Aflatoxin M1 in Raw Milk,Pasteurized Milk and Cottage Cheese Collected along Value Chain Actors from Three Regions of Ethiopia

Milk is a highly nutritious and perfect natural food for humans. However, when lactating animals feed on Aflatoxin B₁ (AFB₁)-containing feed, the hydroxyl metabolite aflatoxin M₁ (AFM₁) contaminates the milk and dairy products. The objective of the current study was to assess the level of AFM₁ in raw milk, normally pasteurized milk and Ethiopian cottage cheese collected from value chain actors (producers, collectors, processors and retailers). Cross-sectional study and simple random techniques were used to collect primary samples. A total of 160 composite samples was collected; raw milk (n = 64), pasteurized milk (n = 64) and cheese (n = 32) was analyzed. Quantitative analysis of AFM₁ was conducted using enzyme-linked immunosorbent assay (ELISA). The results indicate that AFM₁ was detected in all milk products. Results along value chains show that the concentration of AFM₁ in raw milk from collectors was significantly higher than from producers, and in pasteurized milk from processors and retailers (p < 0.05). However, no significant (p > 0.05) difference was observed in cottage cheese value-chain actors in all regions. Comparison of AFM₁ mean values among all dairy products shows that raw milk had a significantly higher concentration of AFM₁ followed by pasteurized milk and cottage cheese. However, there was no significant difference between raw and pasteurized milk (p > 0.05). The mean AFM₁ contamination in milk products ranged from 0.137 to 0.319 µg/L (mean value 0.285 µg/L). The contamination percentages of AFM₁ in raw milk (62.50%), pasteurized milk (67.20%) and cottage cheese (25%) were above the regulatory limit set by the European Union (EU) (0.05 µg/L). According to USA/Ethiopian Standard (US/ES) (0.50 µg/L), 21.87%, 25% and 1% exceeded the regulatory limit for the above products, respectively. The overall prevalence (56.88%) was above the EU regulatory limit and 19.38% over US/ES regulations. Therefore, to provide accurate information about the health risk to consumers, there is a need to conduct risk assessment studies in consumers of milk and dairy products at different age groups.     

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.     

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.     

Aspergillus flavus and Total Aflatoxins Occurrence in Dairy Feed and Aflatoxin M1 in Bovine Milk in Aguascalientes,Mexico

Contamination of food chains by toxigenic fungi and aflatoxins is a global problem that causes damage to human health, as well as to crop and livestock production. The objective is to evaluate Aspergillus flavus and total aflatoxins (AFs) occurrence in totally mixed rations (TMRs) for dairy cows and aflatoxin M₁ (AFM₁) in milk for human consumption. Ninety-nine dairy production units located in Aguascalientes, Mexico, were randomly selected, and samples were collected from TMRs, raw milk, and milk marketed in the city in two consecutive agricultural cycles. AFs were quantified in TMRs and milk by indirect enzyme immunoassay and HPLC; aflatoxigenic and molecular (PCR) capacity of monosporic A. flavus isolates in the feed was characterized. All feed, raw, and pasteurized milk samples showed aflatoxin contamination (26.0 ± 0.4 µg/kg, 32.0 ± 1.0, and 31.3 ± 0.7 ng/L, respectively), and a significant proportion (90.4, 11.3, and 10.3%) exceeded the locally applied maximum permissible limits for feed and milk (20.0 µg/kg and 50 ng/L). Aflatoxin contamination in both TMRs and milk indicated a seasonal influence, with a higher concentration in the autumn–winter cycle when conditions of higher humidity prevail. The results obtained suggest the existence of contamination by aflatoxigenic A. flavus and aflatoxins in the diet formulated for feeding dairy cows and, consequently, in the dairy food chain of this region of the Mexican Highland Plateau.     

