AFM1 Concentrations Research Articles

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

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

Fluorimetric Immunoassay for Multianalysis of Aflatoxins

A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25–50 pg/mL. AFM1 as low as 1 pg/mL was detected by this method with assay volume 40 μL. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.

Effect of experimental feed additives on aflatoxin in milk of dairy cows fed aflatoxin‐contaminated diets

Three studies were conducted to determine the potential of experimental feed additives (EFAs), clays or non-digestible yeast oligosaccharides, to reduce milk aflatoxin (AFM1) concentrations in lactating Holstein cows consuming aflatoxin-contaminated diets. All studies included a pre-treatment period and a 2-week experimental period in a randomized block design. During the pre-treatment period, cows received a total mixed ration (TMR) with no aflatoxin contamination. During both experimental weeks, all cows were fed a TMR containing aflatoxin-contaminated corn. During experimental week 1, cows received no EFA's in the TMR, but EFA's were included in the TMR for the second experimental week. In studies 1 and 2, the experimental period consisted of 2 weeks each lasting 7 days with 12 cows per treatment. Aflatoxin M1 concentrations were analysed by HPLC for milk samples collected on days 5-7 and days 11-14. In various experiments, treatments included control (no EFA), 100 g/cow daily of experimental Lallemand(®) product, 10 g/cow daily of MTB-100(®) -2004, (Alltech, Inc.), 10 g/cow daily of MTB-100(®) -2006, (Alltech, Inc.), 10 g/cow daily of experimental Alltech(®) product (Alltech, Inc.) and 227 g/cow daily of Astra-Ben 20(®) (AB-20(®) ; Prince Agri Products, Inc.). In study 3, the experimental period of 2 weeks each lasting 8 days and milk samples were collected from day 4 to 8 and day 11 to 16. Milk samples from study 3 were analysed for AFM1 concentrations by ELISA. For all experiments, changes in AFM1 concentrations because of the addition of EFA's were calculated. Four of the five EFAs tested in this study had no significant effect on AFM1 concentrations. However, the addition of AB-20(®) resulted in a significant decrease in AFM1 concentrations (60.4%). In summary, the addition of AB-20(®) to the diet of cattle appears to be effective for significantly reducing AFM1 concentrations in the milk of cows fed an aflatoxin-contaminated diet.

A Survey of Aflatoxin M1 Contamination in Bulk Milk Samples from Dairy Bovine, Ovine, and Caprine Herds in Iran

A total of 150 bovine (60), ovine (42), and caprine (48) bulk milk samples were analyzed using a commercially available competitive ELISA kit. Overall, AFM1 was found in 46.7% of the analyzed samples by an average concentration of 40.3±22.2ng/L. The incidence rates of AFM1 contamination in bovine, ovine, and caprine bulk milk samples were 66.7, 31.0, and 35.4%, respectively. The concentration of AFM1 in 37.5% of AFM1-positive bovine milk samples and 5.9% of AFM1-positive caprine milk samples were higher than 50ng/L.

Determinação de aflatoxina B1 em rações e aflatoxina M1 no leite de propriedades do Estado de São Paulo

The occurrence of aflatoxin B 1 (AFB 1 ) in animal feed and aflatoxin M 1 (AFM 1 ) in raw milk was evaluated in dairy farms located in the Northeast region of Sao Paulo state, Brazil, from October 2005 to February 2006. The Aflatoxin analysis was performed using immunoaffinity clean-up with high performance liquid chromatography for quantification. AFB 1 was found in 40% of the animal feeds at the levels of 1.0 to 19.5 µg.kg –1 . The concentration of AFM1 in raw milk (36.7%) ranged from 0.010 to 0.645 µg.L –1 . Only one single sample of raw milk presented values above the tolerance limit adopted in Brazil (0.5 µg.L –1 ) for AFM 1 . In conclusion, the concentrations of aflatoxins in the animal feed and milk samples studied were relatively low although the high frequency of mycotoxins in the both analysed samples indicates the necessity of continuous monitoring in order to prevent mycotoxin contamination of animal feed ingredients for dairy cattle.

