Occurrence of aflatoxin M1 in raw milk collected from traditional dairies in Morocco

A survey of the presence of aflatoxin M1 (AFM1) was carried out on eight traditional dairies belonging to four sectors of Fez city situated in the northern center of Morocco. Raw milk samples were collected between October 2009 and September 2010, and analyzed by LC-fluorescence detection after immunoaffinity purification. AFM1 was detected in 13 out of 48 samples (27%) at concentrations ranged between 10 and 100 ng/l. Within these positive samples, four (∼8% of the total) were above the European legislation limit of 50 ng/l. This study revealed a variation of contamination from one sector to another with a higher incidence in milk samples collected in autumn compared to those collected in other seasons suggesting a link between feeding practices, such as the use of silage and AFM1 contamination. This is the first report on AFM1 contamination in raw milk directly collected from Moroccan traditional dairies. The levels of contamination found justify more detailed and continuous monitoring to assess the public health implication and reduce consumers’ exposure to AFM1.     

Occurrence of aflatoxin M1 in raw cow,goat and sheep milk during spring and autumn in Croatia during 2016

Raw cow, goat and sheep milk samples were collected in different regions of Croatia during spring and autumn 2016. Aflatoxin M1 (AFM1) concentrations were measured in the following ranges (ng/kg): cow milk 0.93–85.4; goat milk 2.0–18.6; sheep milk 2.27–11.2. AFM1 levels exceeding the European Union maximum residue levels of 50?ng/kg were found in two cow milk samples in spring and 10 cow milk samples in autumn. The probable daily intakes (PDIs) for all three dairy species were from 0.0108 to 0.0165?ng/kg b.w./day and contributing 1.08–1.65% to the proposed provisional maximum tolerable daily intake (PMTDI; 1?ng/kg b.w./day), indicating low risk exposure for consumers. The mean positive cow milk PDI was 0.15?ng/kg b.w./day, contributing 15.2% to the PMTDI.     

Assessment of aflatoxin M1 levels in pasteurised milk,raw milk,and cheese in Arak,Iran

Aflatoxins are fungal toxins, which may be found in food and due to negative health effects, they are of major concerns for human health and food industries. Aflatoxin M₁ contamination in dairy products in Arak, Iran, 2013 was evaluated using ELISA method. Sample groups were comprised of 111 samples, including pasteurized milk (n?=?56), raw milk (n?=?16) and cheese (n?=?39), and were analyzed for AfM₁ content. The results showed that 93.7% of the samples were positive with the total average concentration of 85.8?µg/L in milk and 30.39?µg/kg in cheese. Moreover, AfM₁ of 94.4% of milk samples and 92.3% of cheese samples was above the standards of Iran and EU.     

Occurrence of aflatoxin B1 contamination in dairy cows feed in Iran

Aflatoxins (AFs) are secondary toxic metabolites produced by fungal species. Occurrence of aflatoxin B1 (AFB1) was determined in 210 dairy cow feed samples consisting of concentrate (n=?70), corn silage (n =?70) and alfalfa hay ( n=?70) obtained from dairy farms in central part of Iran during July–September 2014. AFB1 analysis was carried out by using the competitive enzyme-linked immunosorbent assay technique for screening and high performance liquid chromatography for confirmatory purpose. AFB1 was detected in 88 analyzed samples (41.9%), ranging from 1.87 to 19.41?μg/kg; and 70 samples (33.3%) had levels of the toxin above the Iranian national standard and European Commission limits (5?μg/kg). The frequencies of AFB1 contamination in the samples were as follows: alfalfa hay, 55.7%; concentrate, 44.3% and corn silage, 25.7%. The high occurrence of AFB1 contamination may be attributed to poor harvesting technique, environmental conditions, insect infestation or unsuitable storage conditions. Hence, the contamination with AFs could be a potential hazard for human and animal health.     

Assessment of aflatoxin M1 contamination in pasteurized and UHT milk marketed in central part of Iran

This study was conducted as a screening survey to determine the occurrence of aflatoxin M1 (AFM1) in 225 commercial liquid milk samples composed of pasteurized milk (116 samples) and UHT milk (109 samples) obtained from popular markets in central part of Iran. The competitive enzyme-linked immunosorbent assay method was employed to determine level of the toxin in the samples. Aflatoxin M1 was detected in 151 (67.1%) samples, consisted of 83 (71.5%) pasteurized milk samples (mean: 52.8 ng/l; range: 5.8–528.5 ng/l) and 68 (62.3%) UHT milk samples (mean: 46.4 ng/l; range: 5.6–515.9 ng/l). Considering the US FDA and Iranian national standard limits for AFM1 in milk (500 ng/l), 2 samples (1.7%) of pasteurized and 3 samples (2.7%) of UHT milk had levels above the maximum tolerance limit. However, according to European Commission limit (50 ng/l), this figure increased to 31 (26.7%) and 19 (17.4%) samples for pasteurized and UHT milk, respectively. The results indicated that the contamination of the samples with AFM1 in such a level could be a serious public health problem at the moment. This paper represents the data of the first survey on the occurrence of AFM1 in commercial liquid milk marketed in central part of Iran.     

