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Safety Assessment of some Traditionally Fermented Foods Produced in Nigeria and South Africa

A Thesis Submitted to the Faculty of Sciences, University of Johannesburg, South Africa, In Fulfilment of the Requirements for a Doctorate Degree in Food Technology

By

OLOTU IFEOLUWA OMOBOLANLE (Student number: 215085652)

Supervisor: Prof Patrick Njobeh Co-Supervisor: Dr Adewale Obadina Co-Supervisor: Prof Sarah De Saeger

FEBRUARY 2018 EXECUTIVE SUMMARY

Traditional fermented foods from cereals and leguminous oil seeds contribute significantly to the energy and protein requirements of many households across Africa. Their production in many sub-Saharan African countries is still a household art and is influenced by chanced inoculants, which in some cases, compromise their quality and safety. Different locations within South-west Nigeria and Gauteng Province of South Africa were sampled between February 2015 and July 2016 to establish the quality and safety of traditionally fermented products (ogi, ogi baba, ugba, iru, ogiri, mahewu and umqombothi). During this period, a descriptive cross-sectional study was carried out within the sampling regions amongst 86 fermented food sellers using open and close-ended questionnaires to establish their perceived attitudes, practices, and knowledge of fungal colonization, being an antecedent to mycotoxin contamination. Ninety-eight percent of the respondents could not link fungi colonization to mycotoxin contamination and associated health risks while majority (61%) of the respondents only had primary education. Furthermore, 11% of the respondents had no formal education and their educational levels slightly correlated (r = 0.308, p < 0.01) with the level of awareness.

The chemical properties of the fermented foods as well as their microbiological quality were investigated including the occurrence of bacteria, mycotoxigenic fungi, endotoxins and mycotoxins. The pH, total titratable acidity and moisture content of the samples ranged from 3.62 - 8.07, 0.12 - 1.20% lactic acid and 27.5 - 94.7%, respectively, and umqombothi samples had the highest water activity (mean: 0.91) and moisture content (mean: 94.7%). The mean total aerobic plate counts of the samples were between 5.50 x 105 and 6.59 x 1010 CFU/g, 450 bacteria isolates were identified amongst which 42% (190) were Gram-negative. Sphingomonas paucimobilis, the most frequently occurring bacterium was detected in 24% of the samples, while the model organism for endotoxin production Escherichia coli, was isolated from 9% of the samples. The presence of endotoxins in the samples was assayed by the Limulus Amebocyte Lysate (LAL) method and the lowest endotoxin contamination level occurred in ogi from Nigeria (42.90 EU/g) while the highest was from iru from Nigeria (5.49 x 104 EU/g).

Cronobacter sakazakii and Acinetobacter haemolyticus species were only isolated from ugba and umqombothi samples. Gram-positive bacteria were isolated less frequently and included: Paenibacillus polymyxa, Bacillus oleronius, Enterococcus durans and Enterococcus

ii casseliflavus. Other unreported bacteria isolated included: V. vulnificus, and P. raistrickii in iru, Aeromonas haemolyticus, and Rhizobium radiobacter in ugba. The mycobiota of the food materials was also characterized by a diversity of fungal species and 804 of them were isolated with the predominance of toxigenic Aspergillia (240), Penicillia (96) and Fusaria species (49). Other fungal genera (419) isolated included Saccharomyces spp. (128) and Geotrichum spp. (44). Aspergillus flavus and A. parasiticus occurred in 42 and 11% of the samples respectively, with the highest occurrence in ogiri than ogi, ogi baba, ugba, iru, mahewu and umqombothi. The most common Fusarium species isolated was F. verticillioides, while the prevalent ones amongst the Penicillia were: Penicillium expansum, P. chrysogenum, and P. crustosum.

The potential of Aspergillus, Penicillium and Fusarium species recovered from the fermented products to produce 49 secondary fungal metabolites including aflatoxin B1 (AFB1), ochratoxin A (OTA) and cyclopiazonic acid (CPA) using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was investigated. Of the 385 fungal strains tested, over 41% were toxigenic producing different mycotoxins. Strains of A. flavus tested were aflatoxin (AF) producers with A. flavus producing AFB1, sterigmatocystin (STE), versiconol (VOH), flavacol (FLV) and kojic acid (KA). In establishing the pairwise associations between the secondary fungal metabolites, AFB1 was found to have a positive pairwise association with CPA while Penicillium chrysogenum was the only Roquefortine C (ROQ C, range: 13 - 1,260 µg/kg) producer found in the study. The occurrence of multiple metabolites by single fungal species and vice-versa was also noted.

For the mycotoxicological survey, 399 fermented products were screened for the presence of

23 mycotoxins including AFB1, FB1, deoxynivalenol (DON), zearalenone (ZEN) and OTA.

Aflatoxin B1, FB1 and DON were present in 50% of ogiri (mean: 4 µg/kg), 51% of ogi (range: 42 - 2,492 µg/kg) and 73% of mahewu (range: 1 8 - 32 µg/L) samples, respectively. Deoxynivalenol was the dominant mycotoxin in 84% of the umqombothi samples. Aflatoxin contamination was highest in ogiri and AFB1 levels in all positive samples exceeded the 2 µg/kg limit. A significant fraction of the samples (272/399) had mycotoxins occurring singly or in combination though mostly at low contamination levels. It was also found that a 60 kg adult consuming 1- 6 L/day of umqombothi was exposed to FB1 + FB2 contamination at an estimated rate of 2.20 - 13.20 µg/kg body weight/day. These values were above the maximum tolerable daily intake of 2 µg/kg bw/day established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA).

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The cytotoxic potential of mycotoxin (AFB1, OTA, DON, ZEA, STE, ROQ C and FB1) extracts of fungal species from the fermented products were evaluated in vitro on human lymphocyte cells via methylthiozol tetrazolium (MTT) assay. All the extracts tested induced prominent mortality on lymphocyte cells as demonstrated by the reduced % cell viability recorded after exposures particularly when the concentration levels (20 to 80 µl) and times of incubation (24, 48 and 72 hrs) were increased. Increasing the concentration level of STE from

20 to 80 µl significantly (p < 0.01) decreased cell viability from 92 to 82%. Aflatoxin B1 extract induced the highest decrease in cell viability (48.3%) amongst the mycotoxin extracts tested which may be due to its high level of concentration as well as its toxicity in comparison with other mycotoxins. Furthermore, the interactive effect of toxin concentration and duration of exposure was significant (p < 0.05) only in the case of FB1.

This study reports the diversity of bacteria, fungi and their respective toxins (endotoxins and mycotoxins) in seven locally processed and commonly consumed fermented products that are not regulated by national and international regulatory agencies. Data generated herein provides information on the safety and quality of these products, and highlighted some unreported microbial species and also identified an array of metabolites of toxicological importance. Although low levels of mycotoxins were noted, the simultaneous occurrence of multiple mycotoxins and endotoxins within some samples may pose significant health risk amongst consumers. There is need to develop and implement multiple and sustainable food control measures both at local and international levels to mitigate potential risks of consumers.

Keywords: Fermented foods, toxigenic fungi, bacteria, safety, mycotoxin, endotoxin, Nigeria, and South Africa

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DECLARATION

I, ADEKOYA Ifeoluwa Omobolanle (Nee Olotu) of the University of Johannesburg hereby declare that this dissertation has been solely written by me and is a record of my own research work. It has not been submitted to any other University or institution for degree purposes. The contributions made by others have duly been acknowledged.

… …………………… OLOTU, Ifeoluwa Omobolanle

Date……27th February 2018…...……

v DEDICATION

To God all mighty, the giver of every good gift, my source of strength, my shield, my portion, deliverer, strong tower and my very present help in time of need.

To my beloved parents, Chief and Mrs Bamidele Olotu, thank you for being good role models and creating an enabling environment for me to pursue my dreams.

To my siblings and their spouses, Mr and Mrs Olanrewaju Lawani, Mr and Mrs Bayodele Olotu, Mr and Mrs Taiwo Babalola and Mr and Mrs Oladapo Olotu, thank you for the love, support, motivation and guidance you made available to me throughout my study.

To my late brother, Ayodeji Olotu, and late teacher, Mr Fakanmi Adesina, your memories make me to strive to be the best in all I do and your fierceness even in the face of life challenges is enough for me not to give up.

To my awesome husband, Adekoya Adedapomola Adebankole, no words can describe my appreciation for all the love, care, patience, motivation and support you gave during the course of my study. I am forever grateful and indebted to you.

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ACKNOWLEDGEMENTS

I am profoundly grateful to my supervisors; Prof. Patrick Berka Njobeh, Dr. Adewale Obadina and Prof. Sarah De Saeger for their moral support, constructive criticism, attention, provision, guidance, and encouragement which facilitated the completion of this research. I particularly and sincerely thank Prof. P.B. Njobeh (University of Johannesburg, UJ) from whom I have gained a lot, for his invaluable advice, unwavering, timely and endless assistance towards my study. To Dr Marthe De Boevre, Dr Jose Di Mavungu, Mr M. Van De Velde, Mr F. Dumoulin, and Mrs C. Detavernier (Laboratory of Food Analysis, Ghent University, Belgium) and Dr J.Z. Phoku (Toxicology and Ethnoveterinary Medicine Unit, Agricultural Research Council, South Africa), I wish to thank you for your timely intervention and excellent contribution towards the achievement of my research goals.

My deepest gratitude also go to Mr Erick Van Zyl (retired) and Prof. E. Green, Heads of Department, Food Technology and Biotechnology, UJ, for providing a strong support to Department, which facilitated the completion of this study.

It is with immense pleasure that I thank my main sponsors and funders; the Organisation for Women in Science in the Developing World (OWSD), Italy and the Swedish International Cooperation Agency (SIDA), Sweden. I also thank the Centre of Excellence (CoE) for Food Security co-hosted by University of Pretoria and University of the Western Cape, South Africa; African Women in Agricultural Research and Development (AWARD), Kenya; MYTOX-SOUTH hosted in the Laboratory of Food Analysis, Ghent University, Belgium; L’Oreal UNESCO for women in science, South Africa; Global Excellence and Stature (GES) fellowship, and the Faculty of Science, UJ.

My earnest appreciation also goes to Prof. S. Okoth (Department of Biological Sciences, University of Nairobi, Kenya), Prof. E. Akinlabi (Department of Mechanical Engineering, UJ) and Prof. G.O. Adegoke (Department of Food Technology, University of Ibadan, Nigeria) for their invaluable advice and encouragement. I wish to also thank Prof. O. Nwinyi (Department of Microbiology, Covenant University, Nigeria) for his assistance in registering the microorganisms identified in this study with the genbank (NCBI, USA)

I thank Dr E. Kayitesi, Dr V. Mavumengwana and Mr W. Qaku of the Department of Biotechnology and Food Technology for use of their laboratory facilities. I am also grateful to Mr S. Mandla (Department of Biomedical Technology), Mr Alista Campbell (Department

vii of Microbiology), Mr A. Pieterse (Water and Health Research Unit) and Miss L. Viljoen (Department of Microbiology) for their technical support.

I would also like to express my genuine appreciation to the members of the Food, Environment, and Health Research Group (FEHRG), UJ, many of whom I worked with particularly Mrs M. Bello, Mr S. Gbashi, Mr S. Tamufon, Ms. M. Olorunfemi, Mrs J. Akinola, Mrs M. Areo and Mr H. Garba for their support. The tremendous contributions of Mr O. Adebo of the FEHRG group and Mr I. Azeez of the Chemistry department, UJ is also acknowledged for their professional advice and willingness to assist at all times amidst thier tight schedules.

My sincere appreciation further goes to Mr and Mrs A. Oladejo and Mr and Mrs K. Asuni for accepting me as part of their family throughout my stay in UJ. To all my friends, Ms K. Owolabi, Dr and Dr Mrs Jude Obidegwu, Mr M. Abdullah, Mr and Mrs Timothy Ewuola, Miss Grace Daji etc., I say thank you for your friendship and support. I want to say a very big thank you to those who have served as a pillar of spiritual support during my study, Prophet G. Ogunleye, Pastor A. Kayode, Pastor and Mrs G. Oluwadairo and all members of Springs of Mercy Church, Johannesburg, South Africa.

My wholehearted appreciation is also extended to the fermented food sellers in Nigeria and South Africa who participated in this study.

I also thank, my parents, in laws: Mr and Mrs Adekoyejo Adekoya and siblings for their financial and spiritual support, love, patience and sacrifice. My most heartfelt gratitude goes to my Son, Mofiyinfoluwa and Husband, Adedapomola; the completion of this study would not have been possible without thier support.

Finally to GOD ALMIGHTY, for giving me life, guidance and granting me supernatural open doors to many opportunities during my study in South Africa.

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PUBLICATIONS AND PRESENTATIONS

Articles published or written for publication: The following articles have either been published in or submitted to refereed journals for publication.

Adekoya I.O., Obadina A.O., Phoku J.Z., Nwinyi O.A., and Njobeh P.B. (2017). Contamination of Fermented Foods in Nigeria with Fungi. LWT-Food Science and Technology. 86, 76-84.

Adekoya, I.O., Njobeh, P.B., Obadina A.O., Chilaka, A.C., Okoth, S, De Boevre M., and De Saeger, S. (2017). Perception and Prevalence of Mycotoxin Contamination in Selected Nigerian Fermented Foods. Toxins. 9, 363.

Adekoya, I.O., De Saeger, S., Chilaka, A.C., De Boevre S., Obadina A.O., and Njobeh P.B. (2018). Mycobiota and Co-occurrence of Mycotoxins in South African Maize- Based Opaque Beer. International Journal of Food Microbiology 270, 22-30.

Adekoya, I.O., Obadina A.O., Phoku, J.Z., De Boevre M., De Saeger, S., and Njobeh, P.B. Fungal and Mycotoxin Contamination of Fermented Foods from Selected South African Markets. Food Control, 90, 295-303.

Adekoya, I.O., Njobeh, P.B., Obadina A.O., Chilaka, A.C., Okoth, S, De Boevre M., and De Saeger, S. Metabolite Profiling and Toxigenicity of Fungi Isolated in Fermented Foods from Selected Nigerian and South African Markets. Submitted (Food and Chemical Toxicology).

Adekoya, I.O., Obadina A.O., Phoku, J.Z., De Boevre M., De Saeger, S., and Njobeh, P.B. Cytotoxic Effects of Mycotoxin Extracts of Fungal Isolates in Fermented Foods from Nigeria and South Africa on Human Lymphocyte Cells. Submitted (Food and Chemical Toxicology).

Adekoya, I.O., Obadina A.O., Olorunfemi, M., De Saeger, S., and Njobeh, P.B. Pathogenic Bacteria and Endotoxins in Fermented Foods and Beverages from Selected Nigerian and South African Markets. Submitted (International Journal of Food Microbiology).

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Conference articles/presentations: The following research outputs have been presented in local and international conferences and published as conference proceedings.

Adekoya, I.O., Njobeh, P.B., Obadina A.O., Chilaka, A.C., Okoth, S, De Boevre M., and De Saeger, S. (2017). Multi-mycotoxin Contamination in Fermented Locust Beans (Parkia biglobosa) and the Perception of Mycotoxin Contamination in Nigerian Markets. Oral Paper presented at the 1st MYCOKEY International Conference, Ghent, Belgium. 11th to 14th of September, 2017.

Adekoya, I.O., De Saeger, S., De Boevre S., Obadina A.O., and Njobeh P.B. (2017). Toxigenic Potential of Fungal Species Occurring in Fermented Foods from Nigeria. Poster presentation at the Society for Applied Microbiology Conference. Newcastle, United Kingdom. 3rd to 6th of July, 2017.

Adekoya, I.O., Obadina A.O., Phoku, J.Z., and Njobeh, P.B. (2017). Incidence and Mycotoxigenic Potentials of Fungi Isolated from some Traditionally Fermented Foods in Nigeria. Oral Paper presented at Food Innovation Conference, Cesena, Italy. 31st January to 3rd of Feburary, 2017.

Adekoya, I.O., Obadina A.O., De Boevre S., De Saeger, S., and Njobeh P.B. (2016). Safety Assessment of some Traditionally Fermented Foods Produced in Nigeria and South Africa. Poster presented at the Ghent African Platform Symposium, Ghent, Belgium. 8th to 9th of December, 2016.

Adekoya, I.O., Obadina A.O., Phoku, J.Z., and Njobeh, P.B. (2016). Mycotoxigenic Potentials of Fungi Isolated from some Traditionally Fermented Foods in South Africa. Poster presented at the ICFMH - Food Microbiology Conference, Ireland. 19th to 22nd of July, 2016.

Adekoya, I.O., Obadina A.O., Phoku, J.Z., and Njobeh, P.B. (2016). Fungi Occurrence in some Traditionally Fermented Foods in South Africa. Poster presentation at the International Association of Food Protection Conference, Missouri, USA. 29th July to 4th of August, 2016.

Adekoya, I.O., Obadina A.O., Phoku, J.Z., and Njobeh, P.B. (2016). Incidence and Mycotoxigenic Potentials of Fungi Isolated from Some Traditionally Fermented

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Foods in Nigeria. Poster presentation at the International Food Safety and Security Conference, Johannesburg, South Africa. 16th to 18th of May, 2016

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TABLE OF CONTENTS

EXECUTIVE SUMMARY ...... II DECLARATION...... V DEDICATION...... VI ACKNOWLEDGEMENTS ...... VII PUBLICATIONS AND PRESENTATIONS ...... IX TABLE OF CONTENTS ...... XII LIST OF TABLES ...... XVIII LIST OF FIGURES ...... XX LIST OF ABBREVIATIONS ...... XXII LIST OF UNITS AND SYMBOLS...... XXVII THESIS OUTLINE ...... XXIX CHAPTER ONE ...... 1 1.0 GENERAL INTRODUCTION ...... 1 1.1 BACKGROUND ...... 1 1.2 PROBLEM STATEMENT ...... 3 1.3 JUSTIFICATION OF STUDY ...... 3 1.4. AIM AND OBJECTIVES OF THE STUDY ...... 5 1.4.1 Aim ...... 5 1.4.2 Objectives ...... 5 CHAPTER TWO ...... 7 2.0 LITERATURE REVIEW ...... 7 2.1 INTRODUCTION ...... 7 2.2 FOOD FERMENTATION ...... 7 2.2.1 Microflora in Fermented Foods ...... 9 2.2.2 Africa Indigenous Fermented Foods ...... 10 2.2.2.1 Fermented vegetable proteins ...... 11 2.2.2.2 Fermented cereal based foods ...... 13 2.2.2.3 Fermented starchy root products ...... 14 2.2.2.4 Alcoholic beverages ...... 14 2.2.2.5 Fermented animal proteins ...... 15 2.2.3 Benefits of Food Fermentation ...... 15 2.2.4 Features of Food Fermentation in Africa ...... 17 2.2.5 Safety of Fermented Foods ...... 18 2.3 BACTERIA ...... 20 2.3.1 Overview, Structure, Metabolism and Significance...... 20 2.3.2 Bacteria Toxins ...... 21 2.3.2.1 Endotoxins: overview, history, structure and clinical association ...... 22 2.3.2.2 Detection methods ...... 25 2.3.3 Occurrence of Gram-negative Bacteria and Endotoxins in Foods ...... 26 2.4 FUNGI ...... 27 2.4.1 An Overview ...... 27

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2.4.2 Natural Occurring Toxigenic Fungi ...... 28 2.4.2.1 Aspergillus species ...... 28 2.4.2.2 Fusarium species...... 29 2.4.2.3 Penicillium species ...... 30 2.4.2.4 Alternaria species ...... 31 2.4.2.5 Stachybotrys ...... 32 2.4.2.6 Claviceps species ...... 33 2.4.3 Factors Influencing Fungal Colonization and Production of Mycotoxin ...... 33 2.4.3.1 Environmental factors ...... 34 2.4.3.2 Biological factors ...... 36 2.4.3.3 Chemical factors ...... 37 2.5 MYCOTOXINS ...... 38 2.5.1 Definition and Concepts ...... 38 2.5.2 Nature, Chemistry, Distribution and Health Implications of Mycotoxins ...... 40 2.5.2.1 Aflatoxins ...... 41 2.5.2.2 Ochratoxins ...... 43 2.5.2.3 Zearalenone ...... 44 2.5.2.4 Fumonisins ...... 44 2.5.2.5 Patulin ...... 46 2.5.2.6 Trichothecenes ...... 46 2.5.2.7 Citrinin ...... 47 2.5.2.8 Ergot alkaloids ...... 48 2.5.2.9 Sterigmatocystin ...... 49 2.5.2.10 Alternariol and Alternariol Monomethyl Ether ...... 49 2.5.2.11 Emerging mycotoxins ...... 50 2.5.2.12 Masked mycotoxins ...... 51 2.5.2.13 Miscellaneous mycotoxins ...... 52 2.5.3 Mycotoxin Regulations ...... 55 2.5.4 Mycotoxin Control and Prevention ...... 57 2.5.4.1 Pre-harvest measures ...... 58 2.5.4.2 Post-harvest measures ...... 60 2.5.5 Socio-economic Impact of Mycotoxin Contamination ...... 62 2.5 CONCLUDING REMARKS ...... 63 REFERENCES ...... 63 CHAPTER THREE ...... 103 CONTAMINATION OF FERMENTED FOODS IN NIGERIA WITH FUNGI ...... 103 ABSTRACT ...... 103 3.1 INTRODUCTION ...... 104 3.2 MATERIALS AND METHODS ...... 106 3.2.1 Sampling ...... 106 3.2.2 Methodology ...... 106 3.2.2.1 Determination of moisture, pH and Total Titratable Acidity (TTA) contents of fermented foods ...... 106 3.2.2.2 Isolation and identification of fungi ...... 107 3.2.2.3 Molecular studies ...... 107 3.2.2.4 Phylogenetic analysis ...... 108 3.2.3 Data analysis ...... 108

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3.3 RESULTS ...... 108 3.4 DISCUSSION ...... 115 3.5 CONCLUSION ...... 118 REFERENCES ...... 119 DATA REFERENCES ...... 124 CHAPTER FOUR ...... 127 AWARENESS AND PREVALENCE OF MYCOTOXIN CONTAMINATION IN SELECTED NIGERIAN FERMENTED FOODS ...... 127 ABSTRACT ...... 127 4.1 INTRODUCTION ...... 128 4.2 RESULTS AND DISCUSSION ...... 130 4.2.1 Perception Studies ...... 130 4.2.2 Method Performance Characteristics ...... 137 4.2.3 Mycotoxin Contamination ...... 139 4.3 CONCLUSION ...... 144 4.4 MATERIALS AND METHODS ...... 145 4.4.1 Sampling ...... 145 4.4.2 Awareness Studies ...... 145 4.4.3 Mycotoxin Analysis ...... 146 4.4.3.1 Materials and Chemicals ...... 146 4.4.3.2 Mycotoxin Standards...... 146 4.4.3.3 Sample Preparation ...... 146 4.4.3.4 Liquid Chromatography-Tandem Mass Spectrometry ...... 147 4.3.5. Method Validation ...... 148 4.4.4 Data Analysis ...... 148 REFERENCES ...... 148 CHAPTER FIVE ...... 154 FUNGAL AND MYCOTOXIN CONTAMINATION OF FERMENTED FOODS FROM SELECTED SOUTH AFRICAN MARKETS ...... 154 ABSTRACT ...... 154 5.1 INTRODUCTION ...... 155 5.2 METHODOLOGY ...... 157 5.2.1 Sampling ...... 157 5.2.2 Chemical properties ...... 157 5.2.3 Isolation and identification of fungi ...... 158 5.2.3.1 Molecular identification of fungal isolates ...... 158 5.2.4 Mycotoxin analysis ...... 158 5.2.4.1 Reagents and standards ...... 158 5.2.4.2 Sample preparation ...... 159 5.2.4.3 Liquid chromatography tandem mass spectrometry ...... 159 5.2.5 Data Analysis ...... 160 5.3 RESULTS AND DISCUSSION ...... 160 5.4 CONCLUSION ...... 171 REFERENCES ...... 172 CHAPTER SIX ...... 178

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MYCOBIOTA AND CO-OCCURRENCE OF MYCOTOXINS IN UMQOMBOTHI: A SOUTH AFRICAN CEREAL-BASED OPAQUE BEER ...... 178 ABSTRACT ...... 178 6.1 INTRODUCTION ...... 179 6.2 MATERIALS AND METHODS ...... 180 6.2.1 Beer samples ...... 180 6.2.2 Methodology ...... 181

6.2.2.1 pH, MC, aw and TTA determination ...... 181 6.2.2.2 Mycobiota of fungi ...... 181 6.2.2.3 Molecular identification of fungi isolates ...... 181 6.2.2.4 Phylogenetic analysis ...... 182 6.2.2.5 Mycotoxin analysis ...... 182 6.2.2.6 Data analysis ...... 184 6.3 RESULTS AND DISCUSSION ...... 184 6.3.1 Chemical properties and fungal load ...... 184 6.3.2 Fungal incidence and dominant fungal genera ...... 185 6.3.3 Phylogenetic analysis ...... 187 6.3.4 Mycotoxin occurrence in South African cereal-based opaque beer samples ...... 188 6.3.5 Estimation of mycotoxin dietary intakes among beer consumers ...... 191 6.4 CONCLUSION ...... 193 REFERENCES ...... 194 DATA REFERENCES ...... 199 CHAPTER SEVEN ...... 202 METABOLITE PROFILING AND TOXIGENICITY OF FUNGAL ISOLATES IN FERMENTED FOODS FROM SELECTED NIGERIAN AND SOUTH AFRICAN MARKETS ...... 202 ABSTRACT ...... 202 7.1 INTRODUCTION ...... 203 7.2 MATERIALS AND METHODS ...... 205 7.2.1 Materials ...... 205 7.2.1.1 Reagents ...... 205 7.2.1.2 Standards ...... 205 7.2.1.3 Mycotoxigenic potential of fungal isolates ...... 206 7.2.2 Methods ...... 206 7.2.2.1 Multi-mycotoxin extraction ...... 206 7.2.2.2 Liquid chromatography-tandem mass spectrometry ...... 207 7.2.2.3 Data Analysis ...... 209 7.3 RESULTS AND DISCUSSION ...... 209 7.4 CONCLUSION ...... 220 ACKNOWLEDGMENTS ...... 220 CONFLICT OF INTEREST ...... 220 REFERENCES ...... 221 CHAPTER EIGHT ...... 227 CYTOTOXIC EFFECTS OF MYCOTOXIN EXTRACTS OF FUNGAL ISOLATES IN FERMENTED FOODS FROM NIGERIAN AND SOUTH AFRICAN ON HUMAN LYMPHOCYTE CELLS ...... 227 ABSTRACT ...... 227 8.2 MATERIALS AND METHODS ...... 230

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8.2.1 Mycotoxin standards and reagents ...... 230 8.2.2 Isolation, molecular characterisation and mycotoxin analysis of isolates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) ...... 230 8.2.3 Isolation and purification of mononuclear cells ...... 231 8.2.4 Enumeration of cells ...... 231 8.2.4.1 Cell enumeration with Neubauer haemocytometer ...... 231 8.2.4.2 Cell enumeration with Muse analyser...... 232 8.2.5 Methyl thiazol tetrazolium assay ...... 232 8.2.6 Data analysis ...... 233 8.3 RESULTS ...... 233 8.4 DISCUSSION ...... 241 8.5 CONCLUSION ...... 244 CHAPTER NINE ...... 252 PATHOGENIC BACTERIA AND ENDOTOXINS IN FERMENTED FOODS AND BEVERAGES FROM SELECTED NIGERIAN AND SOUTH AFRICAN MARKETS ...... 252 ABSTRACT ...... 252 9.1 INTRODUCTION ...... 253 9.2 MATERIALS AND METHODS ...... 255 9.2.1 Materials ...... 255 9.2.1.1 Reagents ...... 255 9.2.1.2 Fermented food and beverage ...... 255 9.2.2 Methodology ...... 256 9.2.2.1 Microbiological analysis ...... 256 9.2.2.2 Microbial identification with the VITEK 2 compact instrument ...... 256 9.2.2.3 DNA Extraction, Polymerase Chain Reaction (PCR) and Sequencing ...... 257 9.2.2.4 Endotoxin analysis ...... 258 9.2.2.5 Data analysis ...... 259 9.3 RESULTS ...... 259 9.4 DISCUSSION ...... 265 9.4.1 Bacterial flora of fermented foods from Nigerian and South African markets ...... 265 9.4.2 Endotoxin levels of fermented foods from Nigeria and South Africa markets ...... 267 9.5 CONCLUSION ...... 268 REFERENCES ...... 268 CHAPTER TEN ...... 273 10.0 GENERAL DISCUSSION AND CONCLUSIONS ...... 273 10.1 General discussion ...... 273 10.2 General Conclusions ...... 276 REFERENCES ...... 277 APPENDICES ...... 280 APPENDIX 3.0 ...... 280 Appendix 3.1 Agar preparations ...... 280 Appendix 3.2 Some fungal species from fermented foods based on macroscopic characteristics (A-C): A. flavus, F. verticilliodes, and P. expansum ...... 280 Appendix 3.3 Microscopic view of A. parasiticus (A: magnification X63) and F. verticilliodes (B: magnification X40) isolated from ogiri ...... 281 APPENDIX 4.0 ...... 282

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Appendix 4.1 Questionnaire on demographics, practices, understanding and perceived health risk of fungal and mycotoxins contamination amongst fermented food sellers in Nigeria ...... 282 APPENDIX 5.0 ...... 284 Appendix 5.1 Calibration curves ...... 284 Appendix 5.2 Chromatograms for standards ...... 285 Appendix 5.3 Chromatograms of some mycotoxins ...... 286 Appendix 5.4 Method performance parameters of the fermented food matrixes ...... 287 APPENDIX 7.0 ...... 288

Appendix 7.1 Multiple Reaction Monitoring (MRM) transitions of Aflatoxin G1 and B2 standards indicating their precursor ions, product ions and retention times ...... 288 Appendix 7.2 Aspergillus species in fermented foods as shown in Figure 7.1 and 7.2 ...... 289 APPENDIX 8.0 ...... 292 Appendix 8.1 Ethical clearance for cytotoxicity experiment ...... 292 APPENDIX 9.0 ...... 293 Appendix 9.1 Agar preparations ...... 293 Appendix 9.2 Results of microbial identification on VITEK 2 Compact instrument ...... 294

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LIST OF TABLES

Table 2.1 Microorganisms of public health importance detected in some traditional fermented foods………………………………………………………………………………………….19

Table 2.2 Temperature range and aw requirement for growth of some fungi………………35

Table 2.3 Temperature range and aw requirement for production of mycotoxins by some fungi………………………………………………………………………………………….35 Table 2.4 Occurrence of mycotoxins in some African foods………………………………...53 Table 2.5 Maximum allowable limits (µg/kg) of mycotoxins in different countries………...56 Table 3.1 Mean pH, TTA and moisture content of some Nigerian fermented foods ……....108 Table 3.2 Correlation coefficient of the pH, Total TTA, moisture content and total fungal count of some Nigerian fermented foods…………………………………………………...109 Table 3.3 Total fungal load and isolated genera of fungi from Nigerian fermented foods...109 Table 3.4 Incidence rates of fungal contamination of Nigerian fermented foods with Aspergillus, Penicillium and Fusarium species………………………………………….....111 Table 3.5 Incidence rate of fungal contamination of Nigerian fermented foods with other fungal species……………………………………………………………………………….113 Table 4.1 Descriptive statistics and knowledge of fungal and mycotoxin contamination of fermented food sellers……………………………………………………………………....132 Table 4.2 Kendall’s tau-b correlation between education and awareness level of fungi and mycotoxins contamination by respondents………………………………………………....136 Table 4.3 Method performance parameters of fermented food matrices…………………...138 Table 4.4 Multi-mycotoxin profile of fermented foods from South-west Nigeria…………140 Table 5.1 Mean pH, water activity, total titratable acidity and moisture content of fermented foods obtained from South African markets………………………………………………..161 Table 5.2 Mean fungal load and isolated fungal genera of fermented foods obtained from South African markets……………………………………………………………………....161 Table 5.3 Correlation coefficients of the pH, water activity, total titratable acidity and moisture content of ogiri, ugba and ogi…………………………………………………………...162 Table 5.4 Incidence rates of fungal contamination of fermented foods from South African markets.…………………...... 164 Table 5.5 Incidence rates of fungal contamination of fermented foods from South African markets with other fungal species…………………………………………………………..166

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Table 5.6 Incidence and mycotoxins levels of fermented foods from South African markets……………………………………………………………………………………...168 Table 6.1 Chemical properties of South African cereal-based opaque beer………………..185 Table 6.2 Occurrence of fungi in South African cereal-based opaque beer (Umqombothi)..186 Table 6.3 Natural incidence and mycotoxin levels of South African cereal-based opaque beer and method performance characteristics…………………………………………………....189 Table 6.4 Deoxynivalenol and fumonisin exposure based on the consumption of different volumes of umqombothi in μg/kg bw/day………………………………………………..…193 Table 7.1 Mass spectrometric parameters for different target analytes………………….....208 Table 7.2 Production of mycotoxins by Aspergillus, Penicillium, and Fusarium spp. isolated from lactic acid fermented products produced in Nigeria and South Africa………………210 Table 7.3 Production of mycotoxins by Aspergillus, Penicillium and Fusarium spp. isolated from alkaline fermented products produced in Nigeria and South Africa...... 212 Table 8.1 Effects of extracts of Aspergillus species isolated from fermented foods on the viability of human mononuclear cells………………………………...…………………….234 Table 8.2 Effects of extracts of Penicillium species isolated from fermented foods on the viability of human mononuclear cells ……………………………………………………...235 Table 8.3 Effects of extracts of Fusarium species isolated from fermented foods on the viability of human mononuclear cells ……………………………………………………...236 Table 8.4 Mean cell viability (%) of fungal isolates of fermented foods as influenced by exposure time and concentration of fungal extracts………………………………………..238 Table 9.1 Mean bacterial load of fermented foods from Nigerian and South African markets in CFU/g or CFU/mL of sample…………….……………………………………………...260 Table 9.2 Incidence of Gram-negative and Gram-positive bacteria isolated from fermented foods from Nigerian markets…………………………………………………………..……261 Table 9.3 Incidence of Gram-negative and Gram-positive bacteria isolated from fermented foods and beverages from South African markets………………………………………….262 Table 9.4 Gram-negative bacteria isolated from fermented foods by VITEK biochemical tests……………………………………………………………………………………..…...263 Table 9.5 Mean endotoxin levels of fermented foods from Nigeria and South African markets……………………………………………………………………………………...264

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LIST OF FIGURES

Figure 2.1 Major pathways of carbohydrate fermentation in lactic acid bacteria through the Embden-Meyerhof and 6-phosphoketolase pathways………………………………………....8 Figure 2.2 Fermented African locust beans (Iru)………………………………………….…11 Figure 2.3 Fermented melon (ogiri)...... 12 Figure 2.4 Fermented African oil bean seed (ugba)……………………………………...... 12 Figure 2.5 Fermented cereal gruel (ogi) from maize and sorghum…………………………..13 Figure 2.6 Fermented cassava product (garri)...... 14 Figure 2.7 General structure of bacteria lipopolysaccharides………………………………..23 Figure 2.8 A model of diseases potentially associated with bacteria/endotoxin……………..24 Figure 2.9 Distinctive structures of Aspergillus species……………………………….…….29 Figure 2.10 F. oxysporum spores; a: microconidia; b: macroconidia; c: chlamydospores…..30 Figure 2.11 Conidiophore branching patterns of Penicillium species……………………...... 31 Figure 2.12 Alternaria species conidia and conidiophores………………………………..…32 Figure 2.13 Microscopic features of Stachybotrys fungi………………………………….....33 Figure 2.14 A simplified representation of some general relationships in mycotoxicosis…...40 Figure 2.15 Molecular structures of aflatoxins………………………………………………42 Figure 2.16 Molecular structure of ochratoxin A………………………………………….....43 Figure 2.17 Molecular structure of zearalenone……………………………………………...44 Figure 2.18 Molecular structures of fumonisins……………………………………………..45 Figure 2.19 Molecular structures of trichothecenes………………………………………….47 Figure 2.20 Molecular structure of citrinin…………………………………………………..48 Figure 2.21 Molecular structure of sterigmatocystin………………………………………...49 Figure 2.22 Molecular structure of enniatin B…………………………………………….....50 Figure 2.23 Molecular structure of deoxynivalenol-3-glucoside…………………………….51 Figure 2.24 Mycotoxin regulations within African countries……………………………...... 57 Figure 3.1 Neighbour-joining phylogenetic tree fungal species from ogiri………………...114 Figure 4.1 Percentage co-occurrence of mycotoxins in fermented foods from South-west, Nigeria…………………………………………………………………………………...... 143 Figure 5.1 Percentage of co-occurring mycotoxins in fermented foods from South African markets……………………………………………………………………………………...171 Figure 6.1 Phylogenetic analysis showing relationships of the 16S rRNA gene sequences of fungi isolated from South African cereal-based opaque beer………………………………188

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Figure 6.2 Spider plot of co-occurring mycotoxins in South African tradtional cereal-based opaque beer………………………………………………………………………………....191 Figure 7.1 Hierarchical clustering based on metabolites profile of Aspergillus species isolated from fermented foods from Nigeria………………………………………………………...215 Figure 7.2 Hierarchical clustering based on metabolites profile of Aspergillus species isolated from fermented foods from South Africa…………………………………………………...216 Figure 7.3 Co-occurrence matrix of metabolites of Aspergillus species isolated from fermented foods from Nigeria……………………………………………………………....217 Figure 7.4 Co-occurrence matrix of metabolites of Aspergillus species isolated from fermented foods from South Africa………………………………………………………....218 Figure 8.1 Mean toxicity induction (%) at different concentrations and times of exposure of different fungi extract……………………………………………………………………….240 Figure 9.1 Percentage of endotoxin contamination in fermented foods from Nigeria and South Africa markets……………………………………………………………………………....264

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LIST OF ABBREVIATIONS

AA Aspergillic acid ACN Acetonitrile ADON Acetyl-deoxynivanelol AF Aflatoxin AFs Aflatoxins

AFB1 Aflatoxin B1

AFB2 Aflatoxin B2

AFG1 Aflatoxin G1

AFG2 Aflatoxin G2

AFM1 Aflatoxin M1

AFM2 Aflatoxin M2 AFN Aflavanine AFTR Aflatrem AFV Aflavarin AFV-1 Aflavarin analog 1 AFV-2 Aflavarin analog 2 AIDS Acquired Immuno-Deficiency Syndrome ALT Altenuene AME Alternariol monomethyl ether ANOVA Analysis of Variance

AOH Alternariol AR Apparent recovery ASPT Aspertoxin 1 ASPT-2 Aspertoxin 2 ATA Alimentary toxic aleukia ATX Altertoxins aw Water activity BEA Beauvericin CAST Council for Agricultural Science and Technology CDC Centers for Disease Control and Prevention CFU/g Colony forming units/gram CFU/ml Colony forming units/millilitre

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CIT Citrinin CPA Cyclopiazonic acid CYA Czapek yeast agar DAS Diacetoxyscirpenol DCM Dichloromethane DH-AF Dihydro-aflavine DHOMST Dihydro-O-methylsterigmatocystin DHoxyHOMST Dihyroxyl-O-methylsterigmatocystin DMSO Dimethylsulphoxide DNA Deoxyribonucleic Acid DOM Deepoxy-deoxynivalenol DON Deoxynivalenol DON3G Deoxynivalenol-3-glucoside DYT Dytryptohenaline EC European Commission EFSA European Food Safety Authority ENN Enniatin (s) ENN B Enniatin B ESI+ Electrospray ionization EU European Union FA Fusaric acid FB Fumonisin FBs Fumonisins

FB1 Fumonisin B1

FB2 Fumonisin B2

FB3 Fumonisin B3 FBS Foetal bovine serum FDA Food Drug and Administration FLV Flavacol FUSA Fusaproliferin FUS X Fusarenon X GRAS Generally Regarded as Safe HAIs Hospital-acquired infections HCl Hydrochloric acid

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HEP Human hepatoma HIV Human Immuno-Deficiency Virus HPLC High performance liquid chromatography HT-2 HT-2 toxin IAC Immunoaffinity column IARC International Agency for Research on Cancer IITA International Institute of Tropical Agriculture ITS Internal Transcribed Spacer JECFA Joint FAO/WHO Expert Committee on Food Additives KA Kojic acid LAB Lactic acid bacteria LAL Limulus Amoebocyte Lysate LC-MS/MS Liquid chromatography tandem mass spectrometry LEO-C Leporin C LPS Lipopolysaccharides LOD Limit of detection LOQ Limit of quantification MC Moisture content MEA Malt extract agar ME-ISOC Methylisocoumarin MMC Matrix matched calibration curve MON Moniliformin MS Mass spectrometry MTT Methyl thiazole tetrazolium assay N number of samples NaOH Sodium hydroxide NAFDAC National Agency for Food and Drug Administration and Control NCBI National Center for Biotechnology Information NEO Neosolaniol NIV Nivalenol NORA Noranthrone OD Optical density OH-AA Hydroxyneoaspergillic acid OMST O-methylsterigmatocystin

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OTA Ochratoxin A OTs Ochratoxins OxyHOMST Oxy-O-methylsterigmatocystin P P value PA Penicillic acid PAS Paspaline PASL Paspalinine PAT Patulin PBS Phosphate buffer saline PCR Polymerase chain reaction PDA Potato dextrose agar pH Hydrogen/ hydroxyl ion concentration PHA Phyto-haemagglutinin PMTDI Provisional maximum tolerable daily intake PNA p-nitroaniline PTWI Provisional tolerable weekly intake PVDF Polyvinylidene fluoride R Correlation co-efficient R2 Co-efficient of determination RASFF Rapid Alert System for Food and Feed RBCA Rose Bengal chloramphenicol agar RNA Ribonucleic Acid ROQ C Roquefortine C RPMI Roswell park memorial institute SD Standard deviation SE Standard error of mean SmF Submerged state fermentation SPE Solid phase extraction SPRE Speradine A Sp. Specie Spp. Species SPSS Statistical package for social scientists SSA sub-Saharan Africa SSF Solid-state fermentation

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STE Sterigmatocystin STE-A Sterigmatocystin analogue T-2 T-2 toxin TAPC Total aerobic plate count TCBS Thiosulfate citrate bile salts sucrose agar TCs Trichothecenes TDI Tolerable daily intake TEA Tenuazoic acid TFC Total fungal count TFF Traditionally fermented foods TTA Total titratable acidity TVPC Total viable plate count UPLC Ultra-high performance liquid chromatography VHA Versiconal hemiacetal acetate VOH Versiconol WHO World Health Organization YES Yeast extract sucrose agar ZAN Zearalanone ZEN Zearalenone

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LIST OF UNITS AND SYMBOLS

% Percent ˚C Degree Celsius < Less than > Greater than ± Plus or minus µg/mL Microgram/millilitre µg/kg Microgram/kilogram µg/L Microgram/litre mg/mL Milligram/ millilitre ng/mL Nanogram/millilitre µL Microlitre µm Micrometre µM Micromolar pM Picomolar mM Millimolar g Gram Hrs Hours kg Kilogram CFU/g Colony forming unit/gram CFU/mL Colony forming unit/millilitre EU/g Endotoxin unit/gram L/day Litres/day U/mL Units/millilitre µg/kg bw/day Microgram/kilogram body weight/day L Litre m/z Mass to charge ratio V Volts eV Electron volts kV Kilovolt L/h Litre per hour M Molar % lactic acid Percentage lactic acid

xxvii min Minute(s) mm Millimetre mg/kg Milligram/kilogram ng/kg Nanogram/kilogram ng/g Nanogram/gram nm Nanometre Sec Second(s) v Volume g/L Gram/litre psi Pounds per square inch rpm Revolutions per minute ° S Degree south ° E Degree east ß Beta γ Gamma α Alpha USD United States Dollars

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THESIS OUTLINE

This thesis covered studies on the safety of some traditionally fermented foods produced in Nigeria and South Africa. A brief outline of the chapters presented in this thesis is provided below.

Chapter One: General introduction This chapter gave a general overview of the research subject, provided relevant background information as well as described the problem investigated. The chapter also highlighted the justification, aim and objectives of the study.

Chapter Two: Literature review The chapter presented a detailed appraisal of the research focus. It gave a description of fermentation and its significance, and highlighted the types and safety of fermented foods. It further reviewed the role of different microorganisms in fermentation, considered different microbial toxins (endotoxins and mycotoxins), their occurrences and health implications relative to human exposure. Measures to mitigate the occurrence of these toxins and their producers were also appraised.

Chapter Three: Contamination of fermented foods in Nigeria with fungi Chapter Three described the fungal contamination of some traditionally fermented foods in Nigeria markets. The work described in this chapter had been published in LWT-Food Science and Technology, and accordingly presented herein following the specific guidelines of the journal.

Chapter Four: Awareness and prevalence of mycotoxin contamination in selected Nigerian fermented foods Chapter Four described the level of awareness of fungal and mycotoxin contamination amongst fermented food sellers in Nigeria and the occurrence of mycotoxins in the products they offered for sale. The work in this chapter had been published in Toxins and is presented in the format of the journal.

Chapter Five: Fungal and mycotoxin contamination of fermented foods from selected South African markets

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Chapter Five provided information on the prevalence of fungi and multiple mycotoxins in fermented food products obtained from selected market outlets in South Africa. The work presented in this chapter had been published in Food Control.

Chapter Six: Mycobiota and co-occurrence of mycotoxins in umqombothi: a South African cereal based opaque beer Chapter Six provided information on the fungal microflora, and mycotoxin levels of umqombothi-a South African cereal-based opaque beer. This chapter had been published in the International Journal of Food Microbiology.

Chapter Seven: Metabolite profiling and toxigenicity of fungal isolates in fermented foods from selected Nigerian and South Africa markets Chapter Seven described the toxigenic potential of fungal species isolated from traditionally fermented foods obtained from Nigerian and South African markets and the chapter is written in the format of Food and Chemical Toxicology.

Chapter Eight: Cytotoxic effects of mycotoxin extracts of fungal isolates in fermented foods from Nigeria and South Africa on human lymphocyte cells In this chapter, the effect of different mycotoxin extracts from fungal species isolated from fermented food products on human lymphocyte cells was established. This chapter was written according to the guidelines of Food and Chemical Toxicology.

Chapter Nine: Pathogenic bacteria and endotoxins in fermented foods and beverages from selected Nigerian and South African markets This chapter highlighted the presence of Gram-negative and Gram-positive bacteria as well as endotoxins in fermented foods and beverages in Nigeria and South Africa. The work presented in this chapter had been presented following the detailed requirements of the International Journal of Food Microbiology.

Chapter Ten: General discussion and conclusions This chapter reaffirmed the research focus (i.e., problem statement and aim) of the thesis, presented an overall discussion of the issues addressed in Chapters Three to Nine and reached a conclusion. Recommendations and prospects for future studies were also provided.

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CHAPTER ONE

1.0 GENERAL INTRODUCTION

1.1 Background

Africa being the home to a large number of people with different ethnic and cultural backgrounds reflects its diversity in many local culinary traditions in relation to ingredients of choice, food preparation styles and techniques of cooking. There are wide arrays of foods that are eaten across the continent and one of the major food groups is fermented food. The biological conversion of complex substrates into simple compounds through microbial action is known as fermentation (Obadina et al., 2008; Subramaniyam and Vimala, 2012). Fermentation as a food processing technology originated from the Middle East and dates as far back as 6000 BC (Ross et al., 2002; Subramaniyam and Vimala, 2012).

Fermented foods represent a significant part of the daily diet of people around the world, with its provision of 20 – 40% of the food supply (Abdel et al., 2009) and are reported to be beneficial to human health. In addition to this, fermentation enhances the nutritional and organoleptic properties of foods, reduces toxicity, and increases shelf life as well as acceptability of foods (Oyewole, 1997; Ogunshe and Olasugba, 2008). In Africa, fermented foods are mostly produced traditionally in homes under spontaneous conditions at room temperature. Most African fermented foods are derived from substrates such as legumes, roots, cereals, oil seeds, fish, meat, milk and palm tree and some traditionally fermented foods (TFF) peculiar to Africa are iru, garri, amasi, banku, ogi, injera, mahewu, and meriss to mention a few. Fermentation techniques vary from the very simple spontaneous fermentation that is completed within a few hours to a day, to the very complex and sometimes long fermentation, which can last for a few days to several months.

Iru or dawadawa from African locust bean (Parkia biglobosa) is the most important condiments used to flavour soups and stews in Nigeria (Aworh, 2008; Onyenekwe et al., 2012). Mahewu is a fermented non-alcoholic beverage produced from maize and popularly consumed in South Africa, while banku is a fermented dough and staple indigenous to Ghana and its substrate is majorly maize and cassava. Majority of the organisms that ferment foods are acid-forming bacteria such as lactic acid bacteria (LAB) (Gadaga et al., 1999; Agarry et al., 2010), obligate fermenters and aromatic compound microorganisms (Adebayo-Tayo and Onilude, 2008). Some fungi such as Rhizopus spp. also play key role in fermentation and are

1 the principal microorganisms involved in the production of tempeh, a soy-based product which originates from Indonesia. Africans have a particular affection for fermented foods and this has increased their demand amongst consumers at home and in the diaspora.

Despite the important role microorganisms play in fermentations, some are harmful as they produce secondary metabolites that compromise food quality, causing a wide range of health complications. For example, bacteria generate exotoxins and endotoxins. Exotoxins are extracellular protein toxins which are usually secreted by bacteria and in some cases released by lysis of the bacterial cell (Bhadoria et al., 2015), whereas endotoxins are ubiquitous lipopolysaccharide (LPS) complexes instituted at the outer cell membrane of Gram-negative bacteria (Adam et al., 2014). Endotoxin is made up of a distinct core polysaccharide chain, a polysaccharide side chain (O-specific) and a lipid part, which accounts for its toxicity. Escherichia coli, Salmonella spp., Pseudomonas spp. and Bordetella pertussis are some of bacteria that have been found to produce endotoxins (Bhadoria et al., 2015). Their presence in human body causes fever, shock, disseminated intravascular coagulation and even death in severe cases (Hurley et al., 2015). Endotoxins are not only present in foods but are also present in the environment (Spaan et al., 2007; Bhadoria et al., 2015).

Additionally, mycotoxins are toxic secondary metabolites produced by a widespread of fungal species (Tuner et al., 2005) that are synthesized particularly by species of Aspergillus, Fusarium, Penicillium, Claviceps and Alternaria (Tuomi et al., 2000; Njobeh et al., 2010; Atanda, 2011). Out of over 400 known mycotoxins, the most agriculturally important ones are aflatoxins (AFs), ochratoxins (OTs), zearalenone (ZEN), fumonisins (FBs), trichothecenes (Wagacha and Muthomi, 2008; Makun et al., 2011) and more recently emerging (enniatins, beauvericin, moniliformin) and masked mycotoxins (α-zearalenol-14-β- D-glucopyranoside, β-zearalenol-14-β-D-glucopyranoside).

Mycotoxins have drawn global attention due to their significant threat to food and feed safety. Their impact on the health of humans and animals and their impact on the economy, especially in sub-Saharan Africa (SSA) are enormous (Shephard, 2008; Makun et al., 2011). Quite a number of fungal species and mycotoxins have been detected in a range of food commodities in South Africa (Mashini and Dutton, 2006), Cameroon (Njobeh et al., 2009), Malawi (Matumba et al., 2014), Nigeria (Chilaka et al., 2016; Adekoya et al., 2017b), and Zimbabwe (Hove et al., 2016). Consumption of food and feed commodities contaminated by these fungal toxins seriously compromise health and several outbreaks of mycotoxicosis 2

(disease caused by mycotoxins) have been recorded. An aflatoxicosis outbreak which occurred in 2005 in Kenya led to the death of 125 people out of over 400 reported cases (Lewis et al., 2005) and more recently in the Manyar and Dodoma regions of Tanzania, 65 acute cases of AF food poisoning with 17 fatalities occurred in July 2016 (Buguzi, 2016).

1.2 Problem Statement

Food insecurity has been a great challenge in developing countries for centuries. Unfortunately, individuals in these countries are not only food insecure but are constantly exposed to high levels of toxins through their diets (Turner et al., 2007; Adetunji et al., 2014a). Food safety is of growing global concern, not only for its continuous importance to public health, but also because of its impact on international trade. Food safety entails proper handling, processing and storage of foods in ways that will prevent food-borne hazards (microbiological, physical and chemical). In most parts of Africa, food safety is given little attention as people’s need for sufficient food supply supersedes food safety, thus, food-borne hazards have become a serious problem. Toxigenic microorganisms including bacteria and fungi synthesize toxins as secondary metabolites that have been reported to promote infections and diseases and consequently destroy the tissues, organs and immune systems of the host (Ghali et al., 2008; Neil et al., 2012; Malangu, 2014). The incessant proliferation of food commodities including fermented foods by these toxigenic microorganisms and their metabolites is of importance, hence, there is need to assess their prevalence and levels in African fermented food products.

1.3 Justification of Study

Fermented foods constitute a major part of the African diet, however, irrespective of their benefits there are concerns about their safety because of the continual and unpredictable growth of microorganisms during and after fermentation of the products (Oyewole, 1997; Adekoya et al., 2017a) as well as pre- and post-processing contamination. Due to these microbial contamination, TFF may harbour some bacterial pathogens and their toxins, which can be hazardous to human health (Aloys and Angeline, 2009; Okeke et al., 2015; Tamang et al., 2016). Pathogenic Gram-negative bacteria such as E. coli, Salmonella spp., Vibrio cholerae and Klebsiella spp. have been isolated from some TFF and various studies have shown the possibility of pathogens surviving and growing in some fermented foods (Gadaga et al., 1999; Ogunshe and Oladugba, 2008; Aworh, 2008). Despite these studies, there is no 3 adequate information on the spectra of microorganisms associated with these foods since their identification were done biochemically. However, a more rapid molecular method involving the use of nucleotide sequence data from 16S ribosomal RNA genes needed to be employed to identify microorganisms present in these products in a more accurate manner with the possibility of obtaining information of previously unreported pathogens. In molecular analysis, 16S rRNA gene sequencing is a more accurate identification technique for microorganisms (Tamang et al., 2016). Moreover, it is not influenced by variation of phenotypes, or technological sidedness, and it has the capability to minimize laboratory errors (Petti et al., 2005; Tamang et al., 2016).

Foods can be a vehicle for endotoxin production and they can be released into the system via food-borne pathogenic Gram-negative bacteria. The endotoxins produced by these bacteria also have the capability to simulate the immune system in an uncoordinated manner, thereby causing inflammations, fever, fatigue and leg pains (Porter et al., 2010; Bhadoria et al., 2015). However, endotoxins have been reported to persist in some foods including infant milk formula causing undesirable health effects (Townsend et al., 2007; Sipka et al., 2015) but no work has been done to establish their presence or absence in TFF til date.

Fungi are salient organisms in food industries that are as preservers, spoilers and toxin producers. They have been used in the fermentation of cheese and milk some of which are Penicillium, Mucor, Geotrichum, and Rhizopus spp. (Chelule et al., 2010a; Pensupa et al., 2013). Certain fungi produce undesirable toxins and their presence in foods have been attributable to their sporulating ability which makes them contaminate the environment and food products easily (Pitt and Hocking, 2009). Even with the perception of the safety of TFF, some authors have reported the presence of toxigenic fungi in TFF (Adebayo et al., 2014; Tamang et al., 2016; Adekoya et al., 2017a). Previous workers also reported the predominance of these organisms in other food systems, i.e., mawe (Hounhouighan et al., 1994), burukutu (Sanni et al., 1999), dried sausages (Mataragas et al., 2002) and fermented beverages (Odhav and Naicker, 2002). These studies highlights that the assumption that TFF are safe by consumers is deleterious as such foods could be potential sources of mycotoxicosis. This study was therefore expedient to clarify this assumption and establish the microbial safety of TFF in terms of the presence of fungi and mycotoxins. In addition, many studies in several countries have focused on the assessment of mycotoxin contamination particularly AFs in foods (Zinedine et al., 2006; Ghali et al., 2008; Moreno et al., 2009) but

4 there is little information on the presence of multiple mycotoxins in most of the selected TFF, which should be addressed.

