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                                   UNIVERSITY OF JOHANNESBURG




           AN INVESTIGATION OF FUNGI AND MYCOTOXINS IN BARLEY
                         GRAIN AND MATERIALS USED FOR BREWING




                                     PHOLO WILSON MAENETJE
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           AN INVESTIGATION OF FUNGI AND MYCOTOXINS IN BARLEY
                           GRAIN AND MATERIALS USED IN BREWING




                                                  By


                                       PHOLO WILSON MAENETJE


                                            DISSERTATION


                          Submitted in compliance of the requirement for the


                                   MASTERS’S DEGREE IN TECHNOLOGY


                                          in the Department of


                                          BIOTECHNOLOGY


                                                 at the


                                     UNIVERSITY OF JOHANNESBURG


           SUPERVISOR AND PROMOTOR: PROFESSOR M. F DUTTON


           CO-SUPERVISOR:                           MR E. VAN ZYL



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                                                 ABSTRACT
           Mycotoxins, secondary metabolites of filamentous fungi, are associated with foods due to
           the ubiquitous nature of certain fungi that infect crops during harvesting or storage. These
           toxins have been implicated as chemical agents of acute and chronic diseases in animal
           and man. The most commonly acute effects of mycotoxin poisoning is the deterioration
           of the liver and kidney functions, allergic responses and immunosuppresion, whereas
           chronic effects include mutagenicity, teratogenicity and carcinogenicity. The most
           common toxigenic fungal genera include Aspergillus, Fusarium and Penicillium.
           Aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), trichothecenes and zearalenone
           (ZEA) are the most important mycotoxins in terms of occurrence on food.


           A study was conducted to evaluate and quantify the occurrence of mycotoxins in barley
           as well as barley-producing beer products in South Africa. A total of 86 barley samples
           were randomly obtained from Gauteng retail outlets, Maltsters and South African
           Breweries and were screened for toxigenic fungi. Two fungal genera, Aspergillus,
           Penicillium occurred regularly whereas Fusarium and Mucor were detected at low
           incidences. High levels of fungal contamination were found in barley obtained in
           Gauteng as compared to Maltsters barley samples, however, most of the fungal strains
           isolated from Gauteng purchased barley were non-toxigenic as compared to Maltsters.


           Barley samples were further screened for mycotoxins by multi-mycotoxins extraction
           coupled with thin layer chromatography (TLC). Mycotoxins detected in the barley
           extracts were aflatoxins, ochratoxins, deoxynivalenol (DON) and zearalenone at trace
           levels on the thin layer chromatograms. However, TLC only indicated qualitative results.
           The presence of the toxins were confirmed by techniques that a highly sensitive and
           quantitative,    such   as   gas   chromatography-mass     spectroscopy    (GC-MS)      and
           immunoaffinity analysis.


           The presence of deoxynivalenol in the barley fractions was confirmed by GC-MS at
           mean concentration levels ranging from 0.0628 to 0.832 ppm. Barley samples from
           Maltsters, however, showed to be highly contaminated with DON compared to barley


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           obtained in Gauteng (p < 0.05). As barley is known to be one of the major ingredients of
           beer, a total of 48 beer samples were also randomly collected from retail outlets in the
           Gauteng region and were surveyed for the presence of AFB1, AFB2 and ochratoxin A.
           Trace levels of AFB1 were detected in some of the beer samples, whereas AFB2 was not
           detected. Ochratoxin A contamination, however, in beer ranged from 0.07 to 0.081 ppb.


           The level of mycotoxins contamination in barley samples analysed by immunoaffinity
           analysis ranged from: 0.0 to 3.9 ppb AFs, 5.0 to 10.0 ppb OTA, 0.0 to 10.0 ppm DON,
           0.0 to 5.0 ppm FBs and 0.4 to 2.9 ppm ZEA in Maltsters barley, whereas in Gauteng
           samples mycotoxin contamination levels ranged from 0.0 to 6.0 ppb OTA, 2.0 to 2.0 ppm
           DON, 0.0 to 2.0 ppm FBs and 0.5 to 3.4 ppm ZEA. Although high fungal infection was
           found in Gauteng samples, Maltsters samples were found to be more contaminated with
           mycotoxins (p < 0.05).


