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1 Mycotoxins Introduction. 2 Mycotoxins are secondary metabolites ( secondary metabolite: A compound that is not necessary for growth or maintenance of.

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Presentation on theme: "1 Mycotoxins Introduction. 2 Mycotoxins are secondary metabolites ( secondary metabolite: A compound that is not necessary for growth or maintenance of."— Presentation transcript:

1 1 Mycotoxins Introduction

2 2 Mycotoxins are secondary metabolites ( secondary metabolite: A compound that is not necessary for growth or maintenance of cellular functions but is synthesized, generally, for the protection of a cell or micro-organism, during the stationary phase of the growth cycle. Many are used in foods, pharmaceuticals, and other industrial applications.) of fungi that are recognized as toxic to other life forms. 1.Fungal growth a. Field fungi : fungi that attack plants that grow in the field (occurring prior to harvest) grow under special conditions. ( Fusarium ) b. Storage fungi : Storage fungi usually invade grain or seed during storage and are generally not present in large quantities before harvest in the field. The most common storage fungi are species of Aspergillus and Penicillium. Contamination occurs through spores contaminating the grain as it is going into storage from the harvest. The development of fungi is influenced by the: Moisture content of the stored grain Temperature Condition of the grain going into storage Length of time the is grain stored and Amount of insect and mite activity in the grain

3 3 2. Characteristics of mycotoxin induced disease a. not transmitted among animals b. Pharmaceutical treatment does not alter the course of disease c. Mycotoxicosis most often presents as a uncertain, sub-acute or chronic condition

4 4 3.Treatment of mycotoxin- induced disease a. For most mycotoxins, there is no specific treatment or antidote b. Supplement with vitamins & selenium may be helpful, and provision of adequate high-quality protein

5 5 4.Prevention of mycotoxin- induced disease a. Avoiding b. Diluting c. Cleaning d. Testing e. Drying f. Adding (organic acids will prevent mold growth)

6 6 A- Aflatoxin 1. Sources : Aspergillus flavus & A.paraciticus : Corn, peanuts 2. Factor favoring production of aflatoxins a. Temperature : ๐ c b. Grain moisture

7 7 3. Chemical characteristics Exhibit intense blue or green fluorescence under UV. : aflatoxins B1, B2, G1 and G2 also : aflatoxin M1 is a metabolites of AFB1 found in animal urine, milk or tissues.

8 8 5.Mechanism of toxicologic damage Also called steatosis, fatty liver can be a temporary or long-term condition, which is not harmful itself, but may indicate some other type of problem. Left untreated, it can contribute to other illnesses. It is usually reversible once the cause of the problem is diagnosed and corrected. The liver is the organ responsible for changing fats eaten in the diet to types of fat that can be stored and used by the body. Triglycerides are one of the forms of fat stored by the body and used for energy and new cell formation. The break down of fats in the liver can be disrupted by alcoholism, malnutrition, pregnancy, or poisoning. In fatty liver, large droplets of fat, containing mostly triglycerides, collect within cells of the liver. The condition is generally not painful and may go unnoticed for a long period of time. In severe cases, the liver can increase to over three times its normal size and may be painful and tendermalnutritionpoisoning

9 9 a. Loss of enzyme b. Lack of formation of lipid acceptor protein in liver c. Decreased cellulose digestion, volatile fatty acid formation & proteolysis (breakdown of proteins ) d. Necrosis

10 10 6.Toxicity a. Young animals are more susceptible than adult. b. Nutrition deficiency increase susceptibility

11 11 7. Diagnosis Clinical sign : decreased growth rate, reduced feed efficiency,,, mild anemia, and increased susceptibility to infectious disease.

12 12 8.Treatment & Prevention a. Detoxification : Hydrated sodium calcium aluminosilicate (HSCAS) can absorb aflatoxins b. Supportive : Vitamin.E & selenium c. Prevention - Mold inhibitor - Treatment of grain with anhydrous ammonia for days.

13 13 B- Zearalenone 1. Sources : Fusarium roseum ( F.graminearum ): corn, wheat, barley, oats 2. Factor favoring production a. High moisture 22% - 25% b. Alternating high and low temp. (7-21 ๐ c)

14 14 3. Mechanism of toxicological damage a. initiating specific RNA synthesis b. Function as a weak estrogen. 4.Toxicity a. Swine are most susceptible b. low for all effects except reproductive function.

