Aflatoxin M1 and B1 in Colombian milk powder and estimated risk exposure

ABSTRACT

Aflatoxin M₁ (AFM₁) and aflatoxin B₁ (AFB₁) were determined in 51 milk powder samples purchased from different grocery stores located in the Caribbean region of Colombia. Analysis was conducted using QuEChERS extraction and high-performance liquid chromatography with fluorescence detection. Results from the analytical method showed recovery ranges from 65% to 110% and relative standard deviations lower than 20%. AFM₁ was detected in 100% of the milk samples (0.20–1.19 µg/kg) and 55% exceeded the maximum level in milk (0.5 µg/kg) set by the Colombian and European regulations. AFB₁ was not detected in any of the analysed samples. Considering the measured contamination the maximum AFM₁ level that can be ingested by consumption of milk powder is 0.007–0.013 µg/person/day. These values are above the average dietary intake estimated in Latin America according to the Joint FAO/WHO Expert Committee, which is 0.0035 µg/person/day.     

定性测定羊奶中黄曲霉毒素M1残留高灵敏度检测卡的研制

研制定性测定羊奶中黄曲霉毒素M1残留的高灵敏度检测卡.在硝酸纤维素膜上预包被黄曲霉毒素M1全抗原作为检测线,以及预包被羊抗鼠二抗作为质控线.将抗黄曲霉毒素M1单克隆抗体用胶体金标记作为金标垫.将样品垫、金标垫、预包被黄曲霉毒素M1的硝酸纤维素膜以及吸水垫依次粘贴在PVC板上,切割成试纸条并组装成检测卡.结果表明:该检测卡的检测限为0.5μg/L,对黄曲霉毒素M1的交叉反应率为100％,而与同族的其他物质的交叉反应率小于5％.假阳性率小于5％,假阴性率为0％,检测卡常温储存,保质期18个月.本研究的检测卡,可快速准确地应用于羊奶中黄曲霉毒素M1残留的定性检测,为快速准确地测定羊奶中黄曲霉毒素M1残留提供了依据.     

ELISA and HPLC determination of the occurrence of aflatoxin M 1 in raw cow's milk

Raw cow's milk collected from dairy farms in the province of Leon, Spain, was examined for aflatoxin M 1 (AFM 1 ). The samples were analysed with a commercial competitive enzyme-linked immunosorbent assay (ELISA) kit and high-performance liquid chromatography (HPLC). The concentrations of AFM 1 in the milk extracts were initially estimated by ELISA, with recovery rates of 74.6-109% for artificially contaminated milk at levels of 10-80 ng l 1 . Samples found to contain more than 10 ng l 1 were further quantified with HPLC. The mean recovery for this method was 89.3%. The quantification limit was 10 ng l 1 for both ELISA and HPLC. Although AFM 1 was confirmed in only 3.3% of the samples, the concentrations in all these cases were lower than the maximum limit applicable to these products pursuant to European Union legislation. Both methods were validated with reference material certified by the Community Bureau of Reference.     

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。     

Survey of the occurrence of Aflatoxin M1 in ovine milk by HPLC and its confirmation by MS

During the period of October-July 2000, 240 samples of dairy ewes milk, obtained from farms of Enna (Sicily, Italy), were checked for Aflatoxin M(1) (AFM(1)) by HPLC using a fluorimetric detector. The limit of detection and the limit of quantification were 250 ng/L for AFM(1). All the positive milk samples for AFM(1) were confirmed by LC-MS. AFM(1) was detected in 81% of milk samples, ranging from 2 to 108 ng/L. Three samples were over the legal limits (50 ng/L). Mean contamination of samples obtained from stabulated ewes was higher than that from grazing ewes (35.27 vs. 12.47 ng/L). Furthermore, samples collected in the period September-October showed higher contamination than samples collected during the other months (42.68 vs. 10.55 ng/L). Both differences are related to the administration of compound feed. Based on current toxicological knowledge we concluded that the AFM(1) contamination levels recorded in ewe milk did not present a serious human health hazard. However, as ewe milk is exclusively used to produce cheese due to its higher protein content, and also considering the preferential binding of AFM(1) to casein during coagulation of milk, a potentially high concentration effect could occur, thus the surveillance of contamination levels should be more continuous and widespread.     