           An investigation was also conducted to confirm the natural occurrence of fumonisin B1
           (FB1) in barley samples at levels of up to 5 ppm, as determined by Vicam immunoaffinity
           analysis. The HPLC analysis was used to determine FB1 in these barley samples. HPLC
           analysis of the barley samples previously found to be positive for fumonisins revealed
           detectable levels of    0.21 ppm FB1 in only 7 samples of the 24 samples analysed.


           Materials found to contain fungi and mycotoxins were further examined for cytotoxicity
           using human lymphocytes for possible chronic effects. Pure mycotoxins and selected
           barley fractions were found to be toxic to the lymphocytes. A study was also conducted
           to determine whether cytotoxicity testing could be used as additional tool for estimating
           the amount of toxin present in a commodity.


           The differences in the level of fungal and mycotoxins contamination between Gauteng
           and Maltsters could have been due to the difference in the environmental conditions,
           which the barley was harvested, or the varying degree of handling and storage within the
           companies. This study may also present the general picture on the quality of products in
           the brewery industry. Although some of the barley samples were of low quality in regards



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           to food safety, the issue of upgrading quality control measures in the barley producing
           regions in South Africa will be of paramount importance.




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                                             DECLARATION

           I hereby declare the dissertation, which I herewith submit for the research qualification


                                                                   ..
           to the University of Johannesburg is, apart from recognized assistance, my own work and
           has not previously been submitted by me to another institution to obtain a research
           diploma or degree.




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                                       ACKNOWLEDGEMENTS
           There are a number of people to whom I am indebted for their contribution to this study
           and I wish to express my gratitude and sincerest thanks to them.


           Thanks firstly, to my supervisor Professor M. F Dutton, for his timely acceptance as my
           supervisor, giving me the opportunity to conduct this study and providing professional
           guidance and suggestions regarding all aspects of the study.


           To Mr Eric Van Zyl, my Head of Department for all the assistance he offered during my
           studies through the provision of financial assistance as well literature materials relevant
           to my research. I am eternally grateful for the assistance you have given to me.


           I am sincerely grateful to Dr I. Meijering of South African Maltsters and Dr P. Toline of
           South African Breweries for the supplying me with barley samples.


           My sincerest thanks to Mr Neil De Villiers, of The Department of Biomedical
           Technology for his invaluable assistance with statistical analysis of data and with the use
           of Bench Mark plate reader as well as other aspects of the laboratory.


           I am also grateful to Mr Patrick Njobeh for his assistance with the preparation of this
           manuscript. His insightful, relevant and critical assessment of the writing was most
           helpful.


           Thanks to Mr. Jan Voster and Mr. Rui Krause of The Department of Chemistry for their
           assistance with the use of the Gas Chromatography-Mass Spectroscopy and the High
           Performance Liquid Chromatography.


           Thanks to Prof. A. Chuturgoon of the Department of Physiology in Natal University for
           the assistance with analysis of samples using High Performance-Liquid Chromatography.




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           To Mr Alaister Campbell, of the Department of Biotechnology for his assistance is
           drawing blood necessary for the lymphocytes studies and valuable technical advise and
           assistance with many aspects of my study, I offer my sincerest thanks.


           My profound gratitude to all members of Food, Health and Environmental Research
           Group, for their invaluable advice and assistance with various aspects of this study.


           I would like to offer my sincerest gratitude to my friends, particularly Mr Josef Rakoma,
           Mr Lebo Mmatli, Mr Lucas Ledwaba, Mr Thabo Makena, Miss Mabusha Mabetoa and
           Miss Thokozile Ledwaba for their encouragement in the darker hours, of they were
           many.