Adsorbents Reduce Aflatoxin M1 Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B1 in Diet and Its Exposure Risk for Humans

This study investigated the effect of moderate risk level (8 µg/kg) AFB₁ in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM₁ content of healthy lactating cows and the AFM₁ residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB₁ and adsorbents (CON), CON with 8 μg/kg AFB₁ (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB₁-challenge phase (day 1 to 14) and an AFB₁-withdraw phase (day 15 to 19). Results showed that both AFB₁ and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM₁ concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB₁ into milk AFM₁ (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2–4 years old had the highest exposure risk to AFM₁ in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB₁ in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM₁ residue in milk and its exposure risk for humans.     

Aflatoxin M1 contamination and antibiotic residue in milk in Khorasan province,Iran

The aim of this study was to evaluate aflatoxin M₁ (AFM₁) contamination and antibiotic presence in milk samples in Khorasan province in Iran. During 4 months (March to June 2008), one hundred ninety-six milk samples were collected from seven dairies. The occurrence and concentration range of AFM₁ in the samples were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA) method. Antibiotic presence was determined using Copan test, a broad-spectrum test capable of detecting beta-lactams, tetracyclines, sulfonamides, aminoglycosides and macrolydes. AFM₁ was found in 100% of the examined milk samples by average concentration of 77.92 ng/kg. The concentration of AFM₁ in all of the samples were lower than Iranian national standard and FDA limit (500 ng/L), but 80.6% of the samples had AFM₁ greater than the maximum tolerance limit (50 ng/L) accepted by European Union and Codex Alimentarius Commission. Statistical evaluation showed no significant difference between the mean concentrations of AFM₁ of milk samples taken from different factories (P > 0.05). Copan milk test was positive for 40.8% of the samples.     

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.     

Infants exposure to aflatoxin M1 as a novel foodborne zoonosis

Occurrence of aflatoxin M₁ (AFM₁) in infant formula milk powder (IFMP) and maternal breast milk (MBM) was investigated as a risk factor affects the health of newborns in Egypt. A total of 125 IFMP and 125 MBM samples were collected and examined for the presence of AFM₁ using competitive ELISA test. The results indicated that the relative risk (RR) of exposure to AFM₁ via consumption of MBM was higher than IFMP (RR; 1.6, 95% CI; 1.28–2.03, p = 0.0001). The mean concentrations of AFM₁ were significantly differed (p < 0.0001) between MBM (74.413 ± 7.070 ng/l) and IFMP (9.796 ± 1.036 ng/l). High frequency distributions were detected within the range of 5–25 ng/l and >50–100 ng/l in IFMP and MBM, respectively. The average daily exposure of newborns to AFM₁ via consumption of MBM and IFMP was 52.684 and 8.170 ng, respectively, with a significant difference at p < 0.0001. Consumption of raw milk by lactating mothers exhibited a significant correlation (p < 0.0001) with the presence of AFM₁ in their milk. In conclusion, this work established a pioneering concept that AFM₁ may be considered as an etiological factor for a novel foodborne zoonosis identified as Aflatoxicosis M₁.     

Decrease in Aflatoxin M1 Concentration in Milk during Cholesterol Removal by Application of β-Cyclodextrin

Approximately one-third of humankind is chronically exposed to the carcinogenic aflatoxin M₁ contained in milk. As β-cyclodextrin is frequently used in the food industry, its effect on aflatoxin M₁ concentration was investigated during cholesterol removal from milk due to the similarity among the physicochemical properties of aflatoxin M₁ and cholesterol. Moreover, the elimination of cholesterol using β-cyclodextrin has been successfully applied in many studies without any substantial effect on the quality of the treated milk. Therefore, milk samples were spiked with aflatoxin M₁ within the range from 0.20 to 2.00 µg/kg, and cholesterol removal was carried out by 2.0% (w/w) β-cyclodextrin addition, as this concentration is enough for the sufficient removal of cholesterol. It was found that the mean cholesterol concentration decreased by 92.3%, while the aflatoxin M₁ concentration decreased to 0.53 ± 0.04 µg/kg, i.e., by 39.1% after treatment (n = 2). This mitigation procedure itself is easy and inexpensive and thus is fully applicable with a high potential for complete decontamination of aflatoxin M₁ milk. This method will therefore considerably improve the food safety issues associated with aflatoxin M₁ presence in milk and dairy products.     

