Review of aflatoxin M1 survey in dairy products from different countries between 2001 and 2011
Aflatoxin M1 is the 4-hydroxylated metabolite of aflatoxin B1 that can be found in animal and human breast milk and dairy products . Aflatoxin M1 presence in milk is considered as a potential risk for human health because of its carcinogenicity potential and thus a need of regular monitoring in milk and dairy products. Unpredicted climatic and environmental variations as well as poor economic and agriculture practices could easily influence the increase of AFM1 in milk and dairy products. Aflatoxins are particularly known to be mainly produced in food and feed materials by
The aim of this book chapter therefore was to review the incidence of AFM1 in milk, dairy products as well as in human breast milk and in addition it was to look into advances of techniques applied for extraction and detection of AFM1 globally and developing countries in particular during the last decade (2001-2011) and to evaluate extraction and detection methods improvement.
2. Chemistry and metabolism of aflatoxin M1
 reviewed extensively on the biochemistry and metabolism of aflatoxins and AFM1 in particular and it was shown that the metabolism of aflatoxin B1 was more carcinogenic as compared to AFM1. It has also been shown that human do not form as much aflatoxin B1 8, 9-epoxide as rats, but suggested that human do not have glutathione-
3. Stability of aflatoxin M1
Several studies have been done regarding AFs and particularly AFM1 stability in milk and dairy sub products. Yousef et al  extensively reviewed information on the stability of AFM1. Studies have shown that there was no significant changes of AFM1 concentration after heat processing (Pasteurisation or boiling) or Ultra-high temperature processing (UHT) technique [21, 11, 22]. The stability measurements on powder milk showed no significant trends for both short- and long-term stability studies . In addition, studies done on AFM1 concentration changes in cheese showed no significant change of concentration even after 3 months of storage . However, Khoury et al.  investigated the binding ability of AFM1 by Lactic acid bacteria (LAB) such as
4. Aflatoxins and Aflatoxin M1 contamination
Most of mycotoxins poisoning problems occur in developing countries and particularly in sub-Saharan African region where maize and groundnuts are the staple foods . It is estimated that about 250.000 hepatocellular carcinoma related deaths occur annually in Africa and around the world . Acute aflatoxicosis has been reported in countries such as: Taiwan, Uganda, Kenya and Thailand [29, 30]. Ciegler et al. reported that between 1974 and 1975, there was a disease outbreak affecting humans, killing about 106 among the 397 registered cases caused by the consumption of badly moulded corn contaminated with aflatoxin (between 6.5 and 15.6ppm). In July 2004, over 100 people died in Kenya due to acute aflatoxicosis after eating maize contaminated with aflatoxins . Human aflatoxicosis (acute hepatitis) in Kenya is problematic among 20 hospitalized, 20% mortality was associated with the consumption of maize highly contaminated with AF , while during 2004, 125 deaths were recorded from a total of 317 reported cases in an outbreak of acute aflatoxicosis associated with Reye’s syndrome . Although such mortalities are viewed as a direct consequence of mycotoxin poisoning, Sharma et al.  indicated that these mortalities might have resulted from a predisposition to infectious diseases resulting from immunosuppressive effects probably caused by mycotoxins. In Malaysia, exposure to aflatoxins contaminated foodstuff was strongly implicated in the death of 13 children in 1988 . Contamination occurs through exposure to contaminated AFM1 milk, milk products such as cheese, yoghurt because as said earlier, AFM1 is mostly excreted in milk of lactating animals and women exposed to diets previously contaminated with aflatoxin B1 and B2 [26, 27]. Aflatoxin M1 can also be found in the organs, e.g., kidney, liver, and excreta of animals exposed to AFB1 . Aflatoxin B1 is excreted into milk of lactating dairy cows primarily in the form of AFM1 with residues approximately equal to 1-3% of the dietary concentration . It has been shown experimentally to present high hepatotoxic and mutagenic risk .