           To Miss Nomfanelo Matiwane, you have been a constant source of love, support and
           assistance. Thank you so much for everything that you have done. You are my guiding
           light and I don t know what I would have done without you!


           To all the students and staff in The Department of Biotechnology and throughout the
           University of Johannesburg, past and present. Many of you have contributed in some way
           towards this study, that any assistance received was truly appreciated.


           My utmost thanks and appreciation go to my parents for their sacrifice, support,
           motivation and encouragement. They have stood by me, prayed for my success and
           supported me all my life. God s protection and love for me is due to their prayers. I also
           thank my brothers; Tshepo Maenetje and Samuel Maenetje and my sisters; Maurine
           Maenetje and Margate Maenetje for the trust and support. Thanks to my grandmother
           Elsie Maenetje (Blessed memory) for her love and guidance in my life.


           I thank TCR funding of Technikon Witwatersrand for the financial assistance of this
           research.


           Finally, TO THE ALMIGHT GOD, for giving the strength to overcome obstacles I had.



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                                           TABLE OF CONTENTS


           ABSTRACT                                                      . III
           DECLARATION                                                 ..... VI
           ACKNOWLEDGEMENTS                                                   VII
           TABLE OF CONTENTS                                           .. X
           LIST OF FIGURES                                             .... XIV
           LIST OF TABLES                                             ...... XVIII


           CHAPTER ONE                                                   .. 1
           1.0 Introduction                                              .. 1
                  1.1 Mycotoxins.                                         .1
                  1.2 Aims of this study                                      2


           CHAPTER TWO                                                    .3
           2.0 Literature review                                         .. 3
                  2.1 Introduction                                       .. 3
                  2.2 Toxigenic fungi                                         4
                            2.2.1 Aspergillus spp.                        .6
                            2.2.2 Fusarium spp.                         ... 7
                            2.2.3 Penicillium spp.                        .8
                  2.3 Occurrence of mycotoxins                          ... 8
                            2.3.1 Aflatoxins                             ... 10
                            2.3.2 Ochratoxins                                 10
                            2.3.3 Fumonisins                              . 12
                            2.3.4 Zearalenone                                 13
                            2.3.5 Trichothecenes                        ... 14
                            2.3.6 Other significant mycotoxins            . 15
                  2.4 Mycotoxin intake and their biological effects       . 19
                            2.4.1 Aflatoxins                             ... 19
                            2.4.2 Ochratoxins                                 20


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                            2.4.3 Fumonisins                                                                                     . 21
                            2.4.4 Zearalenone           ............................................................................ 22
                            2.4.5 Trichothecenes                                                                               .. 22
                            2.4.6 Other mycotoxins                                                                             ... 23
                    2.5 Control of mycotoxin contamination in the food industry                                                 .. 25
                            2.5.1 Concept of quality control                                                                     . 25
                                     2.5.1.1 Fungal control                                                                          25
                                     2.5.1.2 Mycotoxin control                                                                  . 26
                    2.6 Agricultural and food commodities as reservoir for mycotoxin                                            .. 27
                            2.6.1 Barley as a reservoir of mycotoxins                                                           ...27
                                     2.6.1.1 The brewing process                                                                .. 28
                                     2.6.1.2 Fungal contamination in South African barley and malt                             ... 29
                                     2.6.1.3 Mycotoxins in barley, malt and beer                                                 ..30
                    2.7 Legislation                                                                                                31
                    2.8 Conclusion                                                                                                   33


           CHAPTER THREE                                                                                                         . 34
           3.0 Morphological studies on fungal screening                                                                         . 34
                    3.1 Fungal contamination                                                                                    ...34
                    3.2 Methodology                                                                                                  34
                            3.2.1 Sample collection and preparation                                                                34
                            3.2.2 Evaluation of toxigenic fungi                                                                    35
                    3.3 Statistical analysis                                                                                       35
                    3.4 Results                                                                                                 .. 36
                            3.4.1 Fungal screening                                                                                   36
                            3.4.2 Toxigenic potential of the fungal isolates                                                     . 40
                    3.5 Discussion                                                                                                 42
                            3.5.1 Fungal screening                                                                             ... 42
                            3.5.2 Evaluation of toxigenic fungi                                                                    46
                    3.6 Conclusion                                                                                             ... 48




