POTENTIAL OF LACTIC ACID FERMENTATION IN REDUCING AFLATOXIN B1 AND
FUMONISIN B1 IN TANZANIAN MAIZE-BASED COMPLEMENTARY GRUEL
By
Frida Albinusi Nyamete

A THESIS
Submitted to
Michigan State University
in partial fulfilment of the requirements
for the degree of
Food Science - Master of Science
2013

ABSTRACT
POTENTIAL OF LACTIC ACID FERMENTATION IN REDUCING AFLATOXIN B1 AND
FUMONISIN B1 IN TANZANIAN MAIZE-BASED COMPLEMENTARY GRUEL
By
Frida Albinusi Nyamete
Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are the most carcinogenic and heat-stable
mycotoxins and they are harmful to both humans and animals. This study investigated the effect
of fermentation on AFB1 and FB1 reduction in maize-based gruel fermented at 30°C for up to 24
h using four monocultures (Lactobacillus plantarum, Pediococcus pentosaceus, Lactobacillus
casei and Lactobacillus fermentum) and two co-cultures (one composed of Enterococcus
faecium, Lactobacillus plantarum and Lactobacillus buchneri (AS), and the other consisting of
Enterococcus faecium, Lactobacillus plantarum and Lactobacillus lactis (SR3.54) (MC)) of
lactic acid bacteria (LAB), natural fermentation and back-slopping. Generally, the bacteria
increased by two log units and the acidity (expressed as lactic acid) ranged from 3.30 to 3.95 ×
100µg/mL after 24 h of fermentation. Back-slopping and natural fermentation removed 68 and
56% of AFB1 and 30 and 20% of FB1, respectively after 24 h. Lactobacillus strains removed
between 45 – 55% of AFB1 and 14 – 27%, of FB1. Co-culture of (AS) was superior at removing
both AFB1 (68%) and FB1 (27%) than co-culture of (MC) which only removed 54% of AFB1
and 17% of FB1 respectively. Comparatively, back-slopping was the most convenient,
economical and effective method for detoxification of mycotoxins in fermented gruel.

To my beloved uncle, Mr. Alex Mgaya, who raised, cared and supported me in everything. Many
thanks for educating me. And to the loving memory of my parents, the late Mr. and Mrs.
Albinusi Nyamete. May God rest their souls in peace

iii

ACKNOWLEDGMENTS
I would like to express my profound gratitude to my advisors Prof. Maurice Bennink, Prof. John
E. Linz and Prof. Jovin K. Mugula. The work presented in this thesis is a testament of their
invaluable advice, foresight and dedication. I would like to wish them unending success in their
exemplary professional and family lives. I would also like to thank my committee members Prof.
Leslie D. Bourquin and Prof. K. W. Ng Perry for their invaluable assistance and input in the
modelling phase of the study.
I would also like to sincerely thank the United States Agency for International Development
(USAID), through the Innovative Agricultural Research Initiative (iAGRI) programme, for
financial support, which enabled this study to be done. I would like to express my sincere
gratitude to my employer, the Sokoine University of Agriculture, for granting me permission to
pursue the graduate studies. I would like to thank Dr. James Swezey and Dr. O'Donnell of the NRRL
Culture Collection Centre, National Center for Agricultural Utilization Research, Agricultural Research
Service, United States Department of Agriculture, Peoria, Illinois, USA for provision of lactic acid
bacteria strains.

I would like to thank my colleagues at the Michigan State University and sponsored iAGRI first

cohort students for their support and would like to wish them success in their individual
endeavours.

I would like to acknowledge the unwavering love, support, patience, humour and continuous
encouragement of my best friend Dr. Leonnard Ojwang. I would also like to thank my siblings
Aloyce, Angel, Jennifer and Godfrey for their unconditional love and for being there for me.
iv

In fair honesty, it is impossible to mention everyone. I am immensely indebted to for their help
in and outside of the study, but to those I didn’t mention, thank you and may you be blessed with
only the best in your lives.

v

TABLE OF CONTENTS

LIST OF TABLES....................................................................................................................... ix
LIST OF FIGURES...................................................................................................................... x
LIST OF ABBREVIATIONS AND ACRONYMS................................................................... xi
CHAPTER ONE............................................................................................................................1
GENERAL INTRODUCTION.......................................................................................................1
1.1 Background................................................................................................................................1
1.2 Problem statement and study justification................................................................................ 3
1.3 Significance and objectives...................................................................................................... 4
1.3.1 Significance and overall objective of the project.................................................................. 4
1.3.2 Specific objectives..................................................................................................................4
1.3.3 Research hypothesis...............................................................................................................4
BIBLIOGRAPHY.......................................................................................................................... 5
CHAPTER TWO..........................................................................................................................8
LITERATURE REVIEW...............................................................................................................8
2.1 Overview of maize production and handling in Tanzania....................................................... 8
2.1.1 Origin of maize..................................................................................................................... 8
2.1.2 Tanzania maize production and consumption........................................................................8
2.2 Mycotoxins................................................................................................................................9
2.2.1 Overview of mycotoxins........................................................................................................9
2.2.2. Major groups of mycotoxins...............................................................................................13
2.2.2.1 Aflatoxins......................................................................................................................... 13
2.2.2.2 Ochratoxin........................................................................................................................ 13
2.2.2.3 Citrinin............................................................................................................................. 14
2.2.2.4 Ergot alkaloids................................................................................................................. 14
2.2.2.5 Patulin.............................................................................................................................. 15
2.2.2.6 Fusarium mycotoxins....................................................................................................... 15
2.3 Occurrence of mycotoxins in cereal grains............................................................................ 16
2.3.1 Aflatoxin B1........................................................................................................................ 16
2.3.1.1. Biochemical mode of action............................................................................................ 18
2.3.2 Fumonisins.......................................................................................................................... 21
2.3.2.1 Fumonisin B1 mechanism of action................................................................................. 22
2.3.2.2 Toxic effects.....................................................................................................................23
2.3.2.3 Toxic effects in humans................................................................................................... 24
2.3.2.3.1 Neural tube defects....................................................................................................... 24
2.3.2.3.2 Oesophageal cancer...................................................................................................... 25
2.4 Overview of mycotoxins in African countries....................................................................... 25
2.4.1 Current status of mycotoxins in Africa............................................................................... 26

vi

2.4.2 Factors that affect mycotoxins production in Africa......................................................... 27
2.4.2.1 Climatic conditions......................................................................................................... 27
2.4.2.2 Pest infestation................................................................................................................ 28
2.4.2.3 Fungicides and/or fertilizers............................................................................................ 28
2.4.2.4 Post-harvest handling....................................................................................................... 29
2.5 Possible mitigation/intervention strategies for mycotoxins in Africa ................................... 29
2.5.1 Pre-harvest interventions..................................................................................................... 29
2.5.2 Storage................................................................................................................................. 30
2.5.3 Biological control strategies................................................................................................ 30
2.5.4 Physical methods................................................................................................................. 31
2.5.5 Food surveillance................................................................................................................. 31
2.5.6 Community education.......................................................................................................... 31
2.6 Fermentation........................................................................................................................... 32
2.6.1 Traditional fermentation...................................................................................................... 32
2.6.2 Tanzanian fermented gruel.................................................................................................. 32
2.6.3 Lactic acid fermentation...................................................................................................... 33
2.6.3.1 Lactic acid bacteria........................................................................................................... 33
2.6.3.2 Potential of lactic acid bacterial in reducing aflatoxins and fumonisins.……................. 34
BIBLIOGRAPHY......................................................................................................................... 35
CHAPTER THREE.................................................................................................................... 45
EFFECT OF LACTIC ACID FERMENTATION ON AFLATOXIN B1 AND FUMONISIN B1
IN FERMENTED MAIZE-BASED GRUEL.............................................................................. 45
3.1 Introduction............................................................................................................................ 45
3.2 Materials and methods............................................................................................................ 49
3.2.1 Starter culture preparation.....................................................................................................49
3.2.2 Gruel sample preparation..................................................................................................... 50
3.2.3 Sample spiking and inoculation........................................................................................... 51
3.2.4 pH determination................................................................................................................. 52
3.2.5 Lactic acid content determination........................................................................................ 53
3.2.5.1 Lactic acid quantification…….......................................................................................... 53
3.2.6 Microbial analysis................................................................................................................ 54
3.2.7 Aflatoxin B1 detection by thin layer chromatography........................................................ 55
3.2.7.1 Sample extraction.............................................................................................................. 55
3.2.7.2 Sample clean-up using SPE column................................................................................. 55
3.2.7.2.1 Procedure description.................................................................................................... 55
3.2.7.3 Aflatoxin B1 detection on TLC........................................................................................ 55
3.2.8 Aflatoxin B1 and fumonisin B1 quantification using Neogen Reveal Q+ assay.……....... 56
3.2.8.1 Assay principle..................................................................................................................56
3.2.8.2 Aflatoxin B1 and fumonisin B1 recovery........................................................................ 57
3.2.8.3 Quantification of AFB1.................................................................................................... 57
3.2.8.4 Fumonisin B1 extraction and quantification..................................................................... 57
3.2.9 Statistical data analysis........................................................................................................ 58
3.3 Results..................................................................................................................................... 58

vii

3.3.1 Thin layer chromatography assay for aflatoxin B1.............................................................. 58
3.3.2 Quantitative analysis of aflatoxin B1 and fumonisin B1 concentration in
fermented maize gruel....................................................................................................................61
3.3.2.1 Aflatoxin B1 and fumonisin B1 recovery assay............................................................... 61
3.3.2.2 Quantification of AFB1 in fermented gruel...................................................................... 61
3.3.2.3 Effect of fermentation on fumonisin B1 levels................................................................. 66
3.3.3 pH......................................................................................................................................... 68
3.3.4 Lactic acid production during fermentation of maize based gruel...................................... 70
3.3.5 Effect of pH and lactic acid on aflatoxin B1 reduction....................................................... 72
3.3.6 Effect of pH and lactic acid on reduction of fumonisin B1................................................. 75
3.3.7 Microbial load during LAB fermentation............................................................................ 78
3.4 Discussion............................................................................................................................... 81
3.5 Conclusion.............................................................................................................................. 85
APPENDICES.............................................................................................................................. 87
Appendix A: ANOVA output for LAB and fermentation time on aflatoxin B1.......................... 88
Appendix B: ANOVA output for LAB and fermentation time on fumonisin B1........................ 89
Appendix C: ANOVA output for LAB and fermentation time on pH levels............................... 90
Appendix D: ANOVA output for LAB and fermentation time on lactic acid levels................... 91
Appendix E: ANOVA output for LAB and fermentation time on microbial count..................... 92
BIBLIOGRAPHY........................................................................................................................ 93
CHAPTER FOUR...................................................................................................................... 99
GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS......................... 99
4.1 General discussion................................................................................................................. 99
4.2 Conclusion............................................................................................................................ 102
4.3 Recommendations................................................................................................................ 103
BIBLIOGRAPHY...................................................................................................................... 105

