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.     

Stability of aflatoxin M in milk.

This three-part study was designed to determine aflatoxin M recovery from pasteurized and/or stored cow's milk. (a) Aflatoxin M was added to samples of raw Holstein milk at a concentration of 2.0 mug/liter. Half of each sample then was pasteurized at 63 C for 30 min, and both raw and pasteurized portions were stored at 4 C up to 17 days. (b) Samples of raw milk, pasteurized (77 C, 16 s) skim milk, dry cottage cheese curd, and cottage cheese whey were taken from a commercial operation in an area in which natural contamination had been encountered. (c) Milk from a cow dosed with aflatoxin B1 was stored frozen (-18 C) in bulk and in assay-size sample containers for 120 days. Aflatoxin M was recovered completely after either storage or pasteurization in (a) and (b). In (c), a recovery deficiency was detectable after 68 days of storage, which increased to 45% of the original value by 120 days. These observations differ from those of others in that loss of aflatoxin M was significant after pasteurization or storage of raw milk, totaling 87% loss after 120 days of frozen storage. Aflatoxin M partitioning between curd and whey in the preparation of cottage cheese agrees with more recent studies, but differs from previous reports. Three possible explanations for the differences are offered.     

Robotic analysis of aflatoxin M1 in milk

An automated aflatoxin M1 assay system capable of performing multiple unattended extractions and chromatographic analyses has been developed. A six-axis anthropomorphic laboratory robot and a flexible computer system are combined to operate a sample turntable, a multisolvent dispensing facility, a solid-phase extraction station, a vacuum manifold, an automatic HPLC sample preconcentration unit, an HPLC, a fluorescence detector and a computing integrator. The system is capable of handling bulk milk samples and can determine aflatoxins at the sub-micrograms/kg level with an accuracy and precision comparable to those of the manual methods of analysis. Time of analysis is reduced. The system can be run in an unattended mode of operation.     

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。     

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.     

Excretion pattern of aflatoxin M1 in milk of goats fed a single dose of aflatoxin B1

The feedstuffs used in dairy animals must be able to give consumers confidence about the wholesomeness of milk with regard to aflatoxin contamination. The aim of this study was to determine the excretion patterns of aflatoxin M(1) (AFM1) in the milk of dairy goats fed a single dose of pure aflatoxin B(1) (AFB1), which can occasionally occur if feeds are infected by hot-spot growth of molds that produce aflatoxins. Five dairy goats in midlactation were administered 0.8 mg of AFB1 orally. Individual milk samples were collected for 84 h after AFB1 dosage. Aflatoxin M(1) was found in milk in the highest concentration. In all goats, AFM1 was not detected in milk before AFB1 administration, but was detected in the first milking following AFB1 administration. The excretion pattern of AFM1 concentration in milk was very similar in all goats even if the values of the concentration differed between animals. The peak values for AFM1 concentration in milk was observed in milk collected during the milking at 3 and 6h. After the peak, the AFM1 in milk disappeared with a trend that fitted well a monoexponential decreasing function, and the toxin was not detected after 84 h. Only about 0.17% of the amount of AFB1 administered was detected as AFM1 in milk, and about 50% of this was excreted in the first liter of milk yielded after AFB1 intake. Correct procedures to prevent growth of molds, and consequent AFB1 contamination, on the feedstuffs for lactating goats represent the key to providing consumers a guarantee that milk is not contaminated by AFM1.     

