Mycotoxins in Foods and Feeds in Morocco: Occurrence, Sources of Contamination, Prevention/Control and Regulation

Introduction

My'cotoxins are secondary' metabolites, which are very' harmful to human and animal health. They' are produced by' molds mainly belonging to the genera Aspergillus, Penicillium and Fusarium. Park et al. (1999) reported that the Food and Agriculture Organization (FAO) of the United Nations estimated that at least 25% of the world's food crops are contaminated with mycotoxins. According to Eskola et al. (2019), the origin of the statement that the FAO estimated global food crop contamination with my'cotoxins to be 25% is largely' unknown. To assess the rationale for it, the authors examined the relevant literature and data of around 500,000 analyses from the European Food Safety Authority (EFSA) and large global survey for aflatoxins (AFs), fumonisins В (FB), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT- 2), zearalenone (ZEA) and ochratoxin A (OTA) in cereals and nuts. This study seems to confirm the value of 25% estimated by' FAO, although this figure underestimates, according to the study', the occurrence of my'cotoxins above the detection limits (up to 60-80%). These figures only' point out that, in terms ^[1]

of health, contamination of food with mycotoxins is one of the main global health concerns. The European Union's rapid alert system for food and feed reports shows that mycotoxins are hi first place according to the total number of danger notifications (RASFF 2018). In Morocco, the mycotoxin problem has been identified as one of the major challenges for food safety (Montet et al. 2020).

Several mycotoxins are thermally stable and are not easily eliniinated during food processing or by physical and chemical treatments. Currently, more than 400 mycotoxins have been reported but only a limited number have toxic characteristics for humans and/or animals (Pitt et al. 2000, Shi et al. 2018). Certain mycotoxins can be produced by several species belonging to different genera. Likewise, a species can develop several mycotoxins. However, within the same species known to be toxigenic, not all strains have this capacity (Hussein and Brasel 2001, Reboux 2006). The number of contaminated products and emerging mycotoxins continues to increase due to the evolution of extraction and analysis techniques. Indeed, since the first detection of mycotoxins by TLC, several analysis techniques have been developed (ELISA/HPLC, GC, GC-MS, LC-MS, LC-MS/MS ...) leading to multi-detection and quantification of mycotoxins in foodstuffs.

The presence of mycotoxins has been strongly studied in various foods such as cereals and derivatives (Lee and Ryu 2017), coffee and tea drinks (Pallares et al. 2017, Garcia-Moraleja et al. 2015), vegetables (Dong et al. 2019), fruit juices and cooked foods (Sakurna et al. 2013, Carballo et al. 2018). In addition to the human and animal health problem, mycotoxins also cause significant economic losses (Pitt and Miller 2017, Wu and Mitchell 2016). Currently, the most important mycotoxins from a food safety and regulatory point of view are AFs, DON, T-2, HT-2, ZEA, FB, OTA, ergot alkaloids (EA), patulin (PAT) and citrinin (CIT) (Eskola et al. 2019).

