Aflatoxin has been provided in this review, its

Aflatoxin M1 in milkand dairy products: A Review Aroosa ZamanUniversityof HaripurDepartmentof MicrobiologyAbstract: Milkconsists of all macro- and micronutrients that are necessary for developmentand growth as well as for the maintenance of good human health.

Milk is highlynutritious food. However, natural food contaminants can also be present in themilk that can be the cause of disease. In milk and dairy products the presenceof aflatoxin M1 (AFM1) has been reported throughout the world since twenty tothirty year ago.

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Presence of aflatoxin M1in milk and dairy products is seriousproblem throughout the world for the last ten to twenty years. These mycotoxinpresences in milk and milk products cause serious health issues especially tochildren and infants, who are more susceptible than adults. Information aboutoccurrence of AFM1 in milk and its products in several parts of the world hasbeen provided in this review, its stability during processing and somereduction strategies. In this review the toxicity, occurrence of AFM1 in milkand dairy products (preferably for the last 5 years), regulations, strategiesfor its reduction,  seasonal variation,detoxification of aflatoxin M1 by using probiotics and lactic acid bacteria,latest developments in detection methodologies and future challenges aredescribed.Keywords: Aflatoxin M 1, Milk and dairyproducts, Occurrence, Probiotics, Lactic acid bacteria Detoxification andseasonal variation1. Introduction:Milk consists of all macro- andmicronutrients that are necessary for development and growth as well as for themaintenance of good human health.

Human health is often reflected the foodproducing environmental conditions. The quality and quantity of food isdirectly linked with the implementation on food regulations. Therefore,developing countries are facing a big issue related to food safety and securitybecause they depend on locally produced foods. So, the presence of aflatoxin M1(AFM1) in milk and dairy products is one of the major issues, especially for underdeveloping countries.2.

Aflatoxins:Aflatoxins (AF) are produced assecondary metabolites of fungal strains Aspergillusflavus, Aspergillus parasiticusand Aspergillus nomius. During growth,processing, storage and transportation they grow on a variety of food and feedproducts. Due to their properties like mutagenic, carcinogenic, teratogenic,hepatotoxic and immunosuppressive they cause harmful effects on animal andhuman health. The primary classes of mycotoxins are AF, zearalenone,trichothecenes, fumonisins, ochratoxin A and the ergot alkaloids.Among 20 different types ofaflatoxins, only the aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2)are associated with acute liver damage and cirrhosis. Different factors play animportant role in fungal growth and the synthesis of AFs such as prolongeddrought, substrate composition, high temperatures, and storage time and conditions.Aflatoxin B1 is ranked I carcinogen by the International Agency for Research onCancer because it is the most toxic, carcinogenic, teratogenic and mutagenicclass of AFs. After the discovery of AFs, researchers suggested that the residuesof AFs might occur in milk and dairy products taken from animal that ingestedcontaminated feedstuff.

3. Aflatoxin M1 andits toxicity:Aflatoxin M1 (AFM1) is a4-hydroxy derivative of Aflatoxin B1 (AFB1) and hepatocarcinogen, formed inliver and excreted into the milk in the mammary glands of human and lactating animalsthat ingested AFB1 through contaminated diet. Many factors influence theconversion of AFB1 into AFM1 such as type of diet, health, breed and also rateof digestion. Approximately 0.3-6.2% ingested AFB1 is transformed to AFM1 inmilk which is dependent on contamination level.

AFM1 is about ten times lesstoxigenic than AFB1. AFM1 is preferentially linked to the milk casein fractionand this could result in dairy products contaminated at higher AFM1concentration than the original milk.Aflatoxin B1 is considered as themost toxic subtype, due to its both toxicity and occurrence.  However their metabolite aflatoxins M1 grabmore attention. The conversion of AFB1 to the AFM1 is considered as a detoxificationprocess because the carcinogenicity and of AFM1 is only 10% of that for AFB1 invivo while, in vitro metabolic activation, AFM1 only has 10% of themutagenicity of AFB1. However, in ducklings and rats the acute toxicities ofthe two toxins are very similar.

Acute aflatoxicosis symptoms that are normallyobserved in mammals include lethargy, ataxia, lack of appetite, rough and/orpale coat hair, and fatty and enlarged liver. While, the symptoms of chronicaflatoxicosis include jaundice, decreased milk production and feedingeffeciency. AFs may lower resistance against diseases and interruptvaccine-induced immunity. In 1979, Guthrie has reported that in a farmsituation when the lactating dairy cattle were fed a diet containing 120 mg/kgof AFs, their reproductive efficiency decreased, and when these cattle were fedwith non AFs contaminated feed, their milk production increase. Milk productiondecreased in cows consuming AFs produced by culture, however if pure AFs wasingested their production was not markedly affected.4. Occurrence:4.

