A study on the occurrence of aflatoxin M1 in milk consumed in Kosovo during 2009–2010

To assess public health hazards associated with the occurrence of AFM1 residues in milk a survey was carried out, during 2009–2010 in six different major regions of Kosovo (Prishtina, Prizren, Peja, Gjakova, Mitrovica and Gjilan). In the present study a total of 895 milk samples were collected, 656 raw milk and 39 UHT milk were analyzed in 2009, while in 2010 the research materials consisted of 170 raw milk, and 30 UHT milk. The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunosorbent assay (ELISA) method. From 895 samples examined, 25(2.8%) samples were contaminated with AFM1; none of contaminated samples did not exceed the maximum tolerable limit of the EC according to the European Union regulation limits of 50 ng/L. In 2009, 20 out of 695 samples (2.9%) were positive, and in 2010, 5 out of 200 samples examined (2.5%) were positive. In UHT milk, AFM1 was detected in two samples (2.6%). The positive incidence of AFM1 in milk samples collected in 2009 and in 2010 ranged from 5.2 to 26.6 ng/L and from 6.3 to 9.81 ng/L, respectively. In UHT milk levels of AFM1 were 7.2 and 9.9. In terms of regional distribution of positive samples during 2009 and 2010, Peja was the region with most of positive samples 12 or (63.2%), and 5 or (100%), respectively. Due to the unpredictability of climatic and environmental conditions, it is essential that the program of monitoring of AFM1 in milk in Kosovo should continue, to avoid an undetected problem, like occurred in other countries.     

Occurrence of aflatoxin M1 in UHT milk and pasteurized milk in China market

In the present study, the contamination of aflatoxin M1 (AFM1) in 153 UHT milk samples collected in July and September 2010, and 26 pasteurized milk samples collected in September 2010, were assessed using the ELISA method. The AFM1 was detected in 54.9% of UHT milk samples with a concentration of 0.006–0.160 μg/L. Moreover, 96.2% of pasteurized milk samples tested positive for AFM1 with concentration levels of 0.023–0.154 μg/L. The occurrence of AFM1 in all positive samples was far below China's national legal limit of 0.5 μg/L. Meanwhile, the AMF1 content in 20.3% of UHT milk samples and 65.4% of pasteurized milk samples exceeds the European Union's legal limit of 0.05 μg/L.     

Occurrence of aflatoxin M1 in pasteurized and UHT milks in China in 2014–2015

This survey was performed to assess the safety of milk in China, specifically by assessing the presence of aflatoxin M1 (AFM1) residues in pasteurized and ultra high temperature (UHT) milk. In 2014–2015, 193 samples of UHT milk were collected from different cities in China. In 2015, 38 samples of pasteurized milk were collected from different cities in China. AFM1 was detected using an enzyme-linked immunosorbent assay (ELISA). AFM1 positivity was defined as a concentration exceeding the detection limit of the assay (0.005 μg/kg). Other cut-offs that were used were the legal AFM1 limits in the European Union (EU) and China (0.05 and 0.5 μg/kg, respectively). In 2014 and 2015, 88.6% and 59.6% of UHT milk samples were AFM1-positive, respectively. The pasteurized milk samples were less frequently AFM1-positive (47.4%). In 11.9% of the 2014–2015 UHT milk samples, the AFM1 levels exceeded the EU limit. This is lower than the frequency we recorded in 2010 (20.3%). None of the pasteurized milk samples exceeded the EU limit in 2015. The UHT milk samples from the north of China were less likely to be contaminated than the samples from the south in both 2014 and 2015. None of the samples exceeded the Chinese legal limit.     

A survey on the occurrence of aflatoxin M1 in raw and processed milk samples marketed in Lebanon

A survey was conducted to determine the occurrence of AFM1 in 77 cow and goat milk samples (38 raw milk, 25 pasteurized milk and 14 powder milk samples); obtained either from local small farms, or markets. The competitive enzyme – linked immunosorbent assay (ELISA) method was applied for this purpose positively detecting AFM1 in 64.9% of all tested milk samples. The revealed rates of AFM1 contamination were 73.6%, 68.0%, 35.7% for the raw, pasteurized and powder milk samples, respectively. The individual values, within each category of milk samples, ranged from 2.63 to 126 ng/l (average = 60 ng/l), 3.27–84.4 ng/l (average = 30.6 ng/l) and 9.18–16.5 ng/l (average = 13.7 ng/l) for the raw, pasteurized and powder milk samples, respectively. Of the positive samples, 29 were still below the permitted limit (50 ng/l) set by the European Commission whereas 21 exceeded the permissible limit. This work represents the data of the first survey on the occurrence of AFM1 in raw and processed milk marketed and consumed in Lebanon.     

