Distribution of aflatoxin M1 during Grana Padano cheese production from naturally contaminated milk

The distribution of aflatoxin M₁ (AFM₁) has been investigated in samples of whey, curd and a typical hard and long maturing cheese like Grana Padano (ripened for twelve months), produced with naturally contaminated milk in a range of 30–98 ng AFM₁/kg. AFM₁ determinations were carried out on 25 samples of each product by reverse-phase HPLC and fluorescence detection with post-column derivatisation, after a preliminary C18-SPE clean-up. Experimental results show that, in comparison to milk, AFM₁ concentration levels increased both in curd (3-fold) and in long maturing cheese (4.5-fold), while AFM₁ occurrence in whey decreased by 40%.     

Efficacy of Solis, NovasilPlus, and MTB-100 to reduce aflatoxin M1 levels in milk of early to mid lactation dairy cows fed aflatoxin B1

An experiment was conducted to determine the efficacy of 3 adsorbents, Solis (SO; Novus International Inc.), NovasilPlus (NOV; Engelhard Corp.), and MTB-100 (MTB; Alltech), in reducing aflatoxin (AF) M₁ concentrations in milk of dairy cows fed an AF-contaminated diet. Twelve early to mid lactation dairy cows averaging 163 d in milk were used in a 4 × 4 Latin square design with 3 replications. Cows were blocked by parity, body weight, and milk production and were provided ad libitum access to feed and water. Within each replicate, cows were randomly assigned to the 4 dietary treatments for 4 consecutive 7-d periods. Dietary treatments included AF [112 μg of AFB₁/kg of diet dry matter (DM)]; AF + 0.56% SO; AF + 0.56% NOV; and AF + 0.56% MTB. Milk samples were collected on d 6 and 7 of each of the experimental periods. Feed intake, milk production, milk fat percentage, milk protein percentage, and linear somatic cell scores were not affected by dietary treatments and averaged 22.20 kg/d of DM, 33.87 kg/d, 3.78%, 2.95%, and 1.60, respectively, across all treatments. Transfer rates of AF from feed to milk averaged 2.65, 1.48, 1.42, and 2.52% for cows fed AF, AF + SO, AF + NOV, and AF + MTB, respectively. Daily AFM₁ excretion in milk averaged 66, 37, 35, and 63 μg/d for cows fed AF, AF + SO, AF + NOV, and AF + MTB, respectively. The addition of SO and NOV to the AF diet resulted in a significant reduction in milk AFM₁ concentrations (SO, 45%; NOV, 48%) and AFM₁ excretion (SO, 44%; NOV, 46%). In contrast, MTB was not effective in reducing milk AFM₁ concentrations (4%), AFM₁ excretion (5%), or AF transfer from feed to milk (2.52%). Results indicated that SO and NOV at 0.56% of the diet were effective in reducing milk AFM₁ concentrations in cows consuming a total mixed ration containing 112 μg of AFB₁/kg of diet DM.     

Short communication: Investigation of aflatoxin M1 levels in infant follow-on milks and infant formulas sold in the markets of Ankara,Turkey

Aflatoxins are fungal toxins known to be carcinogenic and are classified as food contaminants. This study was performed to investigate aflatoxin (AF) M₁ levels in baby foods sold in Ankara (Turkey) and to evaluate the obtained results according to the Turkish Food Codex (TFC). For this purpose, a total of 84 baby food samples (50 follow-on milks and 34 infant formulas) were obtained from different markets in Ankara and the presence of AFM₁ in the samples was analyzed by ELISA. In 32 (38.1%) of 84 infant food samples, the presence of AFM₁ was detected in concentrations ranging between 0.0055 and 0.0201 µg/kg. The mean level (±standard error) of AFM₁ was found to be 0.0089 ± 0.0006 µg/kg in positive infant follow-on milks. Aflatoxin M₁ was detected in only 1 infant formula sample (2.94%) at a concentration of 0.0061 µg/kg. The extrapolated levels of AFB₁ contamination in feedstuffs were calculated based on levels of AFM₁ in baby food samples. The data estimating AFB₁ contamination in dairy cattle feedstuff indicate that contamination may range from 0.3410 to 1.2580 µg/kg, with the mean level (±standard error) being 0.5499 ± 0.0385 µg/kg, which is lower than the level set by the TFC and European Union regulations (5 µg/kg). According to the obtained results, the levels of AFM₁ in analyzed samples were within the allowed limit (0.025 µg/kg) set in the TFC. Low levels of AFM₁ in infant follow-on milks and infant formula samples obtained during the study do not pose a health risk to infants.     

Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA

Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes.     

Degradation of aflatoxin B1 by fungal laccase enzymes

The enzymatic degradation of aflatoxin B₁ (AFB₁) by white rot fungi through laccase production was investigated in different liquid media. A significant (P < 0.0001) correlation was observed between laccase activity and AFB₁ degradation exhibited by representatives of Peniophora and Pleurotus ostreatus cultivated in minimal salts (MSM) (r = 0.93) and mineral salts — malt extract (MSB–MEB) (r = 0.77) liquid media. Peniophora sp. SCC0152 cultured in MSB–MEB liquid medium supplemented with veratryl alcohol and sugarcane bagasse showed high laccase activity (496 U/L), as well as 40.45% AFB₁ degradation as monitored using high performance liquid chromatography. P. ostreatus St2-3 cultivated in MSM liquid medium supplemented with veratryl alcohol resulted in laccase activity of 416.39 U/L and 35.90% degradation of AFB₁. Aflatoxin B₁ was significantly (P < 0.0001) degraded when treated with pure laccase enzyme from Trametes versicolor (1 U/ml, 87.34%) and recombinant laccase produced by Aspergillus niger D15-Lcc2#3 (118 U/L, 55%). Aflatoxin B₁ degradation by laccase enzyme from T. versicolor and recombinant laccase enzyme produced by A. niger D15-Lcc2#3 coincided with significant (P < 0.001) loss of mutagenicity of AFB₁, as evaluated in the Salmonella typhimurium mutagenicity assay. The degradation of AFB₁ by white rot fungi could be an important bio-control measure to reduce the level of this mycotoxin in food commodities.     

Development of a class-specific monoclonal antibody-based ELISA for aflatoxins in peanut

Three class-specific monoclonal antibodies against aflatoxins were screened by a designed strategy in which aflatoxin G₂ was used as competitor in the screening ELISA system. With a high cross-reactivity (65%) to aflatoxin G₂, antibody 10C9 had the most similar sensitivity for five aflatoxins (AFB₁, AFB₂, AFG₁, AFG₂ and AFM₁), whose I₅₀ values were in a range of 2.1–3.2 ng ml⁻¹. So, antibody 10C9 was selected to develop an ELISA for determination of aflatoxin B₁, B₂, G₁, G₂ and total of them in peanut samples. And spiked recoveries were from 87.5% to 102.0%. The results indicate that the ELISA developed can accurately determine total aflatoxins in samples of peanuts after the simple and rapid extraction procedure.     

Characterisation of monoclonal antibody against aflatoxin B1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli

An anti-aflatoxin B₁ monoclonal antibody (anti-AFB₁ mAb) from the hybridoma 2C12 was established and its inhibition concentration fifty (IC₅₀) for AFB₁ and relative cross-reactivities (CRs) to other mycotoxins were estimated to be 8 ng/mL and less than 4% compared with AFB₁ by a competitive direct enzyme-linked immunosorbent assay. For production of anti-AFB₁ single-chain variable fragment (anti-AFB₁ scFv) in recombinant Escherichia coli, its scFv-coding genes were cloned from the hybridoma 2C12. The anti-AFB₁ scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding process and hence recovered to retain binding activity successfully. Surface plasmon resonance analysis resulted that anti-AFB₁ scFv possessed 1.16 × 10⁻⁷ M of equilibrium dissociation constant (K_D), which was about 17 times higher than the parental anti-AFB₁ mAb of 6.95 × 10⁻⁹ M.     

Aflatoxin M1 in raw milk and aflatoxin B1 in feed from household cows in Singida,Tanzania

Aflatoxin M₁ (AFM₁) contamination in raw milk from household cows fed with sunflower seedcakes or sunflower-based seedcake feeds was determined in 37 milk samples collected randomly from different locations in Singida region, Tanzania. Aflatoxin B₁ (AFB₁) contamination in sunflower-based seedcake feed was determined in 20 feed samples collected from the same household dairy farmers. The samples were analysed by RP-HPLC using fluorescent detection after immunoaffinity column clean-up. Recoveries were 88.0% and 94.5%, while the limits of detection (LOD) were 0.026 ng mL^?1 and 0.364 ng g^?1 for AFM₁ and AFB₁, respectively. Of the analysed cow’s milk samples, 83.8% (31/37) contained AFM₁, with levels ranging from LOD to 2.007 ng mL^?1, exceeding both the European Commission (EC) and Tanzania Food and Drug Authority (TFDA) limit of 0.05 ng mL^?1. Of the contaminated samples, 16.1% exceeded the Codex Alimentarius limit of 0.5 ng mL^?1. AFB₁ was present in 65% (13/20) of the feed samples with levels ranging from LOD to 20.47 ng g^?1, 61.53% exceeding the TFDA and EC maximum limits of 5 ng g^?1 for complete dairy animal feed. The observed AFM₁ and AFB₁ contamination necessitates the need to raise awareness to dairy farmers in Tanzania to safeguard the health of the end-users.     