15 15 C-Ergot 1.Source : Claviceps purpurea : barley, wheat & oats 2. Factor favoring : Warm & humid

16 16 3.Mechanism of toxic a. potent initiators of contraction in smooth muscle b. mimic the action of dopamine. a. necrosis of the feet, ears and tail b. increased temperature., pulse & respiration rate c. lactation does not occur d. hyper-excitability & tremors e. heat intolerance in cattle 4.Clinical sign

17 17 E.Treatment a. animals should be provided with a warm, clean, stress-free environment b. Control secondary bacterial infection c. milk supplement

18 18 D- Ochratoxin & Citrinin 1.Sources : Aspergillus orchraceus & Penicillium viridicatum 2. Mechanism of toxic : target the renal proximal tubule - Disrupt protein synthesis -Bind strongly to protein (albumin) -Interfere with synthesis of tRNA & mRNA -Disrupt carbohydrate metabolism -Increase the generation of free radical

19 19 4.Clinical sign a. Acute : vomiting, diarrhea, dehydration & depression b. Subacute to chronic : weight loss, feed efficiency, & dehydration. Immunosupression, teratogenicity, carcinogenesis & hemorrhage

20 20 Mycotoxins

21 21 Mycotoxin Mycotoxin is a convenient generic term describing the toxic secondary metabolites produced by fungi. “Myco” means fungal (mold) and “toxin” represents poison. They encompass a considerable variety of low molecular weight compounds with diverse chemical structures and biological activities. Some mycotoxins could also be toxic to plants or other microorganisms; but these compounds are not classified as antibiotics of fungal origin. Like most microbial secondary metabolites, the benefit of mycotoxins for the fungi themselves is still not clearly defined.

22 22 In considering the effects of mycotoxins on animals, it is important to distinguish between “mycotoxicosis” and “mycosis.”: Mycotoxicosis is used to describe the action of mycotoxin(s) and is frequently mediated through a number of organs, notably the liver, kidney, lungs, and the nervous, endocrine, and immune systems. Mycosis” refers to a generalized invasion of living tissue(s) by growing fungi.

23 23 Due to their diverse chemical structures, mycotoxins may exhibit a number of biological effects, including both acute and chronic toxic effects as well as carcinogenic, mutagenic, genotoxic, and immunotoxic effects. The interaction of mycotoxins with cellular macromolecules plays a dominant role in their toxic actions. Recent studies on the effect of mycotoxins on apoptosis have further revealed their mode of action at the cellular level.

24 24 Historical Modern mycotoxicology was not developed until the discovery of aflatoxins in the early 1960s as the causative agent in the peanut meal causing the “Turkey X” disease that killed more than 10,000 turkeys fed with the contaminated meal. Because aflatoxins are a series of highly potent carcinogens produced by commonly occurring Aspergillus flavus and A. parasiticus, research has focused new attention on mycotoxins. In the last 40 years, many new mycotoxins have been identified and characterized, and their biosynthetic origin in various fungi elucidated. It has been estimated that at least 25% of the world’s agricultural product is contaminated with mycotoxins and certain diseases have been linked to ingestion of food and feed contaminated with mycotoxins.

25 25 Economic Impact of Mycotoxin Contamination The most obvious negative economic impact of mycotoxins is an outright loss of crops and affected animals. Also, humans may encounter severe health hazard or high mortality rates in countries with less regulation or monitoring programs. Thus, the negative economic impact resulting from mycotoxin contamination is certainly very significant and estimated to be $932 million annually.

26 26 PRODUCTION OF MYCOTOXINS BY TOXICOGENIC FUNGI Invasion by fungi and production of mycotoxins in commodities can occur under favorable conditions in the field, at harvest, and during processing, transportation and storage Fungi that are frequently found in the field include: A. flavus, Alternaria longipes, A. alternata, Claviceps purpura, Fusarium verticillioides (previously called moniliforme), F. graminearum, and a number of other Fusarium spp. Species most likely introduced at harvest include: F. sporotrichioides, Stachybotrys atra, Cladosporium sp., Myrothecium verrucaria, Trichothecium roseum, as well as A. alternata. Most penicillia are storage fungi. These include: Penicillium citrinum, P. cyclopium, P. citreoviride, P. islandicum, P. rubrum, P. viridicatum, P. urticae, P. verruculosum, P. palitans, P. puberulum, P. expansum, and P. roqueforti. All of which are capable of producing mycotoxins in grains and foods.