Mycotoxins

2.1 Aflatoxins

Aflatoxins were first identified in 1960 as the causative agents of "Turkey X disease" killing 100,000 turkeys in England. In humans, the first aflatoxicoses were reported in India in 1974 causing the death of 100 people (Krishnamachari et al. 1975) and in Kenya in 2004, resulting in the death of 125 people (Muture and Ogana 2005). In both cases, the consumption of corn contaminated with high aflatoxins levels seems to have caused the epidemic. Aflatoxins are toxic metabolites synthesized mainly by the species of Aspergillus parasiticus, A. flavus and A. nomius (Van den Broek et al. 2001, Richard 2007). Four types of aflatoxins can be produced by these species: AFB1, AFB2, AFG1 and AFG2. The species A. flavus produces only aflatoxins В while the other two species can produce both aflatoxins В and G (Creppy 2002, Bennett and Klich 2003). The growth of Aspergillus strains and the production of four aflatoxins (AFB1, AFB2, AFG1 and AFG2) are highly dependent on environmental factors such as temperature, humidity, aeration and the nature of the environment. The major proliferation risk is linked to transport and storage conditions. Aflatoxin- producing fungi grow on a wide variety of foods such as cereals (corn, rice, barley, oats and sorghum), dried fruits (grapes and figs), peanuts, pistachios, almonds, nuts and cotton seeds (Bennett and Klich 2003, De Boevre 2012, Soubra et al. 2009). Milk can also be contaminated with aflatoxin Ml (AFM1) produced by hydroxylation of AFB1 by the hepatic microsomal cytochrome P450 in cows fed with a diet contaminated with AFB1 (Bennett and Klich 2003). AFM1 can also be detected in cheese with a higher concentration than that of raw milk. AFM1 is thermostable, can bind to casein and therefore is not affected by the cheese-making process (Barbiroli et al. 2007). AFs have carcinogenic, teratogenic, hepatotoxic, mutagenic and immunosuppressive properties, with AFB1 being the most toxic. According to the International Agency for Research oir Cancer (IARC), AFB1 is classified in group 1 with high risks of hepatocellular carcinoma while AFM1 being less toxic, it is classified in group 2B (possible carcinogen for humans). AFB1 forms DNA adducts by covalent binding to N7-guanine, resulting in persistent DNA lesions and AFB1 induces oxidative stress including modulation of antioxidant (EFSA 2020). The pharmacokinetics of aflatoxin is not yet fully understood (Dohnal et al. 2014). The liver is the primary site of aflatoxin metabolism, where they are coxwerted to the 8,9-epoxide form by cytochrome P450 (CYP) enzymes (Wild and Turner 2002, Verma 2004). In fact, these enzymes oxidize AFB1 in the liver to form AFB1-8,9-exoepoxide and AFBl-endo-epoxide. AFBl-8,9- exoepoxide can bind to DNA mainly forming the adduct (AFB1- N7-Gua) which is responsible for the mutagenic properties of AFB1. The endo-epoxide AFB1 caxmot bind to nucleic acids, it is less toxic (Wild and Turner 2002, Verma 2004, Dolmal et al. 2014).

2.2 Ochratoxine A

Ochratoxin A (OTA) is a secondary metabolite produced by several species of mold belonging to the genera Aspergillus and Penicillium (Abarca et al. 2003, Varga et al. 2003). OTA was first identified in South Africa in cereals (Van der Merwe et al. 1965), but the cereal sector is not the only one affected by this mycotoxin.

Indeed, the presence of OTA has been reported in many other products such as vegetables, coffee, beer, wine, grape juice, raisins as well as cocoa products, nuts and spices (EFSA 2006). It has also been found in the blood and tissues of animals and in the serum of people who have eaten contaminated food (EFSA 2006, Marquardt and Frohlich 1992). The kidney is the main target organ of exposure to OTA, in fact it causes nephrotoxicity in animals. OTA has also been associated with Endemic Balkan Nephropathy (Krogh et al. 1987), although the causality of OTA in human nephropathy remains unclear. OTA has carcinogenic, teratogenic, immunotoxic and possibly neurotoxic properties; IARC (1993) classified it in group 2B as a possible human carcinogen. Recently, Ostry et al. (2017) reported that new data on the formation of OTA-DNA adducts, on the role of OTA in oxidative stress and the identification of epigenetic factors involved in OTA carcinogenesis could lead to the reclassification of OTA.