Aflatoxins in dairy cow feed,raw milk and milk products from Turkey

This study aims to detect aflatoxins (AFs) in dairy cow feed, milk and milk products using a high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method. All the validation parameters met the method performance criteria of the European Union. The samples comprised 76 dairy cow feeds and 205 milk and milk products (including yoghurt and yoghurt-based beverage, ayran). AFs were present in 26.3% of the feed samples. Two feed samples exceeded the maximum limit (ML) of 5 µg kg^?1 for AFB₁ as established by the EU. Nineteen milk samples (21.1%) contained aflatoxin M₁ (AFM₁) of which three exceeded the EU ML of 0.05 µg l^?1. In addition, only two yoghurt samples and one ayran sample contained AFM₁, but the levels were lower than the EU ML.     

Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia

Chronic exposure to aflatoxins, and especially to aflatoxin B₁ (AFB1), causes hepatocellular carcinoma with prevalence 16–32 times higher in developing compared with developed countries. Aflatoxin M₁ (AFM1) is a monohydroxylated metabolite from AFB1 that is secreted in milk and which can be used as a biomarker of AFB1 exposure. This study aimed to determine AFM1 levels in human breast milk using immunoaffinity column clean-up with HPLC and fluorescence detection. Breast milk samples were obtained from 50 nursing mothers. Volunteers filled in a questionnaire giving their consent to analyse their samples as well as details of their socioeconomic, demographic and clinical data. The possible dietary sources of aflatoxins were assessed using a food frequency questionnaire. A total of 90% of the samples tested positive for AFM1, with a mean of 5.2 ng l^?1 and a range of 0.9–18.5 ng l^?1. The study demonstrated a high frequency of exposure of mothers and neonates to AFB1 and AFM1 in Colombia, and it points out the need to regulate and monitor continuously the presence of aflatoxins in human foods. Further research is needed in order to determine the presence of other mycotoxins in foods and in human samples as well as to devise protection strategies in a country where mycotoxins in human foods are commonly found.     

Simultaneous determination of aflatoxin B1 and M1 in milk,fresh milk and milk powder by LC-MS/MS utilising online turbulent flow chromatography

A novel, fully automated method based on dual-column switching using online turbulent flow chromatography followed by LC-MS/MS was developed for the determination of aflatoxin B₁ and M₁ in milk, fresh milk and milk powder samples. After ultrasound-assisted extraction, samples were directly injected into the chromatographic system and the analytes were concentrated on the clean-up loading column. Through purge switch, analytes were transferred to the analytical column for subsequent detection by mass spectrometry. Different types of TurboFlow^TM columns, transfer flow rates and transfer times were optimised. Method limits of detection obtained for AFB₁ and AFM₁ were 0.05 μg kg^–1, and limits of quantification were 0.1 μg kg^–1. Recoveries of aflatoxin B₁ and M₁ were in range of 81.1–102.1% for all samples. Matrix effects of aflatoxin B₁ and M₁ were in range of 63.1–94.3%. The developed method was successfully used for the analysis of aflatoxin B₁ and M₁ in real samples.     

Reduction of aflatoxin B1 contamination in wheat by various cooking treatments

To investigate the effects of various cooking treatments such as washing, heating and steaming on the reduction of aflatoxin toxicity, a simultaneous analytical method for aflatoxin B₁, B₂, G₁, G₂ was established using high performance liquid chromatography (HPLC) with a fluorescence detector. The levels of aflatoxin B₁ (AFB₁) spiked in wheat—three varieties of United States (US) wheat and two varieties of Korean wheat—were analyzed according to washing time and heating temperature. Reduction of AFB₁ toxicity was directly proportional to washing time in both Korean and US wheat. The concentration of AFB₁ was reduced more by heating than washing treatment. The level of AFB₁ in dried wheat was decreased to 50% and 90% by heating at 150 and 200 °C, respectively. However, the reduction of AFB₁ in wet wheat in which water (10%) was intentionally added was higher by heating than in dried wheat. The reduction of AFB₁ was increased by 8% and 23% in 10% water-added US wheat (soft red white wheat) and Korean wheat (Anbaekmil) compared to dried US and Korean wheat, respectively, through heat treatment. Traditional processing used in Korean foods such as Sujebi (a soup with wheat flakes) and steamed bread caused 71% and 43% decrease in aflatoxin B₁ content.     