27 27 Other toxicogenic storage fungi are: Aspergillus. parasiticus, A. flavus, A. versicolor, A. ochraceus, A. clavatus, A. fumigatus, A. rubrum, A. chevallieri, Fusarium verticillioides, F. tricinctum, F. nivale, and several other Fusarium spp. It is apparent, most of the mycotoxin producing fungi belong to three genera: Aspergillus, Fusarium, and Penicillium. However, not all species in these genera are toxicogenic

28 28 Factors Affecting Mycotoxin Production Genetics and environmental and nutritional factors greatly affect the formation of mycotoxins. Depending on the susceptibility of the crop, geographic and seasonal factors, as well as cultivation, harvesting, storage, and transportation practices, mycotoxins are found worldwide. In the field, weather conditions, plant stress, invertebrate vectors, species and spore load of infective fungi, variations within plant and fungal species, and microbial competition all significantly affect mycotoxin production.

29 29 Physical factors such as time of exposure, temperature during exposure, humidity, and extent of insect or other damage to the commodity prior to exposure determine mycotoxin contamination in the field or during storage. Chemical factors including the nutritional status of the crops or chemicals (such as fungicides) used in crop management could affect fungal populations, and consequently toxin production Continue Factors Affecting…….

30 30 In general, mycotoxins are optimally produced at 24–28C, but some toxins such as T-2 toxin is maximally produced at 15C. Contamination during crop storage may be affected by changes in temperature and water activity, that allow ecological succession of different fungi as water activity and temperature of stored grain changes. Continue Factors Affecting……. Water activity = It is defined as the vapor pressure of water above a sample divided by that of pure water at the same temperature;vapor pressuretemperature

31 31 During storage and transportation, water activity (aw), temperature, crop damage, and a number of physical and chemical factors, such as aeration (O2, CO2 levels), types of grains, pH, and presence or absence of specific nutrients and inhibitors are important.aw Continue Factors Affecting…….

32 32 Aflatoxins R1R1 R2R2 R3R3 R4R4 A B DC R1R1 R1R1 R1R1 (A)The B-type aflatoxins are characterized by a cyclopentane E- ring. These compounds have a blue fluorescence under long- wavelength ultraviolet light. (B) The G-type aflatoxins, with a green fluorescence, have a xanthone ring in place of the cyclopentane. (C) Aflatoxins of the B2 and G2 type have a saturated bis-furanyl ring. Only the bis-furan is shown. (D) Aflatoxin of the B1a and G1a type have a hydrated bis-furanyl structure. Chemical structure of flatoxins

33 33 At least 16 structurally related toxins in this group are produced by Asparagillus flavus and A. parasiticus and infrequently by A. pseudotamarii and A. nominus A. ochraceoroseus has also been found to produce aflatoxins The optimal temperatures and water activity (aw) for the growth of A. flavus and A. parasiticus are around 35–37C (range from 6–54C) and 0.95 (range from 0.78–1.0), respectively; whereas for aflatoxin production, they are 28–33C and 0.90–0.95 (range from 0.83–0.97), respectively.aw

34 34 Aflatoxin B1 is most toxic in this group and is one of the most potent naturally occurring carcinogens). Other significant members of the aflatoxin family, such as M1 and M2, are metabolites of AFB1 and AFB2, respectively, and originally isolated from bovine milk.

35 35 Natural Occurrence Aflatoxins have been found in corn, peanuts فول سودانيّ and peanut products, cotton seeds, peppers, rice, pistachios, فستق tree nuts, pumpkin قرع seeds, sunflower seeds and other oil seeds, copra, جوز هند spices, and dried fruits (figs, raisins). Among these products, frequent contamination with high levels of AF in peanuts, corn, and cottonseed, mostly due to infestation with fungi in the field, are of most concern. Soybeans الصويا, beans الفاصولياء, pulses (Pea)البازلاء, cassava منيهوت, sorghum الذرة, millet الدخن, wheat القمح, oats القطن, barley الشعير, and riceرز are resistant or only moderately susceptible to AF contamination in the field. It should be reiterated that resistance to AF contamination in the field does not guarantee that the commodities are free of AF contamination during storage. Inadequate storage conditions, such as high moisture and warm temperatures (25–308C), can create conditions favorable for the growth of fungus and production of AF.