1South Asia:Several studies have beenconducted in South Asia for the presence of AFM1 in milk. Most of the reportsare from Iran and Pakistan, and some of the studies presented very high meanconcentrations of AFM1 in milk. Fallah, Rahnama, Jafari, and Saei-Dehkordi(2011) conducted a study to determine the AFM1 contamination in milk and milkproducts and found the mean levels of AFM1 in milk (0.323 mg/L), cheese (0.085 mg/kg),yogurt (0.016 mg/L), kashk (0.044 mg/kg), and doogh (0.005 mg/L).

Similarly,the incidence of AFM1 in milk (0.252 mg/L) was reported by Sadia et al. (2012)from Pakistan. Moreover, they discovered that AFM1 in sweets produced from milkwas present at an average mean concentration of 0.

48 mg/kg. In India reported amean AFM1 level that ranged from 0.1 to 3.8 mg/L in milk.

Additionally, meanaflatoxin M1 levels in milk (0.212 mg/L), yogurt (0.147 mg/L), cheese (0.189mg/kg), and butter (0.

156 mg/kg) were reported in Pakistan. The levels of AFM1were comparatively high in milk and other dairy products and would be a serioushealth hazard for consumers.4.2East Asia:There have been reports of AFM1contamination in fresh milk from Indonesia, Japan, Thailand, and the Republicof Korea as well as milk and dairy products from China. AFM1 levels in milk(0.04-0.16 mg/L range), powdered milk (0.

16-0.32 mg/ L) and milk products(0.32-0.5 mg/L) were reported from China. Similarly, the levels of AFM1 range fromthe LOD to 0.114 mg/L in raw milk, as reported by Ruangwises and from Thailand.

In China, 0.01-0.42 mg/L in milk was reported.

Moreover, the incidence of AFM1in milk from Thailand is considerably high relative to other countries.4.3Middle East, Africa, Latin America: Aflatoxin M1 in milk and dairy products hasalso been documented in Syria, Egypt, Lebanon, Sudan, Morocco, Serbia and Brazil.

The highest concentration (2.07 mg/L) and incidence (42/44) of milk samplescontaminated with AFM1 were in Sudan. A total of 42 samples were above therecommended limit of 0.05 mg/L. In Morocco, found that 89% of the milk sampleswere contaminated with AFM1 at a mean level of 0.0186 mg/L, and 3 samples werefound above the EU recommended limit. Similarly, a high mean level (0.062 mg/L)of AFM1 in milk was reported by Oliveira and in Brazil, and the level rangedfrom 0.

011 to 0.161 mg/L. Mean levels of less than 0.018 to greater than 0.

250mg/L were reported in milk from Egypt. These data show a high incidence of AFM1in milk and dairy products, especially from African countries such as Sudan.The lack of awareness and constraints in analytical facilities are major causesof the high incidence of this toxin.4.4Europe: The occurrence of AFM1 inEuropean milk and dairy products has been reported in Turkey, France, Italy,Spain, and Croatia and from Greece.

Mean AFM1 level of 0.284 mg/kg in whitebrined cheese with the concentration ranging from 0.052 to 0.860 mg/kg. Inanother, analyzed 100 milk and 132 cheese samples and reported that 67 and 83%of these milk and cheese samples, respectively, were contaminated with AFM1.

The levels of AFM1 in milk and cheese ranged from 0.010 to 0.630 mg/L and from0.05 to 0.690 mg/kg, respectively. The range of AFM1 levels from Turkey,followed by Croatia, is considerably higher compared to other countries.

Generally, the levels and incidence of AFM1 in milk and dairy products fromEurope is less than the South Asian countries, which may be the result ofstrict regulations on these mycotoxins in feed and milk products and from theadoption of good storage practices. Thus, the occurrence of AFM1 in milk anddairy products could be minimized by applying strict regulations and usingstate of the art analytical techniques. Over time, major developments are oftenreflected in changes in food analysis.5. Stability of AFM1 in milk anddairy products:At high temperature, AFM1 is very stable. Manystudies have discovered the stability/distribution of AFM1 from milk to milkproducts.

In 2006, Oruc et al., investigated that AFM1 was stable in kasharcheese for over in 60 days and for over 90 days in traditional white picklecheese. Their results indicated that the toxins was stable during cheesestorage and ripening. In another study, the researcher showed that theAFM1  was also stable in yogurtartificially contaminated with concentration of 0.100 and 0.050 mg/L duringstorage at 4 C for 4 weeks and at pH values of 4.

0 and 4.6.In 2001, Bakirci hasfound 13% higher level of AFM1 in yogurt product as compared to bulk-tank milkproduct, but statistically the differenceof AFM1 was not significant.Cattaneoet al., (2013) found the stability of AFM1-deproteinized whey and contaminatedwhey subjected to different technological treatments.

During production ofricotta cheese the majority of AFM1, 94% on average, was removed in discardedwhey, so only remained 6% in the curd. After this the use of infiltration andultrafiltration removed more than 90% of the toxins in the remaining wheyduring production of ricotta cheese production. The spray-drying was alsoefficient in decreasing AFM1 contamination in whey, where toxin retention wasapproximately 60%, while AFM1 retention was approximately 39% in deproteinizedwhey.