In order to develop sustainable national strategies, laws and regulations to control mycotoxins and endotoxins in fermented foods, which are currently lacking in most African nations, some of the factors to be considered are the availability of data on occurrence and availability of toxicological data. It is important therefore, to generate information on toxin occurrence in fermented foods that could contribute to the establishment of regulatory standards for fermented foods. Furthermore, a significant number of people in SSA are not informed or aware about mycotoxins, endotoxins and their associated health effects and only few studies have been conducted to establish this (Ncube, 2010; Ezekiel et al., 2013; Aboloma, 2014; Matumba et al., 2016). Besides, one of the key intervention strategies for the control and management of mycotoxins in SSA is the creation of awareness (Strosnider et al., 2006; James et al., 2007). This should serve as a basis to conduct a baseline assessment amongst TFF sellers to investigate their practices, understanding and perceived health risk of fungal and mycotoxin contamination.

A significant number of studies have been reported on mycotoxins in Africa (Williams et al., 2004; Strosnider et al., 2006; Njobeh et al., 2009; Topcu et al., 2010; Njobeh et al., 2010; Makun et al., 2013; Ezekiel et al., 2013; Atanda et al., 2013; Adetunji et al., 2014b) and endotoxins in foods (Gehring et al., 2008; Sipka et al., 2015). However, there is a dearth of information on the assessment of both mycotoxins and endotoxins as safety indicators in fermented foods.

1.4. Aim and Objectives of the Study

1.4.1 Aim

The study aimed at assessing the health risk associated with the occurrence of bacteria, mycotoxigenic fungi and their toxins in some traditionally fermented foods from Nigeria and South Africa.

1.4.2 Objectives

The objectives of the research were to:

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 Carry out a baseline assessment survey of traditionally fermented food sellers in Nigeria in respect of their practices, general knowledge and perceived associated health risks of fungal and mycotoxin contamination;  Assess the occurrence of pathogenic bacteria and fungi in selected fermented foods from Nigeria and South Africa;  Evaluate the production potentials of secondary metabolites, including mycotoxins by fungi isolated from the traditionally fermented foods in Nigeria and South Africa;  Detect and quantify the levels of endotoxins and mycotoxins in the selected traditionally fermented foods; and  Determine the cytotoxic effects of mycotoxin extracts obtained from the traditionally fermented foods on human lymphocyte cells.

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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Introduction

Since the inception of human advancement, there has been a close association between man, his sustenance and fermentative enterprise of microorganisms, which has been used in the manufacture of fermented beverages and foods. Fermentation is one of the ancient and most efficient methods of preserving and producing foods. The fermentative organisms may be introduced to the substrates as starter culture or maybe indigenously available on the substrate. This chapter gives an overview of fermented foods, different fermentation processes, roles and benefits of fermentation, classes of fermented foods, and features of African fermented foods as well as their microflora. Although fermentation is a key player in the lives of people all over the world, food safety is still a major challenge to consumers and producers of fermented foods particularly the indigenous ones. This chapter also gives an insight into the safety of fermented foods in relation to the presence of microorganisms such as fungi and bacteria as well as their toxins (mycotoxins and endotoxins, respectively) being current threats to global food safety and security.

2.2 Food Fermentation

The fermentation of food has philosophical, religious, historical and archaeological significance (Steinkraus, 1997). A significant number of fermented foods particularly those derived from root crops in the tropics have advanced with time. By the means of definition, fermented foods are those foods that have been subdued to enzymatic or microbial action to give rise to beneficial biochemical transformation that leads to desirable modification of the food (Campbell-Platt, 1994). The foods are permeated or invaded by beneficial and edible microorganisms whose enzymes specifically lipases, proteases, and amylases breakdown proteins, lipids and carbohydrates, respectively, into non-harmful products with textures, aromas, and flavours that are acceptable and pleasing to consumers. In relation to fermentation, there exist four main fermentation processes namely: lactic acid, alcoholic, alkaline and acetic acid fermentation (Soni and Sandhu, 1990; Steinkraus, 1997). In alcoholic fermentation, the principal organisms responsible are yeasts and the primary product is ethanol (e.g., beer and wine). Lactic acid fermentation is majorly executed by lactic acid bacteria that plays a critical role in the production and preservation of nutritious fermented 7 foods (e.g., fermented cereals, legumes, oil seeds), thereby sustaining global population (Steinkraus, 1997; Reddy et al., 2008). This type of fermentation is cheap and has been utilized in producing several foods and beverages, e.g., cabbage (Sauerkraut), cucumbers (pickles), sourdough breads (Philippine puto, & Indian idli), fermented cereals (Nigerian ogi, & Kenyan uji), etc. (Aderiye and Laleye, 2004). Lactic acid bacteria are group of non-motile, Gram-positive bacteria which are catalase negative, make use of carbohydrates and give rise to lactic acid as their principal end product (Onilude et al., 2005; Reddy et al., 2008). They are categorised into homofermentative and heterofermentative based on carbohydrate metabolism (Figure 2.1).

Figure 2.1 Major pathways of carbohydrate fermentation in lactic acid bacteria through the Embden-Meyerhof and 6-phosphoketolase pathways (Adapted from Reddy et al., 2008)

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Homo-fermenters produce lactic acid as the main product of glucose fermentation through glycolysis (Embden-Meyerhof pathway), while hetero-fermenters produce lactic acid, carbon dioxide, acetic acid and ethanol from glucose fermentation via the 6- phosphogluconate/phosphoketolase pathway (Caplice and Fitzgerald, 1999; Reddy et al., 2008). Streptococcus, Lactobacillus and Pediococcus spp. are homo-fermenters, while Leuconostoc and Weissella spp. are hetero-fermenters (Ross et al., 2002). Another category of bacteria of significance in fermentation belongs to the Acetobacter spp. Acetobacter transform alcohol to acetic acid in the presence of surplus oxygen (Blandino et al., 2003). Vinegar is produced as a result of acetic acid fermentation. In alkaline fermentation, the protein present in the raw materials is hydrolysed into peptides and amino acids whereas ammonia is given off, increasing the pH of the resultant product and resulting into food with pungent ammoniacal odour. A large number of alkaline fermentations are carried out naturally by mixed bacteria cultures, principally influenced by Bacillus subtilis (Wang and Fund, 1996; Parkouda et al., 2009). Common foods in this category are tempeh, iru, ugba, natto and kinema.

In addition to these, solid-state (SSF) and submerged-state fermentation (SmF) are also two broad categories of food fermentation techniques. Solid-state fermentation denotes a process whereby product formation and microbial growth materialize on the surface of a solid substrate. Solid-state fermentation is carried out in the absence of unbound water, where the moisture is assimilated into the solid substrate (Pandey et al., 2000; Ray et al., 2008). Tuber crops including cassava and sweet potato and their wastes have been transformed into diverse products via SSF approaches (Ray et al., 2008). Submerged-state fermentation entails the anaerobic/partially anaerobic disintegration of carbohydrate by microorganisms in a liquid substrate in the presence of free water (Pandey et al., 2000). Yoghurt, wine, curd and beer are examples of SmF products. Solid-state fermentation is advantageous over SmF in terms of improved product attributes, easier product recovery rate, lower cost, higher product yield and reduced energy requirement (Ray et al., 2008).

2.2.1 Microflora in Fermented Foods

By practice, the most frequently used food preservative microorganism is LAB due to their harmless metabolic actions while growing in food using free sugar for the manufacture of organic acids and additional metabolites. Their long-term use and occurrence in foods

9 contributed to their acceptability as Generally Regarded as Safe (GRAS) (Aguirre and Collins, 1993; Reddy et al., 2008), though their microbiology remains unexploited and complicated. However, fermentation is carried out in a number of foods by a mixture of inherent enzymes and other microorganisms but often, mixed cultures arising from the natural native microflora of the substrate are involved. Some microorganisms, take part in this process in a parallel or sequential manner, which is accompanied by continuous change in the prepotent biota. However, under industrial conditions, a starter culture is often employed to maintain a consistent quality. The typical fermenting bacteria genera are Leuconostoc, Streptococcus, Lactobacillus, Pediococcus, Bacillus and Micrococcus, while the fermenting fungi widely belong to Aspergillus, Cladosporium, Penicillium and Trichothecium, Paecilomyces, and Saccharomyces genera (Jespersen, 2003).

Fungi particularly yeast have been associated with a variety of traditionally fermented foods (TFF) (Gadaga et al., 2001). Nevertheless, irrespective of their occurrence, the role of moulds in these products has been poorly investigated. Fungi play an important role in fermentation, they can also add vitamins, fibre and protein to foods (Chelule et al., 2010a; Bourdichon et al., 2012; Pensupa et al., 2013). Aspergillus sojae and A. oryzae are used in the production of miso and soya sauce, while A. niger and A. oryzae are used in the manufacture of sake and awamori liquors, respectively (Bourdichon et al., 2012). Mogensen et al. (2009) highlighted the role of A. acidusis in the fermentation of Puerh tea and Hachmeister and Fung (1993) stated the role of Rhizopus oligosporus in the fermentation of tempeh.

2.2.2 Africa Indigenous Fermented Foods

Africa has a pertinent history in the production of fermented foods and is perhaps a continent with ample diversity of fermented foods. The existence and production of fermented foods in Africa have a great influence on the health, nutrition and socio-economic position of its population, which are often afflicted with drought, conflict, famine, diseases and political instability. Africa indigenous fermented foods are obtained from legumes, cereals, oil seeds, fish, meat, etc. and are manufactured in villages, homes and small-scale cottage industries (Obadina et al., 2008). In view of convenience, Odunfa and Oyewole (1998) classified fermented foods in Africa following major group’s viz.: fermented cereal based foods, fermented starchy roots, fermented vegetables proteins, alcoholic beverages, and fermented

10 animal proteins. Some fermented food products common to Africa, their processing methods and modes of consumption are discussed subsequently.

2.2.2.1 Fermented vegetable proteins

Iru is a type of fermented and processed locust beans (Parkia biglobosa) used as a condiment. Amongst the Manding speaking people of West Africa, iru is known as sumbala and can be found in fresh or dried form. Iru contributes largely to the intake of essential fatty acids, B vitamins, especially riboflavin and protein, being consumed amongst the rural poor as a low cost meat replacement (Aworh, 2008). Being a product of alkali fermentation, it has a pungent smell. Onyenekwe et al. (2012) stated iru to be the foremost natural condiment used in savouring stews and soups in Nigeria. Iru production like other African indigenous fermented foods has not significantly risen beyond cottage level. In order to produce iru, locust been seeds are boiled for 15 hrs, dehulled and boiled again for 30 mins to 2 hrs, after which they are shaped into small balls and packaged in banana or paw-paw leaves (Aworh, 2008). Recently, iru seeds (Figure 2.2) are also packaged in polyethylene films before being offered for sale.

Figure 2.2 Fermented African locust beans (Iru) Ogiri is an oily grey and pungent paste produced principally from melon (Colocynthis citrullus) seeds and eaten across West African countries. Its fermentation is by chanced inoculation and its production remains a local art. Like iru, it is a cheap protein source amidst rural populace and can be obtained from fluted pumpkin (Telfairia occidentalis), castor oil seeds (Ricinus cummunis) (Omafuvbe et al., 2004), and other varieties of melon which are readily obtainable. In order to prepare ogiri, melon seeds are boiled until they are tender, 11 mashed, covered firmly in banana leaves and allowed to ferment for five days to a week. After which, the resultant product is relocated to and enclosed in a jute bag to facilitate low oxygen tension (Odunfa and Oyewole, 1998; Omafuvbe et al., 2004). Thereafter, the mashed fermented melon is positioned on a wire mesh, smoked across a charcoal heat for 2 hrs and crushed before use (Achi, 2005). Ogiri is depicted in Figure 2.3.

Figure 2.3 Fermented melon (ogiri) Ugba is a protein rich fermented food product with a meaty taste and desirable sensory qualities. It is prepared by an ancient process of SSF of the seeds of the African oil bean tree (Pentaclethra macrophylla Benth). The oil bean seed is highly proteinous and calorific and the seeds are rendered eatable by fermenting for 3-5 days (Enujiugha et al., 2012; Olotu et al., 2014). It is a popular delicacy in the Nigerian diet and serves as a snack, side dish or is used as a food condiment. Ugba is an essential food item for various traditional ceremonies that is consumed by all socio-economic groups; it contributes to its consumer’s protein, calorie and vitamin intake. Published research has indicated Bacillus spp. as the principal microorganisms responsible for ugba (Figure 2.4) fermentation. The dominant species is B. subtilis but other species such as B. megaterium, B. lichenformis and B. pumilus have also been found (Okorie and Olasupo, 2013). The same group of organisms has been implicated in the fermentation of other fermented food vegetable proteins including iru and ogiri.

Figure 2.4 Fermented African oil bean seed (ugba) 12

2.2.2.2 Fermented cereal based foods

Ogi is a fermented cereal gruel produced from maize (Zea mays), though sorghum (Sorghum bicolor) or millet (Pennisetum glaucum) are also used as the fermentation substrate. It is also known as akamu or koko in other parts of Africa. The traditional ogi preparation involves immersion of maize, millet or sorghum in water for 1-2 days, thereafter wet milling, sieving and fermenting for 2-3 days. Principal participatory microorganisms are LAB and yeasts (Olasupo et al., 1995; Aworh, 2008). Other microorganism such as Corynebacterium breakdown the starch present, and yeasts such as Saccharomyces and Candida spp. impart flavour (Caplice and Fitzgerald, 1999). Ogi (Figure 2.5) is commonly consumed amongst infants as weaning food and by adults as breakfast (Gadaga et al., 2001). A more viscous form of ogi is consumed as mawe in Benin Republic, kenkey in Ghana and agidi in Western Nigeria. Ogi is utilized in the control of diarrhoea and other gastrointestinal tract related illnesses (Olasupo et al., 1995).

Figure 2.5 Fermented cereal gruel (ogi) from maize and sorghum

Mahewu is indigenous to southern Africa and it is a non-alcoholic sour beverage derived from maize meal. Similar to ogi, it is used as both adult and weaning food with diverse names including amahewu, amarehwu, emahewu, metogo, machleu, and maphulo (Katongole, 2008). Mahewu is locally made by boiling thin maize gruel containing 12-14% maize meal (Solange et al., 2014). After this process, the gruel is allowed to cool and placed in a fermentation vessel with the addition of wheat flour (2-4%) as inoculum. This inoculated gruel is allowed to undergo spontaneous fermentation in a warm place for 24-48 hrs. Mahewu can also be prepared by crushing excess or unconsumed pap into slurry that is then left overnight to ferment (Gadaga et al., 1999). Streptococcus lactis is the chief fermenting microorganism in traditionally made mahewu (Odunfa and Oyewole, 1998).

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2.2.2.3 Fermented starchy root products

Cassava (Manihot esculenta) is a major tuber crop processed into variety of food products including fufu, garri, high quality cassava flour, lafun, kivunde, and cingwada via lactic acid fermentation (Padonou et al., 2009). Cassava has a short shelf life and deteriorates within 24 hrs of harvesting and fermentation offer a good means of overcoming this impediment. In addition to the prolonged shelf life, fermentation enhances the safety of cassava by drastically reducing the concentration of cyanogenic glucoside. Cyanogenic glucoside is a toxic substance inherent in cassava that can lead to several health disorders (Ferraro et al., 2016). Garri (Figure 2.6) is derived by cleaning and grating fresh cassava roots, dewatering, fermenting at room temperature for about 4 days and roasting the resultant mash (Kostinek et al., 2005).

Figure 2.6 Fermented cassava product (garri) Lafun unlike garri is produced through SmF of cassava roots for 4 days, followed by washing, dewatering, drying and milling. Research has shown L. plantarum, L. fermentum, and W. confusa to be the dominant LAB population during lafun production, while prepotent yeasts are Hansenia guilliermondii, Pichia scutulata, S. cerevisiae and Kluyveromyces marxianus (Padonou et al., 2009).

2.2.2.4 Alcoholic beverages

Umqombothi is prominent amidst the black citizens of South Africa. It is an effervescent opaque, pink coloured and yoghurt like flavoured beer with a creamy and thick consistency. It has about 3% alcohol content, consumed in its active fermentative state, and characterised with a short shelf life of 2 to 3 days (Katongole, 2008). The beer is produced from maize, water, maize malt, sorghum malt and yeast. For its production, maize flour is inoculated with sorghum malt, steeped in water for a day, thereafter the mixture is cooked into a soft gruel

14 and allowed to cool for 6 hrs. Sorghum malt is futher added, the gruel is stirred intermittently and thoroughly, umqombothi from a former batch is added, fermentation is allowed to proceed for 18 hrs and the mixture is sieved as umqombothi. The beer plays a central role in the social context and is commonly served during weddings, meetings and funerals.

Burukutu and pito are indigenous, light brown, slightly bitter alcoholic beverages from Nigeria and Ghana that are derived through the fermentation of malted, mashed sorghum or maize (Sunday and Aondover, 2013; Onyenekwe et al., 2016). They are frequently consumed daily as nutritive beverages and served in ceremonies and festivals. For their production, sorghum or maize grains are steeped in water for 2 days, dewatered, allowed to undergo germination for 5 days and sun dried before grinding. Water is added to the grounded flour (mashing stage), which is then boiled for 6-12 hrs, allowed to cool and filtered. For burukutu production, during the mashing stage, adjuncts are added e.g., garri but for pito, adjuncts are not included. Again, fermentation of the filtrate is done overnight followed by 12 hrs boiling and cooling. To the cooled concentrate, sediments from a previous batch is added and incubated for 12-24 hrs to obtain pito and burukutu.

2.2.2.5 Fermented animal proteins

Raw milk is fermented in stone jars or calabashes produced from gourds for several days to obtain amasi - an indigenous fermented milk consumed in Zimbabwe and South Africa (Chelule et al., 2010b). Also in Kenya and Tanzania, the traditional maasai fermented milk, kule naoto, is a critical part of their daily diet. Kule naoto is obtained through spontaneous fermentation of unpasteurized whole milk for at least 5 days. The product is appreciated based on its fresh taste, aroma, and peculiar for its functionality against constipation and diarrhoea (Mathara et al., 2004). Mathara et al. (2004) in their study isolated over 300 LAB strains from Kule naoto predominantly Lactococcus and Leuconostoc followed by Enterococcus. Interestingly, L. plantarum was the most frequently occurring species while L. fermentum and L. acidophilus groups were also found.

2.2.3 Benefits of Food Fermentation

Fermented foods and the microorganisms that take part in the process of fermentation have been associated with many benefits that are highlighted below.

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 Fermentation as a means of preservation: Some of the fermented products have extended shelf life due to the presence of organic acids generated during fermentation (Odunfa and Oyewole, 1998; Chilton et al., 2015). An example can be seen in case of ogi, which can be kept for more than two weeks through decantation and replacement of its supernatant water. In addition to this, the organic acids formed reduce the pH, which inhibit the development of spoilage and harmful organisms.  Fermentation offers variety in flavour/taste: The sour/tart/acidic flavour developed during cassava/cereal fermentation through LAB produces much more distinct flavour than other unfermented counterparts thereby making different flavours attainable from the same substrate.  Fermentation renders inedible foods edible: Some legumes such as African oil bean seed and locust bean are inedible in their natural state but are made edible by the breakdown of their indigestible components and antinutrients (Odunfa and Oyewole, 1998; Aworh, 2008).  Fermentation improves the nutritive value of foods: fermentation enhances the digestibility of foods, net protein utilization, biological value and protein efficiency ratio amongst others. The increased digestibility is partly due to the complete breakdown of protein to amino acids and the fragmentation of galacto- oligosaccharides into simpler sugars, which also make vitamins and minerals more available. For example, phosphate and calcium are released from iru through the breakdown of oxalate and phytate (Omafuvbe et al., 2004).  Fermentation decreases toxicity: fermentation reduces and or annihilate toxic constituent of some seeds/root crops. For example, the toxicity of cassava and African oil bean decreases with fermentation.  Fermented foods serves as probiotic sources: probiotics are live microorganisms, which confer health benefit on the host upon consumption in the adequate amount. Most probiotics organisms are LAB and are consumed through fermented milk, yoghurt or other foods (Parvez et al., 2006). Probiotics improves intestinal microflora, energizes the immune system, promotes nutrient bioavailability, and reduces allergies, lactose intolerance and risk of some cancers (Ayodeji et al., 2017).

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2.2.4 Features of Food Fermentation in Africa

The production of many traditional or indigenous fermented foods and beverages including the ones discussed above persists as a household technique as they are manufactured in villages, homes, and small-scale industries. On the other hand, the production of others such as soy sauce, yoghurt, sauerkraut, and pickles has emerged to a biotechnological state and are done on a large scale. Below are characteristic features of their art of production in Africa.  Their processing is carried out with crude equipment, which has not facilitated increased production over time;  Fermentation is by chanced inoculation and starter cultures are uncommonly used thereby making quality and safety to vary;  The most commonly used type of fermentation is lactic acid fermentation followed by alkaline fermentation;  Little emphasis is placed on the packaging of foods after fermentation and the use of sub-standard and unhygienic materials are common which makes the foods more prone to contamination;  Post-fermentation contamination constitutes a major challenge to their safety and quality and the preservation of the products is not usually complemented with other preservation methods;  The level of production has not been optimized beyond the indigenous method of production;  Processing methods and techniques vary from one production batch or one processor to another, thereby quality and safety is inconsistent;  There is little or no consideration for good manufacturing practices and sanitation;  The sector is originally occupied by women, which have contributed significantly to the substinence of the economy through the production and sale of TFF. Research has shown a correlation of the indigenous knowledge of women and recognized their skills in creating products that are inexpensive and proteinous (Tamang et al., 2008); and  Amidst the benefits of TFF, their lies a perception that they are safe and often unaccompanied with health implications.

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2.2.5 Safety of Fermented Foods

Fermented foods largely have an excellent safety record (Oyewole, 1997; Steinkraus, 1997) and the enactment of the safety principles for fermented foods could enhance the universal quality and the nutritive value of the foods and reduce diseases. Factors that contribute to the safety of fermented food are related to many theories such as that of the lactic acid fermentation, which facilitates a habitat that is unsuitable for pathogenic and spoilage organisms. Also inclusive, are steeping and cooking processes, which decreases microbial populations, salting where salt is used as a preservative, anaerobic fermenting conditions as well as reduced moisture contents particularly during SSF (Aderiye and Laleye, 2004). However, it should be noted that fermentation itself does not address the underlying problems of contaminated raw materials, dirty environment, uncontrolled fermentation process, post process contamination, poor personal hygiene, etc., and any of these factors or more can render fermented foods unsafe.

In sub-Saharan Africa (SSA), Nigeria for example, fermented foods are still prepared and preserved under poor hygienic conditions. In view of this, they are not under any control for their conformation to national set standards (Adeyeye, 2017). Therefore, their ingestion is expected to put public health at risk, unfortunately, health risk associated with these foods have not been evaluated indepth from a scientific standpoint due to inadequate epidemiological data, consumption patterns, and absence of surveillance programs in most SSA countries. It is not surprising that the widespread occurrence of pathogenic bacteria and toxigenic moulds in SSA traditional foods have been reported while a limited number of food intoxications cases have also been linked to their consumption.

The isolation of these pathogens thus indicates that they are able to grow and survive the process of fermentation (Gadaga et al., 1999; Inatsu et al., 2004; Ogunshe and Oladugba, 2008), hence the perception that these foods are safe remains a dangerous one. Nyatoti et al. (1997) delineated the presence of enteropathogenic E. coli in naturally fermented milk consumed as weaning foods. In South Africa, Kunene et al. (1999) found 40% of the fermented sorghum meal samples analysed was contaminated with B. cereus and 8% with E. coli. On the other hand, Klebsiella spp. and S. aureus were isolated from wara, while E. coli, Klebsiella spp. and Salmonella spp. were recovered from nono by Olasupo et al. (2002). Table 2.1 shows some traditional fermented food products from where microorganisms of public health significance were isolated. 18

Table 2.1 Microorganisms of public health importance detected in some traditional fermented foods

Pathogens Food products References B. cereus, & S. aureus Banku, kenkey Mensah (1997) E. coli Mahewu Simango & Rukure (1991) Enteropathogenic E. coli Sour milk Nyatoti et al. (1997) B. cereus, & E. coli Ogi baba Kunene et al. (1999) S. aureus, & Klebsiella sp. Wara Olasupo et al. (2002) E. coli, Salmonella sp., & Klebsiella sp. Nono Olasupo et al. (2002) B. subtilis, E. coli, S. aureus, Klebsiella sp., & Ogi, kunuzaki Olasupo et al. (2002) Enterococcus faecalis B. cereus, Shigella, & Enteropathogenic E. coli Ogiri, iru Oguntoyinbo& Oni (2004) E. coli O157:H7, S. aureus, Shigella flexneri, & Borde Tadesse et al. (2005) Salmonella spp. A. niger, A. flavus, P. citrinum,& F. subglutinans Ogi Omemu (2011) Articulospora inflate A. niger, A. rapens A. Lafun Ijabadeniyi (2007) flavus,& Lemonniera aquatica A. niger, Geotrichum candidum, & Penicillium Eko Adebayo et al. (2014) spp. A.flavus, A. fumigatus, A. minisclerotigenes, A. Umqombothi Adekoya et al. (2017a) niger, A. parasiticus, A. sclerotiorum, A. sydowii, A. versicolor, & A. tritici A. fumigatus A. flavus, A. parasiticus, A. Ugba Adekoya et al. (2017a) sclerotiorum, P. chrysogenum, P. expansum, & F. andiyazi A. amstelodami, candidus A. clavatus, A. flavus, Iru Adekoya et al. (2017a) P. polonicum, P. chrysogenum, & F. verticillioides P. chrysogenum, A. niger, & F. eguseti Ogiri Akinyele & Oloruntoba (2013) A. clavatus, A. niger, A. parasiticus, A. sydowii, Ogi baba Adekoya et al. (2017a) A. tritici P. citrinum, & F. fujikuroi

Some fermented foods may also be contaminated by moulds, which produce mycotoxins. Ogi contained A. niger, A. flavus, P. citrinum, and F. subglutinans according to Omemu (2011). Adekoya et al. (2017a) also identified a series of fungi belonging to the Aspergillus, Monascus, Talaromyces, Saccharomyces, Rhodotorula, Cladosporium, Geotrichum, Fusarium, Candida, Rhizopus, Penicillium, and Mucor genera from Nigerian fermented foods. Aside from the occurrence of these fungi, possible presence of their toxic secondary metabolites (mycotoxins) in fermented foods consumed in Africa also raises an increasing concern regarding public health safety even though they are poorly studied. The presence of multiple mycotoxins in their raw materials (Makun et al., 2009; Njobeh et al., 2010; Somorin et al., 2016; Chilaka et al., 2016) are reported. Subsequent sections of this review focuses on bacteria, fungi, and their respective metabolites (endotoxins and mycotoxins).

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2.3 Bacteria

2.3.1 Overview, Structure, Metabolism and Significance

Bacteria comprises of a broad domain of ubiquitous single celled microscopic organisms, which were part of the foremost form of life that emerged on earth. They occur in different shapes and sizes and are present in water, soil, air, radioactive wastes, and indepth segments of the earth crust (Fredrickson et al., 2004; Young, 2006). Bacteria are beneficial to life and are implicated as causative agents of several deadly diseases. Despite their wide occurrence, the type bacteria found in each environment varies and they often form complex relationships with other living organisms be it fungi, yeasts, animals, etc. Bacteria are divided into various groups based on their habitat, metabolism, morphology, cell structure, staining methods, cell components (Thomson and Bertram, 2001), etc. In terms of morphology, most bacterial species are spherical (cocci), rod-shaped (bacilli) or spiral-shaped (spirilla) while a few species can be tetrahedral, cuboidal, star shaped (Wanger et al., 2008) or variants of all these shapes. This extensive variant of shapes is influenced by the cytoskeleton and cell wall of the bacteria which is significant because it can impact on the movement, survival and nutrient acquisition ability of the bacteria (Cabeen and Jacob-Wagner, 2005; Young, 2006).

Bacterial metabolism entails how bacteria obtain nutrients for various biological activities. In relation to this, they can be autotrophs, heterotrophs, aerobic, anaerobic, chemotrophs, phototrophs amidst other categories (Nealson, 1999). Some bacteria that are heterotrophs form parasitic relationship with other organisms: hence, they are grouped as pathogens. Pathogenic bacteria are major drivers of diseases and cause both acute and chronic infections including typhoid fever, cholera, syphilis, leprosy, tuberculosis, and diphtheria. Bacteria diseases have also been implicated in agriculture to cause leaf spots, leaf rots, wilts, and fire blights in plants as well as anthrax, mastitis and blackquater in animals. Pathogenic bacteria can be associated with raw materials or instituted into foods during processing from contaminated water, insanitary equipment and utensils, air, dirty hands, sewage or by cross contamination.

However, some bacteria can cause diseases but others are also constituents of human microflora and can be beneficial and exist in humans without causing diseases e.g., L. acidophilus, L. iners and L. crispatus. Bacteria, often LAB such as Lactococcus and Lactobacillus in combination with some fungi, have been utilized for decades to manufacture 20 fermented foods both traditionally and industrially such as pickles, soy sauce, wine, vinegar and yoghurt. Some are utilized in bioremediation and waste processing and often used to clean oil spills because of their ability to breakdown organic substances (Cohen, 2002). Bacteria are also used as biocontrol agents and in drug manufacture amongst other uses (Liese and Filho, 1999; Cleveland et al., 2003).

The bacterial cell is enclosed by a cell membrane that functions as a barrier to secure nutrients, proteins and other important constituent of the cytoplasm within the cell. A cell wall that is made-up of peptidoglycan can be present. Peptidoglycan contains cross-linked peptide chains containing amino acids (Heijenoort, 2001). In broad terms, two type of cell walls exist in bacteria, a thick one with many layers of teichoic acids and peptidoglycan in the Gram-positives and a thinner one with few layers of peptidoglycan surrounded by a lipid membrane containing lipoprotein and lipopolysaccharides (LPS) in the Gram-negatives. These names were developed from the response of cells to Gram-staining, which is a well- established bacteria species classification criterion (Coico, 2005). Streptococcus pneumoniae, S. aureus and B. cereus are examples of Gram-positive bacteria.

Lipopolysaccharides are also called endotoxins and their structures are often peculiar to individual bacteria strains, which determine many of their antigenic characteristics. Thus, many Gram-negative bacteria species are pathogenic based on their LPS layer (Adam et al., 2014; Dowhan, 2014). The Proteobacteria are principal Gram-negative bacteria group and includes: E. coli, Salmonella, Pseudomonas, Helicobacter, Acetobacter, Moraxella, Klebsiella, Stenotrophomonas, Legionella, etc. Other notable groups of Gram-negative bacteria include the Cyanobacteria, Spirochaetes, Neisseria, and Chlamydia. They are often associated with respiratory (Klebsiella pneumoniae, and Pseudomonas aeruginosa), urinary (E. coli, and Serratia marcescens), and gastrointestinal (Helicobacter pylori and Salmonella typhi) problems.

2.3.2 Bacteria Toxins

The ability of bacteria to synthesize toxins has been widely demonstrated. These toxins can be lethal and are capable of restraining the physiological activities of the cells either by acting on the cell membrane or on targets organs within the cells (Silverman and Ostro, 1999; Porter et al., 2010; Bhadoria et al., 2015). Mostly, they work in association with other virulent components that enable the bacteria to be rooted in the host and evade or resist their

21 defensive processes (Ramachandran, 2014). Bacteria generate toxins that are categorized as exotoxins and endotoxins (Moscone et al., 2017). Exotoxins are heat labile proteins produced within pathogenic bacteria, most often Gram-positive bacteria that are released after lysis (Silverman and Ostro, 1999). On the other hand, endotoxins are heat stable LPS complexes, which are integral part of the cell wall of Gram-negative bacteria and are released during cell lysis or upon death of the bacteria (Lubran, 1988). Bacteria toxins are also grouped by their target cells or organs e.g., enterotoxins (Clostridium perfringes produce enterotoxins, which cause diarrhoea), neurotoxins (Clostridium perfringens produce neurotoxins that cause paralysis of respiratory muscles) and cytotoxins produced by Clostridium difficile, which cause cell death (Lubran, 1988).

2.3.2.1 Endotoxins: overview, history, structure and clinical association

Lipopolysaccharides as a structural component makes up 70-90% of the surface area of the Gram-negative bacteria cell wall regardless of the pathogenicity of the cell (Adam et al., 2014; Dowhan, 2014). Lipopolysaccharide is a mutagenic and pyrogenic molecule that plays active role in antibacterial drug resistance (Rosenfeld and Shai, 2006). They are mostly found in pharmaceutical items, food products, laboratory utensils and equipment (Das et al., 2014) and are commonly associated with unclean water (Kalita et al., 2017). The study of LPS commenced at the culmination of the 19th century by Richard Pfeiffer (Bacteriologist, 1858-1945), who discovered that the lysate of heat-immobilized Vibrio cholerae could instigate shock and death in laboratory animals (Bayston and Cohen, 1990; Rietschel and Cavaillon, 2003). He called this heat-stable toxin “endotoxin” to differentiate it from exotoxins, which were heat labile and secreted by live V. cholerae.

Within the same period, Eugenio Centanni (Pathologist, 1863-1948) delineated the isolation of endotoxin from other Gram-negative bacteria and made the outstanding pyrogenic characteristics of endotoxins to be recognized, while Hans Buchner (Bacteriologist, 1850- 1902) established the relationship between damaged host immunity, leucocytosis and endotoxins (Bayston and Cohen, 1990). Then in 1935, Andre Boivin (Microbiologist, 1895- 1949) and Lydia Messrobeanu (Microbiologist, 1908-1978) found that endotoxic activities are carried out in the outer membrane that consists of macromolecular protein complex, protein, polysaccharide and lipid (Bayston and Cohen, 1990). After two decades, Otto Westphal (Immunologist, 1913-2004) and Otto Luderitz (Immunologist, 1920-2015) began

22 detailed studies on endotoxin biochemistry and discovered the biological activity of endotoxin was resident in the lipid moiety, now referred to as Lipid A (Bayston and Cohen, 1990). This entire discovery elucidated the chemical structure of endotoxin as shown in Figure 2.7.

Figure 2.7 General structure of bacteria lipopolysaccharides: Lipid A, internal oligosaccharides and specific O-chain (Adapted from Silverman and Ostro, 1999)

Thus, the LPS structure is commonly made of a hydrophobic Lipid A region, O-antigen polysaccharide and an oligosaccharide core (Raetz and Whitfield, 2002). Lipid A anchors LPS to the microbial membrane and it is responsible for the endotoxic properties of LPS (Ramachandran, 2014). Current investigations on endotoxins are based on elucidating their mode of action, developing rapid and sensitive detection methods and proffering solution for the treatment of endotoxin associated diseases.

In humans, LPS can enter the bloodstream principally by bacterial insult, wounds or intestinal hyperpermeability via consumption of contaminated food, water or incompletely purified parenteral drugs (Davies and Cohen, 2011). Endotoxins are heat stable and thus, can be biologically active in foods including those that have been heat-treated and processed. Endotoxin exposure can potentiate many adverse health effects with septic shock as the most common (Kalita et al., 2017). Septic shock is depicted by oliguria, hypotension, hypoxia, acidosis, microvascular abnormalities development, multiple organ failure and disseminated intravascular coagulation. Widespread damage of tissues (oedema, fibrin thrombi, haemorrhage) and organs such as liver, kidneys, etc. have also being demonstrated through

23 necropsy and a correlation of these pathological and physiological conditions have been seen in laboratory animals taking lethal doses of endotoxin (Kalita et al., 2017). The immunological reaction to the appearance of endotoxin in the blood stream is termed endotoxemia (Hurley et al., 2015). Manifestations include low blood pressure, fever, leucocytosis, coagulopathy and thrombocytopenia. Deitch et al. (1987) demonstrated that the ingestion of endotoxin might promote the invasion of bacteria, increase permeability of the intestinal wall, weaken blood brain barrier and cause necrotizing enterocolitis. Silverman and Ostro (1999) also associated endotoxins with sepsis, heart diseases, burns, trauma, etc. in their model shown in Figure 2.8.

Figure 2.8 A model of diseases potentially associated with bacteria/endotoxin (Adapted from Silverman and Ostro, 1999)

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2.3.2.2 Detection methods

Currently the approved and validated method for the detection of endotoxin is the Limulus Amoebocyte Lysate (LAL) test (FDA, 1987). The test was established in 1956 by Jack Levin (Hematologist, 1932-Till date) and Fredrick Bang (Pathologist, 1916-1981), and it is based on an enzymatic coagulation cascade of lysate acquired from the horseshoe crab blood (Mitsumoto et al., 2009). Endotoxin is measured in unit (EU) and regulations have only been established for medicines, drugs and clinical devices (FDA, 1987). The LAL test is based on three techniques namely: the gel-clot (based on formation of gel clot); turbidimetric (based on development of turbidity after cleavage of an endogenous substance) and the chromogenic technique (based on colour development after cleavage of an artificial peptide-chromogen complex). Instead of measuring the end-point for endotoxin detection as outlined in the gel clot method, endotoxin detection is now performed by measuring the release of p-nitroaniline (PNA) from an artificial peptide chromogen through a change in absorbance at 405 nm (Iwanaga et al., 1978).

The quantity of PNA liberated is equivalent to the quantity of endotoxin present and this method is reported to be highly sensitive than the typical gelation method and use E. coli 0111 B4 as LPS source (Harada-Suzuki et al., 1982). According to Yin et al. (1972) and Mitsumoto et al. (2009), the turbidimetric and chromogenic LAL techniques are more selective, sensitive and quantitative compared to the conventional LAL assay. In the turbidimetric technique, the turbidity is evaluated by optical density (OD) with time. As such, increase in OD and time needed to initiate a distinct increment in OD is a resultant of LAL clottable protein concentration. Although these detection methods are widely implemented, some debilitating factors to their application including, the presence of metal ions, antibiotics, and protease inhibitors, have been delineated to affect LAL reagent sensitivity (Donovan and Laue, 1991). Therefore, appropriate sample dilution is required to limit these interferences (Cooper, 1990), as well as increased sensitivity.

Batch-to-batch variability is also an associated problem, therefore new endotoxin detection methods are needed and continuous efforts are being made in this regard. Mitsumoto et al. (2009) describe a novel endotoxin assay based on a particle-counting method using laser light scattering. Abdul-Rahman (2013) also developed new types of planar interdigital sensors for the detection of endotoxins in food, while Kalita et al. (2017) demonstrated a portable, simple

25 and cost-effective strategy to measure endotoxin levels in human serum in 5 min using a flow-through assay.

2.3.3 Occurrence of Gram-negative Bacteria and Endotoxins in Foods

The occurrence of pathogenic Gram-negative bacteria in foods has been widely studied as well as their association with food poisoning and food-borne illnesses (Nyatoti et al., 1997; Kunene et al., 1999; Motarjemi, 2002). Tamang et al. (2016) reported the presence of Klebsiella pneumoniae, Pseudomonas, Enterobacter cloacae, Haloanaerobium, Halococcus, Halobacterium, Propionibacterium and Pseudomonas in many fermented foods. Unhygienic practices, unsanitised utensils, dirty environment, poor handling practices, cross contamination, contaminated raw materials, poor processing and storage conditions are some of the factors responsible for their occurrence in foods. As such, there are heightened chances of the presence of Gram-negative bacteria toxins (endotoxins) in foods in addition to the fact that they are heat stable.

The LAL test has been used in the area of food microbiology for years. Watson et al. (1977) used the LAL test to detect LPS in water. The LAL assay was used to access meat spoilage by Jay et al. (1979) and Jay (1981). Through this assay, viable concentrations of Pseudomonas spp. were determined and LPS was found to increase as the storage period of the meat increased. Suedi et al. (1981) employed the same technique to determine LPS in Ultra High Temperature treated milk while Hansen et al. (1982) used the LAL test to estimate Gram-negative bacteria loads in foods. Sullivan et al. (1983) established a significant relationship between LAL and volatile bases in fish in order to determine lean fish quality. Endotoxin levels from 40 to 5.5 x 104 EU/g was found in infant milk from nine countries (South Africa, Holland, Spain, Switzerland, USA, Belgium, Ireland, Slovenia, and United Kingdom) by Townsend et al. (2007) but these values did not correlate with the population of the viable bacteria found in their study. Gehring et al. (2008) evaluated for the presence of endotoxins in milk samples from farming and non-farming families across five European regions (Germany, Finland, Austria, Switzerland and France), higher levels of endotoxins were found in the milk consumed by non-farming families compared to the farming families. Sipka et al. (2015) also analysed LPS levels in milk.

Relative to other microbial toxins such as mycotoxins, the occurrence of endotoxin in food is poorly researched and most studies have only focused on limited food categories and with no 26 current report on their presence within categories of food such as cereals, legumes or fermented foods. More research needs to be conducted on the presence of endotoxins in food, as they are contaminants that pose a threat to food safety and security. There are currently no regulations for endotoxins in food.

2.4 Fungi

2.4.1 An Overview

Fungi are eukaryotic group of organisms that are either unicellular or multicellular. They lack chlorophyll, possess chitinous cell walls and are heterotrophic. Fungi are saprobic, symbiotic or parasitic and they play important roles in breaking down organic matter and transference of nutrients in the environment (Ingold and Hudson, 1993). Fungi are utilized in food, antibiotic and enzyme production. They also play important roles in their use as biological control of pests and disease. Irrespective of their benefits, their infestation on plants is accompanied with significant losses, which has great impact on the economy as well as food supplies. According to Hawksworth (1991), about 1.5 million species of fungi exist, but Blackwell (2011) estimated the fungal kingdom to consist of over 5 million species. Fungi are separated into two large classes: yeasts and moulds. Moulds have thread-like lengthy cells known as hyphae that grow and branch to form a network called mycelium. The mycelium may form fruiting bodies that produce spores that are released (Penalva et al., 2002; Redecker and Raab, 2006). Yeasts, on the other hand are single celled, microscopic, oval or round shaped organism that do not form hyphae.

Fungal classification is based on their biochemical, physiological and morphological attributes and in recent years, the utilization of molecular tools such as phylogenetic analysis and deoxyribonucleic acid (DNA) sequencing has contributed largely to their taxonomy and genetic diversity (Hibbett et al., 2007; Petti et al., 2005; Tamang et al., 2016). Based on taxonomy, there are seven phyla: Basidiomycota, Chytridiomycota, Glomeromycota, Microsporidia, Neocallimastigomycota, Ascomycota and Blastocladiomycota (Hibbett et al., 2007). The Ascomycota is the largest taxonomic group within the Eumycota (Gams, 2002) and it consist of truffles, morels, a few mushrooms, unicellular yeasts (Saccharomyces and Candida), and many filamentous fungi belonging to the genera Penicillium, Aspergillus, Fusarium, Claviceps, etc., some of which will be considered in details in the next section. 27

2.4.2 Natural Occurring Toxigenic Fungi

Toxigenic fungi include those that are proficient in producing mycotoxins. Many of them are ubiquitous and possess intense ecological association with the food chain. The native fungal flora that are closely linked with human food supplies belong to three main genera: Aspergillus, Penicillium and Fusarium. Toxigenic moulds are known to produce several toxic secondary metabolites but it is well proven that not all secondary metabolites from moulds are toxic and not all moulds are toxigenic. Human exposure to these toxigenic fungi may be from the consumption of contaminated plants (CAST, 2003; Hove et al., 2016) or through dust and air (Jarvis, 2002). Suttajit (1989) divided toxigenic fungi into three: field fungi, which invade plants before harvest such as Alternaria, Fusarium and Cladosporium. Some field fungi grow on stressed plants e.g., F. verticilliodes, while some partly invade plants prior to harvest and predispose them to mycotoxin contamination e.g., P. verrucosum. Another division is the storage fungi, possessing lower humidity requirements and grows only after harvest e.g., Aspergillus and Penicillium species. The third group being the advanced deterioration fungi, usually do not invade wholesome food but attack damaged ones and require high water activity and moisture content e.g., Rhizopus, Scopulariopsis, Absidia and Mucor. Furthermore, human diseases linked with harmful toxins synthesized by these fungi have been demonstrated and studied indepth for several years. Typical examples are toxins from food contaminated with fungi from the genera: Aspergillus, Fusarium, Penicillium, Alternaria, Acremonium, Stachybotrys, Claviceps, Cladosporium, Bipolaris and Aureobasidium as some of them are described below.

2.4.2.1 Aspergillus species

Aspergillus is a large and ever evolving genus, which is made up of over 180 species (Bennett and Klich, 2003). They possess heavy and huge walled stipes with puffy apices known as vesicles (Figure 2.9) and they procreate through the formation of mitotic spores towards the end of the conidiophore. They are easily recognized by their peculiar conidiophore at genus level, but species characterisation and identification is intricate, based on a number of morphological attributes (Rodriguez et al., 2009). Aspergillus spp. are often soil fungi or saprophytes, however some also cause decay of stored foodstuffs and diseases in plants, or can be human and animal pathogens. These fungi are difficult to control, spreading efficiently through the production of asexual spores called conidia.

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Figure 2.9 Distinctive structures of Aspergillus species (Adapted from Klich, 2002)

Some of the Aspergillus spp. that produce mycotoxins of principal economic and health concerns are A. parasiticus, A. flavus, A. ochraceus, A. niger and A. fumigatus. The mycotoxins produced by these species include aflatoxins (AFs), cyclopiazonic acid (CPA), sterigmatocystin (STE) and ochratoxin (OTs) (Samson and Varga, 2007). Aspergillus flavus is the major producer of AFs in crops worldwide; other Aspergillus spp. known to produce AFs principally are A. parasiticus and A. nomius. A. flavus have a particular affinity for cereals and nuts, while A. ochraceus and associated species are widely present in dried foods of various kinds (Pitt and Hocking, 2009). Cyclopiazonic acid producers are A. flavus together with A. oryzae, A. versicolor and A. tamari. The other Aspergilli that are known to produce STE include A. flavus, A. parasiticus, A. rugulosus, A. chevalieri, A. ruber, A. amstelodami, A. aurantobrunneus, A. quadrilineatus, A. sydowii and A. ustus (Versilovskis and De Saeger, 2010).

2.4.2.2 Fusarium species

Fusarium is also an extensive genus and they are widely affiliated with plants and distributed in the soil, their conidia is waterborne and airborne while their chlamydospores are generally soil-borne (Smith, 2007). Figure 2.10 shows the conidia and chlamydospores of F. oxysporum.

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Figure 2.10 F. oxysporum spores; a: microconidia; b: macroconidia; c: chlamydospores (Adapted from Hatai, 2012)

Fusarium spp. are known to cause detrimental infections such as Fusarium head blight in many plants of economic importance and their role as mycotoxin producers was recognized only in the 1970s after an outbreak of Alimentary Toxic Aleukia (ATA) in the USSR, which lead to the deaths of over 100,000 people between 1942 and 1948 (Joffe, 1978). From this period henceforth, Fusarium spp. were unveiled to be instrumental in the production of at least 50 mycotoxins including fumonisins (FBs), T-2 toxin (T-2), HT-2 toxin (HT-2), deoxynivalenol (DON), zearalenone (ZEN), nivalenol (NIV), etc. (Joffe, 1978). Deoxynivanelol can be produced by F. crookwellense, F. graminearum, and F. culmorum while ZEN can be produced by F. equiseti and F. oxysporum (Hatai, 2012). T-2 and HT-2 toxins are produced by F. sporotrichioides and F. langsethiae (Thrane et al., 2004).

2.4.2.3 Penicillium species

The Penicillium genera is made up of over 300 species (Pitt and Hocking, 2009), some members of the genus are used in food fermentation e.g., P. camembertii, while some produce penicillin, an antibiotic that is used to promote human and animal health. Penicillium spp. produce paintbrush-like stalk and heads known as conidiophore, whereas each branch end is organized as clusters with sporulating cells known as phialides as shown in Figure 2.11.

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Figure 2.11 Conidiophore branching patterns of Penicillium species (Adapted from Visagie et al., 2014)

Chains of spores are usually formed from the tip of each phialide. Penicillium spp. also produce wide range of mycotoxins such as patulin (an unsaturated lactone) which is associated with apple and apple products. Penicillium expansum and P. citrinum produce citrinin (CIT), while P. verrucosum also synthesizes OTA in stored grains particularly wheat in temperate regions, and may be associated with A. ochraceus (Frisvad, 1995). Sterigmatocystin may also be synthesized by P. camembertii, P. commune and P. griseofulvum. Other mycotoxins produced by Penicillium spp. are OTA, CPA, roquefortine C (ROQ C) and penicillic acid (PA) (Bernhoft et al., 2004; Bouhet and Oswald, 2005) which will be discussed in detail in further sections.

2.4.2.4 Alternaria species

The genus Alternaria captures plant pathogens and saprobes that have been found globally infecting crops on field and facilitating decay of many crops after harvest (Thomma, 2003). Alternaria spp. spores are pigmented, multi-celled and exists as dark dividing chains as shown in Figure 2.12.

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Figure 2.12 Alternaria species conidia and conidiophores (Adapted from Lawrence et al., 2016) They can be easily identified because of the transverse and longitudinal division of their cells, which gives them a peculiar facade (Larone, 2011). Alternaria spp. reside in food, feed, plants, soil and produce over 70 phytotoxins and mycotoxins with only few occurring in foodstuffs or being of major health significance. Alternaria alternata is the most significant toxin producing fungal species (Battilani et al., 2003). The presence of Alternaria mycotoxins has been reported in sorghum, wheat, pecans, tomato, cotton, and sunflower (Scott, 2001). Some important Alternaria toxins are alternariol monomethyl ether (AME), alternariol (AOH), tenuazoic acid (TEA), altenuene (ALT), and altertoxins (ATX-I, II, III) (Logrieco et al., 2009). Altenuene and AME may also be produced by A. brassicae, A. tenuissima, A. citri, A. dauci, A. cucumerina, A. kikuchiana, A. porri and A. longipes (Andersen and Frisvad, 2004; Pose et al., 2004).

2.4.2.5 Stachybotrys

Stachybotrys are also examples of hyphomycetes filamentous fungi, which thrive within cellulose rich materials. The genus contains about 50 species (Gams et al., 2002; Larone, 2011) with the ability to produce mycotoxins suspected to cause immune suppression and cancer (Corrier, 1991). It has gained public notice sequel to the investigation of its link with idiopathic pulmonary haemorrhage in children (Vesper et al., 2000). A significant percentage (67%) of Stachybotrys spp. produce stachybotryotoxins and S. chartarum is the most closely associated species with trichothecenes (TCs). Interestingly, not every Stachybotrys spp. synthesizes TCs and some lose their ability to produce under certain conditions (Pitt, 2000; Tuomi et al., 2000). Mycotoxin poisoning by this fungus is referred to stachybotryotoxicosis and the toxins may be assimilated through food, inhalation of Stachybotrys or absorbed

32 through the eyes and skin after which they find their way to the blood stream. The colonies of Stachybotrys are fast growing and have cotton-like appearances. When examined under the microscope, they have septate hyphae, cylindrical phialides, branched conidiophores and conidia, which are oval, pigmented and clustered (Larone, 2011; Haugland et al., 2014). The microscopic attributes of Stachybotrys fungi is shown in Figure 2.13.

Figure 2.13 Microscopic features of Stachybotrys fungi (Adapted from Larone, 2011)

2.4.2.6 Claviceps species

Claviceps spp. are found majorly in the tropics and economically important species are C. paspali (grass), C. purpurea (cereals and grasses), C. fusiformis (pearl millet), C. Lutea (paspalum) and C. africana (sorghum) (Bandyopadhyay et al., 1998). Rye is the most common host of C. purpurea; but they also present in triticale, barley and wheat where they produce alkaloids that cause egotism in humans upon consumption of grains contaminated with their sclerotia. Some of these alkaloids are also beneficial and known to be useful in the manufacture of pharmaceuticals used in curing headache, migraine, or psychiatric disorders (Jackson, 2006).

2.4.3 Factors Influencing Fungal Colonization and Production of Mycotoxin

Toxigenic fungi grow under series of conditions and the non-appearance of mould does not translate to the absence of mycotoxins since they can be present in the substrate long after the disappearance of the producing fungus. Even though, it is often difficult to prevent mycotoxin formation, the elimination of condition that influences fungal colonization can

33 largely contribute to the prevention of mycotoxin contamination. Hence, there are different factors associated with fungal growth and mycotoxin production in foods. They include environmental factors, including substrate characteristics (e.g., water activity, temperature, pH and oxygen content); chemical factors (e.g., presence of antifungal agents and nutritional factors), and biological factors (e.g., insect damage, microflora, associated growth of other fungi or microbes and strain variability).

2.4.3.1 Environmental factors

The ability of fungi to infect or attack many agricultural commodities is largely dependent on diverse environmental requirements such as temperature, pH, water activity, light and oxygen availability, some of which are discussed subsequently.  Temperature: Most fungi have the ability to survive under a wide range of environmental temperature usually between 10 and 35 oC, while a few can grow above or below this range (Sweeney and Dobson, 1998; Ramos et al., 1998; Pitt and Hocking, 2009). Fungi can be mesophilic, psychrophilic, thermotolerant, psychrotolerant or thermophilic but generally, the peak temperature for their growth is above the peak temperature for mycotoxin production. Multiple mycotoxins production by single species is also associated with temperature variation. In light of this, temperature can be utilized as a measure to control fungal growth and mycotoxin production. According to Sweeney and Dobson (1998), Fusarium moulds can grow abundantly at between 25 and 30 oC without synthesizing any mycotoxin, but close to freezing temperatures, they are able to produce plenteous mycotoxins with limited mould growth. Temperature requirements for the growth of some fungi and their mycotoxin production are displayed in Tables 2.2 and 2.3, respectively.

 Water activity: Water activity (aw) denotes the amount of water available for the

activities of enzymes and microbial growth (Lacey and Magan, 1991). The aw that is required in a fungal domain may be achieved by equilibrating the substrate with an humidified atmosphere and keeping the water content or the concentration of the solute in the culture substrate constant (Atanda, 2011). Most food-borne moulds are

able to grow at aw of 0.85 or less, although yeasts largely need a higher aw. Water activity levels of 0.6 represent the limit for cell growth, but Penicillium and

Aspergillus spores, can thrive at lower aw for lengthy years (Ramos et al., 1998). The control of moisture is the cheapest and most appropriate way of environmental control

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towards preventing fungi and mycotoxin development (Magan et al., 2011; Okoth et

al., 2012). The aw requirements for fungal growth and mycotoxin production are shown in Tables 2.2 and 2.3, respectively.

Table 2.2 Temperature range and aw requirement for growth of some fungi

Fungal species Temperature range for fungal Water activity (aw) for fungal growth growth (°C) Minimum Optimum Maximum Minimum Optimum Maximum A. flavus 10 25-35 43 0.80 0.95-0.99 - A. parasiticus 10 32-35 43 0.83 0.95-0.99 - A. ochraceus 8 24-37 37 0.77 0.95-0.99 - F. verticilliodes 2 23-30 37 0.87 - 0.99 F. proliferatum 4 30 37 0.9 - - F. culmorum 0 20-25 35 0.90 0.98-0.995 - F. poae 5 20-25 35 0.90 0.98-0.995 - F. avenaceum 5 20-25 35 0.90 0.98-0.995 - F. tricinctum 5 20-25 35 0.90 0.98-0.995 - F. graminearum - 24-26 - 0.90 - 0.99 F. sporotrichioides -2 21-28 35 0.88 - 0.99 P. verrucosum 0 20 35 0.80 0.95 -

Source: Sweeney and Dobson (1998); Ramos et al. (1998)

Table 2.3 Temperature range and aw requirement for production of mycotoxins by some fungi

Fungal species Temperature range for mycotoxins Water activity (aw) for mycotoxins formation (°C) formation Minimum Optimum Maximum Minimum Optimum Maximum A. flavus 12 30-33 40 0.82 0.99 0.998 A. parasiticus 12 33 40 0.87 0.99 - A. ochraceus 12 25-31 37 0.80- 0.98 - P. verrucosum 4 20-25 - 0.83 0.90-0.95 - F. verticilliodes 10 15-30 37 - - - F. proliferatum 10 15-30 37 0.92 - - F. culmorum 11 29-30 - 0.93 - - F. graminearum 11 29-30 - 0.9 0.98 -

Source: CAST (2003); Sanchis and Magan (2004); Ribeiro et al. (2006)

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 Hydrogen/hydroxyl ion concentration (pH): pH could influence fungal growth indirectly by its impact on the surface of the cells or directly through its influence on nutrient availability. The alkaline/acid demand for mould and yeast growth ranges from 3 to 8, with optimum around pH 5, if nutrient conditions are met. Generally, Penicillium spp. are more accustomed to acidic pH while Aspergillus spp. tolerates alkaline pH (Wheeler et al., 1991). Within a neutral pH, they have to compete with bacteria to thrive. Fungi such as P. funiculosum can grow at pH 2 or less (Wheeler et al., 1991) while F. verticillioides can grow at a pH 7.5 (Wang et al., 2005) making each fungi to have different pH requirement.  Availability of oxygen: Nearly all filamentous fungi and yeasts need oxygen but a large number of species appear to be efficient oxygen scavengers, in such a way that their growth is determined by the available oxygen instead of oxygen tension. Pitt and Hocking (1997) stated that the concentration of dissolved oxygen in the substrate has more impact on fungal proliferation than atmospheric oxygen tension. The most oxygen urging moulds will invade food surfaces while the less demanding ones would be found inside the food. Patulin and PA production decreases drastically at reduced oxygen concentration (Pitt and Hocking, 1997). Likewise, the growth of Aspergillus is limited at low oxygen levels of < 1% (Pitt and Hocking, 1997).