viii

LIST OF TABLES
Table 1: Validation of aflatoxin B1 and fumonisin B1 recovery in gruel ..................................... 62
Table 2: Effect of fermentation on reduction of aflatoxin B1 in maize gruel............................... 63
Table 3: Effect of LAB on fumonisin B1 levels in fermented maize based gruel ........................ 67
Table 4: pH levels during lactic acid fermentation of maize based gruel ..................................... 69
Table 5: Lactic acid production during fermentation of maize based gruel ................................. 72
Table 6: Microbial count (log CFU/ml) during fermentation of maize based gruel ..................... 80
Table 7: ANOVA for effect of LAB fermentation on aflatoxin B1 reduction in gruel ................ 88
Table 8: ANOVA for effect of LAB fermentation on fumonisin B1 reduction in gruel .............. 89
Table 9: ANOVA for effect of LAB fermentation on pH levels in gruel ..................................... 90
Table 10: ANOVA for effect of LAB fermentation on lactic acid levels in gruel ....................... 91
Table 11: ANOVA for effect of LAB fermentation on microbial counts in gruel ....................... 92

ix

LIST OF FIGURES
Figure 1: Chemical structures of mycotoxins found in food ........................................................ 12
Figure 2: Overview of biotransformation pathways for aflatoxin B1........................................... 19
Figure 3: Sphingolipid metabolism showing the inhibition of ceramide synthase (x) by
fumonisins and the changed concentrations of other compounds caused by this inhibition ........ 23
Figure 4: Overall study design and sample collection procedure ................................................. 52
Figure 5: Thin layer chromatographic plate of the maize gruel samples spiked with AFB1 ....... 60
Figure 6: Thin layer chromatographic plate showing the effect of fermentation on aflatoxin B1
after 24 hours of fermentation....................................................................................................... 60
Figure 7: Effect of pH and lactic acid on aflatoxin B1 reduction during LAB fermentation of
maize gruel .................................................................................................................................... 74
Figure 8: The effect of pH and lactic acid concentration on reduction of fumonisin B1 ............. 77

x

LIST OF ABBREVIATIONS AND ACRONYMS

ANOVA

Analysis of Variance

AOAC

Association of Official Analytical Chemists.

AFB1

Aflatoxin B1

BHA

Butylated Hydroxy Anisole

BHT

Butylated Hydroxy Toluene

ECS

European Committee for Standardization

FB1

Fumonisin B1

GRAS

Generally Regarded as Safe

GAP

Good Agricultural Practices

GMP

Good Manufacturing Practices

HCC

Hepatocellular Carcinoma

IITA

International Institute of Agricultural Research

IPCS

International Programme on Chemical Safety

IARC

International Agency for Research on Cancer

NTD

Neural Tube Defect

OTA

Ochratoxin A

OTB

Ochratoxin B

OTC

Ochratoxin C

PMTDL

Provisional Maximum Tolerable Daily Limit

PPB

Part per billion

PPM

Part per million

PCA

Plate Count Agar

SD

Standard Deviation
xi

TFDA

Tanzania Food and Drug Authority

USEPA

US- Environmental Protection Agency

USFDA

US- Food and Drug Administration

xii

CHAPTER ONE
GENERAL INTRODUCTION
1.1 Background
Mycotoxins are toxic secondary metabolites produced by fungi, especially by species of
Aspergillus, Penicillium and Fusarium [1]. Factors related to environmental and agricultural
practices favours mycotoxins contamination of food and feed in much of the African continent.
Most African staple diets are based on crops susceptible to mycotoxins, such as maize, leading
high risk of mycotoxins exposure. Tanzania lies between 40ºN and 40ºS latitude; a tropical
location that is associated with high humidity and temperatures that favour fungal growth.
Mycotoxins adversely affect the health of humans and animals when ingested or inhaled.

Mycotoxins, such as aflatoxins, are known to be highly potent mutagenic and carcinogenic
substances [2]. Mycotoxins are also immunosuppressive, and they can cause stunting, digestive
problems, and nerve defects [2, 3]. In addition, mycotoxins are synergistic in the development of
diseases that are common in Africa – malaria and HIV/AIDS for instance. A striking association
between mycotoxins (AB1 and FB1) exposures and growth faltering has been reported in
Tanzania [4,5]. The symptoms and severity of the symptoms depend upon the type and amount
of mycotoxin ingested. Because of the adverse health effects of mycotoxin contamination, fungal
contamination of staple crops can cause wastage of large quantities of food. In developing
countries where food availability is already a problem, this waste can be extremely detrimental.
It is estimated that mycotoxins affect a quarter of the world’s food crops [2].
The majority of sub-Saharan Africa relies on subsistence farming and there is often food
insecurity. Food scarcity may force consumption of mycotoxin-contaminated staples particularly
1

if the people are ignorant concerning the adverse effect of eating mycotoxin contaminated foods.
Due to the adverse effects associated with consuming mycotoxin, many countries have
developed maximum tolerable limits for mycotoxin contamination. However, the standards are
mostly enforced for exported or imported commodities and seldom in market places and
probably never at the household level. For example, Kenya has maximum tolerable limits in
place, but fatalities occurred anyway from consumption of mycotoxins contaminated maize in
2004 [6] because of food insecurity, ignorance of the severe consequences that can occur from
consuming mouldy maize, and the inability to enforce standards.

In Tanzania, maize is grown in all agro-ecological zones in the country. It is a major staple, but it
also serves as a cash crop for about 85% of Tanzania’s population. Annually, more than two
million hectares of maize are planted in the country. The annual average yield is 1.2–1.6 tonnes
per hectare, accounting for 31% of the total food production [7-10] and more than 75% of the
country’s cereal consumption. However, maize is highly susceptible to mycotoxins
contamination, particularly by aflatoxin and fumonisin.

Decontamination and good agricultural practices are some of the possible intervention strategies
that have been proposed to reduce mycotoxin exposure [11, 12]. Because mycotoxins are heat
stable, cooking maize does not reduce mycotoxins contamination. There is an urgent need to find
affordable, inexpensive approaches for mycotoxin decontamination that could be used by rural
communities.
One of the strategies used for mycotoxin biodegradation includes the use of lactic acid bacteria
(LAB) in an appropriate fermentation process [13]. The mechanism of decontamination by LAB

2

is reportedly via binding of mycotoxins to the cell wall of bacteria and/or conversion of
mycotoxins into less or non-toxic forms [13, 14]. However, the specific mechanisms by which
indigenous strains of LAB reduce mycotoxin toxicity in cereals is still unknown; and their
potential to reduce aflatoxin B1 and fumonisin B1 during fermentation of maize based gruel has
never been investigated.
1.2 Problem statement and study justification
In Tanzania, maize contributes as much as 41% of the dietary calorie intake nationally. The overreliance on maize-based gruel during a child’s weaning stage increases its exposure to foodborne mycotoxins, particularly aflatoxin and fumonisin. Crops such as soybeans and groundnuts
can be added to maize to improve the nutritive value of complementary foods, but soybeans and
groundnuts are also prone to mycotoxins contamination. Thus, infant food mixes may be
inadvertently providing mycotoxin exposure beyond the provisional maximum tolerable daily
limit (PMTDL) [9]. The Tanzanian Food and Drug Authority (TFDA) is mandated with
enforcing the standards set by the Tanzania Bureau of Standards (TBS) for mycotoxins, but
enforcement focuses primarily on formally packaged foods, which constitutes a small fraction of
maize consumption in the country.

Fermented maize-based gruel is one popular form of maize consumption in Tanzania.
Fermentation by lactic acid bacteria can be used as one practical and novel approach to reduce
the mycotoxin content of maize-based foods. This approach could be easily adapted by the
Tanzanian society because it is one of the traditional practices among rural communities. It is a
low cost method of food preservation and it contributes to improvement of nutritional value and
digestibility. No study has been conducted in the country to investigate the potential for
3

fermentation of maize-based gruel to reduce aflatoxin and fumonisin concentrations by using
lactic acid bacteria.

1.3 Significance and objectives
1.3.1 Significance and overall objective of the project
The overall objective of the research is to investigate the potential for lactic acid fermentation to
reduce aflatoxin B1 and fumonisin B1 in a Tanzania maize-based complementary food. This
objective is significant because the process can be used as a novel approach to reduce mycotoxin
bioavailability in cereal-based weaning foods and improve the health status of children and
potentially, even for adults.

1.3.2 Specific objectives
The specific objectives are
i. To determine the effect of lactic acid fermentation on aflatoxin B1 concentrations in
maize-based gruel.
ii. To determine the effect of lactic acid fermentation on fumonisin B1 concentrations in
maize-based gruel.

1.3.3 Research hypothesis
Aflatoxin B1 and fumonisin B1 concentrations in foods prepared from maize are reduced by
fermenting these grains with specific prebiotic strains of lactic acid bacteria.

4

BIBLIOGRAPHY

5

BIBLIOGRAPHY

1.

Shephard, G. S. (2006). Mycotoxins in the context of food risks and nutrition
issues. Barug, D., Bhatnagar, D. Van Egmond, HP, Van Der Kamp, JW, Van
Osenbruggen, WA und A. Visconti (Editors): The Mycotoxin Factbook: Food and Feed
Topics. Wageningen Academic Publishers, 21-36.

2.

Zain, M. E. (2011). Impact of mycotoxins on humans and animals. Journal of Saudi
Chemical Society, 15(2), 129-144.

3.

Wagacha, J. M., & Muthomi, J. W. (2008). Mycotoxin problem in Africa: current status,
implications to food safety and health and possible management strategies. International
Journal of Food Microbiology, 124(1), 1-12.

4.

Kimanya, M. E., Meulenaer, B. D., Baert, K., Tiisekwa, B., Van Camp, J., Samapundo,
S., ... & Kolsteren, P. (2009). Exposure of infants to fumonisins in maize‐based
complementary foods in rural Tanzania. Molecular nutrition & food research, 53(5), 667674.

5.

Egal, S., Hounsa, A., Gong, Y. Y., Turner, P. C., Wild, C. P., Hall, A. J., ... & Cardwell,
K. F. (2005). Dietary exposure to aflatoxin from maize and groundnut in young children
from Benin and Togo, West Africa. International Journal of Food Microbiology, 104(2),
215-224.

