Survey of aflatoxin B1 and ochratoxin A occurrence in traditional meat products coming from Croatian households and markets

The aim of this study was to investigate the occurrence of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in different traditional meat products circulating on Croatian markets, produced by a large number of households situated in different Croatian regions. The study involved a total of 410 samples of traditional pork meat products in terms of hams (n = 105), dry fermented sausages (n = 208), bacon (n = 62) and cooked sausages (n = 35), collected over four years period (2011–2014). Mycotoxin concentrations were quantified and confirmed using validated immunoassay method (ELISA) and high performance liquid chromatography with fluorescence detection (HPLC-FLD), respectively. The maximal observed OTA level in the fermented sausages and hams was around 5 times (5.10 μg/kg) to 10 times (9.95 μg/kg) higher than the maximal recommended level (1 μg/kg) stipulated for pork products in some EU countries. AFB₁ levels found in any given meat product analysed within this frame were not significantly higher (p > 0.05) than the applied method limit of detection. The results showed an occasional mycotoxin contamination of traditional meat products, especially that by OTA, pointing that to avoid such contamination meat and meat products on households should be produced and processed under standardised and well-controlled conditions.     

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.     

Inhibition of aflatoxin B1, B2, G1 and G2 production by Aspergillus parasiticus in nuts using yellow and oriental mustard flours

Aflatoxins (AFs) are naturally occurring mycotoxin compounds produced by several species of Aspergillus, as Aspergillus flavus and Aspergillus parasiticus and are mutagenic, teratogenic, and carcinogenic compounds that have been implicated as causative agents in human hepatic and extra hepatic carcinogenesis. In this study, the reduction of the AFs present in dried fruits (peanut, cashew, walnut, almond, hazelnut and pistachio), produced by A. parasiticus CECT 2681, by isothiocyanates (ITCs) generated by the enzymatic hydrolysis of the glucosinolates (GLCs) present in oriental and yellow mustard flours was evaluated. The AFs reduction activity through ITCs application in dried fruits was carried out using a model and food system experiments. The quantification of the AFs in the food products analyzed was carried out employing the technique of the liquid chromatography (LC) coupled to the mass spectrometry detection in tandem (MS/MS). The ITCs produced through GLCs hydrolysis reduced the A. parasiticus growth in the food products tested and in particular in the model system experiments the AFs B₁, B₂, G₁ and G₂ reduction ranged meanly from 83.1 to 87.2% using the oriental mustard flour, whereas employing the yellow flour the mean reduction observed ranged from 27.0 to 32.5%. In the food system experiments carried out employing only the oriental mustard flour the mean AFs reduction observed ranged from 88 to 89%.     

Occurrence,seasonal variation and risk assessment of exposure to aflatoxin M1 in Iranian traditional cheeses

A total of 360 traditional cheeses consisted of Lighvan (n = 62), Koozeh (n = 62), Siahmazgi (n = 58), Khiki (n = 58), Talesh (n = 58) and Lactic (n = 62) collected from different parts of Iran were analyzed for aflatoxin M₁ (AFM₁) using an enzyme linked immunosorbent assay (ELISA). Frequency of AFM₁ and its concentration ranges of all the ELISA positive samples were determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD). AFM₁ was detected in 60.3%, 75.8%, 72.4%, 43.5%, 38.7% and 35.4% of Siahmazgi, Khiki, Talesh, Lighvan, Koozeh and Lactic cheeses, respectively with concentration ranging from 50.5 to 308.7 ng/kg, respectively. HPLC analyses confirmed the ELISA results although the rates of contaminated cheese samples were lower than that of ELISA. There was significant difference in AFM₁ level between various cheese types and samples collected from summer and winter seasons (P < 0.05). By comparing our findings with the EU limit, about 10.5% of cheese samples had exceeding values for the toxin. The results of the present study indicates that there is no health risk in consumption of Iranian traditional cheeses due to the presence of AFM₁.     

