Slaven Zjalić,

Department of ecology, agronomy and aquaculture,

University of Zadar, Croatia

Mycotoxins are secondary metabolites produced by some moulds

Most of mycoxtoxins have no odor and no taste

Through whole human history mycotoxinswere present in food and feed

Mycotoxins were discovered only in the last century after the outbreak of Turkey X desease in UK in 1960

In different farms more than 100.000 turkeys and ducklings died

The only thing in common was peanut based feed contaminated by Aspergillus flavus

The discovered toxin was named aflatoxin

Few years later the definition “mycotoxins” was assigned to the toxins produced by fungi

Up to day about 400 fungal metabolites are considered mycotoxins

Only a dozen of them of constant interest of scientist and authorities

Aflatoxins are among the most studied and controled mycotoxins

WHY?

Aflatoxins has genotoxic and cytotoxic effects on human and animal cells

They are teratogenic, cancerogenic and immnosuppressors

Aflatoxins can be transmitted allong the food chain, unchanged or modified in more or less toxic compounds

Moulds produce 4 aflatoxins B1, B2, G1, G2

Aspergillus flavus

Aspergillus parasiticus

More than 70% of produced aflatoxins

When iradiated with UV light aflatoxins have fluorescence blue (B) or green (G)

In the same organism different tissues are differently affected by aflatoxins

Detoxifications of animal cells from aflatoxins is operated by cytochrome P450 family

In mammalians part of AFB1 is transformed in less toxic M1 secreted in milk. The carryover is up to 6%

In 70´s different authors have hypothesized possible influences of aflatoxins, and mycotoxins, on human history:

- short life expectans of medieval man

- pest epidemies

- collective hysteria

Due to their toxicity the legislations of almost all the countries have limited the concentration of aflatoxins in food and feed commodities

EU

g kg-1

USA

g kg-1

China

g kg-1

Almonds 8 (B1)

12 (tot)

20 (tot) 30 (tot)

Animal feed 2 (B1) 20 (B1) 20 (B1)

Milk 0.05 (M1) 0.5 (M1) 0.5

Infant formulae 0.025 (M1) - -

Spices 5 (B1)

10 (tot)

20 (tot) -

Contamination with aflatoxins is a worldwide problem

Different commodities can be contaminated: cereal seeds, spices, dry fruits, meat products, cheese

Aflatoxigenic moulds requires relatively low aw (>0.7)

Contamination can occur in preharvest, in field, and postharvest period during any phase of transformation process or stocking

Producer

(farmer)

storage

transformation

Feed storage

Dairy cows milk

Aflatoxin control

Official control

Oxidative stress is considered a prerequisite for aflatoxin biosynthesis

Oily seeds, at same humidity, have higher aw and lipid oxidation triggers aflatoxin biosynthesis

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

afl

ato

xin

s (

g/k

g)

15 30 45

days

wheat

mais

peanuts

sunflower

For contamination in field high temperatures and drought or insect attack are needed

In Europe contamination with aflatoxins were considered more a problem of import or bad conservation

Due to global climatic changes in some years aflatoxin contamination in field is a serious problem in some European countries

Aflatoxin control is needed worldwide

Zone with perennial contamination risk

35°S

35°N

Mehl H.L., CORN UTILIZATION & TECHNOLOGY CONFERENCE (CUTC), INDIANAPOLIS, 2012

2003 and 2012 were “black” years for aflatoxin contamination in Europe

In 2013 Gafta (Grain and Feed Trade Association) has signalized reported high levels of aflatoxins in maize grains from:

Spain, Italy, Serbia, Bulgaria, Romania, Croatia

0

5

10

15

20

25

2009 2010 2011 2012 2013

%

wheat

maize

maize products

other cereals

Aflatoxin B1 concentration over EU limits in Croatia

Average temperatures, humidity and rain (1961-1990 and 1971-2000) compared to year 2012

Role of aflatoxins still not clarified

Gens are clustered, part of the signaling network that regulates expression of the cluster described