2.3 Trichothecenes

Trichothecenes belong to a family of around 200 structurally related mycotoxins. These are cyclic sesquiterpenoids characterized by a stable C12-C13 epoxy cycle and a double bond between C9 and CIO. Trichothecenes are divided into four groups (A-D) according to their functional groups (acetoxy and hydroxyl). Type A is represented by toxin HT-2 and toxin T-2, and type В is often represented by deoxynivalenol (DON) and nivalenol (NIV) (Marin et al. 2013). Types C and D include some lesser important trichothecenes (Marin et al. 2013). Trichothecenes type A arid В are produced by several Fusarium species (Nielsen and Thrane 2001), but also by certain Trichoderma species (Nielsen et al. 2005). The most important T-2 and HT-2 producing species are F. sporotricliioides, F. langsethiae, F. acuminatum and F. poae while the main DON producers are F. graminearum, F. culmorum and F. cerealis (Marin et al. 2013). In addition to DON, its acetylated derivatives, 3-acetyl-deoxynivalenol (3-Ac-DON) and 15-acetyl-deoxynivalenol (15-Ac-DON) can be produced by these fungi and have been detected with DON but at levels generally 10% lower than those reported for DON (FAO/WHO 2011). These fungi that grow on crops in fields are phytopathogens and can develop in temperate climates (Marin et al. 2013). Some species are responsible for Fusarium wilt (FHB), one of the most serious plant diseases that results in quality loss and reduced grain yield (Krnjaja et al. 2011). Trichothecenes are generally very stable compounds during storage, grinding, cooking of food and are not degraded by high temperatures (EFSA 2011). Animal exposure to TCs leads to in vitro and in vivo apoptosis in several organs such as lymphoid organs, hematopoietic tissues, liver, intestinal crypts, bone marrow and thymus (Pestka 2007, EFSA 2011). They interact with ribosomes and mitochondria causing inhibition of protein synthesis, their cytotoxic effects on cell membranes and inside the cell are facilitated by their amphiphilic nature (Pace et al. 1988). Acute high dose toxicity of trichothecenes is characterized by diarrhea, vomiting, leukocytosis, hemorrhage, circulator}' shock and death, while chronic low dose toxicity is characterized by anorexia, weight gain suppression, neuroendocrine and immunological changes (Pestka 2007, Larsen et al. 2004). With regard to human exposure, cereals and cereal-based products were the main sources of DON, while for farm and companion animals, cereals, cereal by-products and feed corn were the main sources of DON and contribute the most to their exposure (EFSA 2017).

The main sources of exposure to T-2 and HT-2 toxins are cereals and cereal-based foods, especially bread, fine baker}' products, cereal millings and breakfast cereals (EFSA 2011). The T-2 toxin is rapidly metabolized into a large number of products, the HT-2 toxin being its major metabolite. Estimates of chronic human dietary exposure to the sum of T-2 and HT-2 toxins based on the occurrence data are below the tolerable daily intake (TDI), these toxins do not constitute a health problem (EFSA 2011).

2.4 Zearalenone

Zearalenone is a mycotoxin produced by species of the genus Fusarium and in particular F. graminearum, F. culmorum, F. semitectum, F. ecjuiseti, and F. verticillioides (EFSA 2011). It was first isolated in 1962 from corn contaminated with Giberella zea (Stob et al. 1962). It is a natural contaminant of grains, especially corn, but can also be found in other crops such as wheat, barley, sorghum and rye. ZEA is considered a field mycotoxin but its production can also take place under poor storage conditions. It is thermostable and withstands a temperature of 120°C for 4 h (Yiannikouris et al. 2002). ZEA is a macrocyclic lactone derived from resorcyclic acid. It is described as a mycotoxin which induces obvious estrogenic effects in humans and animals, given its structural similarity to natural estrogens (Bennett and Klich 2003). The reduction of zearalenone to zearalenol, a key step in its bioactivation, is catalyzed by hepatic 3-a-hydroxysteroid dehydrogenase. Zearalenone and its derivatives will bind competitively to estrogen receptors (ERa and ERp) in various animal species and promote the synthesis of RNA, proteins, as well as cell proliferation, increasing the mass of organs and causing changes and lesions in the female animal reproductive system (Hussein and Brasel 2001). Thus, the public health concern regarding the ZEA is associated mainly with its strong estrogenic activity. IARC (1993) has classified this mycotoxin in Group 3 (not classifiable as their carcinogenicity to humans).

