Mycotoxins
 

 

 


 

Presented by  Dr. Shoeb Akhtar Khan on 23.9.02

 

( shoebakin@yahoo.co.in)

 

·                    Introduction

·                    Historical aspect

·                    Mycotoxins in food and feeds

 

-                     Aflatoxin                                                                   

-                     Ochratoxin

Ergot alkaloids

-                     Trichothecenes

-                     Zearalenoxie                                                 

-                     Fumonisines

-                     Moniliformin

-                      

·                    Factors affecting contamination of food

·                    Methods of detection

·                    Methods of detoxification

-                     Physical

-                     Chemical

-                     Microbiological

·                    Regulations

·                    Economic impact

·                    Conclusion

 

Introduction

 

Varity of contaminants are found naturally occurring in foods and feeds like

 

i.                   Chemical : pesticides, herbicides, fertilizers, artificial colours, preservations.

 

ii.                 Toxic plants and there products : as use have been epidemics of lathyrism and dropsy in recent past.

 

iii.              Bacterial contaminations

 

iv.               Mycotoxin : there are basically secondary metabolites of moulds (not yeasts) and contaminants food grains during pre and post harvestation period but can also affects other food commodities.

 

Mycotoxicosis : Ingestion of toxin

 

Mycosis : Fungal infection

 

Mycotoxins in very small amount is very common but under certain environmental conditions these amount increase significantly result in mycotoxicosis which remains unrecognized by medical professionals except when large number of persons or animals are involved.

 

Mycotoxicosis has adverse effect on human health and farm animals and crops as well as has a very significant impact on economy.

 

Specially, in developing countries like India which have raw food grains as one of important export commodity.

 

 

Historical aspect

 

Though mycotoxicosis gain much interest in last few decals the untoward effect of moulds were known from ancient times.

 

7th and 8th century – festival Robigalia was popular to honour the God Robigus to protect grains and trees. It was celebrated in April, high time for crops to be attack by rust.

 

In middle ages out break of Ergotism were common in Europe, mutilating and killing thousand of peoples. It was k/a “St Anyhony’s fire”. Due to instance burning sensation felt by patient and belief that it would relieve by visit to St. Anthony’s church.

Interest in mycotoxins rise again in 1960. Following a feed related mycotoxicosis is farm animals  known as Turkey’s  X disease.

 

1959 : Groundnut meal cargo in UK from Brazil to be incorporated as protein supplement for form animals.

 

1960 : Extensive loss of ducks and other form animals (common factor traced to be groundnut meal from Brazil).

 

Coincidentally out of proportion cases of hepatoma reported in Trouts in US.

 

On extensive study etiological agents was detected which was common at both places. It was identified on Aflatoxin since then along with various reports of epidemics of different mycotoxins thousands of papers published revealing various aspects of disease.

 

 

Mycotoxins in food and feeds

 

Mycotoxins have adverse effects on human and animal health mainly by affecting crops, cereal grains.

 

Crops are commonly affected by toxins producing moulds during pre and post harvest period mostly following unseasonal rain, are also affected due to faculty storage techniques.

 

One mycotoxin may be produced by many different species of moulds on the other hand one species may produces different type of mycotoxins under different conditions.

 

So far over 300 mycotoxins have been characterized, but few are common and important.

 

Table 1.

COMMON MYCOTOXINS

 

Mycotoxins

Moulds associated

Aflatoxins

Aspergillous .flavus, A.parasiticus

Ochratoxins

A.ochraceus, Penicillum.viridicatum. P.cyclopium

Ergot alkaloids

Claviceps.purpurea, C.fusiformis,

Aspergillous spp.

Penicillum spp.

Rhizopus spp.

Trichothecenes

Fusarium sporotrichosis,F.graminaream,F. poae

Myrothecium,Tricothecium

Zearalenone

F. culmorum, F.graminarium

Fumonisins

F.moniliform,F. proliferatum, F.verticilloideae

Moniliformin

F.proliferatum

3-nitropropionic acid

Arthrinium spps.