Incompleate aflatoxin cluster has been found in non producing strains

No method for total prevention of their presence in food and feed has been achieved

Control can be divided in:

prevention

detoxification

adsorption

Prevention

good agriculture practice (avoiding stress, quick postharvest drying)

aflatoxigenix fungi need relatively low aw, in corn dried to 14% of moisture aw could be on the limit for fungal growth

use of fungicides

Environmental problem

Aflatoxins are response to a stress, especially oxidative stress

Major growth of mould doesn’t mean higher production of aflatoxins

0

50

100

150

200

250

300

growth (erg) ATOX

g k

g-1

control

treated

use of antioxidants and organic acids

use of natural products (plant and fungal extracts) cont BHA 1 ppm

0

20

40

60

80

100

120

140

5 days

10 days

15 days

control BHA

15 30 45 15 30 45

Aflatoxins produced on maize seeds treated and non with BHA

Effect of extracts of different strains of Trametes

versicolor, an edible mushroom, on aflatoxin synthesis

by A. flavus

18S

hsf2-like

yap1-like

skn7-like

24h 36h 48h 60h 72h 4d 7d

SOD

0

2

4

6

8

10

24 h 36 h 48 h 60 h 72 h 96 h 7d

U m

g p

rote

in-1

Cont-pH

7,8

LF-pH

7,8

Cont-pH

10 ,0

LF-pH

10 ,0

CAT

0

2

4

6

8

10

12

24 h 36 h 48 h 60 h 72 h 96 h 7d

U m

g p

rote

in-1

Cont

LF

GPX

0

400

800

1200

24 h 36 h 48 h 60 h 72 h 96 h 7d

U m

g p

rote

in -1

Cont

LF

24h 36h 48h 60h 72h 96h 7d

18S

hsf2-like yap1-like

skn7-like

Cont LF

Inhibition seems to be related to the stimulation of

Aspergillus antioxidant system

Biocontrol

Use of non toxigenic strains as competitors

Use of resistant maize cultivars

Use of maize cultivars that stimulates less aflatoxin production (Burow et. al, 1997)

Comparison of constitutive and inducibile maize kernel proteins of genotype resistant or susceptible to aflatoxin production.

Chen ZY, Brown RL, Cleveland TE, Damann KF, Russin JS. J Food

Prot. 2001 Nov;64(11):1785-92.

A USA-Africa collaborative strategy for identifying, characterizing, and developing maize germplasm with resistance to aflatoxin contamination.

Menkir A.., Brown RL, Bandyopadhyvay R, Chen ZY, Cleveland

TE. Mycopathologia 2006, Sept; 162(3): 225-32

Molecular Plant-Microbe Interactions

MPMI Vol. 10, No. 3, 1997, pp. 380-387. Publication no. M-1997-0122-02R. © 1997

Seed Lipoxygenase Products Modulate Aspergillus Mycotoxin Biosynthesis

G. B. Burow, T. C. Nesbitt, J. Dunlap and N. P. Keller

Decontamination

removal of husks can significantly lowers the quantity of aflatoxins

chemical treatments

use of microbial enzymes

Most cultural filtrates (xylidine stimulated) degrade more than

90% of AFB1 on maize seeds after 72 hpi

A. flavus

inoculation on

maize seeds (1 kg) At 10 dpi, 1 ppm of

AFB1 is produced

Toxin residual/ g maize kernels

Isola

tes

Adsorption

different hydrated sodium calcium aluminosilicates (HSCAS) used to adsorb aflatoxins in animals

possible negative effects on adsorption of some nutrients

new generation of adsorbants based on cell walls

Estimated food contamination in Europe

Leblanc et al., 2005. and 2013. has estimated exposure to mycotoxins in French population

Results were encouraging, aflatoxin exposure was lower than 1ng kg-1 day-1

The most important sources were cereals followed by eggs, milk,

Aflatoxins represent high health hazard for humans and animals

No method for total prevention of their presence in food and feed has been achieved

Nevertheless the presence of aflatoxins in food and feed in EU countries is under control