Recent Advances in the Detection of Aflatoxin M1 in Milk and Dairy Products

Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies

Immobilization of Bacillus megaterium spores on Eppendorf tubes through physical adsorption has been used in the detection of aflatoxin M1 (AFM1) in milk within real time of 45 ± 5 min using visual observation of changes in a chromogenic substrate. The appearance of a sky-blue colour indicates the absence of AFM1 in milk, whereas no colour change indicates the presence of AFM1 in milk at a 0.5 ppb Codex maximum residue limit. The working performance of the immobilized spores was shown to persist for up to 6 months. Further, spores immobilized on 96-well black microtitre plates by physical adsorption and by entrapment on sensor disk showed a reduction in detection sensitivity to 0.25 ppb within a time period of 20 ± 5 min by measuring fluorescence using a microbiological plate reader through the addition of milk and fluorogenic substrate. A high fluorescence ratio indicated more substrate hydrolysis due to spore-germination-mediated release of marker enzymes of spores in the absence of AFM1 in milk; however, low fluorescence ratios indicated the presence of AFM1 at 0.25 ppb. Immobilized spores on 96-well microtitre plates and sensor disks have shown better reproducibility after storage at 4 °C for 6 months. Chromogenic assay showed 1.38% false-negative and 2.77% false-positive results while fluorogenic assay showed 4.16% false-positive and 2.77% false-negative results when analysed for AFM1 using 72 milk samples containing raw, pasteurized, and dried milk. Immobilization of spores makes these chromogenic and fluorogenic assays portable, selective, cost-effective for real-time detection of AFM1 in milk at the dairy farm, reception dock, and manufacturing units of the dairy industry.

Aflatoxin M1 in milk and dairy products, occurrence and recent challenges: A review

Assessment of aflatoxin M1 in milk and milk products from Punjab, Pakistan

As milk provides both micro- and macronutrients, it is an important component in the diet. However, the presence of aflatoxin B1 (AFB1) in the feed of dairy cattle results in contamination of milk and dairy products with aflatoxin M1 (AFM1), a toxic metabolite of the carcinogenic mycotoxin. With the aim to determine AFM1 concentrations in milk and milk products consumed in Bangladesh, in total, 145 samples were collected in four divisional regions (Sylhet, Dhaka, Chittagong, and Rajshahi). The samples comprised these categories: raw milk (n = 105), pasteurized milk (n = 15), ultra-high temperature (UHT)-treated milk (n = 15), fermented milk products such as yogurt (n = 5), and milk powder (n = 5). AFM1 levels in these samples were determined through competitive enzyme-linked immunosorbent assay (ELISA). Overall, AFM1 was present in 78.6% of milk and milk products in the range of 5.0 to 198.7 ng/L. AFM1 was detected in 71.4% of raw milk (mean 41.1, range 5.0–198.7 ng/L), and in all pasteurized milk (mean 106, range 17.2–187.7 ng/L) and UHT milk (mean 73, range 12.2–146.9 ng/L) samples. Lower AFM1 levels were found in yogurt (mean 16.9, range 8.3–41.1 ng/L) and milk powder samples (mean 6.6, range 5.9–7.0 ng/L). About one-third of the raw, pasteurized, and UHT milk samples exceeded the EU regulatory limit (50 ng/L) for AFM1 in milk, while AFM1 levels in yogurt and milk powder samples were well below this limit. Regarding regions, lower AFM1 contamination was observed in Chittagong (mean 6.6, max 10.6 ng/L), compared to Sylhet (mean 53.7, max 198.7 ng/L), Dhaka (mean 37.8, max 97.2 ng/L), and Rajshahi (mean 34.8, max 131.4 ng/L). Yet, no significant difference was observed in AFM1 levels between summer and winter season. In conclusion, the observed frequency and levels of aflatoxin contamination raise concern and must encourage further monitoring of AFM1 in milk and milk products in Bangladesh.

A survey of aflatoxin M1 in milk and sweets of Punjab, Pakistan

Rapid and automated analysis of aflatoxin M1 in milk and dairy products by online solid phase extraction coupled to ultra-high-pressure-liquid-chromatography tandem mass spectrometry

This study examined the presence of Aflatoxin M1 (AFM1) in raw milk, cheese, and cheese halva samples collected from the Çanakkale province of Turkey. Raw milk, Ezine cheese, and cheese halva samples (n 120 for each group, total 360) were collected from bazaars, local manufacturers, and supermarkets in the Çanakkale province of Turkey during 2014 and 2015. ELISA was used to detect AFM1 in these products. AFM1 mean values were 5.14-78.69, 19.43-158.30, and 50.25-213.50 ng/kg in raw milk, Ezine cheese, and cheese halva samples, respectively. The levels of AFM1 in four raw milk samples (3.3%) were above the legal limits, whereas AFM1 levels in Ezine cheese and cheese halva samples were within the legal limits according to the requirements of the Turkish Food Codex and the European Commission. Turkey has a wealth of cultures and local delicacies. Over previous decades, gastronomic tourism in Turkey has become popular owing to an increase in visitor demand, and traditional foods are being exported abroad. Our research investigated the presence of AFM1 in these types of food products. Çanakkale is one of the best representative cities in the Marmara Region in Turkey as a source of these unique products.