An ELIME-array for detection of aflatoxin B1 in corn samples

The aim of the present work was the development of an ELIME-array to achieve simple and rapid detection of AFB₁ in corn samples. The system is based on an indirect competitive ELISA format using magnetic beads as immobilisation support and eight magnetised screen-printed electrodes as electrochemical transducers.After an optimisation study, a corn sample treatment, employing an extraction in acetonitrile/water followed by a clean-up step and solvent evaporation, was selected.For the construction of the calibration curve, which was used to evaluate both evaluation of the matrix effect on the performances of the ELIME-array and for the analysis of Certified Reference Materials (CRMs), standard solutions of AFB₁ were added to blank dried corn extracts reconstituted in PBS. The detection limit and the sensitivity of the assay were calculated to be 0.6 ng mL^?1 and 1.5 ng mL^?1, respectively.Precision (11–26%) and recovery (95–114%) data of the ELIME-array, determined by analysing four CRMs, have shown that the proposed system appears suitable as a screening tool for the analysis of AFB₁ in corn samples.     

Seasonal variation of aflatoxin M1 contamination in industrial and traditional Iranian dairy products

This study aimed to determine the occurrence of aflatoxin M₁ (AFM₁) contamination in 682 dairy product samples consisting of raw milk of cow, goat and sheep; Lighvan cheese; and industrial and traditional yoghurt, Kashk and Doogh samples collected from popular markets and dairy ranches in four large Iranian cities. Thin layer chromatography (TLC) technique was used for analysis of the samples. Results showed that the incidence and levels of AFM₁ contamination in raw cow milk and industrial products (manufactured from cow milk) were higher than raw goat or sheep milk, and traditional products (made from goat and sheep milk), respectively. Moreover, seasonal variations influenced the concentration of AFM₁ in most of the analyzed dairy products. Owing to the abundance and popularity of the industrial products, contamination of these products in such a level could be a potential hazard for public health.     

Detection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilization platform

We report an aptamer-based biosensor for detection of aflatoxin B₁ (AFB₁), a mycotoxin identified as contaminant in food. The sensor is assembled in a multilayer framework that utilizes cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) for acquiring the signal response by means of redox indicators: K[Fe(CN)₆]^−3/−4. Poly (amidoamine) dendrimers of fourth generation (PAMAM G4) immobilized on gold electrode covered by cystamine, were employed for attachment of single stranded amino-modified DNA aptamers specific to AFB₁. The cystamine-dendrimers (Cys-PAMAM) layers were compared with other immobilization platforms such as cystamine (Cys), 11-mercaptoundecanoic acid (MUA) and 11-mercaptoundecanoic acid-dendrimers (MUA-PAMAM), being the first approach the most appropriate for producing sensitive and reproducible signal in the range of concentrations 0.1–10 nM AFB₁. The sensor was validated in certified contaminated peanuts extract as well as in spiked samples of peanuts-corn snacks and the sensing response was evaluated and compared in terms of the matrix effect. The aptamer specificity was analyzed by testing the sensor in other mycotoxins such as aflatoxin B₂ (AFB₂) and ochratoxin A (OTA). The limit of detection achieved by this sensor was LOD = 0.40 ± 0.03 nM, it was regenerable in 0.2 M glycine-HCl and it did not lose its stability up to 60 h storing at 4 °C. Atomic Force Microscopy (AFM) studies were also performed for illustrating individual steps of biosensor assembly.     

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.     

Probiotic biological strategies to decontaminate aflatoxin M1 in a traditional Iranian fermented milk drink (Doogh)

Aflatoxin M₁ is an important mycotoxin mostly found in milk and dairy products. The main objective of this work was to study the effects of probiotic strains, a probiotic inoculated population, the physiology of probiotic bacteria and final fermentation pH at four consecutive stages on the reduction of 0.500 ppb of free aflatoxin M₁ (AFM₁) in Doogh (a traditional Iranian fermented milk drink). Samples’ biochemical, microbial and AFM₁ binding characteristics were monitored during fermentation and storage (5 °C for 28 days). An immunoaffinity column was used to extract AFM₁ and a high-performance liquid chromatograph with a fluorescence detector was used to measure it. Results showed that Lactobacillus acidophilus was probiotic strain that most reduced free AFM₁. Inoculation of L. acidophilus at 9 log cfu/mL, despite the higher cost, revealed significantly higher free AFM₁ binding capacity than 7 log cfu/mL. Heat-killed (dead) L. acidophilus bacteria reduced less free AFM₁ at the end of storage than viable. Samples with a final fermentation pH of 4.5 bound more free AFM₁ during fermentation and storage than those with a pH of 4.2. It is concluded that inoculation of 7 log cfu/mL L. acidophilus viable cells in Doogh with a final fermentation pH of 4.5 supplied a safety- and health-promoting and cost-effective drink.     