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           CHAPTER FOUR                                                                                                               49
           4.0 Multi-Mycotoxin screening............................................................................................. 49
                     4.1 Thin layer chromatography                                                                                .. 49
                     4.2 Methodology                                                                                                  50
                                 4.2.1 Extraction of toxins                                                                      ... 50
                     4.3 Statistical analysis                                                                                         51
                     4.4 Results                                                                                                  .. 51
                                 4.4.1 Determination of spots on thin layer chromatograms                                        . 51
                                 4.4.2 Determination of spots on thin layer chromatograms using spray
                                      reagents                                                                                     . 54
                     4.5 Discussion                                                                                                   58
                     4.6 Conclusion                                                                                                   61


           CHAPTER FIVE                                                                                                           . 62
           5.0 Gas Chromatography- Mass Spectrometry (GC-MS) and High Performance Liquid
               Chromatography (HPLC)                                                                                               . 62
                     5.1 Overview                                                                                                 .. 62
                     5.2 Methodology                                                                                                  62
                                 5.2.1 Gas-Chromatography-Mass Spectroscopy determination                                          .62
                                         5.2.1.1 Derivatisation of mycotoxins                                                      . 63
                                         5.2.1.2 Analysis                                                                         ..63
                                 5.2.2 High Performance Liquid Chromatography determination                                       .. 64
                                         5.2.2.1 Aflatoxin B1 analysis                                                                64
                                         5.2.2.2 Ochratoxin A analysis                                                           .. 64
                     5.3 Statistical analysis                                                                                         65
                     5.4 Results                                                                                                  .. 65
                                 5.4.1 Gas-Chromatography-Mass Spectroscopy                                                           65
                                 5.4.2 High Performance Liquid Chromatography                                                      .70
                                         5.4.2.1 Aflatoxin B1                                                                     .. 70
                                         5.4.2.2 Ochratoxin A                                                                     .. 73
                     5.5 Discussion                                                                                                   75



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                            5.5.1 Gas-Chromatography-Mass Spectroscopy                                     75
                            5.5.2 High Performance Liquid Chromatography                               . 77
                                     5.5.2.1 Aflatoxin B1                                             ...77
                                     5.5.2.1 Ochratoxin A                                              .. 78
                    5.6 Conclusion                                                                         80


           CHAPTER SIX                                                                                     81
           6.0 Quantification of mycotoxins using Vicam                                                . 81
                    6.1 Immunoaffinity analysis                                                       .. 81
                    6.2 Methodology                                                                        81
                            6.2.1 Spiking of barley samples                                             . 81
                            6.2.2 Extraction and immunoaffinity clean-up of barley samples             . 81
                                     6.2.2.1 Summary of clean-up procedures                           .. 82
                    6.3 Confirmation of Fumonisin B1 in barley samples using High-Performance
                        Liquid Chromatography                                                         ... 82
                            6.3.1 HPLC analysis                                                       .. 82

                    6.4 Statistical analysis                                                               82

                    6.5 Results                                                                        . 83
                            6.5.1 Quantification of mycotoxins in barley samples                      .. 83
                            6.5.2 Comparison between samples from Gauteng region                        .86
                            6.5.3 Comparison between samples from Maltsters                  ............. 88
                            6.5.6 Confirmation of FB1 in barley fractions                           .. 89
                    6.6 Discussion                                                                         90
                            6.6.1 Confirmation fumonisin in barley                                  .. 94
                    6.7 Conclusion                                                                         95


           CHAPTER SEVEN                                                                               ..97
           7.0 Cytotoxicity testing                                                                    .. 97
                    7.1 Methyl tetrazolium analysis                                                     . 97
                    7.2 Methodology                                                                        97
                            7.2.1 Sample collection                                                    .. 97


