Incidence OfIncidence Of

AFLATOXIN M1AFLATOXIN M1

in Milkin Milk

Prof. Mohamed Abu SalemProf. Mohamed Abu Salem

HassaneinHassanein Prof. Faten Prof. Faten

Introdution

**General BackgroundAflatoxins can be produced by three species of Aflatoxins can be produced by three species of AspergillusAspergillus——A. A. flavusflavus, , A. parasiticusA. parasiticus, and the rare , and the rare A. nomiusA. nomius—which contaminate —which contaminate plants and plant products.plants and plant products.

  A. flavusA. flavus produces only B aflatoxins, while the other two species  produces only B aflatoxins, while the other two species produce both B and G aflatoxins. Aflatoxins Mproduce both B and G aflatoxins. Aflatoxins M11 and M and M22 are the  are the

hydroxylated metabolites of aflatoxins Bhydroxylated metabolites of aflatoxins B11 and B and B22 and can be found  and can be found

in milk or milk products obtained from livestock that have in milk or milk products obtained from livestock that have ingested contaminated feed. The main sources of aflatoxins in ingested contaminated feed. The main sources of aflatoxins in feeds are peanut meal, maize and cottonseed meal.feeds are peanut meal, maize and cottonseed meal.

**BIOLOGICAL DATA

11 – – Chemical StructureChemical Structure

**BIOLOGICAL DATA22 - - Biochemical aspects:Biochemical aspects:A- BiotransformationA- Biotransformation::

B- B- Effects of oltipraz and ethoxyquinEffects of oltipraz and ethoxyquin

**ANALYTICAL METHODS

11 - -Screening testsScreening tests: : Early detection of aflatoxin MEarly detection of aflatoxin M11 and  and

removal of small lots of removal of small lots of contaminated milk can prevent contaminated milk can prevent contamination of much larger contamination of much larger volumes. Screening methods are volumes. Screening methods are particularly useful if they can be particularly useful if they can be carried out quickly, easily, and carried out quickly, easily, and economically. They should allow economically. They should allow detection of concentrations of detection of concentrations of aflatoxin Maflatoxin M11 in milk as low as those  in milk as low as those

detected by the ultimate detected by the ultimate quantitative methodsquantitative methods

**ANALYTICAL METHODS: : 22 - -Quantitative methodsQuantitative methods

The quantitative methods that have been developed The quantitative methods that have been developed and validated for aflatoxin Mand validated for aflatoxin M11 in milk and milk  in milk and milk

products were originally designed to analyse milk products were originally designed to analyse milk powder. Milk was spray-dried or lyophilized to powder. Milk was spray-dried or lyophilized to preserve its shelf life and to reduce sample bulkpreserve its shelf life and to reduce sample bulk . .

**A more recent advance in quantitative extraction of A more recent advance in quantitative extraction of aflatoxin M1 and subsequent clean-up is use of aflatoxin M1 and subsequent clean-up is use of immunoaffinity cartridges. These columns are immunoaffinity cartridges. These columns are composed of monoclonal antibodies specific for composed of monoclonal antibodies specific for aflatoxin Maflatoxin M11, which are immobilized on Sepharose® , which are immobilized on Sepharose®

and packed into small cartridges (see Figure 2). The and packed into small cartridges (see Figure 2). The first published method for aflatoxin Mfirst published method for aflatoxin M11 with  with

immunoaffinity columns was that of Mortimer et al. immunoaffinity columns was that of Mortimer et al. (1987)(1987)..

**ANALYTICAL METHODS

33 - -Analytical quality Analytical quality assuranceassuranceThe availability of collaboratively studied The availability of collaboratively studied ‘official’ methods of analysis for aflatoxin M‘official’ methods of analysis for aflatoxin M11, ,

with acceptable performance characteristics, with acceptable performance characteristics, is no guarantee of accurate results. Check is no guarantee of accurate results. Check sample programmes for aflatoxins, including sample programmes for aflatoxins, including aflatoxin Maflatoxin M11 in milk, organized by IARC  in milk, organized by IARC

(Friesen & Garren, 1982) have shown that (Friesen & Garren, 1982) have shown that there can be wide variation in results. In there can be wide variation in results. In compliance with the principles of analytical compliance with the principles of analytical quality assurance, measurements of the quality assurance, measurements of the mycotoxin by different laboratories should be mycotoxin by different laboratories should be reliable and comparable. A quality assurance reliable and comparable. A quality assurance programme includes, when possible, use of programme includes, when possible, use of (certified) reference materials and many other (certified) reference materials and many other

elementselements . .