36 36 Aflatoxins are mutagenic, teratogenic, and hepatocarcinogenic. Aflatoxin B1 is one of the most potent naturally occurring carcinogen, extensive research was primarily done on this toxin. The main target organ of AF is the liver. AFB1 also affects other organs and tissues including the lungs and the entire respiratory system. For the carcinogenic effects, rats, rainbow trout, monkeys, and ducks are most susceptible and mice are relatively resistant.rainbow trout Consumption of AFB1-contaminated feed by dairy cows results in the excretion of AFM1 in milk. AFM1, a hydroxylated metabolite of AFB1, is about 10 times less toxic than AFB1; but its presence in milk is of concern for human health. Toxic Effects

37 37 Impact on Human Health Whereas AFB1 has been found to be a potent carcinogen in many animal species, the role of AF in carcinogenesis in humans is complicated by hepatitis B virus (HBV) infections in humans). Epidemiological studies have shown a strong positive correlation between AF levels in the diet and primary hepatocellular carcinoma. Since multiple factors are important in carcinogenesis and environmental contaminants such as AFs and other mycotoxins may, either in combination with HBV or independently.

38 38 Ochratoxins Ochratoxins, are produced by a number of fungi in the genera Aspergillus and Penicillium. The largest amounts ochratoxins are made by A. ochraceus and P. cyclopium. A. ochraceus and P. viridicatum (reclassified as P. verrucosum), two species that were first reported as ochratoxin A (OA) producers, occur most frequently in nature. Other fungi, such as Petromyces alliceus, A. citricus, and A. fonsecaeus (both in A. niger group), have also been found to produce OA. Most of the OA producers are storage fungi and preharvest fungal infection.

39 39 Although most OA producers can grow in a range from 48C to 37C and at aw as low as 0.78, optimal conditions for toxin production are narrower with temperature at 24–25C and aw values Ochratoxins are produced primarily in cereal grains (barley, oats, corn, wheat) and mixed feed during storage in temperate climatic conditions, with levels higher than 1 ppm being reported. OA has been found in other commodities, including beans, coffee, nuts, olives, raisin, cheese, fish, pork, milk powder, fruit juices wine beer, peppers. OA can be carried through the food chain because of the presence of OA residues in animal products as result of its binding with serum albumin. Natural occurrence of OA in kidneys, blood serum, blood sausage.

40 40 Structure of the ochratoxins. These metabolites form different classes depending on the nature of the amide group (a–c), and the presence or absence of a chlorine moiety at R2 in the phenyl group.

41 41 Ochratoxin A, the most toxic member of this group of mycotoxins, has been found to be a potent nephrotoxin causing kidney damage as well as liver necrosis and enteritis in many animal species The OA inhibits carboxypeptidase A, renal phosphoenolpyruvate carboxykinase, phenylalaninetRNA synthetase, and phenylalanine hydroxylase activity. Formation of free radicals has been considered as one of the mechanisms for the carcinogenic/toxic effects of OA.

42 42 Fumonisins Fumonisins (Fm) are a group of toxic metabolites produced primarily by F. verticillioides, F. proliferatum and other related species readily colonize corn all over the world. Although F. anthophilum, F. nupiforme, and F. nygamai are capable of producing Fms. More than 11 structurally related Fms (B1, B2, B3, B4, C1, C4, A1, A2, etc.), have been found since the discovery of FmB1. Fumonisins are most frequently found in corn, corn-based foods, and other grains (such as sorghum and rice). The level of contamination varies considerably with different regions and year, ranging from negligible to more than 100 ppm; but is generally reported to be between 1 and 2 ppm. FmB1 is the most common Fm in naturally contaminated samples; FmB2 generally accounts for 1/3 or less of the total. Although production of the toxin generally occurs in the field, continued production of toxin during postharvest storage also contributes to the overall levels.