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                                7.2.2 Isolation and purification of peripheral blood mononuclear cells                                     ..98
                                                     7.2.2.1 Cell enumeration by Trypan blue method                                       ...98
                                                     7.2.2.2 Dose response to individual mycotoxins                                           .99
                                                     7.2.2.3 Methyl thiazole tetrazolium assay                                            ...99
                                7.2.3 Estimation of toxin concentration in extracts using cytotoxicity test                                   100
                     7.4 Statistical analysis                                                                                                 100
                     7.5 Results                                                                                                          .. 100
                                7.5.1 Toxicity of mycotoxin standards and barley fractions                                                ..100
                                7.5.2 Estimation of toxin concentration in barley fractions using
                                        cytotoxicity test                                                                       .             105
                     7.6 Discussion                                                                                                           107
                                7.5.1 Toxicity of mycotoxin standards                                                                    ...107
                                7.5.2 Toxicity of mycotoxin standards in comparison to barley fractions .109
                                7.5.3 Estimation of toxin concentration in barley fractions using
                                        cytotoxicity test                                                                                  . 110
                     7.7 Conclusion........................................................................................................... 111


           CHAPTER 8                                                                                                                       . 112
           8.0 General Conclusion...                                                                                                     ... 112


           REFERENCES                                                                                                                      . 115


           APPENDICES                                                                                                                     .. 146
           Appendix I                                                                                                                         146
           Appendix II                                                                                                                   ...147
           Appendix III                                                                                                                    . 150
           Appendix IV                                                                                                                    . 151
           Appendix V                                                                                                                    ... 153
           RAW DATA......................................................................................................................... 157




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                                           LIST OF FIGURES
           CHAPTER TWO
           Fig. 2.1 Penicillium (left), Fusarium (middle) and Aspergillus (right) (Kendrick, 1986)     .5
           Fig. 2.2 Chemical structure of Aflatoxin B1 (Sweeney and Dobson, 1998)                     .. 10
           Fig. 2.3 Chemical structure of Ochratoxin A (Sweeney and Dobson, 1998)                          11
           Fig. 2.4 Chemical structure of Fumonisin B1 (Sweeney and Dodson, 1998)                       .13
           Fig. 2.5 Chemical structure of Zearalenone (Bhatnagar et al., 2002)                             14
           Fig. 2.6 Chemical structure of Deoxynivalenol (Moss, 1996)                                 .. 15
           Fig. 2.7 Chemical structure of Ergotamine (Moss, 1996)                                      ..16
           Fig. 2.8 Chemical structure of Sterigmatocystin (Sweeney and Dobson, 1998)                ... 17
           Fig. 2.9 Chemical structure of Patulin (Sweeney and Dobson, 1998)                          ... 17
           Fig. 2.10 Chemical structure of Citrinin (Sweeny and Dobson, 1998)                          . 18
           Fig. 2.11 Chemical structure of Moniliformin (Pineda-Valdes and Bullerman, 2000)            . 19
           Fig. 2.12 The simplified scheme for brewing (Linko et al., 1998)                                28


           CHAPTER THREE
           Fig. 3.1 Level of fungal infection in barley samples from Gauteng and Maltsters           .. 39
           Fig. 3.2 Level of fungal infection in barley and malted barley samples from Maltsters           40


           CHAPTER FOUR
           Fig. 4.1 A flow diagram representing an overview of multi-mycotoxin extraction
                   procedure (Patterson and Roberts, 1979)                                            . 50
           Fig. 4.2 Diagrammatic representation of the overall spots occurring on TLC plates from
                   barley and malted barley neutral fractions under UV light                          .. 51
           Fig. 4.3 Diagrammatic representation of the overall spots occurring on TLC plates from
                   barley and malted barley neutral fractions under UV light                       ...... 52
           Fig. 4.4 A thin layer chromatogram indicating AFB1 (A) and AFB2 (B) and OTA (C)
                   under UV light                                                             ..     ... 54
           Fig. 4.5 A thin layer chromatogram indicating pure T-2 toxin (A) and DON (B). Mobile
                   phases: CEI (1st dimension) and TEF (2nd dimension)                                     55