Assessment of aflatoxin M1 levels in selected dairy products in north‐western Iran

The purpose of this survey was to evaluate the natural occurrence and content of aflatoxin M1, AFM1, in dairy products marketed in Urmia. During September 2007, 40 samples of pasteurised milk, 40 samples of ultra high temperature‐treated (UHT) milk, 40 samples of creamy cheese and 40 samples of Iranian Feta cheese were collected from different supermarkets in Urmia city. AFM1 contents were determined by the competitive enzyme‐linked imunosorbent assay (ELISA) technique. All milk samples analysed showed a mean of AFM1 concentrations lower than the permissible level of 50 ng/kg in Iran (23.22 and 19.53 ng/kg in pasteurised milk and UHT milk respectively). The mean levels of AFM1 contamination were 43.31 ng/kg in Feta cheeses and 21.96 ng/kg in creamy cheeses. The potential risk of human exposure to aflatoxin M1 via consumption of milk and milk products is well known. Dairy products must therefore be evaluated for aflatoxin and kept free from fungal contamination as much as possible.

Efficacy of Solis, NovasilPlus, and MTB-100 to reduce aflatoxin M1 levels in milk of early to mid lactation dairy cows fed aflatoxin B1

An experiment was conducted to determine the efficacy of 3 adsorbents, Solis (SO; Novus International Inc.), NovasilPlus (NOV; Engelhard Corp.), and MTB-100 (MTB; Alltech), in reducing aflatoxin (AF) M1 concentrations in milk of dairy cows fed an AF-contaminated diet. Twelve early to mid lactation dairy cows averaging 163 d in milk were used in a 4×4 Latin square design with 3 replications. Cows were blocked by parity, body weight, and milk production and were provided ad libitum access to feed and water. Within each replicate, cows were randomly assigned to the 4 dietary treatments for 4 consecutive 7-d periods. Dietary treatments included AF [112μg of AFB1/kg of diet dry matter (DM)]; AF + 0.56% SO; AF + 0.56% NOV; and AF + 0.56% MTB. Milk samples were collected on d 6 and 7 of each of the experimental periods. Feed intake, milk production, milk fat percentage, milk protein percentage, and linear somatic cell scores were not affected by dietary treatments and averaged 22.20kg/d of DM, 33.87kg/d, 3.78%, 2.95%, and 1.60, respectively, across all treatments. Transfer rates of AF from feed to milk averaged 2.65, 1.48, 1.42, and 2.52% for cows fed AF, AF + SO, AF + NOV, and AF + MTB, respectively. Daily AFM1 excretion in milk averaged 66, 37, 35, and 63μg/d for cows fed AF, AF + SO, AF + NOV, and AF + MTB, respectively. The addition of SO and NOV to the AF diet resulted in a significant reduction in milk AFM1 concentrations (SO, 45%; NOV, 48%) and AFM1 excretion (SO, 44%; NOV, 46%). In contrast, MTB was not effective in reducing milk AFM1 concentrations (4%), AFM1 excretion (5%), or AF transfer from feed to milk (2.52%). Results indicated that SO and NOV at 0.56% of the diet were effective in reducing milk AFM1 concentrations in cows consuming a total mixed ration containing 112μg of AFB1/kg of diet DM.

Zearalenone, deoxynivalenol and aflatoxin B1 and their metabolites in pig urine as biomarkers for mycotoxin exposure

Methods to determine zearalenone (ZEA), deoxynivalenol (DON), aflatoxins (AF) and their metabolites in pig urine were developed as biomarkers for pig exposure to the mycotoxins in feed. Urine samples were incubated with β-glucuronidase to cleave conjugates, extracted and cleaned-up with solid phase and immunoaffinity columns, followed by HPLC with UV and fluorescence detection. Good recoveries (83-130%), low variation (2-10%), and low detection limits (0.3-9.9ng/ml) were obtained. The results of controlled AFB1 feeding trials found no difference in urine concentrations of AFB1 or AFM1 from pigs fed three different levels (127, 227, 327 µg/kg) of AFB1 in diets. The excretion of AFB1 and AFM1 in urine was on average 30% of the oral dose and the ratio AFB1 to AFM1 was around 23%. The analysis of 15 Vietnamese pig urine samples indicate a relatively high exposure of ZEA, DON and AF, which were found as toxin or metabolites in 47, 73, and 80% of the urine samples, respectively.

Determination of Aflatoxin M1 Levels in Some Cheese Types Consumed in Erzurum - Turkey

Summary In this study the total 304 cheese samples (85 white cheese, 75 kashar cheese, 62 civil cheese, 82 cream cheese) were examined in terms of AFM1. The AFM1 content and concentrations of the samples were researched by competitive ELISA method. Determinable limit was 50 ng/kg and it was determined that white cheese samples included 82.4% AFM1, kashar cheese samples 80%, civil cheese samples 19.4% and cream cheese samples 84.2%. According to European Commission limit (250 ng/kg), the sample incidence exceeding the acceptable limits were 27.1%, 34.7%, 17.1% in white cheese, kashar cheese and cream cheese samples, respectively. The sample ratio exceeding the limits regulated by Turkish Food Codex (500 ng/kg) was determined in white cheese, kashar cheese and cream cheese samples as 16.5% (14/85), 14.7% (11/75) and 6.1% (5/82) respectively, any sample exceeding these limits was not met in civil cheese samples. As understood from these results, high AFM1 level determined in some cheese types is an important problem threatening the public health in Turkey.