5. Health implications of AFM1
Aflatoxins are classified as mutagen and carcinogen  and their exposure and exact effects on human and animals are difficult to determine with precisions due to lack of experimental data, in addition due to co-occurrence with other mycotoxins in food and feed. Aflatoxin M1 has been demonstrated to be cytotoxic on human hepatocytes in vitro and its acute toxicity in several species is similar to that of aflatoxin B1 and liver cancer has been related to dietary intake of aflatoxins [11, 38]. Aflatoxin M1 exhibits a high level of genotoxic activity and certainly represents a health risk because of its possible accumulation and linkage to DNA [39, 38]. Moreover, AFB1 contamination at higher levels has also been correlated with reduced birth weight and jaundice in neonates . The capacity of biotransformation of carcinogens in infants is generally slower than that of adults, this result in a longer circulation time of the chemicals .
6. Update on aflatoxin M1 extraction, detection and quantification methods
Several methods of extraction and detection have been used or developed for detection of AFM1 in milk dairy products during the past decade. It is however important that to consider the type of matrix (fresh, stored, pasteurised milk, liquid or powder milk, cheese) as this can affect the final results . In addition, most of commercial kits or rapid tests are designed for specific matrix. This makes the extraction of mycotoxins and AFM1 from different matrices a challenge and costly. The detection of AFM1 in milk or milk products remains a challenge because of the very low concentrations. Therefore there is a need of sensitive methods for extraction and detection. Among screening methods, the enzyme-linked immunosorbent assay (ELISA) has been the most used as a screening method for AFM1 . The method is invariably based on the competition enzyme-linked immunosorbent assay (competition ELISA). The ELISA microtiter plate is coated with a bound antibody against AFM1 and the detecting reagent is a covalent complex of this mycotoxin and an enzyme, usually horseradish peroxidase or alkaline phosphatase. The reagent is mixed with a sample of the mycotoxin extract and the mixture is placed in the well. In the control well (absence of mycotoxin in the sample), the mycotoxin-enzyme conjugate can saturate the bound antibody, and addition of a chromogenic substrate results in the development of colour. In the test well, free mycotoxin molecules in the extract compete with the conjugate on the bound antibody . The higher the concentration of mycotoxin, the less the conjugate can react with the bound antibody, leading to fainter colour development . The competition ELISA approaches that of the LC-MS method . However, ELISA has been noticed to be not fully reliable due to cross-reaction interferences, especially at concentrations lower than 0.05 µg/L . Magliulo et al.  reported a more specific chemiluminescent assay reaching 0.001µg/L. Another new screening assay using a headspace sensor array obtains results comparable to those of ELISA . While Goryacheva et al.  have developed Immunoaffinity pre-concentration combined with on-column visual detection for rapid screening of AFM1 in milk. This method showed a 2% of false negative results. Kanungo et al.  also developed an ultra-sensitive sandwich for the detection of AFM1 in milk. The assay involved the immobilization of rat monoclonal antibody of AFM1 in 384 microtiter plate to capture AFM1 antigen. The miniaturised assay (10µL) enabled ultra-trace analysis of AFM1 in milk with much improved lower limit of detection at 0.005pg/mL . Sensitive magnetic nanoparticles (MNPs) based ELISA has been also developed and coupled with micro plate ELISA for analysis in milk. The hybrid-assay, by coupling the 1 antibodies (Ab) immobilized MNPs column with microwell plate assay enabled simultaneous measurement of low (0.5 pg/mL) and high AFM1 contamination (200 pg/mL). The MNPs-ELISA advantages were that it had a small column size, high capture efficiency and lower cost over other reported materials .