Aflatoxin M1 levels in UHT milk and kashar cheese consumed in Turkey

In the present study, 100 UHT milk and 132 kashar cheese samples were analyzed for aflatoxin M1 (AFM1). They were obtained from retail outlets in five big cities (Istanbul, Izmir, Konya, Tekirdag, Edirne). The occurrence and concentration range of AFM1 in the samples were investigated by competitive enzyme-linked immunosorbent assay (ELISA) method. Sixty-seven percent of the UHT milk samples and 82.6% of the kashar cheese samples contained AFM1. The positive incidence of AFM1 in the UHT milk samples and the kashar cheese samples ranged from 10 to 630 ng/kg and from 50 to 690 ng/kg, respectively. AFM1 levels in 31 (31%) UHT milk and 36 (27.3%) kashar cheese samples exceeded the maximum tolerable limit of the EC and the TFC. AFM1 levels in the samples show that there is a presence of high aflatoxin level that constitutes a human health risk in Turkey. Therefore milk and dairy products have to be controlled continuously for presence of AFM1 contamination by the Turkish public health authorities.     

Evaluation of Mycotoxins in Infant Breast Milk and Infant Food,Reviewing the Literature Data

In this review, an analysis focusing on mycotoxin determination in infant breast milk and infant food has been summarised for the last fifteen years of research focused on the intended population group of 1–9 months. The objective was to know the level of exposure of the child population to an estimated daily intake (EDI) of mycotoxins from the consumption of habitual foods. The EDI was compared with the tolerable daily intake (TDI) established by EFSA to estimate risk. In breast milk, the high prevalence and levels were for samples from Africa (Egypt and Tanzania) with aflatoxin M1 (1.9 μg/L and 10%), and Asia (Iran) with ochratoxin-A (7.3 μg/L and 100%). In infant formulas, high incidences and values were for samples with aflatoxin M1 from Burkina Faso (167 samples, 84%, 87 μg/kg). In cereal products, the highest incidence was for DON from the United States (96 samples), and the highest value was an Italian sample (0.83 μg/kg of enniatin B). In fruit products, patulin was the most detected in Italian (78) and Spanish (24) samples. The highest risk was observed in breast milk during the first month of age, the highest EDI for aflatoxin M1 was reported for Egypt (344–595 ng/kg bw/day) and ochratoxin-A for Iran (97–167ng/kg bw/day), representing a public health problem.     

AFM1 in Milk: Physical,Biological, and Prophylactic Methods to Mitigate Contamination

Aflatoxins (AFs) are toxic, carcinogenic, immunosuppressive secondary metabolites produced by some Aspergillus species which colonize crops, including many dietary staple foods and feed components. AFB₁ is the prevalent and most toxic among AFs. In the liver, it is biotransformed into AFM₁, which is then excreted into the milk of lactating mammals, including dairy animals. AFM₁ has been shown to be cause of both acute and chronic toxicoses. The presence of AFM₁ in milk and dairy products represents a worldwide concern since even small amounts of this metabolite may be of importance as long-term exposure is concerned. Contamination of milk may be mitigated either directly, decreasing the AFM₁ content in contaminated milk, or indirectly, decreasing AFB₁ contamination in the feed of dairy animals. Current strategies for AFM₁ mitigation include good agricultural practices in pre-harvest and post-harvest management of feed crops (including storage) and physical or chemical decontamination of feed and milk. However, no single strategy offers a complete solution to the issue.     

