Food Additives and Contaminants, September 2007; 24(9): 9931000

Survey of maize from south-western Nigeria for zearalenone,a- and b-zearalenols, fumonisin B1 and enniatins produced byFusarium species

T. O. ADEJUMO1, U. HETTWER2, & P. KARLOVSKY2

1Department of Microbiology, Adekunle Ajasin University, Akungba-Akoko, Nigeria and 2Molecular Phytopathology

and Mycotoxin Research, Department of Crop Sciences, University of Gottingen, Gottingen, Germany

(Received 24 October 2006; revised 13 February 2007; accepted 28 February 2007)

AbstractA survey for the natural occurrence of Fusarium mycotoxins in maize for human consumption in four south-western states ofNigeria using High Performance Liquid Chromatography coupled with Mass Spectroscopy (HPLC/MS) showed that93.4% of the samples were contaminated with zearalenone (ZON), - and -zearalenols (- and -ZOL), fumonisin B1(FB1) or enniatins (ENNs). The fractions of contaminated samples were 73% for FB1 (mean:117 mg kg

1,range:10760 mg kg1); 57% for ZON (mean:49 mg kg1, range:115779 mg kg1) and 13% for -ZOL (mean:63.6mg kg1, range:32181 mg kg1), while ENNs A1, B and B1 were present in 3, 7 and 3% of the samples respectively.There was no -ZOL present above the quantification limits of 50 mg kg1. Only the FB1 content was significantly differentat the 95% confidence level among the four states. The Fusarium species most frequently isolated from maize seeds wereF. verticillioides (70%), followed by F. sporotrichioides (42%), F. graminearum (30%), F. pallidoroseum (15%), F. compactum(12%), F. proliferatum (12%), F. equiseti (9%), F. acuminatum (8%) and F. subglutinans (4%). This is the first report of theoccurrence of -zearalenol and enniatins in Nigerian maize.

Keywords: Fumonisin B1, zearalenone, -zearalenol, -zearalenol, enniatins, Fusarium species, maize, Nigeria

Introduction

Maize (Zea mays L.) is the most frequently con-sumed staple crop in all agroecological zones inNigeria with 20% of the population consuming itvarious times in a week (Maziya Dixon et al. 2004),either as roasted, boiled grain or processed to flour assnacks. Maize is prepared and consumed in amultitude of ways, which vary from region toregion or from one ethnic group to another. Forinstance, maize grains are prepared by boiling orroasting as paste (eko), agbado, and elekute in Nigeria,or as popcorn which is eaten all over West Africa(Abdulrahaman and Kolawole 2006). It also con-stitutes a major foodstuff for all classes of livestock.Fusarium species are widespread in nature occur-

ring as saprophytes in many plant materials and aspathogens of various crops, especially maize. Theyhave a direct effect on corn yields by causing a

variety of diseases and sometimes produce mycotox-ins in the infected ears and kernels. The mycotoxinsproduced by Fusarium spp. in cereal grains aresecond to the aflatoxins and ochratoxin A inattracting the attention of scientists and farmers;these include the zearalenone (ZON), trichothe-cenes, and fumonisins (Tseng et al. 1985).Fumonisins are produced mainly by F. verticillioides,F. proliferatum and F. nygamai on cereals (Nelsonet al. 1991, 1992, 1994). They are believed to beresponsible for a variety of animal diseases, e.g.equine leukoencephalomalacia (ELEM) in horses,pulmonary edema in swine (Harrison et al. 1990;Kellerman et al. 1990), and they are hepatotoxic andcarcinogenic to rats (Gelderblom et al. 1991).Fumonisins have carcinogenic properties; the majortarget organs are believed to be liver and kidney(Engelhardt et al. 2006). Fumonisin B1 (FB1) has

Correspondence: T. O. Adejumo. E-mail: toadejumo@yahoo.com

ISSN 0265203X print/ISSN 14645122 online 2007 Taylor & FrancisDOI: 10.1080/02652030701317285

been found to be a potent inhibitor of sphinganineN-acyltransferase causing an elevation of the sphin-ganine : sphingosine ratio which results in the dis-ruption of cell membrane function (Wang et al.1991; Riley et al. 1994; Ramasamy et al. 1995). FB1has been suggested as a cause of esophageal and livercancers in humans (Chu and Li 1994; Rheeder et al.1992; Thiel et al. 1992; Ueno 2000). F. verticillioideshas been associated with human esophageal cancerrisk in the Transkei region of southern Africa(Marasas et al. 1981, 1988; Marasas 1982) and inChina (Li et al. 1980; Yang 1980). Recent workproved carcinogenicity of FB1 in rodents(Commission of the European Communities(CEC) 2005a).

