffee treated with spouted bed roasting significantly reduced the OTA level from 8% to 98%. The spouted bed roasting proved to be a very efficient procedure for OTA reduction in coffee, and its reduction depended directly on the degree of roasting. OTA degradation during coffee roasting followed first order reaction kinetics. Using the apparent activation energy of OTA degradation and the temperature-dependent reaction rate, there was a compliance with the Arrhenius equation. This model was capable of predicting the thermal induced degradation of OTA and may become an important predicting tool in the coffee industry. The present study was also able to propose roasting conditions appropriate to destroy OTA and maintain most of the CGA at the same time.     

Aflatoxin B1 production by Aspergillus parasiticus and strains of Aspergillus section Nigri in currants of Greek origin

Aflatoxin B₁ (AFB₁) mostly produced by Aspergillus flavus and Aspergillus parasiticus, is an extremely toxic and carcinogenic metabolite. Currants are used in the Mediterranean diet as a food with antioxidant properties. Four strains of Aspergillus section Nigri have been isolated from currants originated from Crete and Corinth. In this study AFB₁ production by A. parasiticus and the four strains of Aspergillus section Nigri in Cretan and Corinthian currants (Vitis vinifera L.) is investigated. AFB₁ determination was performed by HPLC–FID. Results revealed that the four strains Aspergillus section Nigri, as well as the aflatoxigenic strain A. parasiticus produced AFB₁ (0.0052–1.31 μg AFB₁ 15 g⁻¹, corresponding to 0.0003–0.087 μg AFB₁ g⁻¹) in both type of currants (Cretan and Corinthian) on the 12th day of observation. Moreover, AFB₁ production, by A. parasiticus in the synthetic Yeast Extract Sucrose (YES) medium was also studied. The ability of AFB₁ production has been affected by the special characteristics of each isolate and the currants substrate.