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.     

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).     

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.     

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.     

Evaluation of latex agglutination inhibition reaction test for rapid detection of aflatoxin B1

Aflatoxin B₁ (AFB₁), a mycotoxin mainly produced by some Aspergillus species, has been found in a wide range of agricultural products. To avoid the risk of AFB₁ consumption, many agricultural commodities, foods and feeds should be analyzed and a rapid and non-instrumental method for the detection of AFB₁ is needed. In this study, a rapid, inexpensive and user-friendly latex agglutination inhibition reaction test (LAIRT) for on site testing of AFB₁ had been established. At first, carboxylated polystyrene latex particles (CPLP) were prepared by soap-free polymerization and sensitized with aflatoxin B₁ oxime-BSA (AFB₁O-BSA) for the detection of AFB₁. In LAIRT, the agglutination reaction with AFB₁O-BSA-sensitized CPLP and anti-AFB₁ antiserum mixture was inhibited by 5 ng/mL AFB₁ and the analysis time for 6 samples on one glass slide was less than 10 min. Subsequently, 10 rice and peanut samples were analyzed by LAIRT and ELISA, and the results showed that 1 rice sample and 2 peanut samples were positive and in agreement with those of ELISA. However, the results could be obtained more rapidly by LAIRT than ELISA. This easy and rapid LAIRT might be useful for screening AFB₁ of agricultural commodities, foods and feeds in the field.     

Development and validation of an accurate and rapid LC-ESI-MS/MS method for the simultaneous quantification of aflatoxin B1, B2, G1 and G2 in lotus seeds

An accurate and rapid LC-ESI-MS/MS analytical method was developed and validated for the simultaneous determination of aflatoxin B₁, B₂, G₁ and G₂ in lotus seeds. The samples were firstly extracted with methanol-water solution (80:20, v/v), and then cleaned up by immunoaffinity columns. The mass spectrometer was operated in the positive ionization electrospray (ESI+) mode using multiple reaction monitoring (MRM) for analysis of four aflatoxins. The transitions of m/z 313 → 285 (AFB₁, CE 33 eV), m/z 315 → 259 (AFB₂, CE 37 eV), m/z 329 → 243 (AFG₁, CE 37 eV) and m/z 331 → 257 (AFG₂, CE 37 eV) were used to quantify these four aflatoxins, respectively. The limits of detection (LODs) of aflatoxin B₁, B₂, G₁ and G₂ were 0.007, 0.005, 0.003 and 0.005 μg kg^?1 based on a signal-to-noise ratio of 3:1, respectively. The limits of quantification (LOQs) of aflatoxin B₁, B₂, G₁ and G₂ were 0.02, 0.015, 0.01 and 0.015 μg kg^?1 based on a signal-to-noise ratio of 10:1, respectively. Recoveries for samples of spiked lotus seeds were all above 66% with relative standard deviation all below 15% for all compounds. Nineteen out of twenty batches of lotus seeds collected from different drug stores or markets in China were found to be contaminated with aflatoxins at different levels ranging from 0.02 to 688.4 μg kg^?1.     

Incidence of aflatoxin M1 in human breast milk in Tehran, Iran

This study examined the exposure of infants to aflatoxin M1 (AFM1) and of lactating mothers to aflatoxin B1 (AFB1), using AFM1 in breast milk as a biomarker for exposure to AFB1. An enzyme-linked immunosorbent assay (ELISA) was modified for the analysis of AFM1 in breast milk samples from 160 women in Tehran, Iran. AFM1 was detected in 157 samples by average concentration of 8.2 ± 5.1 ng/kg (range 0.3–26.7 ng/kg).The concentration of AFM1 in one sample was higher than the maximum tolerance limit accepted by European Union and USA (25 ng/kg), but in 55 samples was higher than the maximum concentration recommended by Australia and Switzerland (10 ng/kg).Logistic regression Analysis failed to show significant correlation between AFM1 and gestational age, education, postnatal age, gender, nationality, clinical condition, the number of family member, the number of children, type and amount of dairy consumption, vegetable, fruits, oil and meat. But it was significant relation to the cereal consumption, also to the height at birth.     

