**5. Occurrence of Aflatoxin in Milk and Dairy Products**

Most of AFB1 and AFB2 ingested by mammals are eliminated through urine and faeces, however a fraction is biotransformed in the liver and excreted together with milk in the form of AFM1 and AFM2, respectively. AFM1 could be detected in milk 12-24 h after the first AFB1 ingestion, reaching a high level after a few days. The ratio between AFB1 ingested and AFM1 excreted has been estimated to be 1-3% [5].

The system responsible for the biotransformation of AFB1 basically has five mechanisms, represented by reactions of reduction, hydration, epoxidation, hydroxylation and ortho-de‐ methylation. The aflatoxicol is produced by reduction of AFB1 by an NADPH-dependent cytoplasmic enzyme present in the soluble fraction of the liver. The toxicity of aflatoxicol is apparently much smaller than AFB1, but the conversion is reversible and the aflatoxi‐ col can serve as a reservoir toxicity of AFB1 in the intracellular space, it can be converted in this mycotoxin by microsomal dehydrogenase. The aflatoxicol can also be metabolized to AFM1 and AFH1. The hydration process results in a metabolite AFB2a. This com‐ pound has the main action the inhibition of enzymes, in the liver and other tissues, caus‐ ing a reduction in proteic synthesis AFM1 and AFQ1 are results of hydroxylation reaction of AFB1. These compounds have a hydroxyl group, allowing their conjugation with glucur‐ onic acid, sulfate and glutathione, making them very water-soluble substances that can be excreted in the bile, urine and milk. Most of the aflatoxins are excreted between 72 to 96 h after the exposure, with the liver and the kidney retaining the waste for a longer period com‐ pare to other tissues [63].

reported that AFM1 concentration in cheese was about four times higher than the cheese milk. The increase in AFM1 concentration in cheese has been explained by the affinity of

The Commission of the European Communities established a limit for AFM1 of 50 ng kg-1 for milk and a variable limit for cheese, depending on concentration caused by drying proc‐ ess or processing. Milk containing AFM1 concentrations above the action level must be dis‐ carded, causing significant economic loss for the dairy producer. Similar regulations exist in

In this Regulation the Commission stated that ''even if AFM1 is regarded as a less danger‐ ous genotoxic carcinogenic substance than AFB1, it is necessary to prevent the presence in milk, and consequently in milk products, intended for human consumption and for young children in particular''. The Commission has also set a limit for AFB1 of 5 µg kg-1 for supple‐ mentary feedstuffs for lactating dairy cattle. However this tolerance level is difficult to ob‐ serve because the average daily individual intake in a herd should be limited to 40 µg AFB1

Many factors may affect the formation of aflatoxins in animal feeds. Geographic and climate changes can affect the farm management practices and feed quality. These effects can lead to the wide variations in AFM1 levels in milk (Table 4). The preserved fodder such as silage and hay might have been contaminated by aflatoxin producing fungi and the improper stor‐ age led to aflatoxin production. The level of AFM1 in feed in rainy seasons is more than in dry seasons. It can be also probable to use higher amounts of contaminated concentrates in

> **Aflatoxin Concentration (ppb)**

AFM1 0.0006-0.059 ELISA [79]

**Method Reference**

Occurrence of Aflatoxins in Food http://dx.doi.org/10.5772/51031 153

per cow, in order to produce milk with less than 50 ng AFM1 per kg [65].

**Country Contaminated/**

Raw Milka Croatia NMa

**Total examined**

/61 (one sample exceeded limit EU)

Raw Milka Italy 125/161 AFM1 <0.023 HPLC [72] Raw Milka Greece 40/58 AFM1 0.005-0.055 HPLC [73] Raw Milka North African35/49 AFM1 0.03-3.13 HPLC [74] Raw Milka Italy ?/310 AFM1 0.002-0.09 HPLC [75] Raw Milka Trinidad 13/212 AFM1 NMa Charm II [29] Raw Milka Slovenia 0/60 AFM1 NDb HPLC [76] Raw Milka Indonesia 65/113 AFM1 5-25 ELISA [77] Raw Milka China 12/12 AFM1 0.16-0.5 ELISA [78]

Raw Milka Turkey 43/50 AFM1 <0.03 ELISA [80]

AFM1 for casein.

most developed countries.

the cold months [71].