Seasonal effect on aflatoxin M1 contamination in raw and UHT milk from Croatia

Aflatoxin M1 contamination was examined in raw milk (3716) and UHT milk (706) samples collected from farms of eastern Croatia and markets of central Croatia from February to July 2013. A maximal mean AFM1 of 1135.0 ng/L was measured in raw milk in March. The AFM1 levels exceeded the European Union maximum residue permitted amount (EU MRL) in 45.9% raw and 36.2% UHT milk samples in February. In total, AFM1 levels exceeded the EU MRL values in 27.8% of raw and 9.64% of UHT milk samples. A slight decrease in the number of samples exceeding the EU MRLs was recorded in the period March to June. The results showed significant statistical differences between the mean AFM1 concentrations of raw and UHT milk samples collected during February, March, May and June (P < 0.05, all). Also, statistical differences in AFM1 concentrations were found between months for raw and UHT milk (P < 0.001, both). In conclusion, the frequency of control of feed and milk samples should increase and should strive to educate breeders and those involved in milk production about the harmful effect of aflatoxins to animal feed.     

Aflatoxin M1 contamination in pasteurized milk in Shiraz (south of Iran)

The aim of this study was to evaluate Aflatoxin M1 (AFM1) contamination in pasteurized milk samples in Shiraz city (Iran). Six hundred and twenty four pasteurized milk samples from different supermarkets in Shiraz city were collected during 6 months (April to September 2003). AFM1 was found in 100% of the examined milk samples. 17.8% of the samples had AFM1 greater than the maximum tolerance limit (50 ng/l) accepted by European Union. Contamination with AFM1 is a serious problem for public health. To achieve a low level of AFM1 in milk, cows’ feed samples from various cowsheds must be evaluated routinely for aflatoxin and kept away from fungal contamination as much as possible.     

Aflatoxin M1 in raw,pasteurized and powdered milk available in the Syrian market

The incidence of contamination of aflatoxin M1 (AFM1) in milk samples collected from the Syrian market was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total of 126 samples composed of raw cow milk (74 samples), raw sheep milk (23), raw goat milk (11), pasteurized cow milk (10) and powdered milk (8) showed that 80% of tested samples were contaminated with various levels of AFM1 ranging from >20 to 765 ng/l. Percentages of AFM1-contaminated samples exceeding the American, Syrian and European tolerance limits were 22%, 38% and 52%, respectively.The range of contamination was relatively higher in pasteurized milk than in raw cow and sheep milk. 80% of AFM1-contaminated pasteurized cow milk samples exceeded the European tolerance limit with a range of contamination between 89 and 765 ng/l. Percentages of contaminated raw cow, sheep and goat milk exceeding the European tolerance limit were 59%, 24% and 14%, respectively.Milk powder was almost free of AFM1 contamination with only one sample containing a concentration lower than the European tolerance limit (12 ng/l).Extrapolation of aflatoxin B1 (AFB1) from AFM1 levels of contamination in milk samples indicates that contamination in dairy cattle feeds may range from 0.5 to 47.8 μg/kg.     

Aflatoxin M1 in raw and UHT cow milk collected in Bosnia and Herzegovina and Croatia

Aflatoxin M1 (AFM1) concentrations were determined in raw and UHT cow milk samples collected in different regions of Bosnia and Herzegovina and Croatia during the autumn months of 2014. The mean AFM1 levels in the raw milk samples were (ng/kg): 6.22 in Bosnia and Herzegovina, 5.65 in Croatia. In all except one milk sample, AMF1 levels were below the LOQ value of 34.2 ng/kg (ELISA method). In four milk samples, AFM1 concentrations exceeded the EU MRL of 50 ng/kg. Samples were subjected to LC-MS/MS analysis which confirmed elevated values determined by ELISA. Elevated levels were in the range 56.6–132.6 ng/kg. Two positive milk samples from Bosnia and Herzegovina originated from Una Sana Canton, two from Croatia from eastern Croatia. The highest AFM1 levels of 132.6 ng/kg was measured in milk from eastern Croatia. In 214 samples of processed UHT milk from Bosnia and Herzegovina and Croatia, AFM1 ranged from 2.29 ng/kg to 21.4 ng/kg, all below the LOQ value. AFM1 exceeded the EU MRL value in only 0.62% of milk samples, indicating the sporadic use of contaminated feedstuff at farms in both countries.     

