Occurrence,seasonal variation and risk assessment of exposure to aflatoxin M1 in Iranian traditional cheeses

A total of 360 traditional cheeses consisted of Lighvan (n = 62), Koozeh (n = 62), Siahmazgi (n = 58), Khiki (n = 58), Talesh (n = 58) and Lactic (n = 62) collected from different parts of Iran were analyzed for aflatoxin M₁ (AFM₁) using an enzyme linked immunosorbent assay (ELISA). Frequency of AFM₁ and its concentration ranges of all the ELISA positive samples were determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD). AFM₁ was detected in 60.3%, 75.8%, 72.4%, 43.5%, 38.7% and 35.4% of Siahmazgi, Khiki, Talesh, Lighvan, Koozeh and Lactic cheeses, respectively with concentration ranging from 50.5 to 308.7 ng/kg, respectively. HPLC analyses confirmed the ELISA results although the rates of contaminated cheese samples were lower than that of ELISA. There was significant difference in AFM₁ level between various cheese types and samples collected from summer and winter seasons (P < 0.05). By comparing our findings with the EU limit, about 10.5% of cheese samples had exceeding values for the toxin. The results of the present study indicates that there is no health risk in consumption of Iranian traditional cheeses due to the presence of AFM₁.     

Occurrence of aflatoxin M1 in white cheese samples from Tehran, Iran

This study was undertaken to determine the occurrence of aflatoxin M₁ (AFM₁) in 50 white cheese samples from 2 dairy factories in summer 2008 and winter 2009. Enzyme-linked immunosorbent assay (ELISA) method was used for analysis of the samples. Aflatoxin M₁ was found in 60% of the cheese samples, ranging from 40.9 to 374 ng/kg. Toxin levels in 6% of the samples exceeded the Iranian national standard limit i.e. 200 ng/kg. Considering seasonal variability, mean concentration of AFM₁ in the samples collected in winter was significantly (P < 0.03) higher than those collected in summer. Therefore, high occurrence of AFM₁ in cheese samples could be a potential hazard for public health.     

Aflatoxin M1 levels of Turkish white brined cheese

This study was undertaken to determine the presence and levels of aflatoxin M₁ (AFM₁) in Turkish white brined cheese consumed in the province of Erzurum, Turkey. For this purpose, a total of 193 cheese samples were randomly obtained from retail outlets and Enzyme Linked Immunosorbent Assay (ELISA) technique was used to determine the presence and levels of AFM₁. AFM₁ at detectable level (50 ng/kg) was found in 82.4% of the samples. The concentration of AFM₁ in samples ranged from 52 to 860 ng/kg. Of the samples, 26.4% exceed the legal limit of 250 ng/kg established by Turkish Food Codex. It was concluded that widespread occurrence of AFM₁ in Turkish white brined cheese samples were considered to be possible hazards for public health especially children.     

Transfer of aflatoxin M1 from milk to ripened cheese in three Italian traditional production methods

Robiola and Primosale, two fresh cheeses, and Maccagno, an hard-type cheese, were produced using milk that was naturally and artificially contaminated with aflatoxin M₁ (AFM₁) at the levels of 10, 50 and 200 ng/l. Concentrations of AFM₁in milk and cheeses were determined by liquid chromatography and fluorimetric detection, coupled with immunoaffinity column extraction. In the Robiola production method, AFM₁ levels in whey ranged between 30% and 65% of the total amount of the toxin present in the milk, while Primosale and Maccagno, that share the same rennet based cheesemaking procedure, showed an higher percentage of AFM₁ partitioning to whey.For each cheese-making method, the concentration of AFM₁ on fresh matter was higher in the cheese compared to the original milk. The fresh cheeses showed a concentration factors of 1.43 and 2.20 for Primosale and Robiola, respectively, whereas the Maccagno cheese showed a value of 6.71. For all the production methods considered, when using milk not exceeding the maximum acceptable level of 0.05 μg AFM₁/kg set by EU, the resulting cheese also complied with current Italian recommendations for AFM₁ contamination (450 ng AFM₁/kg).     