**Food Type**

A tolerable daily intake of 0.2 ng kg-1 b.w. for AFM1 was calculated by Kuiper-Goodman [64] and this toxin has been categorized by the International Agency for Research on Cancer (IARC) as a class 2B toxin, a possible human carcinogen. In the assessment of cancer risk, the infants are more exposed to the risk because the milk is a major constituent of their diet. It must be also considered that young animals have been found to be more susceptible to AFB1 (and so probably AFM1) than adults. Therefore the presence of AFM1 in milk and milk products is considered to be undesirable [65].

The carcinogenicity of AFM1 may be influenced by the duration and level of exposure. Ex‐ posure is most likely to occur through the frequent consumption of milk and milk by-prod‐ ucts (infant milk, cheese, butter, yoghurt). Several studies in different countries have reported high or low contamination levels of AFM1 in different categories of milk and dairy samples. These significantly variable AFM1 levels may be due to several influencing factors such as cheese manufacturing procedures and storage, types of cheese, conditions of cheese ripening, analytical methods and finally the geographical and seasonal effects [6].

The concentration of AFM1 is relatively increased in cheese samples because of its affinity to proteins. During cheese making, AFM1 can be decreased in cheese by increasing renneting temperature from 30 to 40°C, decreasing cutting size of curd and increasing press time from 1 to 2 h, which causes more loss of AFM1 in the whey [66]. On a weight basis, however, AFM1 concentration in cheese actually increases. In soft cheese, it becomes 2.5 to 3.3 times higher and in hard cheese, 3.9 to 5.8 times higher than in the milk from which the cheeses were made. Converting milk that may contain aflatoxin into a cheese, such as feta cheese, reduces the exposure of the consumer to this toxin. During pasteurization of milk, about 90% or more of the AFM1 is retained in the milk but during cheese manufacturing, there is a partitioning of AFM1 between the cheese, whey, and brine. During cheese manufacturing, results on the distribution of AFM1 between curd and whey can be variable. This variability has been associated with the type of cheese, the particular cheese-making process applied, the type and degree of milk contamination, and the analytical method employed. Lopez et al. [67] manufactured cheese using artificially AFM1 contaminated milk and found that the greatest proportion of toxin (60%) was in whey, while 40% AFM1 remained in cheese. Some researchers also reported that the greatest proportion of AFM1 was in the curd ranging be‐ tween 66-80% [68]. About 37% of the AFM1 in milk is lost from the cheese into the whey, and another 30% diffuses from the cheese into brining solution during storage. Thus, the amount that would be ingested in a 30 g serving of cheese made from milk containing 500 ng AFM1/L would be only 35 ng AFM1 compared to 125 ng AFM1 from a 250 g serving of fluid milk. Thus, consumers in a region where there are high aflatoxin levels in milk would be at less health risk if the milk is pasteurized and converted into a cheese such as feta or other white pickled cheese before it is delivered to the consumer [69]. Applebaum et al. [70] reported that AFM1 concentration in cheese was about four times higher than the cheese milk. The increase in AFM1 concentration in cheese has been explained by the affinity of AFM1 for casein.

of AFB1. These compounds have a hydroxyl group, allowing their conjugation with glucur‐ onic acid, sulfate and glutathione, making them very water-soluble substances that can be excreted in the bile, urine and milk. Most of the aflatoxins are excreted between 72 to 96 h after the exposure, with the liver and the kidney retaining the waste for a longer period com‐

A tolerable daily intake of 0.2 ng kg-1 b.w. for AFM1 was calculated by Kuiper-Goodman [64] and this toxin has been categorized by the International Agency for Research on Cancer (IARC) as a class 2B toxin, a possible human carcinogen. In the assessment of cancer risk, the infants are more exposed to the risk because the milk is a major constituent of their diet. It must be also considered that young animals have been found to be more susceptible to AFB1 (and so probably AFM1) than adults. Therefore the presence of AFM1 in milk and