Natural occurrence of aflatoxin B1, ochratoxin A and citrinin in Croatian fermented meat products

When domestic animals are exposed to mycotoxins, significant amounts of the latter shall be carried over into animal products such as milk, eggs and meat. This study was carried out in order to determine the possible presence of aflatoxin B₁ (AFB₁), ochratoxin A (OTA) and citrinin (CIT) in game sausages (n = 15), semi-dry sausages (n = 25) and fermented dry-meat products (n = 50), randomly taken from individual producers and the Croatian market. AFB₁ and OTA were quantified using ELISA, while CIT was quantified using HPLC-fluorescence detector. Out of 90 samples, the fungi most frequently isolated from dry-cured meat products were of Penicillium species, while Aspergillus was isolated from only one sample. As much as 68.88% of the samples were positive for mycotoxins. Finally, the analysis of different types of meat products resulted in OTA identification in 64.44%, CIT identification in 4.44% and AFB₁ identification in 10% of the samples. The maximum OTA concentrations established in the commercial sausage samples equalled to 7.83 μg/kg, while that of AFB₁ amounted to 3.0 μg/kg. Generally, although OTA was detected in all three types of products in different percentage shares, mutual differences were not statistically significant (P > 0.05).     

Diminution of aflatoxin B1 production caused by an active packaging containing cinnamon essential oil

The antifungal and antimycotoxigenic action of an active package containing cinnamon essential oil have been evaluated against the mold Aspergillus flavus on the aflatoxin B1 production. Two independent experiments were carried out, the first one with cinnamon on a paper diffusion disc placed in vapor phase and the second one with an active PP (Polypropylene) films containing the essential oil. The culture media, exposure time, closure of the Petri dish and cinnamon concentration were evaluated. The first experiment revealed an important reduction on mycotoxin, even when the mold grew, and the action remained for 15 days. The second experiment highlighted the importance of cinnamon concentration on the antimycotoxigenic action, achieving a strong reduction with the sub-inhibitory concentration (2% of cinnamon) and a complete reduction with fungicidal concentration (4% and 6% cinnamon). The UPLC system coupled to a fluorescence detector was optimized for analysis of aflatoxin B1.     

Development of an immunochromatographic assay for detection of aflatoxin B1 in foods

A rapid, signal step and sensitive immunochromatographic (IC) test for detection of aflatoxin B₁ (AFB) in foods was described. Colloidal gold-labeled polyclonal antibody specific to AFB was used as the marker; AFB–BSA conjugate was the competitive antigen and goat-anti-rabbit antibody as control. Conditions for the analysis of AFB to be completed in less than 10 min were optimized. With visual observation, the lower limit was found to be around 2.5 ppb. For quantitative analysis by adopting a photometric strip reader, the lower detection limit was found to be around 0.05–0.1 ppb. The detection limit for AFB in food extract was around 2 μg/kg (ppb). The analytical recoveries for AFB added to extracts of rice, corn, and wheat and spiked directly to these foods range in 2–50 ppb were found to be 80.79% (CV, 10.92%) to 110.56% (CV, 7.95%). The CVs of all the assays were between 6.27% and 14.63%. Sixty seven naturally contaminated food samples were subjected to both IC strip and commercial ELISA kit test for AFB. Results showed a good correlation between these two methods with a square of correlation coefficient of 0.93 and slop of 1.13.     

Transfer of aflatoxin M1 from milk to ripened cheese in three Italian traditional production methods

Robiola and Primosale, two fresh cheeses, and Maccagno, an hard-type cheese, were produced using milk that was naturally and artificially contaminated with aflatoxin M₁ (AFM₁) at the levels of 10, 50 and 200 ng/l. Concentrations of AFM₁in milk and cheeses were determined by liquid chromatography and fluorimetric detection, coupled with immunoaffinity column extraction. In the Robiola production method, AFM₁ levels in whey ranged between 30% and 65% of the total amount of the toxin present in the milk, while Primosale and Maccagno, that share the same rennet based cheesemaking procedure, showed an higher percentage of AFM₁ partitioning to whey.For each cheese-making method, the concentration of AFM₁ on fresh matter was higher in the cheese compared to the original milk. The fresh cheeses showed a concentration factors of 1.43 and 2.20 for Primosale and Robiola, respectively, whereas the Maccagno cheese showed a value of 6.71. For all the production methods considered, when using milk not exceeding the maximum acceptable level of 0.05 μg AFM₁/kg set by EU, the resulting cheese also complied with current Italian recommendations for AFM₁ contamination (450 ng AFM₁/kg).     

