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Techniques for Detection, Quantification and Control of Mycotoxins in Dairy Products

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Abstract

A number of mycotoxins resist rumen degradation, causing distinct clinical signs of intoxication. Most mycotoxins are chemically stable so they tend to remain potent during storage and processing, and even high temperatures, such as those reached during baking bread or breakfast cereal production. The choice of extraction solvent is dependent on the matrix from that the extraction is required, as the differing chemical mixtures can affect it. Liquid–liquid extraction (LLE) involves exploiting the different solubility of the toxin in aqueous phase and in immiscible organic phase, to extract the compound into one solvent leaving the rest of the matrix in the other. Supercritical fluid extraction (SFE) uses supercritical fluid, such as carbon dioxide (CO2) to extract the required compound from the matrix. Thin layer chromatography (TLC) is a popular method used for both quantitative and semi-quantitative mycotoxins analysis. The study developed a quantitative Monte Carlo exposure assessment model for mycotoxins in dairy milk, and assessed the potential human exposure levels.

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... The highest abundance of micromycetes is found in the substrate at aw = 0.99 (Hines et al. 2000). Donkor et al. (2017) report that Asperillus flavus grows from aw = 0.73. According to Ushkalov et al. (2020), the minimum aw required for the growth of micromycetes is 0.78 and the optimal aw is 0.95. ...
... Immunoassay techniques or methods, which are used to determine the immunochemical detection of the aflatoxins, in which the antigen-antibodies reaction (Ag-Ab) is carried out [58]. The method which is used to determine the aflatoxins on basis of antibodies-antigen interaction is ELISA (Enzyme-linked Immunosorbent Assay). ...
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... Immunoassay techniques or methods, which are used to determine the immunochemical detection of the aflatoxins, in which the antigen-antibodies reaction (Ag-Ab) is carried out [58]. The method which is used to determine the aflatoxins on basis of antibodies-antigen interaction is ELISA (Enzyme-linked Immunosorbent Assay). ...
Article
Full-text available
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In this study the levels of aflatoxin M1 (AFM1) in UHT milk samples were determined in May, August and November and February. Two hundred and ten UHT milk samples were obtained from supermarkets in Tehran, Iran. The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA) method. AFM1 was found in 116 (55.2%) of 210 UHT milk samples examined. The levels of AFM1 in 70 (33.3%) samples were higher than the maximum tolerance limit (0.05 μg/l) accepted by some European countries while none of the samples exceeded the prescribed limit of US regulations. The highest mean concentration of AFM1 was recorded in February (0.087 μg/l). The lowest mean concentration of AFM1 was recorded in August (0.021 μg/l). Statistical evaluation showed that there were significant difference (P < 0.01) between the mean concentrations of AFM1 of UHT milk samples taken in February with May and August. AFM1 contents of milk samples taken in February were not higher than UHT milk samples taken in November (P < 0.01). The AFM1 incidence of exceeding legal limit in UHT milk samples (33.3%) was relatively much higher than some other countries. It was therefore concluded that, the levels of AFM1 in UHT milk samples consumed in Iran were high and seemed to pose a threat to public health.
Article
Aims: To compare the biosynthetic gene cluster sequences of the main aflatoxin (AF)-producing Aspergillus species. Methods and Results: Sequencing was on fosmid clones selected by homology to Aspergillus parasiticus sequence. Alignments revealed that gene order is conserved among AF gene clusters of Aspergillus nomius, A. parasiticus, two sclerotial morphotypes of Aspergillus flavus, and an unnamed Aspergillus sp. Phylogenetic relationships were established using the maximum likelihood method implemented in PAUP. Based on the Eurotiomycete/Sordariomycete divergence time, the A. flavus-type cluster has been maintained for at least 25 million years. Such conservation of the genes and gene order reflects strong selective constraints on rearrangement. Phylogenetic comparison of individual genes in the cluster indicated that ver-1, which has homology to a melanin biosynthesis gene, experienced selective forces distinct from the other pathway genes. Sequences upstream of the polyketide synthase-encoding gene vary among the species, but a four-gene sugar utilization cluster at the distal end is conserved, indicating a functional relationship between the two adjacent clusters. Conclusions: The high conservation of cluster components needed for AF production suggests there is an adaptive value for AFs in character-shaping niches important to those taxa. Significance and Impact of the Study: This is the first comparison of the complete nucleotide sequences of gene clusters harbouring the AF biosynthesis genes of the main AF-producing species. Such a comparison will aid in understanding how AF biosynthesis is regulated in experimental and natural environments.