2.4.3.2 Biological factors

Biological factors involve activities or processes that influence the function and behaviour of fungi and subsequent mycotoxin production within a substrate. Biological factors include:  Competing microflora: The consequent occurrence of different microorganisms as fungi and bacteria within the same substrate could inhibit fungal growth and toxin production. The use of microorganisms as biocontrol relies on this principle for e.g., Trichoderma harzianum produce enzyme - chitinase that has antifungal activity against diverse fungi such as A. niger (Nampoothiri et al., 2004). Bae et al. (2004) in their study also demonstrated the restraint of the proliferation of A. carbonarius and other fungi by B. thuringiensis.  Strain variability: Mycotoxin production is not only species dependent but also strain dependent (Huwig et al., 2001). If an organism does not produce mycotoxin e.g., OTA under certain condition, this does not rationalize its inability to produce OTA. Furthermore, the grouping of such organism as an OTA producer or non-producer 36

will be deceptive. The biosynthesis of OTA by ochratoxigenic Aspergillus spp. is influenced more by environmental factors than by their innate ability to produce OTA. In contrast, the biosynthesis of OTA amongst Penicillium spp. seems to be more steady and equally distributed (Muhlencoert et al., 2004).  Insects and other vectors: The proliferation of food crops by insects and pests is a widespread problem particularly in the tropics, which occur more on the field than in storage. The activities of insect and pests makes crops to be more susceptible to fungal infestation and resultant mycotoxin production whereas they basically acts as vectors and introduce fungal spores into the foods by the contusion they produce in them (Atanda, 2011). Payne (1998) highlighted that insects are capable of infecting food commodities by transporting inoculum around the commodity, by disseminating spores or by burrowing the commodity. The timing of insect infestation is also critical to the levels of mycotoxin found whereas wind and water also favours the distribution of fungal spores within agricultural commodities.

2.4.3.3 Chemical factors

Chemical factors often reveal the interplay between fungal invasion, mycotoxin production and the chemical properties of plants. They include:  Nutritional factors: moulds need organic compounds for the synthesis of biomass and production of energy being heterotrophs (Smith and Moss, 1985). They can utilize diverse carbon sources to fulfil their carbon needs in order to produce lipids, proteins, nucleic acids and carbohydrates which are oxidised to produce energy. In relation to the above, the ability of each fungus towards utilizing carbon sources varies and this factor is used with their morphology to differentiate them. Fungi also require a nitrogen source to produce amino acids, pyrimidines, glucosamine, vitamins, etc. and subject to the fungus, it can obtain nitrogen as nitrite, nitrate, organic nitrogen or ammonium. Hence, the presence and form of nutritional element such as nitrogen and carbon source available can influence morphological variation and the production of mycotoxin (Gadd et al., 2001; Carmichael et al., 2015). Other principal nutrients required by fungi are magnesium, phosphorus, sulphur, and potassium, which are accessible to a large percentage of fungi as salts (Russell et al., 1991). Aspergillus ochraceus gave the highest OTA yield in a medium that contained

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maltose amidst other carbohydrate sources (fructose, lactose and glucose) (Engel 1976).  Antifungal agents: Diverse antifungal agents or fungicides are utilized in tackling bio- deterioration, terminating fungi and averting or treating fungal diseases in animals and plants. Their right application reduces fungal microflora and this can facilitate decrease in mycotoxins production but some authors have reported that their application at sub-lethal concentration can expedite toxin production (Moss and Frank, 1987; Cuperlovic-Culf et al., 2016).

The occurrence of secondary metabolites in plants and foods intended for human consumption has been studied particularly those that affect human health. These metabolites are diverse in their structures and properties. To lay more emphasis on their degree of exposure, modes of regulation, control and occurrence in foods, an overview of these toxic metabolites are discussed in the next section.

2.5 Mycotoxins

2.5.1 Definition and Concepts

The word mycotoxin is formed from the Greek word: “mukes” meaning “fungi” and the Latin word “toxicum” meaning “poison” (Bhat et al., 2010). Mycotoxins are low molecular weight metabolic substances produced by filamentous fungal species, which have harmful effects when present in foods and feeds. These substances are produced during mould growth on plants in the field or during storage. Mycotoxins are structural molecules, which vary from simple heterocyclic rings to groups with irregularly organised heterocyclic rings (Edite et al., 2014). Globally, fungal toxins takes a central stage amongst toxins produced by microorganisms that naturally contaminate several foods or feeds, not limited to cereals but fruits, grains, nuts, forage and compound foods meant for human and animal consumption. Indeed, about 25% of crops produced worldwide are contaminated with mycotoxins (Ostry et al., 2017) and many studies have demonstrated high frequencies of contamination of agricultural produce from farm to fork (Njobeh et al., 2010; Makun et al., 2013; De Boevre et al., 2013; Ezekiel et al., 2013; Matumba et al., 2016).

In addition, the tendency of most feed and food products to allow fungal growth and mycotoxin formation during production, processing, transport, and storage has been 38 established (Frisvad and Samson, 1991; Pitt, 2000; Pitt and Hocking, 2009; Njobeh et al., 2012) though their occurrence differ between geographic regions. For instance, toxins produced by Aspergillus species are mostly encountered in zones with hot climate whereas Fusarium toxins are more frequent in temperate zones (Frisvad and Samson, 1991). Mycotoxins can penetrate the human and animal food chains either directly or indirectly. Direct contamination, arises when feed or food is infected by toxigenic fungus, which subsequently leads to mycotoxin formation. On the other hand, food or feed can be indirectly contaminated when an ingredient that is laden with mycotoxin is used in manufacturing a product. In this case, the toxigenic fungi may be present or absent from the ingredient.

Furthermore, their ingestion occurs mainly through the consumption of contaminated foods derived from both animals and plants (CAST, 2003). Diseases caused by mycotoxins are known as mycotoxicosis (Nelson et al., 1994; Bennett and Klich, 2003), with less exposure occurring in developed countries than developing countries mainly due to established food laws and utilization of modern technologies for food processing and preservation (Bhat et al., 2010). Nevertheless, the austerity of mycotoxicosis relies on the lethality of the mycotoxin involved, length of exposure, dosage, genetics, nutritional status, age of the individual amidst other factors (Bhat et al., 2010; Zain, 2011) though the synergy between some of these factors and mycotoxicosis is yet to be fully understood. For instance, in a certain individual, the deficiency of a particular mineral, alcohol consumption, depletion of calories or the possession of certain diseases, might aggravate the effects of ingestion of mycotoxin-laden foods (Bennett and Klich, 2003). In Figure 2.14, a schematic of the general association of mycotoxicosis with some factors is shown.

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GENETIC FACTORS

Ethnicity

PHYSIOLOGICAL FACTORS

Age

Hormonal Status Absorption

Nutrition MYCOTOXIN Distribution

Intestinal Microflora METABOLISM Biotransformation

Infection and Parasitism Excretion

TOXICITY

ENVIRONMENTAL FACTORS Climate conditions Biochemical defect

Pollution and Chemicals

Housing Functional defect

Socio-economic

Microscopic anatomical defect

Microscopic defect

Death defect Figure 2.14 A simplified representation of some general relationships in a mycotoxicosis (Adapted from Bryden, 2007)

2.5.2 Nature, Chemistry, Distribution and Health Implications of Mycotoxins

In fact, the word mycotoxin was created in 1962, following the famous death of turkey’s poults in England, after they ingested peanut meal originating from Brazil and Africa (Edite 40 et al., 2014). After confirmation that a secondary metabolite produced by A. flavus was responsible for the bird deaths, a race for the study of these toxins ensued. The most significant mycotoxin group are produced by the genera Penicillium, Aspergillus, Alternaria, Fusarium and Claviceps. Ochratoxins are produced by some Penicillium and Aspergillus spp., AFs are produced by Aspergillus spp. while AOH, ALT, AME, TEA and ATX are produced by Alternaria spp. (Marin et al., 2013). Zearalenone, FBs, TCs (type A: T-2 and HT-2 and type B: DON) and emerging mycotoxins (enniatins (ENNs), moniliformin (MON), fusaproliferin (FUSA) and beauvericin (BEA)) are produced majorly by Fusarium spp. whereas ergot alkaloids are known to be synthesized by Claviceps (Barkai-Golan et al., 2008; Marin et al., 2013; Berthiller et al., 2013).

2.5.2.1 Aflatoxins

The term aflatoxin (AF) was created based on the name of its main producer (A. flavus). The widely known AFs of significance include AFB1, AFB2, AFG1 and AFG2, due to their fluorescence under ultraviolet light (B-Blue, G-Green) and their movement on thin layer chromatography (Zain, 2011; Kumar et al., 2017). The molecular structures of AFB1, AFB2,

AFG1 and AFG2 are shown in Figure 2.15.

Aflatoxin B1 Aflatoxin B2

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Aflatoxin G1 Aflatoxin G2

Figure 2.15 Molecular structures of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 (Adapted from Quiles et al., 2015)

AFs are difuranocoumarins mainly produced by A. flavus particularly in humid and hot climates, A. flavus are ubiquitous and mainly colonise the leaves and flowers (aerial parts) of plants. A. parasiticus synthesizes B and G AFs, and they are mainly found in soil environment with minimal distribution (EFSA, 2007). In addition, some species of A. bombycis, A. nomius and A. pseudotamari have been found to be aflatoxigenic but with limited occurrence in nature (Peterson et al., 2001). The most potent and prevalent AF is

AFB1 and has been categorised as a group 1, human carcinogen (IARC 1993a). Moreover,

AFB1 supresses the immune system (Jiang et al., 2005, 2008), aggravates inflammation and supresses animal and human growth (Turner et al., 2007; Mahdavi et al., 2010; Gong et al., 2016). Aflatoxin ingestion immensely increases liver cancer risk amongst chronic hepatitis B patients (Groopman et al., 2008) and is associated increased incidence of hepatocellular cancer particularly in Asia and Africa (Scholl and Groopman, 2008).

Aflatoxin B1 and AFB2 when ingested undergo hydroxylation in the liver through cytochrome p450-associated enzyme and metabolises into AFM1 and AFM2, respectively (Jiang et al., 2005, 2008). Some substrates favours AF formation and fungal growth, and natural contamination of oil seeds, cereals, spices, legumes, nuts and other commodities are frequently encountered based on their resistance to some processing methods. Aflatoxins are somewhat stable and may restrain severe processes such as extrusion, roasting, baking and cooking (Marin et al., 2013). Based on this, they can be an obstacle in processed foods such as bakery products, roasted nuts, etc. (Marin et al., 2013). Due to their capacity to cohere with DNA of cells, AFs influence the synthesis of protein aside from its contribution to the

42 development of thymic aplasia (congenital privation of the parathyroid and thymus, with a resultant weakness in the immunity of cells otherwise known as DiGeorge’s syndrome) (Raisuddin, et al., 1993).

2.5.2.2 Ochratoxins

According to Poland et al. (2012), OTs belong to a category of associated pentaketide metabolites, made-up of a dihydroisocoumarin attached to phenylalanine. Their chemical structure is comparable to that of AFs, but with an isocoumarin bound replaced with an L- phenylalanine group (Figure 2.16).

Figure 2.16 Molecular structure of ochratoxin A (Adapted from Mally et al., 2005)

OTA, the most popular isoform was first isolated from A. ochraceus, and from this, its name was derived (Van der Merwe et al., 1965). Major OTA synthesizing fungi are A. nigri, P. nordicum, P. verrucosum, A. circumdati, A. meleus, A. alliaceus, A. auricomus, A. carbonarius, A. auricomus, A. glaucus, and A. niger (Larsen et al., 2001). The occurrence of OTA in barley, coffee, beer, oats, wheat and other products meant for human and animal consumption is widely documented (Pitt, 2000; Marquardt and Frohlich, 2016). Ochratoxins can be found singly, simultaneously, and/or as a concurring metabolite with other mycotoxins as AFs particularly in cereals and nuts (Marin et al., 2013). Generally, they are associated with the kidney but at high concentration can cause liver damage (CAST, 2003) and OTA has been linked to nephropathy during in vivo animal studies (Edite et al., 2014). In humans, OTA is majorly found in serum (Reddy and Bhoola, 2010), since it has a lengthy half-life relative to its eradication (Creppy, 1999). Besides being nephrotoxic, OTA is immunosuppressive, carcinogenic, hepatoxic and teratogenic (Schlatter et al., 1996) categorized as a 2B carcinogen (possible human carcinogen) (IARC, 1993b).

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2.5.2.3 Zearalenone

Several species of Fusarium such as F. graminearum and F. culmorum also produce estrogenic mycotoxin, ZEN that may co-occur with DON because, both mycotoxins are produced by F. graminearum and F. culmorum. Maize has been shown to contain the highest level of ZEN amongst cereals (Marques et al., 2008). Nevertheless, ZEN has also been found in sorghum, wheat, rye and barley in various countries around the world (CAST, 2003). Fungal species that produce ZEN majorly thrive in temperate regions that favour their growth in crops associated with wet temperate conditions or those stored in moist environments (Marroquin-Cardona et al., 2014). Nonetheless, ZEN can be formed comparatively under cool temperatures that favour fungal growth and formation of mycotoxin (Richard, 2007). Exposure to ZEN has been related to the manifestation of precocious puberty in girls (Massart et al., 2008; Chilaka et al., 2016) seemingly due to their estrogenic actions with the 17-b-estradiol receptors. Though not classified as a human carcinogen (IARC, 1993a), ZEN and its metabolites continue to receive attention due to its estrogenic action together with their anabolic effects (Edite et al., 2014). In addition to these, reproductive complications such as abortion have been recorded in cows, pigs and ovine species (El-Nezami et al., 2002). The molecular structure of ZEN is shown in Figure 2.17.

Figure 2.17 Molecular structure of zearalenone (Adapted from Ouanes et al., 2003)

2.5.2.4 Fumonisins

Fumonisins (FBs) are often produced by Fusarium spp. mainly F. proliferatum and F. verticillioides (syn. F. moniliforme). Other fungal producing species includes F. napiforme, F. nygamai, and F. dlamini (EFSA, 2005). Not less than 12 fumonisin (FB) analogues are recognized, with the most significant being the B series (FB1, FB2, and FB3) (Figure 2.18).

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(1)

(2)

(3)

Figure 2.18 Molecular structures of fumonisin B1 (1), fumonisin B2 (2) and fumonisin B3 (3) (Adapted from Bryla et al., 2013)

From a toxicological point, FB1 is the most consequential FB and it is chemically identified as 1, 2, 3-propanetricarboxylic acid, 1, 10-(1-(12-amino-4, 9, 11-trihydroxy-2-methyltridecyl) - 2-(1-methylpentyl)-1, 2-ethanediyl) ester (EFSA, 2005). Fusarium verticillioides and F. proliferatum can thrive over a wide temperature range but relatively at high water activities (aw > 0.9), these makes crops like maize grown in warmer regions to be susceptible to FBs contamination during pre-harvest and storage (Sweeney and Dobson, 1998). They are quite heat-stable, but levels may reduce during food processing when temperature surpasses 150 oC

(Sweeney and Dobson, 1998). Fumonisin B1 being the most widely studied congener, is hepatotoxic and nephrotoxic and has been classed together with OTA as a group 2B, possible human carcinogen (IARC, 2002; JECFA, 2011). Together with its association with liver and oesophageal cancers in high-risk populations (Alizadeh et al., 2012), it has been implicated as 45 a risk factor for neural tube defects (Gelineau-van Waes et al., 2009; Phoku et al., 2012). Fumonisins are also responsible for the hydrothorax and pulmonary edema in pigs (Harrison et al., 1990); leukoencephalomacia in equine species and rabbits (Fandohan et al., 2003); and hepatotoxic, carcinogenic and apoptosis effects in rats (Da Silva et al., 2000).

2.5.2.5 Patulin

This metabolite was first isolated as a substance with antimicrobial properties, in around 1940, from P. griseofulvum (Ciegler et al., 1977). Patulin (PAT) was later isolated from other fungal species and received different names, such as clavacin, expansin, micoine C and penicidin (Ciegler et al., 1977). It was used in treating common cold and skin infections until it was found to be toxic to animals and plants in the 1960s and was classified as a mycotoxin (Bennett and Klich, 2003). Chemically, PAT is known as 4- hydroxy-4H-furo (3, 2-c) pyran- 2(6H)-one and widely produced within the Eupenicillium, Penicillium, Byssochlamys, Aspergillus and Paecilomyces genera with P. expansum as the most significant producer (Morales et al., 2007; Puel et al., 2010). Aspergillus clavatus, A. giganteus and A. terreus are also producers of PAT (Morales et al., 2007). It is commonly found in fruits such as grape, apple and its juice and can alter immune response (Puel et al., 2010).

2.5.2.6 Trichothecenes

They constitute a group of nearly 170 metabolites produced by fungi of the genera Myrothecium, Fusarium, Phomopsis, Trichoderma, Stachybotrys, Verticimonosporium and possibly others (McCormick et al., 2011). Trichothecenes are grouped by the replacement pattern of the tricyclic 12, 13-epoxytrichothec- 9-ene (EPT) structure that is shared by all TCs (Figure. 2.19) and critical to their toxicity (Marroquin-Cardona et al., 2014).

Diacetoxyscirpenol Deoxynivalenol

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Nivalenol T-2 toxin Figure 2.19 Molecular structures of trichothecenes (Adapted from Li et al., 2012)

Based on their functional groups, they are categorized into four (A-D): type A is comprised of mycotoxins like HT-2 and T-2, while type B is mostly represented by DON. The most important TCs aside these are NIV and diacetoxyscirpenol (DAS) (Marroquin-Cardona et al., 2014). Trichothecenes inhibit protein synthesis and their fungi causes damp building related illnesses (Dearborn et al., 2002). Deoxynivalenol is most commonly found TCs in grains with F. graminearum and F. culmorum, as its main producers (Pitt and Hocking, 2009; Phoku et al., 2012; Marin et al., 2013). These fungi have been implicated as soil fungi and significant plant pathogens that develop on field crops (Eriksen and Alexander, 1998). Albeit less potent than other TCs, DON occurs more frequently in cereals including barley, wheat, rye, etc. (Miller et al., 2001). Deoxynivalenol is not carcinogenic (IARC, 1993a), but causes detrimental health problems like endocrine dysfunction, weight loss, anorexia, immune alterations and malnutrition and known as vomitoxin or food refusal factor (Pestka, 2010). Fusarium sporotrichioides is the major fungus associated with the production of T-2 (CAST, 2003). Some strains of F. sporotrichioides also synthesize some closely associated mycotoxins (DAS and HT-2). Maize, barley, rice, oats, etc. have been documented to contain T-2 (CAST, 2003). T-2 toxin production peaks at high moisture contents and temperatures ranging from 6 to 24 °C. T-2 toxin negatively affects dividing lymphoid and erythroid cells and decreases the level of cytokines, antibodies and immunoglobulins (Adhikari et al., 2017).

2.5.2.7 Citrinin

Citrinin (CIT) was first isolated from secondary metabolites of P. citrinum, well before the Second World War (Iwahashi et al., 2007; Edite et al., 2014). Subsequently, other species of Penicillium (P. expansum and P. viridicatum) and even of Aspergillus (A. niveus and A. terreus) also showed the ability to produce CIT. Certain strains of P. camemberti, employed

47 in cheese manufacture, and A. oryzae used in the production of Asian foods such as sake, miso and soy sauce, can also synthesize CIT. Corn, wheat, barley, rye, etc. are excellent substrates for their formation (Abramson et al., 2009). This mycotoxin, which is present in the structure of polyketide, has also been found in products with naturally coloured pigments and fermented foods. They are considered to be nephrotoxic and usually co-occur with OTs (Flajs and Peraica, 2009). The chemical structure of CIT is shown below (Figure 2.20).

Figure 2.20 Molecular structure of citrinin (Adapted from Iwahashi et al., 2007)

2.5.2.8 Ergot alkaloids

Ergot alkaloids comprise of compounds produced by several species of the genus Claviceps that infects small grains and grasses on field. They are grouped as peptide alkaloids, lysergic acids, clavine alkaloids and simple lysergic acid amides (Schiff, 2006; Hulvova et al., 2013). The effects of these alkaloids on humans have been known since the middle ages, a period in which some symptoms were called “Holy Fire” or “St Anthony’s Fire (Edite et al., 2014). Outbreaks of ergotism have also occurred amongst humans populations causing gangrene and loss of limbs (Krishnamachari and Bhat, 1975; Demeke et al., 1979). Some of the ergot alkaloids that occur are ergotamine, ergotoxine, ergometrine, etc. with C. purpurea as the major producing species (Schiff, 2006; Hulvova et al., 2013). Other ergot alkaloids producers are C. paspali, C. fusiformis, C. gigantea and Sphacelia sorghi (anamorphic form of Claviceps) (Hawksworth et al., 1996). With the modern techniques of grain cleaning, the problem of ergotism has been practically eliminated along the human food chain. However, it remains a threat from the veterinary perspective. Animals, which are susceptible to intoxication, include cattle, sheep, goats, ovine species, pigs and birds (Edite et al., 2014).

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2.5.2.9 Sterigmatocystin

Sterigmatocystin is produced by A. versicolor, A. nidulans and species affiliated with the genera Chaetomium, Bipolaris and Emiricella (Bertuzzi et al., 2017). They share similar biosynthetic pathway and structure with AFs (Figure 2.21).

Figure 2.21 Molecular structure of sterigmatocystin (Adapted from Versilovskis and De Saeger, 2010)

Sterigmatocystin can be metabolised into O-methylsterigmatocystin, a precursor of AFB1 and

AFG1 but A. versicolor and A. nidulans cannot carry out this action (Bertuzzi et al., 2017). Therefore, STE can be present at high levels in food invaded by these fungi (A. versicolor and A. nidulans) but contrary to this, infestation by A. parasiticus and A. flavus can result in reduced levels of STE, as majority is metabolised into AFs (Yu et al., 2004). It can be found in grain-based products and other foods including spices, cheese, nuts, etc. (Versilovskis et al., 2008; Versilovskis and De Saeger, 2010). Sterigmatocystin has been found to possess mutagenic, genotoxic, carcinogenic and teratogenic properties and it is a group 2B possible human carcinogen (IARC, 1987). In recent times, research has shown that STE is more genotoxic than AFB1 in three human cell lines types (Bertuzzi et al., 2017). Until date, there is no European regulation for STE in food but regulations exist in Slovakia and Czech Republic at 5 µg/kg for rice, vegetables, potatoes, flour, poultry, meat, milk and 20 µg/kg for other foods (Bertuzzi et al., 2017).

2.5.2.10 Alternariol and Alternariol Monomethyl Ether

Alternaria alternata principally produce two mycotoxins namely alternariol and alternariol monomethyl ether. They were first recognised and structurally distinguished as 3,7,9 trihydroxy-1-methyl-6H-dibenzo(b,d)pyran-6-one and 3,7-dihydroxy-9-methoxy-1-methyl-

49

6H-dibenzo(b,d)pyran-6-one, respectively, over a century. Phomopsis spp. and Stagonospora nodorum are also producers of AOH and AME (Ostry, 2008; Logrieco et al., 2009). They both have scarce toxicological evidence but possess carcinogenic properties during experimental assays (Brugger et al., 2006; Ostry, 2008; EFSA, 2011a). Their natural occurrence in fruits and processed fruits, lentils, tomatoes, wheat and oil seeds have been documented (Logrieco et al., 2009). Recently, TEA, another Alternaria mycotoxin was detected in beer and some cereal foods (Siegel et al., 2010; Asam et al., 2011). At this time, the presence of Alternaria toxins in food or feed is unregulated worldwide.

2.5.2.11 Emerging mycotoxins

In recent times, mycotoxins that are neither conventionally determined nor legislatively controlled but with increasing occurrence are known as emerging mycotoxins (Jestoi, 2008; Malachova et al., 2011). Some emerged Fusarium toxins are: MON, BEA, ENNs, and FUSA. Nevertheless, in comparison with the regulated mycotoxins, their prevalence is considered less significant with more focus placed on the regulated mycotoxins. Since the fungal species particularly Fusarium, which have the capacity to produce these emerging mycotoxin are wide spread over a series of geographical zones, their extent of contamination has been depicted to be as high as mg/kg (Logrieco et al., 2002). Santini et al. (2012) studied the occurrence of FUSA, ENNs and BEA in maize, small grains, processed grain-based food, and observed a link between their pattern of contamination and climate change. Going forward, research focusing on the occurrence of emerging mycotoxins in foods needs to be prioritised. The chemical structure of enniatin B is presented in Figure 2.22.

Figure 2.22 Molecular structure of enniatin B (Adapted from Ivanova et al., 2014)

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2.5.2.12 Masked mycotoxins

Another category of mycotoxins is masked mycotoxins, which are derivatives of mycotoxins that cannot be detected by regular analytical methods due to the modification of their structure in plants (Trans and Smith, 2011). Mostly, enzymes associated with the detoxification processes in plants are responsible for the chemical alteration that leads to their formation (Berthiller et al., 2013). This alteration can also be caused by different food processing techniques e.g., fermentation that often give rise to less toxic compounds compare to the precursors. Fermenting organisms can convert fungal metabolites into undetectable compounds through activities of enzymes but this area is underexploited. Masked mycotoxins, are grouped as extractable conjugated and bound, the former are detectable by suitable analytical techniques when their structures are known and subject to the availability of analytical standards (Berthiller et al., 2013). The latter are bonded to polymeric protein or carbohydrate matrixes and cannot be accessed directly but have to be released from the matrix by enzymatic or chemical treatment before being analysed (Berthiller et al., 2009; 2011; 2013).

Masked mycotoxins are often underestimated in matrixes due to: changes in the physicochemical attributes of molecules that lead to modification in their chromatographic behaviour as well as decreased extraction ability (Berthiller et al., 2013). The masked form of DON: deoxynivalenol-3-glucoside (DON3G) (Figure 2.23) has been reported in some foods in association with DON itself and its acetylated derivatives: 3-acetyl-deoxynivalenol (3- ADON) and 15-acetyl- deoxynivalenol (15-ADON).

Figure 2.23 Molecular structure of deoxynivalenol-3-glucoside (Adapted from Berthiller et al., 2011)

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So far, the occurrence of DON3G in maize (Berthiller et al., 2009), oats (Desmarchelier and Seefelder, 2011), beer, barley and malt (Lancova et al., 2008) has been delineated. Zearalenone can also undergo modification into α-zearalenol (α-ZEL) and ß-zearalenol (ß- ZEL) (Poppenberger et al., 2006). To date, only limited research has shown the presence of these metabolites in foods, therefore, there is need for the development of new methods or the expansion of currently multi-mycotoxin methods to incorporate the detection of mycotoxin derivatives in foods.

2.5.2.13 Miscellaneous mycotoxins

Penicillium camemberti and P. roqueforti used in cheese production synthesize a significant number of fungal metabolites namely: ROQ C, PA, isoflumigaclavines, CPA and PR toxin (Scott, 1981). Cyclopiazonic acid has been implicated in the inhibition of ion movement across the cell membranes (Riley and Goeger, 1992). Rao and Husain (1985) reported the consumption of kodo millet that was laden with CPA to cause kuodo poisoning, which is distinguished by nausea and loss of balance. Some tremogenic mycotoxins (mycotoxins that have specific impact on the central nervous system) are also produced by some species of Penicillium, Claviceps and Aspergillus. They include paspalinine, janthitrems, paspalicine, paspaline, penitrems, lolitrems, paxilline, aflatrem and paspalitrem A and B (Bennett and Klich, 2003). Penitrem A, which is associated with various incidences of tremor, bloody diarrhoea and vomiting, is produced by P. crustosum (Hocking et al., 1988; Bennett and Klich, 2003). Other significant mycotoxins with less reported incidence in foods include citreoviridin, gliotoxin, mycophenolic acid, xanthomegnin, griseofulvin, b-nitropropionic acid, kojic acid, vioxantin, viomellein, and walleminols (Marroquin-Cardona et al., 2014). Table 2.4 shows the occurrence of some of the mycotoxins that have been discussed above in some foods consumed in Africa.

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Table 2.4 Occurrence of mycotoxins in some African foods

Country Mycotoxin Type Sample Type Concentration References µg/kg or µg/mL Egypt AFs Meat products 2-150 Aziz & Youssef (1991) AFs Spices 2-35 Selim et al. (1996) AFs Milk 50-270 El-Tras et al. (2011) Fusaric acid (FA) Sugar cane juice 25.4-2,214 Abdallah et al. (2016) Burkina Faso FB1 Maize 22.5 -1,343 Warth et al. (2012) FB2 Maize 11.3 – 589 Warth et al. (2012) DON Maize 31.4 Warth et al. (2012) NIV Maize 11.0-15.8 Warth et al. (2012) Ghana FBs Maize 70-4,222 Kpodo et al. (2000) FBs Kenkey 15-1,000 Kpodo et al. (2000) Benin AFs Chips 2.2-220 Bassa et al. (2001) DAS Cassava flour 0-5 Ediage et al. (2011) FB1 Cassava flour 4-24 Ediage et al. (2011) FB1 Maize 51-836 Ediage et al. (2011) AFs Dried kernels 22-190 Hell et al. (2000) Kenya 3-A DON Wheat kernels 80-1,703 Wagacha et al. (2010) ENN B Wheat kernels 2-256 Wagacha et al. (2010) BEA Wheat kernels 13-15 Wagacha et al. (2010) AFs Milk >5 Kangethe & Langa (2009) ZEN Wheat kernels 7-55 Wagacha et al. (2010) Uganda AFs Groundnut, 0-55 Kitya et al. (2010) cassava, millet and sorghum flour AFs Dried kernels 0-435 Probst et al. (2014) Sudan AFs Peanut butter 21-170 Elshafie et al. (2011) Ethiopia AFs Sorghum, 0-26 Ayalew et al. (2006) barley, teff and Wheat OTA Sorghum, barley 54.1-2,106 Ayalew et al. (2006) and wheat DON Sorghum 40-2,340 Ayalew et al. (2006) FB1 Millet 0 -49.2 Chala et al. (2014) ZEN Millet 0-459 Chala et al. (2014) Tunisia & NIV Cereal &cereal 135-961 Serrano et al. (2012) Morocco products BEA Cereal & cereal 2.1-844 Serrano et al. (2012) products AFs Cereal & cereal 5.5-66.7 Serrano et al. (2012) products OTA Cereal & cereal 75-112 Serrano et al. (2012) products FBs Cereal & cereal 121-176 Serrano et al. (2012) products Zambia FBs Maize 33,500-192,000 Mukanga et al. (2010) AFs Peanut butter 0-130 Njoroge et al. (2016) Mozambique FB1 Maize 159-7,615 Warth et al. (2012) FB2 Maize 27.7-3,061 Warth et al. (2012) BEA Groundnut 0.1-24 Warth et al. (2012) ZEN Maize 10.9-18.1 Warth et al. (2012) 53

Country Mycotoxin Type Sample Type Concentration References µg/kg or µg/mL Malawi ZEN Maize 0-2,025 Matumba et al. (2015) DON Maize 0-2,328 Matumba et al. (2015) NIV Maize 0-2,220 Matumba et al. (2015) AFs Maize based 0-185 Matumba et al. (2014) beer FBs Maize based 493-3,303 Matumba et al. (2014) beer AFs Dried kernels 5-20 Probst et al. (2014) Tanzania AFs Dried kernels 3-1,081 Kamala et al. (2015) AFs Maize 158 Kimanya et al. (2008) FBs Maize 11,048 Kimanya et al. (2008) South Africa PAT Apple juice <10-1,650 Shephard et al. (2010) DON Wheat <10-100 Shephard et al. (2010) AFM1 Cow milk 0.04-1.32 Dutton et al. (2012) AFs Peanut 0.26–131.03 Ncube (2010) DON Maize meal <10-960 Shephard et al. (2010) FBs Maize 1840–142,800 Mogensen et al. (2011) AFB1 Maize 0–741 Chilaka et al. (2012) Zimbabwe FB1 Maize 4,000-8,000 Gamanya & Sibanda (2001) FB1 Sorghum 2,500-6,000 Gamanya & Sibanda (2001) FB1 Peanut 200-1,400 Gamanya & Sibanda (2001) DON Maize 0-492 Hove et al. (2016) NIV Maize 0-530 Hove et al. (2016) AFs Peanut 6.6-622 Mupunga et al. (2014) AFs Maize 0-123 Probst et al. (2014) Cameroon DON3G Maize beer 0.3-27 Abia et al. (2013) α-ZEL Maize beer 4-90 Abia et al. (2013) BEA Soybean 12-19 Abia et al. (2013) ZEN Maize 28-273 Njobeh et al. (2010) ZEN Peanut 31-186 Njobeh et al. (2010) FB1 Maize 37-24,225 Njobeh et al. (2010) Nigeria AFs Rice 28-372 Makun et al. (2011) BEA Maize 0.1-120 Adetunji et al. (2014b) ZEN Maize 115-779 Adejumo et al. (2007) DON Maize 11-749 Adetunji et al. (2014b) AFB1 Egusi 2.3-15.4 Bankole et al. (2006) AFB1 Peanut cake 13-2,824 Ezekiel et al. (2013) DON3G Sorghum 24 (mean) Chilaka et al. (2016) AFB1 Ogiri 3-4 Adekoya et al. (2017b) AME Iru 19-77 Adekoya et al. (2017b) ZEN Ugba 39-117 Adekoya et al. (2017b) FB1 Ogi 68-2,492 Adekoya et al. (2017b) DON Ogi baba 32-112 Adekoya et al. (2017b)

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2.5.3 Mycotoxin Regulations

The corroboration of the presence of mycotoxins in food and the assertion of their ability to cause various diseases led to the establishment of mycotoxins regulations in foods to limit their exposure. Notwithstanding the endeavours made by various agencies including country associated agencies such as the World Health Organization (WHO), the Food and Agriculture Organization (FAO), United States Food and Drug Administration (FDA), European Food Safety Authority (EFSA), Joint FAO/WHO Expert Committee on Food Additives (JECFA), National Agency for Food and Drug Administration and Control (NAFDAC), etc. in putting regulations in place to restrict mycotoxin levels in foods, until date, several countries are deficient in establishing suitable guidelines to control mycotoxins (particularly in Africa) and regulations are not universal. For example 100 countries, which include 15 African countries, have established regulations (Fellinger, 2006).

However, the choice of mycotoxin to regulate and limits is dependent on the health officers and the scientific community in each country, although, nearly all mycotoxin levels are established through worldwide standards (Marroquin-Cardona et al., 2014). Amidst countries worldwide, levels of AF ranging from 2 to 30 µg/kg are allowed in food meant for human consumption (Henry et al., 1999, FDA, 2004). EU has the most stringent mycotoxin standard globally, in foods meant for human consumption asides milk, total AF level of 4 µg/kg is the maximum permissible level (EC, 2008; 2010; 2011) contrary to 20 µg/kg set in the United

States (FAO, 2004). In South Africa, AFB1 limit in foods is 5 µg/kg and total AFs is set at 10

µg/kg (FAO, 2004). Maximum levels for FB1 + FB2 in some food commodities as regulated in the United States are shown in Table 2.5. The permissible level of ZEN has not been set in all countries but some nation in collaboration with WHO have established the maximum tolerated levels to be between 50 and 1,000 µg/kg in foods (FAO/WHO, 2011) with a provisional maximum tolerable daily intake (PMTDI) of 0.5 µg/kg body weight (bw) (JECFA, 2001). However, other recognized agencies have fixed a stricter tolerable daily intake (TDI) of 0.25 µg/kg bw (EFSA, 2011b). Due to their deleterious effects, DON has received attention and its PMTDI and that of its acetylated form is 1 µg/kg bw for conventional exposure, and 8 µg/kg bw for acute exposure (JECFA, 2001). OTA also has a provisional tolerable weekly intake (PTWI) of 100 ng/kg bw (JECFA, 2001). As previously highlighted, there are no regulations established to mitigate the presence of emerging

55 mycotoxins such as ENN, FUSA and BEA in foods. Table 2.5 shows the maximum allowable limits of some mycotoxins in different countries.

Table 2.5 Maximum allowable limits (µg/kg) of mycotoxins in different countries

Country Mycotoxin Max. limit Food Type References

EU* AFB1 2 Maize, cereals EC (2008; 2010; 2011) 8 Unprocessed peanuts 0.1 Cereals & other complementary foods for infants & children OTA 5 Unprocessed cereals ZEN 100 Maize intended for direct human consumption ZEN 20 Processed maize based food for infants & children 0.2 2 Algeria AFB1 10 Peanuts, nuts & cereals Ferrante et al. (2012) China AFB1 20 Maize & maize products, peanut & Ferrante et al. (2012) peanut products Egypt AFB1 5 Peanut & cereals Ferrante et al. (2012) Malawi AFB1 5 Peanut for export Ferrante et al. (2012) Nigeria Total AF 20 All foodstuffs Ferrante et al. (2012) South Africa 5 All food stuffs Ferrante et al. (2012) Tanzania AFB1 5 Cereals & oil seeds Ferrante et al. (2012) Kenya Total AF 20 Groundnut FAO (2004) USA Total AF 20 All foods except milk FAO (2004) FB1 + FB2 4,000 Unprocessed maize Ferrante et al. (2012) FB1 + FB2 200 Processed maize based foods for Ferrante et al. (2012) infants & children AFM1 0.5 Milk Creppy (2002) Canada Total AF 15 Nuts & nuts products FAO (2004) Russia DON 1,000 Grains Creppy (2002) ZEN 1,000 Grains & vegetable oils T2 100 All foodstuffs Malaysia PAT 50 Apple juice (includes apple juice as Ferrante et al. (2012) ingredients in other beverages) Singapore PAT 10 Juices Ferrante et al. (2012) Vietnam PAT 50 Fruit & fruit juices Ferrante et al. (2012) Iran OTA 20 Legumes Ferrante et al. (2012) ZEN 400 Barley AFMI 0.2 Cheese Sudan OTA 15 Wheat Ferrante et al. (2012) Chile ZEN 200 All foods Ferrante et al. (2012) Morocco ZEN 200 Cereals & vegetable oil Ferrante et al. (2012) AFM1 0.05 Milk & milk products OTA 30 Cereals Ferrante et al. (2012) Venezuela AFM1 0.5 Milk Ferrante et al. (2012)

EU* = 27 countries (Austria, Belgium, Bulgaria, Croatia, Republic or Cyrus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latva, Lithuania, Luxembourg, Malta, Netherlands, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, and United Kingdom.

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More specifically, 15 African countries including Nigeria, Morocco, South Africa and Kenya have fixed regulations. For other countries such as Burkina Faso, Cameroon, Uganda, Ethiopia, Zambia, Zimbabwe, etc. fixed regulations are probably not in existence (Ferrante et al., 2012). Irrespective of the absence of regulations or the regulation of only few mycotoxins in these African countries, mycotoxins contamination is seen as a major challenge that needs urgent attention. It is also worthy to mention that, a significant number of the existing regulations in Africa is that of AFs though Morocco currently has the most detailed mycotoxin regulations (FAO, 2004). Continuous efforts are being made by regulatory bodies particularly in Africa to institute more regulations to mitigate contamination in feeds and foods. Figure 2.24 shows the existence of mycotoxin regulation in African countries.

Figure 2.24 Mycotoxin regulations within African countries

2.5.4 Mycotoxin Control and Prevention

Though the simplest approach to avoiding mycotoxin exposure is the non-consumption of mould infested foods, but this might not be the case considering that mycotoxins are invisible and the absence of moulds does not necessarily translate to the absence of mycotoxins. Some populations are also forced to eat contaminated foods due to food insecurity and those that 57 solely rely on a single stable crop that are susceptible to fungal contamination are more prone to mycotoxin exposure (Marroquin-Cardona et al., 2014; Hove et al., 2016). Fungi and mycotoxin mitigation strategies are categorized into pre-harvest or post-harvest.

2.5.4.1 Pre-harvest measures

The safety and quality of final produce depends largely upon the safety and quality of incoming raw materials, hence the critical control point of a significant amount of biological, physical and chemical hazards including mycotoxins are at the primary agricultural stage. Hence, control actions have to be taken on the farm before crops are harvested and sold in form of pre-harvest measures. Proven pre-harvest measures for mycotoxin mitigation are:  Early harvesting: early harvesting reduces fungal infestation and subsequent mycotoxin contamination of harvested crops. Increased moisture levels of crops on field due to over exposure to rainfall through late harvesting also aggravate contamination. According to Rachaputi et al. (2002), early harvesting and threshing of groundnut facilitated reduced AF levels and increased gross returns by 27% compared to prolonged harvesting.  Insect management: the extent of insect damage accentuates the level of mycotoxins contamination. Insects such as weevils stem borers, etc. disseminate toxigenic fungal spores to kernels or stalks from plant surfaces or burrow through plants by their feeding habits (Munkvold, 2003) and allow fungi to infest crops on field. The management of insects during plant growth is therefore expedient to reducing mycotoxin contamination.  Biological control: important progress have been made in the utilization of biocontrol strategies aimed at utilizing atoxigenic fungi or microorganism to out-compete or supress associated toxigenic fungal strains on field thereby lowering mycotoxins level crops (Cleveland et al., 2003). Biocontrol agents such as Lactobacillus spp., B. subtilis, Ralstonia spp., Pseudomonas spp. and Burkholderia spp. have been found to be efficient in the management of AF (Palumbo et al., 2006; Zain, 2011). As reported by Dorner and Cole (2002), post-harvest contamination of AF decreased by 96% upon in-field employment of non-toxigenic strains of A. parasiticus and A. flavus. Reduction of AF (20-90%) by Trichoderma spp. was also recorded by Anjaiah et al. (2006). The use of biocontrol agents has been approved in some countries e.g., in the United States, Afla-guard and AF36 are two commercialised products based on

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atoxigenic A. flavus strains (Dorner, 2009). In addition, in Africa, Aflasafe is a widely recognized biocontrol that is being used to control AF contamination in maize.  Chemical control: proper utilization of pesticide during plant growth has the capacity to mitigate fungal and insect invasion in crops. Fungicides such as amphotericin B and itraconazole have been depicted to successful mitigate aflatoxigenic Aspergillus spp. (Ni and Streett, 2005). Fumonisins contamination could be lowered through the application of fungicides that have been employed to control Fusarium head blight e.g., cyproconazole, prochloraz, epoxyconazole, tebuconazole, and azoxystrobin (Haidukowski et al., 2005). Nevertheless, heighted concerns about environmental sustainability and food safety have suppressed the utilization of fungicides.  Breeding for resistance: this strategy appears to be a positive long-term intervention strategy for mycotoxin mitigation. Sites on plants that are resistant to A. flavus and Fusarium spp., majorly F. verticillioides have been recognized and integrated into breeding programs (Munkvold, 2003). Prospective genetic resistance and biochemical markers have been distinguished in crops, specifically maize and are used as selectable markers in breeding for resistance to AF contamination. Organisations such as International Institute of Tropical Agriculture (IITA) are working effortlessly on these programs in Africa. In recent years, development in genomic technology based decoding and research on fungi genome particularly A. flavus have enhanced the isolation and identification of genes responsible for mycotoxin production and modification (Bhatnagar et al., 2003; Cleveland et al., 2003; Ehrlich, 2009; Adegoke and Letuma, 2013). Kumar et al. (2017) also highlighted the reduction of AF contamination through the utilization of Bt maize that has been incorporated with traits that are insect resistant thereby preventing insect damage, which is a major facilitator of fungal attack.  Other pre-harvest methods: good agricultural practices including tillage, timely planting, crop rotation, weed control, provision of adequate plant nutrition, irrigation, and fertilization management also have influence on prevention of fungal infection and subsequent mycotoxin contamination (Ehrlich and Cotty, 2004).

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2.5.4.2 Post-harvest measures

These involves managerial and technological strategies linked to post-harvest handling, primary production, processing activities etc. integrated from harvest to consumption in order to reduce or eliminate mycotoxins from food. They include:  Proper drying: the reduction of moisture in agricultural produce through proper drying is a critical factor that create less suitable environment for fungal development and proliferation, insect invasion and mycotoxin formation (Lanyasunya et al., 2005). Turner et al. (2005) in their survey reported 60% reduction in AF levels with proper drying of groundnut in Guinea. It has been established that the reduction of maize moisture content to 15.5% or less within 24-48 hrs reduces fungal growth risk and subsequent AF production (Hamiton, 2000). Awuah and Ellis (2002) also observed the absence of fungi when groundnut was dried to 6.6% moisture level and stored for 6 months.  Sorting: this technique is solely dependent on dissociation of contaminated grain from the whole lot and is based on substantial contamination of a small portion of the grain, to reduce the overall contamination level of the bulk. This can be done manually or automatically with sorters. Contaminated grains are often identified with their characteristic colour and other physical properties. Aflatoxin levels in lots of raw groundnut in Philippines were reduced from 300 ng/g to less than 15 ng/g with manual sorting (Galvez et al., 2003). Filbert and Brown (2012) demonstrated 98% AF reduction in groundnut kernels purchased at indigenous markets in Kenya. In a study from Benin by Fandohan et al. (2005), 40% reduction was recorded within the same commodity while Kimanya et al. (2008) and Van der Westhuizen et al. (2010) reported 20% FB reduction during hand sorting of maize in Tanzania and South Africa, respectively. Nevertheless, this control strategy might not be the best option considering the tendency of mycotoxins to accumulate without visible changes in grains (Karlovsky et al., 2016).  Sanitation: primary sanitation procedures like eradication of dirts from former harvest would assist in reducing on field fungal infestation and infection. Hell et al. (2000) showed a correlation between cleaning of stores prior to loading of new batches and lower AF concentrations.  Proper storage: agricultural products need to be stored under appropriate conditions for their quality and safety to be assured. Storage structures and premises must be in 60

good condition with proper aeration. It is important for all moisture sources to be avoided with appropriate control of temperature and humidity since these factors largely influence microbial stability of crops. In Nigeria, shelled maize grains are commonly placed in polyethylene bags and placed on bare floors. Adetunji et al. (2014b) found higher levels of AF (range: 13.25 – 656.24 µg/kg) and DON (33.26 – 91.06 µg/kg) in these maize grains compared to other storage methods used by farmers in Nigeria.  Chemical treatments: many chemical treatment methods for mycotoxin detoxification are not yet approved in most countries due to the ability of chemicals to transform mycotoxins to other compounds (Karlovsky et al., 2016) though some methods have been found to be useful. Some chemical treatment processes involves acid treatments, treatment with bases, reducing agents, oxidizing agents, etc. Treatment of contaminated corn with 2% ammonia reduced AF levels according to Park et al. (1988), however this treatment changed the nutritional value of the seed, caused an ammoniacal odour and colour change. Aflatoxins were treated with diluted lactic acid, citric acid and acetic acid by Aiko et al. (2016) under conditions imitating cooking.

Lactic acid was most efficient, in transforming AFB1 into traces of AFB2. In addition,

AFB1 and AFG1 levels were reduced by 77 and 80 %, respectively, in peanuts by 10 mins ozone treatment at 75 °C (Proctor et al., 2004).  Awareness creation: awareness creation has been advocated as a long-term strategy for mycotoxin mitigation (James et al., 2007). It is expedient for people particularly in SSA to be aware of what mycotoxins are, and their associated health effects. A study by Ezekiel et al. (2013) provided information on AF awareness amongst peanut cake consumers in Nigeria. Also, Aboloma (2014) assessed mycotoxin awareness amongst traders and farmers in Ekiti State, Nigeria. Both studies established that respondents under study were unaware of the existence of mycotoxins but were knowledgeable of the existence of fungi. Similar reports were also obtained by Matumba et al. (2016) amongst Malawian populations. Awareness creation can be embarked upon by private establishment, government organisations, non-governmental organization, media networks such as television, radio, newspapers. Workshops and seminars could also be utilized as a means of exchanging information between researchers and community members.

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2.5.5 Socio-economic Impact of Mycotoxin Contamination

Diverse economic losses are associated with mycotoxin contamination and losses incurred by developed nations are usually trade related whereas Africa tends to incur both economic losses and additional costs related to health. Socio-economic impact associated with mycotoxin contamination worldwide include: increased cost of health care, loss of lives of humans and animals, decline in productivity of livestock, loss of markets, crop losses, reduced food availability, exports rejections, loss of integrity amongst trading partners, forced alternative uses, product recalls, reduced prices of contaminated products, etc. (Zain, 2011; Udomkun et al., 2017). Loss due to AF contaminated maize and peanut, DON contaminated wheat and FB contaminated maize accounted for over $1.5 billion USD in the U.S as reported by Robens and Cardwell (2003). Within a decade, India also lost over $10 million USD due to the exportation of contaminated groundnuts (Vasanthi and Bhat, 1998). Largely, producers are affected through limited storage choices, cost of product testing and loss of local end markets. Companies also take up more costs due to more product losses, cost of monitoring and limited end markets while farmers experience reduced income due to feed and food losses as well as reduction in the selling prices. On the other hand, consumers as the end users in the chain incur higher prices because of heightened surveillance at all stages of handling and in more severe scenario faces health challenges because of the consumption of contaminated foods.

Moreover, this impact does not exclude the high cost of research and regulatory activities aimed at reducing health risks because of the existence of causal relationships between mycotoxins and their influence on health. For example, AFs intoxication even at a dose of 1 µg/kg/bw/day could contribute to cancer development, immune suppression and growth inhibition (JECFA, 2001). It is also overwhelming that in Africa, an annual cost of over $750 million USD has been accrued to AF contamination of crops while the EU regulation of AFs reportedly costs food exporters $670 million USD yearly (Udomkun et al., 2017). Misdiagnosis, poor infrastructures, undependable and inconsistent data amongst other factors makes it difficult to account for the additional and indirect costs associated with mycotoxin exposure in Africa. If the scale of economic and health influence of mycotoxin contamination is well understood, it will hasten policy makers towards imposing regulations and supporting affected populations.

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2.5 Concluding remarks

Upon a comprehensive appraisal of gram-negative bacteria, mycotoxigenic fungi, endotoxins and mycotoxins, their prevalence, together with their deleterious effects on human and animal health and the economy at large, it is important to conclude that their surveillance is critical to food safety and security. With contamination of traditionally fermented foods often overlooked, studies to evaluate the safety of these vulnerable food products are of necessity based on their high consumption levels for the purpose of interventions. These studies could produce toxicological based data that can serve as useful tools for increased awareness creation, establishment of regulations, policy formulations or amendments and development of adaptable strategies for the management and control of food hazards. It is therefore expedient to make effort to continually assess the safety of traditionally fermented foods within producing nations such as Nigeria and South Africa.

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CHAPTER THREE

CONTAMINATION OF FERMENTED FOODS IN NIGERIA WITH FUNGI

Ifeoluwa Adekoyaa*, Adewale Obadinab, Judith Phokuc, Obinna Nwinyid and Patrick Njobeha aDepartment of Biotechnology and Food Technology, University of Johannesburg, South Africa bDepartment of Food Science and Technology, Federal University of Agriculture, Abeokuta, Nigeria cDepartment of Biomedical Technology, University of Johannesburg, South Africa dDepartment of Microbiology, Covenant University, Ota, Nigeria *Corresponding author: Ifeoluwa Adekoya; Email Address: [email protected]

Abstract

This study assessed the safety and quality of some fermented foods in Nigeria. Cluster sampling was used to obtain different fermented foods: maize gruel (ogi), locust beans (iru), sorghum meal (ogi baba), dried locust beans (dried iru), African oil bean seed (ugba) and melon (ogiri) from Southwest, Nigeria. The moisture content, Total Titratable Acidity, pH, and fungal diversity of the samples were determined. The identity of the isolates was established through macroscopic, microscopic and molecular biology means. The moisture content and pH of the analysed samples ranged from 12 – 56% and 3.60 – 8.08, respectively. The overall data on the mycobiota of the fermented foods revealed that total fungal loads of ugba and ogiri were 1.05 x 105 and 7.9 x 105 CFU/g, respectively. The fungal isolates belonged to 17 genera including Aspergillus, Fusarium, Candida, Saccharomyces and Penicillium. The dominant fungi detected were A. flavus and all analysed samples were contaminated with F. verticillioides with the exception of ogi baba. The study led to the discovery of new fungal strains and previously unreported fungal species in the selected fermented foods. The analysed fermented foods were highly contaminated with different fungal species that could potentially be toxigenic in producing various types of mycotoxins.

Keywords: Mycobiota, fermentation, moisture content, fungi, and Nigeria

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Highlights

 The fungal profile of some fermented foods in Nigeria was assessed for their safety.  There was high prevalence and co-contamination of different potentially toxigenic fungi.  New fungal strains and several unreported fungi were discovered in the fermented foods.

3.1 Introduction

Fermented foods are one of the major food groups in Africa and the process of fermentation involves the breakdown of organic compounds into acids or alcohol through enzymatic action of microorganisms particularly yeasts and bacteria under anaerobic conditions (Chilton, Burton, and Reid, 2015). Fermentation has been found to enhance the nutritional, health promoting, organoleptic and preservative properties of food (Oyewole and Isah, 2012). Fermentation plays a significant role in developing economies as a technology that increases income sources, food availability, food diversity and reduces post-harvest losses. In Africa, fermented foods are mostly produced traditionally thus, there is variation in the substrates, processing conditions, packaging materials, handling and storage practices (Babajide et al., 2006). Examples of such foods include iru, garri, amasi, banku, ogi, injera, mahewu and meriss. Iru from fermented African locust bean (Parkia biglobosa), ugba from African oil bean seed (Pentaclethra macrophylla) and ogiri from melon (Colocynthis citrullus) are important condiments used to flavour soups and stews in West Africa particularly in Nigeria.

Enujiugha et al. (2008) cited that ugba is consumed by more than 40 million people in Africa and asides from being a condiment, ugba also serves as a snack (Olotu et al., 2014). Ugba, iru and ogiri are examples of vegetable proteins that are also used as meat substitutes in diets and processed by wild solid-state fermentation, which gives rise to extensive hydrolysis of its carbohydrate and protein constituents (Achi, 2005). In addition to their protein contents, they are rich in fats, carbohydrates and are good sources of calcium. Their typical processing methods involve dehulling, boiling, draining, fermentation, salting, drying (optional) and packaging. Maize and sorghum are converted to ogi and ogi baba by steeping in water (±3days), washing, wet milling and sieving. The products are the resultant sediments from this process. Ogi and ogi baba are products of lactic acid fermentation used mostly for infant

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The microbial quality of these foods is influenced by intrinsic and environmental factors such as hygiene during processing and composition of substrates. The fermentation of melon, African oil bean seed and locust bean into ogiri, ugba and iru, respectively is predominantly by Bacillus species (Olasupo et al., 2016). Other bacteria species that can be isolated during various fermentation processes are Staphylococcus, Escherichia, Micrococcus, Leuconostoc, Pseudomonas and Corynebacterium (Achi, 2005). However, fungal species have not been associated with the fermentation of ugba, iru and ogiri. Lactobacillus species such as L. plantarum and yeasts have been reported to be the fermenting organisms for ogi production (Okeke et al., 2015). Irrespective of the benefits derived from fermented foods, there are concerns about their safety because of the continual and unpredictable pre- and post- processing contamination by pathogenic microorganisms some of which can be toxigenic fungi that produce harmful secondary metabolites including mycotoxins.