6.

Lewis, L., Onsongo, M., Njapau, H., Schurz-Rogers, H., Luber, G., Kieszak, S., ... &
Rubin, C. (2005). Aflatoxin contamination of commercial maize products during an
outbreak of acute aflatoxicosis in eastern and central Kenya. Environmental Health
Perspectives, 113(12), 1763.

7.

Temu, A. E., Manyama, A., & Temu, A. A. (2010). 12. Maize trade and marketing policy
interventions in Tanzania. Food Security in Africa: Market and Trade Policy for Staple
Foods in Eastern and Southern Africa, 317.

8.

Sassi, M. A. R. I. A. (2004). Improving efficiency in the agricultural sector as an answer
to globalization, growth and equity: the case of Tanzania. Pavia: Università degli Studi di
Pavia, Facoltà di Economia, Dipartimento di Ricerche Aziendali, Quaderni di ricerca,
(4).

9.

Amani, H. K. R. (2004). Agricultural Development and Food Security in Sub-Saharan
Africa Tanzania Country Report. Economic and Social Research Foundation (ESRF) Dar
es Salaam, Tanzania.

10.

Isinika, A. C., Ashimogo, G. C., & Mlangwa, J. E. (2003). Africa in Transition Macro
Study: Tanzania. Afrint Working Paper.
6

11.

Turner, P. C., Sylla, A., Gong, Y. Y., Diallo, M. S., Sutcliffe, A. E., Hall, A. J., & Wild,
C. P. (2005). Reduction in exposure to carcinogenic aflatoxins by postharvest
intervention measures in west Africa: a community-based intervention study. The
Lancet, 365(9475), 1950-1956.

12.

He, J., Zhou, T., Young, J. C., Boland, G. J., & Scott, P. M. (2010). Chemical and
biological transformations for detoxification of trichothecene mycotoxins in human and
animal food chains: a review. Trends in Food Science & Technology, 21(2), 67-76.

13.

Gourama, H., & Bullerman, L. B. (1995). Inhibition of growth and aflatoxin production
of Aspergillus flavus by Lactobacillus species. Journal of Food Protection®, 58(11),
1249-1256.

14.

Thyagaraja, N., & Hosono, A. (1994). Binding properties of lactic acid bacteria from
‘Idly’towards food-borne mutagens. Food and chemical toxicology, 32(9), 805-809.

7

CHAPTER TWO
LITERATURE REVIEW
2.1 Overview of maize production and handling in Tanzania
2.1.1 Origin of maize
Maize (Zea mays L), originally domesticated by indigenous peoples in Mesoamerica in
prehistoric times, is a grain plant that quickly spread to the rest of the world because of its ability
to grow in diverse climatic condition [1]. More maize is produced annually than any other grain
worldwide. Maize varieties exist in different phenotypes and genotypes. The white and yellow
varieties are the most preferred, and are region specific.

Since its introduction into Africa in the 1500s, maize has become the most dominant food crop in
the continent [2]. Maize grain is rich in vitamins A, C and E, carbohydrates, essential minerals,
dietary fibre and contains about 9% protein. About 785 million tonnes of maize is produced
worldwide, with the United States and Africa producing 42 and 6.5%, respectively. Therefore,
Africa imports 28% of the required maize from other countries [3,4]. The largest African
producer is Nigeria with nearly 8 million tons, followed by South Africa.

2.1.2 Tanzania maize production and consumption
In Tanzania, the maize crop is cultivated on an average of two million hectares, which is about
45% of the cultivated area in the country [5,6]. The crop is grown mainly during the two main
planting rain seasons: “masika” and “vuli”. Though maize is grown almost in all regions in the
country, productivity is more favourable in the high rainfall areas in Tanzania, such as, southern
highlands, the Lake Victoria zone, and the northern zone [7]. Over 80% of the maize growers in

8

Tanzania are peasant farmers that hold up to 10 hectares of farm land per household, accounting
for about 85% of the maize produced in the country. Their farming system is, however,
characterized by low use of improved technologies (fertilizers, seeds and crop husbandry
practices) and poor post-harvest managements, resulting in the observed slow growth in
productivity, low yield and increased mycotoxins proliferation [8-10].

As a major cereal grown and consumed in Tanzania, maize is the main staple crop in many
households, with over 80% of population depending on it as food [6]. It is estimated that nearly
3 million tons of maize is consumed in Tanzania per year, contributing about 41% of dietary
calories to Tanzanians [6].

2.2 Mycotoxins
2.2.1 Overview of mycotoxins
The term “mycotoxin” refers to a toxic chemical by-product readily produced by moulds in crops
[21]. Different mycotoxins can be produced by one mould species. Similarly, different mould
species can produce the same mycotoxins [11].

Generally, moulds are ubiquitous due to their minute size. Most are aerobic and reproduce by
spore-formation. Moulds grow in organic matter wherever humidity and temperature are
favourable, and thus are able to proliferate and produce high levels of mycotoxins. The reason
for mycotoxin production by moulds is not yet fully understood; however, they are not critical
for their growth or the development of the moulds [12]. But because mycotoxins are toxic to

9

other organisms in the environment, the fungus may utilize them as a strategy to destroy other
competing organisms and thus promote fungal proliferation.
Various intrinsic and extrinsic factors, such as organism infected, its susceptibility, metabolism,
and defence mechanisms affect the types and levels of mycotoxins production and their severity
[13,14]. Mycotoxin toxicity to animals and humans cause death and other health problems
including weakened immune systems and allergies. Some mycotoxins, such as penicillin, cause
harm to other micro-organisms including other fungi and even bacteria [15,16].

In the food chain, mycotoxins occur following fungal contamination of crops consumed by
humans or when used as animal feed. Mycotoxins are generally resistant to microbial
decomposition as well as digestion, Some mycotoxins, unfortunately, cannot be destroyed by
common processing methods, such as cooking and freezing [17].

Mycotoxin levels in food therefore need to be regulated and many international agencies are
working toward achieving this goal. Most countries recognize the potency of mycotoxin in
human and animal health, and some have already placed regulatory measures in their feed
industry [18]. The procedure for assessing a need for mycotoxin regulation is not simple. It
includes complicated laboratory protocols such as careful sample extraction, clean-up and
separation techniques, which require training and experience, as well as sophisticated
instruments such as HPLC analysis [19] to determine concentration. It is important to note that
regulations and control methods of these mycotoxins must be universal and in agreement with
any other countries with which a trade agreement exists [20].

10

The European Committee for Standardization (CEN) is widely involved in setting the standards
for performance for analytical methods for mycotoxins [19]. However, regional differences can
be a challenge. For example, cultural and political influence may have significant impact on
robustness and effectiveness of universal scientific risk assessment procedures, as well as on
trade regulations of food containing mycotoxins [20].

Around the 20

th

century, many food-based mycotoxins were reported around the world.

Following this, different countries begun regulating mycotoxins levels in food and animal feed
[21]. For example, in Europe, the European Directives and Commission regulations set and
define the statutory levels of various mycotoxins permitted in food and animal feed.

In the U.S., the Food and Drug Administration regulate and enforce limits on concentrations of
mycotoxins permitted in foods and animal feeds, and various compliance programs have been
put in place to monitor and guarantee that mycotoxins are kept at a minimum level in these
industries [21].

Typical tropical conditions (e.g. high temperatures and humidity), harvesting as well as poor
post-harvesting practices (e.g. improper storage) promote mould proliferation and mycotoxins in
foods and animal feeds [22]. Deoxynivalenol (DON) and ergot alkaloids (produced before
harvest), fumonisins and ochratoxins (produced during and immediately following harvest), and
aflatoxins (predominantly produced during storage), are the most potent mycotoxins to humans
[23].

11

Figure 1: Chemical structures of mycotoxins found in food
O

O

O

O

OH

HO

O

O

O
O

O

O
O

CH3
CH3

OCH3

Aflatoxin B1

CH3

OH

Citrinin
O

Patulin

COOH
COOH

O

R2

OH

R1

O

NH2

COOH
O

COOH

Fumonisin B1: R1 = R2 = OH
Fumonisin B2: R1 = H; R2 = OH
O

OH
O

OH

N
H

OH

O

O

CH3
O

O
CH3

HO

Cl

Ochratoxin A

O
Zearalonone

O
O

OH

CH2 C

N

O

O
O

S

NH

CH3
CH3

O
COOH

OMe
Penicillin

Sterigmatocystin

12

2.2.2. Major groups of mycotoxins
2.2.2.1 Aflatoxins
Aflatoxins are naturally occurring mycotoxins of fungal origin that are mainly produced by
several species of Aspergillus, particularly, Aspergillus flavus and Aspergillus parasiticus [24].
The four major types of aflatoxins produced in nature include aflatoxins B1, B2, G1, and G2 [25].
In humans, aflatoxin B1, is the most potent toxin and carcinogenic mycotoxin, and can cause
cancer of the hepatic system [24]. Human foods that are particularly susceptible to aflatoxin
contamination are those cultivated in the tropics and subtropics including maize and peanuts
[25].

2.2.2.2 Ochratoxin
Ochratoxin is a group of mycotoxins produced as secondary metabolites mainly by Penicillium
and Aspergillus species. These metabolites, namely, Ochratoxin A, B, and C, are typically
carried by spores, and can even be detected in air samples at high spore concentrations. The three
forms differ from each other by their substituent groups: Ochratoxin B (OTB) is a dechlorinated
analog of Ochratoxin A (OTA) and Ochratoxin C (OTC) is an ethyl ester form of OTA. OTA has
proven carcinogenic properties and has been associated with tumourogenes is in the human
urinal system [26]. In drinks, Aspergillus ochraceus and its toxin can be found in beer and wine;
while Aspergillus carbonarius and its toxin can be found in the vine fruit juice [26].

13

2.2.2.3 Citrinin
Citrinin is a mycotoxin that is produced by several species of Penicillium and Aspergillus
species. Even though citrinin was first isolated from Penicillium citrinum, it has since been
identified in other species such as Penicillium camemberti and Aspergillus oryzae that are used
for making cheese and soy sauce, respectively. Moreover, despite the fact that in Japan,
consumption of foods contaminated with citrinin was associated with yellow rice disease, its full
impact in the rest of the world is still unknown, yet it has also been identified in many other food
commodities such as wheat, rice, corn, barley, oats and rye. Citrinin has been proven to depress
RNA synthesis in murine kidneys, particularly in the presence of ochratoxin A [11].