2.5 Fumonisins

Fumonisins are natural contaminants of corn and corn products; there are currently four types of fumonisins (FA, FB, FC and FP) and the most important of which are type В (FBI, FB2 and FB3). These are mycotoxins produced by species of the genus Fusarium and mainly Fusarium verticillioides (syn. Fusarium moniliforme) and Fusarium proliferatum. Other fungal species, including F. napiforme, F. dlamini and F. nygamai, also produce fumonisins (EFSA 2005). The putative fumonisin biosynthesis gene cluster and the production of FB2 and FB4 have been demonstrated in certain strains of Aspergillus sect. Nigri (Frisvad et al. 2011, Mogensen et al. 2010). Only A. niger and A. welwitschiae species are able to produce FB (Frisvad et al. 2007, 2011, Mogensen et al. 2010) and can contribute to the accumulation of FB2 in corn kernels. Recently, Ferrara et al. (2020) have developed a rapid test based on loop-mediated isothermal amplification (LAMP) for the rapid and selective detection of Aspergillus strains producing FB (A. niger and A. welwitschiae) among the non-producing strains. This rapid molecular test is based on the detection of the fumlO gene, a structural gene of the fumonisin cluster in the toxigenic species of Aspergillus. Their results showed that very small quantities of conidia are necessary to detect the presence of the fumlO gene giving information on the presence of Aspergillus species producing FB2 and on the possible contamination of fumonisins in corn.

FBs can react during food processing, resulting in the formation of modified Maillard-like forms. FBs can strongly interact by non-covalent bond with macro constituents of the matrix, giving rise to the so-called hidden FBs. Hidden forms can release unchanged parent forms of FB into the gastrointestinal tract. Marin et al. (2013) reported that certain heat treatments or extrusions reduce the presence of FB in food and that in general the levels of FB in products intended for direct human consumption such as corn flakes are low (Marin et al. 2013). According to the IARC, fumonisins are classified in category 2B (possible carcinogenicity). They produce a wide range of toxic effects in animals such as encephalitis (or leukoencephalomalacia), a serious and generally fatal disease in horses, pulmonary edema in pigs, an often-fatal disease (Bolger et al. 2001, Haschek et al. 2001) as well as nephrotoxicity and liver cancer in rats (EFSA 2005).

This mycotoxin has a cytotoxic effect; it inhibits the synthesis of proteins and DNA and promotes oxidative stress. It also induces DNA fragmentation and stops the cell cycle (Abado-Becognee et al. 1998). In humans, there is a link between high consumption of fumonisin-contaminated com and the development of esophageal cancer in some parts of the world, and fumonisins have been reported as potential risk factors for malformations of the neural tube, craniofacial and other congenital anomalies originating from neural crest cells (Marasas et al. 2004). Fumonisins are structural analogues of sphingoid bases and they inhibit ceramide synthase. This induces a disturbance in the metabolism of sphingolipids and pathological changes (Riley and Merrill 2019).

2.6 Patulin

Patulin (PAT) is a mycotoxin produced by certain species of the genera Penicillium, Aspergillus, Paecilomyces and Byssochlamys (Steiman et al. 1989). It has often been associated with Penicillium expansum, but other fungal species of Penicillium are able to produce PAT (Morales et al. 2007, Frisvad et al. 2004). It mainly contaminates apples arid derivatives (Beltran et al. 2014, Zhong et al. 2018, Saleh and Goktepe 2019). It has also been detected in other fruits and derivatives, in vegetables and in cereals and cereal products (Shephard et al. 2010). The concentrations of PAT observed in Europe are generally low whereas certain samples of products from other countries have shown higher concentration levels (Vidal et al. 2019). Although classified by the IARC in group 3 as non-carcinogenic, recent reviews on PAT have reported that this mycotoxin has been linked to neurological, gastrointestinal and immunological adverse effects, mainly causing liver damage and renal problems (Puel et al. 2010, Pal et al. 2017). Puel et al. (2010) reported that the affinity of patulin for sulfhydryl groups explains its inhibitory effect on many enzymes. In addition, the WHO considers patulin as a possible genotoxic compound (WHO 2005). Recently, the harmful effects of mvcotoxins in general and of patulin in particular on the sensitive structures of the intestines have been widely studied arid the toxicity of patulin on the function of the intestinal barrier has beeii demonstrated (Akbari et al. 2017).