A survey on the occurrence of aflatoxin M1 in milk samples in Ardabil,Iran

Mycotoxins are natural food and feed toxic contaminants produced as a result of fungal growth on agricultural materials during storage and/or transportation. The purpose of this study was survey on the occurrence of aflatoxin M1 (AFM1) in milk samples from Ardabil, Iran.Ninety samples were collected from local shops of the Ardabil city during different seasons in order to address representative data on AFM1 in milk consumed by people in this city. AFM1 was analyzed by Enzyme Linked Immuno Sorbent Assay (ELISA).All of the samples (100%) were contaminated with AFM1 in concentration levels ranged from 2.9 to 85 ng/kg. In 30 samples (33% of total samples) the AFM1 level was higher than 50 ng/kg, the maximum residue limit (MRL) accepted by European Union for AFM1 in milk. The difference of AFM1 level between seasons was statistically significant (P < 0.05).The highest and lowest levels of contamination were detected in winter and summer seasons which were 17.4 and 56.3 ng/kg, respectively.This is the first report on the occurrence of AFM1 in milk samples of Ardabil, one of the main milk-production regions in Iran. The contamination with AFM1, in this area is most probable because of contaminated feed consumed by cattle, which needs prompt effort to reduce the risk of contamination.     

The seasonal variation of aflatoxin M1 in milk and dairy products and assessment of dietary intake in Punjab,Pakistan

Total 372 samples (169 sample from summer and 203 samples from winter) of milk and dairy products were collected from major cities of Punjab, Pakistan, during 2014–2015. The aflatoxin M₁ (AFM₁) levels were analyzed using HPLC, equipped with floresceⁿce detector. The results have showed that AFM₁ was detected in 77 (45.5%) samples of milk and dairy products from summer at the range of LOD to 229.6 ng/L, and 114 (56.1%) samples from winter at a range of LOD to 345.8 ng/L. The highest mean of AFM₁ i.e. 94.9 ± 5.4 ng/L from summer and 129.6 ± 8.4 ng/L from winter was detected in raw milk samples. The results have shown that 37.5% of milk, 32% of UHT milk, 12.5% of powdered milk, 20% of flavored milk, 20% of yogurt and 16% of flavored milk samples from summer were found exceeded the recommended levels of AFM₁ i.e 50 ng/L (European Union permissible limit), as compared to 38.1%, 37.1%, 15.6%, 21.4%, 27.7% and 40% samples of milk, UHT milk, powdered milk, flavored milk, yogurt and flavored yogurt from winter, respectively. The mean level of AFM₁ in samples of milk, UHT milk and powdered milk samples from winter season has significantly (p˂ 0.05) higher as compared to the mean levels in samples from summer season. The dietary exposures of AFM₁ in milk and dairy products were calculated in five different age groups and the highest level of 3.42 and 3.33 ng/L/day in summer and 4.7 and 4.6 ng/L/day in winter were found in 1–5 years of male and female infants, respectively. The results of present study have shown significant levels of AFM₁ in milk and dairy products from Punjab, Pakistan and could pose considerable thereat to the health of local population.     

Aflatoxin M1 contamination in raw milk from major milk-producing areas of China during four seasons of 2016

This survey was performed to determine the frequency with which raw milk from the major milk-producing areas of China was contaminated with aflatoxin M1 (AFM1) in 2016. In total, 5650 raw milk samples produced during the four seasons of 2016 were collected from the major milk-producing areas of China, including Hebei, Heilongjiang, Henan, Inner Mongolia, Shandong, and Xingjiang provinces. Contamination of AFM1 was detected in 267 of the 5650 raw milk samples in totally, with the incidence of 4.7%. Only 1.1% of raw milk samples exceeded the European Union legal limit (50 ng/L), and none of samples exceeded the Chinese and United States legal limit (500 ng/L). The incidence of AFM1 contamination in raw milk samples was much higher during winter (10.2%) than in spring, summer, or autumn (3.0%, 2.1%, and 4.4%, respectively) in China. Thus, it is particularly important to monitor AFM1 contamination in raw milk during the winter season. This comprehensive study will facilitate future risk analysis and the management of AFM1 contamination in raw milk in China.     