2.2.2.4 Ergot alkaloids
The ergot alkaloids are mycotoxins produced by several species of Claviceps, particularly
Claviceps purpurea that infect many grasses as well as cereals such as rye, wheat, barley,
triticale, oats, millet, sorghum and maize. The main ergot alkaloids are ergotamine, ergocornine,
ergocristine, ergocryptine, ergometrine and agroclavine, produced in the sclerotia of claviceps.
Ingestion of these ergot alkaloids cause ergotism, the human disease also known as St.
Anthony’s fire [27]. Ergotism can be classified into two classes; the gangrenous form which
affects the vascular system and the convulsive form which affect the central nervous system.
Significant reduction of ergotism as a human disease can be achieved through good
manufacturing practices such as grain cleaning [11].

14

2.2.2.5 Patulin
Patulin, reported to damage the immune system in animals [28], is a mycotoxin that is produced
by Penicillium, Aspergillus, Paecilomyces and Byssochylamysspecies. It has been associated
with a variety of mouldy flour, malt feed, vegetables, cheeses, grains and fruits, in particular
rotting apples, pears and figs [29,30]. However, the manufacturing processes such as
fermentation and thermal processes destroy patulin, with the exception of apple juice [30].
Consumption practices, such as removal of the rotten portions of most fruits and grains, seem to
reduce risk of patulin poisoning, and so patulin does not appear to pose a safety concern. The
intrinsic components of a food are also keys in controlling patulin. For instance, the cysteine in
cheese has been proven to inactivate patulin [31]. Even though the carcinogenic properties of
patulin are still unknown, it has been proven to interfere with the immune system in vivo [28].
Some food products now have set limits of concentrations of patulin set by the European
Community. These include fruit juice concentrates, solid apple products for direct consumption,
and children’s apple products set at 50, 25 and 10 μg/kg, respectively [32].

2.2.2.6 Fusarium mycotoxins
The two main mycotoxins produced by Fusarium, a member of the filamentous moulds widely
distributed in soil, are fumonisins and trichothecenes. Because of the relative abundance of
Fusarium in the soil, these mycotoxins can enter the food chain through commonly consumed
cereals such as maize and wheat [33]. Ingestion of fumonisins has been reported to cause
dysfunction of the nervous systems and cancer in horses and rats and oesophageal cancer in
humans respectively. On the other hand, ingestion of trichothecenes causes fatal toxic effects in

15

both animals and humans. Other less prominent Fusarium toxins have also been identified,
including zearalenone, beauvercin, enniatins, butenolide, equisetin, and fusarins [34].

2.3 Occurrence of mycotoxins in cereal grains
Due to the relative abundance of mycotoxins-producing moulds in the soil, crops grown under
conventional and/or organic agriculture are both not immune to mycotoxins contamination [35].
However, post-harvest handling of crops, especially cereal grains; play an important role is
determining the extent of fungal proliferation on human foods. For example, maize stored under
poor conditions may be infected by Aspergillus flavus and Fusarium verticillioides, which have
great potential of producing toxic such as aflatoxins and fumonisin. Aflatoxin B1 and fumonisin
B1 will be covered in this study.

2.3.1 Aflatoxin B1
Of the four naturally occurring aflatoxins (B1, B2, G1 and G2), aflatoxin B1 (AFB1) is the most
abundant and toxic. The US Environmental Protection Agency (USEPA) has classified aflatoxin
B1 as a group 1 carcinogen (that means carcinogenic to humans) [36] and a Class A or group 1
carcinogenic agent due to the exposure to hepatitis B virus [37].

AFB1, the most predominant aflatoxin in cases of aflatoxicosis illness, is found in many staple
foods including cereals such as maize and wheat, in products made from these cereals, in
oilseeds such as cotton seed, in nuts such as pistachio, and in other foods which are commonly
consumed by humans such as chillies and peppers, wines, legumes, fruits, milk, and milk

16

products. Exposure to high levels of AFB1 is lethal due to its acute toxicity, chronic toxicity,
carcinogenicity, teratogenicity, genotoxicity and immunotoxicity. Aflatoxin M1 (AFM1), is a
metabolic derivative of AFB1, and aflatoxin M2 (AFM2), is a metabolic derivative of AFB2;
both occur in milk as well as milk-based and meat based products (hence the designation M1)
[38, 39]. High incidence rates of aflatoxin M1 have been reported in several regions of the world;
however, the reported contamination levels have not caused any serious public health problem
[36].

Populations of developing countries in tropical regions, which rely on these commodities as their
staple food source, are the most susceptible to aflatoxicosis [38] through contaminated foods
[40]. This is because available economic resources cannot ensure proper regulation and
implementation of aflatoxin control measures at pre-harvest and post-harvest stages to limit
aflatoxin exposure in the food supply. Because of high incidences of aflatoxin contaminated food
crops in the tropics, aflatoxin poisoning is now considered an important public health issue.
Chronic incidence of aflatoxin in diets is evident from the presence of aflatoxin M1 in breast
milk and in umbilical cord blood samples in several human subjects sampled in Africa [41].
Generally, concentration and toxicity of aflatoxins vary with the amount of aflatoxin ingested
and the physiological state of the body. Adult humans usually have a high aflatoxin tolerance,
however, children exposed to acute poisoning may die [42].

17

2.3.1.1. Biochemical mode of action
Aflatoxins are enzymatically converted by the microsomal mixed function oxidase (MFO)
primarily present in the liver. Depending on affinity and expression, the main enzymes involved
in aflatoxin metabolism are CYP3A4, 3A5, 3A7 and 1A2; the most important of these is
CYP3A4 [43]. Therefore, following AFB1 ingestion and transportation to the liver, it is
enzymatically metabolized by the cytochromes p450 (CYP) of hepatocytes to form its major
carcinogenic metabolites AFB1-8,9-exo-epoxide and AFB1-8,9-endo-epoxide (AFBO), or to less
mutagenic forms such as AFM1, Q1 or P1 [44,45].

Figure 2 shows the pathways that AFBO takes, leading to cancer and toxicity. AFB1-8,9-exoepoxide is highly unstable, and readily binds to cellular macromolecules such as proteins and
DNA, to form adducts, such as 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-AFB1 (AFB1–N7-Gua)
adduct, which is then excreted in the urine of those exposed [46]. AFB1–N7-Gua adduct is the
most important product that establishes the mutagenic properties of the compound. The AFBO
not only reacts with DNA but also with guanine residues of specific sites, in particular the third
base position of codon 249 of the human p53 tumour suppression gene [47]. Indeed, the
conversion from G (guanine) to T (thymine) at the 3

rd

nucleotide of the codon induced by AFBO

makes it a critical locus for mutation, and has predominantly been reported among patients with
hepatocellular carcinomas who have been exposed to high levels of aflatoxins [48].

18

Figure 2: Overview of biotransformation pathways for aflatoxin B1

AFB

1

AFM1, AFP1, AFQ

1

UDP-GT, ST Glucuronide &
sulphate conjugate

CYP450(s)
Lipoxygenase
PHS

AFB1-8,9-epoxide
Oxo, endo-stereoisomer
DNA
repair

DNA
adduct

GST(S)
GSH conjugate
Epoxide
hydrolase

Protein
binding

Dihydrodiol

Mutation
Cell death
CANCER

TOXICITY

Enzymes systems such as glutathione-S-transferase (GST) facilitate metabolic detoxification of
AFBO, thus reducing the damage to DNA and other cellular constituents caused by AFBO; this
is an important strategy for prevention of mutations [48].

Levels of GST (effective in neutralizing the toxic effects of AFBO), in both animals and humans,
vary significantly. For example, mice have 3-5 times more GST activity than susceptible species
such as rats; as such, mice are more resistant to aflatoxin carcinogenesis than rats.
Unfortunately, humans have lower GST activity or AFBO conjugation than rats or mice,
suggesting that humans are inferior at detoxifying AFBO [49]. However, lower aflatoxin intake
is still the best strategy to reduce hepatocellular carcinoma (HCC) in humans.

19

AFB1 is a strong potentiating factor for human HCC [50]. For example, a cohort study of more
than 18,000 individuals in China found that a person exposed to AFB1 is 3.4 times more likely to
develop HCC [50]. However, human clinical trials of this population showed that excretion of
AFB1-mercapturate (a metabolite of GST reaction with AFB1) significantly increased when
subjected to a daily dose (125 mg/d) of oltipraz. However, long term effects of oltipraz in
lowering human risk for AFB1 carcinogenesis are still unknown. Therefore, epidemiological
studies investigating such intervention strategies and mechanisms underlying overall
susceptibility are still required.

In Africa, Gambia and Kenya are among the areas with reported high incidences of HCC. A
seven day study of the Gambian population revealed that urinary aflatoxin metabolites reflected
day-to-day variations in aflatoxin intake, demonstrating the critical dose-response relationships
for various aflatoxin metabolites, including the major nucleic acid adduct, serum aflatoxinalbumin adduct, and AFM1 [51]. Such relationships are typically dependent on the specific
urinary marker under study. For instance, AFB1-N7-Gua and AFM1 were strongly correlated
with intake, whereas urinary AFP1, a different oxidative metabolite, had no such correlation.
Therefore, it is evident that measuring dietary aflatoxin intake and biomarkers at the individual
level is crucial in validating a biomarker for aflatoxin exposure assessment [52].

In Kenya, however, acute human aflatoxicoses have already caused serious fatal problems. For
example, in 2004, a severe outbreak, causing a 39% death rate, was reported in some districts in

20

Kenya, in which up to 12% of half of the maize food samples tested contained AFB1 levels
ranging from 1000 – 8000 ppb, which is significantly higher than 20 ppb (the upper safe limit set
by Kenya). Out of the 317 cases reported, 125 of the deaths were caused by acute hepatotoxicity
[53].

2.3.2 Fumonisins
Fumonisins are mycotoxins produced by different species of Fusarium, of which Fusarium
proliferatum and Fusarium verticillioides are the most dangerous contaminants on crops in the
field and during storage [54]. More than 15 types of fumonisins have been isolated and
characterized. Group B fumonisins (i.e. B1, B2 and B3) are believed to be the most toxic and the
most predominant, constituting about 70% of fumonisins found naturally in food and feeds [55].
Fumonisins are highly water soluble; but since they are not aromatically structured, they are
usually hard to detect and identify [56].

Pathogenic effects of fumonisins include fatal diseases to farm animals. Major outbreaks of
equine leukoencephalomalacia in horses were reported in 1989 and 1990 in the United States as
the result of ingestion of fumonisin contaminated maize [57]. Pigs have also been shown to
suffer from pulmonary oedema after ingesting fumonisin contaminated feeds [58].