A limited survey of aflatoxin M1 in milk from Indonesia by ELISA

This paper presents a limited survey of aflatoxin M1 (AFM1) in Indonesian milk. AFM1 concentrations of 113 fresh milk samples, collected in 2006 from farms in five different areas of the Yogyakarta Province were analysed. The fresh milk samples were taken directly from dairy farms before pasteurisation. Enzyme-linked immunosorbent assay (ELISA) was used for the analysis of milk samples. Results show that in 48 samples (42.5%) the AFM1 concentrations were less than 5 ng/L and in 31 samples (27.4%) AFM1 was found between 5 and 10 ng/L. In 34 samples (30.1%) the concentrations were above 10 ng/L. None of contaminated samples exceeded the European Union regulation limits of 25 and 50 ng AFM1/L for infant and adult consumption, respectively. Since AFM1 is derived from aflatoxin B1 (AFB1) contained in cow feedstuffs, based on the contamination levels of AFM1 found in this study, the exposure of animals to AFB1 seems to be low.     

Survey of aflatoxins and ochratoxin a contamination in food products imported in Italy

In this study the levels of aflatoxins (AF B₁, B₂, G₁ and G₂) and ochratoxin A (OTA) were monitored in several food products imported in Italy with a high contamination risk. A total of 345 samples were collected from the Maritime Authority of Salerno Customs Port during the period from January 2008 to December 2009 and analyzed by immunoaffinity chromatography as clean-up, high performance liquid chromatography with fluorescence detection for quantification and tandem mass spectrometry for confirmation. The analytical methods were validated on different food matrices and meet the performance criteria set by EC Regulation No. 401/2006 for mycotoxin analysis. The results obtained in this survey showed that 7% of the total samples contained detectable levels of AFs and OTA, and 1.2% had AFs concentrations exceeding the maximum limits set by EU regulation. OTA was the most prevalent mycotoxin, with an incidence of 17.6% of samples analyzed for OTA. The highest detected levels were 23.70 μg kg^?1 of OTA in a green coffee sample and 70.69 μg kg^?1 of AFs in an apricot kernels sample. Among the food products analyzed, hazelnuts paste and dried vine fruits were the commodities mainly contaminated with AFs and OTA, respectively.     

Detection of aflatoxin M1 levels by ELISA in white-brined Urfa cheese consumed in Turkey

Aflatoxin M1 (AFM1) is the hydroxylated metabolite of aflatoxin B1 found in a variety of foods. In this study, 127 samples of white-brined Urfa cheese produced mainly in the southeast of Turkey from raw ovine and bovine milks were surveyed for the presence of AFM1 using a competitive Enzyme Linked Immunosorbent Assay (ELISA) technique. The results showed that at detectable levels (≥50 ng/kg), 36 cheese samples (28.3%) were contaminated with AFM1 ranging from 70.61 to 770.97 ng/kg. Of the 36 cheese samples, 13 (10.2%) were found to have levels that exceeded the legal limits of 250 ng/kg established by the Turkish Food Codex. Consequently, the AFM1 contamination levels determined in this study in white-brined Urfa Cheese, which is commonly consumed in the southeast part of Turkey, were not considered to be a serious public health hazard. It was considered to be a potential risk for customers, particularly for infant health.     

Occurrence of aflatoxin M1 in dairy products in Brazil

The objective of this study was to investigate the incidence and occurrence of aflatoxin M₁ (AFM₁) in dairy products produced in Brazil. A total of 123 samples of three different groups of dairy products (cheese, yoghurt, and dairy drinks) consumed by Brazilians were collected during 2010. All samples including 58 cheese samples, 53 samples of yoghurt and 12 dairy drinks were purchased from grocery stores in the Ribeirão Preto-SP area. Cheese samples were classified into three categories depending on their moisture and fat contents: Minas Frescal cheese, Minas Frescal light cheese and Minas Padrão cheese. Samples were analyzed for AFM₁ by a published method. The method comprised aqueous methanol extraction, immunoaffinity column purification and isolation, reversed phase liquid chromatography separation and fluorescence detection. AFM₁ was detected in 84% of the analyzed cheese samples (>3 ng/kg) with levels ranging from 10 to 304 ng/kg in 67% of the samples. AFM₁ was detected in 95% of the yoghurt and dairy drink samples with levels ranging from 10 to 529 ng/kg in 72% of the samples. Despite the lack of a Brazilian regulatory limit for AFM₁ in yoghurt and dairy drinks the survey data of this study may offer information useful in the determination of whether the occurrence of AFM₁ in Brazilian dairy products may be considered as a possible risk for consumer health and whether Brazilian regulatory guidelines for AFM₁ in dairy products are needed.     