In addition, commercial portable devices such as Ridascreen ELISA kits (R-Biopharm Germany); ROSA aflatoxin M1 SL, Charm (Charm Sciences Inc. 2009); Lateral Flow Immunoassay (LFIA) or Gold-Colloid Based Immunoassay  have since been developed to speed up the detection of AFM1. Typical lateral flow immunochromatography (LFIA) strip is composed of a loading pad where a sample of the extract is applied; a zone containing coloured particles (e.g., latex, gold) coated with a mouse monoclonal anti-mycotoxin antibody; a zone of nitrocellulose membrane that allows the migration of the particles together with the mycotoxin sample; a test line that contains immobilized mycotoxin; a positive control line that contains a secondary anti-mouse antibody, and an absorbent pad . In principle the LFIA is that AFM1 extract is applied and migrates along the strip and once the conjugate zone is reached, the mycotoxin binds the anti mycotoxin–particle complex. Free and mycotoxin-containing particles now migrate to the test line. The immobilized mycotoxin captures only the free particles that form a visible coloured line, whereas mycotoxin-containing particles continue to migrate .
The presence of a mycotoxin in the sample at higher concentration than the cut-off point of the strip (saturation of the particles with mycotoxin) will fail to bind to the test line, and vice versa. Thus, the intensity of the colour in the test line is inversely proportional to the concentration of the mycotoxin. Upon reaching the positive control line, both free and mycotoxin-containing particles can bind the anti-mouse antibody, thus forming a strongly coloured line regardless of the presence or absence of mycotoxin. The sensitivity of LTF is very high, and is comparable to those of sophisticated methodologies such as LC-MS-MS and surface plasmon resonance (SPR) (see below). The use of fluorescent reagents can bring the LOD to 50–200 ppt, as has been shown with other toxins . The highest sensitivity in the detection of a mycotoxin by LTF was 5 ppb of AFB2 in pig feed, using a commercial immunaffinity column for the purification and concentration of the extract .
It is important to mention that the use different of devices available on the market depend on the objectives intended to be achieved by the analysis. It may be screening, confirmatory or both. The most reported methods conventionally used for AFM1 extraction and detection are liquid-liquid extraction, silica gel, SPE cartridges (C18) cartridges  or immunoaffinity column (IAC), . The inconvenience of this method is excessive use of chlorinated solvent in liquid-liquid extraction . recently multifunctional clean-up columns (MFC) have been successfully applied to the clean-up of aflatoxins B1, B2, G1, and G2 when analysed by LC-FLD. MFC entrap matrix materials of cereal extracts but let the analytes pass through. This is simple, quick, and more stable than IAC  will probably be used for AFM1 in the future as there have been no reports on this. Reports have shown that the detection and quantification of AFM1 can be done by separation using thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC) coupled with fluorescence detector [6, 55-57). The inconvenience of the TLC method is that it is challenging in the determination of mycotoxin concentrations with exactitude. In order to enhance AFM1 fluorescence, pre or post derivatization with trifluoroacetic acid with detection limits (LOD) (0.01- 0.3 µg/L) or post column derivatization with pyridinium hydrobromide perbromide and lowered the detection limit to 0.001 µg/L on HPLC . The inconvenience of these methods is that they are time consuming as samples are run singularly to ensure the validity of results. In addition, the derivatizing agents (tirfluoroacetic and pyridinium hydrobromide) are corrosive and therefore dangerous for the technician and have corrosive effect on the HPLC column which reduces its longevity.
There are reports on the use of mass spectrophotometer (MS) for AFM1 detection and quantification [51, 52]. The advantage of this is the low sensitivity. Plattner et al.  and Kokkonen  also reported on the use of tandem MS (MS/ MS) for AFM1 detection in dairy products. However, by their sample preparation procedures these two methods did not eliminate matrix effects and must use matrix standards for calibration. Blank matrixes free of the analytes are not easy to obtain, and their storage time is short. As a result, there would be considerable practical benefit from further work on sample preparation and methods for separation prior to MS. Patel et al.  reported on AFM1 detection 1 in milk and milk powder using an LC-MS/MS method and this contrasted the clean-up efficiencies of IAC and MFC. Dutton et al.  reported the use of IAC clean-up and HPLC coupled with a fluorometer detector coupled with a Coring cell (Kobra cell) for AFM1 detection. The advantage of this method is the avoidance the derivatization procedure which is labour cost and health risky for the technician and corrosive for the column. It is important also to mention that data presented in this study cannot be compared because of the use of different extraction and analytical methods. The use of different analytical methods (TLC, HPLC, LC-MS, ELISA) also affected reported concentrations of aflatoxin M1 in milk and therefore may affect intake estimates; therefore there is a need for trained people to give correct estimations of intake or AFM1 in milk for better evaluation of exposure and anticipation of health problems.
