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.     

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.     

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.     

Presence of aflatoxin M1 in milk and infant milk products in Tehran,Iran

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic compounds. The purpose of this survey was to determine natural occurrence and level of AFM₁ in pasteurized liquid milk, infant formula and milk-based cereal weaning food consumed in Tehran, Iran.A total of 328 branded milk products and liquid milk samples were collected and investigated by Enzyme Linked Immuno Sorbent Assay (ELISA).The samples of pasteurized liquid milk (n = 128), infant formula (n = 120) and milk-based cereal weaning food (n = 80) showed that the incidence of contamination with AFM₁ is 96.3%, the presence of AFM₁ in each group was 72.2 ± 23.5, 7.3 ± 3.9 and 16.8 ± 12.5 ng/kg, ranging between 31–113, 1–14 and 3–35 ng/kg, respectively.In general, the amount of AFM₁ in 100 (78%) of liquid milk samples and 24 (33%) of milk-based weaning food was higher than the maximum tolerance limit accepted by European Union, but in all of the infant formula samples was lower (European Communities and Codex Alimentarius has prescribed a limit of 50 ng/kg for AFM₁ in milk and 25 ng/kg in infant milk products).     

A study on the occurrence of aflatoxin M1 in raw milk produced in Sarab city of Iran

The presence of aflatoxin M1 (AFM1) in 111 samples of raw milk obtained from dairy plants of Sarab city of Iran were analysed. In 85 of the 111 samples (76.6%) the presence of aflatoxin M1 was detected in conc. ranging between 0.015 and 0.28 μg/l. AFM1 level in (40%) of positive samples were higher than the maximum tolerance limit (0.05 μg/l) accepted by some European countries.The lowest mean of AFM1 concentration was found as 0.024 μg/l on August, and the highest AFM1 value as 0.118 μg/l on December. Incidence level of AFM1 on January, February, April, and December, was higher than the other months.Statistical evaluations showed that mean contamination level of AFM1 in autumn and winter samples were significantly higher (P < 0.01) than those of spring and summer.In addition the results showed that there were no statistical differences between AFM1 contents of spring and summer samples.     

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.     

Levels of aflatoxin M1 in milk,cheese consumed in Kuwait and occurrence of total aflatoxin in local and imported animal feed

A total of 321 milk samples (177 fresh, 105 long-life and 27 powdered milk, and 12 human milk), 40 cheese samples and 84 feed samples were analyzed for aflatoxin M1 (AFM1) and total aflatoxin. The samples were collected randomly during January 2005–March 2007, from Kuwaiti markets. The method used was ELISA technique. Results showed that all fresh milk samples except one were contaminated with AFM1 ranging from 4.9 to 68.7 ng/kg. Eight samples exceeded the EC’s regulatory limit. For the long-life milk samples, the ranges of AFM1 were from below the detection limit to 88.8 ng/kg, with four samples above the action limit of the EC. In the powdered milk samples, AFM1 ranged from 2.04 to 4.14 ng/kg. Of the human milk samples, only five were contaminated, with AFM1 levels ranging from 8.83 to 15.2 ng/kg with a mean 9.7 ng/kg. The cheese samples recorded 80% contamination with AFM1 with a range 23.8–452 and mean of 87.6 ng/kg, with one sample being above the regulatory limit (250 ng/kg) while the feed samples, showed 79.8% contamination with total aflatoxin.     