Presence of aflatoxin M1 in milk and infant milk products in Tehran,Iran

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic compounds. The purpose of this survey was to determine natural occurrence and level of AFM₁ in pasteurized liquid milk, infant formula and milk-based cereal weaning food consumed in Tehran, Iran.A total of 328 branded milk products and liquid milk samples were collected and investigated by Enzyme Linked Immuno Sorbent Assay (ELISA).The samples of pasteurized liquid milk (n = 128), infant formula (n = 120) and milk-based cereal weaning food (n = 80) showed that the incidence of contamination with AFM₁ is 96.3%, the presence of AFM₁ in each group was 72.2 ± 23.5, 7.3 ± 3.9 and 16.8 ± 12.5 ng/kg, ranging between 31–113, 1–14 and 3–35 ng/kg, respectively.In general, the amount of AFM₁ in 100 (78%) of liquid milk samples and 24 (33%) of milk-based weaning food was higher than the maximum tolerance limit accepted by European Union, but in all of the infant formula samples was lower (European Communities and Codex Alimentarius has prescribed a limit of 50 ng/kg for AFM₁ in milk and 25 ng/kg in infant milk products).     

Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies

A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) method using monoclonal antibody for measuring aflatoxin M1 (AFM1) in milk and milk products has been described. One monoclonal antibody was isolated and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with AFM1 carboxymethyl oxime conjugated with bovine serum albumin (BSA). Cross-reactivities of the anti-AFM1 monoclonal antibody clone were 100, 13.9, 6.7 and <1% against AFM1, aflatoxin B1 (AFB1), aflatoxin G1 (AFG1) and deoxynivalenol (DON), respectively. Assays of milk samples mixed with AFM1 ranging in concentration from 0.1 to 3.2 ng/ml gave mean ELISA recovery of 98%. The limit of detection concentration of AFM1 was 0.04 ng/ml. AFM1 contamination was measured in 12 samples of raw milk, 15 samples of powdered milk, 104 samples of liquid milk and four cheese samples collected from different supermarkets in Northeast of China. Of 135 milk samples tested, 55 (41%) samples contained AFM1 at levels that ranged from 0.32–0.50 ng/ml, 24 (18%) samples contained 0.16–0.32 ng/ml, and 18 (13%) samples contained 0–0.16 ng/ml; in 38 (28%) samples AFM1 was not detected. The results indicate that the necessary precaution will have to be taken to minimize the AFM1 contamination in milk and milk products from Northeast of China.     

Development of novel monoclonal antibodies-based ultrasensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for aflatoxin B1 detection

Monoclonal antibodies (mAbs) that are specific to aflatoxin B1 (AFB1) were produced from hybridoma cell lines 3F6G11 and 9C7C11 by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells that were isolated from a BALB/c mouse that was immunized with AFB1-bovine serum albumin (BSA). Both 3F6G11 and 9C7C11 mAbs are the immunoglobulin G1 isotypes. Competitive direct enzyme-linked immunosorbent assays (cdELISA) were established to characterize these antibodies. In the 3F6G11 mAb based cdELISA, the concentrations causing 50% inhibition of binding of AFB1-horseradish peroxidase to the antibody by AFB1, AFB2, AFG1, and AFG2 were found to be 0.051, 0.050, 1.820, and 1.270 ng/ml, respectively. Using 9C7C11 mAbs, similar IC₅₀ values for AFB1, AFB2, AFG1 and AFG2 were obtained as 0.045, 0.057, 2.530 and 2.120 ng/ml, respectively. A rapid and sensitive gold nanoparticle immunochromatographic strip (immunostrip) was also established for these antibodies. This strip has a detection limit of 1.0 ng/ml for AFB1 and the whole assay can be completed within 10 min. Extensive analysis of 20 samples by 3F6G11 mAb and 9C7C11 mAbs cdELISAs revealed that six samples were slightly contaminated by AFB1 at concentrations from 0.160 to 16.10 ng/g. Results of analyses of 20 samples with an immunostrip assay correlate well with those obtained using cdELISA. The proposed cdELISA and immunostrip methods are highly sensitive for the rapid screening of AFB1 in food samples.     