The objective of this network meta-analysis was to determine the efficacy of different mycotoxin binders (MTB) to reduce aflatoxin M1 (AFM1) in milk. A literature search was conducted to identify in vivo research papers from different databases. Inclusion criteria were in vivo, dairy cows, description of the MTB used, doses of MTB, aflatoxin inclusion in the diet, and concentration of AFM1 in milk. Twenty-eight papers with 131 data points were selected. Binders used in the studies were hydrated sodium calcium aluminosilicate (HSCAS), yeast cell wall (YCW), bentonite, and mixes of several MTB (MX). The response variables were AFM1 concentration, AFM1 reduction in milk, total AFM1 excreted in milk, and transfer of aflatoxin from feed to AFM1 in milk. Data were analyzed with CINeMA and GLIMMIX procedures with the WEIGHT statement of SAS (SAS Inst. Inc.). The AFM1 concentration in milk decreased for bentonite (0.3 µg/L ± 0.05; mean ± SE) and HSCAS (0.4 µg/L ± 0.12), and tended to decrease for MX (0.6 µg/L ± 0.13) but was similar for YCW (0.6 µg/L ± 0.12), compared with control (0.7 µg/L ± 0.12). The percentage reduction of AFM1 in milk was similar for all MTB and different from control with a range of reduction from 25% for YCW to 40% for bentonite. The excretion of AFM1 in milk was lower in YCW (5.3 µg/L ± 2.37), HSCAS (13.8 µg/L ± 3.31), and MX (17.1 µg/L ± 5.64), and not affected by bentonite (16.8 µg/L ± 3.33) compared with control (22.1 µg/L ± 5.33). The transfer of aflatoxin B1 from feed into AFM1 in milk was lowest in bentonite (0.6% ± 0.12), MX (1.04% ± 0.27), and HSCAS (1.04% ± 0.21), and not affected in YCW (1.4% ± 0.10), compared with control (1.7% ± 0.35). The meta-analysis results indicate that all MTB reduced the AFM1 transfer into milk, where bentonite had the highest capacity and YCW the lowest.

Aflatoxin M1 in milk and dairy products: The state of the evidence for child growth impairment

Excretion of Aflatoxin M1 in Milk of Dairy Ewes Treated with Different Doses of Aflatoxin B1

A quantitative fluorescence-labeled immunosorbent assay and qualitative on-site column tests were developed for the determination of aflatoxin M1 in milk products. The use of liposomes loaded with quantum dots as a label significantly increased the assay sensitivity by encapsulating multiple quantum dots in a single liposome and, therefore, amplifying the analytical signal. Two different techniques were compared to obtain aflatoxin-protein conjugates, used for further coupling with the liposomes. The influence of nonspecific interactions of the liposome-labeled conjugates obtained with the surface of microtiter plates and column cartridges was evaluated and discussed. The limit of detection for fluorescence-labeled immunosorbent assay was 0.014 μg kg(-1). For qualitative on-site tests, the cutoff was set at 0.05μg kg(-1), taking into account the EU maximum level for aflatoxin M1 in raw milk, heat-treated milk, and milk for the manufacture of milk-based products. The direct addition of labeled conjugate to the milk samples resulted in an additional decrease of analysis time. An intralaboratory validation was performed with sterilized milk and cream samples artificially spiked with aflatoxin M1 at concentrations less than, equal to and greater than the cutoff level. It is shown that milk products can be analyzed without any sample preparation, just diluted with the buffer. The rates for false-positive and false-negative results were below 5% (2.6% and 3.3%, respectively).