酶联免疫吸附法测定云南铁皮石斛中黄曲霉毒素B1

目的建立酶联免疫吸附法测定铁皮石斛中黄曲霉毒素B_1(afatoxin B_1, AFB_1)的分析方法,并研究酶联免疫吸附法(enzyme-linkedimmunosorbentassay,ELISA)和高效液相色谱法(highperformanceliquid chromatography, HPLC)测定云南铁皮石斛中AFB_1的结果差异性。方法用甲醇+水溶液(50+50, V/V)提取石斛中AFB_1,采用酶联免疫吸附法和高效液相色谱法同时检测云南铁皮石斛(铁皮枫斗)中AFB_1,进行准确性、精密度和回收率等方法学实验,比较2种方法的差异性。结果 ELISA法在测定范围内AFB_1与其对应的吸光度值之间呈良好的线性关系,回归方程Y=-34.798X+18.134,相关系数r~2为0.9967;添加1.0、4.0、10.0μg/kg3个浓度的平均加标回收率为85.3%～94.7%,相对标准偏差为2.57%～4.88%。2种方法的结果符合率为93.2%～117%,相对标准偏差均小于10%。结论 ELISA法具有操作简单、检测速度快、灵敏、特异性好等优点,可同时检测大批量样品中AFB_1的含量。     

An electrochemical enzyme immunoassay for aflatoxin B1 based on bio-electrocatalytic reaction with room-temperature ionic liquid and nanoparticle-modified electrodes

A new electrochemical immunosensor for the determination of aflatoxin B₁ (AFB₁) based on bio-electrocatalytic reaction was proposed. An imidazolium cation room-temperature ionic liquid (RTIL), 1-ethyl-3-methyl imidazolium tetrafluoroborate ([EMIm][BF₄]), was initially immobilized on the surface of a glassy carbon electrode (GCE) through titania sol and Nafion film, then nanogold particles were adsorbed onto the titania surface, and then horseradish peroxidase (HRP)-labeled anti-AFB₁ antibodies (HRP-anti-AFB₁) were attached on the nanogold surface. With a non-competitive immunoassay format, the formation of the antibody–antigen complex by a simple one-step immunoreaction between the immobilized HRP-anti-AFB₁ and AFB₁ in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the electrode surface, thus local current variations could be detected by the HRP bio-electrocatalytic reaction in 0.1 M PBS (pH 6.8) containing 0.28 M H₂O₂. Under optimal conditions, the electrochemical immunosensor exhibited a good current response relative to AFB₁ concentration in a linear range from 0.1 to 12 ng/mL with a relatively low detection limit of 0.05 ng/mL at 3δ. The inter-assay coefficients of variation are 7.1% and 5.4% for 1.0 ng/mL and 8.0 AFB₁, respectively. Naturally contaminated samples were screened with the developed immunosensor, and results were compared with those obtained by validated ELISA method. The assay was demonstrated to be accurate and reliable giving no false compliant and only a low percentage of false non-compliant results. The described method offers a simple, rapid and cost-effective screening tool, thus contributing to a better consumers’ health protection.     

Development of a new monoclonal antibody based direct competitive enzyme-linked immunosorbent assay for detection of brevetoxins in food samples

Brevetoxin B (PbTx-2) was covalently linked to carrier protein bovine serum albumin and human gamma globulin. A monoclonal antibody against PbTx-2, which showed high cross-reactivity values with PbTx-1, PbTx-3 and PbTx-9 (more than 89%) was obtained from ascites and some characteristics of monoclonal antibody were studied. An direct competitive enzyme-linked immunosorbent assay (ELISA) for detection of PbTxs was developed, which showed an IC50 value of 5.3 ng mL⁻¹ with a detection limit of 0.6 ng well⁻¹. The recoveries of PbTxs from cockle (88.4%–102.3%) and oyster (89.4%–104.3%) demonstrated that the matrices of cockle and oyster where PbTxs are found do not interfere with the assay. The newly developed competitive ELISA appears to be a reliable and useful method for mass monitoring of PbTxs in mollusk.     