43 43 Toxicologic Effects Fumonisin B1 is primarily a hepatotoxin and carcinogen in rats. Feeding culture material from F. verticillioides or pure FmB1 to rats resulted in cirrhosis and hepatic nodules, carcinoma. Kidney is also a target organ. Mechanistically, Fms are inhibitors of ceramide synthase (sphinganine/sphingosine N-acyltransferase), a key enzyme involved in the biosynthesis of sphingolipids, which are heavily involved in cellular regulation, including cell differentiation, mitogenesis and apoptosis تليّف كبديّ The ability of FmB1 to alter gene expression and signal transduction pathways are considered necessary for its carcinogenic and toxic effects. FmB1 is a good example of an apparently non-genotoxic (non-DNA reactive) agent producing tumors through the regulation of apoptosis

44 44 Other fungal genera elaborate TCTCs are: Myrothecium, Trichoderma, Trichothecium, Cephalosporium, Verticimonosporium, and Stachybotrys. In addition to fungi, extracts from a Brazilian shrub, Baccharis megapotamica, also contain macrocyclic TCTCs. The term TCTCs is derived from trichothecin, the first compound isolated in this group. Several species of Fusaria infect corn, wheat, barley, and rice. Under favorable conditions, they elaborate a number of different types of mycotoxins (look figure). (TCTCs) are generally classified as macrocyclic (Type C) or nonmacrocyclic (Types A and B). Although more than 100 TCTCs have been identified, only a few frequently found in foods and feeds are potentially hazardous to human and animal health. Trichothecenes (TCTCs) Trichothecenes

45 45 The TCTC mycotoxicoses affect many organs, including the gastrointestinal tract, hematopoietic, nervous, immune, hepatobiliary, and cardiovascular systems. Mechanistically, inhibition of protein synthesis is one of the earlier events in manifestation of TCTC toxic effects and they act at different steps in the translation process. Inhibitory effects of these mycotoxins vary considerably with the chemical structure of the side chain. T-2 toxin, a highly toxic type A TCTC, is produced by F. tricinctum, F. sporotrichioides (major), F. poae, F. sulphureum, F. acuminatum, and F. sambucinum. Unlike most mycotoxins, which are usually synthesized near 25C, the optimal temperature for T-2 toxin production is around 15C. a) T-2 toxin,

46 46 Almost all the major TCTCs, including T-2 toxin, are cytotoxic and cause hemorrhage, edema, and necrosis of skin tissues. b) Deoxynivalenol (DON) The DON is a major type B TCTC mycotoxin produced by F. graminearum (major) and other related fungi such as F. culmorum and F. crookwellense. Because DON causes feed refusal and emesis in swine, the name “vomitoxin” is also used for this mycotoxin.

47 47 Worldwide frequent natural occurrence of DON in cereal grains has been reported. Contamination of this toxin in corn and wheat is generally high. Also, contamination of barley, oats, sorghum, rye, safflower seeds, and mixed feeds has also been reported. Although inadequate storage may lead to the production of some TCTC mycotoxins, infestation of fusaria in wheat and corn in the field is of most concern for the DON problem. With wet and cold weather during maturation,grains are especially susceptible to F. graminearum infection, which causes so-called “scabby wheat” and simultaneously produces the toxin. The optimal temperature for DON production is about 248C.

48 48 Toxicologically, DON induces anorexia and emesis both in humans and animals. Swine are most sensitive to feed contaminated with DON. Whereas most TCTCs are immunosuppressors, DON is a hyperinducer of cytokines.anorexiaemesis

49 49 Other Selected Mycotoxins In addition to the mycotoxins discussed above, a number of other mycotoxins occur naturally. Other Mycotoxins Produced by Aspergillus: Sterigmatocystin (ST) is a naturally occurring hepatotoxic and carcinogenic mycotoxin produced by fungi in the genera Aspergillus, Bipolaris, and Chaetomium as well as P. luteum. Structurally related to AFB1 ST is known to be a precursor of AFB1. ST is a mutagen and genotoxin and has been found in cereal grains (barley, rice, and corn), coffee beans, and cheese. Structure of sterigmatocystin. The bis-furanyl structure is similar to that of the aflatoxins except that the E-ring is a substituted phenol.

50 50 A. terreus and several other fungi (e.g., A. flavus and A. fumigatus) have been found to produce the tremorgenic toxins, territrems, aflatrem, and fumitremorgin. A. terreus, A. fumigatus, and Trichoderma viride also produce gliotoxin, In addition, A. flavus, A. wentii, and A. oryzae, are capable of producing nitropropionic acid (NPA), a mycotoxin causing apnea, convulsions, congestion in lungs and liver.apneaconvulsions Production of NPA in sugarcanes by Arthrinium sacchari, Arth. saccharicola, and Arth. Phaeospermum has been found to be involved in fatal food poisoning in humans