**SAMPLING PROTOCOLSTwo effective methods for Two effective methods for controlling controlling aflatoxin Maflatoxin M11 in the food supply are to sample  in the food supply are to sample

dairy feed for aflatoxin Bdairy feed for aflatoxin B11 or to sample the milk  or to sample the milk

directly for aflatoxin Mdirectly for aflatoxin M11..

but there has been little evaluation of sampling but there has been little evaluation of sampling plans to detect aflatoxin Mplans to detect aflatoxin M11 in milk. It might be  in milk. It might be

difficult to design an effective programme to difficult to design an effective programme to control aflatoxin in granular feed, particularly control aflatoxin in granular feed, particularly at low concentrations, because of its at low concentrations, because of its heterogeneous distribution inheterogeneous distribution in

these commodities, which results in wide these commodities, which results in wide sampling variationsampling variation..

In the absence of information on the efficacy In the absence of information on the efficacy of sampling plans for the determination of of sampling plans for the determination of aflatoxin Maflatoxin M11 in milk, it is recommended that the  in milk, it is recommended that the

European model, in which a 500-g sample European model, in which a 500-g sample composed of five 100-g portions of milk is composed of five 100-g portions of milk is taken from a batch, be used for the minimum taken from a batch, be used for the minimum sample size and sample selection methodsample size and sample selection method..

**FOOD CONSUMPTION AND FOOD CONSUMPTION AND DIETARY INTAKE ESTIMATESDIETARY INTAKE ESTIMATESDietary intake of aflatoxin Dietary intake of aflatoxin BB11 was estimated from  was estimated from

data on the concentrations data on the concentrations of aflatoxin Mof aflatoxin M11 in milk  in milk

submitted to FAO/WHO, submitted to FAO/WHO, from selected reports in from selected reports in the literature, and from the literature, and from data on milk consumption data on milk consumption in the GEMS/Food in the GEMS/Food Regional Diets (WHO, Regional Diets (WHO, 1998)1998)..

**FOOD CONSUMPTION AND FOOD CONSUMPTION AND DIETARY INTAKE ESTIMATESDIETARY INTAKE ESTIMATES

* *Effect of proposed maximum levels Effect of proposed maximum levels in in ::foods on dietary intake of aflatoxin Mfoods on dietary intake of aflatoxin M11

**ToxicityAflatoxin M

1 is cytotoxic, as demonstrated in human

hepatocytes in vitro and its acute toxicity in several

species is similar to that of aflatoxin B1

. In ducklings

and rats, the acute and short-term toxicity of aflatoxin

M1

 was similar to or slightly less than that of aflatoxin

B1

. In studies of carcinogenicity, aflatoxin M1

 was

about one order of magnitude less potent than

aflatoxin B1

, even in sensitive species like the rainbow

trout and the Fischer rat. The in vitro genotoxic

potency of aflatoxin M1

 was similar to that of aflatoxin

B1

 in some test systems and between one-half and

one-sixth that of aflatoxin B1

 in other test systems.

**EFFECTS OF PROCESSING

**EFFECTS OF PROCESSINGFate of aflatoxin MFate of aflatoxin M11 during  during

processing of milkprocessing of milk::Treatments that are common in Treatments that are common in the dairy industry can be the dairy industry can be separated into two distinct separated into two distinct processes: those that do not processes: those that do not involve separation of milk involve separation of milk components, such as heat components, such as heat treatment, low-temperature treatment, low-temperature storage, and yoghurt preparation; storage, and yoghurt preparation; and processes that involve and processes that involve separation of milk components, separation of milk components, such as concentration, drying, such as concentration, drying, and cheese and butter productionand cheese and butter production..

**EFFECTS OF PROCESSINGThe stability of aflatoxin M1 during heat processing, such as pasteurization and heating milk directly on a fire for 3–4 h has been studied

Studies of the stability of aflatoxin M1 in milk during cool or frozen storage gave variable results ,but storage of frozen contaminated milk and other dairy products for a few months did not appear to affect the aflatoxin M1 content.

