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 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.     

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.     

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.     

Occurrence of aflatoxin M1 in randomly selected North African milk and cheese samples

Forty-nine samples of raw cow's milk and 20 samples of fresh white soft cheese were collected directly from 20 local dairy factories in the north-west of Libya and analysed for the presence of aflatoxin M₁ (AFM₁). The samples were analysed using a high-performance liquid chromatography technique for toxin detection and quantification. Thirty-five of the 49 milk samples (71.4%) showed AFM₁ levels between 0.03 and 3.13 ng ml^-1 milk. Multiple analyses of five milk samples free of AFM₁ artificially contaminated with concentrations of AFM₁ at 0.01, 0.05, 0.1, 1.0 and 3.0 ng ml^-1 showed average recoveries of 66.85, 72.41, 83.29, 97.94 and 98.25%, with coefficients of variations of 3.77, 4.11, 1.57, 1.29 and 0.54%, respectively. Fifteen of 20 white soft cheese samples (75.0%) showed the presence of AFM₁ in concentrations between 0.11 and 0.52 ng g^-1 of cheese. Multiple assays of five cheese samples free of AFM₁ spiked with different concentration of AFM₁ (0.1, 0.5, 1.0 and 3.0 ng g^-1) showed average recoveries of 63.23, 78.14, 83.29 and 88.68%, with coefficients of variation of 1.53, 9.90, 4.87 and 3.79%, respectively. The concentrations of AFM₁ were lower in the cheese products than in the raw milk samples.     

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.     

Natural occurrence of aflatoxins (B1 and M1) in feed,plasma and raw milk of lactating dairy cows in Beja,Tunisia, using ELISA

Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal consumption. A small number of studies on mycotoxin occurrence in feedstuffs and raw milk from lactating dairy cows in this region are available. Therefore, 226 samples were collected from farms and local markets during November 2008 until April 2010. Samples consisted of 112 raw cow milk, 56 blood from lactating cows and 58 feed destined for dairy cows. Plasma and feed were analysed for aflatoxin B₁ (AFB₁). Milk samples were analysed for aflatoxin M₁ (AFM₁). All samples were treated using a simultaneous methanolic-aqueous extraction, followed by immunoaffinity column clean-ups and were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA). Recoveries were 80%–95% and 81%–92% for AFB₁ and AFM₁, respectively, while the limit of detection (LOD) was 0.01?µg/kg or µg/l for both mycotoxins. Results revealed the presence of AFB₁ in 84.4% of the feed samples (mean 18.7?±?1.4?µg/kg), and 39.2% of the plasma-examined samples (median 7.1?±?1.0?µg/l) were found to be contaminated at levels higher than the Tunisian and the European Union (EU) limit for dairy animals, which are 20 and 5?µg/kg in animal feed, respectively. AFM₁ was detected in 60.7% of the cow raw milk samples examined (median 13.6?±?1.4?µg/l). Contaminated levels were higher than the EU limit of 0.05?µg/l. It was concluded that more precaution should be taken on hygiene controls in order to prevent fungal contamination.     

乳品中黄曲霉毒素M1检测方法研究进展

黄曲霉毒素M1是动物摄入黄曲霉毒素B1后的代谢产物,主要分布在动物的乳汁、尿液中。黄曲霉毒素M1毒性很大,经乳制品摄入会对人体产生巨大的危害。本文主要对乳品中黄曲霉毒素M1毒性、危害、检测方法进行综述。对主要检测方法的特性及适用范围进行分析与概括,并且对未来黄曲霉毒素M1的检测方法的发展进行了合理展望。     

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.     

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.     

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.     

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%.     

Multiresidue method for simultaneous analysis of aflatoxin M1, avermectins,organophosphate pesticides and milbemycin in milk by ultra-performance liquid chromatography coupled to tandem mass spectrometry

A method developed for the simultaneous analysis of aflatoxin M₁, abamectin, doramectin, eprinomectin, ivermectin, moxidectin, acephate, azinphos-ethyl, azinphos-methyl, diazinon, methamidophos, methidathion, mevinphos, pirimiphos-ethyl and pirimiphos-methyl in whole raw milk, based on the QuEChERS method for extraction and clean-up, with detection and quantification by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) is described. The method was validated according to parameters of the Analytical Quality Assurance Manual from the Brazilian Ministry of Agriculture and Commission Decision 2002/657/EC, and proved suitable for analysis of these analytes within the proposed working range, with recovery values between 77% and 110%, a standard deviation lower than 20%, limits of detection between 0.05 and 0.99 µg l^?1, and limits of quantification between 0.15 and 1.98 µg l^?1. Samples from animals treated with abamectin, doramectin, ivermectin and diazinon were analysed by the validated method. Residues of aflatoxin M₁ were also found in field samples at levels below the established maximum residue limit.     