Some fungal species belonging to the Penicillium, Mucor, Geotrichum, and Rhizopus genera have been used in the fermentation of cheese and milk (William and Dennis, 2011), while others produce undesirable toxins and their presence in foods have been attributed to their sporulating ability, which makes them to easily contaminate the environment (Frisvad and Samson, 2007) and the food products therein. Hence, the assumption among consumers that fermented foods especially those processed traditionally are safe is a dangerous one as such foods could be potential sources of mycotoxin exposure and accompanied health complications. There has also been evidence of multiple mycotoxins in different food categories including fermented foods. Chilaka et al., (2016) reported the co-occurrence of Fusarium mycotoxins such as fumonisins, nivalenol, HT-2 and deoxynivalenol-3-glucoside in fermented maize from Nigeria. Colak et al., (2012) detected aflatoxin B1 within the range of 0.2 and 13.2 μg/kg in Tarhana, a Turkish fermented cereal.

Nevertheless, most microbiological studies on African fermented foods have been based on the isolation and characterisation of microorganisms that enhance the fermentation processes and only studies with limited scope have been conducted to establish the incidence of pathogenic organisms like fungi (Olasupo et al., 2016). Even though some studies have reported the presence of fungi in some fermented foods consumed in Nigeria, there is however little adequate information on the spectrum of microorganisms associated with these

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3.2 Materials and Methods

3.2.1 Sampling

Cluster sampling was used to obtain fermented foods that included maize gruel (ogi), locust beans (iru), sorghum meal (ogi baba), dried locust beans (dried iru), African oil bean seed (ugba) and melon seeds (ogiri) that are indigenous to Nigeria. A total of 108 samples (18 each) with an equivalent weight of 30±5g were purchased from selected fermented food sellers in South-west, Nigeria between Feburary and March 2015. Upon their collection, samples were placed in sterile containers in cooler boxes and airfreighted to the University of Johannesburg, South Africa where they were analysed. Iru and ugba samples were milled using a sterile mechanical blender (LB10G, ITM Instrument, Alberta, Canada) prior to analysis.

3.2.2 Methodology

3.2.2.1 Determination of moisture, pH and Total Titratable Acidity (TTA) contents of fermented foods

Moisture content of the samples was determined using the method described by AOAC (2005). The pH of the food samples was measured using a pH meter (Jenway, Model 3510, Essex, UK) after calibration using standard buffers. Titratable acidity was determined according to AOAC (2005). The amount of acid (lactic acid) in each sample was determined by using the following equation:

%Lactic acid = Volume of the base used x Normality of alkali x Molecular equivalent of lactic acid (90) Sample weight (g) x 10

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3.2.2.2 Isolation and identification of fungi

The samples were blended using a sterile laboratory blender for 60 secs, 1 g of each sample was weighed and diluted in 9 mL of sterile 0.1% peptone water solution, vortexed and serially diluted to 10−10. Solidified Rose Bengal Chloramphenicol, Czapek Yeast Extract and Malt Extract agar (Appendix 3.1) plates were spread-plated with aliquot of 0.1 mL of the sample. The inoculated plates were checked for fungal colonies after incubation at 25 oC for 5 days. The colonies were counted with a colony counter, mean of fungal colonies was calculated, and results were expressed as CFU/g. Cultures were streaked onto Czapek Yeast Extract agar and Malt Extract agar and plates were incubated at 25 oC for 5 days. Pure isolates were identified based on their macroscopic (Appendix 3.2) and microscopic characteristics (Appendix 3.3) according to keys of Klich (2002), Samson and Varga (2007) and Pitt and Hocking (2009). Prior to this, the fungal isolates were stained with lactophenol blue, mounted on slides, overlaid with cover slides and placed on the stage of an optical microscope (Olympus CX40, Micro-Instruments, New Zealand) to observe the micro morphological attributes for identification at species level.

3.2.2.3 Molecular studies

Genomic DNA was extracted from the fungal cultures using the ZR fungal DNA kit (Zymo Research D6005, California, USA). After DNA extraction, Polymerase Chain Reaction was performed to amplify the DNA of interest within the Internal Transcribed Spacer (ITS) region using EconoTag Plus Master Mix (Lucigen), ITS 1 forward and ITS 4 reverse primers with sequences TCCGTAGGTGAACCTGCGG and TCCTCCGCTTATTGATATGC. After amplification, the PCR products were run on a gel and the gel extracted using ZymoClean Gel DNA recovery clean-up kit (Zymo Research, D4001). The extracted fragments were sequenced in the forward and reversed directions (Applied Biosystems, Thermofisher Scientific, Big Dye terminator kit v3.1, Carlsbad, California, USA) and purified using ZR-96 DNA sequencing clean-up kit (Zymo Research, D4050). The purified fragments were run on an ABI 3500 xL Genetic Analyser (Applied Biosystems, Thermofisher Scientific) for each reaction of every sample. CLC Bio Main Workbench v7.6 was used to analyse the data (.abi files) generated by the ABI 3500 xL Genetic Analyser (Applied Biosystems, Thermofisher Scientific). The similarities of the fragments with previously published sequence data were examined with BLASTN 2.2.31+ version (Stephen et al., 1997) and the sequences generated in this study were submitted to the genbank.

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3.2.2.4 Phylogenetic analysis

The phylogenetic relationship of fungal sequences from ogiri including reference strains were obtained from a Neighbour-Joining analysis of Saitou and Nei (1987). The bootstrap consensus tree was inferred from 1000 replicates (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed and the percentages of the replicate trees were shown over the branches as bootstrap values. The evolutionary distances were computed with the Maximum Composite Likelihood method (Tamura et al., 2004). Nineteen nucleotide sequences were analysed and the final data set had 390 positions after the elimination of gaps and missing values. The phylogenetic and molecular evolution analyses were conducted in MEGA7 (Kumar et al., 2016).

3.2.3 Data analysis

The pH, TTA and moisture content of the fermented food samples were evaluated in triplicates and subjected to one-way Analysis of Variance. Differences among the means were separated with Tukey’s test and significances were accepted at 5% confidence level. In addition, the Pearson's correlation coefficient (two-tailed) was carried out between the pH, moisture content, TTA and fungal load of the fermented food samples. The statistical software used was SPSS version 23.0 for Windows (IBM Corporation, New York, USA).

3.3 Results

The mean pH of the samples ranged from 3.60 to 8.08 with iru having the highest value and ogi baba the least. The mean TTA of the samples were significantly different from each other except for ugba, ogiri and ogi baba (Table 3.1).

Table 3.1 Mean pH, TTA and moisture content of some Nigerian fermented foods Fermented pH Total Titratable Acidity Moisture Content (%) Foods (% lactic acid) Mean + S.D Range Mean ± S.D Range Mean ± S.D Range

Ogi 3.83a±0.05 3.80-3.86 0.56d±0.05 0.53-0.59 46.00c±0.97 46.00-47.50 Ogi baba 3.62a±0.19 3.60-3.69 0.50d±0.05 0.46-0.53 46.00c±3.00 36.00-49.00 Ogiri 8.06d±0.02 8.05-8.08 0.39c±0.02 0.38-0.41 43.33b±1.16 42.00-44.00 Ugba 6.07b±0.01 6.06-6.09 0.12a±0.02 0.10-0.14 44.67b±2.97 41.50-43.00 Dried iru 6.53b±0.03 6.49-6.56 0.22b±0.04 0.21-0.25 13.33a±1.16 12.00-14.00 Iru 7.46c±0.22 7.18-7.76 0.35c±0.01 0.34-0.36 55.33d±1.54 54.00-56.00

Note: sample size per sample type is 18. a-d mean values along the same column with different letters are significantly different. S.D: Standard deviation

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The mean moisture content of the samples ranged from 12 - 56% with dried iru and iru respectively having the lowest and highest moisture content. Total fungal load of ugba, iru and ogiri were 9.4 x 103, 1.05 x 105 and 7.9 x 105 CFU/g, respectively. As shown in Table 3.3, ogiri had the highest mean fungal load and the ogi samples were heavily contaminated with fungi belonging to 14 genera, while ogiri was the least contaminated sample type that consisted of 9 fungal genera. There was a significant but slightly negative correlation between pH and TTA (r = -0.574, p < 0.05) (Table 3.2).

Table 3.2 Correlation coefficient of the pH, Total TTA, moisture content and total fungal count of some Nigerian fermented foods pH TTA Moisture Total fungal content (MC) count (TFC) pH Pearson Correlation 1 -0.574a -0.057 0.132a Sig. (2-tailed) 0.013 0.822 0.600 N 18 18 18 18 TTA Pearson Correlation -0.574a 1 -0.540a 0.276 Sig. (2-tailed) 0.013 0.021 0.267 N 18 18 18 18 MC Pearson Correlation -0.057 -0.540a 1 0.008 Sig. (2-tailed) 0.822 0.021 0.975 N 18 18 18 18 TFC Pearson Correlation 0.132a 0.276 0.008 1 Sig. (2-tailed) 0.600 0.267 0.975 N 18 18 18 18 a Correlation is significant at the 0.05 level (2-tailed). N = Sample size (18)

Table 3.3 Total fungal load and isolated genera of fungi from Nigerian fermented foods Fermented Total Fungal Isolated Fungal Genera Foods Load (CFU/g ) Ogi 8.0 x 103 Aspergillus, Fusarium, Cytobasidium, Rhodotorula, Penicillium, Gibberella, Rhizopus, Saccharomyces, Mucor, Cladosporium, Talaromyces, Pichia, and Candida Ogi baba 6.76 x 104 Aspergillus, Monascus, Talaromyces, Cytobasidium Saccharomyces, Rhodotorula, Cladosporium, Geotrichum, Fusarium, Candida, Rhizopus, Penicillium, and Mucor Ogiri 7.90 x 105 Aspergillus, Penicillium, Talaromyces, Fusarium, Geotrichum, Rhizopus, Saccharomyces, Candida and Mucor Ugba 1.05 x 105 Aspergillus, Cochliobolus, Curvularia, Monascus, Rhodotorula, Geotrichum, Fusarium, Candida, Rhizopus, Penicillium, Mucor, and Talaromyces Dried iru 1.87 x 105 Aspergillus, Fusarium, Monascus, Geotrichum, Candida, Rhizopus, Saccharomyces, Penicillium, Mucor, Talaromyces, and Geotrichum Iru 9.40 x 103 Aspergillus, Penicillium, Fusarium, Curvularia Monascus, Rhizopus, Saccharomyces, Mucor, Rhodotorula, Geotrichum, and Talaromyces

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As observed in Table 3.4, 102 Aspergillus, 49 Penicillium and 23 Fusarium isolates were found in the fermented foods. A. flavus had the highest frequency of occurrence in ogi (26%), ogi baba (38%), ogiri (37%), ugba (44%) and iru (33%) whereas A. parasiticus was only isolated from 11% of ugba samples. A. sydowii and A. tritici were only found in ogi baba while A. ustus was only recovered from ogiri.

P. chrysogenum was the most frequently occurring Penicillium species, P. aethiopicum was detected in 11% of ugba samples, while P. citrinum was recovered in 6% of the ogi baba samples. The two Penicillium species detected in ogi samples analysed were P. chrysogenum and P. crustosum. F. andiyazi, F. chlamydosporum, F. fujikuroi and F. proliferatum were amongst the 23 Fusarium isolates (with distinct genetical profiles) found in the fermented foods. All the samples had F. verticillioides with the exception of ogi baba and iru (Table 3.4). Besides the Aspergillus, Fusarium and Penicillium genera in the fermented foods, 14 other fungal genera were also recovered. Saccharomyces cerevisiae was the most frequently occurring species (Table 3.5) and was found in 100% of the ogi samples. Cochliobolus hawaiiensis was found in 6% of the ugba samples while Cytobasidium slooffiae was found in 11% of the ogi samples, 11% of ogi baba samples had Curvularia lunata and Talaromyces islandicus and 17% of the samples had Monascus ruber, Rhizopus stonolifer, Rhodotorula mucilaginosa and Geotrichum candidum.

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Table 3.4 Incidence rates of fungal contamination of Nigerian fermented foods with Aspergillus, Penicillium and Fusarium species

Isolated species Accession OGI (n=18) OGI BABA (n=18) OGIRI (n=18) UGBA (n=18) IRU (n=18) DRIED IRU (n=18) Number % positive Frequency % positive Frequency % positive Frequency % positive Frequency % positive Frequency % positive Frequency samples of species samples of species samples of species samples of species samples of species samples of species isolated (%) isolated (%) isolated (%) isolated (%) isolated (%) isolated (%) Aspergillus species A. amstelodami 6 1 (4) ------6 1 (10) AY373885.1 A. candidus ------6 1 (10) KT223337 A. clavatus - - 9 2 (13) 6 1 (5) - - - - 6 1 (10) KU052566 A. flavus 33 6 (26) 33 6 (38) 39 7 (37) 39 7 (44) 33 6 (33) 22 4 (40) KR611584 A. fumigatus 17 3 (13) - - 17 3 (18) 6 1 (6) 33 6 (33) - - KX215145.1 A. minisclerotigenes - - 6 1 (6) 6 1 (5) ------JF412778 A. niger 17 3 (13) 6 1 (6) - - 6 1 (6) 6 1 (6) - - KX215115.1 A. niger 11 2 (9) - - 11 2 (11) ------KX215112.1 A. parasiticus 28 5 (22) 9 2 (13) 17 3 (18) 11 2 (13) 17 3 (17) 11 2 (20) DQ467988.1 A. ruber 6 1 (4) ------6 1 (10) KX215127.1 A. sclerotiorum ------6 1 (6) - - - - KT717312 A. species - - 6 1 (6) - - 6 1 (6) - - - - KX215117.1 A. sydowii - - 6 1 (6) ------KR611596 A. tritici - - 6 1 (6) ------KP780810 A. tubingensis ------11 2 (13) - - - - KT717311 A. ustus - - - - 6 1 (5) ------HQ607918.1 A. versicolor 11 2 (9) 6 1 (6) 6 1 (5) 6 1 (6) 11 2 (11) - - LC105698

Penicillium species P. chrysogenum 11 2 (50) - - 17 3 (30) 11 2 (10) 22 4 (36) KP836338 P. expansum - - 11 2 (33) - - 6 1 (10) 11 2 (25) - - AY818338 P. lanosocoeruleum ------6 1 (13) - - JX997110 P. polonicum ------6 1 (9) KX215146.1 P. aethiopicum ------11 2 (20) - - - - KX215125.1 P. chrysogenum - - - - 6 1 (10) - - 11 2 (25) - - KX215141.1 P. citrinum ------11 2 (25) - - KT315422 P. citrinum - - 6 1 (17) ------KX215134.1 P. crustosum - - - - 17 3 (30) ------KT192315 P. crustosum 11 2 (50) - - - - 11 2 (20) - - - - KT735107.1 P. flavigenum - - - - 11 2 (20) ------LN809058 P. glabrum ------11 2 (20) 6 1 (13) - - JN887323.1 P. mallochi - - 6 1 (17) ------17 3 (27) KX215135.1 P. raistrickii - - 11 2 (33) 6 1 (10) - - - - 11 2 (18) KX215126.1 P. steckii ------6 1 (10) - - 6 1 (9) KX215128.1

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Isolated species Accession OGI (n=18) OGI BABA (n=18) OGIRI (n=18) UGBA (n=18) IRU (n=18) DRIED IRU (n=18) Number % positive Frequency % positive Frequency % positive Frequency % positive Frequency % positive Frequency % positive Frequency samples of species samples of species samples of species samples of species samples of species samples of species isolated (%) isolated (%) isolated (%) isolated (%) isolated (%) isolated (%)

Fusarium species F. andiyazi ------17 2 (40) - - - - KX215140.1 F. chlamydosporum 6 1 (33) ------KP769538.1 F. chlamydosporum ------6 1 (25) - - KX215137.1 F. fujikuroi - - 6 1 (33) ------KX215132.1 F. proliferatum - - - - 11 2 (40) - - 6 1 (25) 6 1 (33) KT581408.1 F. sp. - - 11 2 (67) - - 6 1 (20) - - - - JQ350882 F. verticillioides 6 1 (33) - - - - 11 2 (40) 6 1 (25) 11 2 (67) KP003945 F. verticillioides 6 1 (33) - - 17 3 (60) - - 6 1 (25) - - KX215124.1

Note: Total number of Aspergillus species = 102 (ogi: 23 species, ogi baba: 16 species, ogiri: 19 species, ugba: 16 species, iru: 18 species, dried iru:10 species); Fusarium species = 23 (ogi: 3 species, ogibaba: 3species, ogiri: 5 species, ugba: 5 species, iru: 4 species, dried iru: 4 species); Penicillium species = 49 (ogi:4 species, ogi baba: 6 species, ogiri: 10 species, ugba: 10 species, iru: 8 species, dried iru: 11 species).

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Table 3.5 Incidence rate of fungal contamination of Nigerian fermented foods with other fungal species

Other species OGI (n=18) OGI BABA (n=18) OGIRI (n=18) UGBA (n=18) IRU (n=18) DRIED IRU (n=18) Accession Number % Frequency % Frequency of % Frequency % Frequency % Frequency % Frequency of Positive of species Positive species Positive of species Positive of species Positive of species Positive species sample isolated sample isolated (%) sample isolated (%) sample isolated sample isolated (%) sample isolated (%) Cochliobolus hawaiiensis - (%)- - - - - 6 1(%) (4) - - - - KC288116 Curvularia spicifera ------17 3 (11) - - - - KC999935 Curvularia spicifera ------6 1 (6) - - KX215116. Cytobasidium slooffiae 11 2 (4) ------1K T876712. Cytobasidium slooffiae - - 11 2 (1(4) ------1K X215121. Gibberella moniliformis 11 2 (4) ------1JF 499676.1 Monascus ruber - - 17 3 (6) - - 17 3 (11) 11 2 (13) 6 1 (4) KX215143. Mucor circinelloides 22 4 (8) - - 33 6 (16) 6 1 (4) - - - - 1G U966516 Mucor sp. - - 17 3 (6) - - 6 1 (4) - - - - FJ210516 Talaromyces islandicus - - 11 2 (4) - - 11 2 (7) - - - - KJ783270 Talaromyces islandicus - - 6 1 (2) - - 6 1 (4) - - - - KX215129. Talaromyces positive- - positive- - positive- - positive- - positive- - positive17 3 (13) 1K X215142. Tpualraropurmeoygceesn usra dicus -samples - samples- - -samples - -samples - 6samples 1 (6) -samples - A1 B457007 Talaromyces stollii - - - - 6 1 (3) ------AB910938 Talaromyces verruculosus 11 2 (4) ------KJ413368 aOther identified species Candida parapsilosis 17 3 (6) 6 1 (2) 22 4 (11) ------Candida tropicalis 22 4 (8) 22 4 (8) - - 17 3 (11) - - 17 3 (13) Cladosporium 17 3 (6) 6 1 ------Cladosporioides Cu rvularia lunata - - 11 2(4) ------17 3 (13) G eotrichum candidum 28 5 (9) 17 3 (6) 50 9 (24) 28 5 (19) 17 3 (19) 17 3 (13) Pichia membranifaciens 11 2 (4) ------Rhizopus oligosporus 22 4 (8) 22 4 (8) 28 5 (15) - - - - 22 4 (17) Rhizopus stonolifer - - 17 3 (6) 28 5 (14) - - - - 6 1 (4) Rhodotorula mucilaginosa 17 3 (6) 17 3 (6) - - 6 1 (4) 22 4 (25) 22 4 (17) Rhodotorula rubia 6 1 (2) 22 4 (8) - - 17 3 (11) - - - - Saccharomyces cerevisiae 100 18 (34) 67 12 (25) 39 7 (19) 17 3 (11) 28 5 (31) 11 2 (8)

Note: total number of other species = 207 (ogi: 53 species, ogi baba: 49 species, ogiri: 37 species, ugba: 27 species, iru: 16 species, dried iru: 24 species). a These species were distinguished based on their morphological and microscopic characteristics.

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The evolutionary history for 15 nucleotide sequences isolated from ogiri and 4 other reference sequences representing the Aspergillus and Penicillium species complexes are shown in Figure 3.1. The sequences were grouped into five clades that clustered differently on the rationale of species with their bootstrap frequencies ranging from 61% to 100%. A. minisclerotigenes was shown to be closest relative of A. parasiticus strain CICC 2175 in clade 1 while A. fumigatus AD43 in clade 2 was closely related to A. fumigatus ZL13. Majority of the species (64%) in clade 3, 4 and 5 had excellent bootstrap support.

JF412778.1 Aspergillus minisclerotigenes isolate CS5 18S 61 KJ783263.1 Aspergillus parasiticus strain CICC 2175 99 KX171038.1 Aspergillus flavus strain BN2 Clade 1 88 DQ467988.1 Aspergillus parasiticus strain 2999 KX215112.1 Aspergillus niger strain AD1 KU052566.1 Aspergillus clavatus strain HAk2-M26 66 62 KX215145.1 Aspergillus fumigatus strain AD43 Clade 2 99 KU986653.1 Aspergillus fumigatus strain ZL13 KX215126.1 Penicillium raistrickii strain AD19 KX215141.1 Penicillium chrysogenum strain AD35 99 KP836338.1 Penicillium chrysogenum isolate FU42 Clade 3 90 KT192315.1 Penicillium crustosum strain ZP-2 63 LN809058.1 Penicillium flavigenum 1110TES11K3 KU986652.1 Penicillium chrysogenum strain ZL25 HQ607918.1 Aspergillus ustus isolate ATT280

100 LC105698.1 Aspergillus versicolor strain: DY20.1.1 Clade 4 100 KR611584.1 Aspergillus flavus strain PKM18 KX215124.1 Fusarium verticillioides strain AD16 Clade 5 100 KT581408.1 Fusarium proliferatum strain WS4KK12

0.10

Figure 3.1 Neighbour-joining phylogenetic tree of Aspergillus, Penicillium and Fusarium spp. from ogiri. Taxa names with diamonds are reference species isolated from other fermented products. Bootstrap values (as percentages) are shown at internal nodes. The scale bar below the tree indicates number of substitutions per site.

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3.4 Discussion

The observed acidic pH values of the ogi (3.83) and ogi baba (3.82) were expected since maize and sorghum are known to undergo lactic acid fermentation during processing into these products. The pH values of ogiri and iru were higher than the values reported by Olawuni et al. (2013) and this may be due to differences in fermentation conditions. Ogi had the highest TTA value but did not significantly differ from that of ogi baba: the high TTA could be due to the accumulation of organic acids such as acetic and lactic acid (Almeida et al., 2007) produced during spontaneous fermentation. Dried iru had the lowest moisture content because it is usually dried before packaging to the market. As pH within the samples decreased, TTA increased, indicating a negative correlation. This trend has however, earlier been reported by Michodjèhoun-Mestres et al. (2005) and could be because of sugar utilization by the fermenting organisms accompanied by accumulation of fermentation products such as lactic acid and other compounds including ethanol and carbon dioxide (Zvauya et al., 1997).

Ogi and ogi baba had lower fungal loads and their acidic pH might have restricted the growth of some microorganisms thereby resulting in products with lower fungal counts (Alonso- Calleja et al., 2004). In addition, some other factors that influence fungi growth in foods are water activity, light, temperature and nutrient availability. The predominant microorganisms in foods are usually those that can easily utilize the nutrients (nitrogen, carbon etc.) present, and to utilize these nutrients, the microbes engage in different metabolic processes. For example, some fungal species such as A. niger are able to produce amylolytic enzymes in order to hydrolyse complex carbohydrates such as starch into simpler compounds (Pensupa et al., 2013). However, the extent of starch utilization varies amongst different fungal species and strains and constitutes a determinant factor of fungi growth and diversity (Wang et al., 2016).

Fermented foods by their nature are expected to be nutritious and therefore, can serve as rich substrates for microorganisms including fungi. The microbial diversity of foods under study demonstrates contamination of these food products by several harmful fungi besides those reported to enhance fermentation during food processing. This study revealed the occurrence of members of the genera Penicillium, Aspergillus and Fusarium that are commonly isolated and found to produce mycotoxins in foods. Mycotoxins such as aflatoxins, ochratoxin A, fumonisins, citrinin, patulin, zearalenone and others are known to cause diseases such as

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In Format of LWT-Food Science and Technology cancer, liver damage, and immune suppression. Studies from other parts of the world have also shown the presence of mycotoxins in some fermented foods: Philippines (Sakai et al., 1983), India and Nepal (Thapa, 2016).

Aspergillus species particularly A. flavus were more prevalent in the food samples analysed in this study amongst the other two notable fungal genera (Penicillium and Fusarium).This could be because of their sporulating ability in the environment (Frisvad and Samson, 2007). A. niger, A. fumigatus and A. sydowii were amongst the Aspergillus spp. isolated in this study and this conforms to studies carried out by Nwokoro and Chukwu (2012) and Adebayo et al. (2014) who reportedly found these organisms during ogi, eko and kati fermentation. A. amstelodami, A. candidus and A. sclerotiorum have also been reported to be associated with tropical and subtropical regions including the South-west Nigeria.

Penicillium spp. are known to be ubiquitous and nutritionally undemanding as opportunist saprophytes and their identification in foods to species level have only been done in a few studies (Pitts and Hocking, 2009). In the current study, we were able to isolate and identify P. glabrum, P. expansum, P. chrysogenum, P. aethiopicum and P. raistrickii. The reports on F. proliferatum detected in foods have increased dramatically since the discovery of its mycotoxin, fumonisin. The fungus is not only an important producer of this toxin but synthesizes moniliformin and fusaric acid as well (Palmero et al., 2010). A slightly high contamination level of ogiri with Fusarium spp. including F. proliferatum was established in this study, unlike Akinyele and Oloruntoba (2013) who did not isolate it in fermented melon. Fusarium verticillioides which was isolated from some of our samples including ogi, a fermented product from maize, is the main producer of fumonisin that according to Njobeh et al. (2009) has widely been implicated as a major contaminant of maize and maize-based products in Africa.

Some yeast are known to play roles in the build-up of attributes such as flavours in fermented food products e.g., Candida mycoderma, while others have been reported to potentiate some enzymatic activities in breaking down starch as well as permit accessibility to nutrients (Omemu et al., 2007). As much as yeasts can be advantageous even for their fermenting roles, certain species are known to be spoilage microorganisms. The spoilage yeasts that were found in this study were Saccharomyces cerevisiae, Candida krusei, Candida parapsilosis, Pichia membranifaciens and Rhodotorula mucilaginosa. Saccharomyces cerevisiae was the predominant yeast detected in the fermented foods analysed in this study. Similarly,

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Jespersen (2003) reported that the most dominant yeast species affiliated with traditionally fermented beverages and foods in Africa is Saccharomyces cerevisiae. On the other hand, Greppi et al. (2013) identified Candida krusei as the most frequently isolated yeast in some maize-based products including ogi in Benin, West Africa. Candida parapsilosis is often associated with fatty foods (Pitt and Hocking, 2009) and this might be the reason why it was found in ogiri. The species is an emerging fungal pathogen that was recognised as one of the leading causes of invasive candida disease in humans (Trofa et al., 2008). There was low contamination of the analysed foods by Pichia and Cladosporium species and this could be because they were less prevalent in the environment compared to other species.

Fungi found in stored foods can be classified into two distinct groups, the storage and the field fungi, though cases exist whereby there is no marked distinction of whether fungi growth began in the field or during storage (Atanda et al., 2011). The Alternaria, Cladosporium, Curvulaira and Epicoccum species are often grouped with the Fusarium species as field fungi while the Mucor and Rhizopus spp. are often classified with members of Aspergillus and Penicillium genera as storage fungi (Joshaghani et al., 2013). From the mycobiota of the fermented foods, it was evident that there was pre and post-harvest contamination of the raw materials used in the processing of the fermented foods with more emphasis on post-harvest contamination because of the higher incidence of storage fungi compared with field fungi. Adetunji et al. (2014) detected sixty-two fungal metabolites in stored maize from the agro-ecological zones of Nigeria which also captured the sampling zones of this study, they also reported 92.91% of the maize samples analysed were contaminated with fumonisins.

Somorin et al. (2015) also reported the presence of citrinin and aflatoxin B1 in melon (Colocynthis citullus) which is the raw material/basal ingredient for ogiri production. Moreover, the various abiotic factors associated with the spontaneous fermentation processing and storage conditions of these foods may have contributed to the diversity observed. The use of conventional methods of microbial identification reveals only a limited microbiological profile of any community. In this study, we made use of a molecular-based method of identification that led to the discovery of many unreported yeasts and moulds in fermented foods in particular, new strains of these microorganisms.

This is the first report of F. andiyazi (https://goo.gl/aEF9iU) in ugba, P. raistrickii (https://goo.gl/oNXWae) in dried iru, A. tritici (https://goo.gl/KSwGdn) F. fujikuroi

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(https://goo.gl/nvtdo) in ogi baba, Curvularia spicifera (https://goo.gl/M7mLvO) in ugba, Monascus ruber (https://goo.gl/kcyrjZ) in ogi baba and Talaromyces spp. (https://goo.gl/mwISQk, https://goo.gl/V5I1SM, https://goo.gl/pUaRuj) in all the fermented foods. The phylogenetic analysis deduced from the 16S rRNA genes showed the genetic association, clonal relationship and divergence among the Aspergillus, Penicillium and Fusarium spp. isolated from ogiri. It also revealed the emergence of the species within the clades from a common ancestor before their dispersal and acclimatization into ogiri.

Gladieux et al. (2014) identified recombination and mutation of fungal species as some of the factors responsible for genetic variation in their study on fungal evolutionary genomics. Moreover, the wide diversity of these unreported fungi and variation in their evolutionary relationships showed that fungal identification by conventional means should be complemented with molecular identification. Furthermore, Geiser et al. (2004) stated that fungi are not easy to identify by conventional methods due to their unstable and confused taxonomic history. There is need for more research on fungal diversity especially in fermented foods because of their scarce representation in the genbank. Information of their metabolic profiles as well as their relationships with other microorganisms are important in order to combat their growth and toxin production in foods. It is also worthy to note that the presence of these filamentous fungal species in these fermented foods which are widely consumed in Nigeria and other African communities should be of concern considering the fact that some of the fungi may pose some health risks to consumers, particularly the children and immunocompromised adults. Furthermore, the high prevalence of these co- contaminating fungi coupled with their potential in synthesizing attendant mycotoxins within the same food matrix confirms that such foods may likely contain various mycotoxins. Efforts are currently being made by the authors to determine the fungal metabolites in these foods.

3.5 Conclusion

The fermented foods screened for fungal contamination indicated a wide range of species belonging to the genera Aspergillus, Penicillium, Fusarium, Saccharomyces, Mucor, Talaromyces, Rhizopus, and Cladosporium some of which could be potentially toxigenic. The fungal load of the foods also indicated that these food products could be potential sources of mycotoxin exposure among humans in Nigeria. However, this can only be ascertained if the similar diets are screened for various mycotoxins. The occurrence of several unreported

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In Format of LWT-Food Science and Technology fungal species and new strains of fungi in the selected fermented foods was established. The result of this study is a pointer to the need for the development of sustainable strategies such as improved food fermentation processing technology and implementation of good agricultural practices that may decrease fungal contamination in order to effectively control the occurrence and proliferation of these potentially toxigenic fungi in foods that could be accompanied by mycotoxin contamination

Conflict of Interest

The authors have no conflict of interest to declare.

Acknowledgements

The authors wish to appreciate the Organisation for Women in Science in the Developing World (OWSD), Italy through the Swedish International Development Cooperation Agency (SIDA), Centre of Excellence for Food Security and Safety (COE), South Africa, University of Johannesburg, South Africa via the Global Excellence and Stature (GES) Scholarship and L’oreal UNESCO (For Women in Science) for funding this study.

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CHAPTER FOUR

AWARENESS AND PREVALENCE OF MYCOTOXIN CONTAMINATION IN SELECTED NIGERIAN FERMENTED FOODS

Ifeoluwa Adekoya 1,*, Patrick Njobeh 1,*, Adewale Obadina 1,2, Cynthia Chilaka 3, Sheila Okoth 4, Marthe De Boevre 3 and Sarah De Saeger 3

1 Department of Biotechnology and Food Technology, University of Johannesburg, Doornfontein 2028, South Africa 2 Department of Food Science and Technology, Federal University of Agriculture, Abeokuta 2240, Nigeria; [email protected] (A.O.) 3 Laboratory of Food Analysis, Department of Bioanalysis, Ghent University, Ghent B- 9000, Belgium; [email protected] (C.C.); [email protected] (M.D.B.); [email protected] (S.D.S.) 4 Department of Botany, School of Biological Sciences, University of Nairobi, Nairobi 00100, Kenya; [email protected] (S.O.) * Correspondence: [email protected] (I.A.); [email protected] (P.N.); Tel.: +27- 713399978 (I.A.) Abstract

Fermented food samples (n = 191) including maize gruel (ogi), sorghum gruel (ogi baba), melon seed (ogiri), locust bean (iru) and African oil bean seed (ugba) from South-west

Nigeria were quantified for 23 mycotoxins, including aflatoxin B1 (AFB1), fumonisin B1

(FB1), and sterigmatocystin (STE) using liquid chromatography-tandem mass spectrometry. The practices, perceived understanding and health risks related to fungal and mycotoxin contamination amongst fermented food sellers was also established. Data obtained revealed

that 82% of the samples had mycotoxins occurring singly or in combination. FB1 was present

in 83% of ogi baba samples, whereas 20% of ugba samples contained AFB1 (range: 3 to 36 µg/kg) and STE was present in 29% of the ogi samples. In terms of multi-mycotoxin

contamination, FB1 + FB2 + FB3 + STE + AFB1 + alternariol + HT-2 co-occurred within one sample. The awareness study revealed that 98% of respondents were unaware of mycotoxin contamination, and their education level slightly correlated with their level of awareness (p < 0.01, r = 0.308). The extent of contamination of commodities by mycotoxins coupled with

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Keywords: Fermented foods, mycotoxins, awareness, food safety, and LC–MS/MS

4.1 Introduction

Processing of food relies on a series of preservative technologies developed to enhance quality, safety and acceptability, one of which is fermentation. Fermentation is the oxidation of carbohydrates to produce a wide range of products principally alcohol, organic acids and carbon dioxide through microbial activities [1]. Fermentation being a low-cost technology improves the digestibility and functionality of foods and facilitates food detoxification [2]. So far, most microorganisms involved in the fermentation of foods (cereals, legumes, oil seeds, etc.) belong mainly to the Lactobacillus, Leuconostoc, Lactococcus, Pediococcus, Bacillus and Saccharomyces genera. Iru is a condiment that is produced via the fermentation of African locust bean (Parkia biglobosa) by B. substilis, B. licheniformis and B. pumilis [3], whereas ogiri is from melon (Colocynthis citrullus) seeds with Bacillus, Escherichia and Pediococcus spp. as the fermenting organisms [3]. The solid-state alkaline fermented proteinous product of the African oil bean seed (Pentaclethra macrophylla) is known as ugba [4], while ogi is a product of lactic acid fermentation of maize or sorghum and principally consumed as weaning food. Ogiri and ugba like iru are principal condiments used to flavour stews and soups [5]. Ugba is also consumed as snack and used in the preparation of porridge.

These fermented products amongst variants such as injera, banku, amasi, fufu, garri, kenkey, uji, and mawe are indigenous to Africa. In Africa, they are typically manufactured in homes under spontaneous conditions with little or no process control [2]. Their production is also dominated by informal processing sectors (cottage and rural small-scale processors) that make use of different traditional processing methods thereby, bringing about variation in substrates used, processing conditions (time, temperature, moisture), packaging materials, handling, and storage practices [6]. These factors determine the quality and safety of the final products. However, irrespective of the processing methods employed, it is expedient for foods marked for sale to be of good quality and free from pathogenic and spoilage microorganisms such as fungi and associated toxins.

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A number of strains belonging mainly to the Aspergillus, Fusarium and Penicillium genera that attack various food commodities are toxigenic, producing various types of mycotoxins. About 25% of the global food output is contaminated with mycotoxins, causing significant economic losses [7]. Moreover, they constitute a serious health hazard, as they are known to be carcinogenic, nephrotoxic and immunotoxic. Mycotoxins of significance in sub-Saharan Africa (SSA) in terms of health and economy are fumonisins (FBs), aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEN), and trichothecenes (TCs) [8]. They have been found in different foods mainly in cereals such as maize and oil seeds such as melon that are substrates used for the production of fermented foods. Therefore, the presence of mycotoxins in fermented foods (iru, ogi, ogi baba, ugba and ogiri) cannot be undermined, though fermentation play a role in the degradation or detoxification of mycotoxins in foods [9–11]. Furthermore, there are no in-depth studies that report multiple mycotoxin contamination of fermented foods reported in this work.

On the other hand, a model proposed for the management of mycotoxins in SSA, identified awareness creation and enlightenment of people on mycotoxins as a principal strategy that can contribute to limit mycotoxin contamination of foods [12]. However, in recent years, reports of the prevalence of multiple mycotoxins in foods consumed in SSA [8,9,11,13-17] suggested that only minimal efforts were deployed on mycotoxin management particularly amongst food processors and sellers. Though, their main goal is to generate income, chances are high that adequate understanding of health implications of mycotoxin contamination through practical learning will prompt behavioural changes and the enactment of necessary mitigation actions. Strategies such as sorting of mouldy grains, utilization of adequate packaging materials, proper drying, implementation of appropriate storage methods and facilities can reduce mycotoxin contamination to a large extent. It is therefore crucial to investigate the practices, understanding and perceived health risks of fungal and mycotoxin contamination amongst stakeholders along the food value chain including fermented food sellers in order to ascertain their level of awareness. It is also imperative to establish the magnitude of multiple mycotoxin contamination of fermented foods offered for sale. The objective of this study is therefore to determine the level of awareness of fungal and mycotoxin contamination of fermented food sellers in South-west Nigeria and to assess the level of mycotoxin contamination of the products (iru, ogiri, ogi, ogi baba and ugba) they offered for sale.

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4.2 Results and Discussion

4.2.1 Perception Studies

An appraisal study was carried out in South-west, Nigeria on fermented food sellers perceived attitudes, practices and knowledge of fungal colonization of foodstuffs, being an antecedent of mycotoxins contamination. There was a wide knowledge gap amongst the population studied (n = 86), as 98% could not link fungi to mycotoxin contamination and perceived associated health risks. However, these findings corroborate previous studies [14,18,19]. According to Siegrist and Cvetkovich [20], a significant number of people in both developed and developing nations are not well informed of contaminants in foods. Majority (93%) of participants were females (n = 80) as shown in Table 4.1, which highlights the role of women in food production and processing in Africa.

Amongst the respondents, 57% store their finished products in polyethylene bags and 20% in leaves for an average of seven days. Fermented foods stored in leaves are more predisposed to fungal and mycotoxin contamination because of the indigenous microflora of the leaves and the deployment of little or no effort to clean or sterilize the leaves before usage. In the study of Adegunloye et al. [21], they reported that Thaumatococcus daniellii and Musa paradisiaca leaves which are usually used for wrapping fermented foods have high fungal load and prevalence of toxigenic fungi such as A. niger, A.flavus and P. expansum. Although, fermented foods are perceived to be safe, their mode of storage could predispose them to fungal and mycotoxin contamination if the storage methods are not complemented by other means of preservation.

The sellers (95%) obtained foods from different sources (markets or processors) as retailers while a few process their products themselves, the products were also stored over varying length of time (up to seven days for finished products and up to three months for raw materials). Storage and marketing practices employed amongst the sellers also have the tendency of facilitating variations in the degree of mycotoxin contamination [22]. Knowledge of environmental factors such as humidity, temperature, insect infestation, pre- and post- harvest practices that affect fungal growth and mycotoxin production in foods are particularly important when developing and implementing strategies for the control of fungi and mycotoxins along the food chain [23].

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It was evident in the study that the majority of study participants (92%) could attribute these factors to the persistence of fungi in foods and 22% of respondents reported that they frequently experience fungal contamination. Fungi can thrive on varieties of foods but some foods are better substrates for their growth than others. Ugba for example favours the growth of fungi based on its alkaline pH than ogi which has an acidic pH [24]. We noted that ugba and ogiri were more susceptible to fungal contamination amongst the foods offered for sale. Ogiri upon fungal invasion had a characteristic black colour while ugba overgrown with moulds were posited to be more suitable as an ingredient for porridge than its principal use as condiment. Particularly worthy of note was the willingness expressed by 97% of the respondents to attend training on mycotoxin mitigation. Public awareness trainings drives attitudinal transformation if target groups have confidence in the lessons received and apprehend the problem well enough to be persuaded to revise old practices and habits [25].

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Table 4.1 Descriptive statistics and knowledge of fungal and mycotoxin contamination of fermented food sellers Incidence Incidence Incidence Parameters Parameters Parameters (%) (%) (%) Sociodemographic Variables Gender Education level Age Male 6 (7) None 9 (11) <30 years 6 (7) Female 80 (93) Primary 52 (61) 31–50 years 74 (86) Secondary 23 (27) >50 years 6 (7) Tertiary 2 (2) Fermented Food Characteristics Mode of consumption Food type Food source Direct consumption 28 (33) Ogi 28 (33) Home processed 4 (5) Food Ingredient 46 (54) Iru 21 (24) Market 32 (37) Both 12 (14) Ogiri 19 (22) Processors 50 (58) Ugba 18 (21) Storage Variables Storage method of raw materials Storage duration of raw materials Average shelf life of raw material Bags 13 (15) 1–3 months 13 (15) 1–4 weeks 1 (1) Containers 5 (6) >3 months 5 (6) >4 weeks 17 (20) Not applicable 68 (79) Not applicable 68 (79) Not applicable 68 (79)

Storage method of finished Average shelf life of finished Storage duration of finished product product product Polyethylene bags 49 (57) 1–7 days 86 (100) 1–3 days 14 (16) Containers 14 (16) >7 days - 3–7 days 42 (49) Paper 3 (4) >7 days 30 (35) Leaves 17 (20) Wooden Boxes 3 (4)

Knowledge of Fungi and Mycotoxins Knowledge of fungi Frequency of contamination Identification of fungal contamination in food Yes 63 (73) Rarely 36 (42) No 16 (19) Yes 59 (68) Frequently 19 (22) Not sure 7 (8) No 27(32) Not applicable 31 (36)

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Incidence Incidence Incidence Parameters Parameters Parameters (%) (%) (%)

Perception of reasons of fungi Knowledge of health risks associated with Knowledge of production of occurrence fungal contamination toxins by fungi Storage 21 (24) Yes 7 (8) Yes 3 (4) Bad raw materials 19 (22) No 79 (92) No 83 (96) Insect infestation 18 (21) All of the Above 21 (24) Not sure 7 (8)

Knowledge of mycotoxin Willingness to attend training on mycotoxin contamination mitigation Yes 2 (2) Yes 83 (97) No 84 (98) No 3 (3)

Number of respondents: 86

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Few respondents had no formal education (11%), while most had primary education (61%). Table 4.2 presents important information on the association between the level of education and knowledge on fungi and mycotoxins amongst the respondents. Their knowledge of fungi correlated positively (p < 0.01, r = 0.355) with their ability to identify foodstuffs contaminated with fungi which could be due to their experience of fungal contamination as shown in Table 4.2. Moreover, findings also revealed that the level of education had a significant but slightly positive influence (p < 0.01, r = 0.296) on their apprehension of fungi and mycotoxin contamination (p < 0.01, r = 0.308). Dosman et al. [26] highlighted that individuals with higher education levels are likely to be more knowledgeable and aware of food contaminants than individuals with lesser education because they have more access and tend to seek for more information on food safety and related issues [27].

Also in Nigeria, studies have posited that educational attainment is crucial to public awareness of food safety [28]. For individuals with little or no formal education as observed in this study, more strength lies with this as a conventional skill since mycotoxin related issues are not precisely covered in the curricular of any primary and secondary schools in Nigeria as well as other African countries. Even though our findings revealed that education level correlated positively with awareness, knowledge and recognized benefits, it is expedient to make the problem known to all categories of individuals.

Mycotoxins are in the forefront of chronic food toxicants [19] usually occurring below levels that elicit acute health effects, but such levels could provoke long-term health implications amongst humans and animals [29]. It may therefore be difficult to associate several health complications to mycotoxin exposure, which strongly supports the poor perception of mycotoxin contamination. In addition to this, because mycotoxin can be present in foods after the dissipation of fungi, it was therefore unexpected of respondents to physically discern a food that is contaminated with mycotoxins in addition to fact they do not necessarily alter the taste or flavour of foods. These variations need to be considered and communicated particularly during the implementation of trainings aimed at fungal and mycotoxin mitigation.

The findings of our research have profound implications for strategies directed towards mycotoxin management in fermented foods and other food categories in Nigeria and SSA, based on studies [12,25,28] that have established that awareness and education are critical elements in reducing the menace of mycotoxins contamination in developing countries. The

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Table 4.2 Kendall’s tau-b correlation between education and awareness level of fungi and mycotoxins contamination by respondents Correlations Level of Do You Know Can You Does Fungi Contamination of Do You Know Have You Heard of Education What Fungi Is Identify Food Foodstuffs Cause Health Fungi Produce Mycotoxin with Fungi Problems Toxins Contamination Level of Education 1.000 0.296** −0.172 0.014 0.048 0.308** Do you know what fungi is 0.296** 1.000 0.355** 0.249* 0.075 −0.139 Can you identify food with −0.172 0.355** 1.000 0.069 0.190 0.100 fungi Does fungi contamination of 0.014 0.249 * 0.069 1.000 0.122 0.221* foodstuffs cause health problems Do you know fungi produce 0.048 0.075 0.190 0.122 1.000 0.109 toxins Have you heard of 0.308** −0.139 0.100 0.221* 0.109 1.000 mycotoxin contamination

** Correlation is significant at p < 0.01 (2-tailed); * correlation is significant at p < 0.05 level (2-tailed); n: number of respondents (86); values along each column are correlation coefficients.

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4.2.2 Method Performance Characteristics

Table 4.3 shows the results of method performance characteristics including the LOD, LOQ and AR of different fermented food matrices. The calibration curves for the analytes were linear and the AR of all the analysed mycotoxins varied between 89 and 109%, and aligned within the range set by the EC [30]. The LODs for 3-ADON 15-ADON, AFB1, AFB2, AFG1,

AFG2, DAS, and ROQ C were <6 μg/kg while the LOQs of STE, and ZEN were ≤20 μg/kg in all the tested fermented foods.

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Table 4.3 Method performance parameters of fermented food matrices Mycotoxin Calibration Fermented Melon Fermented Locust Fermented African Oil Fermented Maize Fermented Sorghum Range µg/kg (ogiri) Bean (iru) Bean (ugba) Gruel (ogi) Gruel (ogi baba) LOD LOQ AR LOD LOQ AR LOD LOQ AR LOD LOQ AR LOD LOQ AR Deoxynivalenol 200–800 11 22 100 4.9 9.8 99 15 30 101 7 14 97 12 24 101 Nivalenol 100–400 48 96 100 11 22 100 21 42 103 35 70 101 87 175 99 Neosolaniol 50–200 20 40 95 16 32 99 24 48 96 2.2 4.4 103 3.0 6.0 100 Fusarenon-X 100–400 39 78 97 8.1 16 101 25 50 96 21 42 100 45 90 100 3-Acetyldeoxynivalenol 25–100 2.3 4.6 96 2.0 4.0 102 1.2 2.4 101 5.0 10 105 12 24 97 15-Acetyldeoxynivalenol 12.5–50 1.7 3.5 94 3.9 7.9 101 1.8 3.7 96 10 20 95 7.0 14 99 Aflatoxin B1 10–40 2.0 4.0 96 1.2 3.3 96 1.5 3.0 100 3.8 7.5 100 5.0 10 100 Aflatoxin B2 10–40 2.3 4.6 96 1.8 3.3 94 1.4 2.8 96 1.8 3.5 99 2.5 5.0 102 Aflatoxin G1 10–40 3.9 7.8 99 1.7 3.3 95 1.9 3.9 98 1.8 3.5 98 2.5 5.0 101 Aflatoxin G2 10–40 3.7 7.4 96 1.2 2.3 91 2.2 4.4 94 3.8 7.5 100 5.0 10 99 Diacetoxyscirpenol 2.5–10 0.9 1.8 97 0.7 1.4 97 1.0 2.0 89 0.3 0.6 99 0.5 1.0 94 Alternariol 50–200 6.5 13 98 9.7 20 100 5.9 11 98 40 80 92 40 80 99 Alternariol Methyl Ether 100–400 54 107 96 5.0 10 96 4.6 9.2 98 5.0 10 109 6.3 12 96 HT-2 Toxin 50–200 6.5 13 98 7.4 14 98 15 30 94 6.5 13 85 6.5 13 95 T-2 Toxin 50–200 12 24 98 14 28 94 13 26 100 3.6 7.2 87 8.0 16 94 Fumonisin B1 200–800 24 48 97 22 44 100 38 76 97 8.2 16 87 10 20 98 Fumonisin B2 200–800 11 22 99 9.4 18 99 43 87 95 12 23 89 11 22 100 Fumonisin B3 25–100 13 26 97 21 42 97 33 66 94 14 28 89 14 28 96 Ochratoxin A 25–100 11 22 89 1.2 2.4 93 3.6 7.2 90 1.5 3.0 99 2.5 5.0 95 Sterigmatocystin 25–100 5.5 11 100 1.7 3.3 97 1.9 3.8 95 1.3 2.5 100 2.5 5.0 101 Roquefortine C 5–20 4.9 9.7 101 1.2 2.3 99 1.0 2.0 99 4.0 8.0 97 6.0 12 98 Zearalenone 50–200 9.8 20 96 2.9 5.9 92 4.4 8.8 104 3.3 6.5 102 3.8 7.6 93 Enniatin B 40–160 26 52 93 6.4 13 94 5.6 11 99 6.3 12 82 7.9 16 91

LOD: limit of detection (µg/kg); LOQ: limit of quantification (µg/kg); AR: Apparent recovery (%).

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4.2.3 Mycotoxin Contamination

In this study, the multi-mycotoxin profile of fermented foods from South-west Nigeria including ogiri, ugba and iru, intended for use as condiments, as well as ogi and ogi baba, popularly consumed as breakfast cereals, was delineated (Table 4.4). Generally, 56% of the

34 fermented food samples positive for AFB1 had levels above the maximum regulatory limit of 2 µg/kg in foods according to the EC [31] and ogiri samples had a higher incidence of

AFB1 (48%) (range: 3–4 µg/kg). A co-occurrence of AFB2 and AFG2 was also established in ogiri samples. We also noted the prevalence of other important analogues of AFs (AFB1 and

AFG1), singly or in combination in some of the fermented samples, which was due to the presence of AF-producing [24]. It has been established that chronic exposure to AFs from fermented foods affects close to 4.5 billion persons in the developing countries [32].

The presence of Aspergillus spp. and AFs in some raw materials used in production of the tested fermented foods has been previously reported. Ezekiel et al. [33] recovered AFB1 and total AF from melon seeds used in the manufacture of ogiri at a mean level of 37.5 µg/kg and

142 µg/kg, respectively. Makun et al. [15] also reported the presence of A. flavus and AFB1 in 54% sorghum used for ogi baba production. The biosynthetic pathway of AFB1 has also been studied [9] with averantin, averufin, norsolorinic acid, versicolorin A and STE established as precursors/intermediate compounds. STE and AFB1 are synthesized by the same Aspergillus spp. and the presence of AFs in the fermented foods can be attributed to the presence of such a precursor as STE. Sterigmatocystin was detected in ugba (range: 22–27 µg/kg) and ogi (range: 4–7 µg/kg) but absent in iru.

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Table 4.4 Multi-mycotoxin profile of fermented foods from South-west Nigeria Mycotoxin Fermented Melon Fermented Locust Bean Fermented African Oil Bean Fermented Maize Gruel Fermented Sorghum Gruel (n = 31) (ogiri) (n = 60) (iru) (n = 30) (ugba) (n = 35) (ogi) (n = 35) (ogi baba) % +ve Range Mean % +ve Range Mean % +ve Range Mean % +ve Range Mean % +ve Range Mean Deoxynivalenol 3 (10)

Aflatoxin B1 15 (48) 3–4

Aflatoxin B2 5 (16)

Aflatoxin G1 0 0 0 2 (3) 8–8 8 0 0 0 2 (6) 0 0 1 (3) 0 16

Aflatoxin G2 2 (7)

Fumonisin B1 0 0 0 4 (7) 61–167 113 0 0 0 25 (71) 68–2,492 384 14 (40)

Fumonisin B2 0 0 0 4 (7) 32–42 38 0 0 0 23 (66) 94–659 250 9 (25)

Fumonisin B3 0 0 0 4 (7) 76–89 84 0 0 0 18 (51) 42–404 112 26 (74)

Σ Fumonisin B1, B2 0 0 0 8 (13) 32–167 76 0 0 0 25 (71) 68–3,151 645 18 (52)

Σ Fumonisin B1, B2, B3 0 0 0 12 (20) 32–167 78 0 0 0 27 (77) 42–3,555 672 29 (83)

Only concentrations higher than LOQ are recorded and samples containing concentrations higher than LOD were considered positive/contaminated; % +ve: percentage of positive samples

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The vulnerability of food products such as maize to OTA contamination worldwide is documented [16]. The data presented in this study showed the presence of OTA in ogi baba, iru and ugba at mean levels of 6, 6, and 9 µg/kg, respectively. OTA was not found in ogi, which is contrary to the report of Oyelami et al. [17]. All the ugba and ogiri sample positive for OTA, contained levels that were above the 5 µg/kg recommended for foodstuffs [31]. OTA is a potent secondary metabolite synthesized in foods by more than ten fungal species with A. ochraceus and P. verrucosum as principal producers in the tropics and in the temperate regions, respectively [34]. The same toxin is also associated with kidney and liver impairment, Balkan endemic nephropathy, oxidative DNA damage and has been classified by the International Agency for Research on Cancer (IARC) [35] as a Group 2B carcinogen. ROQ C is regarded as one of the most important fungal contaminants in fermented foods and beverages [36], it was detected in this study in iru at a low incidence rate (range: 10–14 µg/kg). It should be noted however that ROQ C is a potent neurotoxin at high concentrations above 1500 µg/kg [31]. Like AF, STE, and OTA, the occurrence of ROQ C in these analysed foods could be due to the participation of various fungi during fermentation, which is principally by chanced inoculation. Odunfa and Adeyele [37] identified Aspergillus and Penicillium fungi during the fermentation of ogi baba.

The TCs are a large family of over 150 chemically related toxins produced principally by the Fusarium genera [38]. Based on their core structures, they are classified into four types: A, B, C and D. Type A includes mainly T-2 toxin and HT-2 toxin together with NEO and DAS in this list. Deoxynivalenol, NIV, 3-ADON, 15-ADON and FUS-X are the Type B TCs mycotoxins. In relation to toxicity according to Schollenberger et al. [39], type A TCs are more toxic when compared to type B TCs. In terms of geographical locations Type A TCs are not commonly reported in Africa, but in our study, the type A TC- HT-2 was the most frequently occurring, and it levels in ogi (range: 20–21 µg/kg) exceeded the recommended level of HT-2 + T-2 (15 µg/kg) for infant foods by EC [40]. Concerning iru, 9 samples were positive for HT-2 within the range of 17 to 51 μg/kg (mean: 33 μg/kg) (Table 4.4) and T-2 was also detected in iru. Generally, T-2 and HT-2 toxins are of great concern based on their capacity to induce oxidative stress, inhibit DNA, RNA, and protein synthesis as well as mitochondrial performance [40]. Furthermore, the contamination of both TCs mycotoxins can co-occur together with DAS, because of similarity in biosynthesis at the side branch of the pathway of T-2 [41], which was only observed in ogiri. The level of DON in all the positive

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In format of Toxins samples (n = 18) reached a maximum of 118 µg/kg, which was far less than the maximum limit for DON in processed cereals (750 µg/kg). The frequency and contamination level of the acetylated derivative of DON (3-ADON) was low, whereas no analysed samples contained 15-ADON. Chilaka et al. [11] found much higher contamination levels of DON and 3-ADON than that reported in our study.