The manufacture of cultured dairy products, such as kefir and yoghurt, also did not significantly decrease the aflatoxin M1 content

**EFFECTS OF PROCESSINGDegradation of aflatoxin Degradation of aflatoxin MM11 in milk in milk::

The observation that the processes described above do not generally lead to loss of aflatoxin M1 is of considerable practical importance. Several possibilities for eliminating or inactivating aflatoxin M1 in milk, involving chemical and physical treatment, have been investigated. The chemicals that have been studied for their ability to degrade aflatoxin M1 are limited to those that are permitted as food additives: sulfites, bisulfites, and

hydrogen peroxide

**EFFECTS OF PROCESSINGThe chemical and physical The chemical and physical treatments described are not treatments described are not readily applicable in the dairy readily applicable in the dairy industry, at least at present, as industry, at least at present, as little is known about the biological little is known about the biological safety, or the nutritional value of safety, or the nutritional value of the treated products. Moreover, the treated products. Moreover, the costs of the processes may be the costs of the processes may be considerable and prohibitive for considerable and prohibitive for large-scale application. If aflatoxin large-scale application. If aflatoxin MM11 cannot be destroyed or  cannot be destroyed or

removed readily, it can be removed readily, it can be excluded from milk only by excluded from milk only by eliminating aflatoxin Beliminating aflatoxin B11 from the  from the

diet of animalsdiet of animals..

**Adverse Effect

**Adverse Effectaflatoxin can appear in the meat, milk, and eggs of food animals aflatoxin can appear in the meat, milk, and eggs of food animals that have consumed aflatoxin-contaminated feeds. The aflatoxin that have consumed aflatoxin-contaminated feeds. The aflatoxin group consist of B1, B2, G1, and G2. Aflatoxin B1 is the most group consist of B1, B2, G1, and G2. Aflatoxin B1 is the most potent of the group and has been shown to be a potent potent of the group and has been shown to be a potent carcinogen. Aflatoxin M1 is metabolic breakdown product of carcinogen. Aflatoxin M1 is metabolic breakdown product of aflatoxin B1 and can appear in the milk of lactating cows aflatoxin B1 and can appear in the milk of lactating cows consuming significant quantities of aflatoxin B1. consuming significant quantities of aflatoxin B1. Aflatoxin M1 is Aflatoxin M1 is not as carcinogenic as B1 not as carcinogenic as B1 but can be as but can be as toxictoxic. The conversion of . The conversion of aflatoxin B1 in feed to aflatoxin M1 in milk is about 1% to 2%. aflatoxin B1 in feed to aflatoxin M1 in milk is about 1% to 2%. The FDA regulatory level of aflatoxin in milk is 0.5 ppbThe FDA regulatory level of aflatoxin in milk is 0.5 ppb..

**Adverse EffectHigh-level aflatoxin exposure produces an acute hepatic necrosis, resulting later in cirrhosis, or carcinoma of the liver. Acute hepatic failure is made manifest by hemorrhage, edema, alteration in digestion, changes to the absorption and/or metabolism of nutrients, and mental changes and/or coma.

No animal species is immune to the acute toxic effects of aflatoxins; however, adult humans have a high tolerance for aflatoxin exposure and rarely succumb to acute aflatoxicosis.

Chronic, subclinical exposure does not lead to symptoms as dramatic as acute aflatoxicosis. Children, however, are particularly affected by aflatoxin exposure, which leads to stunted growth and delayed development. Chronic exposure also leads to a high risk of developing liver cancer.

Medical research indicates that a regular diet including apiaceous vegetables such as carrots, parsnips, celery and parsley, may reduce the carcinogenic effects of aflatoxin.

Moreover, aflatoxin B1 can permeate through the skin. Dermal exposure to this aflatoxin in particular environmental conditions can lead to serious health risks.

**Adverse Effect

**Prevention & Controlthere are several possibilities for preventing the presence of aflatoxin M1 in dairy products; each has its advantages and disadvantages. Although prevention of contamination of dairy cattle feed is the ideal, it may not be possible in practice. Various decontamination methods have a role to play in preventing and reducing the concentrations of aflatoxin M1 in dairy products.