Occurrence of aflatoxin M1 in dairy products in Brazil

The objective of this study was to investigate the incidence and occurrence of aflatoxin M₁ (AFM₁) in dairy products produced in Brazil. A total of 123 samples of three different groups of dairy products (cheese, yoghurt, and dairy drinks) consumed by Brazilians were collected during 2010. All samples including 58 cheese samples, 53 samples of yoghurt and 12 dairy drinks were purchased from grocery stores in the Ribeirão Preto-SP area. Cheese samples were classified into three categories depending on their moisture and fat contents: Minas Frescal cheese, Minas Frescal light cheese and Minas Padrão cheese. Samples were analyzed for AFM₁ by a published method. The method comprised aqueous methanol extraction, immunoaffinity column purification and isolation, reversed phase liquid chromatography separation and fluorescence detection. AFM₁ was detected in 84% of the analyzed cheese samples (>3 ng/kg) with levels ranging from 10 to 304 ng/kg in 67% of the samples. AFM₁ was detected in 95% of the yoghurt and dairy drink samples with levels ranging from 10 to 529 ng/kg in 72% of the samples. Despite the lack of a Brazilian regulatory limit for AFM₁ in yoghurt and dairy drinks the survey data of this study may offer information useful in the determination of whether the occurrence of AFM₁ in Brazilian dairy products may be considered as a possible risk for consumer health and whether Brazilian regulatory guidelines for AFM₁ in dairy products are needed.     

Aflatoxin M1 in milk and distribution and stability of aflatoxin M1 during production and storage of yoghurt and cheese

The objective of this study was to investigate the incidence and occurrence of aflatoxin M₁ (AFM₁) in Brazilian milk and infant formula. The distribution and stability of AFM₁ in cheese and yoghurt were also determined. Milk samples and infant formula samples were purchased in Ribeirão Preto-SP, Brazil and were analyzed for AFM₁ using immunoaffinity column purification, liquid chromatography separation and fluorescence detection. AFM₁ was detected in 83% of the milk samples (>3 ng/kg) with levels ranging from 8 to 437 ng/kg for fluid milk, and 20–760 ng/kg for powdered milk. No AFM₁ was found in infant formula. Processing and storage was shown to have little effect on AFM₁ content in milk and milk products. Total AFM₁ mass in milk was reduced by 3.2% in cheese and by 6% in yoghurt (pH 4.4). The mean concentration of AFM₁ in curds was 1.9-fold higher and whey was 0.6-fold lower than in unprocessed milk.     

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.     

A study on the occurrence of aflatoxin M1 in Iranian Feta cheese

This study was undertaken to determine the presence and levels of aflatoxin M₁ (AFM₁) in cheeses produced by different plants in the province of Tehran. For this purpose, a total of 80 cheese samples analyzed, and thin layer chromatography (TLC) was used to determine the presence and levels of AFM₁.AFM₁ was found in 82.5% of 80 of the cheese samples examined. The range of contamination levels varied among different months. AFM₁ in May, August, November, February samples ranged from 0.17 to 1.30, 0.15 to 2.41, 0.16 to 1.11, and 0.19 to 2.05 μg/kg, respectively, while the mean values were 0.41, 0.35, 0.36, and 0.52 μg/kg, respectively.The highest mean concentration of aflatoxin M₁ (AFM₁) was registered in February samples (0.52 μg/kg). The lowest mean concentration of aflatoxin M₁ was registered in August samples (0.35 μg/kg).Statistical evaluation showed that there were not significant differences (P > 0.05) between the concentrations of AFM₁ of cheese samples taken in May and August with November and February. In other words, AFM₁ contents of cheese samples taken in November and August were not lower than cheese samples taken in May and February. Almost 60.6% of the contaminated samples exceeded the maximum acceptable levels (0.25 μg/kg) that accepted by some of the countries such as Turkey.It was therefore concluded that, high occurrence of AFM₁ in cheese samples were considered to be possible hazards for human health.     

Screening of aflatoxin M1 occurrence in selected milk and dairy products in Terengganu,Malaysia

The study was conducted to screen the occurrence of aflatoxin M₁ (AFM₁) in 53 selected milk and dairy product samples (11 liquid milk, 12 powdered milk, 8 3-in-1 beverages, 6 condensed sweetened milk, 2 evaporated milk, 7 cultured milk drink, 5 yogurt and 2 cheese samples). These samples were purchased from selected markets in Terengganu, Malaysia in January 2014 based on a questionnaire survey among 212 respondents on the types and brands of milk and dairy products that were frequently consumed. Based on the responses, 53 milk and dairy products were purchased and the competitive enzyme-linked immune-absorbent assay (ELISA) method was used to determine the level of AFM₁ in the samples. Of 53 samples, 19 samples were positive with AFM₁ (35.8%) ranging from 3.5 to 100.5 ng/L. Although 4/53 (7.5%) of the tested samples had the contamination level greater than the European Commission (EC) limit (>50 ng/L), the contamination levels were still below the Malaysia Food Regulation 1985 limit (less than 500 ng/L). This study provided a pioneering data on the occurrence of AFM₁ in milk and dairy products in Malaysia.     

