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The Effect of Time on Mycotoxins in Subsistence Farmed Maize from Zimbabwe

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The Effect of Time on Mycotoxins in Subsistence Farmed Maize from Zimbabwe FACULTY OF PHARMACEUTICAL SCIENCES Department of Bio-Analysis Laboratory of Food Analysis Academic year 2015-2016 THE EFFECT OF TIME ON MYCOTOXINS IN SUBSISTENCE FARMED MAIZE FROM ZIMBABWE Sarah GEHESQUIÈRE First Master of Pharmaceutical Care Promoter Prof. Dr. Pharm. D. S. De Saeger Supervisor Melody Hove Commissioners Prof. Dr. Geert Haesaert Dr. Natalia Beloglazova FACULTY OF PHARMACEUTICAL SCIENCES Department of Bio-Analysis Laboratory of Food Analysis Academic year 2015-2016 THE EFFECT OF TIME ON MYCOTOXINS IN SUBSISTENCE FARMED MAIZE FROM ZIMBABWE Sarah GEHESQUIÈRE First Master of Pharmaceutical Care Promoter Prof. Dr. Pharm. D. S. De Saeger Supervisor Melody Hove Commissioners Prof. Dr. Geert Haesaert Dr. Natalia Beloglazova COPYRIGHT “The author and the promoters give the authorization to consult and to copy parts of this thesis for personal use only. Any other use is limited by the laws of copyright, especially concerning the obligation to refer to the source whenever results from this thesis are cited.” August 16, 2016 Promoter Author Prof. Dr. Pharm. D. S. De Saeger Sarah Gehesquière DANKWOORD Ik had deze masterproef niet kunnen verwezenlijken zonder de hulp van bepaalde mensen. Vooreerst wil ik graag Prof. Dr. Apr. Sarah De Saeger bedanken om mij de kans te geven om mijn masterproef op het Laboratorium voor Bromatologie te mogen uitvoeren. Ook een oprechte dankjewel aan mijn begeleidsters Melody Hove en Marthe De Boevre, om mij steeds bij te staan met goeie raad, steeds een antwoord te geven op mijn vele vragen, mij te steunen, en om deze thesis na te lezen. Verder wil ik nog mijn medestudenten en alle medewerkers van het laboratorium bedanken voor de gezellige sfeer, de leuke babbels, en de motiverende gesprekken toen ik het nodig had. Ook wil ik mijn ouders, oma, zus en vrienden bedanken voor alle steun tijdens de voorbije maanden. SAMENVATTING Maïs is een basisvoedsel dat verbouwd wordt in Afrika ten zuiden van de Sahara, zoals Zimbabwe, maar het is ook graan dat vaak gekoloniseerd wordt door schimmels die mycotoxines kunnen produceren. Mycotoxines zijn de secundaire metabolieten van fungi en hebben een nadelig effect op de gezondheid van mens en dier, en kunnen gewassen aantasten. Dit kan resulteren in ziektes en economische verliezen. De belangrijkste toxigene schimmels en hun mycotoxines zijn de Aspergillus spp. (aflatoxines, ochratoxine A en sterigmatocystine), Fusarium spp. (trichothecenes, fumonisines en zearalenone), Alternaria spp. (alternariol en alternariol monomethylether), en Penicillium spp. (roquefortine C). Deze thesis maakt deel uit van een studie uitgevoerd door Melody Hove. Het doel van deze studie was tweevoudig. Ten eerste werden de aanwezigheid en quantiteit van mycotoxins bepaald in maïs afkomstig van kleine boerderijen in de provincies Mashonaland West en Manicaland. Het tweede doel van deze studie was om het effect van tijd op het mycotoxinegehalte te bestuderen. Hiertoe werden stalen die genomen werden op het moment van de oogst vergeleken met stalen van drie maanden na de oogst, en stalen van zes maanden na de oogst. De verzamelde stalen werden geanalyseerd gebruik makend van vloeistofchromatografie gekoppeld aan tandem massaspectrometrie. Van de 158 geteste maïsstalen was 46% positief voor tenminste één mycotoxine. De toxicologisch relevante mycotoxines FB 1, FB 2, FB 3, DON en ZEN kwamen voor in respectievelijk 40, 22, 11, 6 en 3% van de stalen, aan een gemiddelde van 243,30; 52,22; 13,67; 7,32 en 0,24 µg/kg. In één staal werd 14,80 µg/kg AFG 1 gedetecteerd. Dit was hoger dan de wettelijke limit van 10 µg/kg. Andere mycotoxines die in 1 tot 3% de stalen voorkwamen, waren: FX, 15-ADON, DAS, NIV en STERIG. Bij alle fumonisines werden dezelfde trend in de tijd gezien. Algemeen steeg het gemiddelde gehalte aan fumonisines significant tot minimum drie maanden na de oogst, waarna het gehalte terug significant daalde. Het gehalte aan fumonisines was significant hoger in Mashonaland West dan in Manicaland. Fumonisins zijn het grootste probleem in maïs verbouwd op kleine boerderijen in Zimbabwe. Goede praktijken vóór, tijdens en na het oogsten kunnen het voorkomen van mycotoxines in maïs, en bijgevolg ook de hieraan verbonden risico’s, reduceren. SUMMARY Maize is a staple food that grows in sub-saharan Africa, like Zimbabwe. However, it is often colonized by mycotoxin-producing fungi. Mycotoxins are the secondary metabolites of fungi that have adverse effects on animals, humans and crops, and that result in economic losses and illnesses. Most important toxigenic fungi and their mycotoxins are the Aspergillus spp. (aflatoxins, ochratoxin A, sterigmatocystin), Fusarium