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           Fig. 4.6 A thin layer chromatogram indicating a barley neutral fraction indicating the
                    presence of DON. Mobile phases: CEI (1st dimension) TEF (2nd dimension)             . 55
           Fig. 4.7 A thin layer chromatogram indicating pure zearalenone. Mobile phases: 2 %
                    methanol in dichloromethane (1st dimension) and cyclohexane (2n dimension)         .. 56


           CHAPTER FIVE
           Fig. 5.1 Chromatogram of a silylated barley sample spiked with 50 ppm DON                ...... 66
           Fig. 5.2 Mass spectrum of a silylated barley sample spiked with 50 ppm DON                  ... 66
           Fig. 5.3 Chromatogram of a silylated blank barley fraction                                   .. 67
           Fig. 5.4 Chromatogram of a naturally contaminated barley fraction with DON                   .. 68
           Fig. 5.5 Level of DON contamination in Gauteng and Maltsters barley samples                   . 70
           Fig. 5.6 Chromatogram of a spiked beer sample with 0.05 ppb AFB2 (A) and AFB1 (B)             .70
           Fig. 5.7 UV-spectra and the absorption maxima (nm) of a spiked beer sample
                    (0.1 ppb AFB1                                                 ..                ....... 71
           Fig. 5.8 UV-spectra and the absorption maxima (nm) of a spiked beer sample
                    (0.1 ppb (AFB2                                                                        71
           Fig. 5.9 UV-spectra and absorption maxima (nm) of naturally contaminated beer sample
                    with AFB1                                                                             72
           Fig. 5.10 Chromatogram of a beer sample spiked with 0.05 ppb OTA                              . 73
           Fig. 5.11 UV-spectra and the absorption maxima (nm) of a spiked beer sample.................. 73
           Fig. 5.12 UV-spectra and the absorption maxima (nm) of a naturally contaminated beer
                      sample at estimated concentration of 0.09 ppb OTA                                 .. 74


           CHAPTER SIX
           Fig. 6.1 Level of aflatoxin and ochratoxin contamination in Gauteng and Maltsters
                     barley sample using Vicam fluorometry analysis                                       85
           Fig. 6.2 Level of deoxynivalenol, fumonisin and zearalenone in Gauteng and Maltsters
                    barley samples using Vicam fluorometry analysis                                        85
           Fig. 6.3 Level of aflatoxin and ochratoxin in barley samples from Gauteng using
                    Vicam fluorometry analysis                                                          .. 87




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           Fig. 6.4 Level of deoxynivalenol, fumonisin and zearalenone in barley samples from
                    Gauteng using Vicam fluorometry analysis                                             . 87
           Fig. 6.5 Level of aflatoxin and ochratoxin in barley samples from Maltsters using
                    Vicam fluorometry analysis                                                           ...88
           Fig. 6.6 Level of deoxynivalenol, fumonisin and zearalenone in barley samples from
                    Maltsters using Vicam fluorometry analysis                                            .89
           Fig. 6.7 Chromatogram of a derivatised pure fumonisin at 10 ppm FB1 indicated by              . 90
           Fig. 6.8 Chromatogram of naturally contaminated barley fraction with FB1.........................90


