Slaven Zjalić, Department of ecology, agronomy and aquaculture, University of Zadar, Croatia

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 B 1, B 2, G 1, G 2 More than 70% of produced aflatoxins Aspergillus flavus Aspergillus parasiticus

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 Almonds EU g kg -1 8 (B1) 12 (tot) USA g kg -1 China g kg -1 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 a w (>0.7) Contamination can occur in preharvest, in field, and postharvest period during any phase of transformation process or stocking

Producer (farmer) Aflatoxin control storage transformation Official control Feed storage milk Dairy cows

aflatoxins (g/kg) Oxidative stress is considered a prerequisite for aflatoxin biosynthesis Oily seeds, at same humidity, have higher a w and lipid oxidation triggers aflatoxin biosynthesis 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 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 35 N 35 S Zone with perennial contamination risk 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

% Aflatoxin B1 concentration over EU limits in Croatia 25 20 15 10 wheat maize maize products other cereals 5 0 2009 2010 2011 2012 2013

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 a w, in corn dried to 14% of moisture a w could be on the limit for fungal growth

use of fungicides Environmental problem Aflatoxins are response to a stress, especially oxidative stress g kg -1 300 250 200 150 100 control treated Major growth of mould doesn t mean higher production of aflatoxins 50 0 growth (erg) ATOX

use of antioxidants and organic acids 140 120 Aflatoxins produced on maize seeds treated and non with BHA 100 use of natural products (plant and fungal extracts) cont 80 60 40 20 0 BHA 1 ppm 15 30 45 15 30 45 5 days 10 days 15 days control BHA

Effect of extracts of different strains of Trametes versicolor, an edible mushroom, on aflatoxin synthesis by A. flavus

Inhibition seems to be related to the stimulation of Aspergillus antioxidant system U mg protein-1 SOD 10 8 6 4 2 0 24 h 36 h 48 h 60 h 72 h 96 h 7d Cont-pH 7,8 LF-pH 7,8 Cont-pH 10,0 LF-pH 10,0 CAT U mg protein -1 12 10 8 6 4 2 0 24 h 36 h 48 h 60 h 72 h 96 h 7d Cont LF GPX Cont hsf2-like yap1-like skn7-like 18S U mg protein -1 24h 36h 48h 60h 72h 96h 7d 1200 800 400 0 24 h 36 h 48 h 60 h 72 h 96 h 7d LF hsf2-like yap1-like skn7-like 18S Cont LF 24h 36h 48h 60h 72h 4d 7d

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

Isolates A. flavus inoculation on maize seeds (1 kg) At 10 dpi, 1 ppm of AFB1 is produced Toxin residual/ g maize kernels Most cultural filtrates (xylidine stimulated) degrade more than 90% of AFB1 on maize seeds after 72 hpi

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