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.     

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 factors associated with the presence of aflatoxin M1 in breast milk samples of nursing mothers in central Mexico

Aflatoxin M₁ (AFM₁) is the main mycotoxin frequently found in milk and dairy products. Exposure of infants to AFM₁ is of great health public concern because of its toxic and carcinogenic properties. The aim of this study was to determine the occurrence, levels and factors associated with the presence of aflatoxin M₁ in breast milk samples of nursing mothers in central Mexico. A total of 112 samples were collected and analyzed by a competitive-ELISA assay during January–August 2014. A Semi-Quantitative Food Frequency Questionnaire was used to determine weekly intake of food groups. Results showed that 89% of breast milk samples contained AFM₁ in a range of 3.01–34.24 ng/L. AFM₁ mean level was significantly higher (P < 0.05) in winter (12.78 ng/L) and spring (12.09 ng/L) than summer (7.91 ng/L). It was also detected that 7% of samples exceeded the European Community regulation (25 ng/L) for milk infant formula. The presence of aflatoxin M₁ was significantly associated (P < 0.05) to consumption of egg, cola drink and sunflower oil. The high occurrence and noticeable levels of AFM₁ detected, as well as the mean AFM₁ estimated daily intake (2.35 ng/kg b.w./day) suggested, for the first time, that breast-fed infants in the central region of Mexico, may be exposed to significant levels of this toxin through mother's breast milk.     

Fate of aflatoxin M1 during production and storage of parmesan cheese

The fate of AFM₁ during production of a long maturing cheese (Parmesan cheese) was assessed. Different levels of AFM₁ contamination and of the fat/casein (F/C) ratio of milk were considered, in order to evaluate if these factors can influence the enrichment factor (EF) of AFM₁ in cheese. For this purpose, 24 cheese-makings were carried out using naturally contaminated milk at 3 different AFM₁ levels and at 2 F/C ratios. AFM₁ analysis was performed by HPLC in raw milk, cream, cauldron milk, liquid cattle rennet, whey, curd and cheese at 3, 9, 16 and 24 months of ageing. The mass balances of the cheese-making processes were close to 100%; in whey, AFM₁ concentration was about 40% less than the concentration in cauldron milk. The EF in curd was between 4.0 and 5.2, with an average value of 4.7 ± 0.4; this factor was not significantly affected by either AFM₁ contamination level or F/C ratio. During maturation, AFM₁ concentration and consequently EF increased from curd to 16 ageing months; successively, AFM₁ slightly decreased at 24 months and consequently the EF. At 3, 9, 16 months of maturation, the EF was significantly higher for cheeses prepared using milk with low F/C than those with high F/C milk; on the contrary, EF was not significantly influenced by the AFM₁ contamination level. In cheeses, EF values were between 4.7 and 6.3; from these results, the maximum admissible level for AFM₁ in Parmesan cheese should be about 0.275 μg kg⁻¹.     

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.     

Distribution,stability, and protein interactions of Aflatoxin M1 in fresh cheese

Aflatoxin M₁ (AFM₁), a metabolite resulting from the hepatic metabolism of aflatoxin B₁, is a potential carcinogen that can be found in milk, cheese, and others dairy products. In this work, we aimed to elucidate the distribution and fate of AFM₁ in fresh cheese and whey during cheese manufacturing and storage as well as the level of interaction of this toxin with various milk, cheese, and whey proteins. Additionally, we analyzed the in vitro behavior of casein, α-lactalbumin, β-lactoglobulin, bovine serum albumin (BSA), lactoferrin, and mixtures thereof, with a fixed concentration of AFM₁ after covalent crosslinking. Our results show that up to 70% of the AFM₁ levels present in milk spiked with 0.5 and 1.5 μg L⁻¹ are released in whey during fresh cheese manufacturing. The whey and milk proteins with more AFM₁ molecules bound were α-lactalbumin and casein, with 88% and 81%, respectively. We also observed a substantial decrement in AFM₁ concentration during ripening that correlated inversely with plate counts of lactic acid bacteria and directly with the whey-draining process that occurs during fresh cheese maturation or storage. This knowledge may serve as a basis for interventions in the dairy industry aiming to increase the security of cheese and other dairy products.     

