Aflatoxin has been provided in this review, its

Aflatoxin M1 in milk
and dairy products: A Review


Aroosa Zaman

of Haripur

of Microbiology


consists of all macro- and micronutrients that are necessary for development
and growth as well as for the maintenance of good human health. Milk is highly
nutritious food. However, natural food contaminants can also be present in the
milk that can be the cause of disease. In milk and dairy products the presence
of aflatoxin M1 (AFM1) has been reported throughout the world since twenty to
thirty year ago. Presence of aflatoxin M1in milk and dairy products is serious
problem throughout the world for the last ten to twenty years. These mycotoxin
presences in milk and milk products cause serious health issues especially to
children and infants, who are more susceptible than adults. Information about
occurrence of AFM1 in milk and its products in several parts of the world has
been provided in this review, its stability during processing and some
reduction strategies. In this review the toxicity, occurrence of AFM1 in milk
and dairy products (preferably for the last 5 years), regulations, strategies
for its reduction,  seasonal variation,
detoxification of aflatoxin M1 by using probiotics and lactic acid bacteria,
latest developments in detection methodologies and future challenges are

Keywords: Aflatoxin M 1, Milk and dairy
products, Occurrence, Probiotics, Lactic acid bacteria Detoxification and
seasonal variation

1. Introduction:

Milk consists of all macro- and
micronutrients that are necessary for development and growth as well as for the
maintenance of good human health. Human health is often reflected the food
producing environmental conditions. The quality and quantity of food is
directly linked with the implementation on food regulations. Therefore,
developing countries are facing a big issue related to food safety and security
because 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 under
developing countries.

2. Aflatoxins:

Aflatoxins (AF) are produced as
secondary metabolites of fungal strains Aspergillus
flavus, Aspergillus parasiticus
and Aspergillus nomius. During growth,
processing, storage and transportation they grow on a variety of food and feed
products. Due to their properties like mutagenic, carcinogenic, teratogenic,
hepatotoxic and immunosuppressive they cause harmful effects on animal and
human health. The primary classes of mycotoxins are AF, zearalenone,
trichothecenes, fumonisins, ochratoxin A and the ergot alkaloids.

Among 20 different types of
aflatoxins, only the aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2)
are associated with acute liver damage and cirrhosis. Different factors play an
important role in fungal growth and the synthesis of AFs such as prolonged
drought, substrate composition, high temperatures, and storage time and conditions.
Aflatoxin B1 is ranked I carcinogen by the International Agency for Research on
Cancer because it is the most toxic, carcinogenic, teratogenic and mutagenic
class of AFs. After the discovery of AFs, researchers suggested that the residues
of AFs might occur in milk and dairy products taken from animal that ingested
contaminated feedstuff.

3. Aflatoxin M1 and
its toxicity:

Aflatoxin M1 (AFM1) is a
4-hydroxy derivative of Aflatoxin B1 (AFB1) and hepatocarcinogen, formed in
liver and excreted into the milk in the mammary glands of human and lactating animals
that ingested AFB1 through contaminated diet. Many factors influence the
conversion of AFB1 into AFM1 such as type of diet, health, breed and also rate
of digestion. Approximately 0.3-6.2% ingested AFB1 is transformed to AFM1 in
milk which is dependent on contamination level. AFM1 is about ten times less
toxigenic than AFB1. AFM1 is preferentially linked to the milk casein fraction
and this could result in dairy products contaminated at higher AFM1
concentration than the original milk.

Aflatoxin B1 is considered as the
most toxic subtype, due to its both toxicity and occurrence.  However their metabolite aflatoxins M1 grab
more attention. The conversion of AFB1 to the AFM1 is considered as a detoxification
process because the carcinogenicity and of AFM1 is only 10% of that for AFB1 in
vivo while, in vitro metabolic activation, AFM1 only has 10% of the
mutagenicity of AFB1. However, in ducklings and rats the acute toxicities of
the two toxins are very similar. Acute aflatoxicosis symptoms that are normally
observed in mammals include lethargy, ataxia, lack of appetite, rough and/or
pale coat hair, and fatty and enlarged liver. While, the symptoms of chronic
aflatoxicosis include jaundice, decreased milk production and feeding
effeciency. AFs may lower resistance against diseases and interrupt
vaccine-induced immunity. In 1979, Guthrie has reported that in a farm
situation when the lactating dairy cattle were fed a diet containing 120 mg/kg
of AFs, their reproductive efficiency decreased, and when these cattle were fed
with non AFs contaminated feed, their milk production increase. Milk production
decreased in cows consuming AFs produced by culture, however if pure AFs was
ingested their production was not markedly affected.