Aflatoxin M1 in milk powders: Processing, homogeneity and stability testing of certified reference materials

As part of the certification campaign of three candidate reference materials for the determination of aflatoxin M₁ (AfM₁) in whole milk powders, homogeneity, short- and long-term stability tests of naturally contaminated milk powders have been performed. The homogeneity of two AfM₁-contaminated milk powders was studied by taking samples at regular intervals of the filling sequences and analysing in triplicate for their AfM₁ contents by liquid chromatography with fluorescence detection (LC-FLD) using random stratified sampling schemes. The homogeneity testing of an AfM₁ 'blank' milk powder material was performed by determining the nitrogen content because AfM₁ levels were below the limit of detection of the most sensitive determination method. The short-term stability of AfM₁-contaminated milk powders was evaluated at three different storage temperatures (4, 18 and 40°C). After storage times of 0, 1, 2 and 4 weeks, samples were investigated using LC-FLD. The long-term stability study comprised of measurements after 0, 6, 12 and 18 months after storage at -20 and 4°C. Analyses were done by LC-FLD. Based on the homogeneity tests, the materials were sufficiently homogenous to serve as certified reference materials. Corresponding uncertainty contributions of 0.23-0.89% were calculated for the homogeneity. The stability measurements showed no significant trends for both short- and long-term stability studies. The long-term stability uncertainties of the AfM₁-contaminated milk powders were 7.4 and 6.3%, respectively, for a shelf-life of 6 years and storage at -20°C. Supplementary stability monitoring schemes over a long period of several years are currently ongoing.     

Determination of aflatoxins by enzyme-linked immunosorbent assay with special reference to aflatoxin M1 in raw milk

There is an increasing requirement for the screening of animal feedstuffs, plant products and edible animal tissues for human consumption, for the presence of contamination by aflatoxins. An enzyme-linked immunosorbent assay is described which may be used to determine aflatoxins in raw milk at 0.1μg litre^?1 and in extracted feedstuffs at the level of 5μgkg^?1. The method includes a novel conjugation procedure allowing heterologous conjugates to be prepared resulting in increased sensitivity without interference by sample matrix effects. Suitably cross-reactive antisera were produced using the cyclopentanone ring of the aflatoxin molecule as the principal immunogenic determinant.     

Composition and occurrence of aflatoxin M1 in cow's milk samples from Razavi Khorasan Province,Iran

The composition and occurrence of aflatoxin M₁ (AFM₁) were investigated in 60 samples of cow's raw milk samples from Razavi Khorasan Province of Iran. Only percentages of milk samples protein, lactose and total solids in samples collected during summer were slightly lower than the Iranian reference values. Compared with summer samples, higher percentages of milk samples components were observed in the winter. The overall mean AFM₁ level was 61 ± 8 ng/L, with 24 samples (40%) showing concentrations above the maximum permitted level established in Iran (50 ng/L). Control measures are urgently needed to avoid aflatoxin in milk samples produced in Razavi Khorasan Province.     

Survey of aflatoxin M1 in milk and dairy products consumed in Burdur,Turkey

The aim of this study was to investigate the occurrence of aflatoxin M₁ (AFM₁) in dairy product samples in Burdur city. A total of 315 samples of dairy products were collected during 2008. Of the 315 samples analysed, AFM₁ in 246 samples (78.1%) was found to range from 5.5 to 800 ng/kg. In addition, AFM₁ levels of 16 raw milk, two pasteurised milk, only one milk powder and three white cheese samples were above the Turkish Food Codex. It is concluded that the occurrence of AFM₁ in dairy products may be considered as a possible hazard for public health.     

Occurrence of aflatoxin M1 in raw and market milk commercialized in Greece

From December 1999 to May 2000, 114 samples of pasteurized, ultrahigh temperature-treated (UHT) and concentrated milk were collected in supermarkets, whereas 52 raw milk samples from cow, sheep and goat were obtained from different milk producers all over Greece. Sample collection was repeated from December 2000 to May 2001 and concerned 54 samples of pasteurized milk, 23 samples of bulk-tank raw milk and 55 raw milk samples from cow, sheep and goat. The total number of samples analysed for aflatoxin M₁ (AFM₁) contamination by immunoaffinity column extraction and liquid chromatography was 297. In the first sampling, the incidence rates of AFM 1 contamination in pasteurized, UHT, concentrated and cow, sheep and goat raw milk were 85.4, 82.3, 93.3, 73.3, 66.7 and 40%, respectively, with only one cow raw milk and two concentrated milk samples exceeding the EU limit of 50 ng l^-1. In the second sampling, the incidence rates of AFM 1 contamination in pasteurized, bulk-tank and cow, sheep and goat raw milk were 79.6, 78.3, 64.3, 73.3 and 66.7%, respectively, with only one cow and one sheep raw milk samples exceeding the limit of 50 ng l^-1. The results suggest that the current regulatory status in Greece is effective.     

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.     

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.     