Estimate of aflatoxin M1 exposure in milk and occurrence in Brazil

The presence of aflatoxin M₁ (AFM₁) was investigated in 125 samples of powdered milk, pasteurized milk and ultra high treated (UHT) milk in the city of São Paulo, and estimates of AFM₁ intake were assessed. The samples were analysed using an immunoaffinity column for cleanup and a HPLC-FLD for determining AFM₁. The quantification limit was 10 ng/kg. AFM₁ was found in 119 (95.2%) at levels ranging from 10 to 200 ng/kg with mean concentration of 31 ng/kg. The average daily intake estimated for AFM₁ was 1 ng/kg bw per day for children and 0.188 ng/kg bw per day for adults.     

A limited survey of aflatoxin M1 in milk from Indonesia by ELISA

This paper presents a limited survey of aflatoxin M1 (AFM1) in Indonesian milk. AFM1 concentrations of 113 fresh milk samples, collected in 2006 from farms in five different areas of the Yogyakarta Province were analysed. The fresh milk samples were taken directly from dairy farms before pasteurisation. Enzyme-linked immunosorbent assay (ELISA) was used for the analysis of milk samples. Results show that in 48 samples (42.5%) the AFM1 concentrations were less than 5 ng/L and in 31 samples (27.4%) AFM1 was found between 5 and 10 ng/L. In 34 samples (30.1%) the concentrations were above 10 ng/L. None of contaminated samples exceeded the European Union regulation limits of 25 and 50 ng AFM1/L for infant and adult consumption, respectively. Since AFM1 is derived from aflatoxin B1 (AFB1) contained in cow feedstuffs, based on the contamination levels of AFM1 found in this study, the exposure of animals to AFB1 seems to be low.     

Assessment of aflatoxin M1 contamination in the milk of four dairy species in Croatia

Aflatoxin M1 (AFM1) concentrations were measured in bulk cow milk samples from eastern Croatia, and in cow, goat, sheep and donkey bulk milk samples from other parts of Croatia during the period July–September 2013. AFM1 levels in milk were measured in the ranges (ng/L): cow 3.65–162.3 (eastern Croatia) and 2.69–44.9 (other regions of Croatia); goat 2.78–40.8; sheep 2.11–5.87; donkey 3.43–10.4. The concentration of AM1 exceeded the EU MRL in 6.7% of cow milk samples from eastern Croatia. The highest level measured was 162.3 ng/L. AFM1 levels exceeded the LOQ value (23.2 ng/L) in only 59 samples of cow milk and two samples of goat milk of the total 402 samples analysed. A significant difference was found between the mean AFM1 concentrations of cow milk from eastern and other regions of Croatia (P < 0.05). The elevated AFM1 levels in cow milk from eastern Croatia indicate the use of contaminated supplementary feedstuff in some farms during the study period.     

Contamination of UHT milk by aflatoxin M1 in Iran

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.     

Analysis of aflatoxin M1 and M2 in commercial dairy products using high-performance liquid chromatography with a fluorescence detector

Aflatoxin M₁ (AFM₁) and M₂ (AFM₂) in commercial dairy products were analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector (FLD). To ensure an accurate analysis, two derivatization methods, bromination and aflatoxin–trifluoroacetic acid derivatization (ATD), were compared. The limits of detection (LODs) of the bromination method were 124.42–151.73 ng/kg, and the recovery rates were between 64 and 102%. The detection rates and concentration levels of AFM₁ were 6–74% and 14.48–270.94 ng/kg, respectively. AFM₁ was detected in 74% of milk powder samples and 36% of ice cream samples. The mean values of AFM₁ in milk powder and ice cream samples were 270.94 and 33.16 ng/kg, respectively. In the case of AFM₂, the detection rates were 2–10%, and the concentration levels were 20.62–55.67 ng/kg in milk and milk powder. Among milk and milk powder samples, ultra heat-treated (UHT) milk had lower AFM₁ contamination levels than pasteurized milk.     