ZON is a naturally occurring toxic secondarymetabolite produced by several species of Fusariumfungi on a variety of cereal grains, with specialincidence in corn (Vinas et al. 1985; Trenholmet al. 1991). Fusarium graminearum has been found tobe the major causal agent of ZON contamination ofgrains (Ichinoe et al. 1983). The clinical signs ofexposure to ZON in swine include vulval tumefac-tion, vaginal and rectal prolapses, mammary glandenlargement; conception failure, pseudo-pregnancy,decreased pigs per litter, and abortion in maturecycling females (Chang et al. 1979; Trenholm et al.1988). ZON and some of its metabolites have beenshown to bind competitively to oestrogen receptors(ER) in a number of in vitro systems and it has beendemonstrated in the uterus, mammary gland, liverand hypothalamus of different species. ZON is fairlyrapidly absorbed following oral administration andcan be metabolized by intestinal tissue in pigs andpossibly in humans during its absorption, with theformation of - and -zearalenol (- and -ZOL)and - and -zearalanol, which aresubsequently conjugated with glucuronic acid(Kuiper-Goodman et al. 1987; Eriksen andAlexander 1998). Some strains of Fusarium spp.produce, in addition to ZON, -ZOL which hasalso been found in feeds (Mirocha et al. 1979).-ZOL has about 100-fold more oestrogenicpotency than ZON, and it has been shownto inhibit atherogenesis, lowering plasmaLDL-cholesterol and limiting aortic plaqueformation in ovariectomized rabbits fed with ahigh dose of cholesterol (Dai et al. 2004; Murphyet al. 2006). ZON is mainly degraded to themetabolites - and -ZOLs; this transformationis not regarded as detoxification because bothsubstances are still oestrogenic (Hagler et al.1979; Kuiper-Goodman et al. 1987; Fitzpatricket al. 1989). Enniatins (ENNs) are cyclic hexadep-sipeptides structurally related to beauvericin andare produced by various Fusarium species such asF. avenaceum, F. lateritium, F. scirpi, and

F. oxysporum (Bottalico and Perrone 2002;Logrieco et al. 2002; Nicholson et al. 2004).ENNs are synthesized by the multifunctionalenzyme enniatin synthetase (Haese et al. 1993);they are virulence factors, increasing the ability ofisolates to colonize their host plants (Hermannet al. 1996). ENNs have antimicrobial properties,interfere with cholesterol storage in liver cells, andare found to be phytotoxic (Burmeister and Plattner1987) and toxic in various bioassays (Strongmanet al. 1988; Tomoda et al. 1992). It is reported tohave antibiotic, insecticidal and ionophoric activity(Grove and Pople 1980).

Nigeria has a tropical climate with an all-year-round high ambient temperature and relative humid-ity; these provide optimal condition for growth oftoxigenic moulds. Because of the importance ofmaize as a staple food in Nigeria, it is importantto monitor contamination of maize. FB1 was thepredominant toxin detected in 78.6% of sampleswith concentration range of 701780 mg kg1

(Bankole and Mabekoje 2004). The aim of thepresent study was therefore to determinethe contaminating Fusarium spp. and to examinethe occurrence and level of Fusarium mycotoxin:FB1, ZON, - and -zearalenols, and enniatins inmaize in four south-western states of Nigeria.

Materials and methods

Study areas

The area chosen for this study is south-westernNigeria comprising of Ondo, Ekiti, Osun and Oyostates located between latitudes 60 and 90 N andlongitude 30 and 60 E. The climate is character-ized by two seasons: the wet season, which lasts fromApril to October, with a rainfall distribution rangingbetween 1500 and 2000 mm; and the dry season,which lasts from November to March. The meanmonthly temperatures ranges from 17 to 36C,mean monthly rainfall of 12.0314.6 mm, while themean relative humidity is between 78 and 100% for2005 (Source: Nigerian Meteorological Agency,Abuja).

Sampling and sample preparation

A total of 182 maize samples were collected fromfarmers, markets and grain shops in Ekiti, Ondo,Osun and Oyo states with 35, 57, 40 and 50 samplesrespectively, between May and July 2005. Theseinclude a total of 94 white maize and 88 yellowmaize samples (21 white and 14 yellow for Ekitistate, 22 white and 35 yellow for Ondo state,20 white and 20 yellow for Osun state, and 31white and 19 yellow for Oyo state). Samples were

994 T. O. Adejumo et al.

completely dried, protected from moisture andinsects, and stored in a cold room before analysis.A total of 500 g of seeds per sample were groundwith the milling machine (1 mm sieve) and preparedfor mycotoxin analysis.