Article
A gas chromatographic procedure for the quantitative estimation of T-2 toxin, which utilises a florisil SPE clean-up procedure, has been adapted for the quantitative analysis of both T-2 toxin and deoxynivalenol (DON), but using an high performance thin layer chromatographic quantification step. The method has been validated by spiking uncontaminated rice extracts with T-2 toxin and DON over the range 100 to 1000 μg kg−1. A linear relationship was found for both mycotoxins over the entire range tested. The mean recovery for T-2 toxin was 84% and for DON 91% with mean coefficients of variation of 8.2% and 6.0%, respectively.
Article
A simultaneous reversed-phase HPLC determination of two major mycotoxins, ochratoxin A and citrinin, in soft cheese is proposed. Both mycotoxins are eluted on a C18 RP support (25 × 4.6 mm I.D.) using an isocratic eluent consisting of methanol-water (70:30, v/v) containing tetrabutylammonium hydroxide (10−3M), acidified to pH 5.5 with HCl, and pumped at a flow-rate of 0.8 ml/min. Prior to detection, a butanolic solution of 5·10−3M terbium-5 · 10−4M trioctylphosphine oxide (TOPO)-2.5 · 10−2M triethylamine (TEA) was pumped in a postcolumn mode at a flow-rate of 0.2 ml/min to perform time-resolved luminescence (TRL) detection of the corresponding terbium chelates (λex = 331 nm/λem = 545 nm). The method is linear from 3.5·10−6 to 2·10−5M for citrinin and from 1·10−5 to 5·10−5M for ochratoxin A. The repeatability and reproducibility (R.S.D.) are 1.9 and 2.4% for citrinin (c = 3.5·10−6M; n = 10), and 7.2 and 8.3% for ochratoxin A (c = 1.0·10−5M; n = 10). The limits of detection, for a signal-to-background ratio of 3, are 2·10−6 and 3·10−6M for citrinin and ochratoxin A, respectively. With the proposed method, ochratoxin A and citrin are easily determined in soft cheeses, with a significative increase in selectivity in comparison with direct fluorescence detection.
Article
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.
Article
Some Spanish sweet wines are made from raisins, grapes dried by direct exposure to the sun after picking. This drying process can encourage ochratoxin A (OTA) formation. OTA is a mycotoxin formed by several fungi. It has been linked to nephropathy in humans, and may have a long half-life in humans. The aim of this study is to develop and to apply two procedures for the analysis of OTA in grape musts (during the raisining process) and sweet wines, respectively. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to fluorescence detection (FLD) was employed in both analytical methods. In grape must, the method involves the direct injection of the sample in a HPLC-FLD system without any kind of prior clean-up procedure. The complexity of the sweet wine samples requires a solid-phase extraction (SPE) clean-up on a C18 column which enables the OTA to be isolated from the matrix. The methods used were statistically validated. The validation also included the comparison of the slopes of the curve obtained with standards and the regression curves obtained by the addition of a standard. Two different studies of standard additions were conducted. One method was validated without sample preparation and it was applied to must samples. The other method was validated with SPE extraction and it was applied to sweet wine samples. Recovery was always better than 89.69%. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were established at 0.22 and 0.77 μg l−1, respectively. In general, the analytical data obtained provided good results at the sub-μg l−1 concentration level.
Article
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).
Article
Raw and pasteurised sheep’s, cow’s and goat’s milk, eggs, and beef samples from different local markets in Jordan were collected during a period of 5 months (January through May 2007) and examined for aflatoxins B1(AFB1), B2(AFB2), G1(AFG1), G2(AFG2), M1(AFM1) and M2(AFM2). The samples were analysed with high performance liquid chromatography (HPLC) using UV and Fluorescent detectors. The analysed samples of milk collected in January were found to contain 0.56 μg L−1 AFM1 and 0.1 μg L−1 AFM2 whilst, the concentration of AFM1 and AFM2 was < 0.05 μg L−1 for milk samples collected between March and May. The AFB1, AFB2, AFG1 and AFG2 contents in the analysed food products ranged from 1.10 to 8.32 μg L−1 and 0.15 to 6.36 μg L−1 in imported and fresh meat samples collected during March, respectively. The mean recovery for the HPLC method was 92% to 109% and the quantification levels were 50 ng L−1 for AFM1 and AFM2. The AFM1 was found in 10% of the tested samples with concentrations between 0.08 and 1.1 μg kg−1 and AFM2 was only found in 1.82% of the tested samples with a level of 0.1 μg kg−1. The AFM1 levels in the examined foods were higher than the maximum level of AFM1 in liquid milk set by the European Community and Codex Alimentarius of 50 ng L−1.