4. Occurrence:

South Asia:

Several studies have been
conducted in South Asia for the presence of AFM1 in milk. Most of the reports
are from Iran and Pakistan, and some of the studies presented very high mean
concentrations of AFM1 in milk. Fallah, Rahnama, Jafari, and Saei-Dehkordi
(2011) conducted a study to determine the AFM1 contamination in milk and milk
products 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 milk
was present at an average mean concentration of 0.48 mg/kg. In India reported a
mean AFM1 level that ranged from 0.1 to 3.8 mg/L in milk. Additionally, mean
aflatoxin M1 levels in milk (0.212 mg/L), yogurt (0.147 mg/L), cheese (0.189
mg/kg), and butter (0.156 mg/kg) were reported in Pakistan. The levels of AFM1
were comparatively high in milk and other dairy products and would be a serious
health hazard for consumers.

East Asia:

There have been reports of AFM1
contamination in fresh milk from Indonesia, Japan, Thailand, and the Republic
of 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 from
the 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 AFM1
in milk from Thailand is considerably high relative to other countries.

Middle East, Africa, Latin America:

 Aflatoxin M1 in milk and dairy products has
also been documented in Syria, Egypt, Lebanon, Sudan, Morocco, Serbia and Brazil.
The highest concentration (2.07 mg/L) and incidence (42/44) of milk samples
contaminated with AFM1 were in Sudan. A total of 42 samples were above the
recommended limit of 0.05 mg/L. In Morocco, found that 89% of the milk samples
were contaminated with AFM1 at a mean level of 0.0186 mg/L, and 3 samples were
found 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 ranged
from 0.011 to 0.161 mg/L. Mean levels of less than 0.018 to greater than 0.250
mg/L were reported in milk from Egypt. These data show a high incidence of AFM1
in milk and dairy products, especially from African countries such as Sudan.
The lack of awareness and constraints in analytical facilities are major causes
of the high incidence of this toxin.


The occurrence of AFM1 in
European 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 white
brined cheese with the concentration ranging from 0.052 to 0.860 mg/kg. In
another, 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 from
0.05 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 from
Europe is less than the South Asian countries, which may be the result of
strict regulations on these mycotoxins in feed and milk products and from the
adoption of good storage practices. Thus, the occurrence of AFM1 in milk and
dairy products could be minimized by applying strict regulations and using
state of the art analytical techniques. Over time, major developments are often
reflected in changes in food analysis.

5. Stability of AFM1 in milk and
dairy products:

At high temperature, AFM1 is very stable. Many
studies have discovered the stability/distribution of AFM1 from milk to milk
products. In 2006, Oruc et al., investigated that AFM1 was stable in kashar
cheese for over in 60 days and for over 90 days in traditional white pickle
cheese. Their results indicated that the toxins was stable during cheese
storage and ripening. In another study, the researcher showed that the
AFM1  was also stable in yogurt
artificially contaminated with concentration of 0.100 and 0.050 mg/L during
storage at 4 C for 4 weeks and at pH values of 4.0 and 4.6.In 2001, Bakirci has
found 13% higher level of AFM1 in yogurt product as compared to bulk-tank milk
product, but statistically the differenceof AFM1 was not significant.Cattaneo
et al., (2013) found the stability of AFM1-deproteinized whey and contaminated
whey subjected to different technological treatments. During production of
ricotta cheese the majority of AFM1, 94% on average, was removed in discarded
whey, so only remained 6% in the curd. After this the use of infiltration and
ultrafiltration removed more than 90% of the toxins in the remaining whey
during production of ricotta cheese production. The spray-drying was also
efficient in decreasing AFM1 contamination in whey, where toxin retention was
approximately 60%, while AFM1 retention was approximately 39% in deproteinized
whey. In 2009, FAO and WHO investigated that milk and liquid milk product was
commercially sterilize by Ultrahigh treatment. During heat treatmen some
studies such as Purchase (1967) and Kabak (2012) have showed reduction of upto
37% in AFM1 while in 1996, Galvano et al., investigated that AFM1 was stable in
heat. In other studies researchers had showed 12-50% decrease of AFM1 content
in milk sample during heat treatment. However, in general Aflatoxins are heat