51 51 Other Mycotoxins Produced by Penicillium Penicillia produce many mycotoxins with diverse toxic effects. Cyclochlorotine, luteoskyrin (LS), and rugulosin (RS) have long been considered to be possibly involved in the yellow rice disease during the Second World War. They are hepatotoxins. Several other mycotoxins, including patulin (PT) penicillic acid (PA) citrinin (CT), cyclopiazonic acid (CPA, citreoviridin, and xanthomegnin, which are produced primarily by several species of Penicillia. PT and PA are produced by many species in the genera Aspergillus and Penicillium. Byssochlamys nivea also produces PT penicillic acid Patulin

52 52 Chemical structure of cyclopiazonic acid Stucture of zearalenone Structure of alternariol

53 53 Other Mycotoxins Produced by Fusarium Some fusaria are capable of producing mycotoxins other than TCTCs and Fm. Zearalenone (ZE) a mycotoxin produced by the scabby wheat fungus, F. graminearum (roseum), is of most concern. Also called F-2, ZE is a phytoestrogen causing hyperestrogenic effects and reproductive problems such as premature onset of puberty in female animals,especially swine. ZE has been shown to bind with the estrogen and steroid receptors, and stimulates protein synthesis by mimicking hormonal action. Zearalenone can be toxic to plants; it can inhibit seed germination and embryo growth at low concentrations. Natural contamination with ZE primarily occurs in cereal grains such as corn and wheat

54 54 Fusarium verticillioides and related species, also produce several other mycotoxins, including fusarins A-F, moniliformin, fusarioic, and fusaric acid, fusaproliferin and beauvericin. Although the impact of these mycotoxins on human health is still not known, fusarin C (FC) has been identified as a potent mutagen and is also produced by F. subglutinans, F. graminearium and several other Fusaria. Moniliformin, which causes cardiomyopathy in test animals, may be involved in the Keshan disease in humans in regions where dietary selenium deficiency is also a problem. Among many fungi, F. verticilioides is also most capable of reducing nitrates to form potent carcinogenic nitrosamines. These observations further suggest that the contamination of foods with this fungus could be one of the etiological factors involved in human carcinogenesis in certain regions of the world.

55 55 Zearalenone Alternariol Mycotoxins Produced by Alternaria Species Alternaria has been known for centuries to cause various plant diseases. Species of this fungus are widely distributed in soil and on aerial plant parts. More than 20 species of Alternaria are known to produce about 70 secondary metabolites belonging to a diverse chemical group. However, only alternariol tenuazonic acid, altertoxin-I, alternariol monomethyl ether (AME), altenuene are common contaminants in consumable items like fruits (apples), vegetables (tomato), cereals (sorghum, barely, oat), and other plant parts (such as leaves) The most common species of Alternaria, A. alternata (formerly known as A. kikuchiana) produces all important Alternaria toxins including the five mentioned above and tentoxin, alteniusol, alternaric acid, altenusin, dehydroaltenusin

56 56 Mycotoxins Produced by Other Fungi Sporidesmines, a group of hepatotoxins discovered in the 1960s. These mycotoxins, causing facial eczema in animals, are produced by Pithomyces chartarum and Sporidesmium chartarum and are very important economically to the sheep industry. Slaframine, a significant mycotoxin produced by Rhizoctonia leguminicola (in infested legume forage crops).

57 57 PREVENTIVE MEASURES Management of Mycotoxin Contamination The economic implications of the mycotoxin problem and its potential health threat to humans have clearly created a need to eliminate or at least minimize mycotoxin contamination of food and feed. While an association between mycotoxin contamination and inadequate storage conditions has long been recognized, studies have revealed that seeds are contaminated with mycotoxins prior to harvest. Therefore, management of mycotoxin contamination in commodities must include both pre- and postharvest control measures

58 58 Preharvest Control Mycotoxin contamination can be reduced somewhat by using of resistant varieties (most effective, but not all are successful) and earlier harvest varieties: –crop rotation, –adequate irrigation, –control of insect pests. Significant control of toxin contamination is expected to be dependent on a detailed understanding of the: –physiological and environmental factors that affect the biosynthesis of the toxin, –the biology and ecology of the fungus, –the parameters of the host plant–fungal interactions. Efforts are underway to study these parameters primarily for the most agriculturally significant toxins, namely AFs, Fms, and TCTCs

59 59 Use of atoxigenic biocompetitive, native A. flavus strains to out-compete the toxigenic isolates has been effective in significantly reducing preharvest contamination with aflatoxin in cotton and peanuts. However, the aflatoxin contamination process is so complex that a combination of approaches will be required to eliminate or even control the preharvest toxin contamination problem.