 

 

Table2.MYCOTOXINS IN FOOD IN INDIA (IJMR,108;nov.1998)

  

Food type

Region

mycotoxins

Levels(mcg/kg)

Maize

All India

Aflatoxin B1

5-87

 

Maize 

rain affected

Rajasthan & Gujrat

Aflatoxin B1

250-15600

Rice rain affected

Andrapradesh,

Karnataka

Aflatoxin B1

Traces –1130

Wheat & wheat products

N.W.India

Trichothecene

Not detectable

Wheat & wheat products rain affected

N.W. India

Trichothecene

30-8380

Bajra

Rajasthan

Ergot alkaloids

15000-199000

Sorgum

Deccan Plateau

Fumonisin B1

70-360

Sorgum

rain affected

Deccan Plateau

Fumonisin B1

140-7800

Maize

Deccan Plateau

Fumonisin B1

50-240

Maize

rain affected

Deccan Plateau

Fumonisin B1

250-6470

       

 

 

Aflatoxin (AF)

 

·                    It is most common group of mycotoxins wide spread in many parts and reported. It was characterised after “Turkey’s x disease”.

·                    Also found in most parts of India.

·                    Produced by various sp. and aspergillus

·                    Asp flavus

·                    Asp parasiticus

·                    Food stuffs commonly affected are

-                     Nuts

-                     Cereals

-                     Rice

-                     Wheat

Under conditions and high humidity and temperature

Chemically  dihydro and tetrahydro furano moities fused to coumarin ring

Six types:  B1,B2, G1,G2, M1 and M2.

AFB1 – most toxic

 

Biological effects :

 

Main target organ – liver

-                     Fatty degradation and liver

-                     Jaundice, pruritus, rashes and massive ascitis

-                     Acute hepatotoxicity, mortality, 10-60%

-                     Asso. with hepatic carcinoma

-                     Also immunosuppressive action

-                     Group I carcinogen by IARC

 

Mechanism of action

 

·                    Inhibition and synthesis of DNA and RNA.

·                    With regards to the acute cytotoxic effect. It induce lipid peroxidation leading to oxidative damage to hepatocytes

·                    Also associated with coagulopathy due to decreased synthesis of vit. K.

 

 

Table3 OUTBREAKS OF AFLATOXINS (Bulletin of WHO,77(9);1999)

Country

No. of persons affected

No. of deaths

Source

Toxins

Uganda

1

1

cassawa

_

India

1974-75 raj. ,Gujrat

397

106

Maize

AF-B1

India

1977 Raj. ,Gujrat

994

97

Maize

AF-B1

Kenya

20

12

Maize

AF-B1

USA

1

0

Purified AF-B1

Purified AF-B1

 

 

Octratoxin (OTA)

 

·                    It is common in eastern Europe

·                    It is metabolite of Aspergillus and Penicillium sp

·                     Asp.ochraceus from which it was isolated first and derived its name

·                     it is also produced by P.viridicatum, P.cyclopiam

·                    Chemically 3,4 dihydromethyl isocumarine

·                    Foods involved – Coffee beans, wheat flour, chillis, blue eye in corn & Foods of animal origin

 

Biological effects

·                    Its known for its nephrotoxicity

·                    No acute toxicity

·                    Have constent asso with epidemic nephropathy. Initial sign & symptoms are non-specific----- Fatigue, headache, loss of weight, mild protein urea and hypertension.

·                    Histopathology – bilateral renal cortical lesion with tubular degeneration and hyalinisation of glomeruli.

·                    In advance stage diffuse cortical fibrosis

·                    Disease is endemic in Croatia, Bulgaria, Yugoslavia, Romania, Bosnia . .

·                    Also have carcinogenic potential

·                    The incidence of urothelial tumour of pelvis in these areas 50-100 more than non-endemic area.

·                    Group 2B carcinogen by IARC

 

Mode of action

 

·                    Cellular toxicity – competitive inhibition of ATPase , succinate dehydrogenase and cytochrome oxidase.

·                    Cellular damage also caused by hydroxyl radical formation and lipid peroxidation

·                    Induction of unscheduled DNA synthesis with subsequent repair and lesion – mutation – carcinogenicity.

 

 

Trichothecenes

 

Produced mainly by Fusarium species. F.sporotrichoidis, F.graminearum, F. pore and other genera –             Myrothecium ,Trichothecium, Trichoderma

Over 148 mycotoxins in this group are known but common are

-                     T2 toxin

-                     Diacetoxyscrepenal (DAS)

-                     Deoxyminelemal (DOM) _ Vomitoxin

Ingestion of bread from contaminated wheat and by rice.