Screening of aflatoxin M1 occurrence in selected milk and dairy products in Terengganu,Malaysia

The study was conducted to screen the occurrence of aflatoxin M₁ (AFM₁) in 53 selected milk and dairy product samples (11 liquid milk, 12 powdered milk, 8 3-in-1 beverages, 6 condensed sweetened milk, 2 evaporated milk, 7 cultured milk drink, 5 yogurt and 2 cheese samples). These samples were purchased from selected markets in Terengganu, Malaysia in January 2014 based on a questionnaire survey among 212 respondents on the types and brands of milk and dairy products that were frequently consumed. Based on the responses, 53 milk and dairy products were purchased and the competitive enzyme-linked immune-absorbent assay (ELISA) method was used to determine the level of AFM₁ in the samples. Of 53 samples, 19 samples were positive with AFM₁ (35.8%) ranging from 3.5 to 100.5 ng/L. Although 4/53 (7.5%) of the tested samples had the contamination level greater than the European Commission (EC) limit (>50 ng/L), the contamination levels were still below the Malaysia Food Regulation 1985 limit (less than 500 ng/L). This study provided a pioneering data on the occurrence of AFM₁ in milk and dairy products in Malaysia.     

Storage stability of maize-groundnut composite flours and an assessment of aflatoxin B1 and ochratoxin A contamination in flours and porridges

Defatting of groundnut flour used for composite development can not only improve nutritional quality of its products but also the storage stability. Maize, groundnut and their composite (full fat and defatted) flours were prepared and stored at room temperature over a period of 3 months. Storage stability of these products was assessed based on changes in water activity, peroxide value (PV), free fatty acids (FFA), thiobarbituric acid (TBA), microbiological profiling and levels of mycotoxins that included aflatoxin B₁ (AFB₁) and ochratoxin A (OTA). Overall results revealed that the rate of change of PV, FFA and TBA significantly (p ≤ 0.05) increased with increasing storage time, which was highest for full fat flours than in the defatted flours. For example, PV of the FFG and DFG were respectively, 0.88 and 0.40 mEq/kg, meanwhile TBA was 4.53 and 2.71 mg malonaldehyde/kg. There was a much higher rate of increase in FFA (%) with increasing storage time in full fat and composite flours when compared to that of their defatted counterparts. Generally, microbiological data demonstrated an increase in total microbial counts during storage in these foods possibly resulting in mycotoxins, AFB₁ (range: 9.08–38.48 μg/kg) and OTA (range: 0.33–19.50 μg/kg) in all samples with groundnut and maize having the highest contamination levels. A 127.8% increase in OTA level was noted when maize flour inclusion level in the full fat composite increased from 55 to 85%, but only a 24.7% increase in OTA level was noted in defatted composites during storage. Reducing the inclusion level of groundnut flour, the main source of AFB₁ as found, resulted in a drastic reduction in AFB₁ level in full fat and defatted composite flours by 54.1 and 76.4%, respectively, during storage. The findings highlight that shelf life stability of composites can be maintained upon defatting during the fortification process. It can therefore, be inferred that monitoring quality and safety of the raw materials as well as that of the final products during storage is crucial.     

Occurrence of aflatoxin M1 in pasteurised,UHT milk and milk powder from goat origin

In the present study, 36 samples of pasteurised, ultra-high-temperature (UHT) treated and goat milk powder traded in the city of Campinas, Brazil, were analysed for aflatoxin M₁ (AFM₁), from October to December 2004 and March to May 2005. Results showed 25 (69.4%) positive samples for AFM₁ at levels of 0.011–0.161 μg L⁻¹ of milk, which were below the tolerance limit of 0.500 μg L⁻¹ as adopted for AFM₁ in milk by Brazilian regulations. Mean levels of AFM₁ in pasteurised, UHT and goat milk powder were 0.072 ± 0.048, 0.058 ± 0.044 and 0.056 ± 0.031 μg L⁻¹, respectively. It is concluded that the incidence of AFM₁ in goat milk traded in Campinas is high, but at levels that probably leads to a non-significant human exposure to AFM₁ by consumption of goat milks.