Aflatoxin M1 contamination in pasteurized milk in Tabriz (northwest of Iran)

Aflatoxin M1 (AFM1) appears in milk as a direct result of the ingestion of food contaminated with aflatoxin B1 by cattle. The role of milk in human nutrition is well-known. The aim of this study was to evaluate Aflatoxin M1 contamination in pasteurized milk samples in Tabriz city (Iran) by ELISA (Enzyme Linked Immunosorbent Assay). Fifty pasteurized milk samples from different supermarkets in Tabriz city were collected during 6 months (July to December 2008). AFM1 was found in 100% of the examined milk samples. Sixty-two percent of the samples had AFM1 greater than the maximum tolerance limit (50 ng/l) accepted by European Union. It can be concluded that AFM1 levels in the samples purchased in Tabriz city, appear to be a serious public health problem at the moment. To achieve a low level of AFM1 in milk, dairy cows’ feed samples from various cows’ herds must be controlled periodically for aflatoxin and kept away from fungal contamination as much as possible.     

A survey of aflatoxin M1 in cow milk in Southern Iran

The competitive enzyme-linked immunosorbent assay technique was used to evaluate aflatoxin M1 (AFM1) levels in 168 samples of raw milk (135 samples and 33 samples from bulk tanks of farms and milk collection centers, respectively) and 12 samples of pasteurized milk in Fars province, Southern Iran. AFM1 was found in 55.56% of the samples with a mean concentration of 21.31 ng/L. The concentration of AFM1 in raw milk samples from farms was significantly (p < 0.05) lower than that in samples from collection centers and pasteurized milk. The concentration of AFM1 was not influenced by season, location, or type of farm. The concentrations of AFM1 in all samples were lower than the Iranian national standard limit (100 ng/L), but in 30% of raw cow milk samples they were higher than the maximum tolerance limit accepted by the European Union (50 ng/L); therefore, more effort is needed to control AFM1 levels in milk produced in Southern Iran.     

Determination of aflatoxin M1 levels in Iranian white and cream cheese

A screening survey on the occurrence of aflatoxin M1 (AFM1) was accomplished on 210 cheese samples composed of white cheese (116 samples) and cream cheese (94 samples) purchased from popular markets in central part of Iran (Esfahan and Yazd provinces). The quantitative analysis of AFM1 levels in the samples was performed by using the competitive enzyme-linked immunosorbent assay (ELISA) technique. Aflatoxin M1 at measurable level (50 ng/kg) was detected in 161 (76.6%) samples, consisting of 93 (80.1%) white and 68 (72.3%) cream cheese samples. The concentration of AFM1 in the samples ranged from 52.1 to 785.4 ng/kg. Comparing to legal regulation (250 ng/kg) accepted by some of the countries, 24.2% of the samples exceeded the accepted limit. Among these, the AFM1 levels in 28.4% of white and 19.1% of cream cheese samples were not in accordance with the safety limit. The results indicated that contamination of the samples with AFM1 in such a level appear to be a potential hazard for public health. This paper represents the data of the first survey on the occurrence of AFM1 in cheeses consumed in central part of Iran.     

A clonal cell line (BME-UV1) as a possible model to study bovine mammary epithelial metabolism: metabolism and cytotoxicity of aflatoxin B1

Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial cell line (BME-UV1), monolayers were exposed to aflatoxin B1 (AFB1 - 1.0-8.0 μM). Starting from 4 h of exposure, the hydroxylate metabolite aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased linearly with time for 36-48 h and the percent biotransformation of AFB1 (2-4 μM) at 48 h was about 12-14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxicity with cellular metabolism. MTT assay (from 24 h of cell exposure) and NRU assay (from 16 h of cell exposure) showed time-dependent and time/concentration-dependent decrease of cell viability, respectively, and the former assay being more successful at revealing cytotoxic effects (NRU: CC₅₀ at 48 h = 12.00 ± 2.66 μM; MTT: CC₅₀ at 72 h = 20.42 ± 7.30 μM). The results suggest that BME-UV1 cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.     