Elimination of aflatoxin B1 in vegetable oil based on immuno-magnetosomes probes from a novel magnetotactic bacterium

Here, a magnetotactic bacterium (MTB) existing in freshwaters was acclimated and mass propagations for producing natural magnetosomes with diameter about 15 nm at ordinary ambient. It was found that the magnetosomes which are mainly composed of iron oxide and iron sulfide hold pretty well magnetic features. Furthermore, the developed magnetosomes-AFB₁ antibody immunomagentic probes through chemical bonding AFB₁ polyclonal antibody onto the natural magnetosomes maintain 28 fold capability on collection of AFB₁ toxin in vegetable oil compared to conventional Fe₃O₄ magnetic nanoparticle-AFB₁ antibody probes. Recovery ratio up to 93.7% from commercial vegetable oil with artificial AFB₁ contamination during 15 min collection were demonstrated. Through circular dichroism (CD) spectral measurements, the mechanism of magnetosomes probe was interpreted for proposing the practical way with high-performance on controlling the aflatoxins contamination in liquid foods basing on immunomagentic separation technique.     

Degradation and detoxification of aflatoxin B1 using nitrogen gas plasma generated by a static induction thyristor as a pulsed power supply

Toxic fungal metabolite aflatoxin B₁ (AFB1) is a stable carcinogen that is sometimes found in foods, such as peanuts and peppers. In this study, AFB1 was applied to a coverglass and subjected to treatment with nitrogen gas plasma generated by a plasma apparatus using a short high voltage pulse from a static induction thyristor power supply at 1.5 kpps (kilo pulse per second). Enzyme-linked immunosorbent assay showed that a 20 μL aliquot of a 200 ppb solution of AFB1 was efficiently degraded to less than one tenth of the original level within 15 min. High-performance liquid chromatography confirmed the loss of AFB1 after plasma treatment and the generation of small fragments, possibly originating from the degradation process. Moreover, a cell-based assay using HepG2 as an index to measure the cell-growth promoting properties of AFB1 showed that the gas plasma treatment reduced the biological activity of the mycotoxin. Although the amount of heat and ultraviolet light is insufficient to inactivate the toxin, the reactive chemical products appear to contribute to the degradation of AFB1. Controlling and optimizing the conditions for producing these reactive chemical species during plasma generation would lead to efficient inactivation of AFB1.     

Identification of aflatoxin B1 on maize kernel surfaces using hyperspectral imaging

A shortwave infrared (SWIR) hyperspectral imaging system with wavelength range between 1000 and 2500 nm was used to assess the potential to detect aflatoxin B₁ (AFB₁) contaminants on the surface of healthy maize kernels. Four different AFB₁ solutions were prepared and deposited on kernels surface to achieve 10, 20, 100, and 500 ppb, respectively. A drop of 20% methanol was dipped on the surface of 30 healthy kernels in the same way to generate the control samples. Based on the standard normal variate (SNV) transformation spectra, principal components analysis (PCA) was used to reduce the dimensionality of the spectral data, and then stepwise factorial discriminant analysis (FDA) was performed on latent variables provided by the PCA's. Furthermore, beta coefficients of the first three of four discriminant factors were analyzed and key wavelengths, which can represent AFB₁ and be used to differentiate different level of AFB₁ were indentified. Furthermore, 150 independent samples were used as verification set to test the reproducibility of the proposed method. A minimum classification accuracy of 88% was achieved for the validation set and verification set. Results indicated that hyperspectral imaging technology, accompanied by the PCA-FDA method, can be used to detect AFB₁ at concentrations as low as 10 ppb when applied directly on the maize surface.     