Aflatoxins (AFs) are toxic and carcinogenic metabolites produced by a variety of fungi. Aflatoxin M1 (AFM1) is the major carcinogenic type frequently found in milk and dairy products, thus posing a significant impact on human health. The current study was undertaken to examine milk and some dairy products for contamination with AFM1 in local markets, Sharkia Governorate, Egypt, as well as the effect of manufacture. A total of 75 samples (15, each) of raw milk, pasteurized milk, yoghurt, processed cheese and Domiati cheese were randomly collected. AFM1 was detected in 27 (36%) out of the examined samples in which the level of AFM1 exceeded the limits (0 ng/L, kg) allowed by Egyptian regulation but only 6 (8%) samples exceeded the limits (50 ng/L, kg) allowed by European Commission regulation. Levels of AFM1 contamination in the examined milk and dairy products with mean values of 35.68 ± 10.90, 45.83 ± 7.80, 7.57 ± 1.92, 24.53 ± 3.91 and 42 ± 4.93 ng/L, kg in raw milk, pasteurized milk, yoghurt, processed cheese and Domiati cheese, respectively, were detected. The level of AFM1 decreased after yoghurt manufactur, while, cheese manufacture showed concentration of AFM1 in curd than those in cheese milk. During refrigeration storage of yoghurt, the mean AFM1 toxin decreased after one, two, three, seven days, respectively, then nearly similar level from seven days to fourteen days of storage. In conclusion, widespread presence of AFM1 in raw milk and some dairy products were considered to be possible hazards for public health especially children therefore, continuous monitoring of AFM1 level in commonly marketed raw milk and dairy products in Sharkia markets should be regularly done. Manufacture and storage had little effect on AFM1 content in milk and dairy products, therefore, new or modern technologies for detoxification of milk should be further studied

Background: Mycotoxins produced by yeast and fungi have toxic effects on human and animal health. Aflatoxin B1 (AFB1) is the most toxic hepatocarcinogen to mammals. Aflatoxin M1 (AFM1), which has been found in milk and dairy products, is the hydroxylated metabolite of AFB1. Aflatoxin M1 is formed by the cytochrome P450 enzyme in the liver. Ochratoxin A (OTA) is synthesized by Aspergillus and Penicillium species. Ochratoxin A is known to cause teratogenic, immunotoxic, nephrotoxic and carcinogenic effects. Due to the potential harmful effects on human and animal health, OTA has also been receiving increased attention globally; however, there is limited information on the presence of OTA in milk and dairy products. The aim of this study was to determine how mycotoxins impact the hygienic quality of raw and heat-processed milk.Materials, Methods & Results: In this study, a total of 105 milk samples were analyzed (35 raw, 35 pasteurized and 35 UHT) to identify AFM1 and OTA in raw, pasteurized and ultra-high temperature processing (UHT) milk. The levels of AFM1 were detected by using the enzyme-linked immunosorbent assay (ELISA). The milk samples were centrifugedin order to remove the fat content from the milk. After centrifugation, the upper cream layer was withdrawn with a pipette. The non-fat liquid portion was placed in wells at 100 μL for analysis. The concentration of AFM1 in the milk samples was analyzed by AFM1 test kit.The milk samples with AFM1 levels greater than 50 ng/L were confirmed by using High-Performance Liquid Chromatography (HPLC). An Ochratoxin A Serum / Milk ELISA test kit was used for the analyses of OTA. The analyses were made according to the manufacturer’s instructions, and samples were analyzed in duplicate. The absorbance value of milk samples was obtained from the ELISA plate reader at 450 nm. The mean value of AFM1 was found to be 19.54 ng/L in the milk samples. According to the European Commission (EC), the maximum limit for AFM1 in milk is 50 ng/L. In our study, eight (7.61%) of the 105 samples exceeded this limit. The mean value of OTA was found to be 119 ng/L in the milk samples. The relationship between milk type and levels of AFM1 was found to be significant at (P < 0.01). The mean value of AFM1 in pasteurized milk was found statistically significant and lower than raw milk (P < 0.05). The difference between levels of OTA and milk type was not statistically significant at (P > 0.05).Discussion: Milk is a great protein source especially for children in the age of growth. Yeasts such as Fusarium, Aspergillus and Penicillium produce mycotoxins that cause food, feed contamination. Owing to carcinogenic, mutagenic and teratogenic effects of AFM1, presence of AFM1 in milk samples may adversely affect human health. The presence of AFM1 in different contamination levels can be observed in milk and milk products. Factors such as ration type, climate conditions, feed storage conditions, feeding regime and health status of dairy animals may be effective in the occurrence of these contamination. It is necessary to establish legal limits by conducting effective research on the existence of OTA in animal-derived products. The existence of mycotoxins in milk and dairy products can be reduced by preventing the contamination of feed materials with yeast and molds used in the feeding of dairy cows. Milk is one of the most important protein source for the human, effective hygienic controls should be applied to prevent microbiological and chemical hazards. Our data suggest that heat-treated milk may also be dangerous to human health, mycotoxins contamination should be controled with monitoring programs routinely in milk and feed materials for food safety. Determination of Aflatoxin M1 and Ochratoxin A in Raw, Pasteurized and UHT Milk in Turkey

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M1 (AFM1) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM1 sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM1 in milk. However, if there was no color change, presence of AFM1 at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM1 detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM1 in milk.