 

 

Biological effect

 

-                     Nausea and vomiting, diarrhoea

-                     Immunosuppression

-                     Hemorrhagic manifestation – rashes,gangrenous pharyngitis , laryngitis

·                    First recognised trichotheceas toxicosis was alimentry toxic aleukia out -break in USSR with mortality of >60%.

·                    Toxin are 40 time more toxic when inhaled then ingested have be reported to associated with sick building syndrome.

·                    It was used as chemical warfare agent in Combodia

·                    In India an outbreak of trichothecenes was recorded in Kashmir valley in 1987. Following consumption of mould damaged wheat involving several thousand persons.

 

Mechanism

 

·                    Disruption of membrane transport and amino acids and nucleotides

·                    Disruption of activity of Ca-K channel

·                    Inhibition of protein synthesis

 

 

Ergot alkaloids

 

As we have discuss earlier also, Ergotism was known cause of many epidemics in middle ages the main species of mould responsible are :

-                     Claviceps purpurea

-                     Claviceps fusifornis

Few other moulds like Penicillium, Aspergillus and Rhizopus  also produce Ergot alkaloids.

Wheat, rye, barley and other food grains are affected by these fungi usually following unseasonal rain and form black spur.

If bread were prepared without removing these black spur it result in epidemic.

So far over 40 Ergot alkaloids have been isolated. They are divided in 3 groups.

i.                    Derivatives of lysergic acid e.g. Ergotamine and ergocristine

ii.                 Derivatives and isolysergic acid – Egotaminine

iii.               Derivatives and dimethylegroline – clavines agro clavines

 

Biological effects

 

·                   Application of black spur were described in 16th century. It was described to hasten the delivery but inaccurate doses result in rupture uterus  and  death its nick name changed from

Pulvis adpartun ŕ pulvis admartam

 

It produced 2 type poisoning outbreaks

-                     Gangrenous type

-                     Convulsive type

 

The source of Ergot strongly influences.

 

Type of alkaloid and subsequent clinical picture

C. purpurea – produces Ergotamine and ergocristine with causes gangrenous form due to their vasoconstrictive activity.

Symp:   Edema of leg with severe pain ,Parasthesia ŕ gangrene

Last recorded epidemic of this  Type was in 1977 in ethiopia

 

Other type is convulsive related to intoxication with clavine alkaloids produced by C.fusiformis

Characterised by G.I. symp – nausea, vomiting followed by effect on CNS, drowsiness, twitching convulsion blindness and paralysis within 1-48 hours after exposure.

 

Ergotism is extremely rare today due to :

i.                    Normal grain cleaning and milling process removing most of Ergot.

ii.                 Causative agents are relatively labile and usuall, destroyed during backing and cooking.

 

 

Zearalenone (Zen)

 

Phytoestrogenic compound produced by fusarium sp

F. culnorum

F. gramineream

F. sporotrichoides

Mainly affect wheat and maize, some time Barlay and Sorghum.

 

Biological effects

 

 

ZEN and its derivatives produce estrogenic effects in form animal.

-                     Infertility

-                     Prolapse of vagina

-                     Atrophy of testes and feminisation in male, enlargement of mammory gland

-                     In Porterico it was reported form children with precaucious puberty.

 

Mode of action

 

·                    By binding to estrogen receptors influencing estrogen dependent transcription in nucleus.

·                    Recent studies have show potential of ZEM to stimulate growth of human breast cancer cells containing estrogen response receptors.

 

 

Fumonisin

 

Fumonisins are both cytotoxic and cancer producing matabolities mainly  produced by  F. proliferatum, F. verticilloideae and F.moniliform

When they grow in maize

B1 and B2 are toxic elements, B1 is more toxic.

 

Biological effects

 

Transient abdominal pain and diarrhoea ˝ - 1 hr after consumption of food staffs  recovers fully. No fatality

High incidence of oesophageal cancer with presence F.funonsine in maize.

Promote initial lesion in hepatic malignancy due to AFB1

IARC group 2B carcinogen

In out break of acute toxicity in South India with B1 involving 27 villages following consumption and rain affected maize and sorgum

 

Mode of action

Not known completely

Disrupt sphingo lipid metabolism by inhibiting enzyme sphingocine N-acetyl transferase

Also inhibit other intracellular enzymes.

Carcinogenicity linked to accumulation of sphingoid base that cause unscheduled DNA synthesis.