An effective self-control strategy for the reduction of aflatoxin M1 content in milk and to decrease the exposure of consumers

The study reports the results of testing the sensitivity of an early warning sampling plan for detecting milk batches with high aflatoxin AFM₁ concentration. The effectiveness of the method was investigated by the analysis of 9017 milk samples collected in Italian milk processing plants that applied control plans with different action limits (AL). For those milk processing plants where 30 ng kg^?1 AL has been applied, the AFM₁ contamination was significantly lower at or above the 95th percentile of the milk samples when compared with plants that used 40 ng kg^?1 AL. The results show that the control plan can be used effectively for early warning of occurrence of high AFM₁ contamination of milk and to carry out pro-active measures to limit the level of contamination. Estimation of dietary exposure was also carried out, based on the aflatoxin M₁ content of the milk samples and on Italian food consumption data. Estimated Daily Intakes (EDI) and Hazard Indices (HI) were calculated for different age groups of the population. HIs show that no adverse effects are expected for the adult population, but in the case of children under age three, the approximate HI values were considerably higher. This underlines the importance of the careful monitoring and control of aflatoxin M₁ in milk and dairy products.     

亲水亲脂平衡柱萃取高效液相色谱法快速测定干酪中的黄曲霉毒素M1

研究了利用RP-HPLC对干酪中黄曲霉毒素M1进行测定,分别采用乙腈、甲醇、丙酮作为提取溶剂对干酪样品中的黄曲霉毒素M1进行提取,利用一种亲水亲脂平衡柱萃取黄曲霉毒素M1,然后上机检测,采用质量分数为25％的乙腈水溶液为流动相,流速1 mL/min,利用荧光检测器进行检测.结果表明,采用乙腈作为干酪中黄曲霉毒素M1的提取试剂比其他2种溶剂更为适宜;OASIS HLB固相萃取净化柱对于黄曲霉毒素M1的分离纯化效果很好;干酪中的黄曲霉毒素在8mm内被快速检出,平均同收率较高,相对标准偏差(RSD)为1.4％～5.7％,最低检出限为0.037μg/kg.该方法是一种针对干酪中黄曲霉毒素M1的快速高效的检测方法.     

Aflatoxin M1 in fresh milk collected from local markets of Karachi,Pakistan

During 2016–2017, 156 samples of fresh milk samples were collected from local markets of Karachi, Pakistan and analysed for aflatoxin M₁ (AFM₁) contamination using ELISA technique. AFM₁ was detected in 143 (91.7%) samples, ranged from 20 to 3090 ng L^?1 with a mean level of 346.2 ng L^?1. In 125 (80.1%) samples, the AFM₁ contamination was greater than the maximum limit (ML = 50 ng L^?1) set by EU. However, in 51 (32.7%) samples, the AFM₁ level was higher than the ML of 500 ng L^?1 as assigned by the USA. Statistical analysis showed that the AFM₁ level in milk samples from summer was significantly (p < 0.05) higher than that obtained in winter. It was concluded that the AFM₁ levels in the tested samples appear to be a serious public health problem. Therefore, immediate measures should be taken and re-evaluation done for the procedures for farming, transportation, refrigeration, and storage for the control of AFM₁ level in milk samples.     

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.     

牛奶及奶粉中黄曲霉毒素M1的快速测定

采用免疫亲和柱－荧光光度法快速测定了牛乳及乳制品中黄曲霉毒素M1。试样经过离心、脱脂、过滤后滤液经过键合有黄曲霉毒素M1特殊抗体的免疫亲和柱净化，此抗体对黄曲霉毒素M1具有专一的识别能力，黄曲霉毒素M1键合在分离柱中的抗体上，用甲醇与水之比为10：90的混合液将免疫亲和柱上杂质除去；以甲醇与水之比为80：20的混合液通过分离柱洗脱；加入溴溶液衍生，以提高测定灵敏度，衍生化后的洗脱液于荧光光度计中测定黄曲霉毒素M1，检测低限为0．1μg/kg；在0．1－1．0μg/kg范围内，回收率为89．6％－96．1％，变异系数为0．52％－5．8％，分析一个样品的时间小于30min，分析过程中不使用黄曲霉毒素M1标准物质，结果表明，本方法具有准确、简单、快速、安全等优点，可以满足少量和批量样品的检测需要。     

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.     

Short communication: Aflatoxin M1 in dairy products sold in Şanlıurfa,Turkey

The aim of this study was to detect the presence of aflatoxin M₁ (AFM1) in samples of raw milk (n = 38), UHT milk (n = 12), white pickled cheese (n = 50), and yogurt (n = 50) collected from the ?anl?urfa city markets and locally produced dairy products by ELISA. The mean contamination rates were 56.74 ± 40.32, 43.1 ± 23.19, 103.2 ± 29.13, and 55.28 ± 12.68 ng/kg, respectively, for raw milk, UHT milk, white pickled cheese, and yogurt. According to the data, 21 (55%) raw milk, 3 (25%) UHT milk, 10 (20%) white pickled cheese, and 10 (20%) yogurt samples were contaminated with AFM1 over the acceptable levels (≥50 ng/kg), ranging from 0.82 to 130.89 ng/kg. None of the white pickled cheese samples contained AFM1 levels above the Turkish legal limit (250 ng/kg). Consequently, the AFM1 contamination levels determined in this study in white pickled cheese were not considered to pose a serious public health hazard. However, the AFM1 levels in raw and UHT milk and yogurt samples indicate an increased human health risk in Turkey related to high aflatoxin levels. Therefore, milk and dairy products have to be monitored by the Turkish public health authorities continuously to detect AFM1 contamination.