The FBs were the dominant mycotoxins in ogi and ogi baba, with most of the ogi samples having FB1 and FB2 higher than maximum set limits of 200 µg/kg for FB1 + FB2 in maize- based infant foods[31]. This suggests a high exposure of infants to FB1 and FB2. The high incidence of this toxin in ogi, a maize-based product substantiates the vulnerability of the maize crops to FB producing fungi such as F. proliferatum and F. verticillioides. Additionally, the low levels of FBs observed in ogi baba: a fermented processed sorghum product also correlates well with the findings that sorghum is less susceptible to fungal infestation when compared to maize [11]. While, data on the occurrence of mycotoxins in ogi baba is scarce, studies from Nigeria had reported the presence of FBs in sorghum grains

[11,15]. Amongst the alkaline fermented food studied, only iru (7%) was positive for FB1,

FB2 and FB3 with mean values of 113, 38 and 84 µg/kg, respectively. FB1 has been linked to liver and oesophageal cancer and was classified as a group 2B carcinogen [35]. Recently in Tanzania, Shirima et al. [42] studied child growth during early childhood in relation to AF and FBs exposure and established that exposure to FBs alone or together with AF is a factor responsible for growth impairment in children.

In 90% of the samples positive for ZEN (n = 20), the levels recovered were less than 50 µg/kg, which is insignificant when compared with the maximum limit of 50–1,000 µg/kg in foods in 16 countries where ZEN is being regulated [43]. According to Kpodo et al. [9], ZEN is largely produced by some Fusarium spp. in cool dry climates between 10 °C and 15 °C, whereas temperatures from 27 oC to 40 oC commonly persist yearly in Nigeria. The occurrence of low levels of ZEN in the fermented foods may therefore be due to the persistence of such climatic conditions that are unfavourable for the production of the toxin in Nigeria. Amongst all these fermented food types analysed in this study, 18% (n = 35) was devoid of the tested mycotoxins with ugba recording the least contamination in terms of the number of mycotoxins (10) detected. Ugba is encapsulated in extremely hard seed coats, which make it less prone to fungi and mycotoxin contamination than others with less

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In format of Toxins formidable coats. This might account for the low levels in the number of mycotoxins found in ugba in comparison with other samples. The co-occurrence of several mycotoxins in the fermented foods was also noted (Figure 4.1).

Figure 4.1 Percentage co-occurrence of mycotoxins in fermented foods from Southwest, Nigeria

Out of the 191 samples analysed, 82% (n = 156) had mycotoxins occurring singly or in combination. For the 23 different mycotoxins analysed in each food matrix, 3 different toxins co-occurred in 16% ogiri, 12% iru, 26% ogi and 26% ogi samples. This phenomenon has been demonstrated for several mycotoxins in foods consumed in Africa [11,14,15]. DON usually co-occurs with its acetylated forms but this was not the case in this study. The co- occurrence of DON and ZEN was also established, but ZEN occurred mostly at lower concentrations than DON [38]. This relationship was observed in some ogiri samples that were positive for both mycotoxins. Also, the co-occurrence of up to seven metabolites (FB1 +

FB2 + FB3 + STE + AFB1 + AOH + HT-2) in ogi could be due to its susceptibility to fungal contamination when compared to other samples. The multiple mycotoxins observed within the same sample could exacerbate the health risks amongst humans since they can elicit some synergistic and additive effects especially at levels above those accepted by various regulatory bodies.

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The varying contamination levels, and co-occurrence of mycotoxins in the fermented foods analysed may be due to carry-over of the microbes from the raw materials used in processing them, or from fungal contamination of the processed foods. The ignorance of the sellers as well as the climatic conditions that prevailed in the study sites seemed to play a role in the contamination of the fermented foods. The practice of mixing mouldy food products with high-quality products to maximize profit was also noticed because of non-enforcement of regulatory limits on locally grown crops or locally produced products sold in Nigeria. In addition most of the fermented foods were manufactured in homes under unhygienic conditions. Improper storage practices, which provide optimal conditions for mould development and subsequent mycotoxin accumulation, may also exacerbate the situation. Bearing in mind that these fermented products are not the only dietary sources of mycotoxin exposure amongst humans, the overall daily exposure to these mycotoxins can be high.

4.3 Conclusion

This study gave an insight into the safety of fermented foods produced in Nigeria and equally established the awareness of the sellers towards fungal and mycotoxin contamination and associated health risks. We observed that there existed a wide knowledge gap amongst sellers on this aspect of food safety. Majority of the fermented foods (156/191) had mycotoxins occurring singly or in combination though relatively at low incidence and contamination levels. Ogi was the most contaminated sample based on the total number of positive samples (94%, n = 35) which makes the risk of mycotoxin exposure higher amongst its consumers. Some of the samples exceeded the maximum limit for FB, AF, OTA and ZEN in foods as regulated by the EC. In broad terms, the incidence of type A TCs was slightly higher than type B TCs. All the samples were negative for 15-ADON. AOH, AME, STE, ENN B and ROQ C were also present at low levels in few samples. Ogi baba and ogi had the highest number of co-occurring fungal metabolites. To the best of our knowledge, this is the first study that assessed the presence of mycotoxins in ugba and ogi baba. As well as the first to report a wide range of previously unreported mycotoxins in iru, ogiri and ogi consumed in Nigeria.

Considering the high level of consumption of these fermented foods in Nigeria, strategies towards mycotoxin mitigation should be a priority. Awareness needs to capture good agricultural practices aimed at reducing fungal infestation of the raw materials during growth

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In format of Toxins and storage, while executing training on ways of selecting high-quality raw materials. Hands- on learning activities needs to be integrated with awareness campaigns to create more opportunities for the target groups to adopt the recommendations provided. Awareness can also be created from a gender focal point where women being at the forefront of food production are involved in the formulation of education programs on mycotoxin management. Proper understandings of the economic and health effects of mycotoxins are important drivers as individuals are most likely to take steps towards mycotoxin reduction if effects are known. Proposed strategies should therefore emphasize benefits. To pave the way forward, there is need for enforcement of risk-based food laws, encouragement of dietary diversity, sustained use of intervention technologies and more surveillance programs that could be implemented to provide toxicological and exposure data.

4.4 Materials and Methods

4.4.1 Sampling

The cluster sampling method was used to obtain fermented foods namely; maize gruel (ogi), sorghum gruel (ogi baba), locust bean (iru), African oil bean seed (ugba) and melon seed (ogiri) from various fermented food sellers in Southwest Nigeria between February 2015 and July 2016. Composite samples of each fermented food: ogi (n = 35), ogi baba (n = 35), iru (n = 60), ugba (n = 30) and ogiri (n = 31) were taken to obtain a total of 191 composite samples. Each composite sample of about 270 g was an aggregate of sub-samples of 90 g obtained from three different fermented food sellers. All samples were collected in sterile containers and immediately transported to the laboratory. The composite samples were properly mixed and trisected twice to obtain a representative sample of 30 g, after which they were transported to the Laboratory of Food Analysis, Ghent University, Belgium and stored at −18 °C prior to mycotoxin analysis.

4.4.2 Awareness Studies

A descriptive cross-sectional study was carried out amongst some of the fermented food sellers within the sampling area in February 2015 using a questionnaire that consisted of closed- and open-ended questions. The questionnaire (Appendix 4.1) was designed to capture the demographics, practices, understanding and perceived health risk of food contamination by fungi and mycotoxins amongst fermented food sellers. The sellers were informed about

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4.4.3 Mycotoxin Analysis

4.4.3.1 Materials and Chemicals

Methanol, glacial acetic acid, and acetonitrile (LC-MS/MS grade) were purchased from Biosolve B.V. (Valkenswaard, The Netherlands). Ammonium acetate and acetic acid (analytical grade) were supplied by Merck (Darmstadt, Germany). HPLC grade methanol and n-hexane in addition to Whatman® (Maidstone, UK) glass microfiber filters were obtained from VWR International (Zaventem, Belgium). Ultrafree-MC centrifugal filter devices (0.22 μm) were obtained from Millipore (Brussels, Belgium). MultiSep®226 AflaZon+ immunoaffinity columns and C18 solid phase extraction (SPE) columns were obtained from Romer Labs (Gernsheim, Germany) and Grace Discovery Sciences (Lokeren, Belgium), respectively. Water was purified in a Milli-Q Gradient apparatus (Millipore, Brussels, Belgium). All other reagents and chemicals were of analytical grade.

4.4.3.2 Mycotoxin Standards

Mycotoxin standards consisted of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1

(AFG1), aflatoxin G2 (AFG2), fumonisin B1 (FB1), fumonisin B2 (FB2), deepoxy- deoxynivalenol (DOM), 15-acetyl-deoxynivanelol (15-ADON), neosolaniol (NEO), OTA, alternariol (AOH), alternariol monomethyl ether (AME), zearalenone (ZEN), nivalenol (NIV), deoxynivanelol (DON), 3-acetyl-deoxynivanelol (3-ADON), sterigmatocystin (STE), roquefortine C (ROQ C), enniatin B (ENN B), fusarenon-X (FUS-X), HT-2 toxin (HT-2) and zearalanone (ZAN) which were obtained from Sigma-Aldrich (Bornem, Belgium). Fumonisin

B3 (FB3) was procured at Promec Unit (Tynberg, South Africa), while T-2 toxin (T-2) and diacetoxyscirpenol (DAS) were purchased from Biopure Referenzsubstanzen (Tulln, Austria).

4.4.3.3 Sample Preparation

All samples were dried in a hot air oven (UM200, Memmert, Schwabach, Germany) and milled to a particle size between 0.5 and 1 mm. Milled samples were accurately measured (5 ± 0.005 g), and reinforced with internal standards (ZAN-10 µg/mL and DOM-50 µg/mL), 146

In format of Toxins and allowed to equilibrate for 15 mins in the dark. Extraction of mycotoxins in the sample was done using 20 mL of acetonitrile/acetic acid/water (79/1/20, v/v/v). The mixture was vortexed for 10 secs, placed on an overhead shaker (Agitelec, Paris, France) for 1 hr and centrifuged for 15 mins at 3,500 rpm. All the supernatant was transferred into a pre- conditioned SPE C18 column and defatted (2×) using 10 mL n-hexane. Two cleanup procedures were applied to recover the 23 mycotoxins. First, 27.5 mL of acetonitrile/acetic acid (99/1, v/v) was added to 12.5 mL of the defatted extract, and passed through a MultiSep 226 AflaZon+ immunoaffinity column. Second, using a glass micro filter (General Electric, Coventry, UK), 2 mL of defatted extract was filtered and combined with the MultiSep 226 eluate and evaporated to dryness. The residue was reconstituted in 150 µL of injection solvent consisting of methanol/water/acetic acid (57.2/41.8/1, v/v/v) and 5 mM ammonium acetate (0.385 g/L). The reconstituted extract was placed in an Ultrafree® PVDF centrifuge filter (Merck, Darmstadt, Germany), and centrifuged at 10,000 rpm for 10 mins. The eluent was transferred into an LC-MS/MS injection vial prior to analysis.

4.4.3.4 Liquid Chromatography-Tandem Mass Spectrometry

A Waters Acquity UPLC apparatus paired to a Quattro Premier XE Tandem Mass Spectrometer (Waters, Milford, MA, USA) was utilized for the identification and quantification of the analytes. Data acquisition and processing utilities included the use of the MassLynx™ (V. 4.1) and QuanLynx® (V. 4.1) software (Micromass, Manchester, UK). The column used to separate the analytes of interest was a Symmetry C18 column (150 mm × 2.1 mm i.d. 5 μm particle size) with a guard column (10 mm × 2.1 mm i.d.) of the same material (Waters, Zellik, Belgium). The chromatographic conditions set were similar to those of Ediage et al. [44]. Mobile phase A contained acetic acid/methanol/water (1/5/94, v/v/v) and 5 mM ammonium acetate (0.385g/L), and mobile phase B contained acetic acid/water/methanol (1/2/97, v/v/v) and 5 mM ammonium acetate (0.385 g/L). With a sample injection volume set at 10 μL, the total analytical run time was 28 mins with a pressure that varied between 0 and 5,000 psi. The mass spectrometer was operated using selected reaction monitoring (SRM) channels in positive electrospray ionization (ESI+) mode. Further details on the mycotoxin transitions are reported by De Boevre et al. [45] and Monbaliu et al. [46]. For the identification of the targeted mycotoxins, the criteria of the Commission Regulation 657/2002/EC [47] were followed.

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4.3.5. Method Validation

The Commission Regulation 401/2006/EC [30] was used for the validation studies. Variables including limit of quantification (LOQ), limit of detection (LOD) and apparent recovery (AR) were accessed by spiking mycotoxin-free samples (blank) with the different mycotoxins in triplicates. ZAN and DOM were used as internal standards and matrix matched calibration curves (MMC) were constructed from the ratio of the peak area of each analyte to the internal standard. The linearity of each analyte was estimated graphically using a scatter plot, and the linear regression model evaluated using a lack-of-fit test, while apparent recoveries were established by dividing the calculated concentration by the theoretical concentration.

4.4.4 Data Analysis

A descriptive statistics (mean, range, frequencies, and percentages) of the data generated in this study was performed using Microsoft Office Excel 2010 (Redmond, WA, USA). In addition, the degree of awareness of fungal and mycotoxin contamination amongst the fermented food sellers was correlated with their level of education using Kendall’s tau-b test on SPSS version 23.0 (IBM Corporation, New York, USA).

Acknowledgments: The authors wish to sincerely thank the following: Organisation for Women in Science in the Developing World (OWSD), Italy; Centre of Excellence (CoE) in Food Security co-hosted by the University of Pretoria and the University of the Western Cape, South Africa; African Women in Agricultural Research and Development (AWARD), Kenya; and the MYTOX-SOUTH, hosted in the Laboratory of Food Analysis, Ghent University, Belgium for their financial and technical contributions to this study.

Author Contributions: I.A. and A.O. conceived and designed the experiment; I.A. performed the experiment, analysed the data and wrote the manuscript; C.C. assisted with the experiment and edited the manuscript; P.N., A.O., S.O., M.D.B. and S.D.S. supervised the researched, edited and approved the final manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

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34. Kumar, V.; Basu, M.S.; Rajendran, T.P. Mycotoxin research and mycoflora in some commercially important agricultural commodities. Crop Prot. 2008, 27, 891–905, doi:10.1016/j.cropro.2007.12.011. 35. International Agency for Research on Cancer (IARC). Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Monogr. Eval. Carcinog. Risks Hum. 2002, 96, 1–390. 36. Finoli, C.; Vecchio, A.; Galli, A.; Dragoni, I. Roquefortine C occurrence in blue cheese. J. Food Prot. 2001, 64, 246–251. 37. Odunfa, S.A.; Adeyele, S. Microbiological changes during the traditional production of ogi baba, a West African fermented sorghum gruel. J. Cereal Sci. 1985, 3, 173–180, doi:10.1016/S0733-5210(85)80027-8. 38. Mbundi, L.; Gallar-Ayala, H.; Khan, M.R.; Barber, J.L.; Losada, S.; Busquets, R. Advances in the analysis of challenging food contaminants: Nanoparticles, bisphenols, mycotoxins, and brominated flame retardants. Adv. Mol. Toxicol. 2014, 8, 35–105, doi:10.1016/B978-0-444-63406-1.00002-7. 39. Schollenberger, M.; Müller, H.M.; Ernst, K.; Sondermann, S.; Liebscher, M.; Schlecker, C.; Wischer, G.; Drochner, W.; Hartung, K.; Piepho, H.P. Occurrence and distribution of 13 trichothecene toxins in naturally contaminated maize plants in Germany. Toxins (Basel) 2012, 4, 778–787, doi:10.3390/toxins4100778. 40. European Food Safety Authority (EFSA). Scientific Opinion on the risks for animal and public health related to the presence of T-2 and HT-2 toxin in food and feed. EFSA J. 2011, 9, 1–187. 41. Kimura, M.; Tokai, T.; Takahashi-Ando, N.; Ohsato, S.; Fujimura, M. Molecular and genetic studies of Fusarium trichothecene biosynthesis: Pathways, genes, and evolution. Biosci. Biotechnol. Biochem. 2007, 71, 2105–2123, doi:10.1271/bbb.70183. 42. Shirima, C.P.; Kimanya, M.E.; Routledge, M.N.; Srey, C.; Kinabo, J.L.; Humpf, H.U.; Wild, C.P.; Tu, Y.K.; Gong, Y.Y. A prospective study of growth and biomarkers of exposure to aflatoxin and fumonisin during early childhood in Tanzania. Environ. Health Perspect. 2015, 123, 173–178, doi:10.1289/ehp.1408097. 43. Food and Agriculture Organization of the United Nations. Mycotoxin regulations in 2003 and current developments. In Worldwide Regulations for Mycotoxins in Food and Feed; Food and Agriculture Organization of the United Nations: Rome, Italy, 2004; pp. 9–28.

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44. Ediage, E.N.; Di Mavungu, J.D.; Monbaliu, S.; Van Peteghem, C.; De Saeger, S. A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples. J. Agric. Food Chem. 2011, 59, 5173– 5180 45. De Boevre, M.; Di Mavungu, J.D.; Landschoot, S.; Audenaert, K.; Eeckhout, M.; Maene, P.; Haesaert, G.; De Saeger, S. Natural occurrence of mycotoxins and their masked forms in food and feed products. World Mycotoxin J. 2012, 5, 207–219. 46. Monbaliu, S.; Van Poucke, C.; Van Peteghem, C.; Van Poucke, K.; Heungens, K.; De Saeger, S. Development of a multi-mycotoxin liquid chromatography/tandem mass spectrometry method for sweet pepper analysis. Rapid Commun. Mass Spectrom. 2009, 23, 3–11, doi:10.1002/Rcm.3833. 47. European Commission (EC). Commission Decision 2002/657/EC implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off. J. Eur. Union 2002, L221, 8–36.

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CHAPTER FIVE

FUNGAL AND MYCOTOXIN CONTAMINATION OF FERMENTED FOODS FROM SELECTED SOUTH AFRICAN MARKETS

Adekoya Ifeoluwa*a, Obadina Adewaleab, Phoku Judithc, Marthe De Boevred, Sarah De Saegerd and Njobeh Patricka aDepartment of Biotechnology and Food Technology, University of Johannesburg, Johannesburg, South Africa bDepartment of Food Science and Technology, Federal University of Agriculture, Abeokuta, Nigeria cAgricultural Research Council-Onderstepoort Veterinary Research, Toxicology and Ethnoveterinary Medicine, Public Health and Zoonoses, Onderstepoort, South Africa dLaboratory of Food Analysis, Department of Bioanalysis, Ghent University, Ghent, Belgium *Corresponding authors: [email protected] and [email protected]

Abstract

Foods marked for both local and export markets should be of good quality and free of fungi and their toxins. In this study, five regularly consumed fermented foods: melon (ogiri), locust beans (iru), African oil bean (ugba), maize meal (mahewu) and maize gruel (ogi) purchased from South African markets were evaluated for fungal diversity using 16S rRNA gene sequencing and mycotoxin contamination using liquid chromatography tandem mass spectrometry. In addition, their pH, moisture content, Total Titratable Acidity (TTA) and water activity were accessed. The investigation revealed a mean pH range of 3.60 to 8.14 and a significant negative correlation between the pH and TTA (r = -0.560, p < 0.05). Ogiri had the highest mean fungal load (8.30 x 105 CFU/g) and 340 fungal isolates belonging to 17 genera were recovered from the foods with the dominant fungi genera being Aspergillus and Saccharomyces. In addition, potentially toxigenic species such as A. flavus, A. parasiticus and F. verticilliodes occurred in the fermented foods. A total of 23 mycotoxins were quantified including aflatoxin B1 (AFB1), fumonisins B1 (FB1) and deoxynivalenol (DON).

AFB1, FB1 and DON were present in 50% of ogiri (mean: 4 µg/kg), 37% of ogi (range: 42- 326 µg/kg) and 73% of mahewu (range: 18-32 µg/L) at relatively low contamination levels. Overall, 66% of the samples (n=176) had mycotoxins occurring singly or in combinations

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Keywords: Fermented foods, fungi, mycotoxins, food safety, water activity, and South Africa

Highlights

 Five fermented foods from South African markets were accessed for fungal and mycotoxin contamination and some chemical constituents.  Fungal isolates (n=340) were recovered and A. flavus had the highest incidence of occurrence amongst the mycotoxigenic species.

 Water activity ranged from 0.82 to 0.93 and deoxynivalenol and fumonisin B1 were prevalent in mahewu (73%) and ogi (37%).  Majority of the analysed samples (66%) had mycotoxins occurring singly or in combinations.  This is the first study on fungi and mycotoxins contamination of fermented foods from South African markets.

5.1 Introduction

In several developing countries including South Africa, fermented foods constitute a major part of people’s diet and have significantly contributed to the socio-economic wellbeing of individuals (Oguntoyinbo, 2014). The socio-economic role of fermented foods includes employment creation particularly for women and provision of affordable varieties of foods. With increasing population, the demand for fermented foods has increased both at home and in the diaspora. However, expatriate Nigerians are the main consumers of fermented foods such as maize gruel (ogi), African oil bean (ugba), locust beans (iru) and melon (ogiri), which are sold in South African markets after they are imported from Nigeria. An estimate of $1.1 million was accrued to agricultural imports from Nigeria to South Africa accounting for 12% import from the region (Daya & Steenkamp, 2005). Ogi is produced from maize through lactic acid fermentation, consumed mainly as breakfast and used as weaning foods for infants (Omemu, Oyewole & Bankole, 2007). Ugba and ogiri are derived from oil seeds (African oil bean and melon seeds, respectively) in addition to iru, they are products of alkaline fermentation and are highly proteinous condiments used in food preparations or as meat

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In Africa, mycotoxin contamination has been a hindrance to the safety of foods, international trade and has adversely affected human health causing both acute and chronic effects (Bhat, Rai, & Karim 2010). This is due to the contamination of crops by toxigenic fungal species such as A. flavus, A. niger, and F. verticilliodes during production and processing. Within Africa, fungal contamination is also heightened by the humid tropical climatic condition that creates a suitable environment for mycotoxin production (Bankole, Ogunsanwo, Osho, & Mabekoje, 2006). Some of the substrates of fermented foods particularly maize have been demonstrated to be contaminated with toxigenic fungi and a significant number of mycotoxins such as aflatoxin (AFs), fumonisins (FBs) and deoxynivalenol (DON) (Bankole, Adenusi, Lawal, & Adesanya, 2010; Chilaka, De Boevre, Atanda, & De Saeger, 2016). In the study of Phoku et al. (2012), F. verticilliodes was the prevalent Fusarium species isolated from maize and the contamination level of FB1 in maize samples ranged from 101–

53,863 µg/kg. Adedeji et al. (2017) also detected AFB1 at mean concentrations of 5.6 μg/kg in fermented melon seeds from Nigeria. Thus, oil seeds and emanating products such as ugba and ogiri are significant contributors to human mycotoxin exposures (EFSA, 2007). There have also been cases of border rejections of the substrates of these fermented foods such as melon from importing countries (RASFF, 2012).

Therefore, the presence of both fungi and mycotoxins in these foods cannot be ruled out, even though sanitary and phytosanitary measures might have been put in place in the countries of origin. Also, the processing of these fermented foods has not significantly improved above cottage level, hence, variations in their fermentation processes and unhygienic processing conditions might accentuate contamination (Oguntoyinbo, 2014). Generally, little interest is directed towards the safety of fermented foods, as they are often perceived to be safe whereas many studies focus on microbial succession during fermentation (Omemu, Oyewole, & Bankole, 2007; Olasupo, Okorie, & Oguntoyinbo, 2016). Though, a few studies have highlighted the presence of mycotoxins in some fermented foods from Nigerian markets (Okeke et al., 2015; Chilaka, De Boevre, Atanda, & De Saeger, 2016;

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Adedeji et al., 2017), there has been no reported studies on fungal and mycotoxin contamination of fermented foods in South African markets. This emphasises the need for prompt and continuous monitoring to ensure that consumers are not at a high risk of dietary exposure of mycotoxins. Moreover, it is expedient to; (1) determine if different mycotoxins co-occur in the selected fermented foods and (2) evaluate the chemical constituents (pH,

Total Titratable Acidity (TTA), water activity (aw) and moisture content (MC) that largely influence their safety. Therefore, this study was aimed at assessing the safety of selected fermented foods from South African markets in relation to fungal and mycotoxin contamination.

5.2 Methodology

5.2.1 Sampling

Between April 2015 and July 2016, samples of fermented maize gruel (ogi), fermented maize meal (mahewu), fermented locust beans (iru), fermented African oil bean seed (ugba) and fermented melon (ogiri) were randomly collected from food sellers in Johannesburg (26.2041° S, 28.0473° E) and Tshwane (25.6051° S, 28.3929° E) municipalities, South Africa. Samples of each fermented food (~270 g, ~800mL): ogi (n=33), mahewu (n=21), iru (n=66), ugba (n=25), iru (n=66) and ogiri (n=31) was collected to obtain a total of 176 samples. All samples were collected in sterile plastic bags and transported to the laboratory. Furthermore, each sample was mixed appropriately trisected twice to obtain representative samples of 30 g each for ogi, iru, ugba and ogiri, and 50 mL for mahewu. All the samples (n=176) were subjected to mycotoxin analysis while representative samples ogi (n=18), iru (n=36), ugba (n=18), ogiri (n=18) and mahewu (n=18), were analysed for pH, fungal load,

TTA, aw, and MC.

5.2.2 Chemical properties

The pH of the fermented foods was measured with a pH meter (3510, Jenway, UK). The moisture and TTA contents of the sample were done following the AOAC (2005) method while aw was determined with a water activity meter (Novasina MSI, Switzerland) after calibrating with humidity standards (Novisina SAL-T, Switzerland). All analyses were carried out in triplicates.

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5.2.3 Isolation and identification of fungi

One gram of ugba, iru, ogiri, and ogi samples and 1 mL of mahewu were measured into 9 mL of sterilized peptone solution (0.1%). The solution was vortexed and serially diluted, 0.1 mL of the aliquots was inoculated onto solidified Czapek Yeast Extract agar (CYA), Rose Bengal Chloramphenicol agar (RBCA) and Malt Extract agar (MEA). From the inoculated plates, fungal colonies were counted after incubation at 25 oC for 5 days. The harvested single colonies were sub-cultured on CYA and MEA, and then incubated at 25 °C for 7 days. Pure isolates were identified according to their macro- and microscopic characteristics using the keys of Klich (2002) and Pitt & Hocking (2009). The mean fungal loads were calculated and results expressed as CFU/g and CFU/mL.

5.2.3.1 Molecular identification of fungal isolates

Molecular analysis was performed to confirm the identities of the fungal species recovered from samples as outlined by Adekoya, Obadina, Phoku, Nwinyi, & Njobeh (2017). Concisely, pure fungal DNA was extracted with ZR fungal DNA kit (Zymo Research, D6005, USA). EconoTag Plus Master Mix (Lucigen) was used to amplify the Internal Transcribed Spacer (ITS) target region with ITS 1 and ITS 4 primers. The products were extracted, ran on a gel using ZR Zymoclean Gel DNA recovery kit, purified, sequenced (Applied Biosystems, Thermofisher Scientific, Big Dye terminator kit v3.1, Carlsbad, California, USA) and finally ran on a ABI 3500 xL Genetic Analyser (Applied Biosystems, Thermofisher Scientific). CLC Bio Main Workbench v7.6 was used to analyse the generated data. Results were obtained through a BLAST search on the National Center for Biotechnology Information (NCBI) nucleotide database.

5.2.4 Mycotoxin analysis

5.2.4.1 Reagents and standards

Acetonitrile (analytical grade), methanol (LC-MS/MS grade) and glacial acetic acid (LC- MS/MS grade were procured from Biosolve B.V. (Valkenswaard, Netherlands). Acetic acid and ammonium acetate (analytical grade) were supplied by Merck (Darmstadt, Germany). A Milli-Q gradient apparatus (Millipore; Brussels, Belgium) was used for water purification.

For mycotoxin standards, fumonisin B3 (FB3) was procured from Promec Unit (Tynberg,

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South Africa), while diacetoxyscirpenol (DAS) and T-2 toxin (T-2) were procured from

Biopure Referenzsubstanzen (Tulln, Austria). Fumonisin B1 (FB1), Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), deepoxy-deoxynivalenol

(DOM), fumonisin B2 (FB2), 15-acetyl-deoxynivanelol (15-ADON), ochratoxin A (OTA), neosolaniol (NEO), alternariol (AOH), zearalenone (ZEN), alternariol monomethyl ether (AME), nivalenol (NIV), 3-acetyl-deoxynivalenol (3-ADON) roquefortine C (ROQ C), deoxynivanelol (DON), sterigmatocystin (STE), enniatin B (ENN B), HT-2 toxin (HT-2), fusarenon-X (FUS-X) and zearalanone (ZAN) were purchased from Sigma-Aldrich (Bornem, Belgium). Other chemicals and reagents were of analytical grade

5.2.4.2 Sample preparation

Ugba, iru, ogiri, and ogi were dried at 55 oC in a hot air oven (UM200, Memmert, Schwabach, Germany), and milled. In addition to mahewu, they were weighed (5 ± 0.005 g) and augmented with internal standards (ZAN and DOM). Mycotoxins were extracted from the samples with 20 mL of acetonitrile/acetic acid/water (79/1/20, v/v/v). The mixture was swirled (10 secs), positioned on an overhead shaker (Agitelec, Paris, France) for 1 hr and centrifuged at 3,500 rpm for 15 mins. All the supernatants were transferred into a pre- conditioned SPE C18 column and defatted twice with n-hexane (10 mL). To 12.5 mL of the defatted extract, 27.5 mL of acetonitrile/acetic acid (99/1, v/v) was added, and then passed through a MultiSep 226 AflaZon+ immunoaffinity column. Furthermore, 2 mL of the defatted extract was passed through a glass micro filter (General Electric, Coventry, United Kingdom), merged with the MultiSep 226 eluate, and evaporated to dryness. Injection solvent (150 µL), made up of 5 mM ammonium acetate (0.385 g/L) and methanol/water/acetic acid (57.2/41.8/1, v/v/v), was used to reconstitute the evaporated residue. An Ultrafree® PVDF centrifugal filter (Merck, Darmstadt, Germany) was used to filter the extract whose eluent was conveyed into the LC-MS/MS injection vial for analysis.

5.2.4.3 Liquid chromatography tandem mass spectrometry

For the detection and quantification of the mycotoxin, an ultra-performance liquid chromatography instrument coupled to a Quattro Premier XE tandem mass spectrometer (Waters, Milford, MA, USA) was utilized. MassLynx™ version 4.1 and QuanLynx® version 4.1 softwares (Micromass, Manchester, UK) were used for data acquisition and processing. For the separation of the targeted analytes, a Symmetry C18 (150 mm x 2.1 mm i.d. 5 um

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5.2.5 Data Analysis

The pH, TTA, aw and MC of the samples were subjected to a one-way Analysis of Variance. Tukey’s test was used to separate differences amongst the means at 5% confidence level. Correlation of the chemical parameters was done with Pearson correlation co-efficient (two– tailed). Descriptive statistics (range, mean, frequencies, and percentages) of the fungal and mycotoxin results were also done. SPSS version 23.0 (IBM Corporation, New York, USA) was used as the statistical software.

5.3 Results and Discussion

Chemical properties and fungal load of fermented foods from South African markets

As shown in Table 5.1, the mean pH of the samples ranged from 3.60 to 8.14, with ogiri having the highest pH. Ogi had the highest mean TTA of 0.91% lactic acid (range: 0.78 - 0.96%). The acidic pH of ogi and mahewu is typical based on their formulations via lactic acid fermentation. This substantiates the significant differences (p < 0.05) obtain between their pH values, and that of ogiri, ugba and iru which are formulated via alkaline fermentation. While the production of lactic acid, acetic acid, diacetyl, and acetaldehyde during lactic acid fermentation lowers pH conversely, the hydrolysis of proteins to amino acids, peptides and ammonia during alkaline fermentation increases pH (Steinkraus, 2002). The significant positive correlation (r = 0.727, p < 0.01) observed between total fungal load (Table 5.2) and the pH of the fermented foods corroborates the findings of Kohajdova and Karovicova (2007) that acids formed during fermentation contributes to reduction of

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microbial load. The same trend was also observed between the fungal load and aw (r = 0.860, p < 0.01) (Table 5.3).

Water activity is distinguished as one of the parameters that influence fungal growth and mycotoxin production and most foodborne moulds are able to grow at aw of 0.85 or less

(Carlile, Watkinson, & Gooday, 2001). The minimal, aw for the growth of A. flavus and F. proliferatum for example is 0.80 and 0.90, respectively (Sweeney and Dobson, 1998). Fermented foods had varied fungal loads because of sample and sampling variability, country of origin and differences in storage and processing practices. Higher fungal loads were observed in ogiri (8.30 x 105 CFU/g) and the fermented foods had fungi that mainly belonged to the Aspergillus, Penicillium, Fusarium and Saccharomyces genera.

Table 5.1 Mean pH, water activity, total titratable acidity and moisture content of fermented foods obtained from South African markets

Samples pH TTA (% lactic acid) Moisture Content (%) Water Activity

Mean + S.D Range Mean ± S.D Range Mean ± S.D Range Mean ± S.D Range

Mahewu 3.62a±0.03 3.60-3.66 0.48a±0.08 0.39-0.44 90.67c±1.58 86.29-91.00 0.92a ± 0.03 0.90-0.93

Ogiri 8.07d±0.01 8.03-8.14 0.58ab±0.11 0.47-0.69 41.33b±1.16 40.00-42.20 0.84a ± 0.02 0.82-0.85

Ugba 6.39c±0.06 6.34-6.46 0.74ab±0.20 0.58-0.96 44.67b±1.15 44.00-46.50 0.89a ± 0.06 0.85-0.92

Iru 6.86c±0.03 6.84-6.87 0.65ab±0.26 0.44-0.94 30.00a±2.96 27.50-33.00 0.87a ± 0.02 0.86-0.90

Ogi 4.15b±0.15 4.13-4.16 0.91bc±0.11 0.78-0.96 42.67b±3.43 43.00-47.50 0.89a ± 0.02 0.88-0.91 Note: a-d mean values along the same column with different letters are significantly different.

Table 5.2 Mean fungal load and isolated fungal genera of fermented foods obtained from South African markets

Fermented Fungal Load Isolated Fungi Genera Food Sample CFU/g /CFU/mL Mahewu 4.0 x 103 Aspergillus, Penicillium, Fusarium, Epiococcum, Saccharomyces, Rhodotorula, Moniella, Candida, Rhizopus, Monascus, Curvularia, and Gibberella Ogiri 8.30 x 105 Aspergillus, Fusarium, Candida, Saccharomyces, Talaromyces, Curvularia, Rhizopus, Penicillium, Sarocladium and Mucor Ugba 1.13 x 105 Aspergillus, Rhodotorula, Fusarium, Rhizopus, Penicillium, Mucor, Moniella, Talaromyces, Curvularia, Geotrichum, Zygosaccharomyces, Saccharomyces Iru 1.47 x 105 Aspergillus, Geotrichum, Fusarium, Rhizopus, Candida, Saccharomyces, Penicillium, Sarocladium, Mucor, and Monascus Ogi 9.19 x 104 Aspergillus, Fusarium, Candida, Rhizopus, Penicillium, Mucor, Saccharomyces, and Monascus

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Table 5.3 Correlation coefficients of the pH, water activity, total titratable acidity and moisture content of ogiri, ugba and ogi

PH TTA Moisture Total Water content fungal activity load PH Pearson Correlation 1 -0.560* 0.251 0.727** 0.309 Sig. (2-tailed) .030 .368 .002 .015 N 18 18 18 18 18 Total titratable Pearson Correlation -0.560* 1 -0.567* 0.230 -0.291 acidity (TTA) Sig. (2-tailed) .030 .027 .410 .007 N 18 18 18 18 18 Moisture Pearson Correlation 0.251 -0.567* 1 0.274 0.766* content Sig. (2-tailed) .368 .027 .323 .004 N 18 18 18 18 18 Total fungal Pearson Correlation 0.727** 0.230 0.274 1 0.860** load Sig. (2-tailed) .002 .410 .323 .142 N 18 18 18 18 18 Water activity Pearson Correlation 0.309 -0.291 0.766* 0.860** 1 Sig. (2-tailed) .015 .007 .004 .0142 N 18 18 18 18 18

**Correlation is significant at the 0.01 level (2-tailed), N=number of samples. *Correlation is significant at the 0.05 level (2- tailed), N=number of sample

Fungal diversity in the food samples

Different fungal species were present in the fermented foods. As shown in Table 5.4, members of the Aspergillus genera were more dominant (34%) than those of Fusarium (7%) and Penicillium (12%) genera. A. flavus and A. parasiticus were amongst the significant mycotoxigenic producing Aspergillus species isolated. The isolation of A. niger in ogi in this study corroborates the work of Nwokoro & Chukwu (2012). The high incidence of A. flavus in the fermented foods analysed (ogi: 33%, mahewu: 39%, ogiri: 44%, ugba: 11%, iru: 38%) corresponds with the presence of AFs in the samples particularly ogiri. A. flavus is resident in soil and plant debris as a nutrient recycler, it survives harsh conditions and out- competes other organisms easily (Jaime-Garcia & Cotty, 2004). Thus, its isolation from foods might be based on the microbiota of the soil that the raw materials of the fermented foods were cultivated upon coupled with the prevalence of their air-borne conidia (Adhikari, Sen, Gupta-Bhattacharya, & Chanda, 2004). Similarly, A. fumigatus, which has been reported as the most prevalent species within the Aspergillus genus group (Hedayati, Pasqualotto, Warn, Bowyer, & Denning, 2007) was found in all samples. Other Aspergillus species found but at relative low frequencies were A. minisclerotigenes, A. tritici, A. ustus, A. versicolor and A. oryzae, which have been reported to be frequently isolated from fermented foods (Pitt & Hockings, 2009).

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Furthermore, some important toxigenic Penicillium species present in the foods included P. citrinum that is a ubiquitous fungus in foods (Nwokoro & Chukwu, 2012) though at a relatively low incidences and P. chrysogenum that is usually detected in salty foods (Samson, Houbraken, Thrane, Frisvad, & Andersen, 2010), hence its prevalence in the iru samples (13%). F. verticillioides the predominant Fusarium species in the fermented foods was also isolated with F. proliferatum. As shown in Table 5.5, Monascus ruber was isolated from ogi and mahewu (17%) and the prevalence of Saccharomyces cerevisiae in both samples, accentuates its key role in their fermentation. Zygosaccharomyces bailii which is recognized as spoilage yeast was also found in ugba, its involvement in the production of undesirable properties in foods such as off flavours and haze formation have been reported (Rodrigues, Corte-Real, Leao, Van Dijken, & Pronk, 2001).

Comparable mycobiota were reported by Adekoya, Obadina, Phoku, Nwinyi & Njobeh (2017) in some fermented foods obtained from Nigerian markets. To the best of our knowledge, this is the first documented evidence of fungal diversity of mahewu. It also reports the association of A. clavatus, A. tritici, P. camemberti, and P. steckii with ogi, A. oryzae, and also A. ustus and F. fujiroki with ogiri, A. candidus with ugba and P. rubens, P. verrucosum, P. camemberti and A. tubigensis with iru.

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Table 5.4 Incidence rates of fungal contamination of fermented foods from South African markets

Isolated species Ogi Mahewu Ogiri Ugba Iru Accession number % +ve Frequency of % +ve Frequency of % +ve Frequency of % +ve Frequency of % +ve Frequency of samples spp. isolated samples spp. isolated samples spp. isolated samples spp. isolated samples spp. isolated (%) (%) (%) (%) (%) Aspergillus species A. amstelodami ------6 2 (5) AY373885.1 A. candidus ------11 2 (13) 6 2 (5) KT223337 A. clavatus 11 2 (11) 17 3 (14) - - - - 6 2 (5) KUO52566 A. flavus 33 6 (33) 39 7 (32) 44 8 (35) 28 5 (33) 38 12 (32) KR611584 A. fumigatus 11 1 (6) 17 3 (14) 17 3 (13) 11 2 (13) 13 4 (11) KU684451 A. minisclerotigenes - - - 11 2 (9) - - - - JF412778 A. niger - - 17 3 (14) ------KX215111.1 A. niger 22 2 (11) - - 11 2 (9) 11 2 (13) 6 2 (11) KX215115.1 A. oryzae - - - - 11 2 (9) - - - - KX215113.1 A. parasiticus 22 2 (11) 17 3 (14) 17 3 (13) 11 2 (13) 19 6 (16) DQ467988.1 A. sclerotiorum ------11 2 (13) 6 2 (5) KT717312 A. sydowii 6 1 (6) ------6 2 (5) KX215130.1 A. tritici 22 2 (11) ------KX215119.1 A. tubingensis - - 6 1 (5) - - - - 6 2 (5) KT717311 A. ustus - - - 11 2 (9) - - - - HQ607918.1 A. versicolor 22 2 (11) 11 2 (9) 6 1 (4) - - 3 1(3) LC105698

Penicillium species P. chrysogenum 17 3 (33) 11 2 (40) 11 2 (25) 11 2 (50) 13 3 (27) KX215133.1 P. lanosocoeruleum ------3 1 (9) JX997110 P. aethiopicum - - - - 11 2 (25) - - - KX215125.1 P. camemberti 11 2 (22) ------3 1 (9) KT355012 P. citrinum 17 3 (33) - - 11 2 (25) - - - - KX215122.1 P. verrucosum - - - - 5 1 (13) 11 2 (50) 10 3 (27) KM115130

Note: total number of Aspergillus species is 115 (ogi: 18 species, mahewu: 22 species, ogiri: 23 species, ugba: 15 species, iru: 37 species); Fusarium species is 23 (ogi: 4 species, mahewu: 5 species, ogiri: 5 species, ugba: 3 species, iru: 6 species); Penicillium species is 39 (ogi:9 species, mahewu: 5 species, ogiri: 10 species, ugba: 4 species, iru:11 species). % +ve: percentage positive

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Isolated species Ogi Mahewu Ogiri Ugba Iru Accession number % +ve Frequency of % +ve Frequency of % +ve Frequency of % +ve Frequency of % +ve Frequency of samples spp. isolated samples spp. isolated samples spp. isolated samples spp. isolated samples spp. isolated (%) (%) (%) (%) (%) P. crustosum - - 17 3 (60) ------KT735107.1 P. raistrickii - - - - 17 3 (38) - - 3 1 (9) KX215126.1 P. rubens ------6 2 (18) LC105692 P. steckii 5 1 (11) ------KX215128.1

Fusarium species F. chlamydosporum ------3 1 (17) KP769538.1 F. chlamydosporum ------6 2 (33) KX215114.1 F. fujikuroi - - - - 6 1 (20) - - - - KT192328 F. proliferatum ------11 2 (67) KP773280 Fusarium sp. - - 12 2 (40) - - 3 1 (17) JQ350882 F. sporotrichioides 11 2 (50) 11 2 (40) 6 2 (33) AF404149.1 F. verticillioides 11 2 (50) 6 1 (20) ------KX215136.1 F. verticillioides - - - - 11 2 (40) - - - - KX215131.1 F. verticillioides - - 6 1 (20) - - 6 1 (33) - - KX215138.1 F. verticillioides 6 1 (20) ------JF499676.1

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Table 5.5 Incidence rates of fungal contamination of fermented foods obtained from South African markets with other fungal species

Other fungal species Ogi Mahewu Ogiri Ugba Iru Accession Number % +ve Frequency % +ve Frequency of % +ve Frequency of % +ve Frequency of % +ve Frequency of samples of spp. samples spp. isolated samples spp. isolated samples spp. isolated samples spp. isolated isolated (%) (%) (%) (%) (%) Curvularia spicifera ------11 2 (7) - - KC999935 Monascus ruber 17 3 (8) 17 3 (11) - - - - 22 7 (16) KXX215143.1 Mucor circinelloides 22 4 (11) ------GU966516 Mucor circinelloides ------17 3 (10) 10 3 (7) KX215120.1 Mucor sp. 6 1 (3) ------FJ210516 Sarocladium sp. 17 3 (14) 10 3 (7) KX215139.1 Talaromyces islandicus ------17 3 (10) - - KX215129.1 Talaromyces radicus ------11 2 (7) - - AB457007 Talaromyces stollii - - - - 11 2 (10) - - - - AB910938 Talaromyces stollii ------KX215122.1 Talaromyces verruculosus ------17 3 (10) - - KJ413368 aOther identified species Candida krusei - - 17 3 (11) 28 5 (24) - - 10 3 (7) Candida tropicalis 11 2 (6) ------Curvularia lunata - - 11 2 (7) 11 2 (10) - - - - Epiococcum nigrum - - 17 3 (11) ------Geotrichum candidum ------6 1 (3) 19 6 (14) Moniella suaveolens - - 11 2 (7) - - 17 3 (10) 6 2 (5) Mucor piriformis 17 3 (8) ------Rhizopus oligosporus 11 2 (6) 11 2 (7) 17 3 (14) - - - - Rhizopus stonolifer 17 3 (8) 17 3 (11) - - 17 3 (10) 13 4 (9) Rhodotorula mucilaginosa - - 22 4 (15) ------Rhodotorula rubia ------17 3 (10) - - Saccharomyces cerevisiae 100 18 (50) 56 5 (19) 33 6 (29) 22 4 (13) 50 16 (37) Zygosaccharomyces balli ------17 3 (10) - -

Note: total number of other species is 163 (ogi: 36 species, mahewu: 32 species, ogiri: 21 species, ugba: 30 species, iru: 44 species).aSpecies were distinguished based on their morphological and microscopic. characteristics. % +ve: percentage positive

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Mycotoxin occurrence in the fermented food samples

Our study delineates for the first time a broad array of mycotoxins in fermented products from selected South African markets. Generally, the food products were tainted with different mycotoxins including AFB1, AFB2, FB1, OTA, DON, AME, STE, ROQ C, NIV, ZEN and ENN B as shown in Table 5.6. The incidence of fungi and mycotoxins in these products may be attributed to the microflora composition in addition to pre- and post-harvest contamination of the raw materials, poor processing techniques and improper and marketing practices.

The most incriminated mycotoxins were the AFs with an incidence rate of 26% (45/176). The frequencies of samples contaminated by ENN B, AME, DON, OTA, STE and FBs were 7,

12, 13, 13, 14 and 24% respectively, and the highest level of AFB1 was recorded in ugba

(25 μg/kg). The level of AFB1 in all positive samples exceeded the recommended level for

AFB1 in foods meant for human consumption (EC 2006b). The concentrations of AFB1 found in the ogiri samples (3 - 12 µg/kg) are comparable to the levels reported by Bankole, Adenusi, Lawal, & Adesanya (2010) in ogiri (2-13 µg/kg) obtained from Nigerian markets.

Aflatoxin B1 is the most toxic AF and has been associated with deleterious health issues including immune suppression, kwashiorkor, growth retardation and haemorrhage (Shephard, 2003). Lower incidence of AFs were observed in the two maize products analysed ogi (14%) and mahewu (37%), compared with FBs which respectively occurred in 86% and 61% of the samples. Adetunji et al. (2014) also reported lower incidences of AFs compared to FBs in maize from five agro-ecological zones of Nigeria.

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Table 5.6 Incidence and mycotoxins levels of fermented foods from South African markets

Mycotoxin Fermented Melon Fermented Locust Bean Fermented African Oil Fermented Maize Meal Fermented Maize Gruel (ogiri) N=31 (iru) N=66 Bean (ugba) N=25 (mahewu) N=21 (ogi) N=33 % +ve Range Mean % +ve Range Mean % +ve Range Mean % +ve Range Mean % +ve Range Mean samples samples samples samples samples

Deoxynivalenol 1 (3) 0 61 0 0 0 3 (12) 31-33 32 16 (73) 18-32 22 2 (6) 17-69 43 Nivalenol 0 0 0 0 0 0 1(4) 0 45 5 (24) 94-117 99 0 0 0 Neosolaniol 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Fusarenon-X 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3-Acetyldeoxynivalenol 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15-Acetyldeoxynivealenol 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Aflatoxin B1 15 (50) 3-12 4 9 (14) 3-7 6 4 (16) 4-25 12 2 (10) 6-6 6 9 (27) 4-6 5 Aflatoxin B2 1 (3) 0 3 1 (2) 0 8 5 (20) 4-9 5 1 (5) 0 5 2 (6) 4-4 4

Aflatoxin G1 0 0 0 0 0 0 0 0 0 0 0 0 2 (6) 2-2 2 Aflatoxin G2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ƩAflatoxins 15 (50) 3-12 4 9 (14) 3-15 7 6 (24) 4-25 12 3 (14) 5-6 6 12 (37) 2-8 6 Diacetoxyscirpenol 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Alternariol 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Alternariol Methyl Ether 0 0 0 14 (21) 35-91 79 2 (8) 9-11 10 3(14) 16-37 24 2 (6) 12-12 12 HT-2 Toxin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 T-2 Toxin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Fumonisin B1 5 (16) 53-73 63 0 0 0 0 0 0 14 (67) 23-86 53 12 (37) 42-326 65

Fumonisin B2 0 0 0 0 0 0 0 0 0 7 (33) 31-49 48 11 (33) 31-105 59

Fumonisin B3 0 0 0 0 0 0 0 0 0 1 (5) 0 40 12 (37) 42-326 95

Ʃ Fumonisin B1, B2 0 0 0 0 0 0 0 0 0 18 (86) 23-134 60 15 (46) 31-216 96

Ʃ Fumonisin B1, B2, B3 5(16) 53-73 63 0 0 0 0 0 0 18 (86) 23-134 62 20 (61) 31-325 129 Ochratoxin A 1 (3) 0 22 14 (21) 3-79 24 4 (16) 8-11 9 3 (14) 7-12 7 0 0 0 Sterigmatocystin 4 (13) 15-165 86 15 (23) 6-79 24 0 0 0 1 (5) 0 15 5 (15) 3-5 5 Roquefortine C 4 (13) 11-20 17 3 (5) 17-18 18 0 0 0 0 0 0 0 0 0 Zearalenone 0 0 0 0 0 0 0 0 0 0 0 0 1 (3) 0 17 Enniatin B 0 0 0 0 0 0 3 (12) 13-16 14 9 (43) 16-22 18 0 0 0

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Fumonisins are well known to contaminate maize and its products (Bankole, Ogunsanwo, Osho, & Adewuyi, 2006; El Khoury et al., 2008) more than AFs. Consumption of FBs contaminated foods have been linked to oesophageal cancer (Shephard et al., 2005), abdominal pain, diarrhoea outbreaks and stunted growth particularly in children (Bhat, Rai, & Karim 2010). Asides FBs, DON may also cause stunted growth because of lowered food or feed intake (Pinton, & Oswald, 2014). Deoxynivalenol occurred in the ogiri, ogi, ugba and mahewu at incidence rates of 3, 6, 12 and 73% and maximum concentrations of 61, 69, 33 and 32 µg/kg, respectively. Furthermore, Chilaka, De Boevre, Atanda and De Saeger (2016) detected a higher incidence rate of DON (13%) in ogi from Nigeria.

Furthermore, the level of OTA contamination in ogiri, ugba and mahewu did exceed the maximum permissible limit of 5 µg/kg for OTA in foods (EC, 2006b). Based on literature, OTA is identified as a global natural contaminant, majorly in cereals, grains, fruits and their processed products, coffee and fermented beverages (El Khoury et al., 2008). Other researches correlated their presence to congenital deformity in the foetus of experimental animals with minimal understanding on their modes of action (Raiola, Tenure, Manes, Mecca, & Rotini, 2015). The occurrence of STE contamination was 23% in iru samples and 13% in ogiri with concentrations ranging from 6 to 79 μg/kg and 15 to 165 μg/kg respectively. Enniatin B occurred in ugba (12%) and mahewu (43%) while iru, ogi, ugba and mahewu were positive for AME with concentrations ranging from 9 to 91 µg/kg. Our results substantiates those of previous survey for STE in ogiri (Adedeji et al., 2017), while for ENN B, there is a lack of information on their occurrence in ugba, iru, ogi, mahewu and ogiri.

The incidence rate of ZEN in ogi was very low (3%) whereas it was not detected in other samples. In a comprehensive report on global distribution of mycotoxins by Devegowda, Radu, & Nazar (1998), ZEN was reported to be mainly dominant in agricultural products in Eastern Europe, Western Europe and North America rather than Africa. However, more surveillance data on ZEN needs to be provided in the face of international trade. All the samples were negative for DAS, NEO, FUS-X, T-2, HT-2 and acetylated forms of DON (3- ADON, 15-ADON). Generally, 34% of the samples were not contaminated by any mycotoxin, which demonstrates the possibility of having fermented foods devoid of mycotoxins. Activities of microorganisms such as lactic acid bacteria and Saccharomyces cerevisiae during fermentation have been demonstrated to reduce some mycotoxins, although their mode of activity is still under investigation and no microbial strain has been approved as

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Co-occurrence of mycotoxins in fermented foods

Co-occurrence of mycotoxins within foods creates a supplementary threat to public health. As indicated in Figure 5.1, 54% of ogi and 95% of mahewu were contaminated with a minimum of 2 out of the 23 mycotoxins understudied. Similar result was observed by Chilaka, De Boevre, Atanda and De Saeger (2016) who investigated the co-occurrence of Fusarium mycotoxins in ogi from Nigerian markets. There was simultaneous occurrence of

AFB1 and STE in ogiri (13%) and a significant percentage of iru (17%) was contaminated with AFB1, OTA and AME. Adedeji et al. (2017) studied the occurrence of 7 mycotoxins in iru (n=9) and ogiri (n=9) from Nigeria and only noticed few incidences of multiple mycotoxins in both samples. The distribution of mycotoxins in the fermented foods revealed possible exposure of consumers to multiple mycotoxins through daily consumption. More credible data are needed in respect of the simultaneous occurrence of mycotoxins in foods in order to develop a more robust risk assessment for human health (Juan, Raiola, Manes, & Ritieni, 2014) in addition to studies on their possible synergetic and antagonistic effects (Njobeh et al., 2010; Prosperini, Font, & Ruiz, 2014).

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Figure 5.1 Percentage of co-occurring mycotoxins in fermented foods from South African markets

5.4 Conclusion

The study gave an overview of the diversity of both fungi and mycotoxins in five fermented foods selected from South African markets: ogi, iru, ugba, ogiri and mahewu, as well as their chemical properties. The fermented foods had high fungal loads and were contaminated with different potentially toxigenic fungal species particularly Aspergillus species. A significant fraction of the samples (116/176) had mycotoxins occurring singly or in combination though at low incidences (NIV, ZEN, ROQ C, ENN B, AFB2, AFG1) and contamination levels (FBs, AME, ROQ C, DON). Mahewu was the most contaminated sample (100%, n=21) for the analysed metabolites particularly DON and had the highest number of co-occurring metabolites (n=6), exasperating a higher risk of mycotoxin exposure amongst its consumers.

Majority of the samples positive for OTA (91%, n=22) and AFB1 (100%, n=39) exceeded the maximum limit for OTA in foods as regulated by the EC. All samples were negative for 15-

ADON, 3-ADON, AFG2, ALT, NEO, T-2, HT-2, FUS-X, and DAS. This study emphasises the high health risks and the potential obstacle of international trade that lies with the foods.

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There is need to perform more surveillance studies and enact stringent quality control measures on locally fermented foods consumed or sent to export markets to alleviate contamination that could potentiate marked-health complications amongst consumers.

Acknowledgments

This work was supported by the Organisation for Women in Science in the Developing World (OWSD), Italy, African Women in Agricultural Research and Development (AWARD), Kenya, Centre of Excellence for Food Security and Safety (COE), South Africa and MYTOX-SOUTH, hosted in the Laboratory of Food Analysis, Ghent University, Belgium.

Conflict of Interest

The authors have no conflict of interest to declare.