 

 

 

Moniliformin

 

Produced by Fusarium prolifaratum when it grow on corn kernel.

Can be transverse to next generation of crop remain in soil for years without decomposition

Chemically – Na or K salt of 1-hydroxycyclobut 1-ene 3,4 dione

 

Biological effects

Keshan disease in human

Cardiotoxicity

 

Mode of action

 

Cytotoxic –    inhibition of pyruvate dehydrogenase

Increased cardiac permeability

Inhibition of other enzymes e.g. glutathione peroxidase, glutathione reductase

 

 

Factors affecting contamination of  food and feed and production of mycotoxins.

 

The main difficulty in assessing the risk is multiplicity of factors responsible for fungal contamination and production and mycotoxins

-                     Physical

-                     Chemical

-                     Biological

Physical environmental condition conducive for fungal colonisation and mycotoxin production

-                     Unseasonal rain fall

-                     Temperature

-                     Humidity

-                     Insect infestation

-                     Storage of food stuff

Chemical – use of pesticides, fungicides, fertilizers

Biological – inter action between colonizing mould and substrate

Few plants are susceptible while other are resistant or intrinsic factor of infective fungi – species, strain specificity, strain variation, toxigenic pro parity

Among these temperature and humidity play the major role.

As well as unseasonal  rain which renders crop susceptible to mycotoxin production.

Several studies show optimal condition for fugal growth may not be optimal for toxin production.

 

A.     flavus colonisation can occur between  25-35°C however toxin production occurs  at 30°C.

 

-                     Ideal growth condition for Fuserium sp. are 21°C, while optimum temp for toxin T2, ZEM, and DOM are 6-12, 19-20, and 28°C respectively.

-                     In practical sense mycotoxin contamination of food and feed may result from inadequate storage and handling. The grows must be stored at <14% moisture, must be dry, oxygen free (sealed) fermented or treated with mould growth inhibitors.

 

Methods of detection

 

Various methods based on physical, clinical and biological assay are tested for detection and mycotoxins in foods, and food.

 

However use and many of these are limited prohibitive cost, lack and availability of kits at place and time short shelf life, and need to have infrastructure for regular use.

 

To overcome these problems attempts has been made to develop simple sensitive and rapid method various method which are available.

 

1.                  ELISA kits – only in developed countries.

2.                  Minicolumn chromatography – tested in India with high degree and reproducability and has been used as rapid screening method

3.                  With advent of molecular techniques,  method have been developed to detect biomarkers of  Aflatoxin in urine / blood / tissue – detection of Aflatoxin DNA adducts in urine

4.                  Recently a specific sensitive method has been developed to detected. Alfatoxin B1 – N7 guanine adduct increase by immunoaffinity chramatography, coupled with indirect competitive ELSIA

 

  

Methods of detoxification

 

A large number of method have been investigated for removal  of mycotoxins from food inclusion

-                     Physical

-                     Chemical

-                     Microbiological

 

1.         Physical

·                    Physical removed

·                    Processing and drug milling

·                    Density segregation by Flotation

·                    Adsorption &Filtration

·                    Heat inactivation

·                    Irradiation

·                    Solvents

2.         Chemical

·                    Treatment with amonia

·                    Treatment with sod. bisulphite

·                    Degradation with acids

·                    Degradation with oxidising agents

 

3.                  Microbiological 

 

Microinactivation, fermantation and conversion to less toxic metabolites by

·                    Flavobactcrium

·                    Lactobacillus

·                    Corynebacterium

 

As microbiological methods have certain disadvantages, organism also utilize food, elaborate undesirable compounds. 

 

Chemical methods also has limited application because they may form toxic residues, damage nutrient content, flavour, odour texture or functional property of products. 

 

·                    Most practical approach were found to physical methods which are simple practical not harmful like

·                    Hand picking of visibly mouldy nuts

·                    Removal  of Fumonisine from maize by density segregation in water and NaCl

·                    Removing ergoty pearl milled (BAZRA) by teatment with water and NaCl.