The International Agency for Research on Cancer (IARC) has classified fumonisin B1 as a 2B
group compound, which means that it is potentially carcinogenic [36]. In humans, fumonisin has
been linked with oesophageal cancer in some regions with high maize consumption such as

21

South Africa, China and northern Italy [59]. Fumonisin B1 reportedly causes interruption of
sphingolipid metabolism through inhibition of ceramide synthase enzymes responsible for lipid
synthesis. This in turn affects the uptake of folic acid in the human body which may result in
neural tube defects (NTD) [60]. Different studies in animal have successfully examined the ratio
of sphinganine/sphingosine as a biomarker of fumonisin exposure [61]. However, in human
studies, this correlation could not be established [62].

2.3.2.1 Fumonisin B1 mechanism of action
Fumonisin B1 is known to have a similar structure to sphingoid bases (i.e. sphingosine and
sphinganine) which are the principle components of the sphingolipid molecule. The part of
fumonisin B1 that is structurally similar to sphingoid bases (the aminopentol part) may interact
with the sphinganine binding site of ceramide synthase, with a further interaction between the
negatively charged tricarbyllic acid groups and the fatty acyl-CoA binding site [63]. Thus, the
proposed mechanism of action of the toxin is mainly through disruption of sphingolipid
metabolism (Figure 3). Fumonisin B1 fills the fatty acyl-CoA space, thus is not acylated in the
reaction, and by doing so it inhibits ceramide synthase which is a key enzyme in de novo
sphingolipid biosynthesis (Figure 3) [63]. Subsequent hydrolytic removal of tricarbillic acid
group from fumonisin B1 results in a product known as aminopentol and this compound acts as
both an inhibitor and a substrate for ceramide synthase [64]. Ceramide synthase can then acylate
aminopentol to form N-palmitoyl-AP1 [65]. This supports the suggestion that the aminopentol
part of FB1 occupies the space of sphinganine in the enzyme. N-palmitoyl-AP1 is a highly
potent inhibitor of ceramide synthase and may be responsible for the toxicity of nixtamalized
fumonisins [64].

22

Figure 3: Sphingolipid metabolism showing the inhibition of ceramide synthase (x) by
fumonisins and the changed concentrations of other compounds caused by this inhibition
Palmitoyl CoA+ serine
Serine
palmitoyltra
nsferase
Increase biosythesis of
phosphatidlyethanolamine
and selected fatty acid
Sphinganini
-1-p lyase

Free sphingaine
Sphingaine-1-p

Phosphoethanolamine
+
long chain aldehyde
Lyase
Sphingosine-1-p

CoAdependant
ceramide
sythase
Ceramide

Complex
sphingolipids

x

Kinase
Sphingosine

Ceramide

2.3.2.2 Toxic effects
Due to the toxic effect associated with consumption of fumonisins contaminated foods in both
human and animals, various international organizations (i.e. World Health Organization’s
International Programme on Chemical Safety (IPCS) and the Scientific Committee on Food
(SCF) of the European Commission) have identified recommended maximum fumonisin levels
that are considered adequate to protect human and animal health and that are achievable in
human foods and animal feeds [65]. These organizations evaluated all possible risk associated
with fumonisin exposure and determined the tolerable daily intake (TDI) [65]. TDI ranges from
2 to 4µg/g in maize intended for human consumption, and 1 to 50µg/g in animal feeds [18].

23

Limited information is available on the kinetics and metabolism of fumonisins in humans [63].
However, several possible pathways that may result in fumonisin toxic effect have been
proposed. One mechanism is through alteration of sphigolipid metabolism by blocking the
ceramide synthase enzyme, resulting in reactive oxygen species (ROS) production. This
generates an increase in oxidative stress and induction of lipid peroxidation that damage cells
[66]. To support this argument that (fumonisin produces ROS), studies have shown that
fumonisin treatment decreases the level of glutathione – a small intracellular protein in the body
which has anti-oxidative property [67].

Fumonisin may also induce cytotoxic effects through fragmentation of DNA and caspace-3
activation which result in induction of apoptosis in various cells and tissues. Studies have
demonstrated that fumonisin B1 stimulates apoptosis of several different human and animal cell
types, indicating that treatment can directly lead to apoptosis [68].

2.3.2.3 Toxic effects in humans
2.3.2.3.1 Neural tube defects
A deficiency of folic acid may be a major risk factor for neural tube defect (NTD), i.e., a defect
of the brain and spinal cord in the embryo that results from failure of the neural tube to close
[69]. Disruption of sphingolipid metabolism by fumonisin B1 may affect folic acid uptake and
cause NTD [66]. In 1990 and 1991, a NTD outbreak was reported to occur along the TexasMexico border and it was suggested that the outbreak might have been caused by high levels of
fumonisin B1 that had been reported in corn in the previous season [70]. A similar outbreak was

24

reported in China and South Africa in regions with high corn consumption showing a high
prevalence of NTD [60].

2.3.2.3.2 Oesophageal cancer
Available clinical trials conducted in South Africa and China suggests a correlation between
dietary fumonisin exposure and oesophageal cancer. This was observed in regions with
proportionately high exposure to fumonisin and where environmental conditions promote
fumonisin accumulation in maize, which is an important dietary staple [60]. People living in
such regions with high of FB1, FB2 and F. verticillioides in maize are at higher risk to develop
oesophageal cancer [71]. This was observed in some areas in Italy, Iran, Kenya, Zimbabwe,
United States and Brazil [18]. However, there was no correlation found between sphingolipid
levels in blood cells and cancer incidence with serum sphingolipids and risk of oesophageal
cancer [60].

2.4 Overview of mycotoxins in African countries
Mycotoxin contamination in food products is a serious issue in African countries [3]. Avoiding
consumption of food contaminated with mycotoxins seems to be one of the most fundamental
approaches for minimizing the risks associated with mycotoxicosis risk in humans. However,
avoidance of contaminated food is not feasible for poor communities in sub-Sahara Africa.
Therefore, feasible mitigation strategies to manage mycotoxins contamination are highly
encouraged or sought [72].
At least fifteen African countries, accounting for approximately 59% of the continent’s
population, have had mycotoxin regulations since 2003 [73]. There are numerous reports on

25

aflatoxin levels in commodities such as maize and peanuts, which exceed the allowable limits,
set by the Codex Alimentarius, especially in Africa. Mycotoxicosis has already caused massive
fatalities in the Africa continent. For example, a widespread outbreak of mycotoxicosis in the
North Eastern region of Kenya in 2004 caused 125 deaths out of the 137 cases reported, resulting
from consumption of contaminated maize [74]. The same region experienced a similar outbreak
in 2005 and 2006.

Mycotoxin contamination in grains and other staple foods and feedstuffs has serious implications
to human health. In Tanzania, for instance, growth faltering is affecting infants and young
children in the country. In year 2004–2005 growth failure statistics, stunting, underweight and
wasting affected about 38, 22 and 3% of children under 5 years of age, respectively. Growth
retardation is often experienced during the weaning period, suggesting a possibility of fumonisin
and aflatoxin contaminated complementary foods being a causative agent for stunting of young
children in the country. A study conducted to assess fumonisin exposure for infants consuming
maize in Tanzania revealed the percentage of fumonisin exposure that was significantly higher
than the provisional maximum tolerable daily intake (PMTDI) [75]. Other reports from West
Africa (Benin and Togo) showed that children exposed to higher aflatoxin level had lower height
gains than those exposed to the lowest levels [76].

2.4.1 Current status of mycotoxins in Africa
Exposures to mycotoxins are widespread in Africa. Different food commodities across the
continent have been tested for mycotoxins and reportedly carry mycotoxins levels above the
recommended limit. In some villages in Tanzania, for example, 52% of 120 home-stored maize

26

samples collected in 2005 were contaminated with fumonisins at levels of up to 11,048 μg/kg.
Around the same year, Tanzanian maize-based weaning foods consumed by 131 infants
contained fumonisins at levels ranging between 21 to 3,201 µg/kg [77].

Maize samples collected from home storage facilities in Kenya in 2010 were found to contain
1,776 ppb aflatoxin while those sampled from the markets had 1,632 ppb aflatoxin [78]. In 2010,
about 40% of corn sampled from farms in the Eastern and Western Kenya had aflatoxin level of
>10 ppb; while maize in Benin contained 4,000 ng/g aflatoxin.

Other foods also contain mycotoxins. In Nigeria, peanut oil and yam flour reportedly contained
500and 7,600ng/g of aflatoxin, respectively [79]. A significant number of peanuts harvested in
Mali were also found to contain >10 ppb aflatoxin levels in the 2009 – 2010 season, while peanut
paste had 300 ppb [80]. In Ghana, however, aflatoxin levels in peanuts and peanut paste were
reported as 216 and 3,278 ng/g, respectively. Intervention strategies are urgently needed to
manage/mitigate mycotoxin contamination, particularly aflatoxin and fumonisin, which affect
most of the foods in the region.
2.4.2 Factors that affect mycotoxins production in Africa
2.4.2.1 Climatic conditions
Hot and humid conditions are critical environmental components that favour growth of moulds
and mycotoxins production. These conditions are typical in most parts of Sub-Saharan Africa.
Drought stress and other factors facilitate toxigenic mould infections which promote production
of mycotoxins. Drought stress can happen rapidly if plants are exposed to high ambient
temperatures and low relative humidity [81]. High temperatures, drought, poor fertilization, and

27

strong nutrients competition are some conditions that results into weakening of plant’s natural
defences, thus predisposing them to increased mycotoxins infections. Intrinsic factors such as
moisture content, water activity, substrate type, plant type and nutrient composition may
influence growth of mycotoxigenic fungi as well as toxin production in the field.

2.4.2.2 Pest infestation
Insect infestation is one of the major causes of deterioration of grains in the fields and in storage
facilities since insects act as vectors of mycotoxigenic fungi [82]. Insect damage results into
lowering of cereals’ quality and market value of the crops, and can render crops unsafe for
human consumption. Pest infestation can be caused by poor harvesting and storage practices;
higher levels of insect damage increases risk of mycotoxins contamination [82].

2.4.2.3 Fungicides and/or fertilizers
Fertilizers and fungicides, if properly used, can be effective at inhibiting mould growth and
mycotoxin production [83]. For example, NPK (Nitrogen, Phosphorus and Potassium) fertilizer
has been found to reduce aflatoxin while urea fertilizers increases aflatoxin levels on corn.
However, some fungicides such as Fenpropimorph have actually been observed to not only
significantly increase aflatoxin B1 and aflatoxin G1 production but to also shift production in
favour of the more potent aflatoxin B1 [84].