60 60 Mycotoxins and food chain Fungal contamination Vegetables Man Animal Production Products ( Animal origin) Many accidents rare accidents (cancer) Elimination

61 61 Postharvest Control After harvest, crop should not be allowed to over-winter in the field as well as subjected to birds and insects damage or mechanical damage. Grains should be cleaned and dried quickly to less than 10–13% moisture and stored in a clean area to avoid insect and rodent infestation. Postharvest mycotoxin contamination is prevalent in most tropical countries due to: a hot, wet climate coupled with subadequate methods of harvesting, (handling, and storage practices), which often lead to severe fungal growth and mycotoxin contamination of food and feed.

62 62 Sometimes contaminated food has been diverted to animal feed to prevent economic losses and health concerns. However, this is not a solution to the contamination problem. Irradiation has been suggested as a possible means of controlling insect and microbial populations in stored food, and consequently, reducing the hazard of mycotoxin production under these conditions. Significant emphasis has been placed on detoxification methods to eliminate the toxins from the contaminated lots or at least reduce the toxin hazards by bringing down the mycotoxin levels under the acceptable limits.

63 63 I. Removal or Elimination of Mycotoxins. –Since most of the mycotoxin burden in contaminated commodities is localized to a relatively small number or seeds or kernels removal of these contaminated seeds/kernels is effective in detoxifying the commodity. Methods currently used include: –(a) physical separation by: –identification and removal of damaged seed; – mechanical or electronic sorting; –flotation and density separation of damaged or contaminated seed; – physical screening and subsequent removal of damaged kernels by air blowing; –washing with water – use of specific gravity methods All these methods have shown some effect for some mycotoxins, including DON, FmB, and AFB1 – (b) removal by filtration and adsorption onto filter pads, clays, activated charcoal, etc., –(c) removal of the mycotoxin by solvent extraction

64 64 II. Inactivation of Mycotoxins. When removal or elimination of mycotoxins is not possible, mycotoxins can be inactivated by: (a) physical methods such as thermal inactivation, photochemical or gamma irradiation, (b) chemical methods such a treatment of commodities with acids, alkalies, aldehydes, oxidizing agents, and gases like chlorine, sulfur dioxide, NaNO2, ozone and ammonia, (c) biological methods such as fermentations and enzymatic digestion that cause the breakdown of mycotoxins. The commercial application of some of these detoxifying mechanisms is not feasible because, in a number of cases, the methods will be limited by factors such as the toxicity of the detoxifying agent, nutritional or aesthetic losses of commodities during treatment, and the cost of the sophisticated treatment. Although several detoxification methods have been established for aflatoxins, only the ammoniation process is an effective and practical method. Other chemicals such as ozone, chlorine, and bisulfite have been tested and some effect for some mycotoxins was shown in it. Solvent extractions have been shown to be effective but are not economically feasible.

65 65 III. Removal of Mycotoxins During Food Processing. While cooking generally does not destroy mycotoxins, some mycotoxins can be detoxified or removed by certain kinds of food processing. –For example, extrusion cooking appears to be effective for detoxifying DON but not AFB. FmB1 can form Schiff’s bases with reducing sugars such as fructose under certain conditions and lose its hepato-carcinogenicity; but the hydrolyzed FmB1 was found to be still toxic.

66 66 Avoiding Human Exposure Role of Rigorous Monitoring Programs While it is impossible to remove mycotoxins completely from foods and feeds, effective measures to decrease the risk of exposure depend on a rigorous program of monitoring mycotoxins in foods and feeds. Consequently, governments in many countries have set limits for permissible levels or tolerance levels for a number of mycotoxins in foods and feeds. Over 50 countries of the world have developed such guidelines. For example, levels varying from zero tolerance to 50 ppb have been set for total AFs. A tolerance level of 1 ppm for DON in grains for human consumption has been set by a number of countries, including the United States. The FmB1 levels established by FDA in 2000 are limited to 5, 20, , 30, and 10 ppm, in corn and corn by-products to be used for horse and rabbit, catfish and swine, and mink, poultry,

67 67 Among 77 countries which have regulations for different mycotoxins, eight have specific regulations for OA, with limits ranging from 1 to 20 mg/kg in different foods. Regulatory guidelines to limit the presence of PT to 50mg/kg in various foods and juices have been established by at least ten countries worldwide. Details on worldwide regulatory issues and permissive levels of mycotoxins in foods and feeds have appeared in a number of recent reviews.