Aflatoxin B1 and aflatoxin M1 induced cytotoxicity and DNA damage in differentiated and undifferentiated Caco-2 cells

Aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) are natural mycotoxins that frequently present in food and feed and pose risks to human health. There are few data in the literature regarding the impairment of them in the intestine. Therefore, the present study investigated their cytotoxic effect on Caco-2 cells, especially the differentiated ones that resemble mature small intestinal enterocytes. Both undifferentiated (UC) and differentiated (DC) cells were treated with AFB1 and AFM1 at various concentrations for up to 72 h. Cell viability, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) production and DNA damage were determined. Data showed that AFB1 and AFM1 significantly inhibited UC and DC cell growth, increased LDH and caused genetic damage in a time- and dose-dependent manner (p < 0.05). In comparison, AFB1 was found to be more toxic than AFM1 on both UC and DC. All these cytotoxic outcomes might be associated with intracellular ROS generation, leading to membrane damage and DNA strand break. Additionally, DC was found to be more sensitive to aflatoxins, which might be due to the alteration of enzymes during cell differentiation. The present study provided the first in vitro evidence of DNA damage of DC induced by AFB1 and AFM1.     

Occurrence of aflatoxin M1 in some samples of UHT,raw & pasteurized milk from Indian states of Karnataka and Tamilnadu

Aflatoxin M₁ (AFM₁) is a toxic metabolite found in the milk of lactating animals which have consumed feedstuffs contaminated with aflatoxin B₁. Ultra high temperature treated (UHT) milk is a product which is becoming popular in developing countries like India as there is a lack of proper cold storage or refrigeration facilities. In this study, 45 samples of UHT milk of popular brands prevalent in the market were analyzed for the presence of AFM₁ by reversed phase HPLC using fluorescent detector after cleanup of sample with immunoaffinity columns. All samples of plain UHT milk were positive for AFM₁ and 38% of these contained levels more than 0.5 μg/kg, the maximum permitted limit prescribed by the Codex Alimentarius Commission and by the mandatory regulations of the country, the FSSAI Regulations, 2011. In 62.5% of flavored UHT milk, AFM₁ was below detectable levels (0.02 μg L⁻¹). However, 12.5% of these samples also contained levels exceeding the maximum permitted limits. AFM₁ was present in 61.6% of the 52 raw milk samples analyzed from the two states of Karnataka and Tamilnadu with a range of 0.1–3.8 μg L⁻¹. 17.3% of these samples also exceeded the regulatory limits of the country.     

Production of a monoclonal antibody against aflatoxin M1 and its application for detection of aflatoxin M1 in fortified milk

Aflatoxin M1 (AFM1) is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs) against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1–1, AFM1–3, AFM1–9, AFM1–11, and AFM1–17) were obtained that showed cross-reactivity with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA) using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC₅₀) and the limit of detection (LOD) values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV) and % recovery of the detection was investigated for both intra- (n = 6) and inter- (n = 12) variation assays. The % CV was found in the range of 3.50–15.8% and 1.32–7.98%, respectively, while the % recovery was in the range of 92–104% and 100–103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1–33.0% and 91–109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R² of 0.992 for the concentration of 0.2–5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples.     

Occurrence of aflatoxin M1 in UHT milk in Turkey.

Aflatoxin M1 (AFM1) appears in milk as a direct result of the ingestion of food contaminated with aflatoxin B1 by cattle. The role of milk in human nutrition is well-known. The purpose of the study was to determine the levels of AFM1 in UHT milk samples in Central Anatolia, Turkey. The occurrence of AFM1 contamination in UHT milk samples was investigated by ELISA (Enzyme Linked Immunosorbent Assay) technique. A total of 129 samples of commercial UHT whole milk were analysed. The mean value was 108.17 ng/L. There was a high incidence rate of AFM1, with 75 (58.1%) milk samples being contaminated. Although 68 (53%) were below the limit, the remaining 61 (47%) were well above the limit permitted by the EU. Four of the samples exceeded the prescribed limit of US regulations. It can be concluded that AFM1 levels in the samples purchased in Central Anatolia Region, appear to be a serious public health problem at the moment. Dairy farmers must be educated by the government authorities on potential health consequences of aflatoxins.     

Fate of aflatoxin M1 in Iranian white cheese processing

Aflatoxin M₁ (AFM₁) is an important mycotoxin frequently found in milk and dairy products. AFM₁ is a major metabolic product of Aflatoxin B₁ and is usually excreted in the milk and urine of dairy cattle that have consumed aflatoxin-contaminated feed.

The aim of this study was to determine the AFM₁ concentration in curd and whey of Iranian white cheese. The cheese milk samples were artificially contaminated with AFM₁ in six levels (0.25, 0.5, 0.75, 1, 1.25, and 1.75 μg L⁻¹). Cheese was produced according to Iranian traditional recipe. AFM₁ distribution between curd, whey and cheese was determined by high performance liquid chromatography (HPLC) using immunoaffinity column clean up and florescence detection. AFM₁ was recovered in whey, curd and cheese in the concentrations of 0.43, 1.47 and 1.57 μg L⁻¹,respectively. The level of Aflatoxin M₁ in curd and cheese obtained 3.12- and 3.65-fold more than that in whey that shows the affinity of Aflatoxin M₁ to the protein fraction of milk.     

Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels

This study describes the effect of dietary levels of aflatoxin B1 (AFB1) and age of the birds upon the residue level in liver and muscles of broiler chicks. In three different experiments broiler chicks of 7, 14 and 28 days of age were kept for 7 days on contaminated rations having 1600, 3200 and 6400 μg/kg AFB1. AFB1 residues were detected earlier in younger birds and those fed high AFB1 dietary levels. The highest residue levels in liver and muscles of young chicks fed 6400 μg/kg AFB1 was 6.97 ± 0.08 and 3.27 ± 0.05 ng/g, respectively. Maximum residue concentration was high in birds of young age and those kept on high AFB1 ration. After withdrawal of AF contaminated rations, residues clearance was slow and AFB1 was detectable in liver and muscles of birds for longer duration in younger birds and those fed high AFB1 dietary levels. AFB1 residues in poultry tissues may buildup to high levels in areas with no regulatory limits on AFB1 levels of poultry feed and may pose a risk to consumers health.     

On the occurrence of aflatoxin M1 in milk and dairy products

Aflatoxins are toxic fungal metabolites found in foods and feeds. When ruminants eat AFB₁-feedstuffs, they metabolise the toxin and excrete AFM₁ in milk. To control AFM₁ in foods it is necessary to reduce AFB₁ contamination of feeds for dairy cattle by preventing fungal growth and AFB₁ formation in agricultural commodities intended for animal use. Corn and corn-based products are one of the most contaminated feedstuffs; therefore risk factor analysis of AFB₁ contamination in corn is necessary to evaluate risk of AFM₁ contamination in milk and milk products. During the corn silage production, the aflatoxins production is mostly influenced by: harvest time; fertilization; irrigation; pest control; silage moisture; and storage practices. Due to the lower moisture at harvest and to the conservation methods, the corn grain is mostly exposed to the contamination by Aspergillus species. Therefore, it is necessary to reduce the probability of this contaminant through choice of: hybrids; seeding time and density; suitable ploughing and fertirrigation; and chemical or biological control. Grains harvested with the lowest possible moisture and conservation moisture close to or less than 14% are necessary to reduce contamination risks, as is maintaining mass to homogeneous moisture. Kernel mechanical damage, grain cleaning practices and conservation temperature are also factors which need to be carefully controlled.     

Aflatoxin occurrence in foodstuff supplied to dairy cattle and aflatoxin M1 in raw milk in the North of Paraná state.

Moulds occur in a great variety of foods, including the concentrated and roughage destined for animal feeding, and can produce mycotoxins under certain conditions. The ingestion of mycotoxin AFB(1) by dairy cattle leads to the biotransformation of that substance, which is eliminated via milk as AFM(1), and also causes damage to human health. The present study aimed to analyze the presence of aflatoxins in foodstuff destined for dairy cattle and in the milk produced by these animals. The contamination of the foodstuff by aflatoxins happened mainly in the feeds and the silages did not present contamination by aflatoxins. Out of the 42 samples of milk analyzed, 10 (24%) were contaminated by AFM(1), and 3 (7%) were above the 0.5 microg/l limit.     

Occurrence of Aflatoxin M1 in Cow,Goat, Buffalo,Camel, and Yak Milk in China in 2016

In this present study, 195 cow milk, 100 goat milk, 50 buffalo milk, 50 camel milk, and 50 yak milk samples were collected in China in May and October 2016. The presence of aflatoxin M1 (AFM1) was determined using enzyme-linked immunosorbent assay method. For all cow milk samples, 128 samples (65.7%) contained AFM1 in concentrations ranging from 0.005 to 0.191 µg/L, and 6 samples (3.1%) from Sichuan province in October were contaminated with AFM1 above 0.05 µg/L (EU limit). For all goat milk samples, 76.0% of samples contained AFM1 in concentrations ranging from 0.005 to 0.135 µg/L, and 9 samples (9.0%) from Shanxi province in October were contaminated with AFM1 above 0.05 µg/L. For all buffalo milk samples, 24 samples (48.0%) contained AFM1 in concentrations ranging from 0.005 to 0.089 µg/L, and 2 samples collected in October were contaminated with AFM1 above 0.05 µg/L. Furthermore, 28.0% of samples contained AFM1 in concentrations ranging from 0.005 to 0.007 µg/L in camel milk samples, and 18.0% of samples contained AFM1 in concentrations ranging from 0.005 to 0.007 µg/L in yak milk samples. Our survey study has expanded the current knowledge of the occurrence of AFM1 in milk from five dairy species in China, in particular the minor dairy species.