28

2.4.2.4 Post-harvest handling
After harvest, food commodities may be stored, transported, and processed, before reaching
consumers. Each step in this process has the potential for mycotoxin contamination if appropriate
preventive measures are not taken.

For example, if transport containers are not dry and have free visible mould growth, insects or
any contaminated material; this can promote mould growth which results into mycotoxins
production. Wet storage of cereals, the presence of insects during storage, and the use of storage
facilities which are not well ventilated have been reported to be major reasons for mycotoxigenic
mould growth during storage [22].

2.5 Possible mitigation/intervention strategies for mycotoxins in Africa
Cereals, roots and tubers dominate crop production in most African countries [85]. These food
crops are highly susceptible to mycotoxins contamination, and in particular aflatoxins and
fumonisins. Constantly social- economic challenges accelerate the magnitude of mycotoxins
problem [90]. Good agricultural practices (GAP) during pre-harvest and good manufacturing
practices (GMP) after harvest are proposed as a primary line of defense against contamination of
cereals with mycotoxins [86]. Multidisciplinary approaches involving all actors along the food
chain are critical for mycotoxins reduction and control.

2.5.1 Pre-harvest interventions
Crop management during the period of production is critical for reducing mycotoxins, including
minimizing insect damage and fungal infection and proper use of registered insecticides,

29

fungicides and herbicides. Mechanical injury to the plant during cultivation should also be
reduced [87].

One valuable method to reduce plant stress is the use of irrigation. However, in most African
countries, this practice is still a challenge due to issues related to water availability and cost [88].
Crop rotation should be encouraged; and care must be taken to avoid rotating crops identified to
be susceptible to toxigenic moulds with each other. Grains should be harvested after reaching
maturity, and then dried to safe moisture level (13%) before storage [88]. Cleaning and removal
of infected seeds should follow recommended procedures.

2.5.2 Storage
Storage facilities should be well ventilated and protected from rain. Drainage of ground water
should be achieved, and insect and rodent infestation should be minimized. Crops to be stored
should also be dried to a safe moisture level to ensure prevention of fungal invasion and
mycotoxin production [89].

2.5.3 Biological control strategies
Indigenous atoxigenic strains of A. flavus that outcompete and replace the toxin-producing
strains have been identified as one potential strategy to mitigate mycotoxin contamination in
Africa. This technique, pioneered by the USDA and International Institute for Agricultural
Research (IITA), involves the development of Aflasafe which contains indigenous atoxigenic
strains of A. flavus and has been used effectively in some African countries such as Nigeria in

30

controlling mycotoxins in maize [90]. In Tanzania, atoxigenic strains of aspergilli have been
isolated and atoxigenic cultures are being developed.

2.5.4 Physical methods
Physical separation of visibly contaminated product can be applied as one mycotoxin minimizing
strategy. Sorting and cleaning may lower levels of mycotoxins by removing the contaminated
material. Broken maize kernels have been reported to contain nearly 10 times higher levels of
mycotoxins [91]. Thus, visible sorting of grains can reduce the exposure of subsistence farmers
and consumers to mycotoxins [92].

2.5.5 Food surveillance
Surveillance for mycotoxins is a primary intervention routine done in order to protect consumers
and economic interests of producers and traders in various countries. Surveillance data can be
used to establish regulatory guidelines that define the limits of mycotoxins, especially aflatoxins
and fumonisins, in foods. However, in many developing countries, these guidelines require better
enforcement to help minimize risk for aflatoxicosis to these populations [65].

2.5.6 Community education
Sensitization, or educating the community about mycotoxins, could be the most practical and
fundamental intervention at the subsistence farm level in developing countries. This includes
education on how to use low-energy technologies for food preservation, proper food handling
and storage methods [93]. If these primary approaches can be routinely adapted and implemented

31

it can significantly reduce the level of mycotoxins contamination in post-harvest foods and
associated exposure in human populations.

2.6 Fermentation
2.6.1 Traditional fermentation
Traditional fermentation is one of the oldest methods of food preparation, processing and
preservation. Fermented foods have improved taste and texture, extended shelf life, improved
nutritional value, and in some cases improved levels of safety. Fermentation of locally consumed
foods is considered a cost-effective and nutritionally beneficial technology for communities with
food safety and malnutrition problems, especially in developing countries. Changes that occur
during the fermentation process are predominantly the result of enzymatic activity brought about
by bacteria, yeasts and moulds. Some microorganisms produce desirable characteristics while
others are responsible for spoilage or toxicity. Several microorganisms may be involved in a
particular, single fermentation; and each may be responsible for a specific change or series of
changes in the entire process [94].

2.6.2 Tanzanian fermented gruel
Fermented gruel, commonly known as togwa, is made from maize, sorghum, millet, cassava, or a
blend of two or more of these cereal flours. Basically, togwa can be made by mixing flour with
water. This mixture is then boiled, cooled and left to ferment for at least 18 hours. Togwa can be
served at any time of the day in traditional (rural) society, either alone as a thirst quencher or as a
breakfast drink. Togwa is also widely used as a complementary food for children. It has a long

32

history in Tanzanian tribal custom, that is, as it is consumed as a symbolic drink during
ceremonies such as weddings, funerals, dances and other occasions.

Consumption of togwa in the country has been declining particularly in urban areas. Decline in
consumption is thought to be the result of several factors, including the time needed for
preparation and the availability of cheaper and more easily prepared substitutes such as tea and
coffee.

2.6.3 Lactic acid fermentation
2.6.3.1 Lactic acid bacteria
Lactic acid bacteria (LAB) are Gram-positive, catalase-negative, non-spore forming rods and
cocci. They are generally non-motile and utilize carbohydrates fermentatively to form lactic acid
as the major end product [95]. A few LAB are aero-tolerant Most LAB are used for preservation
of food products such as sauerkraut, fermented cereal gruels and legumes [96], by inhibiting the
growth of spoilage and pathogenic bacteria through production of lactic acid, acetic acid,
diacetyl, and acetaldehyde. Accumulation of these products causes inhibition of spoilage bacteria
in some foods and beverages.
The use of LAB in food preservation and to enhance food safety has recently gained momentum,
as consumers are increasingly becoming more concerned with products containing artificial
preservatives. All LAB used in food fermentation have GRAS (Generally Regarded As Safe)
status [78].

33

2.6.3.2 Potential of lactic acid bacterial in reducing aflatoxins and fumonisins
One practical approach for the prevention of mycotoxins (aflatoxin B1 and fumonisin B1) is the
use of lactic acid producing bacteria. LAB are reported to be able to metabolize or transform
mycotoxins into compounds that are less toxic. The ability of some of the LAB strains to repress
mycotoxins by producing low-molecular-weight metabolites and/or binding of the toxic to
bacteria cell wall has been reported [97].

Microorganisms can either use sorption and/or enzymatic degradation to detoxify mycotoxins.
Lactic acid bacteria can absorb mycotoxins either by attaching them to their cell wall
components or by active internalization and accumulation [98]. During cell rupture, it is
postulated that LAB can release molecules that potentially inhibit mould growth and therefore
lead to a lower accumulation of their mycotoxins [99].

Some LAB, have been identified with the strain-specific ability to reduce mycotoxins, especially
aflatoxins with great efficiency [100]. For example, an in vitro study on probiotic strains such as
Lactobacillus rhamnosus GG and Lactobacillus rhamnosus LC-705, demonstrated that they were
very effective in removing aflatoxin B1, with more than 80% of the toxin removed [101]. More
research is required to establish the mechanisms and extent of reduction of other notorious
mycotoxins, particularly the fumonisins in lactic acid fermented foods such as gruels.

34

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44

CHAPTER THREE
EFFECT OF LACTIC ACID FERMENTATION ON AFLATOXIN B1 AND FUMONISIN
B1 IN FERMENTED MAIZE-BASED GRUEL
3.1 Introduction
Even though maize is known to be highly susceptible to aflatoxin and fumonisin contamination,
lack of adequate economic resources in developing countries such as Tanzania prevent proper
implementation of existing regulations to reduce aflatoxins and fumonisin exposure in the food
supply. Many factors promote mycotoxin contamination of Tanzanian maize including drought
stress, erratic rainfall, insect activity, soil type, minimum and maximum daily temperatures [1],
poor harvesting practices, heavy rains during harvest, improper drying before storage and poor
storage methods (i.e. gunny bags, poor traditional storage structures, etc.) [2]. Additionally,
unprecedented climate change and global warming provide the right conditions where fungi
proliferate and produce high levels of aflatoxins and fumonisins [3]. As such, the production
environment and handling practices increase the risk of toxic contamination of Tanzanian maize,
thus presenting serious health problems to both humans and animals [2]. This is because not only
the grains (generally consumed by people), but also the whole plant from which they are
harvested (generally fed to animals), can be contaminated by mycotoxins.

The danger to humans is exacerbated because maize is a major cereal staple and component in
Tanzanian complementary food, such as gruel, thus increasing the risk of exposure of many
Tanzanian children to high levels of these toxins in their diets at an early stage. This is
particularly problematic because children are highly susceptible to aflatoxins and fumonisins
[4]. In children, these toxins may cause stunted growth, and in severe cases, culminate in liver
failure and even death. Unfortunately, many rural and urban communities in developing
countries such as Tanzania may not be aware of risks associated with mycotoxins [5]. It’s even

45

more alarming that a majority of sub-Saharan Africa inhabitants are at risk for chronic dietary
exposure to mycotoxins [6]. It is also unsettling that the number of people in these countries,
including Tanzania, who are occasionally exposed to high levels of mycotoxins in contaminated
foods, is still unknown. In certain areas where hepatitis B virus (HBV) infection is endemic such
as in China and other parts of Asia, chronic dietary exposure to aflatoxins can cause aflatoxicosis
in episodic poisoning outbreaks, thus HBV patients are considered at risk for fulminant liver
failure [7], and in severe cases, hepatocarcinogenicity [1]. A similar situation in Tanzania would
also be catastrophic.

Due to this largely ignored worldwide health matter, some organizations and institutions such as
the International Agency for Research on Cancer (IARC) have classified aflatoxin B1 (AFB1) as
a group 1 carcinogen (which means it is carcinogenic to humans) and fumonisin B1 (FB1) as a
group2B compound (which means that it is potentially carcinogenic) [8,9]. Thus, a great number
of prevention strategies have been proposed to reduce the risks posed by AFB1 and FB1,
particularly in developing countries. However, the full benefits of efforts to reduce the risk in
local communities have not been realized due to lack of political will and financial commitment
from host governments.

