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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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