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CHAPTER SIX

MYCOBIOTA AND CO-OCCURRENCE OF MYCOTOXINS IN UMQOMBOTHI: A SOUTH AFRICAN CEREAL-BASED OPAQUE BEER

Ifeoluwa Adekoyaa*, Adewale Obadinaab, Chilaka Cynthia Adakuc, Marthe De Boevrec, Sheila Okothd, Sarah De Saegerc and Patrick Njobeha aDepartment of Biotechnology and Food Technology, University of Johannesburg, Johannesburg, South Africa bDepartment of Food Science and Technology, Federal University of Agriculture, Abeokuta, Nigeria cLaboratory of Food Analysis, Department of Bioanalysis, Ghent University, Ghent, Belgium dDepartment of Botany, School of Biological Sciences, University of Nairobi, Kenya *Corresponding author: Ifeoluwa Adekoya; Email Address: [email protected]

Abstract

Beer, an alcoholic beverage consumed throughout the world is mainly derived from cereals. In this study, fungal and mycotoxin contamination, as well as the chemical properties of a cereal-based opaque beer (umqombothi) obtained from the Gauteng province of South Africa, was investigated. The mean water activity, pH and total titratable acidity of the opaque beer were 0.91, 3.76 and 1.20% lactic acid, respectively. The samples had a fungal load of 3.66 x 105 CFU/mL and Aspergillus, Penicillium, Phoma and Saccharomyces were the predominant fungal genera. Aspergillus flavus had the highest incidence of 26% among the isolated mycotoxigenic fungal species and previously unreported fungal strains such as P. chrysogenum strain AD25, A. sydowii strain AD 22 and A. tritici strain AD11 were isolated. Quantitative analysis of the mycotoxin content of the beer by liquid chromatography-tandem mass spectrophotometry showed that deoxynivalenol was the dominant mycotoxin occurring in 84% of the samples. This was followed by enniatin B that occurred in 75% of samples

(range: 12-44 µg/L) and fumonisin B1 (FB1) (range: 112-182 µg/L). The incidence of aflatoxin contamination was low in addition to other Aspergillus mycotoxins. However, they were contaminated by at least two mycotoxins that could pose some additive or synergistic health effects to the consumers. On an average: a 60 kg adult consuming 1-6 L/day of the beer was exposed to FB1 + FB2 at an estimated 2.20 - 13.20 µg/kg body weight /day. These values were far above the maximum tolerable daily intake of 2 µg/kg bw/day established by

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Keywords: Mycotoxins, fungi, beer, cereals, and South Africa

Highlights

 The study confirmed the presence of fungi and 23 mycotoxins in South African cereal based opaque beer (umqombothi).  A. flavus had the highest incidence among the mycotoxigenic species and new strains were identified.  Deoxynivalenol was the predominant mycotoxin in 84% of the samples.  Samples were contaminated with at least 2 mycotoxins and exposure was evident.  This is the first study that reports the presence of fungi and mycotoxins in umqombothi from Gauteng, South Africa.

6.1 Introduction

In Africa, fermented foods and beverages are typically traditionally manufactured in homes under spontaneous conditions, and many of them confer some cultural and traditional values (Matumba et al., 2014). Popular among South Africans is mahewu, a fermented non-alcoholic beverage produced from maize and umqombothi, a traditionally brewed alcoholic beverage made from maize, sorghum, malt and yeast. Other alcoholic beverages consumed are isiqatha, utshwala, and imfulamfula. Umqombothi is an opaque, pink-coloured, B-vitamins rich beer with a distinctively sour aroma and creamy constituency (Nicholas, 2008). It is characterised by a short shelf life (2-3 days) and consumed in its active state of fermentation on daily basis, particularly among the black South African populations that live in rural communities (Nicholas, 2008). Umqombothi is brewed majorly by women either for sale or for social gatherings.

Several studies have outlined the pre- and post-harvest contamination of cereals used for umqombothi production with toxigenic fungal species namely A. flavus, A. parasiticus and F. verticilliodes (Odhav & Naicker, 2002; Shephard et al. 2005; Njobeh et al. 2009). This

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To the best of our knowledge, information on fungi and mycotoxins that may be present in umqombothi produced in Gauteng province is lacking. Therefore, the aim of this study was to assess the safety of umqombothi produced in Gauteng province with respect to fungal and mycotoxin contamination. In addition, quality parameters [pH, moisture content (MC), water activity (aw) and total titratable acidity (TTA)], which largely influences the safety of the beer were determined.

6.2 Materials and Methods

6.2.1 Beer samples

Between April 2015 and July 2016, A total of 32 composite umqombothi samples (n=32) were randomly collected from the Tshwane (25.6051° S, 28.3929° E) and Johannesburg (26.2041° S, 28.0473° E) municipalities in the Gauteng province of South Africa. Each composite sample of about 2,400 mL was an aggregate of sub samples obtained from three umqombothi sellers. All samples were collected in sterile containers and immediately transported to the laboratory. Afterwards, each composite sample was properly mixed and quartered twice to obtain a representative sample of 150 mL, that was divided into three (50 mL) each for chemical, microbiological and mycotoxin analysis. All samples were placed in sterile containers and stored at 4 oC and -18 oC (samples for mycotoxin analysis) until analysed.

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6.2.2 Methodology

6.2.2.1 pH, MC, aw and TTA determination

The pH content of the samples was determined with a pH meter after calibration with standard buffers of pH 4.00 and 7.00. The moisture and TTA contents of the beer were determined as described by the Association of Official Analytical Chemists (AOAC, 2005), while the aw was measured with a water activity meter (Novasina MSI, Switzerland) after calibrating with humidity standards: Salt 11 (91110930), 33 (1110932), 53 (110934), 75 (1110936) and 90 (1110938) (Novisina SAL-T, Switzerland). All analyses were carried out in triplicates.

6.2.2.2 Mycobiota of fungi

For the mycological study of the samples, 1 mL was weighed and suspended in 9 mL of sterilized 0.1% peptone solution in a test tube, vortexed and serially diluted to 10−10. An aliquot of 0.1 mL was each inoculated on solidified Czapek Yeast Extract agar (CYA), Rose Bengal Chloramphenicol agar (RBCA) and Malt Extract agar (MEA) using spread plate technique. The inoculated petri dishes were incubated at 25 °C for 5 days. Thereafter, fungal colonies were counted, the mean fungal load calculated and results expressed as CFU/mL of sample. Thereafter, single colonies were harvested, sub-cultured on MEA and CYA and then incubated at 25 °C for 5 days. Using the keys of Klich (2002) and Pitt and Hocking (2009), the identities of pure isolates was established. To achieve this, both macro- and microscopic characteristics of fungal colonies were carried out following staining with lactophenol blue and placement under a microscope (Olympus B061, Wirsam Scientific, South Africa).

6.2.2.3 Molecular identification of fungi isolates

To confirm the identities of the fungal species recovered from samples, a molecular analysis was performed as described by Adekoya et al. (2017). Briefly, genomic DNA was extracted from each fungal culture using the ZR fungal DNA kit (Zymo Research, D6005, California, USA). The ITS region was amplified using EconoTag Plus Master Mix (Lucigen) with ITS 1 forward and ITS 4 reverse primers. The PCR products were run on a gel, and the gel was extracted using ZymoClean Gel DNA recovery clean-up kit (Zymo Research, D4001). The extracted fragments were sequenced in the forward and reversed directions (Applied Biosystems, Thermofisher Scientific, Big Dye terminator kit v3.1, Carlsbad, California,

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USA) and purified. The purified fragments were run on an ABI 3500 xL Genetic Analyser (Applied Biosystems, Thermofisher Scientific). CLC Bio Main Workbench v7.6 was used to analyse the data. Results were obtained by a BLAST search on NCBI, and the sequence data obtained in this study were submitted to the gene bank.

6.2.2.4 Phylogenetic analysis

The phylogenetic relationship of fungal sequences established in this study including those of reference strains were inferred with the Neighbour-Joining analysis of Saitou & Nei (1987). The bootstrap consensus tree was constructed from 1000 replicates (Felsenstein 1985). Branches analogous to partitions reproduced in < 50% bootstrap replicates were collapsed. The percentages of the replicate trees are shown next to the branches as bootstrap values (Figure 6.1). The Maximum Composite Likelihood method of Tamura, Nei & Kumar (2004) was followed to construct the evolutionary distances. Sixteen nucleotide sequences were analysed. The 1st+2nd+3rd+noncodings were included in the codon positions, and all positions with missing data and gaps eliminated. The final dataset had 156 positions. The evolutionary analysis was conducted in MEGA 7 (Pennsylvania State University, USA).

6.2.2.5 Mycotoxin analysis

6.2.2.5.1 Reagents and standards

Acetonitrile (VWR International, Zaventem, Belgium) was of HPLC grade and methanol of LC-MS grade (Biosolve, Valkenswaard, Netherlands), ammonium acetate (Grauwmeer, Leuven, Belgium) and Acetic acid (Merck, Darmstadt, Germany) were of analytical reagent grade. The mycotoxin standards comprising of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), FB1, fumonisin B2 (FB2), zearalenone (ZEN), nivalenol (NIV), DON, deepoxy-deoxynivalenol (DOM), 15-acetyl-deoxynivalenol (15- ADON), 3-acetyl-deoxynivalenol (3-ADON), neosolaniol (NEO), ochratoxin A (OTA), alternariol (AOH), roquefortine C (ROQ-C), alternariol monomethyl ether (AME), enniatin B (ENN B), HT-2 toxin (HT-2), fusarenon-X (FUS-X) and zearalanone (ZAN) were obtained from Sigma-Aldrich (Bornem, Belgium). Fumonisin B3 (FB3) was procured at Promec Unit (Tynberg, South Africa) while T-2 toxin (T-2) and diacetoxyscirpenol (DAS) were purchased from Biopure Referenzsubstanzen (Tulln, Austria).

6.2.2.5.2 Mycotoxin extraction

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All samples were degassed by ultrasonication in an ultrasonic bath (2800, Branson, Vlierberg, Netherlands). Samples were measured accurately (5 ± 0.005 mL), fortified with internal standards DOM and ZAN at 50 and 10 µg/mL, respectively, and allowed to equilibrate for 15 mins in the dark. 5 mL of the sample was extracted with 24 mL of acetonitrile/acetic acid (99/1, v/v) for 30 mins on an overhead shaker (Agitelec, France), followed by the addition of 6 g of magnesium sulphate and a repetition of the extraction step. The samples were placed in a centrifuge (3-16PK, Sigma, Irvine, United Kindgom) for 10 mins at 4,000 rpm, filtered through a glass microfiber filter (General Electric, Coventry,UK) and the filtrate dried under a stream of nitrogen gas at 40 oC. The residue was reconstituted in 150 µL of injection solvent made-up of methanol/water/acetic acid (57.2/41.8/1, v/v/v) and 5 mM ammonium acetate. The reconstituted extracts were placed in Ultrafree® PVDF centrifuge filters (Merck, Darmstadt, Germany) and centrifuged at 10,000 rpm for 10 mins. The eluent was transferred into 2 mL screw-cap vials prior to analysis.

6.2.2.5.3 Liquid chromatography-tandem mass spectrometry Detection and quantification of mycotoxins was performed on a Waters Acquity UPLC apparatus attached to a Quattro Premier XE Tandem Mass Spectrometer (Waters, Milford, MA, USA). A Symmetry C18 (150 mm x 2.1 mm i.d.) column attached to a guard column (10 mm x 2.1 mm i.d.) (Waters, Zellik, Belgium) was used. The chromatographic conditions followed that of Njumbe Ediage et al. (2011). The sample injection volume was fixed at 10 μL. The total analytical run time was 28 mins and the range of the pressure was between 0 and 5,000 psi. The mass spectrometer was operated using selected reaction monitoring channels in positive electrospray ionization mode. Detailed information of the transitions of the mycotoxins under studied was reported by Monbaliu et al. (2009) and De Boevre et al. (2012).

6.2.2.5.4 Method validation The European Commission (EC) Regulation 401/2006/EC (EC, 2002) was used as guideline for the validation study. Parameters including limit of detection (LOD) and quantification (LOQ) as well as apparent recoveries of mycotoxins were accessed by spiking mycotoxin- free (blank) samples with the different mycotoxins in triplicates. ZAN and DOM were used as internal standards, and matrix-matched calibration (MMC) curves were constructed based on the ratio of the peak area of each analyte to that of the corresponding internal standards. The linearity of each analyte was evaluated using a lack of fit test, while their apparent

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6.2.2.6 Data analysis

Descriptive statistics (mean, range, maximum, frequency) of the data obtained in this study were done with SPSS version 23.0 (IBM Corporation, New York, USA) and Microsoft Office Excel 2007 (Redmond, WA, USA).

6.3 Results and Discussion

6.3.1 Chemical properties and fungal load

The mean pH, TTA, MC and aw of the samples were 3.76, 1.20 % lactic acid, 94.67 % and

0.91, respectively (Table 6.1). The pH, TTA, MC and aw are important quality parameters in fermented beverages which largely influence their safety. The low pH of beer is typical, because of their formulations via lactic acid fermentation. TTA is a measure of the amount of acid and increased acidity in alcoholic beverages such as in umqombothi is a consequence of the acids formed during fermentation which lowers the pH of beverages thereby inhibiting the growth of spoilage and pathogenic organisms. Despite the low pH observed in the beer, fungi were present and a fungal load of 3.66 x 105 CFU/mL was observed. The fungal load however, exceeded the permissible limit of fungi (1 x104 CFU/g) in ready-to-eat foods based on the microbiological criteria (EC, 2005) and values above this limit indicate the potential presence of mycotoxins (EC, 2012). The high fungal load (3.66 x 105 CFU/mL) is probably due to the high MC and aw of the beer as well as contamination of processing environment. The possibility of microbial growth during storage of the beer before they were sold cannot be ruled out as they have a short shelf life and lower keeping quality due to low alcohol content (3%) (Nicholas, 2008).

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Table 6.1 Chemical properties of South African cereal-based opaque beer

Parameters Meana ± S.Db Range pH 3.76±0.02 3.74-3.78 TTA (% Lactic acid) 1.20±0.10 1.13-1.31 Moisture content 94.7 ± 1.25 93.5-96.0 Water activity 0.91± 0.12 0.89 -0.92

a Analysis was carried out in triplicates. b S.D = standard deviation

6.3.2 Fungal incidence and dominant fungal genera

Since the 1980s, fungi and their secondary metabolites have emerged as causative agents of various diseases with these metabolites potentiating some carcinogenic, mutagenic, genotoxic and or teratogenic tendencies in humans (Hussein and Brassel, 2001). Data obtained in this study revealed the presence of different fungal species in beers, predominantly those belonging to the genera Aspergillus, Penicillium, Saccharomyces and Phoma. Members of the Aspergillus genera had a mean load of 4.9 x 105 CFU/mL and they were more prevalent (28%) than those of Fusarium and Penicillium genera as shown in Table 6.2. Aspergillus flavus and A. parasiticus were among the significant mycotoxigenic Aspergillus spp. being producers of AFs.

The Penicillia is a large genus with more than 200 recognized species (Pitt & Hocking, 2009) and the important toxigenic species belonging to these genera included P. crustosum (38%) and P. chrysogenum (38%). Penicillium crustosum synthesizes penitrem A, cyclopiazonic acid and ROQ-C. Incidentally, P. chrysogenum also produces ROQ-C, which was detected in the samples. F. verticillioides was the predominant Fusarium sp. detected in the beer and this correlated well with the presence of FBs in the beer. Bipolaris maydis was detected in 4% of the samples analysed, but at low incidence rate. Phoma sorghina appears in the literature as the main Phoma sp. found in sorghum and its current observation in 62% of the beer could be linked to the contamination of the raw materials, particularly sorghum grains and malt used for beer production. The higher incidence (33%) and microbial load (2.3 x 107) of Saccharomyces cerevisiae, when compared to other fungal species, accentuates its key role in the fermentation of the beer.

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Table 6.2 Occurrence of fungi in South African cereal-based opaque beer (Umqombothi)

Fungal species % positive Frequency of Mean fungal Accession sample species load number isolated (%) (CFU/mL)

Aspergillus species 4.90 x 105 A. flavus 33 26 na KR611584.1 A. fumigatus 11 9 na KU684451.1 A. minisclerotigenes 11 9 na JF412778 A. niger 17 13 na KX215115.1 A. parasiticus 17 13 na DQ467988.1 A. sclerotiorum 11 9 na KT717312 A. sydowii 11 9 na KX215130.1 A. versicolor 6 4 na LC105698.1 A. tritici 11 9 na KX215119.1 Penicillium species 2.30 x 105 P. chrysogenum 17 38 na KX215133.1 P. aethiopicum 17 38 na KX215125.1 P. crustosum 11 25 na KT192315.1 Fusarium species 1.13 x 103 F. verticillioides 17 100 na KX215138.1 Other species Mucor circinelloides 17 6 6.40 x 105 KX215120.1 Bipolaris maydis 22 8 2.20 x 106 Candida ethanolica 17 6 6.00 x 103 Epiococcum nigrum 11 4 2.50 x 104 Geotrichum candidum 28 10 6.50 x 105 Phoma sorghina 62 23 2.40 x 106 Rhodotorula mucilaginosa 17 6 Saccharomyces cerevisiae 89 33 2.30 x 107 Saccharomyces capsularis 11 44 na

Note: total number of fungi isolate is 83; Aspergillus species (23), Fusarium species (3), Penicillium species (8) and other species (49). na= not applicable

This is the first time that PCR in combination with 16S rRNA gene sequencing method will be used to investigate the microbial diversity of South African cereal-based opaque beer. Some new and unreported strains of microorganisms in the beer included P. chrysogenum strain AD25 (KX215133.1), A. sydowii strain AD 22 (KX215130.1), A. tritici strain AD11 (KX215119.1), F. verticillioides strain AD 30 (KX215138.1) and Mucor circinelloides strain AD 12 (KX215120.1). The high fungal load was an indication of post-process contamination of the beer either with contaminated equipment or through subsequent addition of contaminated ingredients. In addition, various abiotic factors such as temperature and humidity associated with the processing and storage of the beer might have contributed to the observed fungal diversity.

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6.3.3 Phylogenetic analysis

The evolutionary history of 13 distinct sequences isolated from the beer and 3 other reference sequences representing the Aspergillus spp. complexes are shown in Figure 6.1. The sequences clustered differently on the rationale of species with their bootstrap frequencies ranging from 28 to 100% and the emanation of species in the clades from a common progenitor was evident. The A. niger strain AD 7 was closely related to the A. niger strain OC3, while the P. crustosum strain ZP-2 and the P. chrysogenum strain AD 25 had a weak bootstrap support (65%). The wide variation in the evolutionary relationships, bootstrap frequencies and the presence of previously unreported fungal isolates indicated the need to complement morphological identifications with molecular identifications. Fungi are not easy to identify via conventional means, because of their unstable and complex taxonomy (Geiser et al. 2004). Information on their clonal relationship, genetic association and divergence are paramount to combat their proliferation and subsequent toxin production in foods.

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KR611584.1 Aspergillus flavus strain PKM18 96 100 LC105698.1 Aspergillus versicolor strain: DY20.1.1

KX215130.1 Aspergillus sydowii strain AD22 99

KX215138.1 Fusarium verticillioides strain AD30 1 Clade 28 44 KX215120.1 Mucor circinelloides strain AD12

KX215119.1 Aspergillus tritici strain AD11

KU684451.1 Aspergillus fumigatus strain IVRI 100 KX171028.1 Aspergillus fumigatus strain DO1

68 KT717312.1 Aspergillus sclerotiorum strain G121802

KX215115.1 Aspergillus niger strain AD7 2 Clade 100 86 KX171023.1 Aspergillus niger strain OC3

DQ467988.1 Aspergillus parasiticus strain 2999 61 100 KX171029.1 Aspergillus flavus strain DO2

JF412778.1 Aspergillus minisclerotigenes strain CS5

42 KX215125.1 Penicillium aethiopicum strain AD17 Clade 3 Clade KT192315.1 Penicillium crustosum strain ZP-2 100 65 KX215133.1 Penicillium chrysogenum strain AD25

0.05

Figure 6.1 Phylogenetic analysis showing relationships of the 16S rRNA gene sequences of fungi isolated from South African cereal-based opaque beer. Taxa names with triangles are reference species isolated from other cereals. Bootstrap values (as percentages) are shown at internal nodes. The scale bar denotes number of substitutions per site.

6.3.4 Mycotoxin occurrence in South African cereal-based opaque beer samples

Data obtained in this study on the occurrence of mycotoxins in umqombothi are presented in Table 6.3. Trichothecenes (TCs) are commonly found as contaminants in feeds and foods, and their consumption can lead to alimentary haemorrhage and vomiting (Bennett & Klich, 2003). The dominating mycotoxin group in the beer was the TCs with DON as the prevalent mycotoxin; being present in 84% of the beers at a maximum level of 72 µg/L and no sample exceeded the EU maximum limit of 750 µg/kg for cereals meant for direct consumption. In Africa, only few studies have reported on the occurrence of DON in beer: Mary et al. (2014) reported the presence of DON in Busaa, a Kenyan traditional beer at levels ranging between

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200 and 360 µg/L with an incidence rate of 23%. Schothorst & Jekel (2003) also detected DON in only 3 of 51 beers from Dutch retailers at levels of 26 - 41 µg/L. Niessen et al. (1993) also reported the carry-over of DON into the final beer with a four-fold increase in levels during mashing. The acetylated derivatives of DON, 3-ADON and 15-ADON and other TCs (NIV, H-T2, T-2 and DAS) and ZEN were not found.

Table 6.3 Natural incidence and mycotoxin levels of South African cereal-based opaque beer and method performance characteristics Mycotoxin Positive Concentration µg/L Method performance samples (%) N= 32 Mean Maximum AR LOD LOQ (%) (µg/L) (µg/L) Deoxynivalenol 27 (84) 47 72 95 8.0 14 Nivalenol 0 na na 101 46 88 Neosolaniol 1 (3) 21 21 98 2.6 4.8 Fusarenon-X 2 (6) 167 173 96 49 96 3-Acetyldeoxynivalenol 0 na na 104 10 20 15-Acetyldeoxynivealenol 0 na na 99 5.0 8.0

Aflatoxin B1 2 (6) 6 7 100 2.5 5.0 Aflatoxin B2 0 na na 99 1.1 2.2 Aflatoxin G1 0 na na 96 1.3 2.6 Aflatoxin G2 1 (3) 5 5 98 1.7 3.4 ƩAflatoxins 2 (6) 6 11 na na na Diacetoxyscirpenol 0 na na 103 0.7 1.2 Alternariol 22 (69) 47 54 98 3.4 6.8 Alternariol Methyl Ether 11 (34) 41 57 100 9.1 18 HT-2 Toxin 0 na na 99 3.0 6.0 T-2 Toxin 0 na na 96 3.0 6.0 Fumonisin B1 17 (53) 151 182 89 8.3 18 Fumonisin B2 10 (32) 96 143 91 11 22 Fumonisin B3 2 (6) 36 37 94 14 28 Ʃ Fumonisin B1, B2 17 (53) 132 270 na na na Ʃ Fumonisin B1, B2, B3 17 (53) 125 303 na na na Ochratoxin A 0 0 0 99 5.2 10 Sterigmatocystin 21 (66) 18 43 95 1.7 3.4 Roquefortine C 5 (16) 3 4 98 0.8 1.6 Zearalenone 0 na na 102 3.6 7.2 Enniatin B 24 (75) 17 44 90 2.4 4.8

a na=not applicable. b N=sample size. AR: Apparent recovery. LOD: Limit of Detection, LOD: Limit of Quantification

Enniatins constitute an emerging food safety issue because of their increased occurrence in foods, particularly cereals (Jestoi, 2008). This is the first documented report on the occurrence of ENN B in umqombothi at an incidence rate of 75% and a maximum contamination level of 44 µg/L. Unlike DON, reports have demonstrated the reduction of ENN B during beer production. During the malting process, levels of ENN B decreased by 30% to below LOD at the beer maturation stage due to the phenomenon of its accumulation in rootlets, which are removed from the malt as side products (Vaclavikova et al. 2013).

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Our study reported FB contamination levels (36 -182 µg/L) in beer samples lower than those reported by Matumba et al. (2014) in Malawian traditional beers (mean: 1,745 µg/L) and Xhosa Beer (max: 1,329 µg/L) in the Transkei region, South Africa (Shephard et al. 2005). Fumonisins like some other mycotoxins in raw materials are carried into traditional beer (Malachova et al. 2010). The occurrence of FBs in food products, particularly maize was linked to the high incidence of oesophageal cancer in the Transkei region of South Africa (Shephard et al. 2005). Although potentially aflatoxigenic Aspergillus spp. were recovered from the beer samples, incidence of AF contamination was fairly low. Only 6% of the 32 beer was positive for AFB1 and values were 5.8 and 7.0 µg/L, higher than the maximum level of 2 µg/kg for individual AFs and 4 µg/kg for total AFs in ready to eat foods (EC, 2008). However, the values were within the permissible levels of total AFs (10 µg/kg) in South African foods (Sibanda et al. 1997).

To the best of our knowledge, we provide the first report on the occurrence of STE, ROQ-C, AME and AOH in umqombothi from South Africa. Some of the fungi (A. versicolor (STE) and P. crustosum (ROQ-C)) that produce these metabolites were also detected in this study. STE was categorised in 1987 as a class 2B possible human carcinogen (IARC, 2002) and was detected in 66% of the samples. The same toxin was however, not found in traditional cereal-based beverages (kunu-zaki and pito) consumed in rural Nigeria (Ezekiel et al. 2015), but estimated low levels were obtained in Irish beers (Versilovskis et al. 2008). AME and AOH are the major benzopyrone mycotoxins produced by Alternaria alternata and both mycotoxins were detected in beer analysed with maximum levels of 54 and 57 µg/L, respectively. Toxicological data on AOH and AME are limited but in vitro studies have established these toxins as being mutagenic and exhibiting some carcinogenic properties (EFSA, 2011). Alternaria toxins are however currently not regulated in food or feed (Scott et al. 2012).

We postulate that the multiple mycotoxins in the beer are a direct consequence of poor and uncontrolled processing conditions, particularly malting, which involves increasing the MC of the grains and interaction with humid environment. Similarly, improper storage, the utilization of contaminated grains, adjuncts such as maize, sorghum, and their malts can significantly contribute to increased mycotoxin formation. Natural occurrences of mycotoxins have been reported in ingredients meant for traditional beer production (Odhav & Naicker, 2002; Mngqawa et al. 2016). Shephard et al. (2005) highlighted the use of homegrown

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The co-occurrence of mycotoxins is significant because they may toxicologically interact with each other potentiating significant synergistic and additive health effects especially among those found at high concentrations (Njobeh et al. 2012). All the samples analysed were contaminated with at least two mycotoxins (Figure 6.2). This phenomenon has been reported for several mycotoxins particularly Fusarium toxins in cereals across Africa (Chilaka et al. 2016). This is because of the co-existence of several fungal species within the same commodity as well as their ability to produce more than one mycotoxin under different conditions (aw and temperature). DON usually co-occurs with its acetylated forms (Jestoi, 2008), but this was not the case in this study. It is important to note that even though there are mycotoxin regulations for most countries in the world, the effect of co-occurring mycotoxins is not taken into account when establishing these limits and needs to be further studied. A particular sample had up to nine mycotoxins (DON, NEO, FUS-X, ENN B, AOH, FB1, FB2, AME, and STE) that co-occurred and this may confer some health risks.

Figure 6.2 Spider plot of co-occuring mycotoxins in South African tradtional cereal- based opaque beer

6.3.5 Estimation of mycotoxin dietary intakes among beer consumers

To estimate the degree of human exposure to mycotoxins via consumption of traditional beer within the study location, the level of beer consumption within Africa was considered based

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132 -792 µg for FB1 + FB2 which translates to mean daily exposure of 2.20 and 13.20 µg/kg bw/day for a 60 kg adult. These values are higher than the TDI of 2 µg/kg bw/day for the sum of FB1 and FB2 (Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2001).

On the other hand, dietary exposure was obtained using upper and lower bound exposure scenarios whereas the lower bound (LB) was derived by assigning a value of zero to samples wherein the targeted analyte were not detected. Upper bound (UB) was obtained by assigning LOD to all samples with non-detected results and LOQ to samples with results that are less than LOQ but greater than LOD. The total mean intake for the upper bound (worst case) scenario for the consumption of 6 L of umqombothi for DON and FB1 + FB2 were 4.09 and 6.12 μg/kg bw/day, respectively. These values are 4 and 3 times greater that the TDI set for

DON (1 μg/kg bw/day) and FB1 + FB2 (2 μg/kg bw/day), respectively. The LB and UB scenario results further showed that the umqombothi samples represented an important dietary exposure of DON and FB amongst populations that consume more than a litre of the beer. This thus represents a health risk particularly amongst heavy drinkers and makes the beer a potential contributor of mycotoxins intake in South Africa as observed by Shephard et al. (2005). There is no TDI for AF because an exposure as little as < 1 µg/kg bw/day can significantly contribute to liver cancer risk (JECFA, 1999). Combined with possible exposure from other diets or other mycotoxins within the same sample, these estimates are of toxicological concern.

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Table 6.4 Deoxynivalenol and fumonisin exposure based on the consumption of different volumes of umqombothi in μg/kg bw/day

Consumption Exposure based on means of Exposure based on non-detect values positive samples

DON FB1 FB1+FB2 DON FB1 FB1+FB2 LB UB LB UB LB UB 1 Litre/day 0.78 2.52 2.20 0.66 0.68 1.34 1.40 0.92 1.02 2 Litres/day 1.57 5.03 4.40 1.32 1.36 2.68 2.80 1.84 2.04 3 Litres/day 2.35 7.55 6.60 1.98 2.05 4.02 4.20 2.76 3.06 4 Litres/day 3.13 10.07 8.80 2.64 2.73 5.36 5.60 3.68 4.08 5 Litres/day 3.92 12.58 11.00 3.31 3.41 6.70 7.00 4.60 5.10 6 Litres/day 4.70 15.10 13.20 3.97 4.09 8.04 8.40 5.52 6.12 aLB: Lower bound bUB: Upper bound

6.4 Conclusion

The present study provides information on the occurrence of fungi and mycotoxins in maize- based opaque beer (umqombothi) consumed in Gauteng, South Africa. The presence of different mycotoxigenic strains and their mycotoxins were evident with A. flavus as the dominant toxigenic species found. Saccharomyces cerevisiae had the highest fungal load as a consequence of its active fermentative role in beer processing. Previously unreported fungal spp. as well as new fungal strains were recovered in this study. DON, AOH, AME, ENN B and FB were found in relatively high number of samples with DON as the dominant mycotoxin whereas HT-2, T-2, NIV, OTA and ZEN were not detected in any sample. The co- occurrence of at least 2 mycotoxins within the same sample is of health concern due to the additive and/or synergistic effect especially when considering that this alcoholic beverage is consumed almost on a daily basis among most rural populated groups in South Africa. The data obtained in this study also reveal that increased mycotoxin exposure arising from the consumption of umqombothi poses a health risk. The levels of contamination of both fungi and mycotoxin in the samples justify the need for regular and in-depth monitoring to assess public health risks and implement strategies to reduce consumers’ exposure to mycotoxins. Moreover, reducing mycotoxigenic fungi and mycotoxins in beer is paramount, largely dependent on the quality of the raw materials used for beer processing and the inherent processing and storage conditions. Therefore, strategies aimed at mitigating mycotoxin contamination and toxigenic fungal colonization need to be enacted.

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Acknowledgments

The authors wish to appreciate the Organisation for Women in Science in the Developing World (OWSD), Italy, African Women in Agricultural Research and Development (AWARD), Kenya, Centre of Excellence for Food Security and Safety (COE), South Africa and the MYTOX consortium, hosted in the Laboratory of Food Analysis, Ghent University, Belgium for their financial and technical contributions to this study.

Conflict of Interest

The authors have no conflict of interest to declare.

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CHAPTER SEVEN

METABOLITE PROFILING AND TOXIGENICITY OF FUNGAL ISOLATES IN FERMENTED FOODS FROM SELECTED NIGERIAN AND SOUTH AFRICAN MARKETS

*Ifeoluwa Adekoyaa, *Patrick Njobeha, Adewale Obadinaab, Sofie Landschootc, Sheila Okothd, Marthe De Boevree, and Sarah De Saegere aDepartment of Biotechnology and Food Technology, University of Johannesburg, South Africa bDepartment of Food Science and Technology, Federal University of Agriculture, Abeokuta, Nigeria cDepartment of Applied Bioscience Engineering, Ghent University, Belgium dDepartment of Botany, School of Biological Sciences, University of Nairobi, Kenya eLaboratory of Food Analysis, Department of Bioanalysis, Ghent University, Ghent, Belgium *Corresponding authors: [email protected] and [email protected]

Abstract

Fungal species recovered from seven fermented foods from Nigeria and South Africa was studied to establish their toxigenic potential in producing an array of secondary metabolites including mycotoxins. In total, 385 fungal isolates (240 Aspergillia, 96 Penicillia and 49 Fusaria) species were grown on solidified yeast extract sucrose agar and their metabolites were extracted and quantified via ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). In order to group the isolates and establish co-occurrence of metabolites, hierarchal clustering and pairwise association analysis were performed. In total,

49 secondary fungal metabolites were screened including aflatoxins B1 (AFB1), ochratoxin A

(OTA), fumonisin B1 (FB1), deoxynivalenol (DON), cyclopiazonic acid (CPA), kojic acid (KA), sterigmatocystin (STE) and versicolorins. The results showed that of the 385 fungal strains tested, over 41% were toxigenic. A. flavus and A. parasiticus strains were the principal producers of AFB1. AFB1 and CPA had a positive association whereas OTA was produced by 67% of the A. niger strains in the range of 28 – 1,302 µg/kg. The STE producers found were A. versicolor (n=12), A. amstelodami (n=4), and A. sydowii (n=6). Amongst the Fusarium species tested, F. verticillioides produced FB1 (range: 77 - 218 µg/kg). The production of multiple metabolites by a single fungal species was also established. Even though the

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Keywords: Toxigenicity, fungi, mycotoxins, fermented foods, metabolites, and LC-MS/MS

Highlights

 Secondary metabolite profiling of 385 fungal species isolated from fermented foods was performed.

 There was a synthesis of some principal mycotoxins (AFB1, STE, OTA, FB1) and some toxic secondary metabolites (CPA, KA).

 A. flavus was the main producer of AFB1 (range of 27 to 7,406 µg/kg), while P. chrysogenum produced ROQ C and maximum level of T-2 from F. sporotrichioides was 1,749 µg/kg.  The mycotoxins levels of some species were high and the toxigenicity of multiple metabolites by a single species was noted.  The toxigenic potential of the isolates heightens the risk of mycotoxin contamination of the fermented foods.

7.1 Introduction

Secondary metabolism in filamentous fungi involves the synthesis of a wide array of chemical compounds including mycotoxins, which are not necessary for normal growth and development. However, a number of them have been widely exploited for biological applications such as antibiotics, anticancer and anti-infective agents to name a few (Fox and Howlett, 2008; Osbourn, 2010). Mycotoxins are secondary fungal metabolites that are harmful substances with deleterious effects on human and animal health. Apart from the production of secondary metabolites, filamentous fungi (Aspergillus awamori, A. niger, and F. oxysporum) play an important role in the production of enzymes of biotechnological significance while others like A. oryzae and A. luchuensis, are used in food fermentation (Jayani et al., 2005).

An important consideration in the safety aspect of foods derived from microorganisms is the safety of the competing and indigenous microbial strains/species due to possible production

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In format of Food Chemical Toxicology of toxic secondary metabolites including mycotoxins. Our previous studies (Adekoya et al., 2017; Adekoya et al., 2018) demonstrate the high prevalence of significant mycotoxin- producing fungi in fermented products viz.: fermented: maize meal (mahewu), sorghum gruel (ogi baba), maize gruel (ogi), locust beans (iru), melon (ogiri), African oil bean seed (ugba), and cereal based opaque beer (umqombothi) from Nigeria and South Africa. Therefore, fungal contamination in these products exasperates the risk of mycotoxin contamination.

Aflatoxins (AFs) are synthesized by members within the Aspergillus genera with A. parasiticus, and A. flavus being the most economically significant producers (Shundo et al., 2009). This group of mycotoxins constitutes the most toxic and carcinogenic compounds

(Lewis et al., 2005) particularly aflatoxin B1 (AFB1) which is the most potent. AFB1 has been classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) (2002). During the biosynthesis of AFs, several metabolites are produced as precursors, which have much lower toxicity (versicolorins, sterigmatocystin (STE), and O- methylsterigmatocystin (OMST)) (Yu et al., 2004; Jiang et al., 2015). Some other toxic secondary metabolites produced by some Aspergillus species are cyclopiazonic acid (CPA), kojic acid (KA), patulin (PAT), and ochratoxin A (OTA).

Ochratoxin A is known to be nephrotoxic and hepatotoxic and mainly produced by P. verrucosum in the warm climates and by A. ochraceus in the tropics but may also be produced by A. niger. Within the Fusarium genus, species that produce trichothecenes (TCs), fumonisins (FBs), and zearalenone (ZEN) have received the highest recognition (Yazar and Omurtag, 2008). F. proliferatum, F. verticillioides, and F. oxysporum are examples of FB producers (Pestka and Smolinski, 2005; Phoku et al., 2017), while F. culmorum, and F. graminearum are notable deoxynivalenol (DON) producers (Phoku et al., 2017). Furthermore, Fusarium mycotoxins exhibit immuno-suppressive, oestrogenic, and mutagenic effects in humans and animals (Yazar and Omurtag, 2008). Due to their impact on health as toxin producers, it is important to investigate the toxigenic properties of fungi commonly isolated in foods. Although, the production of mycotoxins are restricted to only a few fungal species or strains (Huwig et al., 2001), multiple toxin production by a single specie or strain also exists. Furthermore, the toxin-synthesizing ability of mycotoxigenic fungi is influenced by different growth and environmental conditions leading to complicated mycotoxin manifestations (Shi et al., 2017).

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Currently, the polyphasic taxonomic approach which involves morphological, physiological, biochemical and molecular characterization such as use of DNA sequence analysis and ultra- performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is increasingly being used for fungal classification due to their complex taxonomies (Oliveri et al., 2008; Lamboni et al., 2016). Although, numerous secondary metabolites have been poorly investigated (Blumenthal, 2004; Nielsen et al., 2009), multiple toxin production have also not been fully understood but the resultant synergistic effects have been established (Van Egmond et al., 2007; Shi et al., 2017). It is therefore paramount to establish the toxigenic potentials of various Aspergillus, Penicillium, and Fusarium spp. isolated from fermented products. To accomplish this task, several isolates of Aspergillus, Penicillium, and Fusarium spp. previously recovered from various fermented food products, after establishing their identities by molecular means (Adekoya et al., 2017, Adekoya et al., 2018), were investigated for their ability to produce mycotoxins and other secondary metabolites.

7.2 Materials and Methods

7.2.1 Materials

7.2.1.1 Reagents

LC-MS grade methanol and glacial acetic acid were purchased from Biosolve B.V. (Valkenswaard, Netherlands) and analytical grade ethyl acetate, ammonium acetate, acetonitrile (ACN), and dichloromethane (DCM) were purchased from Merck (Darmstadt, Germany; Johannesburg, South Africa). Water was purified using a Millipore Milli-Q gradient system (Brussels, Belgium). All other reagents used were of analytical grade.

7.2.1.2 Standards

Standards used for the quantification of Aspergillus metabolites included AFB1, aflatoxin B2

(AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), OTA, and STE, which were purchased from Sigma-Aldrich (Bornem, Belgium). Quantitative standards for sterigmatocystin analogue (STE-A), OMST, dihydro-O-methyl sterigmatocysitin (DHOMST), Oxy-O-methyl sterigmatocystin (OxyHOMST), dihydroxyl-O-methyl sterigmatocystin (DHoxyHOMST), aspergillic acid (AA), hydroxyneoaspergillic acid (OH-AA), aflavanine (AFN), dihydro- aflavine (DH-AF), aflavarin (AFV), aflavarin analog 1 (AFV-1), aflavarin analog-2 (AFV-2), aflatrem (AFTR), KA, CPA, ß-cyclopiazonic acid (ß-CPA), aspertoxin 1 (ASPT), aspertoxin 2 (ASPT-2), paspaline (PAS) paspalinine (PASL), versiconol (VOH), versiconal hemiacetal

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For the Penicillium metabolites, OTA, and STE standards were purchased from Sigma- Aldrich (Bornem, Belgium), while standards for the quantification of Fusarium metabolites including diacetoxyscirpenol (DAS) and T-2 toxin (T-2) were procured from Biopure

Referenzsubstanzen (Tulln, Austria). Fumonisin B3 (FB3) was obtained from Promec Unit

(Tygerberg, South Africa) while Zearalenone (ZEN), DON, fumonisin B1 (FB1), fumonisin

B2 (FB2), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), neosolaniol (NEO), nivalenol (NIV), HT-2 toxin (HT-2), and fusarenon-X (FUS-X) were purchased from Sigma-Aldrich (Bornem, Belgium).

7.2.1.3 Mycotoxigenic potential of fungal isolates

The ability of the Aspergillus, Penicillium, and Fusarium species previously isolated from the fermented foods to synthesize secondary metabolites was investigated. Pure cultures of the isolates were sub-cultured unto petri dishes containing solidified yeast extract sucrose (YES) agar using the streak plate technique. Afterwards, the plates were incubated at 28 oC for 2 weeks.

7.2.2 Methods

7.2.2.1 Multi-mycotoxin extraction

Aspergillus and Penicillium metabolites were extracted from solid cultures using the agar plug technique as described by Phoku et al. (2017). Pure fungal colonies together with media (1 g) were plugged into an amber vial containing HPLC grade methanol (1 mL) using a cork borer. The content was vortexed for 1 min, filtered through a 0.2 µm Millex syringe filter unit (Merck, Johannesburg, South Africa) into a screw-cap amber vial. To facilitate evaporation, the vials were placed on a heating block set at 60 oC under a stream of nitrogen gas. The dried extracts were reconstituted in 1 mL of injection solvent consisting of 60:40 (v/v) of mobile phase A and B (A: water/methanol (95/5, v/v), 5 mM ammonium acetate and 0.1% formic acid; B: methanol/water (95/5, v/v), 5 mM ammonium acetate and 0.1% formic acid) and vortexed for 1 min. The extract was dispensed into Ultrafree® PVDF centrifugal filters (Merck, Darmstadt, Germany), centrifuged at 14,000 rpm for 5 mins, transferred into vials and injected into the UPLC-MS/MS.

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For Fusarium toxins, they were also extracted from cultures as described by Phoku et al. (2017). Briefly, 50 mL of ACN:water (60:40, v/v) were added to 10 g of macerated agar- containing mycelia in a 250 mL conical flask and placed on a shaker for 1 hr. The mixture was filtered through a Whatman #4 filter paper (Merck, Johannesburg, South Africa), and the pH of the filtrate was adjusted to 6.2 ± 0.3 with 1 M sulphuric acid. The filtrate was emptied into a separation funnel (250 mL) and extracted (x3) with dichloromethane (25 mL). Acetonitrile (25 mL) was added to the previously extracted solution, and filtered through a bed of sodium sulphate anhydrous (Merck, Johannesburg, South Africa) to remove moisture. The solution was dried over a stream of nitrogen gas and the remaining step as previously described for the Aspergillus and Penicillium extracts was followed.

7.2.2.2 Liquid chromatography-tandem mass spectrometry

7.2.2.2.1 Detection and quantification of Aspergillus metabolites The metabolites produced by the Aspergillus species was detected and measured using a Waters Acquity UPLC coupled to a XEVO TQ-S triple quadrupole tandem mass spectrometer (Waters, Milford, MA, USA). The data acquisition and processing were done using the MassLynx™ (V. 4.1) and QuanLynx® (V. 4.1) software (Manchester, UK). The chromatographic separation of analytes was done using the HSS T3 (100 mm x 2.1 mm, 1.8 µm) column (Waters, Zellik, Belgium) with a BEH C18 guard column (2.1 mm x 5 mm, 1.7 µm) (Waters, Zellik, Belgium). Mobile phase A and B were pumped at a flow rate of 0.4 mL/min. The sample injection volume was set at 5 μL, the total analytical run time was 32 mins and the pressure ranged between 0 and 15,000 psi. Ionization was performed in the positive electrospray ionization (ESI+) mode, while data acquisition was carried out in the multiple reaction monitoring (MRM) mode (See Appendix 7.1 for MRM transitions of AFG1 and AFB2 standards). For each of the 34-targeted Aspergillus analytes, two-product ion transitions were selected and their collision energies optimized.

The following instrumental settings were applied: desolvation temperatures 500 °C; capillary voltage 3 kV; cone voltage 10 V; source offset voltage 50 V; cone and desolvation gas flows of 150 and 1,000 L/h, respectively. For the identification of the targeted metabolites, the performance criteria of the European Commission (EC) No. 2006 were met. Information in respect of the transition of the different Aspergillus metabolites is shown in Table 7.1. The method was validated the metabolites according to the guidelines of EC (2002), and more information on this method is provided by Okoth et al. (2018).

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Table 7.1 Mass spectrometric parameters for different target analytes

Component Precursor ion Product Cone Voltage Collision Expected (m/z) ion (V) energy retention time (eV) (mins) Kojic acid 143.1 69.1 35 30 3.70 125.1 Methylisocoumarin 307.1 149.1 35 35 8.68 247.1 Aflatoxin G2 331.1 245.1 25 18 8.95 313.1 Aflatoxin G1 329.1 243.1 35 25 9.00 311.0 Aflatoxin B2 315.1 259.1 25 35 9.43 286.9 Speradine A 367.2 160.1 35 35 9.72 266.1 Aflatoxin B1 313.1 270.1 70 35 9.92 285.1 Sterigmatocystin Analogue 325.1 281.1 35 34 9.95 310.1 Ochratoxin A 214.0 142.0 51 25 9.96 152.0 Oxy-o-methyl Sterigmatocystin 371.1 282.1 35 35 10.10 315.1 Dihydroxyl-o-methyl STE 373.1 322.1 35 35 10.32 355.1 Aflavarin 455.2 379.2 35 40 10.35 413.2 Aspertoxin 1 355.1 322.1 35 30 10.51 340.1 Aspertoxin 2 355.1 322.1 35 40 10.53 340.1 Aspergillic acid 225.2 165.1 35 40 10.70 207.2 Versiconol 361.1 285.1 35 35 11.13 325.1 Aflavarin-Analog 2 425.1 334.1 35 35 11.24 383.1 Dihydro-o-methyl Sterigmatocystin 341.1 285.1 35 35 11.49 326.1 Versiconal Hemiacetal Acetate 401.1 283.1 35 35 11.52 307.1 Flavacol 209.2 123.1 35 40 11.78 137.1 Dehydro-Aflavanine 438.3 285.2 35 40 12.17 402.3 O-methyl Sterigmatocystin 339.1 306.1 35 35 12.69 324.1 Aflavarin-Analog 1 439.1 365.1 35 35 12.93 397.1 Noranthrone 357.2 245.1 35 35 13.02 273.1 Aflatrem 502.3 156.1 35 40 13.17 198.1 Paspalinine 434.2 130.1 35 40 13.78 376.2 Sterigmatocystin 325.0 281.1 35 34 14.28 310.1 Cyclopiazonic acid 337.2 140.1 35 40 14.29 196.1 Hydroxyneoaspergillic acid 241.2 137.1 35 40 14.31 163.1 ß-Cyclopiazonic acid 339.2 154.1 35 40 14.75 198.1

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Dytryptohenaline 693.3 318.2 35 40 14.80 346.2 Paspaline 422.3 158.1 35 35 15.04 386.3 Leporin C 336.2 200.1 35 40 17.40 214.1 Aflavinine 406.3 180.1 35 45 19.38 224.3

7.2.2.2.2 Detection and quantification of Penicillium and Fusarium metabolites An Acquity UPLC (Waters, Milford, MA, USA) instrument attached to a Waters Quattro Premier XE tandem mass spectrometer was used for the detection and quantification of both Penicillium and Fusarium metabolites. The chromatographic and spectrometric conditions used were similar to those described by Ediage et al. (2011). Details on the transition of the different metabolites are described by Monbaliu et al. (2009) and De Boevre et al. (2012). Also, the EC (2006) guideline was followed for the identification of the targeted metabolites.

7.2.2.3 Data Analysis

The R software package (R core Team, 2014) version 2.15.3 was used for statistical evaluation. The secondary metabolites of the isolates were grouped by hierarchical clustering based on the simple matching distance, which measured the dissimilarity between binary sample sets. Furthermore, based on the expected and observed co-occurrence of the metabolites, pairwise associations between metabolites were examined and classified as negative, positive or random according to the probabilistic model of Veech (2013).

7.3 Results and discussion

In recent years, the varieties of foods potentially attacked by mycotoxigenic fungi and number of fungal species have significantly increased. Tables 7.2 and 7.3 show the mycotoxins produced by the isolates of Aspergillus, Penicillium and Fusarium, while Figures 7.1 and 7.2 further show the hierarchical clustering based on metabolite profiles of the Aspergillus spp. previously recovered from fermented foods. Aspergillus spp. (n=240) were screened for 34 secondary metabolites produced by different Aspergillus spp. as described by Larsen (2005), Samson and Varga (2007) and Samson et al. (2014). Up to 70% of the A. flavus isolates produced AFB1 (range: 27 – 4,406 µg/kg), while 77, 64, 31 and 19% of A. parasiticus isolates (n=36), respectively, produced AFB1 (range: 89 – 3,602 µg/kg), AFB2

(range: 37 – 566 µg/kg), AFG1 (range: 36 – 322 µg/kg), and AFG2 (range: 34 - 664 µg/kg).

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Table 7.2 Production of mycotoxins by Aspergillus, Penicillium, and Fusarium spp. isolated from lactic acid fermented products produced in Nigeria and South Africa

Isolated species Ogi Ogi baba Umqombothi Mahewu Nº of Nº of Toxin produced Nº of iso. Nº of Toxin produced Nº of iso. Nº of Toxin Nº of iso. Nº of Toxin iso. spp. Tox. (range: µg/kg) spp. (%) Tox. (range: µg/kg) spp. (%) Tox. produced spp. (%) Tox. produced (%) spp spp spp (range: µg/kg) spp (range: µg/kg) *Aspergillus species A. amstelodami 1 (2) 1 STE (371) ------A. clavatus 2 (5) - - 2 (13) - - - - - 3 (14) - - A. flavus 12 (29) 7, 3 AFB1 (109-231), 6 (38) 4,3 AFB1 (217- 6 (26) 6, 3 AFB1 (127- 7 (32) 4,1 AFB1 (69- STE (434-600) 1,556), STE (49- 1,117), 1,931), STE 701) STE (4-32) (119) A. fumigatus 4 (10) - - - - - 2 (9) - - 3 (14) - - A. minisclerotigenes - - - 1 (6) - - 2 (9) 1 AFB1 (96) - - - AFB2 (106) A. niger 7 (17) 7 OTA (197-1302) 1 (6) 1 OTA (411) 3 (13) 2 OTA (28-55) 3 (14) 2 OTA (89-212) A. parasiticus 7 (17) 5, 5, 4, 3 AFB1 (89- 2 (13) 2,2,1,1 AFB1 (721- 3 (13) 2,2,1 AFB1 (172- 3 (14) 2,2,1 AFB1(109-117), 1,018), AFB2 1,030), AFB2 3602), AFB2(58-192), (118-324), AFG1 (185-498), AFG1 AFB2 (288- AFG1(109) (95-158), AFG2 (48), 566), (978-664) AFG2 (34) AFG1 (322) A. ruber 1 (2) ------A. spp - - - 1 (13) ------A. sclerotiorum ------2 (9) - - - - - A. sydowii 1 (2) 1 STE (91) 1 (13) 1 STE (433) 2 (9) 1 STE (53) - - - A. tritici 2 (5) - - 1 (13) - - 2 (9) - - - - - A. tubingensis ------1 (5) - - A. ustus ------A. versicolor 4 (10) 2 STE (97-477) 1 (13) 1 STE (422) 1 (4) 1 STE (54) 2 (9) - -

Penicillium species P. chrysogenum 5 (39) 1 ROQ C (22) - - - 3 (38) 1 ROQ C (278) 2 (4) - - P. expansum - - - 2 (33) ------P. aethiopicum ------3 (38) - - - - - P. camemberti 2 (15) ------P. citrinum 3 (23) - - 1 (17) ------P. crustosum 2 (15) - - - - - 2 (25) - - 3 (6) - - P. raistrickii - - - 2 (33) ------P. mallochi - - - 1 (17) ------

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Isolated species Ogi Ogi baba Umqombothi Mahewu Fusarium species Nº of iso. Nº of Toxin produced Nº of iso. Nº of Toxin produced Nº of iso. Nº of Toxin Nº of iso. Nº of Toxin spp. (%) Tox. (range: µg/kg) spp. (%) Tox. (range: µg/kg) spp. (%) Tox. produced spp. (%) Tox. produced spp spp spp (range: µg/kg) spp (range: µg/kg)

F. 1 (43) ------chlamydosporum F. fujikuroi - - - 1 (33) ------F. sporotrichioides 2 (29) 1, 1, 2 HT2 (127), ------DAS, NEO Fusarium sp. - - - 2 (67) 2,2, 1 FB3 (79), DON - - - 2 (40) 2 ZEN (180-309) (27-300), 3ADON F. verticillioides 4 (57) 4 FB1 (77-218) - - - 3 (100) 3 FB1 (92-109) 3 (60) 3, 1 FB2 (103 -195) FB3 (88)

Note: Nº:Number; Iso.: isolated; Tox.: toxigenic; *Only Aspergillus metabolites with available standards for quantification (AFB1, AFB2, AFG1, AFG2, STE, OTA) are presented in Table 7.2

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Table 7.3 Production of mycotoxins by Aspergillus, Penicillium and Fusarium spp. isolated from alkaline fermented products produced in Nigeria and South Africa

Isolated species Ugba Ogiri Iru *Aspergillus species Nº of iso. Nº of Toxin produced Nº of iso. Nº of Toxin produced Nº of iso. Nº of Toxin produced spp. (%) Tox. spp (range: µg/kg) spp. (%) Tox. spp (range: µg/kg) spp. (%) Tox. spp (range: µg/kg) A. amstelodami ------3 (5) - - A. candidus 2 (7) - - - - - 3 (5) - - A. clavatus - - - 1 (2) - - 3 (5) - -

A. flavus 12 (39) 10, 12 AFB1 (27-1,889), STE 15 (36) 9, 9 AFB1 (96-7,406), 22 (34) 16, 4 AFB1 (82-1,723), (28-325) STE (94-736) STE (77-128) A. fumigatus 3 (8) - - 6 (14) - - 10 (15) - - A. minisclerotigenes - - - 3 (7) 1 AFB1 (242) - - - A. niger 3 (8) 3 OTA (76-1,265) 4 (10) 2 OTA (118-229) 3 (5) 1 OTA (78) A. oryzae - - - 2 (5) - - - - -

A. parasiticus 4 (13) 4, 4, 1 AFB1 (391-1,132), 6 (14) 5, 5, 1, 1 AFB1 (120-1,470), 11 (17) 8, 3, 2, 2 AFB1 (206-445), AFB (37-504), AFG AFB (83-323), AFB (51-340), 2 1 2 2 (46) AFG1(69), AFG2 AFG1 (36-59), (97) AFG2 (49-71) A. ruber ------1 (2) - - A. spp 1 (3) ------A. sclerotiorum 3 (10) 1 OTA (161) - - - 2 (3) - - A. sydowii ------2 (3) - - A. tritici ------A. tubingensis 2 (7) - - - - - 2 (3) - - A. ustus - - 3 (7) - - - - - A. versicolor 1 (3) 1 STE (101) 2 (5) 2 STE (89-500) 3 (5) 1 STE (89)

Penicillium species P. chrysogenum 4 (29) 4 ROQ C (360 -1,260) 6 (30) - - 9 (30) 6 ROQ C (13-57) P. expansum 1 (7) - - - - - 2 (7) - - P. polonicum ------1 (3) - - P. lanosocoeruleum ------2 (7) - - P. aethiopicum 2 (14) - - 2 (10) - - - - - P. camemberti ------1 (3) - - p. citrinum - 2 (10) - - 2 (7) - p. verrucosum 2 (14) 1 OTA (15) 1 (5) 1 OTA (19) 3 (10) 2 OTA (18-32) p. crustosum 2 (14) - - 3 (15) - - - - - P. flavigenum - - - 2 (10) - - - - - P. raistrickii - - - 4 (20) - - 3 (10) - -

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Isolated species Ugba Ogiri Iru Nº of iso. Nº of Toxin produced Nº of iso. Nº of Toxin produced Nº of iso. Nº of Toxin produced spp. (%) Tox. spp (range: µg/kg) spp. (%) Tox. spp (range: µg/kg) spp. (%) Tox. spp (range: µg/kg)

P. glabrum 2 (14) - - - - - 1 (3) - - P. rubens ------2 (7) - - P. steckii 1 (7) - - - - - 1 (3) - - P. mallochi ------3 (10) - -

Fusarium species F. andiyazi 2 (25) ------F. chlamydosporum ------4 (32) 1 DAS F. fujikuroi - - - 1 (10) - - - - - F. proliferatum 2 (25) - - 2 (20) - - 2 (16) - - F. sporotrichioides 1 (13) 1 NEO 2 (20) 2, 2 T-2 (134), 2 (8) 2, 2, 2 T-2 (139-1,749), DAS DAS, FUS-X Fusarium sp. 1 (13) 1, 1, 1 DON (870), ZEN - - - 1 (8) 1,1 ZEN (139) (197), 3 ADON F. verticillioides 2 (38) 1, 1, 1 FB1 (81), FB2 (63), 5 (50) 1, 3 FB2 (234), FB3 (79- 4 (31) - - FB3 (205) 148)

Note: Nº:Number; Iso.: isolated; Tox.: toxigenic; *Only Aspergillus metabolites with available standards for quantification (AFB1, AFB2, AFG1, AFG2, STE, OTA) are presented in Table 7.3

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Generally, species within the Aspergillus section Flavi exhibit close morphology and phylogenetic relationships and are grouped into two (aflatoxigenic and domesticated species) based on their effects on food quality (Rodrigues et al., 2011). Aspergillus flavus and A. parasiticus are the principal species within the aflatoxigenic group that are associated with AF contamination of agricultural commodities. Aspergillus oryzae belong to the domesticated group of fungi together with A. sojae and A. tamarii, which are frequently used in food fermentation (Rodrigues et al., 2011). This corroborates our present study as the production of AFs was not evident among the A. oryzae species (n=2) isolated from ogiri, but some of its extrolites were identified, particularly CPA and KA. The most widely used extrolites in species identification are AFs, CPA, AA and KA (Samson and Varga, 2007; Varga et al., 2007) and each species is often distinguished by a particular metabolic profile (Larsen et al., 2005) but some might not produce the anticipated metabolites (Vaamonde et al., 2003; Rodrigues et al., 2011). A. parasiticus for example is not associated with CPA synthesis and is also readily distinguishable from A. flavus. Another group of A. flavus closely related isolate - A. minisclerotigenes produced AFB1 (range: 96 – 242 µg/kg), AA, CPA, KA, AFTR, PAS, AFV and AFN (Figures 7.1 and 7.2).