A rapid FT-NIR method for estimation of aflatoxin B1 in red chili powder

The feasibility of measuring Aflatoxin B₁ (AFB₁) in red chili powder was investigated by using Fourier transform near-infrared (FT-NIR) spectroscopy in diffuse reflectance mode combined with appropriate chemometric techniques. Aflatoxin free chili powder samples were spiked with known amount of AFB₁ ranging from 15 to 500 μg/kg and used for calibration model building based on partial least squares (PLS) regression algorithm. Different spectral preprocessing methods were investigated and optimized based on the lowest values of root mean square error of cross validation (RMSECV). Spectral wavenumber range of 6900.3–4998.8 and 4902.3–3999.8 cm^?1 and straight line subtraction preprocessing technique predicted AFB₁ content with best accuracy with lowest RMSECV = 0.654% and maximum correlation coefficient for validation plots (R² = 96.7). The overall results demonstrate that FT-NIR spectroscopy can be used for rapid, non destructive quantification of Aflatoxin B₁ in red chili powder.     

Fungal contamination and aflatoxin B1 of ‘egusi’ melon seeds in Nigeria

One hundred and thirty seven samples of melon seeds (Colocynthis citrullus L.) from randomly selected farmers’ stores in the humid forest and Northern Guinea savanna of Nigeria were analysed for the incidence of diseased seeds, moisture content, associated moulds and levels of aflatoxin B₁ contamination. The proportion of diseased seeds ranged from 2.5 to 37.3% in the forest and 2.1 to 17.9% in the savanna, while the seed moisture content varied from 5.3 to 10.4%, and 4.6 to 9.5% respectively. All the samples contained moulds, with the two genera, Aspergillus and Penicillium predominating, while A. flavus had the highest species count. The other common fungal isolates in order of decreasing incidence were A. niger, P. citrinum, Botryodiplodia theobromae, Cladosporium sp and A. clavatus. Thin layer chromatography analysis showed that 32% in the forest and 21% samples in the savanna contained aflatoxin B₁ with mean levels of 14.8 μg/kg in the forest and 11.3 μg/kg in the savanna respectively. Significant positive correlations were found between number of aflatoxin B₁ positive samples and the percentage of A. flavus infected samples and between the levels of diseased seeds and the levels of aflatoxin B₁ contamination.     

Structure elucidation and toxicity analyses of the degradation products of aflatoxin B1 by aqueous ozone

Earlier researches showed that aflatoxin B₁ (AFB₁) can be effectively degraded using ozonation in the aqueous systems. However, the degradation products have not been identified until today. In this article, the degradation products of AFB₁ were analyzed using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometers (UPLC Q-TOF MS). Under our experimental conditions, six key degradation product structures and possible paths for generating fragment ions were proposed. The proposals were based on the low mass error, high match rate in degradation product speculation, and possible molecular formulas that could be obtained using UPLC Q-TOF MS. UPLC revealed the existence of degradation products, whereas Q-TOF MS exposed the structures of the fragment ions for AFB₁ and its degradation products. Due to the conjugate addition reaction on the double bond of the terminal furan ring for AFB₁, the toxicity of the degradation products was significantly decreased compared with that of AFB₁ through the toxicity analysis of the degradation products according to the structure–activity relationship. The results showed that aqueous ozone treatment was an effective method for degrading AFB₁.     

Kinetics of traditional Turkish sausage quality aspects during fermentation

Changes in chemical (pH, moisture, salt, ash, fat, protein, free fatty acids (FFA), thiobarbituric acid reactive substances (TBARS) and residual nitrite contents), colour (Hunter L*, a* and b*, hue angle, chroma (saturation index), browning index (BI) and total colour difference (ΔE)) and microbiological (total mesophilic aerobic bacterial (TMAB), lactic acid bacteria (LAB), total Enterobacteriaceae (TE) and Staphylococcus and Micrococcus (SM)) quality characteristics of traditional fermented sausage “sucuk” during fermentation were investigated and kinetic modeling of these parameters were performed, in this study. The fermentation of the sucuk lasted 9 days. Analysis of the quality parameters was run on the beginning of the fermentation and on the 1st, 2nd, 3rd, 4th, 5th and 9th days of the fermentation process. Changes in the chemical and colour parameters were represented by zero, first and second order kinetic models. Microbial increments were represented by linear (first order kinetic) models and reductions were represented by both linear and Weibull distribution models.