Aflatoxins and cyclopiazonic acid in feed and milk from dairy farms in São Paulo, Brazil

The occurrence of aflatoxins (AF) B1, B2, G1, G2 and cyclopiazonic acid (CPA) in feeds, and AFM1 and CPA in milk was determined in dairy farms located in the northeastern region of São Paulo state, Brazil, between October 2005 and February 2006. AF and CPA determinations were performed by HPLC. AFB1 was found in 42% of feed at levels of 1.0–26.4 µg kg−1 (mean: 7.1 ± 7.2 µg kg−1). The concentrations of AFM1 in raw milk varied between 0.010 and 0.645 µg l−1 (mean: 0.104 ± 0.138 µg l−1). Only one sample was above the tolerance limit adopted in Brazil (0.50 µg l−1) for AFM1 in milk. Regarding CPA in feed, six (12%) samples showed concentrations of 12.5–153.3 µg kg−1 (mean: 57.6 ± 48.7 µg kg−1). CPA was detected in only three milk samples (6%) at levels of 6.4, 8.8 and 9.1 µg l−1. Concentrations of aflatoxins and CPA in feed and milk were relatively low, although the high frequency of both mycotoxins indicates the necessity to continuously monitor dairy farms to prevent contamination of feed ingredients.

Comparison of a competitive ELISA with an HPLC method for the determination of aflatoxin M1 in Turkish White, Kasar and Tulum cheeses

AFM1 is a hydroxylated metabolite produced when ruminants ingest contaminated feed with AFB1 and transferred to dairy products such as cheese, which represents an important risk factor for consumers. Turkish White, Kashar and Tulum cheeses are traditional cheese types in Turkey. This study was planned to compare the performance of a competitive ELISA for the determination of AFM1 in Turkish White, Kasar and Tulum cheeses against a new HPLC method. For this purpose, different AFM1 concentrations (50–400 ng/kg) were added to the cheese samples and the toxin levels were determined by ELISA and HPLC. Also, both methods were performed in 24 real samples obtained from different markets. In conclusion, the results obtained by ELISA in this study were related to those by HPLC for AFM1.

Survey For Detection and Determination of Aflatoxins M1 and B1 in local Milk and Certain Dairy Products by Thin Layer Chromatographic Method.

90 different type of milk samples, 10 Yogurt Samples, 110 different type of cheese samples and 10 ice cream samples were collected randomly from Giza Governorate during the summer of 1998 – 1999, for detection and determination of Aflatoxins M1& B1 by using thin layer chromatographic method. Results revealed that the average range of Aflatoxin M1 in milk samples amounted from 0.144 to 0.378 ng/ml. About 20 % of cows and buffaloes milk samples contained form 0.378 to 0.342 ng/ml of AFM1, whereas about 10% of other milk samples were contaminated with 0.162, 0.288, 0.324, 0.234, 0.144 and 0.162 ng/ml for skim , Pasteurized , sterilized, UHT, powder, and baby milk, in the same order. Concentrations of AFM1 detected in cheese samples, furthermore, varied due to the type and age of cheese being examined. 20% of cheese samples were contaminated with AFM1 being 5.1, 3.2, 2.99, 2.099, and 2.34 ng/gm for fresh Domiati, aged Domiati, Processed and Karish cheese, respectively, whereas, 30% of the other types of cheese contained 5.88, 6.3 and 3.4 ng/gm for Roquefort, fresh Romi, and Cheddar cheese, respectively. The lowest concentration of AFM1, of 0.116 ng/gm was detected, however, in 10% of yogurt samples. Meanwhile, 20% of ice cream samples were found to be contaminated with 2.7 ng/ml, and 10% of Feta cheese samples contained 3.3 ng/gm. It could also be appeared from results that both of cream and spread cheese were found completely free from this Aflatoxin, the lowest content of Aflatoxin detected in all of the above examined samples was 0.116, 0.162, 0.162 and 0.216 (ppb) in yogurt, skim, baby milk and cream, respectively. On the other hand, results also indicated that all milk samples were free from Aflatoxin B1 except one sample of skim milk (out of 10) which gave positive result.