spp. (trichothecenes, fumonisins, zearalenone), Alternaria spp. (alternariol, alternariol monomethyl ether), and Penicillium spp. (roquefortine C). This thesis is part of a larger study conducted by Melody Hove. The aim of this study was twofold. The first goal was to determine the presence and quantity of mycotoxins in maize obtained from subsistence farms in the provinces Mashonaland West and Manicaland, the two agricultural zones in Zimbabwe. The second goal was to study the effect of time on mycotoxin content by comparing contamination in samples taken at harvest, three months postharvest, and six months postharvest, to see whether there is a difference between the different stages or not. Samples were collected at harvest, three months postharvest, and six months postharvest, in two agricultural zones of Zimbabwe. Analysis was performed using liquid chromatography coupled to tandem mass spectrometry. Of the 158 maize samples tested, 46% were positive for at least one mycotoxin. Of the toxicologically relevant mycotoxins, FB 1, FB 2, FB 3, DON, and ZEN were detected in 40, 22, 11, 6, and 3% of the samples, at a mean level of 243.30, 52.22, 13.67, 7.32, and 0.24 µg/kg, respectively. AFG1 was detected in only one sample, at a level of 14.80 µg/kg, which exceeds the legal limit of 10 µg/kg. Other detected mycotoxins were FX, 15-ADON, DAS, NIV, and STERIG. The percentage of contamination for these mycotoxins ranged between 1 and 3% of the samples. The content of all fumonisins showed a significant change over time. In general, the average amount of fumonisins in the contaminated samples significantly increased until at least three months after harvest, and thereafter significantly decreased again. The fumonisin content in Mashonaland West was significantly higher than in Manicaland. Fumonisins are the major problem in subsistence farmed maize in Zimbabwe. Good pre- and postharvest practices can reduce the occurrence of mycotoxins and the associated risks. TABLE OF CONTENTS 1. INTRODUCTION ............................................................................................................ 1 1.1. Aspergillus spp. Mycotoxins .................................................................................... 2 1.1.1. Aflatoxins ......................................................................................................... 2 1.1.2. Ochratoxin A (OTA) .......................................................................................... 3 1.1.3. Sterigmatocystin ............................................................................................... 4 1.2. Fusarium spp. Mycotoxins ....................................................................................... 5 1.2.1. Trichothecenes ................................................................................................. 6 1.2.2. Fumonisins ....................................................................................................... 9 1.2.3. Zearalenone (ZEN) .........................................................................................11 1.3. Alternaria spp. Mycotoxins .....................................................................................12 1.4. Penicillium spp. Mycotoxins ....................................................................................13 1.5. Legislation ..............................................................................................................13 2. OBJECTIVES ................................................................................................................16 3. MATERIALS AND METHODS .......................................................................................17 3.1. Sampling ................................................................................................................17 3.2. Determination of mycotoxins in maize ....................................................................19 3.2.1. Standards ........................................................................................................19 3.2.2. Reagents and materials ......................................................................................19 3.2.3. Sample preparation .........................................................................................20 3.2.4. Apparatus ...........................................................................................................21 3.2.5. Statistics and data analysis .................................................................................24 4. RESULTS AND DISCUSSION .......................................................................................25 4.1. Investigation of the contamination level of mycotoxins in maize .............................25 4.2. Determination of mycotoxin levels at three stages (harvest, three months postharvest and six months postharvest), and in
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