Determination of Fusarium mycotoxins enniatins, beauvericin and fusaproliferin in cereals and derived products from Tunisia

Abstract

In this study, 51 samples of cereals (wheat, Barley, maize and Sorghum) and by-products (mainly pasta and couscous) purchased from Tunisian supermarkets were examined for contamination with the emerging Fusarium mycotoxins: Enniatins ENs (EN A, EN A₁, EN B and EN B₁), beauvericin (BEA) and fusaproliferin (FUS).

The extraction of the samples was performed with methanol using an Ultra-turrax homogenizer. Mycotoxins were analyzed with a liquid chromatography (LC) coupled to a diode array detector (DAD).

The frequencies of contamination of total samples with ENs were 96%. EN A₁ was the most common EN found with the highest prevalence of 92.1%, levels ranged between 11.1 and 480 mg/kg. EN B was evidenced in 35 samples and levels ranged from 1.5 to 295 mg/kg. EN B₁ was detected in 20 samples (39.2%) and levels varied from 4.8 to 120.1 mg/kg and EN A was detected in 14 samples with contamination levels ranging between 19.6 and 121.3 mg/kg. The maximum concentration of total ENs in a single sample was 683.9 mg/kg (sorghum). The analytical results also showed that all the analyzed samples were free of BEA and FUS.

The present work is the first one ever drafted on the presence of the emerging Fusarium mycotoxins in Tunisian cereals and derived products.

Introduction

Mycotoxins are toxic secondary metabolites produced by filamentous fungi, mainly Aspergillus, Penicillium, and Fusarium species under appropriate environmental conditions. They are common contaminants of many grains such as wheat, barley, maize and rice. Mycotoxins may be present in foods or feeds. According to the International Agency for Research on Cancer (IARC), Mycotoxins contamination may be a serious concern for both human and animal health because of their wide range of harmful effects, including carcinogenicity, teratogenicity, and mutagenicity (IARC, 1993, IARC, 2002). Fusarium species are known to be the most prevalent mycotoxin-producing fungi in the northern temperate regions (Jestoi, 2008). They produce mycotoxins that accumulate either in preharvest grain plants or in stored grains, and thereby unavoidably contaminate processed foods or feeds (Bottalico & Perrone, 2002). “Traditional” Fusarium mycotoxins groups that occur quite often in cereals and derived products are the trichothecenes, zearalenone, and the fumonisins (Kumar, Basu, & Rajendran, 2008), while other groups of “emerging” mycotoxins compounds that include Enniatins (ENs), Beauvericin (BEA) and Fusaproliferin (FUS) can be found in cereal products.

The ENs are six-membered cyclic depsipeptides, which are commonly composed of three D-a-hydroxyisovaleric acid (Hiv) residues linked alternatively to three L-configured N-methyl amino acid residues to give on 18-membered cyclic skeleton (Zhukhlistova, Tishchenko, Tolstykh, & Zenkova, 1999). ENs are described as phytotoxins, with antibiotic and insecticidal activities (Grove & Pople, 1980).

BEA is formed from d-α-hydroxyisovaleryl-(2-hydroxy-3-methylbutanoic acid) alternating with an N-methylphenylalanine moiety. BEA is toxic to several human cell lines (Fornelli et al., 2004, Logrieco et al., 2002) and can induce apoptosis and DNA fragmentation (Ojcius, Zychlinsky, Zheng, & Young, 1991). BEA was shown to be a specific cholesterol acyltransferase inhibitor (Tomoda et al., 1992).

ENs and BEA are produced by several species of Fusarium such as Fusarium verticillioides, Fusarium proliferatum, Fusarium subglutinans, Fusarium oxysporum, Fusarium poae, and Fusarium avenaceum, which are known to be able to contaminate cereals and by-products (Nilanonta et al., 2000, Plattner and Nelson, 2004, Supothina et al., 2004).

These mycotoxins act as ionophores that disturb the physiological ionic balance and pH by forming dimeric structures transporting monovalent ions across the cell membranes (Ivanov et al., 1973, Kamyar et al., 2004, Kouri et al., 2003).

FUS is a bicyclic sesterterpene identified and purified from F. proliferatum cultures (Randazzo et al., 1993). It was also found to be produced by other Fusarium species, like F. subglutinans, F. antophilum, F. begoniae, F. bulbicola, F. circinatum, F. concentricum, F. succisae, and F. udum (Meca et al., 2009, Moretti et al., 2007). During toxicity assays, FUS and its derivatives caused toxic effects on Artemia salina L. brine shrimp larvae and on human nonneoplastic B-lymphocyte cell line IARC/LCL 171 as well (Logrieco et al., 1996, Ritieni et al., 1995). While Ritieni, Monti, et al., 1997 reported that during chicken embryotoxicity bioassays, severe teratogenic effects were observed in 20% of the embryos exposed at 5 mM FUS a day.

The emerging mycotoxins and their occurrence in cereals and derived products from Mediterranean countries are still poorly investigated and only little data is available about their toxicity and their occurrence in foods (Jestoi, 2008).

Tunisia is a North African country whose climate is characterized with high temperature and humidity level that seems to stimulate the toxigenic molds growth and their secondary metabolites production. To our knowledge, no data is available on the presence of these toxic compounds in cereals products. Thus, in the present study, we aimed to investigate the occurrence and contamination levels of the four ENs (EN A, EN A₁, EN B and EN B₁), FUS and BEA in cereals and by-products distributed in Tunisia.