Feb 27, 2018 - within South-west Nigeria and Gauteng Province of South Africa were sampled between ..... Foods from Selected Nigerian and South African Markets. ...... roots, cereals, oil seeds, fish, meat, milk and palm tree and some traditionally fermented ...... Patulin and PA production decreases drastically at reduced.
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How to cite this thesis Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date).
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 - 3 2 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). iii
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…...……
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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 MaizeBased 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 xii
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 xiii
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 xv
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 xvi
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 xx
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 xxii
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 xxiii
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 xxiv
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 xxv
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
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
xxix
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.
xxx
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-βD-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:
5
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.
6
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) 8
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). 13
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.
15
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 galactooligosaccharides 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).
16
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.
17
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 B. cereus, & S. aureus E. coli Enteropathogenic E. coli B. cereus, & E. coli S. aureus, & Klebsiella sp. E. coli, Salmonella sp., & Klebsiella sp. B. subtilis, E. coli, S. aureus, Klebsiella sp., & Enterococcus faecalis B. cereus, Shigella, & Enteropathogenic E. coli E. coli O157:H7, S. aureus, Shigella flexneri, & Salmonella spp. A. niger, A. flavus, P. citrinum,& F. subglutinans Articulospora inflate A. niger, A. rapens A. flavus,& Lemonniera aquatica A. niger, Geotrichum candidum, & Penicillium spp. A.flavus, A. fumigatus, A. minisclerotigenes, A. niger, A. parasiticus, A. sclerotiorum, A. sydowii, A. versicolor, & A. tritici A. fumigatus A. flavus, A. parasiticus, A. sclerotiorum, P. chrysogenum, P. expansum, & F. andiyazi A. amstelodami, candidus A. clavatus, A. flavus, P. polonicum, P. chrysogenum, & F. verticillioides P. chrysogenum, A. niger, & F. eguseti
Food products Banku, kenkey Mahewu Sour milk Ogi baba Wara Nono Ogi, kunuzaki
References Mensah (1997) Simango & Rukure (1991) Nyatoti et al. (1997) Kunene et al. (1999) Olasupo et al. (2002) Olasupo et al. (2002) Olasupo et al. (2002)
Ogiri, iru Borde
Oguntoyinbo& Oni (2004) Tadesse et al. (2005)
Ogi Lafun
Omemu (2011) Ijabadeniyi (2007)
Eko
Adebayo et al. (2014)
Umqombothi
Adekoya et al. (2017a)
Ugba
Adekoya et al. (2017a)
Iru
Adekoya et al. (2017a)
Ogiri
A. clavatus, A. niger, A. parasiticus, A. sydowii, A. tritici P. citrinum, & F. fujikuroi
Ogi baba
Akinyele & Oloruntoba (2013) Adekoya et al. (2017a)
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).
19
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 wellestablished 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, 18501902) established the relationship between damaged host immunity, leucocytosis and endotoxins (Bayston and Cohen, 1990). Then in 1935, Andre Boivin (Microbiologist, 18951949) 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)
24
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. 28
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.
29
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.
30
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.
31
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 a w. 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 34
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 Temperature range for fungal growth (°C)
Water activity (aw) for fungal growth
A. flavus A. parasiticus
Minimum 10 10
Optimum 25-35 32-35
Maximum 43 43
Minimum 0.80 0.83
Optimum 0.95-0.99 0.95-0.99
Maximum -
A. ochraceus F. verticilliodes F. proliferatum F. culmorum
8 2 4 0
24-37 23-30 30 20-25
37 37 37 35
0.77 0.87 0.9 0.90
0.95-0.99 0.98-0.995
0.99 -
F. poae F. avenaceum F. tricinctum F. graminearum
5 5 5 -
20-25 20-25 20-25 24-26
35 35 35 -
0.90 0.90 0.90 0.90
0.98-0.995 0.98-0.995 0.98-0.995 -
0.99
F. sporotrichioides
-2
21-28
35
0.88
-
0.99
P. verrucosum
0
20
35
0.80
0.95
-
Fungal species
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
A. flavus A. parasiticus A. ochraceus P. verrucosum F. verticilliodes F. proliferatum F. culmorum F. graminearum
Temperature range for mycotoxins formation (°C)
Water activity (aw) for mycotoxins formation
Minimum 12 12 12 4 10 10 11 11
Minimum 0.82 0.87 0.800.83 0.92 0.93 0.9
Optimum 30-33 33 25-31 20-25 15-30 15-30 29-30 29-30
Maximum 40 40 37 37 37 -
Optimum 0.99 0.99 0.98 0.90-0.95 0.98
Maximum 0.998 -
Source: CAST (2003); Sanchis and Magan (2004); Ribeiro et al. (2006)
35
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
37
maltose amidst other carbohydrate sources (fructose, lactose and glucose) (Engel 1976). Antifungal agents: Diverse antifungal agents or fungicides are utilized in tackling biodeterioration, 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.