Occurrence and estimation of aflatoxin M1 exposure in milk in Serbia

The occurrence of AFM1 was investigated in 150 cow's, 10 goat's, 5 donkey's, 10 breasts milk and 1 infant formula samples. Analyses were done using Enzyme Linked Immunosorbent Assay (ELISA) method. AFM1 was detected in 98.7% of analyzed cow's milk samples in concentrations ranged from 0.01 to 1.2 μg/kg. Further, even 129 (86.0%) cow's milk samples contained AFM1 in concentration greater than maximum residue levels (MRL) of 0.05 μg/kg defined by European Union (EU) Regulation. Analysis of other types of milk showed that AFM1 was detected in 80.0% goat's, 60.0% donkey's and 60.0% of breasts milk samples.Although Serbian Regulation for MRL of AFM1 in milk has been changed and harmonized with EU Regulation in 2011, occurrence of AFM1 in milk in Serbia during 2013 resulted in Regulation changes, and MRL were changed from 0.05 to 0.5 μg/kg.On the basis of the obtained concentrations of the AFM1 in cow's milk, collected information about average milk intake and mean body weight (bw) for different age's categories, mean ingestion of AFM1 in ng/kg per bw per day were estimated. Obtained results showed that all age's categories, especially children, are exposed with high risk related to presence of AFM1 in milk.There are only a few published data about occurrence of AFM1 in milk in Serbia and none about intake assessment for AFM1.     

Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies

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.     

Occurrence of macrocyclic lactones in milk and yogurt from Brazilian market

Avermectins and milbemycins belong to a family of compounds called macrocyclic lactones (ML) and are highly used as antiparasitic agents in the treatment of cattle for control of gastrointestinal nematodes, ticks and myiasis. In Brazil, there are five substances (ivermectin, abamectin, doramectin, eprinomectin and moxidectin) registered for bovines. The use of these compounds may bring benefits to the cows but indiscriminate use might result in the presence of residues in milk and dairy products. In this context, the objective of the study was to validate an analytical method for determination of five ML in dairy products and verify the occurrence of these compounds in milk and yogurt available in the Brazilian market. The method involved QuEChERS sample preparation, derivatization and determination by high performance liquid chromatography with fluorescence detection. The methodology was validated using organic samples of milk and yogurt for the following parameters: linearity, precision, accuracy, repeatability and limits of detection (LOD) and quantification (LOQ). The method showed good linearity. Average recovery, performed at three different levels varied from 83% to 112% (RSDs < 14%). The method provides LOD ranging from 0.4 to 3.2 μg L⁻¹ for milk and 0.6 to 0.9 μg kg⁻¹ for yogurt. The LOQ was established according to the lower spike level (0.2–10 μg L⁻¹ for milk and 2.5 μg kg⁻¹ for yogurt). Repeatability and within-laboratory reproducibility were in satisfactory for both matrixes. In order to monitor milks and yogurts marketed in Campinas, SP, Brazil, 13 brands of UHT milk (135 samples), 8 brands of pasteurized milk (103 samples) and 13 brands of yogurt (104 samples) were acquired. A total of 342 samples were analyzed in duplicate for the presence of ivermectin, abamectin, doramectin, eprinomectin and moxidectin. Moxidectin was detected in one sample of pasteurized milk. No residue of the analyzed compounds was found in UHT milk or yogurt. Results indicate that the consumption of milk and yogurt does not represent a health risk for Brazilian consumers.     

Assessment of benzoic acid levels in milk in China

Benzoic acid is the most commonly used preservative in foodstuffs. Although benzoic acid is generally recognized as safe (GRAS), adverse effects such as asthma, urticaria, metabolic acidosis and convulsions were observed at low doses in sensitive persons. Some weak clastogenic activity was noted in in vitro assays. The aim of our study was to determine the levels of benzoic acid in milk samples consumed in the city of Guangzhou, China. In this study, 142 samples of pasteurized milk (24), ultra high temperature milk (UHT, n = 45), milk powder (n = 31) and infant formula (n = 42) were analyzed by high performance liquid chromatograph (HPLC) with diode array detector (DAD) from October 2006 to January 2007. Benzoic acid was detected in 109 (76.8%) samples, ranging from 0.51 to 111 mg/kg. The LOD for benzoic acid was, respectively, 0.2 mg/kg in pasteurized and UHT milk, 2 mg/kg in milk powder and infant formula. The mean recoveries of spiked samples at 4 levels were 99.5 ± 3.1% in liquid milk, and 98.5 ± 4.3% in milk powder, respectively. The results show that benzoic acid widely occurs in milk and milk products in China at the low levels. The levels found are not high, and should not affect the general public; however, it may affect the infants and/or sensitive persons. Therefore, it is important to assess the levels of benzoic acid in milk in China. Meanwhile, it should be noted that the lowest percentage of positive samples in pasteurized milk probably results from the difference in the quality of raw milk, processing technical and storage condition of milk products.