Field Monitoring of Aflatoxins in Feed and Milk of High-Yielding Dairy Cows under Two Feeding Systems

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) that can be excreted in milk of cows after consuming contaminated feed. The aim of this study consisted of a field monitoring to assess the contamination levels of AFB1 in 60 feed samples from two feeding systems for high-yielding dairy cows and of AFM1 in the corresponding raw milk samples. The aflatoxins were analyzed by in-house validated methods based on high-performance liquid chromatography (HPLC) with fluorescence detection. AFB1 was detected in 55% of feed samples (mean 0.61 μg/kg, with 2 samples exceeding the European Union (EU) maximum level set at 5 μg/kg), with greater incidence and concentration in compound feed than in unifeed rations (p < 0.05). AFM1 was detected in 38.3% milk samples (mean 12.6 ng/kg, with 5 samples exceeding the EU maximum level set at 50 ng/kg), with a higher occurrence in milk of cows fed compound feed, as well as in spring milk compared to that produced in winter. The overall transfer ratio of aflatoxins from feed to milk was 3.22%, being higher in cows fed with compound feed and in spring milkings. In a selection of positive matched samples (n = 22), the ratio AFM1/AFB1 exceeded the European Food Safety Authority (EFSA) estimated 6% threshold for high-yielding dairy cows.     

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.     

Mycotoxin Co-Occurrence in Milks and Exposure Estimation: A Pilot Study in São Paulo,Brazil

The aim of this study was to conduct a first evaluation on the co-occurrence of aflatoxins (AF) M₁, B₁, B₂, G₁ and G₂; fumonisins (F) B₁ and B₂; deoxynivalenol (DON); de-epoxydeoxinivalenol (DOM-1); ochratoxin A (OTA); zearalenone (ZEN); α-zearalenol (α-ZEL); and β-zearalenol (β-ZEL) in 68 samples of fluid milk consumed in Pirassununga, São Paulo, Brazil. The probable daily intake (PDI) was also calculated for each mycotoxin evaluated. Mycotoxins were determined by liquid chromatography coupled to mass spectrometry. Sixty-two (91.2%) samples contained at least one type of mycotoxin. AFM₁ was found in 6 samples (8.8%), and none of them presented concentrations above the Brazilian maximum permitted level in milk (500 ng/L). Low levels of non-regulated mycotoxins DOM-1, OTA, FB₁, FB₂, α-ZEL and β-ZEL were found in 6 (8.8%), 17 (25%), 10 (14.7%), 3 (4.4%), 39 (57.4%) and 28 (41.2%) samples of milk, respectively. None of the PDIs calculated for the quantified mycotoxins were above recommended values, indicating low exposure through milk consumption in the area studied. However, 21 samples (30.9%) contained 2–4 types of mycotoxins, which warrants concern about the potential adverse effects of mycotoxin mixtures in milks.     

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.     

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.     

Aflatoxin M1 in Cow’s Milk: Method Validation for Milk Sampled in Northern Italy

Aflatoxins (AFs) are mycotoxins produced by some species of Aspergillus. In dairy cows, ingested AFB1 is metabolized into carcinogenic AFM1 which is eliminated through milk, thus posing a risk for consumer health. Here we describe the set, validation, and application of screening (ELISA) and confirmatory (HPLC) tests carried out on milk samples collected through official control of mycotoxin levels in northern Italy over a three-year period (2012–2014). The limit of detection (LOD) was set at 5 ppt (ng/kg) and 2 ppt for ELISA and HPLC, respectively, and the limit of quantification (LOQ) was 10 ppt for confirmatory HPLC. A total of 1668 milk samples were analyzed: ELISA identified 36 (2.2%) positive milk samples that were subsequently confirmed by HPLC. The level of AFM1 in the positive samples ranged between 18 ± 2 and 208 ± 27 ppt. Of the total samples, only eight (0.5%) were found non-compliant with the EU regulatory limit (50 ppt; range 74 ± 10 to 208 ± 27 ppt). Use of ELISA and HPLC tests in series allows for high-volume analysis of samples, thus saving time and money while guaranteeing high analytical precision and accuracy.