2.7 Citrinin

Citrinin (CIT) is a polyketide mycotoxin first described in the species Penicillium citrinum (Hetherington and Raistrick 1931). Since then, it has been isolated from several species of the genera Aspergillus (A. terreus, A. carneus and A. niveus), Penicillium (P. verrucosum and P. expansum) and Monascus (M. ruber and M. purpureus) (Blanc et al. 1995, Hajjaj 2000a, Doughari 2015, de Oliveira Filho et al. 2017). Some of the citrinin-producing fungi are also able to produce other mycotoxins such as OTA, PAT or AFs leading to co-occurrence in commodities of CIT and these mycotoxins (Blanc et al. 1995, EFSA 2012, Doughari 2015). Although described for its antibiotic activities against Gram positive bacteria (Bacillus, Streptococcus) but also against Pseudomonas, Saccharomyces and Candida, its use for this purpose has been rejected because of its toxicity, despite positive results in the treatment of tropical skin diseases (Bastin 1949).

Citrinin is generally formed after harvest under storage conditions and it occurs mainly in grams. Stored gram samples (wheat, oats, barley and rye) associated with lung problems in farmers and silo operators were contaminated by citrinin (up to 80 mg/kg) and ochratoxin A (Scott et al. 1972). The occurrence of CIT has been detected in various commodities worldwide such as cereals, beans, fruits, fruit and vegetable juices, herbs and spices and also in spoiled dairy products (EFSA 2012, Zaied et al. 2012, Doughari et al. 2015). Analysis of biological fluids showed the presence of CIT in urine and plasma (Martins et al. 2019, Ouhibi et al. 2020). CIT has also been isolated from fungal ferment species of the genus Monascus, used for industrial purposes for the production of a red food coloring and commonly used in Asia (Blanc et al. 1995, Bennett and Klich 2003). It has been shown that the addition of short chain fatty acids (C8, CIO and C12) or methyl ketones (2-heptanone, 2-nonanone or 2-undecanone) in the culture medium in Monascus ruber, lowers the titer of the mycotoxin (Hajjaj et al. 2000b). Oxygen is not only the final electron acceptor of the respiratory chain, but also a substrate in certain reactions of secondary metabolism. It is indeed established that oxygen is used thanks to monooxygenases in the biosynthesis of pentacetides (Turner 1971). Discontinuous fermenter cultures carried out in M. ruber showed that when the oxygen is in limiting concentration, the production of citrinin is weak and the stoichiometric balance is far from being balanced with the important production of ethanol (fermentative metabolism) (Hajjaj et al. 2015).

Conversely, under conditions of no oxygen limitation, the production of citrinin is greater (Hajjaj et al. 1999). The nature of the amino acids has been shown to modify the concentration of citrinin in M. ruber, with high levels in the presence of glutamate, alanine or proline as a source of nitrogen (Hajjaj et al. 2012).

Citrinin is acutely nephrotoxic at relatively high doses in mice and rats, rabbits, pigs and poultry, causing swelling and eventual necrosis of the kidneys and affecting the liver function at a lesser extent. Based in the available data, the International Agency for Research on Cancer (IARC) concluded that there is limited evidence for carcinogenicity in animals (EFSA 2012). Recently, Sun et al. (2020) reported that exposure of citrinm at 30 pM disrupts organelle distribution and functions in mouse oocytes.

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