Excretion of aflatoxin M1 in milk of dairy ewes treated with different doses of aflatoxin B1

Two experiments were conducted to study the amount of aflatoxin M1 (AFM1) in milk in response to feeding aflatoxin B1 (AFB1). In experiment 1, four dairy ewes in early lactation received a single dose of pure AFB1 (2 mg). Individual milk samples were collected during the following 5 d to measure AFM1 concentration. The average excretion of AFM1 in milk followed an exponential decreasing pattern, with two intermediate peaks at 24 and 48 h. No AFM1 was detected in milk at 96 h after dosing. The mean rate of transfer of AFB1 into AFM1 in milk was 0.032%, with a high individual variability (SD = 0.017%). In experiment 2, 16 dairy ewes in midlactation were divided into four groups that received different daily doses of AFB1 (0, 32, 64, and 128 microgram for control and groups T1, T2, and T3, respectively) for 14 d. Pure AFB1 was administered to each animal divided in two daily doses. Individual milk samples were collected at 12, 24, 36, 48, 72, 96, 144, 216, and 312 h after the first AFB1 administration, during the intoxication period, and every 24 h for 7 d after the withdrawal of AFB1. AFM1 was detected in the milk of all animals of the treated groups at 12 h after the administration of AFB1. In all treated groups, milk AFM1 concentration increased from 12 to 144 h after the beginning of administration. It then decreased, reaching a stable concentration at 216 and 312 h after the first administration. No AFM1 was detected in milk 3 d after the last administration of AFB1. Milk AFM1 concentration measured at steady-state condition was significantly affected by the AFB1 dose (0.031, 0.095, and 0.166 in T1, T2, and T3 groups, respectively), with a linear relationship between AFB1 dose and milk AFM1 concentration (R2 = 77.2%). The carryover (AFM1/AFB1 ratio) was not significantly affected by treatment, and its mean value was 0.112% (SE = 0.011). The carryover was lower than that reported for dairy cattle and goats, suggesting a better ability of sheep to degrade AFB1.     

Efficiency of Bifidobacterium bifidum and Saccharomyces cerevisiae for detoxification of aflatoxin M1 in skim milk

Aflatoxin M1 (AFM1) is a carcinogenic mycotoxin mostly found in dairy products. The aim of this study was to evaluate the anti-aflatoxin effect of Bifidobacterium bifidum and Saccharomyces cerevisiae with two different concentrations (10⁸ and 10¹⁰ cfu/mL), alone or mixed, in contaminated skim milk with three concentrations of AFM1 (0.1, 0.25 and 0.5 μg/mL) and incubated at 4, 25 and 37°C for different times (30, 60, 120 min and 24 h). We found that the storage time is a key factor that significantly affects AFM1 removal. Also, removal of AFM1 was dependent on other factors such as micro-organisms' concentration, incubation temperature and toxin concentration. The effective strategy for the highest removal of AFM1 (90%) in contaminated milk spiked with 0.5 μg/mL, which was treated with mixed strains at concentration10¹⁰ cfu/mL and incubated at 37°C after 24 h. The presented methodology can be considered as a potential method for reducing the aflatoxin content of polluted dairy products.     

Aflatoxin M1 and ochratoxin A in raw bulk milk from French dairy herds

Mycotoxins in milk are a public health concern and have to be regularly monitored. A survey on the presence of aflatoxin M₁ (AFM₁) and ochratoxin A (OTA) in raw bulk milk was conducted in 2003 in the northwest of France, the main French milk-producing basin. Randomly selected farms (n = 132) were characterized by a diet based on corn silage and containing a large proportion of on-farm produced cereals, feeding sources that are frequently contaminated by mycotoxins. Farms were surveyed twice in winter and in summer. At each sampling time, a trained surveyor completed a questionnaire recording farm management procedures and production traits. The AFM₁ was found in 3 out of 264 samples but at levels (26 ng/L or less) that are below the European legislation limit of 50 ng/L. Traces of AFM₁ (less than 8 ng/L) were also found in 6 other samples. The OTA was detected in 3 samples also at low levels, 5 to 8 ng/L. Farms that tested positive to the presence of mycotoxins, 12 in total including 6 farms that had traces of AFM₁, differed from negative farms by a more extensive use of total mixed rations, 58 vs. 27%. In addition, the positive farms tended to have lower milk yields. Although the incidence of milk contamination with AFM₁ and OTA at the farm level was low during the period studied, production and management data from the surveyed farms suggest a link between feeding management practices and mycotoxin contamination.     

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.     

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.     

酶联免疫法测定婴幼儿配方乳粉中黄曲霉毒素M1的方法研究

采用酶联免疫法建立婴幼儿配方奶粉中黄曲霉毒素M1的测定方法。样品经冷冻离心、脱脂棉去脂等简便的前处理操作,采用黄曲霉毒素M1的酶联免疫试剂盒进行分析。结果显示,黄曲霉毒素M1在0～0.08 μg/L范围内线性关系良好,方法检出限为0.05 μg/kg,回收率为90.0%～105.0%,精密度相对标准偏差（RSD）为8.1%。该方法快速、简便、特异、可靠,适于现代批量检测的需求,为保证乳制品的质量安全提供了一可靠的筛选方式,将更好地配合液相色谱-质谱法作定性和定量分析。