Isolation and identification of Fusarium species

Fungi were isolated by surface-sterilizing 15 seeds persample in 1% sodium hypochlorite solution in a 50 mlbeaker for 1 min and washed with three changes ofsterile distilled water. Five seeds each were placed onpotato-dextrose agar (PDA) (Roth, Karlsruhe,Germany) and incubated for 47 days at 25C. Thefungi were isolated and subcultured to obtain purecultures and the Fusarium species were then inocu-lated to both potassium chloride agar (KClA)(Merck, Darmstadt, Germany) and SpeziellerNarstoffarmer agar (containing 1.0 g KH2PO4, 1.0 gKNO3, 0.5 g MgSO4 7H2SO4, 0.5 g KCl, 0.2 gglucose, 0.2 g saccharose, 20.0 g agar, 1 litre ofdistilled water; Gerlach and Nirenberg 1982), incu-bated for 7 days in the dark and placed under long-wavelength ultraviolet light at 22C for 24 weeks.Fungi were identified by the descriptions of Gerlachand Nirenberg (1982) and Burgess et al. (1994). Thecontamination level of Fusarium species was deter-mined by the presence or absence of fungal species inthe samples and calculated as the percentage ofsamples infected.

Reagents and reference standards

ZON, FB1, and - and -ZOLs standards wereobtained from Sigma (Taufkirchen, Germany) in thehighest purity available. The ENN mixture wasextracted from Locabiosol (Servier, Munchen,Germany) and used only as a reference for masstransitions and retention time. In addition to ENNs,Locabiosol contains isopropylmyristat, ethanol,saccharin and flavours. The pharmaceutical wasextracted with hexane to remove oils and thedefatted solution was used as a qualitative standardfor HPLC-MS. The ratios of ENNs in the extractdetermined by HPLC with ultraviolet light detectionwas as follows: ENN B, 38%; ENN B1, 44%; andENN A1, 18%. Acetonitrile and methanol ofgradient quality grade were obtained from VWR(Darmstadt, Germany); ammonium acetate andformate were from Merck (Darmstadt, Germany).HPLC water was prepared by twofold distillation ina distillation apparatus consisting of only glass andTeflon components.

Mycotoxins analysis

The methods of Royer et al. (2004) were used foranalysis of ZON, - and -ZOLs and FB1, while

those of Monti et al. (2000) were used for enniatins(ENNs) A1, B and B1. The toxins were extractedfrom 4 g of ground maize in 40 ml acetonitrilewater(84:16) in 50 ml Falcon tubes as follows. Sampleswere incubated and shaken vigorously overnight ina wrist-action shaker, centrifuged at 4500 rpm for10 min and decanted. Supernatant (1 ml) waspipetted unto 1.5 ml Eppendorf tubes, evaporatedto dryness at 45C in a speed vacuum chamberand stored in the freezer at 20C. Samples weredissolved in a mixture of 375ml methanol and 375mlbuffer (10 mM ammonium acetate, pH set to 4.5with formic acid, containing 1% acetonitrile) anddefatted with 500ml of hexane. After phase separa-tion (20C, 14 000 rpm for 1 min) the lower phasewas filtered (OPTI-FLOW, 13 mm, 0.2mm; PTFE;WICOM, Heppenheim, Germany) into HPLC vials.

LC analysis was performed using a Varian systemconsisting of two pumps, a degasser, an autosamplerand a column oven. The analytes were separated ona polar modified RP-18 column (Synergi Fusion RP80A column; 4 mm, 100 2 mm i.d.; Phenomenex,Aschaffenburg, Germany). The column was main-tained at a temperature of 40C. The flow rate wasset to 0.2 ml min1, the injection volume was 10 ml.Solvent A was water; solvent B was methanol. ZONand the ZOLs were separated as follows: 07 min2040% B, 710 min 4070% B, followed bywashing and re-equilibration steps. FB1 and ENNswere separated as follows: 01 min 20% B, 110 min2060% B, followed by washing (98% B) andequilibration steps. Since it is possible to ionizeFB1 in positive and negative ESI, the FB1 sampleswere scanned twice. Peak integration was performedfrom the positive mode scan.