In addition to herein mentioned techniques, Stark  in his review on other simple techniques such as the identification of mycotoxins based on molecular techniques for AFs and other mycotoxins detections from fungal strains are being developed and experimented . Bhatnagar et al.  mentioned also that the biosynthesis and regulations of AFs involved at least 25 genes. Shapira et al. ; Chen et al.  used Primers pertaining to sequences of afl-2, aflD, aflM and aflP, (apa-2, nor-2, ver-2, omt-2, respectively) to detect and identify aflatoxigenic strains of
Rojas-Dura´n et al.  also noted that the detection of AFs by studying the fluorescence of fungal colonies remained a challenge because non-aflatoxigenic strains of
The use of Polymerase Chain Reaction (PCR) for the detection of mycotoxigenic fungi has been applied recently to prevent contamination of crops by mycotoxigenic fungi . Bhatnagaret al.  demonstrated also that at least 25 genes were involved in the biosynthesis of AFs and its regulation. To detect and identify aflatoxigenic strains of
Other methods used for mycotoxin detection and not yet applied for AFM1 and will be the future in AFM1 detection and quantification include Molecular Imprinting (MI) in which a pseudo receptor is formed after polymerization of the surrounding molecules and washout of the mycotoxin. The selective binding of other molecules of the same mycotoxin to the imprinted polymer relies on immuno-competition reactions which are enhanced as compared to binding to a non-imprinted polymer . It is important to mention that frequent production of several mycotoxins by a single fungus or in the same sample , and the contamination of crops with several toxigenic fungi are some of the reasons for the development of the arrays . The Arrays biosensors have been developed to perform the simultaneous assays of more than one mycotoxin . Electronic Nose sensors have been developed to identify volatile biomarker compounds such nitrosamines emitted from grains. These volatile biomarkers are dependent also on the types of mycotoxin in the contaminated grains . Solid State SsDNA Odor Sensors with ssDNA 22-mers of ssDNA containing a fluorescent chromophore and dried onto a solid support can interact non-covalently with volatiles resulting in fluorescence increase have been developed. Volatile precursors of AFB2, ochratoxin A and DON  could be used to identify toxigenic fungi by such odour sensors.
Although all the above mentioned methods of evaluating AFs contamination and identification in corn or food are applicable, there is an absolute necessity that the identification and determination of AFs levels with portable kits, and by confirmation of their identity in the laboratory be carried out.
7. Review of a decade survey of AFM1 in dairy milk
Due to AFs and AFM1 health hazard potential, it is therefore important to monitor and regulate the level of AFM1 in milk and milk products through control of animal feed quality for consumer’s safety purposes . Aflatoxin M1 is a metabolite of AFB1 that can occur in milk and milk products from animals consuming feed contaminated with AFB1 . Data from this review (Table 1) revealed that not much is done in regard to AFM1 surveys in many developing countries and particularly in African countries. The list of countries presented in Table 1 is not exhaustive however does represent the trend in AFM1 survey in developing countries. Numerous epidemiological studies have shown in some areas a correlation between high aflatoxin exposure and high incidence of hepatho carcinoma in several countries in Africa . Data in Table 1 show that only few African countries apart from South Africa , Egypt  and Morocco  and Nigeria  were involved in the survey for AFM1. However the following Middle East, Asian and Latin American countries are implementing controls of the toxin in both dairy and human milk Iran  and Kuwait  and Pakistan [80-83] India, Argentina  and Brazil [18, 85-87]
Data obtained clearly show that ELISA immunoassay technique has been the most used analytical method in the past decade to measure AFM1. This has also been confirmed in a survey done in Italy on AFM1 in dairy products by  The use of ELISA immunoassay can be justified by the fact that it is affordable, simple and easy, to use and does not need expensive equipment such as liquid chromatography. However, several researchers mostly from developed countries combined Imunoaffinity column and Liquid Chromatography for specificity and confirmation of results [18, 48, and 57].