39
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
41
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 Lphenylalanine 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).
43
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).
44
(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) pyran2(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 46
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-methyl49
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 (3ADON) and 15-acetyl- deoxynivalenol (15-ADON).
Figure 2.23 Molecular structure of deoxynivalenol-3-glucoside (Adapted from Berthiller et al., 2011) 51
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 (ßZEL) (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
Egypt
Uganda
AFs AFs AFs Fusaric acid (FA) FB1 FB2 DON NIV FBs FBs AFs DAS FB1 FB1 AFs 3-A DON ENN B BEA AFs ZEN AFs
Sudan Ethiopia
AFs AFs AFs
Meat products Spices Milk Sugar cane juice Maize Maize Maize Maize Maize Kenkey Chips Cassava flour Cassava flour Maize Dried kernels Wheat kernels Wheat kernels Wheat kernels Milk Wheat kernels Groundnut, cassava, millet and sorghum flour Dried kernels Peanut butter Sorghum, barley, teff and Wheat Sorghum, barley and wheat Sorghum Millet Millet Cereal &cereal products Cereal & cereal products Cereal & cereal products Cereal & cereal products Cereal & cereal products Maize Peanut butter Maize Maize Groundnut Maize
Burkina Faso
Ghana Benin
Kenya
OTA
Tunisia & Morocco
DON FB1 ZEN NIV BEA AFs OTA FBs
Zambia Mozambique
FBs AFs FB1 FB2 BEA ZEN
Concentration µg/kg or µg/mL 2-150 2-35 50-270 25.4-2,214 22.5 -1,343 11.3 – 589 31.4 11.0-15.8 70-4,222 15-1,000 2.2-220 0-5 4-24 51-836 22-190 80-1,703 2-256 13-15 >5 7-55 0-55
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0-435 21-170 0-26
Probst et al. (2014) Elshafie et al. (2011) Ayalew et al. (2006)
54.1-2,106
Ayalew et al. (2006)
40-2,340 0 -49.2 0-459 135-961
Ayalew et al. (2006) Chala et al. (2014) Chala et al. (2014) Serrano et al. (2012)
2.1-844
Serrano et al. (2012)
5.5-66.7
Serrano et al. (2012)
75-112
Serrano et al. (2012)
121-176
Serrano et al. (2012)
33,500-192,000 0-130 159-7,615 27.7-3,061 0.1-24 10.9-18.1
Mukanga et al. (2010) Njoroge et al. (2016) Warth et al. (2012) Warth et al. (2012) Warth et al. (2012) Warth et al. (2012)
Aziz & Youssef (1991) Selim et al. (1996) El-Tras et al. (2011) Abdallah et al. (2016) Warth et al. (2012) Warth et al. (2012) Warth et al. (2012) Warth et al. (2012) Kpodo et al. (2000) Kpodo et al. (2000) Bassa et al. (2001) Ediage et al. (2011) Ediage et al. (2011) Ediage et al. (2011) Hell et al. (2000) Wagacha et al. (2010) Wagacha et al. (2010) Wagacha et al. (2010) Kangethe & Langa (2009) Wagacha et al. (2010) Kitya et al. (2010)
53
Country
Mycotoxin Type
Sample Type
Malawi
ZEN DON NIV AFs
AFs AFs AFs FBs PAT DON AFM1 AFs DON FBs AFB1 FB1
Maize Maize Maize Maize based beer Maize based beer Dried kernels Dried kernels Maize Maize Apple juice Wheat Cow milk Peanut Maize meal Maize Maize Maize
FB1
Sorghum
2,500-6,000
FB1
Peanut
200-1,400
DON NIV AFs AFs DON3G α-ZEL BEA ZEN ZEN FB1 AFs BEA ZEN DON AFB1 AFB1 DON3G AFB1 AME ZEN FB1 DON
Maize Maize Peanut Maize Maize beer Maize beer Soybean Maize Peanut Maize Rice Maize Maize Maize Egusi Peanut cake Sorghum Ogiri Iru Ugba Ogi Ogi baba
0-492 0-530 6.6-622 0-123 0.3-27 4-90 12-19 28-273 31-186 37-24,225 28-372 0.1-120 115-779 11-749 2.3-15.4 13-2,824 24 (mean) 3-4 19-77 39-117 68-2,492 32-112
FBs
Tanzania
South Africa
Zimbabwe
Cameroon
Nigeria
Concentration µg/kg or µg/mL 0-2,025 0-2,328 0-2,220 0-185
References
493-3,303
Matumba et al. (2014)
5-20 3-1,081 158 11,048