For MS/MS detection a Varian 1200 triplequadrupole was used with positive and negativeelectrospray ionization (ESI). Gas flow andtemperature were 250C and 21 psi respectively.Needle, shield and capillary voltage were4400/600/40 V for negative ESI, and5000/250/80 V for positive ESI. Fragmentation wasperformed by collision-induced fragmentation.System control was done by Varian MSWorkstation 6.42. Quantitative determination wasperformed in single-reaction monitoring (SRM) forFB1 and multiple-reaction monitoring (MRM)for ZON and ZOLs respectively. Transitions usedfor quantification were as follows: FB1 in positiveESI: 7224352; ZON: 3174175; ZOLs: 3194275.The presence of ENN was determined as follows:ENNs were A1: 685.54455, ENN B: 6574445,ENN B1: 671.54427. Calibration curves wereprepared by spiking maize extract prepared frommycotoxin-free grain with a standard solutionin the range 6.252000 mg kg1. Limits of detectionand quantification were estimated as follows.

Nigerian maize and Fusarium species 995

Blank matrix was prepared in ten repetitions. Blankmaize flour was spiked to 5, 10, 20, 50 and200mg kg1 ZON and ZOL, and 20, 50, 200 and500mg kg1 FB1 in three repetitions each. The noiserange was estimated in the peak-to-peak mode.Toxin concentrations generating signals thatexceeded the noise level threefold were set as limitsof detection; concentrations corresponding to signalsexceeding the background noise tenfold were set asthe limits of quantification. Quantification limitswere 30 mg kg1 for -ZOL, 50mg kg1 for -ZOLand 10mg kg1 for FB1.%RSD for both ZOLs were30%, for ZON 20%, and for FB1 8%.

Statistical analysis

Statistical analyses were performed using Statistix8.1 Analytical Software. Data were arcsined trans-formed and analyses of variance (ANOVA) wereperformed and a Tukey HSD all-pairwise compar-isons test at 5% significance level was used tocompare the means of mycotoxin levels.A Spearmans rank correlation coefficient was usedto evaluate the strength of the relationships betweenthe incidence of Fusarium and the mycotoxins as wellas co-contamination of mycotoxins.

Results

Occurrence of fungi

Nine Fusarium species were isolated from maizeseeds: F. acuminatum, F. compactum, F. equiseti,F. graminearum, F. oxysporum, F. pallidoroseum,F. sporotrichioides, F. subglutinans and F. verticillioides.Four Aspergillus spp. (A. flavus, A. niger, A. terreusand A. ochraceus), two Penicillium spp., Cladosporiumsp., Curvularia sp., Mucor sp. and seven unidentifiedfungal species. The highest frequency among theFusarium species was 70% for F. verticillioides,followed by F. sporotrichioides (42%), F. graminearum(30%), F. pallidoroseum (15%), F. compactum (12%),F. proliferatum (12%), F. equiseti (9%), F. acuminatum(8%) and F. subglutinans (4%).

Incidence and levels of Fusarium mycotoxins

The occurrence of Fusarium mycotoxins (FB1,ZON, - and -ZOLs, ENNs A1, B and B1) inNigerian maize is summarized in Table I. At leastone mycotoxin was detected in each of the 170samples (93.4%), while 12 samples were toxin free.FB1 was detected in 133 (73%) of the samples, nineof them below the quantification limit of 10mg kg1.ZON was detected in 103 (57%) of the samples,79 of them below the quantification limit. The meanand maximum levels of ZON were 48.8 and778.9 mg kg1 respectively. -ZOL was detected in23 (13%) of the samples, 12 of them below thequantification limit. The mean and maximum levelsfor -ZOL were 63.6 and 181.3 mg kg1, respect-ively. ENNs A1, B and B1 occurred in five samples,while a further four samples contained only ENN B1.None of the maize samples contained -ZOL abovethe quantification limits of 50mg kg1.

The distribution of Fusarium mycotoxins in maizeseeds in four south-western states of Nigeria isshown in Table II. The highest mean contents of149mg kg1 FB1 were detected in Oyo state in46 (25.2%) of the samples, followed by Ekiti statewith 130mg kg1 in 19 (10.4%) samples, while theleast mean FB1 contents of 66 mg kg

1 was obtainedin Osun state in 31 (17.0%) samples. The means ofFB1 contents across the four states were significantlydifferent at p0.05. The content of the other toxinsdid not differ significantly among the states. Out ofthe 133 samples that showed presence of FB1,52 and 59 samples were in the concentration rangeof

Ondo state, four samples from Ekiti state, twosamples from Osun state and three samples fromOyo sate showed the highest level of 51200 mg kg1.Spearmans rank correlation analysis showed thatties were found between the incidence ofF. verticillioides and FB1 levels (r0.3509,p 0.0000). There was co-existence and multiplecontaminations of six, four, three and two mycotox-ins in two, one, 18 and 48 samples respectively.The study revealed a high frequency but low level ofcontamination of FB1, ZON, -ZOL in maizesamples obtained from four states in Nigeria.In contrast, the incidence of ENNs were low.Table III shows the distribution of mycotoxins in

white and yellow maize. Yellow maize generallyshowed higher concentrations of mycotoxins thanwhite maize. The mean concentration level for whiteand yellow maize are FB1 (115 and 119mg kg

1),ZON (39 and 61mg kg1) and -ZOL (55 and75mg kg1) respectively.