68 68 Detection and Screening of Mycotoxins Because of the diverse chemical structures of mycotoxins, the presence of trace amounts of toxins in very complicated matrices that interfere with analysis, and the uneven distribution of the toxins in the sample, analysis of mycotoxins is a difficult task. Because many steps are involved in the analysis, it is not uncommon that the analytical error can amount to 20–30% To obtain reliable analytical data, an adequate sampling program and an accurate analytical method are both important. To minimize the errors, studies have led to many improved and innovative analytical methods for mycotoxin analysis over the years. New, more sensitive TLC, HPLC, and GC techniques are now available. The MS methods have also been incorporated into HPLC systems. New chemical methods, including capillary electrophoresis and biosensors are emerging and have gained application for mycotoxin analysis.

69 69 After a number of years of research, immunoassays have gained wide acceptance as analytical tools for mycotoxins in the last decade. Antibodies against almost all the mycotoxins are now available. Some quantitative and qualitative immunoassays have been approved. Many immunoscreening kits, which require less than 15 min. per test, are commercially available. Rather than analysis of toxin, PCR methods, based on the primers of key enzymes involved in the biosynthesis of mycotoxins, have been introduced for the determination of toxicogenic fungi present in foods. Detailed protocols for mycotoxin analysis can be seen in several of the most recent reviews and books and the most recent edition of AOAC.

70 70 Dietary Modifications Dietary modification greatly affects the absorption, distribution, and metabolism of mycotoxin and subsequently affect its toxicity. For example, the carcinogenic effect of AFB1 is affected by nutritional factors, dietary additives, and anticarcinogenic substances. Diet containing chemoprotective agents and antioxidants such as ascorbic acid, and even green tea, have also been found to inhibit carcinogenesis caused by AFB1 in test animals. The toxic effect of OA and FmB to test animals was minimized when antioxidants such as vitamins C and E are added to the diet. Ascorbic acid also provided protective effect against AFs. Most mycotoxins have a high affinity for hydrated sodium calcium aluminasilicate (HSCAS) and other related products.

71 71 CONCLUSIONS Mycotoxins are low molecular weight secondary metabolites of fungi that are contaminants of agricultural commodities, foods, and feeds. Fungi that produce these toxins do so both prior to harvest and during storage. Although contamination of commodities by toxigenic fungi occurs frequently in areas with a hot and humid climate, they can also be found in temperate conditions. Production of mycotoxins is dependent upon the type of producing fungus and environmental conditions such as the substrate, water activity (moisture and relative humidity), duration of exposure to stress conditions, and microbial, insect, or other animal interactions.

72 72 Although outbreaks of mycotoxicoses in humans have been documented, several of these have not been well characterized, neither has a direct correlation between the mycotoxin and resulting toxic effect been well established in vivo. Even though the specific modes of action of most of the toxins are not well established, acute and chronic effects in prokaryotic and eukaryotic systems, including humans have been reported. The toxicity of the mycotoxins varies considerably with the toxin, the animal species exposed to it, and the extent of exposure, age, and nutritional status.

73 73 Most of the toxic effects of mycotoxins are limited to specific organs, but several mycotoxins affect many organs. Induction of cancer by some mycotoxins is a major concern as a chronic effect of these toxins. It is nearly impossible to eliminate mycotoxins from food and feed in spite of the regulatory efforts at the national and international levels to remove the contaminated commodities. This is because mycotoxins are highly stable compounds, the producing fungi are ubiquitous, and food contamination can occur both before and after harvest. Nevertheless, good farm management practices and adequate storage facilities minimize the toxin contamination problems.

74 74 A combination of natural biocontrol competition fungi and enhancement of host-resistance against fungal growth or toxin production could prevent toxin formation to a very significant extent. Rigorous programs for reducing the risk of human and animal exposure to contaminated food and feed also include: economically feasible safe detoxification processes dietary modifications.

75 75 Additional, systematic epidemiological data for human exposure is needed for establishing toxicological parameters for mycotoxins and the safe dose for humans. It is unreasonable to expect complete elimination of the mycotoxin problem. But multiple approaches will be needed to minimize the negative economic impact of the toxins on the entire agriculture industry as well as their harmful effects on human and animal health.


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