The carcinogenicity of AFM1 may be influenced by the duration and level of exposure. Ex‐ posure is most likely to occur through the frequent consumption of milk and milk by-prod‐ ucts (infant milk, cheese, butter, yoghurt). Several studies in different countries have reported high or low contamination levels of AFM1 in different categories of milk and dairy samples. These significantly variable AFM1 levels may be due to several influencing factors such as cheese manufacturing procedures and storage, types of cheese, conditions of cheese

The concentration of AFM1 is relatively increased in cheese samples because of its affinity to proteins. During cheese making, AFM1 can be decreased in cheese by increasing renneting temperature from 30 to 40°C, decreasing cutting size of curd and increasing press time from 1 to 2 h, which causes more loss of AFM1 in the whey [66]. On a weight basis, however, AFM1 concentration in cheese actually increases. In soft cheese, it becomes 2.5 to 3.3 times higher and in hard cheese, 3.9 to 5.8 times higher than in the milk from which the cheeses were made. Converting milk that may contain aflatoxin into a cheese, such as feta cheese, reduces the exposure of the consumer to this toxin. During pasteurization of milk, about 90% or more of the AFM1 is retained in the milk but during cheese manufacturing, there is a partitioning of AFM1 between the cheese, whey, and brine. During cheese manufacturing, results on the distribution of AFM1 between curd and whey can be variable. This variability has been associated with the type of cheese, the particular cheese-making process applied, the type and degree of milk contamination, and the analytical method employed. Lopez et al. [67] manufactured cheese using artificially AFM1 contaminated milk and found that the greatest proportion of toxin (60%) was in whey, while 40% AFM1 remained in cheese. Some researchers also reported that the greatest proportion of AFM1 was in the curd ranging be‐ tween 66-80% [68]. About 37% of the AFM1 in milk is lost from the cheese into the whey, and another 30% diffuses from the cheese into brining solution during storage. Thus, the amount that would be ingested in a 30 g serving of cheese made from milk containing 500 ng AFM1/L would be only 35 ng AFM1 compared to 125 ng AFM1 from a 250 g serving of fluid milk. Thus, consumers in a region where there are high aflatoxin levels in milk would be at less health risk if the milk is pasteurized and converted into a cheese such as feta or other white pickled cheese before it is delivered to the consumer [69]. Applebaum et al. [70]

ripening, analytical methods and finally the geographical and seasonal effects [6].

pare to other tissues [63].

152 Aflatoxins - Recent Advances and Future Prospects

milk products is considered to be undesirable [65].

The Commission of the European Communities established a limit for AFM1 of 50 ng kg-1 for milk and a variable limit for cheese, depending on concentration caused by drying proc‐ ess or processing. Milk containing AFM1 concentrations above the action level must be dis‐ carded, causing significant economic loss for the dairy producer. Similar regulations exist in most developed countries.

In this Regulation the Commission stated that ''even if AFM1 is regarded as a less danger‐ ous genotoxic carcinogenic substance than AFB1, it is necessary to prevent the presence in milk, and consequently in milk products, intended for human consumption and for young children in particular''. The Commission has also set a limit for AFB1 of 5 µg kg-1 for supple‐ mentary feedstuffs for lactating dairy cattle. However this tolerance level is difficult to ob‐ serve because the average daily individual intake in a herd should be limited to 40 µg AFB1 per cow, in order to produce milk with less than 50 ng AFM1 per kg [65].

Many factors may affect the formation of aflatoxins in animal feeds. Geographic and climate changes can affect the farm management practices and feed quality. These effects can lead to the wide variations in AFM1 levels in milk (Table 4). The preserved fodder such as silage and hay might have been contaminated by aflatoxin producing fungi and the improper stor‐ age led to aflatoxin production. The level of AFM1 in feed in rainy seasons is more than in dry seasons. It can be also probable to use higher amounts of contaminated concentrates in the cold months [71].