Presence of aflatoxin M1 in pasteurized milk from Morocco

Fifty four samples of pasteurized milk produced by five different dairies from Morocco were surveyed for the presence of aflatoxin M1 (AFM1) using immunoaffinity columns and liquid chromatography coupled to fluorescence detection. Confirmation of AFM1 identity in positive samples was based on the formation of AFM1 hemi-acetal derivative (AFM2a) after derivatization with trifluoracetic acid. Analytical results showed that 88.8% of the samples were contaminated with AFM1; 7.4% being above the maximum level of 0.05 microg/L set by the Moroccan and European regulations for AFM1 in liquid milk. The incidence of AFM1 in milk from these dairies was 100, 92.3, 90, 83.3 and 77.7% respectively, with AFM1 levels ranging from 0.001 to 0.117 microg/L and a mean value of 0.0186 microg/L. Based on the results presented in this study, the estimated daily intake of AFM1 was 3.26 ng/person/day. In this work, data on the natural occurrence of AFM1 in pasteurized milk produced in Morocco is presented for the first time.     

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.     

Distribution of aflatoxin M1 in cheese obtained from milk artificially contaminated

Small-scale manufacture of cheese using artificially AFM1 contaminated milk as raw material to study the distribution of such toxin both in whey and in cheese, was carried out. Whole milk with undetectable levels of AFM1 was used. The toxin was added in concentration that varied from 1.7 to 2.0 microg/l of milk. After the home-made production of cheese, the concentration of AFM1 was determined both in whey and in cheese, using the enzymatic immunoassay technique. The greatest proportion, 60%, was detected in whey while 40% AFM1 remained in cheese.     

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。     

Occurrence of aflatoxin M1 in retail milk samples from Bogotá, Colombia.

A study was conducted to establish the occurrence and levels of contamination of aflatoxin M1 (AFM1) in retail milk from Bogotá, Colombia. A total of 241 samples were analysed during 2004 and 2005. Samples were cleaned up by an immunoaffinity column and AFM1 was quantified by liquid chromatography with fluorescence detection. A total of 69.2 and 79.4% of the samples analysed during 2004 and 2005, respectively, were found to contain levels of AFM1 above 10 ng l(-1). Levels of contamination ranged from 10.7 to 213.0 ng l(-1) in 2004, and from 10.6 to 288.9 ng l(-1) in 2005. Despite the high incidence of AFM1 found in the milk samples analysed, all samples complied with current local regulations, which allow AFM1 content in milk up to 400 ng l(-1). However, due to the high incidence of AFM1 in milk found in the present study, it is recommended that a permanent surveillance programme be established for milk consumed in Bogotá in order to prevent milk lots containing levels above the regulatory level entering the food chain.     

Methods for determination of aflatoxin M1 in milk and milk products--a review of performance characteristics

Among numerous methods that have been published for determination of aflatoxin M1 in milk and milk products, the following have been selected for review of performance characteristics: methods for which interlaboratory testing has been carried out, methods proposed in support of national (Swiss) regulations following inclusion in check sample series, and methods that report detection limits for milk of less than or equal to 5 ng/l (less than or equal to 0.005 microgram/l) or less than or equal to 10 ng/kg (less than or equal to 0.01 microgram/kg) for cheese. It is practical to determine aflatoxin M1 in milk with good accuracy and precision down to low ng/l concentrations using thin-layer chromatography, high-performance liquid chromatography or enzyme-linked immunosorbent assay for quantitation. However, measurement at such low levels has not been tested by a true collaborative study. Confirmation of identity of aflatoxin M1 at low ng/l levels has also been reported. Recent evidence suggests that consideration should be given to inclusion of aflatoxin M4 in methods for aflatoxin M1.     

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.     