Prevalence of Aflatoxins in Blood and Urine of Egyptian Infants with Protein–Energy Malnutrition

The aim of the present work was to study the presence of aflatoxins in blood and urine of infants with protein-energy malnutrition (PEM). The study was conducted on 60 infants, 30 with kwashiorkor and 30 with marasmus, with 10 age-matched healthy infants studied as a control group. Complete blood count, liver function tests, and determination of the level of aflatoxins (B1, B2, G1, G2, M1, M2, G2a, B3, GM1, P, and aflatoxicol R0) in blood and urine were carried out in all studied infants. Serum aflatoxins were detected in more infants with kwashiorkor (80%) than in those with marasmus (46.7%). The mean serum levels of total aflatoxins, AFB1, AFG1, and AFB2a, were significantly higher in infants with kwashiorkor (p <.001). Aflatoxin B1 (AFB1) was the most commonly detected type. The prevalence of aflatoxin excretion in the urine of infants with kwashiorkor was 80%, a higher value than that in infants with marasmus (46.7%). The mean urinary concentration of total aflatoxins followed the same pattern of distribution (p < .052). There were no significant differences between groups in the mean urinary concentrations of AFB1, AFG1, AFB2a, AFM1, and AFG2a. Aflatoxins were not detected in any of the serum or urine samples of the control group. Aflatoxins are highly prevalent in this study population and show a high degree of correlation with severe PEM.

Development and application of an indirect competitive enzyme-linked immunoassay for aflatoxin m(1) in milk and milk-based confectionery.

High-titer rabbit polyclonal antibodies to aflatoxin M(1) (AFM1) were produced by utilizing AFM1-bovine serum albumin (BSA) conjugate as an immunogen. An indirect competitive enzyme-linked immunosorbent assay was standardized for estimating AFM1 in milk and milk products. To avoid the influence of interfering substances present in the milk samples, it was necessary to prepare AFM1 standards in methanol extracts of certified reference material (CRM) not containing detectable AFM1 (< 0.05 ng/g). The reliability of the procedure was assessed by using CRM with AFM1 concentrations of < 0.5 and 0.76 ng/g. Also, assays of milk samples mixed with AFM1 ranging in concentration between 0.5 and 50 ng/L gave recoveries of > 93%. The relative cross-reactivity with aflatoxins (AF) and ochratoxin A, assessed as the amount of AFM1 necessary to cause 50% inhibition of binding, was 5% for AFB1 and much less for AFB2, AFG1, and AFG2; there was no reaction with ochratoxin A. AFM1 contamination was measured in retail milk and milk products collected from rural and periurban areas in Andhra Pradesh, India. Of 280 milk samples tested, 146 were found to contain < 0.5 ng/mL of AFM1; in 80 samples it varied from 0.6 to 15 ng/mL, in 42 samples from 16 to 30 ng/mL, and in 12 samples from 31 to 48 ng/mL. Most of the milk samples that contained high AFM1 concentrations were obtained from periurban locations. The results revealed a significant exposure of humans to AFM1 levels in India and thus highlight the need for awareness of risk among milk producers and consumers.

Distribution of aflatoxin M1 in cheese obtained from milk artificially contaminated

Small-scale manufacture of cheese using artificially AFM1 contaminated milk as raw material to study the distribution of such toxin both in whey and in cheese, was carried out. Whole milk with undetectable levels of AFM1 was used. The toxin was added in concentration that varied from 1.7 to 2.0 μg/l of milk. After the home-made production of cheese, the concentration of AFM1 was determined both in whey and in cheese, using the enzymatic immunoassay technique. The greatest proportion, 60%, was detected in whey while 40% AFM1 remained in cheese.

Fate of Aflatoxin M1 in Cottage Cheese

Two batches of long-set cottage cheese were prepared from milk naturally contaminated with aflatoxin M1. Cottage cheese was stored for 2 weeks at 7°C. Analyses for pH, moisture content and AFM1 were done on days 0, 3, 7, 10 and 14 of storage. In comparison with the initial curd concentrations (18.3 and 20.5 μ of AFM1/kg of cheese for trials 1 and 2, respectively), AFM1 concentrations in the finished product did not decrease appreciably during storage. Overall average concentrations of AFM1 in trials 1 and 2 from day 0 to day 14 were 15.0 and 20.5 μg of AFM1/kg of cheese, respectively.