Development of a McAb-based immunoassay for parathion and influence of the competitor structure

A specific monoclonal antibody (McAb) for parathion was produced. Based on this McAb, a battery of competitors as coating antigens were used to develop homologous and heterologous indirect competitive enzyme-linked immunosorbent assays (ELISAs) for parathion. The relationship between the heterology degree of the competitor and the sensitivity of the corresponding immunoassay was investigated. Results showed that, when the specific McAb was used in the ELISA experiment, competitors should have a certain degree of homology with the immunizing hapten for immunoassays, and the best performance occurred when the competitor hapten was highest heterologous to the target analyte. With the most suitable competitor, a sensitive and selective ELISA was developed. The IC₅₀ value of the ELISA was 2.94 ng/ml with a detection limit (IC₂₀) of 0.70 ng/ml. The average recoveries of parathion in spiked water, soil, cucumber and rice were 88.09%, 93.15%, 91.37% and 83.42%, respectively.     

Seasonal patterns of aflatoxin M1 contamination in commercial pasteurised milk from different areas in Thailand

Aflatoxin M₁ (AFM₁) levels were determined in pasteurised milk from five commercial trademarks produced in different areas in Thailand. One hundred and twenty milk samples were collected from local markets in Chiang Mai province, Thailand, to evaluate AFM₁ concentrations using immunoaffinity columns and high-performance liquid chromatography with fluorescence detection. The overall median AFM₁ level was 0.023?µg?L^?1 ranging from 0.004 to 0.293?µg?L^?1. All trademarks had average AFM₁ concentrations lower than 0.05?µg?L^?1, with those in Trademarks 3 to 5 being higher than Trademarks 1 and 2 (P?<?0.01). All trademarks had different seasonal patterns of AFM₁, even though operating in the same area. However, only Trademark 3 showed significant differences of AFM₁ levels between seasons. The results suggested that farm management factors, rather than environment factors, were likely to be the main cause of AFM₁ contamination in dairy products.     

Aflatoxin M1 in buffalo and cow milk in Afyonkarahisar,Turkey

Potential hazardous human exposure to aflatoxin M₁ (AFM₁) via consumption of milk and milk products has been demonstrated by many researchers. The aim of this study was to investigate the presence of this mycotoxin in buffalo and cow milk samples in the city of Afyonkarahisar, Turkey. For this purpose, 126 buffalo and 124 cow milk samples were collected from dairy farms in Afyonkarahisar province. AFM₁ levels were determined by high-performance liquid chromatography with tandem mass spectrometric detection. Although AFM₁ was not detected in cow milk samples, AFM₁ was found above the limit of detection (<0.008–0.032 µg/L) in 27% (34 out of 126) of the buffalo milk samples. The results of this study indicated the importance of continuous surveillance of commonly consumed milk or milk product samples for AFM₁ contamination in Turkey.     

Binding of aflatoxin M1 to different protein fractions in ovine and caprine milk

The affinity of aflatoxin M₁ toward the main milk protein fractions in ewe and goat milk was investigated by using an ELISA. This study took into account the possible effects of common dairy processes such as ultrafiltration, acidic or rennet curding, and production of ricotta from acidic or rennet whey. Treatments that allowed the separation of casein from whey proteins under conditions that do not alter the physical or chemical status of the proteins (such as ultracentrifugation) were used as a reference. None of the treatments used in typical dairy processes caused significant release of the toxin, in spite of the relevant changes they induced in the interactions among proteins. Only the combined heat and acidic treatment used for production of ricotta cheese altered the structure of whey proteins to the point where they lost their ability to bind the toxin. This study also showed that, regardless of the physical state of the sample, a commercial electronic nose device, in combination with appropriate statistical tools, was able to discriminate among different levels of sample contamination.     

A simple and reliable liquid chromatography-tandem mass spectrometry method for the determination of aflatoxin M1 in milk

A new chromatographic method is proposed for the analysis of aflatoxin M₁ in milk. The method is based on liquid–liquid extraction followed by LC-MS/MS analysis. Liquid–liquid extraction (LLE) is performed on the defatted milk plus sodium chloride by using ethyl acetate as an extraction solvent. Accuracy and precision were evaluated at the LOQ (15?ng?kg^–1) spiked sample as well as with three other different naturally contaminated reference materials. The mean overall recovery (n?=?24) was 95% with a confidence interval of 1.9% and a CV% of 4.5%. The performance of the proposed method was compared with that of the Official ISO Method based on the use of immunoaffinity chromatography columns (IAC): LLE protocol could be considered a valid alternative to the LC-IAC. In general it showed better accuracy with lower data dispersion. Moreover, the sample preparation is very simple and straightforward, potentially being applicable as a high-throughput method which, on account of its simplicity and low cost, may be applied to the analysis of a large number of samples in the occasion of outbreaks of large-scale contamination.