Aflatoxin M1 contamination in dairy products marketed in Iran during winter and summer

In the present study, 298 dairy product samples consisting of pasteurized milk (91 samples), yoghurt (68 samples), white cheese (72 samples), butter (31 samples) and ice cream (36 samples) collected from popular markets in four large Iranian cities were examined for aflatoxin M₁ (AFM₁) by thin layer chromatography (TLC) technique. The toxin was detected in 66 (72.5%) pasteurized milk samples (mean: 0.052 μg/l; range: 0.013–0.250 μg/l), 45 (66.1%) yoghurt samples (mean: 0.032 μg/kg; range: 0.015–0.119 μg/kg), 59 (81.9%) white cheese samples (mean: 0.297 μg/kg; range: 0.030–1.200 μg/kg), 8 (25.8%) butter samples (mean: 0.005 μg/kg; range: 0.013–0.026 μg/kg) and 25 (69.4%) ice cream samples (mean: 0.041 μg/kg; range: 0.015–0.132 μg/kg). The concentration of AFM₁ in 36.2%, 20.6%, 30.5%, 9.6% and 27.7% of pasteurized milk, yoghurt, white cheese, butter and ice cream samples, respectively, were higher than Iranian national standard limits. Levels of AFM₁ in samples of pasteurized milk, yoghurt, butter and ice cream collected in winter were significantly higher (P < 0.05) than those collected in summer. In the case of white cheese, level of AFM₁ was higher in winter than in summer, but the difference was not statistically significant (P > 0.05). The results indicated that the contamination of the dairy products in such a level could be a serious public health problem at the moment.     

Changes in the concentration of aflatoxin M1 during manufacture and storage of White Pickled cheese

In this study White Pickled cheese was produced from cow’s milk contaminated artificially with aflatoxin M₁ (AFM1) at two different levels, 1.5 and 3.5 μg/kg (ppb), and the effects of process stages on the AFM1 contents were investigated. Pasteurization at 72 °C for 2 min caused losses of AFM1 about 12% and 9%, respectively, in milk contaminated with 1.5 μg/kg AFM1, and 3.5 μg/l AFM1. These losses were found to be statistically significant (P < 0.01). After the cheese production, about 56% and 59% of total AFM1 remained in cheese–curd while about 32% of total AFM1 transferred to the whey for both 1.5 μg/kg and 3.5 μg/kg AFM1 contaminated milk. After 3-month storage in brine, AFM1 content of cheeses produced from 1.5 and 3.5 μg/kg AFM1 contaminated milks decreased by 2.9% and 2.8%, respectively. Changes in AFM1 content of cheese samples were found statistically insignificant (P > 0.05 and P > 0.01) for 3-month storage periods.     

Occurrence of aflatoxin M1 in raw milk in South Korea using an immunoaffinity column and liquid chromatography

The level of aflatoxin M₁ (AFM₁) contamination in raw milk produced in South Korea was investigated using immunoaffinity column chromatography and high performance liquid chromatography with fluorescence detector. A total of 100 raw milk samples were collected from 100 cattle ranches located in three different provinces of South Korea. Forty eight out of 100 raw milk samples contained AFM₁ at low level (0.002–0.08 μg/L) with mean value of 0.026 μg/L. Among the AFM₁ contaminated samples, 29 raw milk samples contained only traceable amount of AFM₁ below the limit of LOQ, 0.02 μg/L. None of samples exceeded the maximum level (0.5 μg/L) of Korean regulation for AFM₁ in milk. The limit of detection was 0.002 μg/L. The result of recovery test with 0.5 μg/L AFM₁ in raw milk sample was 96.3% (SD 3.6, n = 5). This is the first pioneering study to investigate the level of AFM₁ in raw milk used in dairy industries in South Korea.     

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.     

Aflatoxin M1 in milk and traditional dairy products from west part of Iran: Occurrence and seasonal variation with an emphasis on risk assessment of human exposure