In 2009, FAO and WHO investigated that milk and liquid milk product wascommercially sterilize by Ultrahigh treatment. During heat treatmen somestudies such as Purchase (1967) and Kabak (2012) have showed reduction of upto37% in AFM1 while in 1996, Galvano et al., investigated that AFM1 was stable inheat. In other studies researchers had showed 12-50% decrease of AFM1 contentin milk sample during heat treatment. However, in general Aflatoxins are heatstable. 6.

Reduction of AFM1 in milk anddairy products:Many studies had reported the reduce level of AFM1in milk and dairy products. Carraro et al., in 2014 removed or attenuated AFM1contamination in bovine milk by using clay. In 2014, Elsanhoty et al.,reducedthe AFM1 level in yogurt by using different strains of lactic acid bacteria. In2013, Serrano-Nino et al., showed reduction in the AFM1 level in milk by usingfive strains of probiotic bacteria in an invitro digestive model.

 Fordetoxification of food containing AFM1 contamination, results shows that somestrains of bifidobacteria and LAB strains are used. There are many studiesshowing the detoxification of AFM1 are important to completely kill the lethaltoxins. Therefore most countries have implemented regulation to reduce thehealth risk related with these toxins.7. Regulations on aflatoxin M1 in milkand dairy products:Themaximum limit for AFM1 in milk and milk products in international regulationsrange from 0-1.0 mg/kg are shown (Table 1). In human food and milk the action level for AF concentrations of 20 and0.5 mg/kg was establish by United States Food and Drug Administration (Chase,Brown, Bergstrom, & Murphy, 2013).

In food the regulatory limits for AFsvary from 0 to 50 mg/ kg (FAO, 2009). According to US regulation, the level of AFM1should not exceed 0.5mg/kg. Similarly, in Switzerland and Austria, for infantfood the maximum limit is 10g/mL. Throughout the world studies showed thepresence of AFM1 in milk and dairy product.  8.

Latest detection methods:Frommilk and dairy products the extraction of AFM1 involves aqueous mixture ofpolar organic solvent such as methanol, acetone or acetonitrile. To minimizethe consumption of chlorinated solvents is an effort due to extraction withchloroform has been replaced or reduced with more friendly environmentalsolvents (Shephard, 2008). To obtain the reliable application procedure, thepresence of interfering compound that contaminated the primary sample extract,which must be removed (Krska, Welzig, Berthiller, Molinelli, & Mizaikoff,2005).

Column chromatography, liquid liquid extraction, solid phase extraction(SPE), one step multifunctional and immunoaffinity columns (IACs)  are currently used common purificationmethod. Sample purification is achieved in 10e 30s. Researchers showed that toanalyze AFM1 in milk and dairy product, the liquid chromatography (LC) with ELISA and florescence detection(FLD) were used in table 6. For detection of AFM1 in milk and milk productsother different methods are fluorometry (Hussain & Anwar, 2008), gel-basedimmunoassays (Goryacheva, Karagusheva, Peteghem, Sibanda, & Saeger, 2009),TLC (Atanda, Oguntubo, Adejumo, Ikeorah, & Akpan, 2007; Fallah, 2010),ultra performance liquid chromatography-tandem mass spectrometry (UHPLCeMS/MS)(Huang et al.

, 2014) and lateral flow immunoassays (Anfossi et al., 2013). Thecommon effective method for analysis of AFM1 is HPLC with florescencedetection. Previous method for the purity assessment, identification andseparation of organic compounds. For AF analysis the separation technique waswidely used.

Normal or reverse-phase HPLC was used for separation of toxinsdepending on their polarity. 9.Future challenges for milk and dairy industry:In several region of world, milk due to itsbeneficial effect to health has positive image among consumers. As the globaltemperature continues to rise, dairy livestock breeding has become increasinglydifficult. In atmosphere the higher concentration of carbon dioxide, volatileweather patterns as well as rising temperature make finding a better system ofanimal husbandry necessary. Global level of production of milk is estimated in2000 will need to double by 2050. 10.

Conclusions:Milkand dairy products could be a highly risk to human as well as animal health dueto the presence of aflatoxin M1. In milk high contamination in feed may resultin a significant AFM1 level when animals fed highly contaminated foodstuffs.Meeting the demands for higher milk yields and striving for increased milkproduction may create such situations. The present study shows that in milk anddairy product AFM1 appears to be a natural contaminant.

The highest level ofAFM1 was found in samples from South Asian countries, followed by Africancountries. This literature shows the importance of monitoring level of  continuous aflatoxin in animal feed and thenecessary implementation of strict regulatory levels for mycotoxins in thesecountries. Due to regulatory measures for these toxins, presence of AFM1 in milk and milk products was lowfrom Europe. The currently common method for AFM1 analysis is HPLC in milk anddairy products.1.