UHT Milka Turkey 50/50 AFM1 0.01-0.244 ELISA [7] UHT Milka Iran 116/210 AFM1 0.012-0.249 ELISA [97] UHT Milka Iran 68/109 AFM1 0.006-0.516 ELISA [91] UHT Milka Brazil 53/60 AFM1 0.015-0.5 HPLC [93] UHT Milka Iran 48/48 AFM1 0.01-0.10 ELISA [88] UHT Milka Turkey 14/24 AFM1 <0.01-0.05 HPLC [98]

Portugal 17/18 AFM1 <0.005-0.059 HPLC [86]

Occurrence of Aflatoxins in Food http://dx.doi.org/10.5772/51031 155

Portugal 20/22 AFM1 <0.005-0.061 HPLC [86]

Portugal 23/30 AFM1 <0.005-0.02 HPLC [86]

Japan 207/208 AFM1 0.001-0.029 HPLC [99]

Italy 85/102 AFM1 0.05-0.25 ELISA [101]

India 76/87 AFM1 0.063-1.012 ELISA [102]

Milk powder Brazil 72/75 AFM1 0.01-0.5 HPLC [90] Milk powder China 15/15 AFM1 Max 0.54 ELISA [78] Milk powder Syria 1/8 AFM1 0.012 ELISA [84]

Ewe's milk Greece 19/27 AFM1 0.005-0.055 HPLC [73] Ewe's milk Greece 27/54 AFM1 <0.005-0.182 ELISA [100] Ewe's milk Syria 13/23 AFM1 0.006-0.634 ELISA [84] Goat milk Greece 12/20 AFM1 0.005-0.05 HPLC [73] Goat milk Syria 7/11 AFM1 0.008-0.054 ELISA [84]

UHT-whole milk

UHT-semi skimmed milk

UHTskimmed milk

UHT-Pasteurized milk

Milk (ewe, goat and buffalo mix)

Infant milk food, Milk based cereal, weaning food, infant formula and liquid milk


Raw Milka Iran 60/60 AFM1 2.0-64.0 HPLC [81] Raw Milka Pakistan 177/232 AFM1 0.002-1.9 ELISA [82] Raw Milka Pakistan 63/120 AFM1 0.004-0.174 HPLC [83] Raw Milka Syria 70/74 AFM1 0.02-0.69 ELISA [84] Raw Milka South Korea 48/100 AFM1 0.002-0.08 HPLC [85] Raw Milka Portugal 25/31 AFM1 <0.005-0.05 HPLC [86] Raw Milka Iran 128/128 AFM1 0.031-0.113 ELISA [87] Raw Milka Iran 117/140 AFM1 <0.01-0.10 ELISA [88] Raw Milka Spain 3/92 AFM1 0.014-0.019 HPLC [89]

Pakistan 153/360 AFM1 0.002-0.087 HPLC [83]

Greece 113/136 AFM1 0.005-0.05 HPLC [73]

Morrocco 47/54 AFM1 0.001-0.117 HPLC [51]

Brazil 7/10 AFM1 0.01-0.02 HPLC [90]

Iran 83/116 AFM1 0.006-0528 ELISA [91]

Iran 624/624 AFM1 0.045-0.08 ELISA [92]

Syria 10/10 AFM1 0.008-0.765 ELISA [84]

Iran 48/48 AFM1 0.01-0.10 ELISA [88]

Brazil 58/79 AFM1 0.05-0.24 HPLC [93]

Argentina 18/77 AFM1 0.01-0.03 ELISA [94]

UHT Milka Greece 14/17 AFM1 0.005-0.05 HPLC [73] UHT Milka Turkey 75/129 AFM1 Max.0.54 ELISA [95] UHT Milka Turkey 67/100 AFM1 0.01-0.63 ELISA [96] UHT Milka Brazil 40/40 AFM1 0.010-0.5 HPLC [90]

Buffalo raw milk

154 Aflatoxins - Recent Advances and Future Prospects

Pasteurized milka

Pasteurized milka

Pasteurized milka

Pasteurized milka

Pasteurized milka

Pasteurized milka

Pasteurized milka

Pasteurized milka

Milka (Raw, pasteurized and powder)


Kashar cheeseb

Tulum cheeseb

Tulum cheeseb

Milk products

Strained yoghurt

Yogurt (whole fat)

Yoghurt (Semi fat)