6. Reduction of AFM1 in milk and
dairy products:

Many studies had reported the reduce level of AFM1
in milk and dairy products. Carraro et al., in 2014 removed or attenuated AFM1
contamination in bovine milk by using clay. In 2014, Elsanhoty et al.,reduced
the AFM1 level in yogurt by using different strains of lactic acid bacteria. In
2013, Serrano-Nino et al., showed reduction in the AFM1 level in milk by using
five strains of probiotic bacteria in an invitro digestive model.

detoxification of food containing AFM1 contamination, results shows that some
strains of bifidobacteria and LAB strains are used. There are many studies
showing the detoxification of AFM1 are important to completely kill the lethal
toxins. Therefore most countries have implemented regulation to reduce the
health risk related with these toxins.

7. Regulations on aflatoxin M1 in milk
and dairy products:

maximum limit for AFM1 in milk and milk products in international regulations
range from 0-1.0 mg/kg are shown (Table 1). 
In human food and milk the action level for AF concentrations of 20 and
0.5 mg/kg was establish by United States Food and Drug Administration (Chase,
Brown, Bergstrom, & Murphy, 2013). In food the regulatory limits for AFs
vary from 0 to 50 mg/ kg (FAO, 2009). According to US regulation, the level of AFM1
should not exceed 0.5mg/kg. Similarly, in Switzerland and Austria, for infant
food the maximum limit is 10g/mL. Throughout the world studies showed the
presence of AFM1 in milk and dairy product.  

8. Latest detection methods:

milk and dairy products the extraction of AFM1 involves aqueous mixture of
polar organic solvent such as methanol, acetone or acetonitrile. To minimize
the consumption of chlorinated solvents is an effort due to extraction with
chloroform has been replaced or reduced with more friendly environmental
solvents (Shephard, 2008). To obtain the reliable application procedure, the
presence 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 purification
method. Sample purification is achieved in 10e 30s. Researchers showed that to
analyze 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 products
other different methods are fluorometry (Hussain & Anwar, 2008), gel-based
immunoassays (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). The
common effective method for analysis of AFM1 is HPLC with florescence
detection. Previous method for the purity assessment, identification and
separation of organic compounds. For AF analysis the separation technique was
widely used. Normal or reverse-phase HPLC was used for separation of toxins
depending on their polarity.

Future challenges for milk and dairy 

In several region of world, milk due to its
beneficial effect to health has positive image among consumers. As the global
temperature continues to rise, dairy livestock breeding has become increasingly
difficult. In atmosphere the higher concentration of carbon dioxide, volatile
weather patterns as well as rising temperature make finding a better system of
animal husbandry necessary. Global level of production of milk is estimated in
2000 will need to double by 2050.


10. Conclusions:

and dairy products could be a highly risk to human as well as animal health due
to the presence of aflatoxin M1. In milk high contamination in feed may result
in a significant AFM1 level when animals fed highly contaminated foodstuffs.
Meeting the demands for higher milk yields and striving for increased milk
production may create such situations. The present study shows that in milk and
dairy product AFM1 appears to be a natural contaminant. The highest level of
AFM1 was found in samples from South Asian countries, followed by African
countries. This literature shows the importance of monitoring level of  continuous aflatoxin in animal feed and the
necessary implementation of strict regulatory levels for mycotoxins in these
countries. Due to regulatory measures 
for these toxins, presence of AFM1 in milk and milk products was low
from Europe. The currently common method for AFM1 analysis is HPLC in milk and
dairy products.



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