Survey in developing countries showed high levels of AFM1 contamination (> 0.05 µg/L) in many milk samples analysed as compared to data obtained from developed countries such as France , Italy , Portugal and Spain  in which the results were mostly low or with very few samples above the European Commission regulation of 0.05 µg/L. The explanation to this might be that, developed countries have imposed strict control on the quality of feed provided to animals which reduces chances for aflatoxin contaminations. Such regulations are not yet implemented or being implemented in developing countries. In addition, climatic conditions mostly tropical; hot and humid conditions favourable for aflatoxin producing fungi contamination in cereals  recorded in most developing countries could be the pivotal reason for AFM1 contamination in milk. In addition, differences noticed between data shown in Table 1 could be also explained by the use of different extraction, and analysis (ELISA, TLC, HPLC, and LC-MS) techniques as said before which could affect significantly the level of mycotoxin detection.
( % )
The investigation of AFM1 contamination in milk according to climatic season variations (winter and summer) showed clear effect of seasons on the occurrence and concentration of AFM1 in milk. Most of the studies showed that higher levels of AFM1 are obtained in winter milks as compared to summer samples [22; 57, 92]. The reason being that during the summer, animals are fed on pasture, grass, weeds and green fodder while during winter, due to shortage or unavailability of fresh green feed, animals are more on concentrate feeding based on corn, wheat, and cotton seeds which could harbour mycotoxins than the fresh fodder . Moreover, green fodder and hay preserved as silage under inadequate storage conditions which is ideal for toxigenic fungi such as
A comparison study was also carried out between milk samples obtained from rural subsistence and commercial farm in South Africa . Analysed samples revealed the incidence of contamination with AFM1 of 86.0% in rural subsistence farms samples while in samples from commercial farms, the incidence of contamination was of 100%. The lower frequency of contamination of AFM1 in rural milk samples, as compared to those from commercial farms was explained by the fact that in subsistence farming animals were not fed with commercial feed on daily basis but mainly with leftovers from harvest season found on poor pastures with very little or no supplementation  while in commercial farms animals were fed on concentrates and silage which were also contaminated with toxigenic fungi and aflatoxins. This explaining the presence of AFM1 in all milk samples analysed [4, 57]. Hence, the low intake of AFs in rural subsistence farm animals as compared to commercial farm animals exposed on feed.
8. Update on Aflatoxin M1 in human breast milk
Human breast milk has nutritional and immunological beneficial components for children and may contain trace amounts of a wide range of contaminants including AFM1 following maternal dietary exposures . Maternal consumption of aflatoxin contaminated food such as grain products, milk and milk products, legumes, meat, fish, corn oil, cottonseed oil, dried fruits, and nuts during breastfeeding can result in the accumulation of aflatoxins and their metabolites in breast milk . Approximately 95% of AFB1 metabolite is excreted in milk as AFM1in breast milk . Studies on possible effects of infant exposure to AFs and AFM1 have revealed growth retardation in human children  and foetal growth retardation in some animals exposed to aflatoxins prenatally . In addition, AFs have been detected in blood of pregnant women . A review done by Weidenborner  (Table 2) revealed the presence of AFM1 in breast milk of lactating women in several countries with contamination levels varying according to countries and type of food exposed to. In addition to this, other studies confirmed the presence of AFM1 in milk of lactating women (Table 2) at varying concentrations with some samples being above accepted limit in the USA and Europe (25ppb) or Australia and Switzerland (10 ppb). Methods used in studies to determine AFM1 contamination in human breast milk have been mainly ELISA and HPLC as observed in studies done with dairy milk. Similar to the situation of dairy milk, few developing countries and in particular African countries have done investigations regarding AFM1 contamination in human breast milk. Such investigations would be indicative of human exposure to AFs through food.