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97 83 15 14 91 12 43 13 53 48 94 89 98 80 79 52 49 8 6 5 65 7 70 4 3 58 41 57 51 95 38 54 88 87 93 42 30 31 66 22 69 82 26 25 24 81 68 17 23 39 90 73 72 67 2 85 84 75 74 78 16 37 29 61 1 20 35 45 33 40 27 32 21 44 55 71 28 9 11 56 100 101 36 34 10 62 59 60 96 76 63 19 86 102 99 92 47 46 50 18 77 64 Flavacol Aflatrem Aflavarin Leporin.C Paspaline Kojic.acid Aflavanine Versiconol Spreaine.A Paspalinine Aflatoxin.B2 Aflatoxin.B1 Aflatoxin.G1 Aflatoxin.G2 Noranthrone Aspertoxin.1 Aspertoxin.2 Ochratoxin.A STE.Analogue O-methyl.STE Aspergillic.acid Dihydro-aflavine Dytryptohenaline Sterigmatocystin Cyclopiazonic.acid Oxy-o-methyl.STE Methylisocoumarin Aflavarin.Analogue.2 Aflavarin.Analogue.1 ß-cyclopiazonic.acid Versiconol.Hemac.C Hydroxyneoaspergillic Dihydro-o-methyl.STE

Dihydroxyl-o-methyl.STE Figure 7.1 Hierarchical clustering based on metabolites profile of Aspergillus species isolated from fermented foods from Nigeria. NB: Colour codes indicate the presence (green) or absence (red) of a certain metabolite or toxin. Numbers listed corresponds with isolated species listed in Appendix 7.2 and 7.3 215

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34 25 93 36 35 66 65 6 40 39 38 37 33 63 67 41 5 118 131 88 19 18 13 73 130 50 112 87 48 111 72 64 125 124 8 7 32 121 116 120 98 81 110 100 75 62 1 22 74 54 71 56 24 31 53 49 59 58 113 70 47 86 107 109 126 127 128 10 9 11 14 26 27 28 29 42 43 44 45 55 57 61 76 77 84 90 91 92 94 95 96 104 105 106 108 115 122 123 132 133 134 136 137 135 69 4 46 117 85 119 82 68 103 78 102 79 80 99 21 3 60 138 52 129 83 89 30 23 2 20 17 16 12 51 15 114 97 101 Flavacol Aflatrem Aflavarin Leporin.C Paspaline Kojic.acid Aflavanine Versiconol Spreaine.A Paspalinine Aflatoxin.B1 Aflatoxin.B2 Aflatoxin.G1 Aflatoxin.G2 Noranthrone Aspertoxin.1 Aspertoxin.2 Ochratoxin.A STE.Analogue O-methyl.STE Aspergillic.acid Dihydro-aflavine Dytryptohenaline Sterigmatocystin Cyclopiazonic.acid Oxy-o-methyl.STE Methylisocoumarin Aflavarin.Analogue.1 Aflavarin.Analogue.2 ß-cyclopiazonic.acid Versiconol.Hemac.C Hydroxyneoaspergillic Dihydro-o-methyl.STE Dihydroxyl-o-methyl.STE Figure 7.2 Hierarchical clustering based on metabolites profile of Aspergillus species isolated from fermented foods from South Africa 216

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Also, the hierarchical clustering revealed STE, OMST, CPA, ß-CPA, AFB1, VOH, ASPT, and ASPT-2 (Figure 7.1), and STE, OMST, ß-CPA, VOH, ASPT, and ASPT-2 (Figure 7.2) as key compounds for chemical clustering. These metabolites have been found to be AF precursors within their biosynthetic pathways (Yu et al., 2004; Jiang et al., 2015), whereas

ASPT is the 12c-hydroxy derivative of OMST, which is a precursor of both AFB1 and AFG1 (Yabe and Nakajima, 2004). As shown in Figure 7.1, isolates having the same origin coincided with iru. However, isolates having the same origin do not necessarily have the same metabolic profile (Pildain et al., 2008). Interestingly, AFB1 and CPA had a positive pairwise association (Figure 7.3 and 7.4), thus denoting that if AFB1 is detected in the fermented foods, it is highly likely that CPA will also be present or vice versa. This conforms to the report of Vaamonde et al. (2003) on the co-contamination of agricultural commodities by both AFB1 and CPA. Zorzete et al. (2013) also reported the co-occurrence of both toxins in 11% of 70 peanut kernel samples from two cultivars.

Figure 7.3 Co-occurrence matrix of metabolites of Aspergillus species isolated from fermented foods from Nigeria

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Figure 7.4 Co-occurrence matrix of metabolites of Aspergillus species isolated from fermented foods from South Africa

According to Mogensen et al. (2010), up to 75% of A. niger isolates produced FBs and only 41% produced OTA. Our present studies revealed that among the 24 species of A. niger tested, 67% produced OTA (range: 28 – 1,302 µg/kg), while none produced FB. Furthermore, we did not observe the simultaneous production of FB and OTA by A. niger as reported by Nielsen et al. (2009) and Lamboni et al. (2016). However, the production of OTA by A. tubingensis has been overrated in previous studies due to the provision of false positives established via HPLC fluorescence detection (Perrone et al., 2006; Storari et al., 2012). Only 20% (n=5) of the isolates tested produced OTA (161 µg/kg) which is ochratoxigenic.

Samson and Varga, (2007) had earlier reported that A. sclerotium (Aspergillus section Circumdati) had ochratoxigenic potential. Sterigmatocystin has been associated with teratogenic and genotoxic effects in experimental animals and considered a potential carcinogen (Versilovskis and De Saeger, 2010). In our study, this toxin was produced by 75% of A. versicolor, 25% of A. amstelodami and 50% of A. sydowii. Aspergillus metabolites produced by A. ruber, A. candidus, A. clavatus, A. tritici, and A. ustus isolates were not

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The commonly occurring Fusarium mycotoxins are - DON, 3-ADON, 15-ADON, NIV, FUS-

X, T-2, HT-2, FB1, FB2, FB3, NEO, ZEN, DAS, and fusaric acid (FA) (Logrieco et al., 2002).

We found that FB1 (range: 77 - 218 µg/kg), FB2 (range: 63 - 234 µg/kg) and FB3 (range: 79 - 205 µg/kg), were produced by 38, 14 and 24% of F. verticillioides (n=21) in our present study. This corroborates the report of Phoku et al. (2017) that the fungus that is mostly linked to FB production is F. verticilliodes. F. fujikuroi also produces FBs, but this was not established in our study and its mycotoxigenic potential have been highlighted to be largely dependent on the inoculation conditions (Wulff et al., 2010). Type A TCs (HT-2, T-2, DAS and NEO) mycotoxins were produced by F. sporotrichioides, and the maximum level of T-2 produced was 1,749 µg/kg. Type A TCs are highly potent in mammals with T-2 being 10 times more potent than DON (Foroud and Eudes, 2009). In addition, a low incidence of DON was observed in the Fusarium isolates. According to Frisvad et al. (2007), secondary metabolite production and profiling is an effective way of identifying fungal species. In our present work, we found out that the Fusarium spp. that were not normally possible to be identified by conventional methods which were present in ugba and iru indicated close relationship to F. graminearum via their capacity to produce 3-ADON, DON and ZEN.

The toxigenic potential of the fungi isolated from the fermented foods could be due to the effect of the food matrices. Vaamonde et al. (2003) observed diversity in AFs produced by strains of Aspergillus section Flavi isolated from wheat, peanut, and soybean. While similar microflora were observed among fermented foods from Nigeria and South Africa, there were differences in the proportion of positive species that produced low, intermediate and increased concentrations of mycotoxins. Mycotoxin production by a species or strain has however, been found to be influenced by geographical locations (Lamboni et al., 2016). Okoth et al. (2012) observed higher prevalence of toxigenic A. flavus strains than non-

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In format of Food Chemical Toxicology toxigenic strains in five out of six locations of Kenya. Generally, 42% of the isolated fungi (n=385) were toxigenic and comparatively, higher incidence of toxigenic strains was observed amongst fungal species isolated from Nigerian fermented foods (47%, n=81/175) than those recovered from South African fermented foods (39%, n=78/200). The inherent tropical climatic conditions that prevail in Nigeria might have favoured the observed higher toxigenic potential of the isolates, denoting a higher risk of mycotoxin contamination and exposure amongst Nigerian consumers than for South Africans. However, Bankole and Adebanjo (2003) had reported that the high level of mycotoxin contamination in Nigerian foods is due to the presence of toxigenic fungi.

7.4 Conclusion

This is the first study in both Nigeria and South Africa that investigates the potential toxigenic capacity of Aspergillus, Fusarium, and Penicillium spp. isolated from fermented foods. This study reports a wide array of metabolites produced by 385 different fungal species that contaminated fermented foods from Nigerian and South African markets. The existence of principal mycotoxins (AFB1, DON, STE, ROQ C, OTA, FB1, and ZEN) and some other toxic metabolites (CPA, KA, and VHA) were established. Even though the level of toxigenic fungal species that we found in our present study was lower than non-toxigenic, the amounts of mycotoxins produced by some of the species were high. Although fungal existence does not necessarily translate to the presence of mycotoxins, the metabolites produced by the isolates can adversely affect the safety and quality of fermented foods. This study is therefore crucial for enhancing mycotoxin monitoring, management and control towards ensuring public health and safety.

Acknowledgments

This project received supports from the Organisation for Women in Science in the Developing World, Italy; Centre of Excellence for Food Security hosted by University of Pretoria and University of the Western Cape, South Africa; African Women in Agricultural Research and Development, Kenya; and MYTOX-SOUTH hosted in the Laboratory of Food Analysis, Ghent University, Belgium. The authors thank Dr Jose Di Mavungu for his technical assistance.

Conflict of Interest

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The authors have no conflict of interest to declare.

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CHAPTER EIGHT

CYTOTOXIC EFFECTS OF MYCOTOXIN EXTRACTS OF FUNGAL ISOLATES IN FERMENTED FOODS FROM NIGERIAN AND SOUTH AFRICAN ON HUMAN LYMPHOCYTE CELLS

Ifeoluwa O. Adekoya*a, Adewale Obadinaab, Judith Phokuc, Sarah De Saegerd and Patrick Njobeh*a aDepartment of Biotechnology and Food Technology, University of Johannesburg, Johannesburg, South Africa bDepartment of Food Science and Technology, Federal University of Agriculture, Abeokuta, Nigeria cAgricultural Research Council-Onderstepoort Veterinary Research, Toxicology and Ethnoveterinary Medicine, Public Health and Zoonoses, Onderstepoort, South Africa dLaboratory of Food Analysis, Department of Bioanalysis, Ghent University, Ghent, Belgium *Corresponding authors: [email protected] and [email protected]

Abstract

Mycotoxin exposure via consumption of contaminated foods can lead to severe health complications amongst exposed and vulnerable human populations. Seven toxigenic mycotoxin extracts of fungi isolated from traditionally fermented foods in Nigeria and South Africa were assessed for their toxicity against human mononuclear cells using methyl thiazol tetrazolium (MTT) salt assay. The mononuclear cells were exposed to 20, 40 and 80 µg/mL of mycotoxin extracts over 24, 48 and 72 hrs incubation period. The results showed that aflatoxin B1 extract induced the highest decrease in cell viability (48.9%) amongst the mycotoxin extracts tested. Fumonisin B1 produced by F. verticilliodes also reduced cell viability by 24% over a 48 hrs period, while lymphocyte cells exposed to zearalenone were the most viable (82%). Mean cell viability for ochratoxin A and deoxynivalenol decreased by 27.3% and 21.3%, respectively. Roquefortine C and sterigmatocystin also induced cytotoxicity up to 26 and 37%, respectively after 72 hrs incubation period. Increase in concentration levels from 20 to 80 µg/mL and time of exposure from 24 to 72 hrs significantly (p < 0.05) induced toxicity and resulted in a significant correlation of the tested

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Keywords: Mycotoxins, cell viability, lymphocytes, health, fermented foods, and cytotoxicity

Highlights

 Extracts of fungi isolated from fermented foods were tested against human lymphocyte cells to determine their toxicity.  Cell mortality was evident even at low doses and some mycotoxin extracts were generally more cytotoxic than others.

 Aflatoxin B1 extracts induced the highest decrease in cell viability (48.9%) amongst the fungal extract tested.  Increased level of extract from 20 - 80 µg/mL and time of exposure from 24 - 72 hrs significantly (p < 0.01) induced toxicity.  All the fungal extracts reduced the viability of the human lymphocytes cells.

8.1 Introduction

Globally, mycotoxins are at the epicentre of microbial toxins that naturally affect crop quality. They are quite stable molecules with accompanied toxicological effects that are modulated by toxigenic fungal strains (Bennett and Klich, 2003) majorly members of the Aspergillus, Fusarium, and Penicillium genera. Their synthesis is determined genetically and relatively linked to prominent metabolic pathways and mediated by various factors such as temperature, competing microflora, water activity and pH. Human exposure to mycotoxins is principally via ingestion of contaminated foods, but respiratory and dermal routes could also be involved (Omar, 2013). A harmful effect can manifest rapidly as acute toxicity from a single exposure, while the same effect can also occur slowly over a lengthy period from multiple exposures as chronic toxicity (Omar, 2013). These adverse effects can be cytotoxic, neurotoxic, oestrogenic, nephrotoxic, immunotoxic, hepatotoxic and manifests as various life threatening health complications in humans and animals (Bennett and Klich, 2003; Surai and Dvorska, 2005).

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Studies have shown that dietary mycotoxin exposure is common in Africa due to the susceptibility of the staple diets of most African populations to fungal and mycotoxin contamination (Shephard, 2008; Wagacha and Muthomi, 2008; Wild and Gong, 2009). In our recent survey, we discovered that majority of the fermented foods produced in Nigeria and South Africa were contaminated with toxigenic fungi as A. flavus, A. niger, A. minisclerotigenes, P. expansum, and F. verticilliodes (Adekoya et al., 2017a; Adekoya et al,

2018) including mycotoxins (aflatoxin B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA), sterigmatocystin (STE), roquefortine C (ROQ C), deoxynivalenol (DON), and zearalenone (ZEN) (Adekoya et al., 2017b; Adekoya et al., 2018).

Amongst the mycotoxins reported in the literature, AFB1 has gained increased popularity because it has been established as an extremely potent naturally occurring carcinogen (Stec et al., 2007; Wild and Gong, 2009; Makun et al., 2011). Upon the entry of AFB1 into cells, it is metabolised either in the endoplasmic reticulum to hydroxylated forms (AFM1), which is further metabolised to sulphates and glucuronides or oxidised into responsive epoxide that is hydrolysed and can bind proteins to thereafter exhibits its cytotoxic ability (Aguilar, 1993; Mwanza and Dutton, 2014).

Similar to AFB1, OTA is a potential carcinogen (Group 2B) that is involved in inhibition of protein synthesis and DNA single-strand damages (IARC, 1993; Pfohl-Leszkowicz and

Manderville, 2007) and its mutagenicity can increase particularly if it co-occurs with AFB1 in the same substrate (Pfohl-Leszkowicz and Manderville, 2007). The mode of action of OTA and its toxic potential in humans have not been fully elucidated (Stoev et al., 2009; Mwanza and Dutton, 2014). While STE shares similar biosynthetic pathway and structure with AFs, it also possesses some mutagenic, carcinogenic and teratogenic properties. Bertuzzi et al.

(2017) reported the toxin to be more genotoxic than AFB1 in three human cell lines.

A wide range of cell lines have been utilized for evaluating the cytotoxic activity of DON and

FB1 (Meky et al., 2001; Jestoi et al., 2008; Yang et al., 2017). The effect of DON on human health is characterised by anorexia, reduced food intake, weight loss and immune alterations

(Pestka, 2010; Flannery et al., 2011), while FB1 exposure is linked to oesophageal cancer (Shephard, 2008). The toxicity of ZEN has also been accrued to its molecular structure, which is similar to naturally occurring oestrogens, but a link between its exposure and human diseases has not been well established (Gromadzka et al., 2008). The interaction of ROQ C

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In format of Food Chemical Toxicology with mammalian cytochrome P450 as a means of evaluating its toxicity has been documented (Aninat et al., 2001), but studies on its toxicity on experimental animals have not conformed to standard laboratory procedures (Vallone et al., 2014).

One of the approaches to determining the effect of toxic secondary fungal metabolites on health includes in vitro cytotoxicity testing of their extracts on lymphocytes. This rapid and cost effective method excludes the use of animal models (Braydich et al., 2005; Maenetje et al., 2008) and involves the use of target cells (Cetin and Bullerman, 2005; Maenetje et al., 2008). The viability of target cells is determined by their ability to reduce methyl tetrazolium bromide to purple formazan dye in the presence of potential toxicants (Meky et al., 2001; Jestoi et al., 2008; Maenetje et al., 2008). Many studies have demonstrated the impact of mycotoxins on human and animal health (Meky et al., 2001; Gromadzka et al., 2008; Shephard, 2008; Stoev et al., 2010) but none have assessed the health implications of fungal metabolites isolated from fermented foods from Nigeria and South Africa on the viability of human lymphocyte cells which is herein reported.

8.2 Materials and Methods

8.2.1 Mycotoxin standards and reagents

Complete culture media (CCM), foetal bovine serum (FBS), phyto-haemagglutinin-p (PHA- p), Roswell Park Memorial Institute (RPMI)-1640 medium (with L-glutamine), phosphate buffered saline (PBS)-pH 7.4, Histopaque-1119, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), dimethyl sulphoxide (DMSO), penicillin, streptomycin, and trypan blue were obtained from Sigma, Missouri, USA. Guava instrument cleaning fluid (MCH100107) as well as count and viability reagent (MCH100102) were purchased from Merck Pty Ltd., Johannesburg, South Africa. For the preparation of MTT solution, 5 mg of MTT was dissipated in 0.14 M PBS (1 mL) and sterilized through a 0.22 µm pore size syringe filter. The stock solution (5 mg/mL) was stored at 4 C in the dark until it was needed. Mycotoxin standards including AFB1, STE, OTA, ROQ C, ZEN, DON and FB1 were purchased from Sigma, Missouri, USA. The other reagents used were of analytical grade.

8.2.2 Isolation, molecular characterisation and mycotoxin analysis of isolates by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

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The isolation and molecular characterisation of members of the Aspergillus, Penicillium and Fusarium genera were carried out as reported by Adekoya et al. (2017a). Thereafter, the mycotoxigenic potential of the isolates on solidified yeast extract sucrose agar using the agar plug technique was determined. The positive mycotoxins were further quantified by the LC- MS/MS method described in Adekoya et al. (2017b).

8.2.3 Isolation and purification of mononuclear cells

Lymphocytic cells were isolated and purified according to the modified method of Meky et al. (2001). Briefly, 15 mL of blood was drawn from two healthy consenting donors (Appendix 8.1) using sterile syringe into heparin tubes and each homogenized with 15 mL of CCM, which consisted of 100 U/mL penicillin, RPMI-1640 medium fortified with 10% FBS, and 100 µg/mL streptomycin. The mixture was carefully placed on an equal volume of Histopaque-1119 in centrifuge tubes and centrifuged at 3,000 rpm for 30 mins. The interface layer that consisted of the lymphocyte cells was gently removed using a sterile pipette. An equal volume of RPMI-1640 was used in washing the cells (3x) and cells (between 1 x 105 and 1 x 106) re-suspended in CCM.

8.2.4 Enumeration of cells

8.2.4.1 Cell enumeration with Neubauer haemocytometer

In order to determine the concentrations of cells, 10 µL of cells was mixed with 40 µL of trypan blue solution (0.2%). Afterwards, 10 µL of the cell suspension was transferred into the chambers of the improved Neubauer haemocytometer and covered with a glass cover slip. The number of cells in 1 mL was calculated as:

Where: n= number of cells counted in all squares v = area (number of squares) x depth (0.1) Dilution factor= 5 (10 μL of blood: 40 μL of trypan blue)

The percentage cell viability was thus calculated as:

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8.2.4.2 Cell enumeration with Muse analyser

The assessment of the cell concentration was also performed using a Muse cell analyser (0500-3115, Merck, South Africa). Briefly, the muse analyser was cleaned with an instrument cleaning fluid (MCH100107) and a system check was done to verify the instrument performance. In a 1.5 mL micro-centrifuge tube (MCH 100-0785, Eppendorf, Germany), 10 µL of cell suspension was added to 450 µL of count and viability reagent (MCH100102). The mixture was incubated for 5 mins at room temperature (25 ± 2 oC) to allow staining of the cells, after which the sample was loaded on the muse analyser and the result was read.

8.2.5 Methyl thiazol tetrazolium assay

In preparation for the MTT assay, the extracts of the fungi isolates were randomly selected based on their mycotoxin (AFB1, FB1, STE, OTA, ROQ C, DON and ZEN) concentrations. Dried fungal extracts that produced low, medium and high concentrations of mycotoxins were re-dissolved in RPMI-1640. Cells with > 96% viability were thereafter transferred into a flask containing CCM (100 mL), gently mixed and incubated at 37 oC for 24 hrs in a 5% humidified and buffered CO2 incubator (3140, Thermoscientific, Massachusett, USA). Upon incubation, the suspension (20 µL) was distributed in triplicates into a 96-well plate, PHA-p (20 µg/ml) was added to the wells a stimulants and the cells exposed to 20, 40 and 80 µL of extracts and individual mycotoxins used as reference. Furthermore, the wells were made-up to 250 µl with CCM, gently mixed and incubated at 37 oC for 24, 48 and 72 hrs and assayed for cytotoxicity. This same procedure was followed for negative control wells devoid of standards or extracts. After the incubation periods (24, 48 and 72 hrs), MTT solution (30 µL) was pipetted into the wells and thoroughly mixed. The 96 well plates were again incubated for 4 hrs, and DMSO (50 μL) added to solubilize the formazan crystals formed and again further incubated for 2 hrs. A microplate reader (18237, Biorad, Japan) was used to measure the absorbance optical density (OD) at wavelengths of 540 and 620 nm. Human mononuclear cell viability was thus calculated as:

Where: ODN = OD value of control (no mycotoxin) PHA-stimulated cells

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ODM= OD value of mycotoxin treated PHA-stimulated cells. Percentage toxicity induction (% toxicity induction) was further calculated as:

8.2.6 Data analysis

Data generated from the study were subjected to a one- and two-way Analysis of Variance. The descriptive statistics and mean values of the data were derived and equal variances were assumed using Duncan, Scheffe and Tukey’s tests. For the multivariate analysis, a full factorial model was used to test for the effects of exposure and concentration on cell viability (%) which were further correlated using Pearson’s correlation. Data were also represented graphically. The statistical software used was SPSS version 23 for windows (IBM Corporation, New York, USA).

8.3 Results

The effect of the fungal extracts on cell viability is shown in Tables 8.1, 8.2 and 8.3, wherein cell viability of the untreated cells (control) was 100%. Compared to the control, exposure to the Aspergillus mycotoxins showed reduced cell viability up to 31% for AFB1, and 21% for STE after 24 hrs of exposure at concentrations of 738 and 7,406 µg/kg from A. flavus culture recovered from ogiri.

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Table 8.1 Effects of extracts of Aspergillus species isolated from fermented foods on the viability of human mononuclear cells

Aspergillus Food source Sample extracts (µg/mL) % Cell viability at different times of exposure (hrs) and levels (µg/mL) toxin 24 48 72 20 40 80 20 40 80 20 40 80 Control 100 100 100 100 100 100 100 100 100 AFB1 Standard 83 77 70 77 72 68 72 67 66 Ugba A. flavus (27) 83 81 79 78 74 69 71 65 61 Ugba A. flavus (69) 84 81 78 81 69 69 69 63 59 Ogiri A. amstelodami (97) 82 80 76 79 66 65 67 61 59 Mahewu A. parasiticus (117) 80 78 77 80 68 68 63 58 57 Ogiri A. minisclerotigenes (379) 78 77 75 73 69 69 63 62 55 Ogi A. parasiticus (507) 77 76 76 73 66 64 60 52 52 Ugba A. flavus (735) 79 78 73 71 66 60 57 51 42 Ogi A. parasiticus (948) 76 76 74 69 67 48 55 54 39 Mahewu A. flavus (1,931) 79 75 68 63 61 51 51 49 45 Ogiri A. parasiticus (3,350) 77 76 68 65 63 63 49 44 42 Umqombothi A. parasiticus (3,602) 75 73 64 59 61 58 49 42 39 Ogiri A. flavus (7,406) 75 72 69 58 56 52 46 42 35 STE Standard 90 85 79 85 82 76 78 76 69 Umqombothi A. flavus (4) 92 89 83 87 82 76 79 77 72 Iru A. flavus (33) 87 82 80 84 81 77 77 76 68 Ogi A. sydowii (91) 88 81 78 83 81 75 74 73 69 Mahewu A. flavus (119) 87 82 78 85 82 75 75 75 64 Ugba A. flavus (237) 87 82 78 85 79 74 75 72 64 Ogi A. amstelodami (371) 85 83 77 85 81 75 73 70 62 Ogi baba A. sydowii (437) 85 83 79 85 77 72 72 69 60 Ogiri A. versicolor (500) 84 82 79 79 77 70 74 65 58 Ogiri A. flavus (736) 79 77 79 74 63 60 62 57 51 OTA Standard 84 79 75 74 76 75 65 63 61 Iru A. niger (78) 86 83 79 84 83 74 74 65 65 Ugba A. sclerotiorum (161) 83 81 76 83 79 73 72 65 64 Mahewu A. niger (212) 82 81 74 80 78 71 68 67 66 Ogi baba A. niger (411) 79 77 73 78 77 72 68 65 65 Ogi A. niger (850) 75 74 72 76 72 70 60 59 59 Ogi A. niger (1,302) 77 74 72 62 62 61 57 54 52

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Table 8.2 Effects of extracts of Penicillium species isolated from fermented foods on the viability of human mononuclear cells

Penicillium Food source Sample extracts % Cell viability at different times of exposure (hrs) and levels (µg/mL) toxin (µg/mL) 24hrs 48 72 20 40 80 20 40 80 20 40 80 Control 100 100 100 100 100 100 100 100 100 OTA Standard 84 79 75 74 76 75 65 63 61 Ugba P. verrucosum (15) 94 92 84 85 83 74 80 77 74 Ogiri P. verrucosum (19) 93 84 78 81 81 77 75 74 74 Iru P. verrucosum (32) 88 83 83 80 80 78 73 72 72 ROQ C Standard 96 87 88 90 88 82 86 81 77 Iru P. chrysogenum (13) 97 95 90 92 88 88 88 84 84 Ogi P. chrysogenum (22) 93 95 85 86 82 82 80 80 77 Iru P. chrysogenum (39) 90 87 83 83 82 83 76 79 76 Iru P. chrysogenum (57) 88 86 83 85 84 83 77 76 73 Umqombothi P. chrysogenum (278) 87 83 83 86 81 79 77 73 70 Ugba P. chrysogenum (360) 85 84 79 85 84 79 76 74 77 Ugba P. chrysogenum (593) 87 84 80 84 84 73 77 75 76 Ugba P. chrysogenum (980) 87 85 80 78 75 76 72 71 72 Ugba P. chrysogenum (1,260) 86 83 80 76 76 75 70 68 64

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Table 8.3 Effects of extracts of Fusarium species isolated from fermented foods on the viability of human mononuclear cells

Fusarium Food source Sample extracts % Cell viability at different times of exposure (hrs) and levels (µg/mL) toxin (µg/mL) 24hrs 48 72 20 40 80 20 40 80 20 40 80 Control 100 100 100 100 100 100 100 100 100

FB1 Standard 95 92 89 92 87 82 86 82 80 Ogi F. verticillioides (77) 97 91 83 90 83 83 83 84 82 Ugba F. verticillioides (81) 95 88 84 87 85 84 79 79 80 Umqombothi F. verticillioides (92) 94 85 80 84 83 82 79 79 75 Umqombothi F. verticillioides (109) 92 84 80 86 83 82 76 76 73 Ogi F. verticillioides (187) 86 82 80 85 82 80 74 73 74 Ogi F. verticillioides (218) 79 78 75 79 77 76 68 67 67 DON Standard 90 89 87 88 87 85 82 74 72 Ogi baba Fusarium sp. (20) 94 92 86 85 83 84 80 77 74 Ogi baba Fusarium sp. (300) 93 84 80 81 81 75 75 74 75 Ugba Fusarium sp. (870) 88 83 80 80 75 75 75 74 75 ZEN Standard 97 93 93 93 88 86 88 84 83 Iru F. sporotrichioides (139) 98 92 88 86 87 82 87 80 78 Ugba Fusarium sp. (197) 93 89 82 87 80 83 76 75 74 Mahewu Fusarium sp. (309) 88 84 80 79 79 74 72 72 73

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For OTA produced by A. niger isolated from ogi, a dramatic decrease of 28, 38 and 48% in cell viability was recorded over a 24, 48 and 72 hrs exposure period (Table 8.1). Roquefortine C produced by P. chrysogenum from ugba induced toxicity by 27% over a 48 hrs period

(Table 8.2), while amongst the Fusarium extracts, FB1 at a concentration of 1,260 µg/kg decreased cell viability by 33% over a 72 hrs period. A similar trend of decreased cell viability was also noticed amongst cells treated with pure standards (AFB1: 66-83%; STE:

69-90%; OTA: 61-84%; DON: 72-90%; ZEN: 83-97%; FB1: 80-95% and ROQ C: 77-96%). Furthermore, the mean viability of human mononuclear cells after exposure to the toxins over a period of time was significantly different (p < 0.05) in respect of the degree and level of exposure (Table 8.4).

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Table 8.4 Mean cell viability (%) of fungal isolates of fermented foods as influenced by exposure time and concentration of fungal extracts

Concentration Time of AFB1 STE OTA ROQ C FB1 DON ZEN µg/mL exposure (hrs) mean +S.D mean +S.D Mean +S.D Mean +S.D Mean +S.D Mean +S.D Mean +S.D a20 a24 78.8±9.2 86.0±6.6 82.6±5.8 88.9±4.0 90.3±7.5 91.3±5.7 92.8±4.4 b48 70.8±12.0 82.9±6.4 79.9±9.4 83.8±5.1 85.2±4.5 82.2±2.9 83.9±4.1 c72 58.4±17.7 73.2±5.5 68.6±8.1 76.9±5.6 76.7±6.4 75.1±5.6 78.4±7.5 b40 a24 76.9±6.6 82.2±5.7 78.3±7.8 86.8±5.2 84.6±5.6 86.1±6.7 88.3±4.6 b48 65.4±11.1 78.1±8.8 77.0±8.1 81.8±4.3 82.2±4.2 79.8±5.4 82.0±3.7 c72 65.3±13.7 70.5±7.6 65.2±6.8 75.7±6.2 76.1±7.3 72.8±5.4 75.4±4.9 c80 a24 73.2±8.6 79.0±4.4 75.5±8.3 82.6±3.6 80.4±4.5 81.9±5.1 83.6±3.9 b48 61.4±9.0 72.6±7.7 72.4±6.7 79.7±5.5 81.1±3.6 76.6±6.6 79.9±4.8 c72 48.9±9.5 63.1±8.3 64.2±6.5 74.3±6.5 78.9±5.4 75.3±9.2 75.1±3.1 Range 48.9-78.8 63.1-86.0 64.2-82.6 74.3-88.9 78.9-90.3 75.3-91.3 75.1-92.8 Mean 65.2 76.4 73.7 81.1 81.3 79.3 82.2 Standard deviation (S.D) 14.5 9.6 9.6 7.0 7.2 9.2 7.1 Standard error 1.0 0.7 0.4 0.6 0.8 1.3 0.9 P. of concentration (C) *** *** *** *** *** *** *** P. of exposure (E) *** *** *** *** *** *** *** P. of C x E ns ns ns ns *** ns ns P: probability, a-c mean values are significantly different at 5% confidence level (p < 0.05), ns: not significant

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The overall mean cell viability of the concentrations of the mycotoxins were: 65.2% (range: 48.9 -78.8%), 73.3% (range: 64.2-82.6%), 79.3% (range: 75.3-91.3%), 82.2% (range: 75.1- 92.8%), 81.1% (range: 74.3-89.0%), 81.3% (range: 78.9-90.3%), and 76.4% (range: 63.1-

86.0%) for AFB1, OTA, DON, ZEN, ROQ C, FB1 and STE, respectively. The interactive effect of toxin concentrations and duration of exposure was significant (p < 0.05) for FB1 only. Exposures to AFB1 extracts at concentrations of 24, 48 and 72 h period induced toxicity up to 51% while for ZEN it was 25% (Figure 8.1). Significant negative correlations were observed between the cell viability, time of exposures and concentrations of different fungal extracts (r = -0.226 to -0.687, p < 0.01). An increase in the level of the extracts from 20 to 80 µg/mL for STE for example as well as time of exposure from 24 to 72 hrs, resulted in a significant (p < 0.01) decrease in cell viability from 92 to 82% and 89 to 77%.

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Figure 8.1 Mean toxicity induction (%) at different concentrations and times of exposure of different fungal extracts

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8.4 Discussion

The significance of toxigenic fungi and attendant metabolites as food-borne contaminants in causing serious health complications cannot be overemphasised. Zearalenone (ZEN), AFB1,

FB1, DON, STE, ROQ C and OTA are the most significant mycotoxins synthesised by the Aspergillia, Penicillia and Fusaria genera. Exposures of human populations to these mycotoxins can be via the consumption of these fermented foods. The most common route of human exposure to mycotoxins is through the ingestion of foods that contains high levels of singly or simultaneously occurring metabolites, capable of stimulating several biological actions, although the biochemical mechanism of action of many secondary metabolites have not been elucidated (Rocha et al., 2005; Nielsen et al., 2009). One of the main biological effects of mycotoxins in humans is the generation of mycotoxin-induced apoptosis, also referred to as programmed cell death (Maenetje et al., 2008; Njobeh et al., 2009; Makun et al., 2011). The effects of mycotoxins on lymphocyte cells have been assayed both in vivo (Jestoi et al., 2008; Devreese et al., 2014; Burn et al., 2008) and in vitro (Meky et al., 2001; Njobeh et al., 2009; Egbuta et al., 2016). However, in vitro studies via MTT assay have the additional advantage of revealing the direct effect of toxins often expressed as cell viability (Meky et al., 2001).

It is evident in our present report that the mycotoxin (ZEN, AFB1, FB1, DON, STE, ROQ C and OTA) extracts tested induced mortality of lymphocyte cells as demonstrated by the reduced % cell viability recorded after exposure and a dose-dependent effect of the tested extracts was observed on cell viability. The cell viability also had a direct inverse relationship with time of exposures. This corroborates the report of Mwanza et al. (2009), who observed a 23.5% reduction in cell viability of human lymphocytes after 96 hrs exposure to a concentration of 20 µg/mL FB1. According to Hanelt et al. (1994), Yiannikouris and Jouany (2002), a number of reasons could account for this variation. Mycotoxins have diverse chemical structures and properties and as such, their biological actions are variable, i.e., ability to bind to cellular receptors, capacity to interfere with protein synthesis, enzymatic actions and rate of transport into the cells (Hanelt et al., 1994; Yiannikouris and Jouany, 2002). For these reasons, to predict their mechanism of action particularly in the case of co- occurrence of mycotoxins becomes complicated and as such, will require various assays to assess these factors.

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In addition, ZEN and DON were found to induce cytotoxic effect on various human cell lines singly and in combination (Visconti et al., 1991; Cetin and Bullerman 2005). The cell mortality was also inversely proportional to cell proliferation and both were affected by the degree of mycotoxin exposures. There is therefore the likelihood of reduction in cell proliferation with increased concentration as reported by Lioi et al. (2004), though cell proliferation was not assayed in this study.

The decreased cell viability caused by AFB1 might be due to it high level of concentration and toxicity. Mwanza et al. (2014) reported that AFs (5 ng/mL) reduced cell viabilities up to

27% after 72 hrs exposure. AFB1 is both water and fat-soluble and this hastens its infiltration into cell membranes and cellular organelles (Stec et al., 2007), inducing apoptosis and causing cell death. Since it has been established to be immunosuppressive, the loss in viability of cells exposed to it may cause decreased immunological defence functions of such cells (Fairbrother et al., 2004). This immunosuppressive ability in addition to its inhibitory capacity against DNA and RNA synthesis (mutation and cancer) as well as its action on cell mediated and phagocytic functions is responsible for its cytotoxic property (Mwanza et al., 2014). This mycotoxin is reputed for forming adducts that result in genotoxicity, carcinogenicity and cytotoxicity (Stec et al., 2007).

Sterigmatocystin a biogenetic precursor of AF biosynthesis (Anninou et al., 2014) was reported to have enhanced up to 24% reduction in cell viability in contrast with the study of

Liu et al. (2014) where STE was observed to have exhibited more cytotoxic action than AFB1 on human hepatoma (Hep) G2 cells. It is important to note that, the concentration of mycotoxin needed to elicit any marked effect varies significantly amongst the toxins, which is why different permissible/tolerance levels are set for different mycotoxins in foods. Sterigmatocystin causes cell death and deregulation by modifying DNA, hence considered cytotoxic (Wang et al., 2013). Anninou et al. (2014) in their study reported that STE, OTA and citrinin (CIT) singly or in combination had cytogenetic and cytotoxic potentials in vitro even at picomolar (pM) on Hep3B cells but STE exhibited the greatest action on cells when compared to OTA in this study.

Ochratoxin A is nephrotoxic, inducing apoptosis in various types of cell lines in vivo (Jestoi et al., 2008; Devreese et al., 2014; Burn et al., 2008) and in vitro (Rahimtula and Chong, 1991; Marin-Kuan et al., 2011; Anninou et al., 2014). Structure-activity studies indicate that OTA toxicity may be associated with its isocoumarin moiety and lactone carbonyl group.

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Other report indicated that OTA impedes enzymes that participate in phenylalanine metabolism, which has an effect on RNA, DNA, and protein synthesis (Swenberg et al., 2008; Marin-Kuan et al., 2011). OTA toxicity had been linked to certain kidney diseases due to the inducement of oxidative stress (Marin-Kuan et al., 2011). The dietary intake of OTA had been linked with the formation of uroepithelium and renal malignancies in countries such as Egypt, France and Tunisia (Omar, 2013). Ochratoxin A like AFB1 is also immunosuppressive since its other biological actions in mammalian species are reduced phagocytosis and lymphocyte markers and antioxidant defence breakdown (Rached et al., 2007).

Roquefortine C is synthesized by Penicillium species such as P. chrysogenum and P. roqueforti. It is a potent neurotoxin usually at high doses but exhibits lower toxicity than

AFB1 and OTA at low concentrations (Fontaine et al., 2015). The cytotoxicity of ROQ C (81.1%) isolated in our present study on the mononuclear cells was lower than that of other mycotoxin extracts with the exception of ZEN (82.2%) but similar to that of FB1 (81.3%) and DON (79.3%). Furthermore, Fontaine et al. (2015) reported that the viability of human intestinal Caco-2 cells decreased significantly after 48 hrs exposure at high ROQ C concentrations. The contribution of other unscreened toxic substance(s) to the observed decrease in cell viability cannot be ruled out.

Oxidative mutilation is presumed to be one of the principal pathways of ZEN toxicity and may lead to genotoxic and cytotoxic actions (Hassen et al., 2007). The dose dependent effect of ZEN in this study corroborates the report of Stec et al. (2007) where it was reported to instigate a significant inhibition of cell lines from kidney fibroblasts of farm animals as compared to the control. Deoxynivalenol had an overall mean cell viability of 79.3% (range: 75.3-91.3%). At cellular level, DON act by inhibiting protein and DNA synthesis as well as instigate cell membrane damage (Rizzo et al., 1992). Its dietary intake facilitates apoptosis both in vitro and in vivo in different organs such as the bone marrow, lymphoid tissues, liver, hematopoietic tissues, intestinal crypts, and thymus (Instanes and Hetland, 2004). Reduced cell viability in the case of DON is in line with the report of Pestka (2010) and Flannery et al. (2011), where it was reported to be a strong inhibitor of lymphocyte proliferation.

Cetin and Bullerman (2005) reported a reduced cell proliferation of 50% upon exposure of lymphocyte cells to FB1 at 94.8 and 98.38 µg/mL. This same trend was also observed by Lioi et al. (2004) and Makun et al. (2011). This decrease is due to the cytotoxic action of FB1,

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Africa primarily due to heavy dietary intake of FB1 contaminated foods (Shephard, 2008).

It is important to state herein that the cytotoxicity of some of the mycotoxin extracts tested was higher than that of the mycotoxin standards used as reference materials. This was not surprising considering their high concentration. Continual ingestion of the contaminated foods is deleterious to health taking into consideration that a complete diet would involve a series of food products that could be contaminated with different mycotoxins including those assayed in the present study. Mycotoxin exposure patterns are predictable in regions where foods are improperly handled and stored, where few or no mycotoxin regulations exist to protect exposed populations especially those with high prevalence of malnutrition and HIV/AIDS.

8.5 Conclusion

The MTT assay facilitated prompt comparability of the toxic effects of different mycotoxins on lymphocytes and a time and dose dependent relationship was evident amongst the exposed cells. All the mycotoxin extracts were highly cytotoxic as substantiated by profound cell mortality even when administered at low doses at varying degrees in the case of extracts from

Aspergillus cultures particularly AFB1. This study further showed evidence that mycotoxins pose detrimental effect on human health and strict modulation of food quality particularly in sub-Saharan Africa need to be prioritised to prevent manifestations of mycotoxicoses amongst the populations.

Acknowledgements

This project obtained support from the Organisation for Women in Science in the Developing World, Italy, Swedish International Cooperation Agency (SIDA), Sweden and Centre of Excellence in Food Security co-hosted by University of Pretoria and University of the

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Western Cape, South Africa. The technical support received from Dr Eugenie Kayitesi, Mr Mandla Sibiya, and Mr Alista Campbell of the University of Johannesburg, South Africa is duly acknowledged.

Conflict of Interest

The authors have no conflict of interest to declare.

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CHAPTER NINE

PATHOGENIC BACTERIA AND ENDOTOXINS IN FERMENTED FOODS AND BEVERAGES FROM SELECTED NIGERIAN AND SOUTH AFRICAN MARKETS

Ifeoluwa Adekoyaa*, Adewale Obadinaab, Momodu Olorunfemic, Sarah De Saegerd and Patrick Njobeha* aDepartment of Biotechnology and Food Technology, University of Johannesburg, Johannesburg, South Africa bDepartment of Food Science and Technology, Federal University of Agriculture, Abeokuta, Nigeria. cDepartment of Botany, University of Ibadan, Ibadan, Nigeria dLaboratory of Food Analysis, Department of Bioanalysis, Ghent University, Ghent, Belgium *Corresponding authors: [email protected] and [email protected]

Abstract

In Africa, fermented foods and beverages play significant roles in contributing to food security. Endotoxins are ubiquitous heat stable lipopolysaccharide (LPS) complexes situated in the outer cell membranes of Gram-negative bacteria. This study evaluated the microbiological quality of fermented foods (ogiri, ugba, iru, ogi and ogi baba) and beverages (mahewu and umqombothi) from Nigerian and South African markets. The bacterial diversity of the fermented foods was also investigated and the identity of the isolates confirmed by biochemical and molecular methods. The samples were further investigated for endotoxin production with the chromogenic Limulus Amoebocyte Lysate assay. The total aerobic count of the samples ranged from 5.50 x 105 to 6.59 x 1010 CFU/g. Fourteen bacteria genera were detected with most of isolates being members of the Enterobacteriaceae family. Sphingomonas paucimobilis and Escherichia coli were the dominant Gram-negative bacterial species detected. There were considerable variations in the concentrations of endotoxins produced and the lowest endotoxin concentration was found in ogi (4.3 x 10 EU/g) and the highest in iru (5.49 x 104 EU/g) while 44% of umqombothi samples had endotoxins. Ogi baba samples had better microbial quality than other samples due to its reduced bacteria load and endotoxin levels. Some previously unreported species of bacteria found in the fermented foods included Aeromonas haemolyticus and Rhizobium radiobacter. This is the first

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Keywords: Fermented foods, Gram-negative bacteria, endotoxins, safety, Nigeria, and South Africa

Highlights

 Bacterial diversity and endotoxin levels in fermented products were investigated.  Fourteen bacteria genera were detected including previously unreported bacteria species.  Escherichia coli and Sphingomonas paucimobilis were the dominant bacterial species detected.  Endotoxin levels in positive samples ranged from 4.3 x 10 to 5.49 x 104 EU/g.  This is the first comprehensive report on the presence of endotoxins in fermented products from Nigeria and South Africa.

9.1 Introduction

The incidence of food-borne infections has increased globally within the past few years with a significant number of human population mostly at risk (Cho et al., 2011) and Gram- negative and Gram-positive bacteria pathogens are at the epicentre of most reported cases (Sudershan et al., 2014). Globally, fermented foods and beverages have proven to be critical in the sustenance of the nutritional status of people due to their availability, richness of nutrients, affordability and ease of processing. They are consumed almost on daily basis across Africa by both infants and adults. They are produced from different substrates; maize (Zea mays), sorghum (Sorghum bicolor), melon (Colocynthis citrullus), African oil bean (Pentaclethra macrophylla Benth), and locust beans (Parkia biglobosa). The examples of fermented foods and beverages indigenous to Africa include ogi, ogi baba, ugba, iru, ogiri, mahewu and umqombothi. Ogi, ogi baba, mahewu, and umqombothi are products of lactic acid fermentation of maize and sorghum with ogi and ogi baba being indigenous to Nigeria and consumed as breakfasts and weaning foods while mahewu and umqombothi are beverages both popularly consumed by black South Africans.

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The microorganisms involved in the fermentation of these products (Ogi, ogi baba, mahewu, and umqombothi) are mainly lactic acid bacteria, Saccharomyces cerevisiae and Streptococcus lactis (Odunfa and Oyewole, 1998). Ogiri (substrate: melon), ugba (substrate: African oil bean), iru (substrate: locust beans) are condiments derived from alkaline fermentation of proteinaceous oily seeds usually consumed in Nigeria and also exported to other countries in the world including South Africa. Ugba is also used as a snack and as a basal ingredient for salad preparation. Despite the benefits of fermented foods, they have been demonstrated to be regularly contaminated with pathogenic microorganisms including Gram-negative bacteria (Aworh, 2008; Ogunshe et al., 2012). A study by Ogunshe and Olasugba (2008) revealed that 66% of iru (n=1125), 82.4% of ogiri (n=148) and 93.9% of ugba samples (n=115) obtained from the Middle belt region of Nigeria were contaminated with coliforms. Similarly, Nwachukwu et al. (2014) recovered Klebsiella, and Pseudomonas spp. from ogiri, thus consumption these foods may pose health risk to consumers.

Apart from the fact that Gram-negative bacteria in fermented foods may cause infections, they can be toxigenic, producing endotoxins in foods. Endotoxins are ubiquitous heat stable lipopolysaccharide (LPS) complexes that can be found at the outer cell membranes of Gram- negative bacteria (Adam et al., 2014). The potency of endotoxins varies amongst different bacterial species but E. coli often used as the model organism produces LPS with extremely high endotoxin activity (Raetz and Whitfield, 2002). Human exposure to endotoxins can lead to many health complications such as septic shock, microvascular abnormalities development, multiple organ failure, disseminated intravascular coagulation and necropsy (Kalita et al., 2017). The incidences of endotoxin in foods have been poorly investigated and most studies conducted on endotoxins cover sparse food categories such as milk. Endotoxin levels from 40 - 5.5 x 104 EU/g were found in infant milk manufactured in nine countries (South Africa, Holland, Spain, Switzerland, USA, Belgium, Ireland, Slovenia, and United Kingdom) by Townsend et al. (2007).

There is a need for surveillance of fermented foods to ensure consumer safety and the interaction of severe factors associated with their production particularly in Africa. Such factors include poor handling practices, unstandardized processing methods, participation of various microbial flora and unhygienic display practices which may facilitate their contamination with pathogenic microorganisms and their toxins. Since endotoxins are contaminants that pose a threat to food safety and security, their presence as well as that of thier causative agents (Gram-negative bacteria) in fermented foods need to be investigated.

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Therefore, the prime objective of this study was to screen for pathogenic bacteria using molecular based methods and quantify the levels of endotoxins in fermented foods and beverages from Nigeria and South African markets.

9.2 Materials and Methods

9.2.1 Materials

9.2.1.1 Reagents

Pierce Limulus Amebocyte Lysate (LAL) chromogenic endotoxin quantification kit (88282) was purchased from Separations Pty Ltd., South Africa. Nutrient agar (CM003), Thiosulfate- citrate-bile salts-sucrose agar (TCBS) (CM0333), Plate count agar (CM0325), Salmonella Shigella agar (CM 0099), peptone water (0009), cephaloridine, fucidin and cetrimide (CFC) selective agar supplement (SR0103), Pseudomonas agar (CM0929) and MacConkey agar (CM0007) were purchased from Oxoid Ltd, Basingstoke Hants, England. Columbia Sheep blood agar plates (M1013) were purchased from BioMerieux, South Africa. 96-well plates were supplied by AEC-Amersham SOC Ltd, Johannesburg, South Africa, while analytical grade acetic acid, sodium hydroxide (NaOH) and hydrochloric acid (HCl) were obtained from Merck, Johannesburg, South Africa. Other reagents used were of analytical grade. Glassware used in the LAL assay was rendered pyrogen free by heating at 140 °C for 4 hrs.

9.2.1.2 Fermented food and beverage

Cluster sampling method was followed to collect different fermented foods and beverages from South-west, Nigeria and Gauteng province, South Africa between Feburary 2015 and July 2016. A total of 399 samples of fermented foods viz.: maize gruel (ogi, n=68), sorghum gruel (ogi baba, n=35), locust bean (iru, n=126), African oil bean seed (ugba, n=55), maize meal (mahewu, n=21), traditional cereal-based opaque beer (umqombothi, n=32) and melon (ogiri, n=62) were collected in sealed sterile containers and maintained on ice bags. The samples were transported to the Food, Environment and Health Research Group (FEHRG) Laboratory, University of Johannesburg, South Africa and stored at 4 oC prior to analysis. The samples were trisected twice and representative sample of each product was 30 g (ogi, ogi baba, iru, ogiri and ugba) and 50 mL (mahewu, umqombothi). Ogi (n=36), ogi baba

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(n=18), iru (=72), mahewu (n=18), ugba (n=36), ogiri (n=36), and umqombothi (n=18) were subjected to microbiological and endotoxin examinations.

9.2.2 Methodology

9.2.2.1 Microbiological analysis

The fermented food samples were blended with a sterile laboratory blender (LB10G, ITM Instrument, Alberta, Canada) and 10 g of each sample was homogenized in 90 mL sterile 0.1% peptone water for 30 secs to a homogenous suspension and the samples serially diluted. For the isolation of total aerobic bacteria and Vibrio spp., 0.1 mL aliquot from each sample was plated on solidified Nutrient and Thiosulfate-citrate-bile salts-sucrose (TCBS) agar, respectively, and plates incubated at a temperature of 37 oC for 24 hrs. For the enumeration of total coliform and Salmonella spp., 0.1 mL of aliquot was plated on MacConkey and Salmonella Shigella agar, respectively. The incubation temperature was 37 °C and plates were examined after 48 hrs. For Pseudomonas spp. isolation, 0.1 mL of aliquot was plated on solidified Pseudomonas agar with CFC selective supplement. The incubation temperature was set at 25 °C and plates examined after 48 hrs.

Control plates which had no inoculum were also prepared. The colonies were counted using a colony counter, and mean bacterial load was calculated and results expressed as CFU/g or CFU/mL of sample. Furthermore, pure isolates were sub-cultured on Nutrient agar plates, incubated at 37 oC for 24 hrs and their micro morphological characteristics examined on an optical microscope (Olympus CX40, Micro-instruments, New Zealand). Purified cultures were maintained on appropriate agar slants, which were kept as stock cultures at 4 oC. For each pure isolate, Gram staining was carried out as described by Brenner et al. (2005). The Gram-negative bacteria isolates were further identified using the VITEK 2 Compact microbial identification system.

9.2.2.2 Microbial identification with the VITEK 2 compact instrument

The VITEK 2 instrument (BioMerieux, North Carolina, USA) utilizes advanced colorimetric measurements, which is dependent on verified biochemical procedures such as carbon utilization, growth resistance and enzymatic actions to establish the identity of a microorganism. Prior to microbial identification, Gram-negative bacteria isolates were sub- cultured on Columbia Sheep blood agar to ensure their viability and purity. Culture

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9.2.2.3 DNA Extraction, Polymerase Chain Reaction (PCR) and Sequencing

9.2.2.3.1 DNA extraction Genomic DNA analysis was performed using a Fungal/Bacterial DNA extraction kit (Zymo Research (ZR) Corporation, Southern California, USA) as recommended by the manufacturer. Bacterial cells (50-100 mg) were re-suspended in a 1.5 mL ZR Bashing BeadTM lysis tube containing 200 µL of phosphate buffer saline (PBS). The tube was inserted into a beater for 5 mins, followed by 1 min centrifugation at 10,000 x g. The supernatant (400 µL) was transferred to a Zymo-SpinTM IV spin filter in a collection tube with the repetition of the centrifugation step. To the resultant filtrate, 1.2 mL of binding buffer was added and 800 µl of the mixture was transferred to a Zymo-SpinTM IIC column in a new collection tube and centrifuged at 10,000 x g for 1 min. This step was repeated twice, followed by the addition of 200 µL pre-wash buffer and 500 µL DNA wash buffer. The column was transferred to a sterile 1.5 mL microcentrifuge tube and 100 µL DNA elution buffer added and centrifuged at 10,000 x g for 30 secs to elute the DNA.