           CHAPTER SEVEN
           Fig. 7.1 Dose- response linear regression curve of pure fumonisin B1 on the human
                     PBMCs (r = 0.958). All plotted dots of the curves represent means
                    of 3 measurements in 3 distinct test procedures                                     ... 101
           Fig. 7.2 Dose-response linear regression curve of pure deoxynivalenol on the human
                    PBMCs (r = 0.713). All plotted dots of the curves represent means
                    of 3 measurements in 3 distinct test procedures                                     ... 102
           Fig. 7.3 Dose- response linear regression curve of pure ochratoxin A on the human
                    PBMCs (r = 0.819). All plotted dots of the curves represent means
                    of 3 measurements in 3 distinct test procedures                                     ... 103
           Fig. 7.4 Dose- response linear regression curve of a neutral barley extract on the human
                    PBMCs (r = 0.811). All plotted dots of the curves represent means
                    of 2 measurements in 3 distinct test procedures                                     ... 104
           Fig. 7.5 Dose- response linear regression curve of acid barley extract on the human
                    PBMCs (r = 0.714). All plotted dots on the curve represent means
                    of 3 measurements in 3 distinct test procedures                                     ... 105
           Fig. 7.6 Linear regression curve of FB1 actual and estimated concentration values
                   (r = 0.946)                                                                           .. 106
           Fig. 7.7 Linear regression curve of DON actual and estimated concentration values
                   (r = 0.802                                                                                106
           Fig. 7.8 Linear regression curve of FB1 actual and estimated concentration values
                   (r = 0.371)                                                                           .. 107



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                                             LIST OF TABLES
           CHAPTER TWO
           Table 2.1 Incidence of mycotoxins in South African Agricultural commodities from
                      1984- 1993 (Dutton and Kinsey, 1996)                                           .9
           CHAPTER THREE
           Table 3.1 Results of fungal species detected in barley samples from Gauteng               . 36
           Table 3.2 Results of fungal species detected in pearl barley samples from Gauteng             37
           Table 3.3 Results of fungal species detected in barley samples from Maltsters                 38
           Table 3.4 Results of fungal species detected in malted barley samples from Maltsters          38
           Table 3.5 Detected fungal species belonging to the genera Penicillium                    .. 40
           Table 3.6 The toxigenic potential of fungal strains isolated from Gauteng barley samples.. 41
           Table 3.7 The toxigenic potential of fungal strains isolated from Maltsters barley samples.42


           CHAPTER FOUR
           Table 4.1 Results of the averaged RF values of the spots that commonly occurred on
                      thin layer chromatograms                                                      .. 53
           Table 4.2 Results of the incidence rate of common spots on thin layer chromatograms .. 53
           Table 4.3 Results of the incidence rate of mycotoxins from barley fractions determined
                      by thin layer chromatograms.                                                   .57
           Table 4.4 Results of the intensity of mycotoxins detected on thin layer chromatograms
                      in Gauteng barley fractions                                                        57
           Table 4.5 Results of the intensity of mycotoxins detected on thin layer chromatograms
                      in Maltsters barley fractions                                                      58

           CHAPTER FIVE
           Table 5.1 Results of the incidence rate of DON from barley fractions determined by
                      gas chromatography- mass spectroscopy                                         ... 68
           Table 5.2 Level of DON contamination in the barley fractions from Gauteng region          . 69
           Table 5.3 Level of DON contamination in the barley fractions from Maltsters                   69
           Table 5.4 The incidence of AFB1 contamination in beer samples obtained from the
                       Gauteng region                                                               .. 72
           Table 5.5 Level OTA contamination in beer samples obtained from the Gauteng region            75


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           CHAPTER SIX
           Table 6.1 Results of barley samples spiked with pure mycotoxins using Vicam
                    fluorometry analysis                                                           .. 83
           Table 6.2 Results of the incident rate of mycotoxin contamination in barley samples
                    using Vicam fluorometry analysis                                                . 83
           Table 6.3 Results of level of mycotoxin contamination in barley samples using Vicam
                    fluorometry analysis                                                           ...84
           Table 6.4 Level of mycotoxin contamination in Gauteng and Maltsters barley samples
                    using Vicam fluorometry analysis                                                . 86


           CHAPTER EIGHT
           Table 8.1 Comparison of analytical results for barley samples from Gauteng and
                    Maltsters                                                                        .113




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