Article
Many methods for AFM1 detection exist, but most are time consuming, employ expensive equipment and require experienced personnel. To overcome these problems a membrane-based flow-through enzyme immunoassay has been developed (patent pending). The assay comprised a nylon Immunodyne ABC membrane spotted with anti-mouse antibodies, a plastic snap-fit device, absorbent cotton wool, mouse anti-AFM1 monoclonal antibodies (Mab), and AFB1–horseradish peroxidase (HRP) conjugate. This assay was coupled to an immunoaffinity column (IAC). The visual detection limit was 0.05 ng/g AFM1 in milk. Assay time for IAC clean-up was 12 min, and that for the flow-through assay was 18 min, hence the total assay time was 30 min. This method allows for a rapid screening of milk consignments which do not conform to the maximum permissible limits of 0.05 ng/g AFM1, hence enabling the rejection of such at the farm level. Laboratory validation was done using certified reference materials (CRM) with AFM1 concentrations of <0.05, 0.09 and 0.76 ng/g. Precision of the assay was high as shown by the high repeatability of the assay results. There were no significant differences in recoveries between standard in buffer and CRM (P>0.05), and assay responses for these two were highly correlated (99.63%).
Article
A flow-injection immunoassay (FI-IA) method with amperometric detection for aflatoxin M1 (AFM1) determination in milk has been developed. The first step consists in an incubation of the sample containing AFM1 (Ag) with fixed amounts of anti-AFM1 antibody (Ab) and of the tracer (Ag∗, AFM1 covalently coupled to HRP) until equilibrium is reached. In this mixture a competition occurs between Ag and Ag∗ for the Ab. The mixture is then injected into a flow system where the separation of the free tracer (Ag∗) and the antibody-bound tracer (AbAg∗) is performed in a column with immobilized Protein G. The antigen–antibody complexes are retained in the column due to the high affinity of the Protein G for the antibody. The activity of the eluted enzyme label is then amperometrically detected.The immunoassay was optimised relative to conditions for antibody–antigen incubation (pH, incubation time, ionic strength, temperature) and enzymatic label detection. This method showed a dynamic concentration range between 20 and 500 ppt AFM1, a low detection limit (11 ppt), good reproducibility (RSD < 8%) and a high throughput (six samples per hour in triplicate). Different milk samples were analysed and the results were in good agreement with those obtained by HPLC using the AOAC 2000.08 method.
Article
Trichothecene mycotoxins are common contaminants of cereal grains and animal feed worldwide. The toxins are toxic to both human and animals. The objectives of this study were to determine the occurrence of trichothecenes in grains and animal feed in Croatia. Total of 465 samples were collected during the seven-year period (1998–2004) from manufactures and small holders farm storage facilities. The samples were analyzed by thin layer chromatography, which proved to be fast, reliable and inexpensive method. T-2 toxin, diacetoxyscirpenol and deoxynivalenol were detected in 16.8%, 27.6% and 41.2%, respectively. The amount of toxins ranged between 0.05 and 3.4 mg/kg. The majority of animal feed samples was poultry feed. Only small number of it contained T-2 toxin and diacetoxyscirpenol levels greater than the Croatian regulatory levels for poultry feed. Positive samples were in correlation with evidenced clinical symptoms of toxicosis in poultry. Since trichothecenes are frequently isolated from animal feed and grains in Croatia, they could have significant economic and safety implications in animal production.
Article
A surface plasmon resonance (SPR) immunosensor is developed to determine concentrations of the mycotoxin, fumonisin B1(FB1), in spiked samples. Polyclonal antibodies produced against FB1are adsorbed onto a thin gold film substrate, which is coupled to a glass prism in the Kretschmann configuration. The output beam of a planar light-emitting diode is focused through the prism to excite SPR at the surface of the gold film. When a sample containing FB1is added to a cell on the outside of the gold film, the angular profile of reflected light intensity shifts. This changes the resonance angle and the reflected beam intensity at a selected angle, both of which are proportional to the FB1concentration. After optimization of the antibody overlayer, a detection limit of 50 ng/mL is obtained for the direct assay with an analysis time under 10 min. Multiple sample additions and large-volume sample circulation can be used with the high-affinity antibodies to achieve lower detection limits.