A significant amount of Tanzania’s maize crop is used for direct human consumption. However,
the prevalence of aflatoxin and fumonisin contamination in Tanzanian maize varies significantly;
more prevalence is reported in the Eastern and Western agricultural zones. Therefore, because of
the enormous significance of AFB1 and FB1 (responsible for acute toxicity, chronic toxicity,

46

carcinogenicity, teratogenicity, genotoxicity and immunotoxicity), the Tanzanian Food and Drug
Authority (TFDA) recently conducted aflatoxin testing for 254 maize samples. They found that
average aflatoxin levels in maize samples from the Eastern zone (Morogoro) and the Western
zone (Shinyanga) were 50 and 28 ppb, respectively [10]. More than 40% of all maize sampled
from both regions had more than 5 ppb aflatoxin (minimum tolerated dose in Europe). From a
global health perspective, these results show that infants and school children that regularly
consume maize-based complementary foods (especially those living in the Morogoro and
Shinyanga regions), are at higher risk of dietary aflatoxin exposure due to their relatively high
energy needs, which can lead to stunting.

Increased incidences of neural tube defects in infants of mothers who consume maize-based
foods contaminated with fumonisins have also been reported [11]. In adults, however, fumonisin
contamination is reportedly associated with oesophageal and liver cancers in South Africa [12]
and China [13], respectively. Around the world, many communities rely on maize as a dietary
staple food. In India, a severe outbreak of abdominal pain and diarrhoea was associated with
consumption of mouldy maize contaminated with fumonisin B1 [14]. It is therefore important
not only to discourage consumption of mouldy crops but also to find better processing methods
that can eliminate or reduce the concentration of food-borne fumonisins.

The Tanzanian Food and Drugs Authority (TFDA) is tasked with enforcing standards for
maximum aflatoxin and fumonisin concentrations that were set by the Tanzanian Bureau of
Standards (TBS). These standards apply to several products that are highly susceptible to
contamination by aflatoxin and fumonisin (e.g. maize and maize products). However, most of

47

their efforts focus on formally packaged foods intended for domestic and export markets. Sadly,
because of the socio-economic and cultural status of the Tanzanian population, a majority of
these toxin-susceptible foods, which are typically unpackaged, are locally consumed or traded
within domestic markets without being tested by TFDA. Thus, there is a significant risk of
mycotoxicosis outbreaks should there be contamination levels similar to those in Kenya in 2004.
Unfortunately, most Tanzanian maize farmers and consumers still have a low awareness about
these toxins and their health impacts primarily due to weak regulatory mechanisms, poor preand post-harvest management practices, and economic pressure during chronic food shortages
[15]. It is therefore critically important to find practical and economical approaches to reduce the
level of mycotoxin in foods and this approaches must be accepted by the local population.

Traditional fermentation, one of the oldest practices for food preparation, could be a practical
and novel approach that can be used in reducing aflatoxins and fumonisins in cereal-based foods.
It is a low cost method of food preservation that not only improves the nutritional value and
organoleptic properties of food but also its digestibility [16].

Traditional lactic acid bacteria (LAB) fermentation, which is known to confer preservative and
detoxifying effects on food and feed, can be used to make fermented gruel. Selected strains of
lactic acid bacteria reportedly reduce mycotoxins to trace levels during fermentation. Thus, it is
expected that when used to ferment maize gruel, fermentation may represent one key strategy to
reduce health risks associated with exposure to aflatoxins and fumonisins. Even though trace
amounts of these mycotoxins in fermented food products should continue to be a cause for
concern, the extent to which LAB fermentation may reduce the biological potency of aflatoxin

48

and fumonisin contaminated maize gruel is still unknown. Therefore, the effect of LAB
fermentation process on maize gruel was investigated in this study in order to elucidate its ability
to reduce the levels of AFB1 and FB1.

3.2 Materials and methods
3.2.1 Starter culture preparation
Strains of lactic acid bacteria (Lactobacillus plantarum B 4496, Lactobacillus casei subsp casei
B 1922, Lactobacillus fermentum B 1840, Pediococcus pentosaceus B 14009, were obtained
from the NRRL Culture Collection Centre at the National Center for Agricultural Utilization
Research, Agricultural Research Service, United States Department of Agriculture, Peoria,
Illinois, USA. Co-cultures composed of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus buchneri (AS), or consisting of Enterococcus faecium, Lactobacillus plantarum
and Lactobacillus lactis (SR3.54) (MC) were obtained from Chr. Hansen, Germany.

The

lyophilized strains were activated by inoculation into MRS broth (Becton) in screw-capped test
o

tubes and incubated at 30 C for 24hours (Becton Dickinson, Cockeysville, MD, USA). The
LAB were single colony isolated by streaking on MRS agar (Fischer) plates which were
o

incubated at 30 C for 48 hours. A colony was picked from each plate, grown in MRS broth, and
then centrifuged at 655×g for 15 min (Kubota 2010, Kubota, Tokyo, Japan). The bacterial cell
pellet was then washed in peptone physiological salt solution (ingredients and concentrations),
centrifuged as above, and re-suspended in physiological salt solution. The procedure resulted
7

into a culture preparation containing 10 colony forming units (CFU, examined as viable count
o

on MRS agar). The remaining LAB were stored in an ultra-freezer at –80 C in sterile cryo-tubes

49

containing MRS broth with 10% (v/v) glycerol (with several acid-washed glass beads) until
required.

3.2.2 Gruel sample preparation
Maize and millet grains for gruel preparation were purchased from Morogoro municipality
markets in Tanzania. Maize grains were ground using a Neogen Grinder #9401 (Neogen, USA)
and the flour was collected, sieved (Retsch, USA) and stored in cool and dry cupboard. Millet
grains were cleaned, weighed and then steeped in water at room temperature for 18 h. The
steeped grains were washed thrice at 6 h intervals to prevent fermentation. After steeping, the
grains were spread on a cheese cloth, covered, sprinkled with water, and left to germinate in a
dark cupboard at room temperature for 48h. Germinated millet grains were sun-dried, milled and
the flour was sieved (mesh size 20 µm) and stored in a dry container before use for preparation
of gruel samples.

Gruel was prepared according to procedure described by Mugula [17]. Maize flour was mixed
o

with water in a ratio of 1:9 (10% w/v), and the slurry was heated (100 C) (Belling,UK) while
stirring continuously for approximately 20 minutes. The mixture was then cooled using water
bath (Memmert, Germany). For naturally fermented samples (NF); the gruel was cooled to
o

around 30 C and then mixed with millet malt flour at a ratio of 9:1. To prepare back slopped
samples (BS), the gruel was mixed with millet malt flour and this was inoculated by addition of
(10% v/v) a successful spontaneously fermented gruel. To prepare control fermentation samples,

50

o

malt flour was added when the gruel reached a temperature of 55 – 58 C, left to cool, sterilized
o

o

by autoclaving at 121 C for 15 minutes, and then cooled to 30 C prior to inoculation.

3.2.3 Sample spiking and inoculation
For natural fermentation and back slopping, 100 mL of prepared gruel sample was spiked with
800 ppb of aflatoxin B1 and 20 ppm of fumonisin B1. For controlled fermentation, 100 mL of
the sterile gruel was spiked with 800 ppb aflatoxin B1 and 20 ppm fumonisins B1 followed by
inoculation with 1mL of LAB cultures. The inoculated samples were completely vortexed
(Gene-2 Model G-560E, Scientific Industries, Behemia, New York) to make a homogenous mix.
In summary, triplicates of the spiked sample were prepared as follows (Figure 5): (i) gruel spiked
with aflatoxin B1 and fumonisins B1, but fermented naturally (NF); (ii) gruel spiked with
aflatoxin B1 and fumonisins B1 and fermented by back slopping (BS); (iii) gruel spiked with
aflatoxin B1 and fumonisins B1, fermented with various LAB cultures. Each tube was screwcapped aseptically, vortexed and allowed to ferment in an incubator set at 30°C for 24 h.
Aflatoxin B1, fumonisin B1, pH and lactic acid concentrations were monitored at 0, 4 and 24 h.
All measurements were taken in duplicate for three replications.

51

Figure 4: Overall study design and sample collection procedure
Maize flour
90% H O + heat+ malt
2

Maize gruel
AFB / FB
1

1

Spiked gruel

Spiked gruel +
back slopping

Control

15 mL

15 mL

15 mL

Spiked gruel +
LAB

AFB , FB , pH,
1

0 hour

1

L.A & CFU
AFB , FB , pH,
1

4 hours

1

& L.A
AFB , FB , pH,
1

24 hours

1

L.A & CFU

3.2.4 pH determination
The pH of the gruel samples was measured using a glass electrode pH-meter (Model HI 9124,
Hanna Instrument Inc, Romania). Prior to reading, the pH was calibrated against standard buffer
solutions at pH 4.0, 7.0 and 10.0.

52

3.2.5 Lactic acid content determination
The standard method and standard curve for lactic acid determination was based on the method
1

developed by Taylor [18] but with minor modifications. Between 0 – 60 µg mL— of standard
lactic acid (in 15 µg increment) were added into borosilicate tubes, and brought to 1 mL volume
by adding double distilled water. Concentrated sulphuric acid (6 mL) was then added to the tubes
and then vortexed to mix. The quantity of acid was defined as 82% acid. The mixed solutions
o

were then incubated at 95 – 100 C for 10 min in a steam water bath, and then cooled to room
temperature using a water bath, upon which 100µL copper sulphate (Sigma Aldrich, USA) was
added immediately, followed by 200 µL of the p-hydroxydiphenyl reagent (Sigma Aldrich,
USA), and then vortexed. The tubes were kept at room temperature for at least 30 minutes and
the absorbance was recorded at 570 nm. Blank samples had values ranging between 0.2 – 0.5
compared to water.

3.2.5.1 Lactic acid quantification
The samples were filtered using Whatman #1 filter paper. The collected filtrate was diluted
(1:100) with de-ionized water. The diluted filtrate was collected in small test tubes. Each diluted
filtrate was treated as follows: The filtrate (1 mL) was placed in a tube (15 mL corning plastic
centrifuge tube) and 6 mL of concentrated sulphuric acid (H2SO4) was added. The tubes were
allowed to stand in boiling water for 5 min and cooled to room temperature in a water bath. To
each tube 100 µL copper sulphate (Sigma Aldrich, USA) was added immediately, followed by
200 µL of the p-hydroxydiphenyl reagent and the contents were vortexed to ensure even
distribution of the insoluble reagent. The tubes were kept at room temperature for about 30

53

minutes and absorbance was measured using a spectrophotometer (Thermo Fisher Scientific
GENESYS 20 Spectrophotometer). Lactic acid concentration was estimated by using the
standard curve.