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 and ochratoxin A in human milk in Ribeirão Preto-SP,Brazil

The objective of this study was to determine the extent of aflatoxin M₁ (AFM₁) and ochratoxin A (OTA) contamination in human breast milk in the city of Ribeirão Preto, São Paulo State, Brazil. During 2012, 100 samples of human milk were collected at the local Human Milk Bank. The method comprised, immunoaffinity column purification and isolation, liquid chromatography separation and fluorescence detection. The average percentage recoveries of AFM₁ and OTA spiked at 20 and 50 ng/L in control human milk were 78.1 ± 11.7% and 73.7 ± 9.6%, respectively. The average relative standard deviations of AFM₁ and OTA spiked at the same levels were 11.7 and 9.6% respectively. The limits of detection was 0.3 ng/L for AFM₁ and OTA. The limit of determination was 0.8 ng/L for both mycotoxins. This method was used to analyze 100 human milk samples, of which, two samples were found to contain AFM₁ at level greater than 0.3 ng/L. OTA was detected in 66 samples (66%), wherein 32 were above the limit of detection and 34 were in the range of from 0.8 to 21 ng/L. Results of our study indicate that breast-fed Brazilian infants had only an insignificant exposure to AFM₁ and OTA.     

Seasonal variation of aflatoxin M1 contamination in industrial and traditional Iranian dairy products

This study aimed to determine the occurrence of aflatoxin M₁ (AFM₁) contamination in 682 dairy product samples consisting of raw milk of cow, goat and sheep; Lighvan cheese; and industrial and traditional yoghurt, Kashk and Doogh samples collected from popular markets and dairy ranches in four large Iranian cities. Thin layer chromatography (TLC) technique was used for analysis of the samples. Results showed that the incidence and levels of AFM₁ contamination in raw cow milk and industrial products (manufactured from cow milk) were higher than raw goat or sheep milk, and traditional products (made from goat and sheep milk), respectively. Moreover, seasonal variations influenced the concentration of AFM₁ in most of the analyzed dairy products. Owing to the abundance and popularity of the industrial products, contamination of these products in such a level could be a potential hazard for public health.     

Seasonal variation of aflatoxin M1 in raw milk from the Yangtze River Delta region of China

The objective of this study was to evaluate the occurrence of aflatoxin M₁ (AFM₁) in raw milk samples from 18 dairy farms in the Yangtze River Delta region during four different seasons. A total of 72 tank milk samples was collected with 18 samples for each season. Milk AFM₁ was detected using LC-MS/MS. The AFM₁ was detected in 43 milk samples (59.7%) ranging in concentration from 10 to 420 ng/L. The concentration of AFM₁ in raw milk was significantly higher during the winter (123 ng/L) than during other seasons (P < 0.05). There was no significant difference between the spring (29.1 ng/L), summer (31.9 ng/L), and autumn (31.6 ng/L) (P > 0.05) seasons. This indicates that raw milk collected during the winter is at high risk for AFM₁ and that seasonal factors should be considered for the management of aflatoxins in both the feed and milk.     

Seasonal study of aflatoxin M1 contamination in milk of four dairy species in Yazd,Iran

This survey was conducted to determine the occurrence of aflatoxin M₁ (AFM₁) in samples of raw milk obtained from cow, sheep, goat, and camel herds in Yazd province during different seasons. Aflatoxin M₁ was analyzed using the competitive enzyme-linked immunosorbent assay technique for screening and high-performance liquid chromatography with fluorescence detection for confirmatory purposes. The detection rates of AFM₁ in cow, sheep, goat, and camel milk samples were 46.5%, 21.6%, 20.1%, and 4.03%, respectively. Levels of the toxin in 15.4% of cow milk, 11.5% of sheep milk, and 9.15% of goat milk samples exceeded the legal limit (0.050 μg/kg) recommended by the Institute of Standards and Industrial Research of Iran; while none of the camel milk samples exceeded the legal limit. The occurrence and levels of AFM₁ in cow milk samples from industrial dairy farms was significantly lower (P ≤ 0.05) than those from traditional ones. Seasonal variations influenced the occurrence and levels of AFM₁ in cow, sheep, and goat milk; however, no statistically significant seasonal effect was found for camel milk. This study indicates a high occurrence of AFM₁ in cow milk especially those obtained from traditional dairy farms. Therefore, more supervision is required on these farms; and traditional dairy farms should be gradually replaced by industrial ones.