The most effective way of controlling The most effective way of controlling aflatoxin Maflatoxin M11 in the food supply is to  in the food supply is to

reduce contamination with aflatoxin Breduce contamination with aflatoxin B11 of  of

raw materials and supplementary raw materials and supplementary feedstuffs for dairy cattle. Specific feedstuffs for dairy cattle. Specific regulations exist in many countries (FAO, regulations exist in many countries (FAO, 1997), and practical programmes are 1997), and practical programmes are being developed; e.g. the Codex being developed; e.g. the Codex Committee on Food Additives and Committee on Food Additives and Contaminants has developed a code of Contaminants has developed a code of practice for reducing aflatoxin Bpractice for reducing aflatoxin B11 in raw  in raw

materials (van Egmond et al., 1997)materials (van Egmond et al., 1997)..

**Prevention & Control

**Prevention & ControlReduction can be achieved by good manufac-turing practices and good storage practices. If preventive measures fail to reduce fungal growth and aflatoxin B1 formation in agricultural commodities intended for use as animal feeds, the last means for avoiding or reducing the occurrence of aflatoxins in feed is to eliminate (part of) the toxins. Feeds that have higher concentrations of aflatoxin B1 may be acceptable for feeding to dairy animals if they are blended with feed that has lower concentrations, provided that the resultant aflatoxin M1 concentration in milk does not exceed levels considered to be safe. In principle, aflatoxin-contaminated consignments of feeds can be decontaminated by removing the toxin (segregation) or by converting it to a non-toxic form (degradation).

**Prevention & ControlDegradation may be achieved by physical, chemical, or biological means.

Attempts have been made to degrade aflatoxins in feed by applying physical treatments such as heat, microwaves, gamma-rays, X-rays, ultra-violet light and adsorption (van Egmond & Speijers, 1999). Degradation of aflatoxin M1 has also been attempted by combined treatments, such as ultra-violet radiation followed by ultrafiltration. In most cases, neither heat treatment not irradiation is effective. Adsorption of aflatoxins from animal feed onto bentonite and hydrated sodium calcium aluminosilicate (Veldman, 1992; Galvano et al., 1996a) has been used in the feed industry to reduce the aflatoxin M1 content of milk (Harvey et al., 1991; Phillips et al., 1995).

** **Prevention & ControlPrevention & ControlA newer approach is use of oltipraz, a substituted dithiolthione that inhibits aflatoxin B1 metabolism by inhibiting the activity of several cytochrome P450 enzymes (Kuilman et al., 2000). No aflatoxin M1 formation was found in bovine hepatocytes incubated with aflatoxin B1 and oltipraz. The findings suggest that oltipraz is highly effective in inhibiting aflatoxin M1 contamination of milk from diary cows exposed to aflatoxin B1-contaminated feeds.

** **Prevention & ControlPrevention & ControlSome chemical procedures have been developed to degrade aflatoxins in animal feed, usually based on addition of oxidizing agents, aldehydes, acids, and bases. The most widely used chemical detoxication reagent is ammonia, as an anhydrous vapour or as an aqueous solution. Treatment of aflatoxin B1 with ammonia opens the lactone ring of the molecule. Ammoniation of agricultural commodities leads to decomposition of 95–98% of the aflatoxin B1 present. This process is used in various countries for the decomposition of animal feedstuffs

**ConclusionAflatoxin M1 have a lot of bad effects Aflatoxin M1 have a lot of bad effects which more toxic for human body and which more toxic for human body and may cause liver of lung may cause liver of lung carcinoma ,degeneration of DNA and carcinoma ,degeneration of DNA and

stunted growth , hepatic diseasesstunted growth , hepatic diseases. .,,,. .,,,

**ConclusionThe most important part of treatment of The most important part of treatment of AFLATOXIN M1 is have a AFLATOXIN M1 is have a good hygienic practicegood hygienic practice in in handling and treating of the animal feed (transfer, handling and treating of the animal feed (transfer, storage,….) to prevent of AFLATOXIN B1 storage,….) to prevent of AFLATOXIN B1 (Most (Most

Carcinogenic) Carcinogenic) which transformation to AFLATOXIN which transformation to AFLATOXIN M1M1(Toxic)(Toxic)which secreted in milk ,urinewhich secreted in milk ,urine..

There are different ways to prevent or treatment of There are different ways to prevent or treatment of reduce of AflaToxin M1 which may reduce of AflaToxin M1 which may physical ,chemical ,biological or physical ,chemical ,biological or ..mechanical mechanical processesprocesses

**Recommendation

**Recommendation

RegardsRegards ,,,,,,