( % )
0.401 - 0.525
9. Worldwide Aflatoxins control and regulations
Tolerable limits worldwide including South Africa for AFs vary between 10-20ppb . Studies done have demonstrated that a concentration of 20 ppb of AFB1 in the total mixed ration dry matter of lactating dairy cattle will result in AFM1 levels in milk below the FDA set up limit of 0.5ppb . European Union and several other countries including South Africa have however, presently set up acceptable level of AFM1 in milk and milk products at 0.05 ppb. It is estimated that safe feed and food is the one in which detection levels may result in mycotoxin concentrations in milk being above limited level because absolute concentrations of mycotoxins in these feed and food are difficult to determine and concentrations may not be uniform throughout a lot of feed and concentrations can change over time . The application of these regulations requires the use of sophisticated, expensive scientific equipment, and highly trained professional personnel commonly not found in developing countries where contamination of cereals by AFs producing fungi are mainly found and observed. To reduce crops contamination by these AFs producing fungi, the trend is the experimentation of molecular enabling detection of their mycotoxins producing potential . In addition, recently researchers are proposing the use of strains of lactic acid bacteria to effectively remove AFB1 and AFM1 from contaminated liquid media and milk (102, 103]. In vitro binding ability of AFM1 by
Binders or sequestering agents added to feed have been another approach to reduce toxicity of mycotoxins by reducing reactivity of bound mycotoxins and reducing their intestinal absorption. Substances used as mycotoxin binders include indigestible adsorbent materials such as silicates, activated carbons, complex carbohydrates and others. Whitlow  extensively reviewed information on mycotoxins binders. The addition of mycotoxin binders to contaminated diets has been considered the most promising dietary approach to reduce effects of mycotoxins . The use of binders offers an approach to salvaging feeds with low levels of mycotoxins and to protecting animals from the background levels of mycotoxins that, although low in concentration, routinely occur and may cause chronic disease problems and losses in performance . Researchers have also noticed that there is no binder product that meets all the desirable characteristics, however, the potential currently exists for practical judicial use of mycotoxins binders for reducing mycotoxin exposure to animals . Aflatoxin and some other mycotoxins which have chemical structure similar to aflatoxin such as sterigmatocystin, can bind to silicate. Silicates vary and bind to mycotoxins depending on the structure of mycotoxins. Chemically modification of silicates can increase binding to mycotoxins such as deoxynivalenol and zearalenone. Activated carbon (charcoal) has produced variable binding results most probably because of differences in physical properties of the test product . Aflatoxin binding by activated charcoal has been variable, but mostly positive . Complex indigestible carbohydrate polymers derived from  yeast cell walls are shown effective in binding aflatoxin and restoring performance to animals consuming multiple mycotoxins (generally
There is an excellent potential for binders to help manage the mycotoxin problem. Various materials can bind mycotoxins in feed and thus reduce toxic exposure to consuming animals. No product currently meets all the characteristics for a desirable binder. Mycotoxin control measures may require many approaches . Animals may also be supplemented with antioxidants and other beneficial substances. In addition, the enforcement of legislation against mycotoxins and public enlightenment on hazards and control of mycotoxins should be emphasized by government in developing countries to ensure the control of mycotoxins contamination in food and milk.
Aflatoxin M1 remains a mycotoxin that is yet to be investigated in most of developing countries including Africa. As long as conditions favourable for aflatoxin contamination in food and animal feed are present, AFM1 in milk and milk products will continue to be an issue that needs constant monitoring because of the serious effects it could cause on human health, particularly children. Developing countries compared to developed nations need to develop and implement regulations and control systems that would regulate AFM1 in milk and its products thus ensuring food quality and safety. The coming decade will definitively focus on development and application of new, quick and low cost technology for aflatoxin detection. This would be key to developing strategies that would improve prevention, promotion awareness with regards to fungi and aflatoxins contamination.