Discussion

This study found that that F. verticillioides was themost prevalent among the nine Fusarium species inNigerian maize, contaminating 71% of samples.This result is in agreement with the reports of

Table II. Distribution of fumonisin B1, zearalenone, - and -zearalenols, and enniatins in four south-western states of Nigeria.

South-western states of Nigeria (mg kg1)

Mycotoxins1 Ondo Ekiti Osun Oyo

Fumonisin B1 (FB1)Number (% of contaminated samples) 37 (20.3%) 19 (10.4%) 31 (17.0%) 46 (25.2%)Mean 115 130 66 149Zearalenone (ZON)Number (% of contaminated samples) 32 (17.6%) 23 (12.6%) 21 (11.5%) 27 (14.8%)Mean 21 63 41 75-Zearalenol (-ZOL)Number (% of contaminated samples) 4 (2.2%) 7 (3.8%) 5 (2.7%) 7 (3.8%)Mean 67 84 60 44Enniatin BNumber (% of contaminated samples) 3 (1.6%) 0 0 2 (1.1%)Enniatin B1Number (% of contaminated samples) 5 (2.7%) 3 (1.6%) 1 (0.5%) 3 (1.6%)Enniatin A1Number (% of contaminated samples) 3 (1.6%) 0 0 2 (1.1%)

1Total number of samples 182.Means across the four states are significantly different at p 0.05 (Turkey HSD all-pairwise comparisons test).

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Oyeniran (1977), Bankole (1994), and Bankole andMabekoje (2004) that F. verticillioides was the mostprevalent fungus in freshly harvested maize.The same observation was reported by Owoladeet al. (2001) and Bankole and Adebanjo (2003) forNigerian stored maize. We confirmed the observa-tion of these authors that F. verticillioides was oftenisolated from symptomless kernels.

Maximum limits for ZON and FB1 have not yetbeen established in Nigeria. Eight samples (threefrom Oyo State, one from Osun State and four fromEkiti state) (Figure 1) were not fit for humanconsumption by European Union (EU) standardsbecause they contained ZON above the permissiblelevel of 200mg kg1 (CEC 2005b). No sampleexceeded the EU maximum limit of 2000mg kg1

for FB1. -ZOL was detected in 14% of themaize samples, with mean levels of 64mg kg1

and a maximum of 181 mg kg1, though recom-mended legal limits for -ZOL have not yet beenestablished.

The percentage of maize samples contaminatedand mean levels of FB1 in this study agree withprevious reports from pre-harvest maize in Nigeria(Bankole and Adebanjo 2003; Bankole andMabekoje 2004). However, the present paperreports a lower FB1 concentration range of10760 mg kg1 probably due to an unsuitableclimate for fungal contamination in the four stateswhere the samples were obtained, though a highlevel of contamination may occur in other years.Spearmans rank correlation analysis showed thatcorrelations were found between ZON and -ZOL(r 0.5197; p0.00001), meaning that as the grainwas being contaminated by the production of ZON,the chances of production of the more potent -ZOLincreases, thereby having a greater impact on thehealth of animals and livestock.

The co-occurrence of FB1, ZON, -ZOL, andENN A1, B and B1 in 69 samples calls for urgentconcern and monitoring of mycotoxins in maize,owing to its importance as a major staple foodin Nigeria. The simultaneous contamination issignificant from the standpoint of potential risks to

human and animal health (Yazdanpanah et al.2001). A more comprehensive survey in all otheragroecological zones is desirable in order to assessthe extent to which the Fusarium toxins are aproblem in Nigeria. The maintenance of fumonisinsat undetectable levels from post-harvest to the dryinginterval is a challenge (Marn et al. 1998). Therefore,efforts to reduce the harvest/drying interval, as wellas the constant monitoring of toxigenic fungi andfumonisin contamination in corn and corn-basedfoods are essential in order to ensure the quality andsafety of products and to minimize the potentialhazards to human and animal health.

Acknowledgements

This work was funded by the Alexander vonHumboldt Foundation (AvH), Germany, throughthe award of a Georg Forster Research Fellowship tothe senior author. Its support is gratefullyacknowledged.

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