·                    It was observed dehusking of rice removes 55-67% toxin  & subsequent, polishing and parboiling reduce toxin to 72-93%

 

 

Table 4. REDUCTION IN MYCOTOXINS CONTENT DUE TO DECONTAMINATION METHODS ( IJMR,108;nov.1998)

 

Food

mycotoxin

Method of decontamination

Level of mycotoxin( mcg/kg)

Before treatment

After treatment

%reduction

Ground nut

Aflatoxin

Segregation by removal visually mouldy seeds

10-1600

Not detected

 

Rice

Aflatoxin

Milling, dehusking & polishing

Perboilingdehusking & polishing

13.3-53.5

 

 

 

13.32-53.5

2.6-18.8

 

 

 

2.1-4.5

65-80

 

 

 

72-93

maize

fumonisin

Density segregation with water

With 30% NaCl

923.5

 

 

 

973.6

242.4

 

 

 

164.4

73.7

 

 

 

82.9

wheat

Deoxynivalenol

Washing with water

With 15% NaCl

93.3

 

93.3

76.2

 

71.3

18

 

23

 

 

  

Regulations

 

There are several legislative control measures for mycotoxins specially Aflatoxins world wide to protect public health and at same time to promote international trade.

 

-                     As for 1994 – 77 countries had regulations for Aflatoxin in food and feeds ranging from 0-50mcg/kg for foods and 1000 mcg/kg for feeds

-                     In India PFA act set a limit and 30µg/kg for Aflatoxin in all food material for human consumption

-                     Regulatory limit for ergot affected food grain is 0.05% by PFA act.

-                     Fusarium mycotoxin have less stringent regulation world wide – in US control limit for Fumonisine is 10µg/kg

-                     However much criticism has been leveled in International circle against tolerance limit as there is lack of uniformity which affects in the international trade

-                     In order to protect international trade codex alimentarius commission has been attempt to  harmonize the regulatory limit for export food commodities

-                     In a recent meeting CCFAC have set a level and 15 mcg/kg for total Aflatoxin is Peanuts

 

 

 Economic impact

 

-                     Contamination of food and feed with mycotoxins is a world wide problem

-                     Several  review studies have shown extensive mycotoxin contamination both in developing as well as in developed countries both in turn of health and economy

There are multiple criteria for assessing economic impact of mycotoxins

Consideration includes

-                     Loss of human and animal life

-                     Health care veterinary care cost

-                     Loss of crops and feeds

-                     Regulatory implementation cost

-                     Trade loss

The variation in regulation for acceptable limit in mycotoxin is adding to economic loss. A lower limit is considered as a trade barrier. And it also has a repercussion on public health 

-                     While rejection of export consignment with contamination above limit would result in economic loss, the distribution of rejected consignment in domestic market increase  the consumers risk on Indian market

-                     The impact of these variation in limit can be observed from data by a recent study conducted by ICMR on Ground nut contamination by AF

30µg/kg          (Indian Limit)            -           21%crop will be rejected

15 µg/kg         (Codex limit)             -           37%crop will be rejected

10µg/kg          (Few countries)         -           >50%crop will be rejected

 

 

For maize

 

30µg/kg          (Indian Limit)            -           26%crop will be rejected

 

15 µg/kg         (Codex limit)             -           47%crop will be rejected

 

 

-                     It was estimated 10 million dollars were last in Indian export due to ground nut contamination with mycotoxins

 

 

Conclusion

 

-                     Occurrence of mycotoxins and resultant mycotoxicosis in human and animals is a complex series of interaction between contaminated food, causative fungi, environmental factors, and intoxicated host

-                     This complexity result in difficulty in achieving control over problem

-                     Newer threats are fore seen in health and economy with newer mycotoxins affecting staple foods

-                     Detection method for various mycotoxins in different food have improved in recent time with emphasis on simplicity, rapidity and sensitivity

-                     International harmonization of regulatory limits have to be viewed with caution specially for staple foods in areas where food availability is crucial factor

-                     Strategies for intervention includes

 

1) Social intervention

 

-                     Education of former and local whole seller and consumers about

-                     Hazards of mycotoxins

-                     Improved harvesting methods

-                     Post harvest handling

-                     Storage techniques

Thorough audiovisual methods and mass media, in this regard National Institute of Nutrition has  published a booklet which is also available in regional languages.

 

2. Technical intervention

 

-                     Development of appropriate programme

-                     Incorporation of antifungal gene into crops and use of these resistant varities

-                     Replacing aflatoxigenic strain with non aftatoxigenic strains of moulds

-                     Development and application of simple rapid cost affective methods of toxin detection in food and feed

-                     Application and combined physical and chemical methods of detoxification effectively

-                     Prevention of mycotoxin biosynthesis