9.2.2.3.2 Amplification of 16S rRNA gene by Polymerase Chain Reaction

The I6S rRNA gene based universal primers: 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-CGGTTACCTTGTTACGACTT-3’) were used for the amplification of the

DNA) according to Turner et al. (1999). Each amplification reaction contained 3 µL MgCl2, 1 µL dNTPs, 2 µL of the primers, 0.2 µL Taq polymerase buffer, 5 µL of template DNA, 5 µL 2X PCR buffer, made-up to a final volume of 50 µL using nuclease free water. A thermocycler (Mastercycler pro384, Eppendorff, USA) was utilized for the amplification with

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9.2.2.3.3 Sequencing of the PCR Products The PCR products were gel extracted using Zymoclean gel DNA recovery kit. The extracted products were sequenced in the forward and reverse directions (Applied Biosystems, Thermofisher Scientific, Big Dye terminator kit v3.1, Carlsbad, California, USA) and cleaned with ZR-96 DNA clean-up kit. The cleaned products were run on an ABI 3500 xL Genetic Analyser (Applied Biosystems) and the data generated (.abi files) were analysed on the CLC Bio Main Workbench 7. This was followed by a BLAST search on the National Center for Biotechnology Information (NCBI) database in order to establish the equivalence of the fragments with existing sequences (Stephen et al., 1997). The sequences generated from this study were further deposited in the Genbank (NCBI, USA).

9.2.2.4 Endotoxin analysis

All samples were homogenised using a sterile mechanical blender. Endotoxins was analysed with the chromogenic LAL test kit that consisted of endotoxin standard, LAL, chromogenic substrate and endotoxin-free water following the manufacturers instruction (Separations Pty Ltd., South Africa). The minimum dilution of each sample was 1:10 with pyrogen free water, while the maximum dilution was 1: 1,000,000. The pH of the samples was adjusted to 7 ± 1 with 0.1 M NaOH or 0.1 M HCl. 96-well plates were placed on a heating block for 10 mins and temperature maintained at 37 oC. Samples and standards (50 µL each) were dispensed into the wells, the plates were covered with the lid and placed in a 37 oC incubator for 5 mins. Thereafter, 50 µL of LAL was added to each well, the plates shaken gently for 10 secs and incubated at 37 oC for 10 mins after which 100 µL of chromogenic substrate solution was added. To the mixture, 50 µL of 25% acetic acid was added and the absorbance of the mixture was measured with a microplate reader (ELX800, Cole Palmer, Illinois, USA) set at a wavelength of 405 nm. Assay range was from 0.1 to 1.0 EU/ml and the concentration of endotoxin in each sample was established using a standard curve which was prepared by spiking known amount of endotoxin standards (0.1-1.0 EU/mL) into endotoxin free water.

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Endotoxin free water (50 µL) was used as a blank. Positive control was also prepared at the midpoint concentration of the standard curve (0.5 EU/mL) as well as the enhancement/inhibition control. The samples were tested over at least five dilutions and values were expressed as endotoxin units (EU) per g or mL of sample. All analyses were carried out in triplicates.

9.2.2.5 Data analysis

A descriptive statistics of bacteria counts and endotoxin levels (mean, frequencies, and percentages) was performed using the Microsoft Office Excel 2010 (Redmond, WA, USA). Data generated were subjected to a one-way Analysis of Variance with the SPSS version 23.0 (IBM Corporation, New York, USA) and the Tukey’s test was used to separate differences amongst the means at 5% confidence level.

9.3 Results

The bacterial flora of the fermented foods from Nigerian and South African markets are shown in Table 9.1. As seen, total aerobic plate counts (TAPC) and total viable plate counts (TVPC) of all the samples were greater than 105 CFU/g, with iru samples from South Africa having the highest mean TAPC (6.59 x 1010 CFU/g) and TVPC (6.87 x 109 CFU/g). Vibrio spp. and Pseudomonas spp. were isolated from the all the analysed umqombothi samples with mean counts of 1.83 x 102 and 8.67 x 102 CFU/mL, respectively. A total of 499 bacterial isolates were identified in the samples (ogi=45, ogi baba=11, ogiri=100, mahewu=22, iru= 191, ugba= 95, umqombothi=35).

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Table 9.1 Mean bacterial load of fermented foods from Nigerian and South African markets in CFU/g or CFU/mL of sample

Source Fermented Total Total Total Samonella Vibrio Pseudomonas food aerobic viable coliform shigella spp. spp. samples count count count count count count Nigeria Ogiri 1.05 x 109 1.68 x 108 1.50 x 103 1.72 x 102 4.19 x 102 1.38 x 102 Iru 6.50 x 1010 1.05 x 109 0.91 x 103 1.10 x 102 2.09 x 102 1.54 x 102 Ugba 5.04 x 106 5.35 x 105 1.07 x 102 1.21 x 101 1.56 x 102 2.81 x 102 Ogi 5. 75 x107 1.24 x106 1.27 x 101 1.91 x 101 3.03 x 102 2.89 x 102 Ogi baba 5.50 x 105 2.30 x 105 1.22 x 101 1.62 x 102 1.73 x 102 1.53 x 101 South Africa Ogiri 2.89 x 108 6.12 x 107 1.87 x 102 1.53 x 102 1.86 x 102 3.79 x 102 Iru 6.59 x 1010 6.87 x 109 1.23 x 103 1.87 x 102 5.97 x 102 4.98 x 102 Ugba 5.37 x108 5.05 x107 2.82 x 102 1.63 x101 3.69 x 102 3.14 x 102 Ogi 1.83 x 107 1.57 x 106 1.53 x 101 1.78 x 102 1.88 x 102 1.47 x 102 Mahewu 2.57 x108 3.30 x 107 1.38 x 102 1.08 x 102 1.26 x 102 7.99 x 102 Umqombothi 2.10 x 108 4.00 x 107 1.47 x102 1.73 x 102 1.83 x102 8.67 x 102

Furthermore, 13 bacteria genera belonging to the Enterobacteriaceae family were detected in the fermented foods from Nigeria (Table 9.2), while 14 genera were found in the South African foods (Table 9.3). Amongst the Gram-negative bacteria isolated, E. coli was detected only in 19% of iru and 45% of ogiri from South Africa, while P. luteola was found in 17% of mahewu samples. Additionally, 78% of ugba samples obtained from Nigeria were positive for B. subtilis but only 3 bacteria genera (Sphingomonas, Pantoea and Bacillus) were found in ogi baba. However, other species within these genera were found in other samples including ogi, mahewu, and iru.

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Table 9.2 Incidence of Gram-negative and Gram-positive bacteria isolated from fermented foods from Nigerian markets

Bacteria Ogi Ogi baba Ogiri Ugba Iru Accession n=18 n=18 n=18 n=18 n=36 Number

Gram-negative bacteria % +ve % +ve % +ve % +ve % +ve samples samples samples samples samples

Acinetobacter haemolyticus - - - 3 (17) - KT027777.1 Acinetobacter iwoffii - - - 2 (11) - AB859068.1 Escherichia coli - - 4 (22) - 7 (19) JF892819.1 Escherichia coli - - 1 (6) - - AJ875445.1 Pantoea spp. - 3 (17) - - 2 (6) EF469213.1 Pasteurella canis - - - - 5 (14) AY634676.1 Serratia marcescens 6 (33) - - 5 (28) - KU894791.1 Vibrio vulnificus 2 (11) - 10 (57) - 4 (11) KT823473.1 Vibrio parahaemolyticus - - 2 (11) - - DQ345442.1 *Aeromonas salmonicida - - - 2 (11) 1 (3) 97% *Pseudomonas luteola - - 2 (11) 3 (17) 3 (8) 95% *Pantoea spp. 4 (22) - 3 (16) - - 94% *Rhizobium radiobacter - - 3 (16) - - 94% *Serratia plymuthica - - - - 3 (8) 93% *Sphingomonas paucimobilis 3 (17) 4 (22) 4 (22) 3 (17) 5 (14) 93%

Gram-positive bacteria Bacillus cereus 3 (17) - - - - CP012691.1 Bacillus altitudinis - - 2 (11) - - MG855711 Bacillus pumilus 3 (17) - - - 3 (8) MG855694 Bacillus spp. - - - 3 (17) - MG855696 Bacillus oleronius - - - - 6 (17) MG855705 Bacillus subtilis - - 4 (22) 14 (78) 24 (67) KP224305.1 Bacillus safensis 3 (17) - - 4 (22) 8 (22) MG855704 Bacillus anthracis - - - 2 (11) - MG855713 Bacillus kochii - - - - 3 (8) MG855709 Bacillus spp. - 4 (22) - - 4 (11) MG855693 Bacillus spp. - - 3 (17) - - MG855714 Enterococcus faecium - - 4 (22) - - KU898955.1 Enterococcus faecalis - - - - 3 (8) MG855691 Enterococcus faecalis - - 2 (11) 4 (22) 3 (8) MG855708 Enterococcus gallinarum - - 3 (17) 1 (6) - KU196084.1 Enterococcus casseliflavus - - 2 (11) - - KT630829.1 Paenibacillus spp. - - - - 2 (6) MG855710 Paenibacillus spp. 3 (17) - - - - MG855699 %: percentage, +ve: positive, n: number of samples, * Gram-negative bacteria with excellent identification on the VITEK 2 compact instrument

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Table 9.3 Incidence of Gram-negative and Gram-positive bacteria isolated from fermented foods and beverages from South African markets

Bacteria Ogi Mahewu Ogiri Ugba Iru Umqomb Accession n=18 n=18 n=18 n=18 n=36 -othi Number n=18 Gram-negative bacteria % +ve % +ve % +ve % +ve % +ve % +ve samples samples samples samples samples samples Acinetobacter haemolyticus - - - 2 (11) - - KT027777.1 Acinetobacter iwoffii - - - 3 (17) - - AB859068.1 Cronobacter Sakazakii - - - - - 8 (44) AB274299.1 Escherichia coli - - 5 (28) - 3 (8) - JF892819.1 Escherichia coli - - 3 (17) - 1 (3) - KU880540.1 Pantoea spp. - 3 (17) 7 (39) 6 (33) - - EF469213.1 Pasteurella canis - - - - 2 (6) - AY634676.1 Serratia marcescens - 2 (11) - 1 (6) - - KU894791.1 Serratia nematodiphila - - - - - 4 (22) FJ662869.1 Shigella flexneri 1 (6) - - - 2 (6) - KX146471.1 Vibrio vulnificus 2 (11) - 3 (17) - 4 (11) 2 (11) KT823473.1 Vibrio parahaemolyticus - - 2 (11) - - - DQ345442.1 Cronobacter dublinensis 2 (11) - - - - - KR347471.1 *Rhizobium radiobacter - - 4 (22) 2 (11) 3 (9) - 94% *Sphingomonas paucimobilis 3 (17) 8 (44) 4 (22) 3 (17) 9 (25) 7 (39) 95% *Serratia plymuthica - - - - 4 (11) - 93% *Pseudomonas luteola - 3 (17) - 5 (28) 7(19) 2 (11) 95%

Gram-positive bacteria Bacillus cereus 2 (11) - - - 13 (36) 3 (17) MG855712 Bacillus spp. 1 (6) - - - - - MG855707 Bacillus cereus - 3 (17) - - - - KR709243.1 Bacillus altitudinis 2 (11) - 3 (17) - 6 (17) - MG855697 Bacillus xiamenensis - - - 4 (11) - 3 (17) MG855692 Bacillus toyonensis - - - 2 (3) - - KX129781.1 Bacillus pumilus 2 (11) 3 (17) 4 (22) - - 4 (22) KU844041.1 Bacillus oleronius - - - - 4 (11) - MG855706 Bacillus halotolerans - - - - 8 (22) - MG855697 Bacillus subtilis - - 3 (17) 10 (56) 28 (78) - MG855715 Bacillus safensis 3 (17) - - - - - MG855700 Enterococcus durans - - 3 (17) - - - MG855702 Enterococcus faecium - - 2 (11) - 2 (11) - MG855695 Enterococcus faecalis - - 5 (28) 8 (44) 3 (8) - MG855701 Paenibacillus spp. - - 3 (17) 3 (17) - - Mg855703 Paenibacillus polymyxa - - - - - 2 (11) MG855716

%: percentage, +ve: positive, n: number of samples, *Gram-negative bacteria with excellent identification on the VITEK 2 compact instrument

The Gram-negative bacteria identified by the VITEK instrument are presented in Table 9.4. Aeromonas salmonicida, S. paucimobilis, and P. luteola were amongst the bacteria identified with excellent identification at 97% (Appendix 9.2).

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Table 9.4 Gram-negative bacteria isolated from fermented foods by VITEK biochemical tests

Biochemical Test Abb. Aeromonas Sphingomonas Serratia Pseudomonas Rhizobium Pantoea salmonicida paucimobilis plymuthica luteola radiobacter spp.

Ala-phe-pro-arylamidase APPA + - - - - - Adonitol ADO ------L-Pyrrolydonyl-arylamidase PyrA - - + - - + L-arabitol IARL ------D-cellobiose dCEL - + + - + + ß-galactosidase BGAL - + - - - - H2S production H2S - - + + + - ß-N-acetyl-glucosaminidase BNAG - - + - + + Glutalyl arylamidase pNA AGLTp ------D-glucose dGLU - + + + + + γ-glutamyl-transferase GGT ------Fermentation/glucose OFF ------ß-glucosidase BGLU - + + + + + D-maltose dMAL - + + + + + D-mannitol dMAN - - + - + + D-mannose dMNE - + + + + + ß-xylosidase BXYL ------ß-alanine arylamidase pNA BAlap ------L-proline arylamidase ProA ------Lipase LIP ------Palatinose PLE - + + - - - Tyrosine arylamidase TyrA - - + - - - Urease URE ------D-sorbitol dSOR - - + - + - Saccharose/sucrose SAC - + + - + + D-tagatose dTAG - - - - + - D-trehalose dTRE - + + + + + Citrate (sodium) CIT ------Malonate MNT ------5-keto-d-gluconate 5KG ------L-lactate alkalinisation ILATk ------α-glucosidase AGLU - - + - - - Succinate alkalinisation SUCT ------ß-N-acetyl galactosiminidase NAGA ------α-galactosidase AGAL - + + - + + Phosphatase PHOS ------Glycine arylamidase GlyA ------Ornithine decarboxylase ODC ------Lysine decarboxylase LDC ------L-histidine assimilation IHISa ------Coumarate CMT - + + + + + ß-glucoronidase BGUR ------O/129 resistance 0129R - - + + - + Glu-gly-arg-arylamidase GGAA ------L-malate assimilation IMLTa ------Ellman ELLM ------L-lactate assimilation ILATa ------

Abb.: Abbreviation, -: Absent, +: Present

The mean levels of endotoxins produced are presented in Table 9.5, which showed considerable variations in the pattern of bacterial contamination. There were significant differences (p < 0.05) in the mean endotoxin levels of iru, ugba and ogiri from both Nigeria and South Africa and the lowest contamination level of 42.90 EU/g was found in ogi samples while the highest level (5.49 x 104 EU/g) was detected in iru samples from Nigeria.

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Most of the positive endotoxin samples were below 1.0 x 10 EU/g whereas the incidence of endotoxin contamination in umqombothi, mahewu and ogi baba was 44%, 28% and 22%, (Figure 9.1). Generally, iru and ogi from Nigeria had higher incidences of aerobic and Gram- negative bacteria and endotoxins contamination than their South African counterpart did.

Table 9.5 Mean endotoxin levels of fermented foods from Nigeria and South African markets

Source Fermented food aMean endotoxin level Range sample (EU/mL/ EU/g) sample (EU/mL/EU/g) sample Nigeria Ogiri e3.87 x 103 8.54 x 102 - 3.82 x 104 Iru f4.65 x 103 9.38 x 102 - 5.49 x 104 Ugba ab9.00 x 101 4.40 x 101 - 2.32 x 102 Ogi a4.78 x 101 4.29 x 101 - 5.67 x 101 Ogi baba a5.05 x 101 4.31 x 101 - 6.60 x 101 South Africa Ogiri c3.19 x 102 1.32 x 102 - 2.04 x 103 Iru ef4.14 x 103 5.70 x 102 - 4.22 x 104 Ugba b1.09 x 102 5.90 x 101 - 1.65 x 102 Ogi a4.61 x 101 4.30 x 101 - 5.06 x 101 Mahewu b1.03 x 102 4.70 x 101 - 4.61 x 102 Umqombothi d5.50 x 102 6.90 x 101 - 9.81 x 102 aMean values with same superscripted letter no not differ significantly at 5% confidence level

Figure 9.1 Percentage of endotoxin contamination in fermented foods from Nigeria and South Africa markets

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9.4 Discussion

9.4.1 Bacterial flora of fermented foods from Nigerian and South African markets

Fermented foods and beverages play a significant role in the diet of African populations based on their availability, affordability and contribution to addressing protein-calorie malnutrition as well as micronutrient deficiencies. These products are commonly manufactured following various traditional processing methods but must still be made safe for human consumption. The mean TAPC of the samples exceeded the microbiological limit of > 104 CFU/g set by the European Union for foodstuffs (EC, 2005). The presence of microorganisms beyond this tolerable level is detrimental and could cause food-borne diseases in form of food poisoning or intoxications. There is a high cost paid for negligence of circumspection to food safety and quality in Africa, as several outbreaks of food poisoning linked to microbial contamination are often not reported (Blumberg et al., 2011; Neil et al. 2012; Malangu, 2014). The outbreaks can manifest as acute gastroenteritis, abdominal discomfort, pain, diarrhoea and even death (Kimmons et al., 1999).

In the study of Olasupo et al. (2002), TAPC and Enterobacteriaceae counts of ogi samples from Nigeria were found to be 3.5 x l06 and 4.0 x l05 CFU/g, respectively, however, none of the samples was positive for Vibrio spp. This is in contrast to the data obtained in our study wherein Vibrio spp. was recovered from some ogi samples analysed. Amongst the Enterobacteriaceae spp. identified, E. coli was the most significant because its presence in samples such as ogiri and iru is an indication of faecal contamination. Furthermore, Nwamaka et al. (2010) detected E. coli during ugba fermentation. In addition, Escherichia coli and Serratia ficaria were the enteric bacteria isolated from mahewu by Simango (2002), while E. coli, Klebisiella pneumoniae, P. mirabilis and P. aeruginosa, were the Gram- negative bacteria mainly recovered from iru by Ogunshe and Olasugba (2008).

It is important to note that most studies on fermented foods in Africa have mainly focused on microflora involved in the fermentation process (Oguntoyinbo et al., 2007; Olasupo et al., 2016) by biochemical analytical methods and only a few (Okeke et al., 2015; Adedeji et al., 2017) like in our present study, have investigated the microbiological status of fermented foods with modern techniques such as DNA sequencing. This facilitated the identification of previously unreported organisms of public health importance such as P. canis and V. vulnificus in iru, and Aeromonas haemolyticus, Aeromonas iwoffii, A. salmonicida, Sphingomonas paucimobilim, and Rhizobium radiobacter in ugba. Serratia marcescens was

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In format of International Journal of Food Microbiology detected in 11% of mahewu and 33% of ogi samples obtained from Nigeria. This Gram- negative bacterium is associated with hospital-acquired infections (HAIs) such as urinary tract infections, and is abundantly found in the environment, with starchy foods reported to be an excellent substrate for its growth (Hejazi and Falkiner, 1997).

The infections induced by C. sakazakii had principally been found amongst infants and neonates, therefore the occurrence of this causative agent in foods as noted in our present study raises new concerns as to the risk that it may pose amongst immunocompromised consumers (Beuchat et al., 2009). The persistence of Sphingomonas paucimobilis in all the samples is a source of concern, which like Serratia marcescens is associated with HAIs and widely distributed in nature (Lugito et al., 2016). Two Vibrio spp. (V. parahaemolyticus and V. vulnificus) which are pathogenic, causing acute gastroenteritis in humans (Newton et al., 2012) were found in ogiri samples. Since its recognition, V. parahaemolyticus had been found to be the leading cause of 20–30% of food poisoning cases reported in Asian countries (Alam et al., 2002) and gastroenteritis in humans, relating as well to seafood consumption in the United States (Newton et al., 2012).

In this study, some Gram-positive bacteria such as Bacillus spp. were detected. This agrees with previous reports that fermentation of vegetable proteins (ugba, ogiri and iru) is predominantly by proteolytic Bacillus spp. particularly B. subtilis (Odunfa and Oyewole, 1998; Okorie and Olasupo, 2013; Eze et al., 2014). With respect to this bacteria genus, its incidence was typically high in ugba (67%) and iru (74%). Bacillus altitudinis, B. pumilus, B. halotolerans, B. oleronius, B. safensis, B. xiamenensis and B. toyonensis were members within the Bacillus genera isolated from the samples analysed, probably causing increased level of hydrolytic enzyme production during fermentation due to their hydrolytic properties (Oguntoyinbo et al., 2007). However, when compared to other species, B. subtilis appeared to be more dominant and adapted to the medium because of its higher protease, amylase, polyglutamic, pyrazine, and subtiliosin production (Oguntoyinbo et al., 2007). The predominance of B. cereus in ogi and umqombothi samples with a % incidence of 17 in each of the two samples might be due to its wide distribution in the environment being spore formers (Okorie and Olasupo, 2013).

Enterococcus durans, E. faecium, E. faecalis, E. gallinarum and E. casseliflavus were also detected in the alkaline fermented foods at varying levels and corroborates the report of Oladipo et al. (2013) who isolated different Enterococcus spp. from ugba, iru and ogiri. The

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In format of International Journal of Food Microbiology lactic acid fermented foods and beverages obtained from markets in both countries (ogi, ogi baba, mahewu and umqombothi) had a better safety profile in terms of their bacterial load and of the types of pathogenic microorganisms found than the alkaline fermented foods (ugba, iru and ogiri) in the present study. This is because alkaline fermentation involves hydrolysis of protein and generation of ammonia and amino acids and its preservative action in condiments seems to be limited (Oguntoyinbo et al., 2007). On the other hand, lactic acid fermentation involves the conversion of carbohydrates to alcohol or organic acids, which creates an antagonistic environment for survival of pathogenic and spoilage organisms, enhancing the safety of such foods and beverages.

The pathogenic bacteria present in these foods can be associated with several factors that lead to the contamination of food products along the food chain. This include poor knowledge of food safety, use of contaminated raw materials, utilization of polluted water, inadequate hygienic practices, unstandardized production processes, mixed-culture processing and deplorable hygiene status of processing environments. Others include, poor packaging, inadequate preservation techniques, meagre storage habits, and unhygienic hawking activities.

9.4.2 Endotoxin levels of fermented foods from Nigeria and South Africa markets

The level of production of endotoxins within the samples was between 4.3 x 10 and 5.49 x 104 EU/g. Some of the inherent medical effects of the latter have been reported (Townsend et al., 2007; Wallace et al., 2016; Kalita et al., 2017). Endotoxins are known to proliferate in the gastrointestinal tract but do not enter the blood-stream of healthy individuals due to permeability barriers. With increased ingestion or collapse of the immune system, the permeability barriers may be disrupted, thereby offering entry of endotoxins into the blood stream leading to serious health complications, even death in severe circumstances (Wallace et al., 2016). Endotoxins take a central stage in the pathogenesis of septic shock in man (Martich et al., 1993; Wallace et al., 2016) and induce a series of acute inflammatory responses including fever and fatigue (Porter et al., 2010). Being a heat stable toxin, endotoxins can persist in foods including fermented foods.

Although, fermentation has been reported to inhibit the growth of pathogenic organisms, some studies have reported their persistence during fermentation, thus the possibility of the production of their toxins in such foods. Shigella spp. and pathogenic E. coli were detected in mahewu after 24 hrs fermentation (Shimago and Rukure, 1992), while Nwokoro and Chukwu

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(2012) detected Serratia sp., E coli and E. aerogenes in ogi after 72 hrs fermentation. Iru samples had the highest mean endotoxin levels while the highest contamination (100%) occurred in ogiri (South Africa) and iru (Nigeria). This may be due to the presence of both viable and non-viable Gram-negative bacteria cells in the samples particularly E. coli, which is a known producer of LPS with extremely high endotoxin activity (Silverman and Ostro, 1999; Wallace et al., 2016). In addition to their alkaline state, they are more susceptible to microbial contamination than ogi, ogi baba, mahewu and umqombothi that are acidic.

9.5 Conclusion

The study established the microbial profile of fermented foods and beverages from Nigerian and South African market. The aerobic plate count of the samples tested exceeded the permissible levels for foodstuffs. Pathogenic microorganisms belonging to 14 genera recovered included Escherichia coli, Vibrio vulnificus, Pseudomonas luteola, Bacillus cereus and Serratia mercences and most of the Gram-negative bacteria isolated belonged to the Enterobacteriaceae family. Endotoxin, a heat stable component of the Gram-negative cell wall was present in the samples at varying concentrations. Iru samples had the highest mean endotoxin levels. To the best of our knowledge, this is the first study that assessed the presence of Gram-negative bacteria and their toxins in fermented foods and beverages from Nigerian and South African markets. The presence of pathogenic bacteria and their toxins in these foods and beverages is undesirable and calls for the development and enactment of adaptable food safety measures.

Conflict of Interest

The authors have no conflict of interest to declare.

Acknowledgements

The authors are grateful for the financial support received from the Organisation for Women in Science in the Developing World, Italy and the Centre of Excellence for Food Security co- hosted by the University of Pretoria and University of the Western Cape, South Africa.

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CHAPTER TEN

10.0 GENERAL DISCUSSION AND CONCLUSIONS

10.1 General discussion

The production and processing of fermented foods and beverages in sub-Saharan Africa has not been given due attention beyond cottage level. Fermentation of most cereals and leguminous oil seeds is often by chanced inoculation, unstandardized and only slight consideration is given to the adoption of good manufacturing and hygienic practices along the process and value chains with their safety being overlooked. Moreover, there are no specific regulations guiding the processing and distribution of traditionally fermented foods and beverages and only limited data on the occurrence of pathogenic microorganisms, their toxins and associated deleterious health effects exists. Based on these, a significant number of various population groups in sub-Saharan Africa have a poor perception on the persistence of microbial toxins along the food chain, coupled to their impact on health and the economy. Therefore, it was expedient to conduct studies that may provide useful information on the safety of traditionally fermented foods and level of awareness of fungal and mycotoxin contamination.

Fungal contamination and mycotoxin production in foods can be mitigated to a significant extent if farmers, processors, marketers and consumers possess adequate food safety knowledge. According to Strosnider et al. (2006) and James et al. (2007), there is a strong correlation between awareness and reduction of mycotoxin contamination in developing countries. The level of education is also a significant factor, and in our study, the low level of education of the food sellers correlated (r = 0.308, p < 0.01) with poor perception of mycotoxin contamination and associated health risks. These findings corroborate with that of Matumba et al. (2016) on the knowledge, attitude and practices concerning the presence of moulds in foods amongst members of the public in Malawi.

The present study examined food safety with respect to bacterial, fungal, mycotoxin and endotoxin contaminations. Our target was to screen for these contaminants and establish their contamination levels in traditionally fermented foods from various market outlets in Nigeria and South Africa beginning with isolating and identifying fungi that were responsible for synthesizing mycotoxins (Chapters Three, Four, Five, Six, and Seven). Within this context, 273

emphasis was placed on the genera that were involved in the production of mycotoxins of agricultural, health and economic importance such as the Aspergillia, Fusaria and Penicillia (Pitt et al., 2000; Samson and Varga, 2007) even though some other genera like Alternaria, Claviceps and Stachybotrys are important. Within the Aspergillus genera, A. flavus was the most dominant species, followed by A. parasiticus. Penicillium chrysogenum was the most frequently occurring Penicillium species preceding P. crustosum. Fusarium verticillioides, being the most commonly reported fungal species in maize, was isolated from the fermented maize samples (ogi and mahewu). New fungal strains were also discovered which have been deposited in the National Centre for Biotechnological Information (NCBI) database for reference purposes.

Representative isolates of these fungi were further investigated for their potential in producing mycotoxins and other secondary metabolites. Aflatoxin (AF) producer’s namely A. flavus and A. parasiticus were isolated from some alkaline fermented foods (ugba, iru and ogiri). Some A. flavus species produced sterigmatocystin (STE), O-methyl STE, cyclopiazonic acid (CPA), ß-CPA, AFB1, versiconol (VOH), aspertoxin (ASPT) 1, and ASPT 2 which are AF precursors in the biosynthetic pathways (Yu et al., 2004; Jiang et al., 2015). The production of these metabolites by A. flavus was further shown through their occurrence within a group through the hierarchal clustering. Furthermore, OTA was associated with A. sclerotiorum, A. niger and P. verrucosum.

Similarly, fumonisins (FBs) were mainly produced by F. verticillioides, whereas polycyclic trichothecenes- HT-2 toxin (HT-2) and T-2 toxin (T-2) were produced by the F. sporotrichioides isolates recovered. According to Chelkowski (1998), F. sporotrichioides appears to be a far more potent and widespread producer of HT-2 and T-2 than other Fusarium species including F. acuminatum. Through secondary metabolite production and profiling, an unidentified Fusarium spp. that was present in ugba and iru indicated close relationship to F. graminearum by their capacity to produce deoxynivalenol (DON), 3- Acetyl DON, and zearalenone (ZEN), and this needs to be further investigated.

Fumonisin contamination was more prevalent in maize products (ogi and mahewu) whereas AF contamination was higher in ogiri being a product of melon. These findings are consistent with that of Phoku et al., (2012) who established that maize and maize products are usually high-risk FB contamination substrates, and EFSA (2007) who delineated the

274

susceptibility of leguminous oil seeds such as melon to AF contamination as found in Chapters Four, Five and Six of this report. Some variations were also observed between the presence of fungi and their respective metabolites in foods and the incidence of some mycotoxins was more than that of producing fungal species. For example, FB producing fungi were completely absent but FB was present in 83% of ogi baba samples. It is probably because some strains after toxin production fail to survive amidst others (Summerell et al., 2003) especially due to competition or that the pH of the fermenting substrate/medium was unfavourable for the survival of FB producing fungi.

Other Fusarium mycotoxins such as nivalenol (NIV), neosolaniol (NEO), 3-ADON, Fusarenon X (FUS-X) and diacetoxyscirpenol (DAS) were present in the samples but at low incidence levels and their presence may be due to some of the Fusarium species isolated. Higher incidence of toxigenic fungi and mycotoxins were observed in the fermented foods collected from Nigeria compared to those from South Africa as seen in Chapters Three, Four, Five, Six, and Seven. The inherent climatic conditions in Nigeria might have favoured the proliferation of fungi and made them to be more viable in producing mycotoxins.

Many studies (Strosnider et al., 2006; Pfohl-Leszkowicz and Manderville 2007; Shephard, 2008; Wagacha and Mutomi 2008) have reported the various health effects associated with mycotoxin contamination and in some countries the debilitating health status of individuals have been linked to acute and chronic exposure of mycotoxins by dietary means. In Chapter Eight, the effects of mycotoxin extracts of fungal species isolated from the fermented products on human lymphocytes were investigated. The mycotoxin extracts tested (AFB1,

OTA, STE, FB1, ZEA, DON and roquefortine C (ROQ C) induced cell mortality significantly (p < 0.05), particularly with increased concentration and exposure time and corroborates previous studies (Meky et al., 2001; Lioi et al., 2004; Njobeh et al., 2009; Mwanza et al., 2014).

Aflatoxin B1, OTA and STE had more impacts on cell viability than other mycotoxin, which may be due to their higher concentrations. Moreover, mycotoxins have various chemical structures and properties, and as such their biological actions are variable (Yiannikouris and Jouany, 2002) making them to have different effects on cell viability. Cell mortality induced by the tested mycotoxins may facilitate carcinogenesis, inhibit protein synthesis as reported for OTA (Pfohl-Leszkowicz and Manderville, 2004), potentiate cell membrane damage or

275

apoptosis in different organs as reported for DON (Rizzo et al., 1992) or lead to other health complications. This study further showed that continual ingestion of contaminated foods is deleterious to human health. With the fact that more mycotoxins are being discovered, there is need to conduct more studies on their cytotoxicity in vitro and in vivo for better understanding of their mechanisms of actions and effects on living systems.

To further establish the safety of traditionally fermented food products, their bacterial flora was assessed by evaluating the occurrence of Gram-negative bacteria and their toxin endotoxins (Chapter Nine). Total aerobic bacteria were present beyond acceptable levels in all the samples and the occurrence of the Gram-positive bacteria particularly Bacillus, and Enterococcus species in ugba, iru and ogiri corroborates the report of Okorie and Olasupo (2013), Eze et al. (2014), and Oladipo et al. (2013), who identified these microorganisms as key players in their fermentation. Amongst the Gram-negative bacteria identified, E. coli was the most significant in iru and ogiri being an indicator of faecal contamination.

Serratia marcescens, Cronobacter sakazakii, Vibrio vulnificus and Pasteurella canis were identified by DNA sequencing in this report.The presence of some previously unreported bacterial species within some fermented products was established. The occurrence of these Gram-negative bacteria may be responsible for increased production of endotoxins in the fermented foods. Endotoxin levels varied considerably within the fermented foods and this may be due to substrate variations and the fermenting microflora. Since endotoxins are heat labile, it is therefore highly probable that their presence in some of these samples may induce some health complications such as septic shock, fever or fatigue (Porter et al., 2010, Wallace et al., 2016) particularly amongst unhealthy and immunocompromised individuals.

10.2 General Conclusions

The present study provides an insight into the safety of traditionally fermented foods from Nigeria and South Africa with respect to the occurrence of bacteria, fungi and their associated toxins. The study highlights that exposure to these microorganisms and their toxins were evident amongst consumers even at unacceptable levels. Furthermore, the study established the low level of awareness of food sellers to fungal and mycotoxin contamination and investigated the likely health risks resulting from such exposures, while proposing possible strategies for mitigation. Several microorganisms including new species strains and microbial

276

toxins were also identified, highlighting the need for continual monitoring and implementation of food control measures.

References

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EFSA (European Food Safety Authority). (2007). Opinion of the scientific panel on contaminants in the food chain [CONTAM] related to the potential increase of consumer health risk by a possible increase of the existing maximum levels for aflatoxins in almonds, hazelnuts and pistachios and derived products. EFSA Journal, 5(3), 446.

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Meky, F. A., Hardie, L. J., Evans, S. W., & Wild, C. P. (2001). Deoxynivalenol-induced immunomodulation of human lymphocyte proliferation and cytokine production. Food and Chemical Toxicology, 39(8), 827–836.

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Njobeh, P., Dutton, M., Chuturgoon, A., Koch, S., Steenkamp, P., & Stoev, S. (2009). Identification of novel metabolite and its cytotoxic effect on human lymphocyte cells in comparison to other mycotoxins. International Journal of Biological and Chemical Sciences, 3(3), 3–6.

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APPENDICES

APPENDIX 3.0

Appendix 3.1 Agar preparations

Czapek Concentrate

NaNO3 (30 g), KCl (5 g), MgSO4.7H2O (5 g), FeSO4.7H2O (0.1 g), ZnSO4.7H2O (0.1 g) and

CuSO4.7H2O were dissolved in 100 mL of sterile distilled H2O and mixed thoroughly.

Czapek Yeast Extract Agar (CYA)

K2HPO4 (1 g), yeast extract agar (5 g), sucrose (30 g), agar powder (15 g) and Czapek concentrate (10 mL) were dissolved in 1 L of sterile distilled H2O and autoclaved for 15 mins at 121 oC.

Malt Extract Agar (MEA) Malt extract powder (20 g), peptone (1 g), glucose (20 g) and agar powder (20 g) were o dissolved in 1 L of sterile distilled H2O and autoclaved for 15 mins at 121 C.

Rose-Bengal Chloramphenicol Agar (RBCA)

Rose-Bengal chloramphenicol agar (16 g) was dissolved in 1 L of sterile distilled H2O and autoclaved for 15 mins at 121 oC.

A B C

Appendix 3.2 Some fungal species from fermented foods based on macroscopic characteristics (A-C): A. flavus, F. verticilliodes, and P. expansum

280

A B

Appendix 3.3 Microscopic view of A. parasiticus (A: magnification X63) and F. verticilliodes (B: magnification X40) isolated from ogiri

281

APPENDIX 4.0

Appendix 4.1 Questionnaire on demographics, practices, understanding and perceived health risk of fungal and mycotoxins contamination amongst fermented food sellers in Nigeria

(Instruction: Tick as appropriate) Food Type: Location: 1. Socio-demographic Variables a) Gender Male Female b) Age <30 Years 31-50 Years >50 Years c) Education Level None Primary Secondary Tertiary 2. Fermented Food Characteristics a) Fermented food source Home processed Purchased in the market Purchased directly from processors b) Storage duration of raw materials of the fermented food 1-3 Months >3 Months Not Applicable c) Storage duration of fermented food before sale 1-7 Days >7 Days d) Storage method of raw materials of fermented food Bags Containers Not Applicable e) Storage method of fermented food before sale Polyethylene bags Containers Paper Leaves Wooden boxes f) Average shelf life of raw material of the fermented food 1 Month >1 Month Not Applicable g) Average shelf life of the fermented food 1-3 Days 3-7 Days >7 Days h) Mode of consumption Directly As food ingredient Both 282

3. Knowledge of Fungi, Mycotoxins and Health Effects

S/N Questions Yes No 1 Do you know what fungi are? 2 Can you identify food/crops contaminated with fungi? 3 Do you experience fungal contamination in the fermented food you offer for sale? 4 How frequent do you experience fungal Rarely Frequently Not contamination? Applicable

5 Why do you think fungal contamination occur?

6 Does fungal contamination of foodstuffs cause any health problem when contaminated foods are consumed? 7 Do you know fungi produce toxins called ‘mycotoxins’? 8 Do you know the health effect associated with mycotoxin contamination? 9 Are you willing to attend training on mycotoxins and how they can be reduced in our foods?

4. Any other comment or suggestion(s)

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THANK YOU

283

APPENDIX 5.0

Compound name: Deoxynivalenol Correlation coefficient: r = 0.997421, r^2 = 0.994849 Calibration curve: 0.00324068 * x + 0.17139 A Response type: Internal Std ( Ref 31 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

2.50

2.00

1.50

Response 1.00

0.50

-0.00 microg/kg -0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800

Compound name: Aflatoxine B2 Correlation coefficient: r = 0.996105, r^2 = 0.992225 B Calibration curve: 0.0191981 * x + -0.0373849 Response type: Internal Std ( Ref 32 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

0.600

0.400 Response 0.200

-0.000 microg/kg -0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0

Compound name: Alternariol Correlation coefficient: r = 0.996652, r^2 = 0.993315 Calibration curve: 0.00242952 * x + -0.0190546 C Response type: Internal Std ( Ref 32 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

0.400

0.300

0.200 Response 0.100

-0.000 microg/kg -0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

Appendix 5.1 Calibration curves

2 2 Calibration curve for A: deoxynivanelol (R =0.994849), B: Aflatoxin B2 (R =0.992225), and C: alternariol (R2 =0.993315) 284

A

05032017_005 Smooth(Mn,2x3) F2:MRM of 6 channels,ES+ Spike 0.75 A 297.1>249.2 Deoxynivalenol;4.21;76780.58;7.68e4;612113;303.78 microg/kg 6.131e+005 100

%

0 min

05032017_005 Smooth(Mn,2x3) F2:MRM of 6 channels,ES+ Spike 0.75 A 297.1>231.2 Deoxynivalenol;4.21;29084.59;2.91e4;230347 2.308e+005 100

%

0 min 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 B

05032017_007 Smooth(Mn,2x1) F5:MRM of 4 channels,ES+ Spike 1.5 A 315>287.2 Aflatoxine B2;7.62;85615.32;8.56e4;675721;29.71 microg/kg 6.775e+005 100

%

0 min

05032017_007 Smooth(Mn,2x1) F5:MRM of 4 channels,ES+ Spike 1.5 A 315>259.2 Aflatoxine B2;7.62;72422.66;7.24e4;575076 5.757e+005 100

%

0 min 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 C

05032017_008 Smooth(Mn,2x2) F9:MRM of 6 channels,ES+ Spike 2A 258.9>185.1 Alternariol;10.18;70078.29;7.01e4;455207;203.39 microg/kg 4.582e+005 100

%

0 min

05032017_008 Smooth(Mn,2x2) F9:MRM of 6 channels,ES+ Spike 2A 258.9>213.1 Alternariol;10.17;46436.49;4.64e4;318278 3.216e+005 100

%

0 min 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60 10.80

Appendix 5.2 Chromatograms for standards

Chromatograms for standards A: deoxynivanelol at 300 µg/kg at retention time of 4.21mins, B: Aflatoxin B2 at 30 µg/kg at retention time of 7.62 mins, and C: Alternariol at 200 µg/kg at retention time of 10.17 mins. 285

A

13042017_017 Smooth(Mn,2x1) F6:MRM of 8 channels,ES+ Ogiri 313>285.1 Aflatoxin B1;7.76;211748.72;2.12e5;1802661;12.42 microg/kg 1.813e+006 100

%

0 min

13042017_017 Smooth(Mn,2x1) F6:MRM of 8 channels,ES+ Ogiri 313>241.2 Aflatoxin B1;7.76;188213.55;1.88e5;1607606 1.624e+006 100

%

0 min 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20 8.30 8.40 8.50 8.60 8.70

B

20042017_010 Smooth(Mn,2x2) F10:MRM of 2 channels,ES+ OGI 10 706.1>336.5 Fumonisin B3;10.45;45197.30;4.52e4;323635;123.24 microg/kg 3.287e+005 100

%

0 min

20042017_010 Smooth(Mn,2x2) F10:MRM of 2 channels,ES+ OGI 10 706.1>688.5 Fumonisin B3;10.45;23207.42;2.32e4;163089 1.710e+005 100

% 9.79 10.05 10.93 0 min 9.60 9.80 10.00 10.20 10.40 10.60 10.80 11.00 11.20 11.40

C

23012017_010 Smooth(Mn,2x3) F13:MRM of 4 channels,ES+ Mahewu 325>310.2 Sterigmatocystine;12.45;1067.75;1.07e3;8459;14.53 microg/kg 8.612e+003 100

%

0 min

23012017_010 Smooth(Mn,2x3) F13:MRM of 4 channels,ES+ Mahewu 325>281.1 Sterigmatocystine;12.45;1103.64;1.10e3;8165 8.299e+003 100

%

0 min 11.20 11.40 11.60 11.80 12.00 12.20 12.40 12.60

Appendix 5.3 Chromatograms of some mycotoxins

Chromatograms of some mycotoxins in fermented foods from South African markets A:

Aflatoxin B1 in ogiri (12 µg/kg), B: Fumonisin B3 in ogi (123 µg/kg), and C: Sterigmatocystin in mahewu (15 µg/L).

286

Appendix 5.4 Method performance parameters of the fermented food matrixes

Mycotoxins Calibration range Melon Locust bean African Oil Bean Maize

µg/kg LOD LOQ AR LOD LOQ AR LOD LOQ AR LOD LOQ AR (µg/kg) (µg/kg) (%) (µg/kg) (µg/kg) (%) (µg/kg) (µg/kg) (%) (µg/kg) (µg/kg) (%)

Deoxynivalenol 200-800 11 22 100 4.9 9.8 99 15 30 101 7 14 97 Nivalenol 100-400 48 96 100 11 22 100 21 42 103 35 70 101 Neosolaniol 50-200 20 40 95 16 32 99 24 48 96 2.2 4.4 103 Fusarenon-X 100-400 39 78 97 8.1 16 101 25 50 96 21 42 100 3-Acetyldeoxynivalenol 25-100 2.3 4.6 96 2.0 4.0 102 1.2 2.4 101 5.0 10 105 15-Acetyldeoxynivalenol 12.5-50 1.7 3.5 94 3.9 7.9 101 1.8 3.7 96 10 20 95 Aflatoxin B1 10-40 2.0 4.0 96 1.2 3.3 96 1.5 3.0 100 3.8 7.5 100 Aflatoxin B2 10-40 2.3 4.6 96 1.8 3.3 94 1.4 2.8 96 1.8 3.5 99

Aflatoxin G1 10-40 3.9 7.8 99 1.7 3.3 95 1.9 3.9 98 1.8 3.5 98

Aflatoxin G2 10-40 3.7 7.4 96 1.2 2.3 91 2.2 4.4 94 3.8 7.5 100 Diacetoxyscirpenol 2.5-10 0.9 1.8 97 0.7 1.4 97 1.0 2.0 89 0.3 0.6 99 Alternariol 50-200 6.5 13 98 9.7 20 100 5.9 11 98 40 80 92 Alternariol Methyl Ether 100-400 54 107 96 5.0 10 96 4.6 9.2 98 5.0 10 109 HT-2 Toxin 50-200 6.5 13 98 7.4 14 98 15 30 94 6.5 13 85 T-2 Toxin 50-200 12 24 98 14 28 94 13 26 100 3.6 7.2 87

Fumonisin B1 200-800 24 48 97 22 44 100 38 76 97 8.2 16 87 Fumonisin B2 200-800 11 22 99 9.4 18 99 43 87 95 12 23 89

Fumonisin B3 25-100 13 26 97 21 42 97 33 66 94 14 28 89 Ochratoxin A 25-100 11 22 89 1.2 2.4 93 3.6 7.2 90 1.5 3.0 99 Sterigmatocystin 25-100 5.5 11 100 1.7 3.3 97 1.9 3.8 95 1.3 2.5 100 Roquefortine C 5-20 4.9 9.7 101 1.2 2.3 99 1.0 2.0 99 4.0 8.0 97 Zearalenone 50-200 9.8 20 96 2.9 5.9 92 4.4 8.8 104 3.3 6.5 102 Enniatin B 40-160 26 52 93 6.4 13 94 5.6 11 99 6.3 12 82 1LOD: limit of detection (µg/kg) 2LOQ: limit of quantification (µg/kg) 3AR: Apparent recovery (AR)

287

APPENDIX 7.0

5: MRM of 4 Channels ES+ % 9.00 329 > 311.2 (Aflatoxine G1) 100 8.44e5

0 50 100 150 200 250 300 350 400 450 500 550 600

5: MRM of 4 Channels ES+ % 8.98 329 > 243 (Aflatoxine G1) 100 1.14e6

0 50 100 150 200 250 300 350 400 450 500 550 600

5: MRM of 4 Channels ES+ % 9.43 315 > 287.2 (Aflatoxine B2) 100 1.05e6

0 50 100 150 200 250 300 350 400 450 500 550 600

5: MRM of 4 Channels ES+ % 9.43 315 > 259.2 (Aflatoxine B2) 100 9.10e5

0 Scan 50 100 150 200 250 300 350 400 450 500 550 600

Appendix 7.1 Multiple Reaction Monitoring (MRM) transitions of Aflatoxin G1 and B2 standards indicating their precursor ions, product ions and retention times 288

Appendix 7.2 Aspergillus species in fermented foods as shown in Figure 7.1 and 7.2

Nigerian Fermented Isolates species as shown in Figure Iru A.ruber 44 Food Samples 7.1 and corresponding numbers Iru A.sclerotium 45 Ogiri A.flavus 1 Iru A.versicolor 46 Ogiri A.flavus 2 Iru A.versicolor 47 Ogiri A.flavus 3 Ugba A.flavus 48 Ogiri A.flavus 4 Ugba A.flavus 49 Ogiri A.flavus 5 Ugba A.flavus 50 Ogiri A.flavus 6 Ugba A.flavus 51 Ogiri A.flavus 7 Ugba A.flavus 52 Ogiri A. fumigatus 8 Ugba A.flavus 53 Ogiri A. fumigatus 9 Ogiri A. fumigatus 10 Ugba A.flavus 54 Ogiri A. minisclerotigenes 11 Ugba A.fumigatus 55 Ogiri A. parasiticus 12 Ugba A.niger 56 Ogiri A. parasiticus 13 Ugba A.parasiticus 57 Ogiri A. parasiticus 14 Ugba A.parasiticus 58 Ogiri A. niger 15 Ugba A.spp 59 Ogiri A. niger 16 Ugba A.sclerotium 60 Ogiri A. ustus 17 Ugba A.tubingensis 61 Ogiri A. versicolor 18 Ugba A.tubingensis 62 Ogiri A. clavatus 19 Ugba A.versicolor 63 Iru A. candidus 20 Ogi A.amstelodami 64 Iru A. clavatus 21 Ogi A.flavus 65 Iru A.flavus 22 Ogi A.flavus 66 Iru A.flavus 23 Ogi A.flavus 67 Iru A.flavus 24 Ogi A.flavus 68 Iru A.flavus 25 Ogi A.flavus 69 Iru A.flavus 26 Ogi A.flavus 70 Iru A.flavus 27 Ogi A.fumigatus 71 Iru A.flavus 28 Ogi A.fumigatus 72 Iru A.flavus 29 Ogi A.fumigatus 73 Iru A.flavus 30 Ogi A.niger 74 Iru A.flavus 31 Ogi A.niger 75 Iru A.fumigatus 32 Ogi A.niger 76 Iru A.fumigatus 33 Ogi A.niger 77 Iru A.fumigatus 34 Ogi A.niger 78 Iru A.fumigatus 35 Ogi A.parasiticus 79 Iru A.fumigatus 36 Ogi A.parasiticus 80 Iru A.fumigatus 37 Ogi A.parasiticus 81 Iru A.niger 38 Ogi A.parasiticus 82 Iru A.parasiticus 39 Ogi A.parasiticus 83 Iru A.parasiticus 40 Ogi A.ruber 84 Iru A.parasiticus 41 Ogi A.versicolor 85 Iru A.parasiticus 42 Ogi A.versicolor 86

Iru A.parasiticus 43 Ogi Baba A.clavatus 87

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Ogi Baba A.clavatus 88 Iru A.candidus 28 Ogi Baba A.flavus 89 Iru A.candidus 29 Ogi Baba A.flavus 90 Iru A.flavus 30 Ogi Baba A.flavus 91 Iru A.flavus 31 Ogi Baba A.flavus 92 Iru A.flavus 32 Ogi Baba A.flavus 93 Iru A.flavus 33 Ogi Baba A.flavus 94 Iru A.flavus 34 Ogi Baba A.minisclerotigenes 95 Iru A.flavus 35 Ogi Baba A.niger 96 Iru A.flavus 36 Ogi Baba A.parasiticus 97 Iru A.flavus 37 Ogi Baba A.parasiticus 98 Iru A.flavus 38 Ogi Baba A.spp 99 Iru A.flavus 39 Ogi Baba A.sydowii 100 Iru A.flavus 40 Ogi Baba A.tritci 101 Iru A.flavus 41 Ogi Baba A.versicolor 102 Iru A.fumigatus 42 Iru A.fumigatus 43 Iru A.fumigatus 44 South African Isolates species as shown in Figure Iru A.fumigatus 45 Fermented Samples 7.2 and corresponding numbers Ogiri A.flavus 1 Iru A.niger 46 Ogiri A.flavus 2 Iru A.niger 47 Ogiri A.flavus 3 Iru A.parasiticus 48 Ogiri A.flavus 4 Iru A.parasiticus 49 Ogiri A.flavus 5 Iru A.parasiticus 50 Ogiri A.flavus 6 Iru A.parasiticus 51 Ogiri A.flavus 7 Iru A.parasiticus 52 Ogiri A.flavus 8 Iru A.parasiticus 53 Ogiri A.fumigatus 9 Iru A.sclerotiorum 54 Ogiri A.fumigatus 10 Iru A.sclerotiorum 55 Ogiri A.fumigatus 11 Iru A.sydowii 56 Ogiri A.minisclerotigenes 12 Iru A.sydowii 57 Ogiri A.minisclerotigenes 13 Iru A.tubingensis 58 Ogiri A.niger 14 Iru A.tubingensis 59 Ogiri A.niger 15 Iru A.versicolor 60 Ogiri A.oryzae 16 Ugba A.candidus 61 Ogiri A.oryzae 17 Ugba A.candidus 62 Ogiri A.parasiticus 18 Ugba A.flavus 63 Ogiri A.parasiticus 19 Ugba A.flavus 64 Ogiri A.parasiticus 20 Ugba A.flavus 65 Ogiri A.ustus 21 Ugba A.flavus 66 Ogiri A.ustus 22 Ugba A.flavus 67 Ogiri A.versicolor 23 Ugba A.fumigatus 68 Iru A.amstelodami 24 Ugba A.fumigatus 69 Iru A.amstelodami 25 Ugba A.niger 70 Iru A.candidus 26 Ugba A.niger 71 Iru A.candidus 27 Ugba A.parasiticus 72 290

Ugba A. parasiticus 73 Umqombothi A.flavus 118 Ugba A.sclerotiorum 74 Umqombothi A.flavus 119 Ugba A.sclerotiorum 75 Umqombothi A.flavus 120 Ogi A.clavatus 76 Umqombothi A.flavus 121 Ogi A.clavatus 77 Umqombothi A.fumigatus 122 Ogi A.flavus 78 Umqombothi A.fumigatus 123 Ogi A.flavus 79 Umqombothi A.minisclerotigenes 124 Ogi A.flavus 80 Umqombothi A.minisclerotigenes 125 Ogi A.flavus 81 Umqombothi A.niger 126 Ogi A.flavus 82 Umqombothi A.niger 127 Ogi A.flavus 83 Umqombothi A.niger 128 Ogi A.fumigatus 84 Umqombothi A.parasiticus 129 Ogi A.niger 85 Umqombothi A.parasiticus 130 Ogi A.niger 86 Umqombothi A.parasiticus 131 Ogi A.parasiticus 87 Umqombothi A.sclerotiorum 132 Ogi A.parasiticus 88 Umqombothi A.sclerotiorum 133 Ogi A.sydowii 89 Umqombothi A.sydowii 134 Ogi A.tritici 90 Umqombothi A.sydowii 135 Ogi A.tritici 91 Umqombothi A.tritici 136 Ogi A.versicolor 92 Umqombothi A.tritici 137 Ogi A.versicolor 93 Umqombothi A.versicolor 138 Mahewu A.clavatus 94 Mahewu A.clavatus 95 Mahewu A.clavatus 96 Mahewu A.flavus 97 Mahewu A.flavus 98 Mahewu A.flavus 99 Mahewu A.flavus 100 Mahewu A.flavus 101 Mahewu A.flavus 102 Mahewu A.flavus 103 Mahewu A.fumigatus 104 Mahewu A.fumigatus 105 Mahewu A.fumigatus 106 Mahewu A.niger 107 Mahewu A.niger 108 Mahewu A.niger 109 Mahewu A.parasiticus 110 Mahewu A.parasiticus 111 Mahewu A.parasiticus 112 Mahewu A.tubingensis 113 Mahewu A.versicolor 114 Mahewu A.versicolor 115 Umqombothi A.flavus 116 Umqombothi A.flavus 117

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APPENDIX 8.0

Appendix 8.1 Ethical clearance for cytotoxicity experiment

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APPENDIX 9.0

Appendix 9.1 Agar preparations

Nutrient Agar

Nutrient agar (28 g) was dissolved in 1 L of sterile distilled H2O and autoclaved for 15 mins at 121 oC.

Plate Count Agar

Plate count agar (23.5 g) was dissolved in 1 L of sterile distilled H2O and autoclaved for 15 mins at 121 oC.

Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar

TCBS agar (88.1 g) was dissolved in 1 L of sterile distilled H2O and boiled to dissolve the medium.

MacConkey Agar

MacConkey agar (49.53 g) was dissolved in 1 L of sterile distilled H2O and autoclaved for 15 mins at 121 oC.

Salmonella Shigella Agar

Salmonella Shigella agar (60 g) was dissolved in 1 L of sterile distilled H2O and boiled to dissolve the medium.

Pseudomonas Agar Pseudomonas agar (24.2 g) and glycerol (5 mL) were suspended in 500 mL of sterile distilled

H2O and autoclaved at 121 °C for 15 minutes. The medium was allowed to cool to 50 °C and 1 vail of Pseudomonas CFC Supplement was added upon rehydration.

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Appendix 9.2 Results of microbial identification on VITEK 2 Compact instrument

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