Infant formula

Infant formula

a.Cow milk, b. Cow cheese

**Table 4.** Aflatoxins in milk and dairy products.

Turkey 85/100 AFM1 0.05-0.80 ELISA [110]

Occurrence of Aflatoxins in Food http://dx.doi.org/10.5772/51031 157

Turkey 16/20 AFM1 <0.378 ELISA [80]

Turkey 81/100 AFM1 0.05-0.80 ELISA [110]

China 66/104 AFM1 Max 0.5 ELISA [78]

Turkey 29/52 AFM1 <0.15 ELISA [116]

Turkey 18/25 AFM1 <0.069 ELISA [80]

Turkey 10/25 AFM1 <0.078 ELISA [80]

South Korea 18/26 AFM1 0.032-0.132 HPLC [103]

Iran 116/120 AFM1 0.001-0.014 ELISA [87]

Dairy dessert Turkey 26/50 AFM1 <0.08 ELISA [80]

Ewe's cheeseTurkey 14/50 AFM1 0.02-2.0 TLC [6] Dairy drinks Brazil 10/12 AFM1 0.01-0.05 IAC/LC [107]

Butter Turkey 92/92 AFM1 0.01-7.0 ELISA [114] Butter Turkey 25/27 AFM1 Max 0.1 ELISA [36] Butter Turkey 66/80 AFM1 0.01-0.12 ELISA [115] Yoghurt Brazil 49/65 AFM1 0.01-0.529 IAC/LC [107] Yoghurt Italy 73/120 AFM1 <0.032 HPLC [72] Yoghurt Turkey 68/104 AFM1 <0.1 ELISA [116] Yoghurt South Korea 31/60 AFM1 0.017-0.124 HPLC [103] Yoghurt Portugal 2/48 AFM1 0.043-0.045 HPLC [117] Fruit yoghurtPortugal 16/48 AFM1 0.019-0.098 HPLC [117] Fruit yogurt Turkey 7/21 AFM1 <0.1 ELISA [115]


**Table 4.** Aflatoxins in milk and dairy products.

Milk powder South Korea 17/24 AFM1 0.083-0.342 HPLC [103] Cheeseb Iran 66/80 AFM1 0.15-2.41 TLC [104] Cheeseb China 4/4 AFM1 0.16-0.32 ELISA [78] Cheeseb Lebanon 75/111 AFM1 0.056-0.315 ELISA [105] Cheeseb Iran 30/50 AFM1 0.041-0.374 ELISA [106] Cheeseb Brazil 39/58 AFM1 0.01-0.304 IAC/LC [107] Cheeseb North African15/20 AFM1 0.11-0.52 HPLC [74] Cheeseb Turkey 14/20 AFM1 <0.155 ELISA [80] Cheeseb Turkey 10/200 AFM1 0.1-0.6 ELISA [108] Cheeseb Iran 93/116 AFM1 0.052-0.745 ELISA [109] Cheeseb Turkey 82/100 AFM1 <0.05-0.8 ELISA [110] Cheeseb Turkey 36/127 AFM1 0.07-0.77 ELISA [111]

Turkey 31/50 AFM1 0.1-5.2 Fluorometri[112]

Turkey 159/193 AFM1 0.052-0.86 ELISA [113]

Turkey 52/60 AFM1 0.16-7.26 Fluorometri[112]

Turkey 44/49 AFM1 Max 0.25 ELISA [36]

Turkey 8/200 AFM1 0.1-0.7 ELISA [108]

Turkey 99/100 AFM1 0.01-4.1 ELISA [114]

Iran 68/94 AFM1 58.3-785.4 ELISA [109]

Turkey 47/53 AFM1 "/0.25 ELISA [36]

Turkey 12/200 AFM1 0.12-0.8 ELISA [108]

Turkey 8/28 AFM1 <0.37 ELISA [80]

Turkey 109/132 AFM1 0.05-0.69 ELISA [96]

White brined cheeseb

156 Aflatoxins - Recent Advances and Future Prospects

White brined cheeseb

Herby cheeseb

Cream cheeseb

Cream cheeseb

Cream cheeseb

Cream cheeseb

Kashar cheeseb

Kashar cheeseb

Kashar cheese

Kashar cheeseb