Global occurrence of aflatoxin M1 in milk with particular reference to Iran

Aflatoxin M₁ (AFM₁) is an hydroxylated derivative of aflatoxin B₁ (AFB₁), which occurs in the milk of lactating animals. The aim of the present study was to determine the concentrations of AFM₁ in raw milk samples collected from 18 dairy farms in Qazvin, Iran, over a period of 1 year and compare them with those found in other countries. Samples (30 per farm) were collected in the four seasons, Spring, Summer, Autumn and Winter occurring between April 2009 and March 2010, giving a total of 2160 samples. They were centrifuged and 100 μl of the resulting skimmed milk were tested for AFM₁ contamination by competitive enzyme immunoassay (EIA). All samples (100 %) were contaminated with AFM₁ with concentrations ranging from 0.04 to 148.01 ng.l^?1 and a mean of 38.82 ng.l^?1. Summer samples with a mean of 64.69 ng.l^?1 and autumn samples with a mean of 0.14 ng.l^?1 had the highest and lowest concentrations, respectively, and differed significantly (P?<?0.05). AFM₁ content in 722 samples (33.4 %) was higher than the maximum tolerance limit of 50 ng.l^?1 accepted by the European Union (EU). As contamination of milk with AFM₁ is a potential risk for human health, raw milk should be monitored for its presence.     

Seasonal study of aflatoxin M1 contamination in milk in five regions in Iran

Between April 2003 and February 2004, 98 samples of raw milk were obtained from milk tanks in one dairy plant in each of five regions in Iran. These were chosen with mean distances apart of 400 km, whereby they have different ecologies (temperature, relative humidity, etc.) and different agricultural products were used for animal feeding. Samples (24-25 per season) were laboratory heat treated and were analyzed for aflatoxin M1 with a validated High Performance Liquid Chromatography (HPLC) system. The overall mean of all samples was 0.041-0.065 microg/L (95% confidence) and the adjusted mean based on statistical modification was 0.039 ppb: 61 samples had 0.000-0.050 microg/L, 29 samples were contaminated with 0.05-0.10 microg/L, and the remaining 8 samples had 0.1-0.39 microg/L. All of the samples were lower than Codex Alimentarius and FDA standards (0.5 microg/L). Levels of aflatoxin M1 were higher in winter and spring than in summer and autumn, but the differences were not statistically significant (P>0.07). However, the level of aflatoxin in milk from one region (Hamedan) was significantly lower (P<0.05) than in those of the other regions (Gorgan, Rasht, Shiraz, Tehran).     

Aflatoxins in acid-treated grain in Sweden and occurrence of aflatoxin M1 in milk

A survey of aflatoxins in acid-treated grain and milk from farms using such grain was conducted in Sweden during 1986. Aflatoxins occurred most frequently (40%) in grain treated with a new formula of diluted (700 g litre^?1) aqueous formic acid, but also in 31% of the samples of grain treated with 850 g litre^?1 aqueous formic acid. The lowest incidence was found in grain treated with propionic acid, where aflatoxins were found in only one sample (3%). Aspergillus flavus/A parasiticus occurred in the same manner, but were more frequent than the aflatoxins. When cultivated on aflatoxin-producing agar, positive reactions were more common (56%) among strains originating from grain treated with formic acid than among strains originating from grain treated with propionic acid (4%). Aflatoxin M¹ in concentrations over 50 ng kg^?1 was mainly found in milk from farms using formic acid, and in most of these cases aflatoxins were also detected in the grain samples. In some cases, milk from a single farm was contaminated enough to generate consumption milk from the dairy with aflatoxin M¹ concentrations above or close to 50 ng kg^?1. The risk of aflatoxin formation after inadequate treatment of grain with formic acid is very high and is considerably lower with propionic acid. Formic acid has now been prohibited for use as a preservative of high moisture grain in Sweden.     

Reduction in aflatoxin M1 concentration during production and storage of selected fermented milks

This study focused on monitoring changes in aflatoxin M1 (AFM1) concentrations during production and storage of different fermented milks using selected probiotic and nonprobiotic combined cultures. Milk samples intended for fermentation were intentionally contaminated by adding a standard of AFM1. All of the tested cultures caused remarkable reductions in AFM1 concentrations during the fermentation process. Probiotic cultures were more effective than nonprobiotic cultures, with Lactobacillus caseiLC‐01 strain being the most efficient, achieving a reduction level of approximately 58%. Among the nonprobiotic cultures, yoghurt culture YC‐380 was the most efficient, with a reduction level of approximately 41%.