Article
SPME, using a carbowax/templated resin fiber, interfaced with HPLC–UV/DAD has been optimized for the determination of the mycotoxin mycophenolic acid (MPA) in cheese samples. All the parameters influencing the efficiency of the analyte extraction and desorption have been carefully explored. The procedure has been applied to the analysis of blue-cheese samples such as Gorgonzola and Danablu. Samples were subjected to a preliminary short sonication in bicarbonate buffer (0.2 M, pH 9.7); the subsequent SPME was capable of a selective extraction of MPA, characterized by high recovery yields and detection limits of 50 and 100 ppb for Danablu and Gorgonzola, respectively. The present method is faster and simpler than any other existing method for the extraction of MPA from cheese and does not involve the use of toxic organic solvents.
Article
Most data dealing with the biopreservative activity of lactic acid bacteria (LAB) are focused on their antibacterial effects. Food spoilage by mould and the occurrence of their mycotoxins constitute a potential health hazard. Development of biological control should help improve the safety of products by controlling mycotoxin contamination. Data have actually shown that many LAB can inhibit mould growth and that some of them have the potential to interact with mycotoxins.This review summarizes these findings and demonstrates that LAB are promising biological agents for food safety.
Article
This study was undertaken to determine the presence and levels of aflatoxin M1 (AFM1) in cheeses consumed in the province of Ankara. For this purpose, a total of 400 cheese samples containing 100 samples each of white, kashar, tulum and processed cheeses were used as the study material. The cheese samples were purchased randomly from different markets. The competitive ELISA (RIDASCREEN Aflatoxin M1, no. R1101) was used to determine the presence and levels of AFM1. Different levels of AFM1 were detected in the 327 (81.75%) cheese samples consisting of white (82%), tulum (81%), kashar (85%) and processed cheese (79%) samples. Altogether, 110 cheese samples (27.5%) were found to have levels that exceed the legal limits of 250 ng/kg established by the Turkish Food Codex. Among these, 27% of white cheese, 24% of tulum cheese, 34% of kashar cheese and 25% of processed cheese exceeded the Turkish safety limits. It was therefore concluded that, widespread occurrence of AFM1 in cheese samples sold in the Ankara province were considered to be possible hazards for human health.
Article
The aim of the study involved evaluation of the presence of aflatoxin M1 in milk for sale in a specific North West Italian region, Piedmont. The study, conducted from November 2003 to July 2005, was linked to the specific emergency situation which arose due to the climatic conditions during the summer of 2003 which encouraged the development of aflatoxin B1 in items used for animal feed. This in turn led to the transfer of the metabolite, aflatoxin M1, into the milk. In total some 316 milk samples were collected during the commercial phase by the official control bodies and analysed. The analysis involved the use of high pressure liquid chromatography (HPLC) combined with fluorimetric measurement, and purification of the extracts using immunoaffinity columns. The results indicated only 2 non conforming samples (0.6%), with limits higher than those set out in the regulations (0.05 μg L−1). In addition, the analyses revealed, in 5 samples (1.6%), threshold values of 0.05 μg L−1. From the data obtained it can be seen that the “aflatoxins” problem only marginally affected Piedmont Region though the trend for average monthly values suggests a return to the use of contaminated animal feed as soon as official controls are less intensive.
Article
A supercritical-fluid extraction (SFE) method has been developed that extracts aflatoxins (B1, B2, G1 and G2) from spiked corn using modified supercritical carbon dioxide. Methanol is added to the SFE extraction cell containing the corn layered between Hydromatrix (a dispersing material) which helps to prevent the clogging of the frits and reduces the effect of moisture on the extraction. The corn is held in static extraction at 65°C and 51.7 MPa for 15 min followed by a dynamic extraction with 20 mL of liquid carbon dioxide. The sample is depressurized and modifier re-added with a 10-min static extraction at 51.7 MPa followed by a dynamic extraction with 20 mL of liquid carbon dioxide. The SFE extract is collected in 10 mL of chloroform and further cleaned up with a Florisil Sep-Pak. The aflatoxins were analyzed by HPLC using fluorescence detection after post-column derivatization with iodine. Recoveries of the aflatoxins B1, B2, G1, and G2) over a range of 3 to 11 ng g-1 averaged 77.3, 82.9, 75.4, and 80.3%, respectively.
Article
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.
Article
Immunoassays are now in routine use for analysis of mycotoxins. This paper presents background information on antibody production and on immunoassay formats, particularly the enzyme-linked immunosorbent assay (ELISA). Applications of immunoassay technology to determination of aflatoxins, trichothecenes, ochratoxin A and others are reviewed and their performance discussed.