In the present study, a total of 358 samples consisting of raw milk of cow (n = 64), goat (n = 56) and sheep (n = 52); traditional cheese (n = 40), yoghurt (n = 42), Kashk (n = 40), Doogh (n = 44) and Tarkhineh (n = 20) were analyzed for aflatoxin M₁ (AFM₁) by using an enzyme linked immunosorbent assay (ELISA). Frequency of AFM₁ and its concentration ranges in the ELISA positive samples were determined by high performance liquid chromatography with fluorescence detection (HPLC-FD). AFM₁ contamination was 84.3%, 44.6% and 65.3% for cow, goat and sheep raw milks, respectively. Moreover, AFM₁ was in 65.5%, 23.8%, 14%, 13.6% and 35.0% of cheese, yoghurt, Kashk, Doogh and Tarkhineh samples, respectively. Percentages of cow milk, goat milk, sheep milk and cheese samples exceeding the EU limit were 35.9%, 11.1%, 26.9% and 10%, respectively. HPLC analyses confirmed the ELISA results although the percentages of AFM₁ contamination in raw milk and dairy products were lower than that of ELISA. There were significant differences (P < 0.05) between the mean AFM₁ contents of raw milk, cheese and yoghurt samples during winter and summer seasons. Our study demonstrated that there is a potential risk for liver cancer due to the consumption of milk and dairy products in Iranian consumers.     

A survey on the Aflatoxin M1 occurrence and seasonal variation in buffalo and cow milk from Southern Italy

This study evaluates the aflatoxin M1 (AFM1) contamination in 804 samples of raw milk from cow and buffalo, collected randomly in Campania and Calabria regions of Southern Italy over a two years period.The competitive enzyme linked immunosorbent assay (ELISA) method was used to analyze AFM1 in the samples. AFM1 levels result above the CCβ value of 0.004 μg/kg in 51 (12.3%) cow milk samples and in 28 (7.2%) buffalo milk samples. Positive results from screening analysis were confirmed by high performance liquid chromatography with fluorimetric detection (HPLC-FLD) after a procedure of centrifugation, extraction and immunoaffinity column clean-up of milk. Only one cow milk sample exceeded the maximum limit (0.05 μg/kg) set by the European Regulation.The occurrence of AFM1 contamination was significantly (p < 0.05) higher in cold season, particularly fall, than in warm season, principally spring.Our results indicate that feedstuff used in the buffalo and cow farms were not highly contaminated with aflatoxins, determining a good quality of the analyzed milk. Therefore, the AFM1 contamination of the milk does not represent a serious public health problem in both regions in Southern Italy.     

A survey of aflatoxin M1 of raw cow milk in China during the four seasons from 2013 to 2015

In the present study, a total of 1550 raw cow milk samples were collected from Southern, Northern, Northeast, and Western regions of China during the four seasons from 2013 to 2015. Samples were analyzed for aflatoxin M1 (AFM1) using high performance liquid chromatography (HPLC). In 2013, AFM1 was detected in 21% of 366 raw cow milk samples with levels ranging between 0.01 and 0.24 μg/L. In 11.7% of samples, AFM1 levels were >0.05 μg/L, which is the legal limit in the European Union. The mean and median of positive samples were 0.069 ± 0.052 μg/L and 0.056 μg/L, respectively. In 2014, AFM1 was detected in 28.5% of 624 raw cow milk samples, with levels ranging from 0.01 to 0.25 μg/L. Of these samples, 7.7% had AFM1 levels exceeding 0.05 μg/L, with a mean of 0.042 ± 0.039 μg/L and median of 0.028 μg/L. AFMI was detected in 14.1% of 560 raw cow milk samples in 2015, with levels ranging from 0.01 to 0.144 μg/L. In 1.8% of these samples, AFM1 levels were above 0.05 μg/L, with a mean of 0.026 ± 0.024 μg/L and median of 0.017 μg/L. Our results demonstrate that AFM1 levels of the samples did not exceed the legal limit of 0.5 μg/L in China, the United States, and Codex Alimentarius Commission. Geographically, AFM1 contamination was more predominant in raw cow milk samples from Southern China than in those from other regions, with a higher number of samples containing AFM1 levels above 0.05 μg/L in 2013, 2014, and 2015. AFM1 levels were higher in autumn than in the other seasons during the entire study period. According to our survey, AFM1 contamination has been well-controlled in China during recent years; however, some samples still exceeded the European Union (EU) legal limit. Better prevention and management of aflatoxins in both feed and milk should be considered especially in Southern regions of China and in autumn.     

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.     

Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese

A polymerase chain reaction, amplifying a fragment of the mitochondrial DNA D loop region was developed for species specific detection of cattle and buffalo milk. The method was simultaneously extended for detection of HTST pasteurized milk samples and cheeses of bovine and buffalo origin. A common forward primer was used with two different species specific reverse primers that resulted amplification of a 126 bp and 226 bp products for cattle and buffalo, respectively, in simplex as well as in multiplex polymerase chain reaction. The primers successfully amplified DNA extracted by conventional protocol from minimal amount of raw milk, heat treated milk and cheese of either bovine or buffalo origin. The primers showed a high degree of specificity. The sensitivity of the assay was excellent with detection level of 0.1 percent adulteration of cow and buffalo milk or cheese (0.15 ng buffalo and 0.04 ng cattle DNA). The assay represents a sensitive and simple method for identification of adulteration in milk and cheese.     

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.     

Characteristics of Batzos cheese made from raw,pasteurized and/or pasteurized standardized goat milk and a native culture

Experimental cheeses were made from raw (R), raw with starter (RS), pasteurized with starter (PS) and standardized, pasteurized with starter (PSS) goat milk to study the influence of the starter and pasteurization on the quality of Batzos cheese. Lactococcus lactis subsp lactis strains from raw milk cheese used as starter proliferated significantly (P < 0.05) only in PSS cheese. Heat treatment lowered (P < 0.05) the levels of most microbial groups. However, TC and LAB counts were higher (P < 0.05) in PSS cheese and this was accompanied by lower (P < 0.05) pH; thus, a higher rate of decrease of undesirable microorganisms in PSS cheese was recorded. Degradation of αs-casein was in R > RS and in PS > PSS, while a small reduction of β-casein during ripening and storage was recorded. The proportions of aminoacids and lipolysis products increased throughout ripening and storage.     

Immunoaffinity pre-concentration combined with on-column visual detection as a tool for rapid aflatoxin M1 screening in milk

A non-instrumental rapid test was developed for the screening of aflatoxin M1 (AfM1) in milk at the 40 ng/L level. The method combines on one immunoaffinity gel layer, AfM1 pre-concentration and direct competitive immunoassay detection with visual evaluation. Aflatoxin B1-horse radish peroxidase (AfB1-HRP) and Sepharose 4B-immobilized anti-aflatoxin B1 monoclonal antibody with a 79% cross-reaction for AfM1 were used. The assay was performed in a standard column for solid phase extraction using a milk sample volume of 10 mL. Raw milk, pasteurized milk, milk powder, kefir and yogurt were analyzed with the developed test. It was shown that pasteurized milk could be analyzed without any sample preparation. The other types of milk were analyzed after centrifugation. For the assay procedure an aliquot of milk was flowed through the immunoaffinity gel layer in the tube by pushing the plunger of a syringe. Further, AfB1-HRP was applied and bound to free antibody binding sites. After addition of the chromogenic substrate the results were visually evaluated. Blue colour developed in case of negative milk samples while no colour appeared for positive samples. This method showed a false negative rate of only 2% and a high throughput (20–35 min for six samples).     

Determination of aflatoxin M1 in dairy cattle milk in Khartoum State,Sudan

Aflatoxins are a group of mycotoxins that contaminate various types of food and feedstuff leading to health risk in both humans and animals. Aflatoxin M1 (AfM1), the major metabolite of AfB1, was determined in dairy cattle milk samples of Khartoum State of Sudan using high-performance liquid chromatography (HPLC) with fluorescence detection. A total of 44 bulk dairy cattle milk samples were collected and analyzed. The percentage of AfM1 contamination has been found in 42/44 (95.45%) samples with contamination level ranging between 0.22 and 6.90 μg L⁻¹ and average concentration of 2.07 μg L⁻¹. AfM1 contamination in the samples of dairy cattle milk of Khartoum State of Sudan appears to be prevalent and may pose a public health problem at the moment. Awareness must be conveyed to producers, handlers and specialists.