3.2.6 Microbial analysis
Using aseptic technique, the initial dilution was made by transferring 1mL of gruel that had been
fermented for 24 h to 9 mL sterile peptone saline (0.1% neutral peptone, 0.9% NaCl), and
homogenized by shaking. Subsequent dilutions were prepared with the same diluents, and in all
cases duplicate plates were prepared from each of the dilutions.
o

The relevant dilutions were surface plated on MRS agar and incubate at 30 C for 48 h. At the
end of incubation period, all the petri plates containing between 30 – 300 colonies were selected.
Number of bacteria (CFU) per sample was calculated by dividing the number of colonies by the
dilution factor multiplied by the amount of sample added to MRS agar as indicated in the
equation below.

54

3.2.7 Aflatoxin B1 detection by thin layer chromatography
3.2.7.1 Sample extraction
Five grams of gruel samples were extracted with 25 mL of 65% ethanol (v/v), vortexed for 3
minutes, and then allowed to settle. Aliquots of the solvent extracts were decanted and filtered
using Whatman #1 filter paper.

3.2.7.2 Sample clean-up using SPE column
3.2.7.2.1 Procedure description
The SPE column was conditioned by adding 5 mL of ethanol followed by 5 mL of double
distilled water. One mL of sample extract was diluted with 5 mL of double distilled water. This
is because ethanol concentration in the sample must be less than 10% to improve retention of
AFB1 and its metabolites on the SPE column. A total of 6 mL of diluted samples was then
passed through the conditioned SPE column followed by 10% ethanol to remove polar
compounds from the column bed. AFB1 was then eluted by passing 2 mL of 80% ethanol
through the column.

3.2.7.3 Aflatoxin B1 detection on TLC
Thin layer chromatography (TLC) is one of the preferred qualitative methods for aflatoxins
detection [19]. TLC of mycotoxins was performed in all instances on pre-coated silica gel G
glass plates (Camlab Limited, UK). Approximately 20 μL of each of the analytes were spotted on
TLC plate, and then air dried. The plate was placed into the TLC chamber saturated with
acetone: chloroform (1:9) solvent. TLC was also performed on standard aflatoxin B1, within the
same chamber, alongside the analytes. Further, the spiking of samples with standard aflatoxin B1
55

was done to confirm the presence of aflatoxin spots on TLC. TLC plates were observed under
UV illuminator at 365 nm.

3.2.8 Aflatoxin B1 and fumonisin B1 quantification using Neogen Reveal Q+ assay
3.2.8.1 Assay principle
Quantitative testing of aflatoxin B1 and fumonisin B1 was performed using Reveal Q+ for
aflatoxin

and

fumonisin

test

methods,

which

utilize

a

single-step

lateral

flow

immunochromatographic principle based on a competitive immunoassay format. Using a
mycotoxin-antibody particle complex coated test strip and the Reveal AccuScan

®

III reader

(Neogen, USA), the presence of aflatoxin and fumonisin in the samples was determined by
following approved test protocols and Neogen’s assay principles. First, the extract was wicked
through a reagent zone containing antibodies specific for the mycotoxin conjugated to colloidal
gold particles. If the mycotoxin of interest such as AFB1, was present, it was captured by the
particle-antibody complex. The mycotoxin-antibody-particle complex was then wicked onto the
membrane containing a zone of mycotoxin conjugated to a protein carrier, which captured any
uncomplexed mycotoxin antibody, thus, allowing the particles to concentrate and form visible
lines. Free mycotoxin in a sample would always complex with the antibody-gold particles, thus
allowing less antibody-gold to be captured in the test zone. Algorithms programmed into the
Reveal AccuScan

®

III reader converted the line densities into quantitative results reported in

parts per billion (ppb). The membrane also contained a control zone to ensuring the strip was
functioning properly. A visible line would always form regardless of the level of mycotoxin
when an immune complex present in the reagent zone was captured by an antibody.

56

3.2.8.2 Aflatoxin B1 and fumonisin B1 recovery
Recovery of AFB1 and FB1 were determined by spiking a series of duplicate maize gruel
samples with AFB1 standards (0, 20, 50, 100 ppb) and FB1 standards (0, 2, 4, 6 ppm). The
spiked gruel was then analysed by Reveal Q+ for aflatoxin and fumonisin. Procedures for
analysis are described in section below.

3.2.8.3 Quantification of aflatoxin B1
Reveal Q+ for aflatoxin was used to quantify aflatoxin B1 during lactic acid fermentation.
Extracts sampled at 0, 4, 12 and 24 h were used for AFB1 detection. Approximately 500 μL of
sample diluent was added to a dilution cup followed with 100 μL of sample extract. The solution
was mixed by pipetting up and down 5 times. A 100 μL aliquot of the diluted sample extract was
transferred into a new clear sample cup. Reveal Q+ for aflatoxin test strip with the sample end
down was placed into the sample for 6 minutes. After 6 minutes the strip was removed from the
sample cup and inserted into the cartridge adapter. The adapter was placed into Reveal
®

AccuScan III reader and concentration of aflatoxin B1 was recorded.

3.2.8.4 Fumonisin B1 extraction and quantification
Fumonisin B1 was extracted from 5 mL of fermented gruel samples by adding 25 mL of 65%
(v/v) ethanol/water and shaken for 3 minutes at 200 rpm in a shaker. The extracts were then
filtered using a filter paper (Whatman #1) and fumonisin B1 concentration was measured using
Neogen Reveal Q+ for fumonisin test kit (Neogen, USA). Exactly, 200 μL of Neogen sample
57

diluent was added into a dilution cup followed by 100 μL of the sample extract. The solution
was mixed by pipetting up and down 5 times. A 100 μL aliquot of diluted sample extract was
transferred into a new clear sample cup. Reveal Q+ for fumonisin test strip was placed with the
sample end-down into the sample cup for 6 minutes. The strip was then removed promptly,
inserted into black cartilage adapter, analysed and quantitative results were displayed by the
Reveal AccuScan III reader in ppm.

3.2.9 Statistical data analysis
The data were analysed by using SAS 9.2 software statistical package for two-way ANOVA to
determine the significant differences and interaction between the factors’ means at (P < 0.05).
Means were separated by Tukey’s Honest Significant Difference at P < 0.05. Results were
presented in tables as means ± SD.

3.3 Results
3.3.1 Thin layer chromatography assay for aflatoxin B1
Even though it is time-consuming, thin-layer chromatography (TLC) is one of the most specific
and sensitive methods for determining levels of aflatoxins in food commodities or in culture
[20]. This method requires extraction procedures that remove interfering substances, by using a
mixture of water and a polar organic solvent [20].

Using this TLC technique in this study, it was observed that all maize gruel extract samples
spiked with AFB1 standard showed visible spots on the TLC plate under UV florescence light
(Figure 5).This screening technique for detection of AFB1 in the gruel sample extracts provided
58

reliable preliminary information about the presence of AFB1 prior to quantitation of the
mycotoxin by Neogen Reveal Q+. Figure 6, on the other hand, shows the effect of fermentation
on aflatoxin B1 in maize gruel after 24 h. As was expected, the standard showed very intense
spots compared to the maize gruel samples. Since TLC is specific for the aflatoxin type, it was
possible to confirm that the fermentation process reduced AFB1 levels in maize gruel fermented
for 24 h. The retention factor (Rf) value for both the standard and the gruel samples was not
expected to differ significantly. However, the Rf value or the standard sample was 0.42, while
that for fermented maize gruel samples was 0.31 (Figure 6), suggesting possible formation of
other metabolites during the fermentation process or other isolates that might have affected
AFB1 affinity, thus reducing elution time.

59

Figure 5: Thin layer chromatographic plate of the maize gruel samples spiked with AFB1

Figure 6: Thin layer chromatographic plate showing the effect of fermentation on aflatoxin
B1 after 24 hours of fermentation

60

3.3.2 Quantitative analysis of aflatoxin B1 and fumonisin B1 concentration infermented
maize gruel
3.3.2.1 Aflatoxin B1 and fumonisin B1 recovery assay
Aflatoxin B1 and fumonisin B1 in the maize gruel samples, analysed by Reveal Q+ for aflatoxin
and reveal Q+ for fumonisin, are presented in Table 1. To validate that Reveal Q+ for aflatoxin
and fumonisin would provide us with accurate and reliable values when determining toxic levels
in the fermented gruel samples, it was critical to establish the mean recovery on samples spiked
with known concentrations of standard aflatoxin B1 and fumonisin B1. The results indicated
that, in each of the gruel samples, more than 93% of the added toxins were recovered. Similar
observations were also reported by other investigators who reported about 92% mean recovery of
AFB1 and FB1 using reveal Q+ for aflatoxin and fumonisins [21, 22]. The device was therefore,
accurate with a low level of variability.

3.3.2.2 Quantification of AFB1 in fermented gruel
Probiotic bacteria have been shown to efficiently bind AFB1 in vitro [23]. Other strategies of
mycotoxin inactivation are reported in the literature [24], but only a handful of them are accepted
for practical use, e.g. ammonia treatment. Toxigenic colonies from contaminated grains would
change colour from yellow to pink upon exposure to ammonia [25], thus colour intensity
correlates with aflatoxin concentration. Other cultural methods are also used to detect and
decontaminate mycotoxins in food [26]. Since none is entirely effective, some researchers
suggest that the best approach for decontamination of mycotoxins in foods should be degradation
by selected bacterial strains [27]. Lactic acid bacteria are well known for their fermentative
activity causing transformation of mycotoxins into compounds that are less toxic.
61

Table 1: Validation of aflatoxin B1 and fumonisin B1 recovery in gruel
AFB1
spike
level
(ppb)

AFB1
detected
(ppb)

Net
b
detected
(ppb)

0

a

19.1

18.7

50

47.1

100

93.7

FB1
spike
level
(ppm)

0.4

20

%
c
Recovery

a

FB1
detected
(ppm)

Net
b
detected
(ppm)

%
c
Recovery

a

0.2

93.3

2

1.88

1.86

93.0

46.9

93.7

4

3.74

3.72

93.0

93.3

93.3

6

5.60

5.58

93.0

0

Controls (samples with no toxin added);

b

Net detected = Corrected for control (by subtracting toxin concentration found in unspiked
samples from the toxin concentration in samples with added toxin);
c
% Recovery, Percentage recovery.

Several lactic acid bacteria strains have been identified that are able to efficiently reduce
mycotoxins in foods during fermentation [28]. In this study, selected lactic acid bacteria strains
including the species that were reported to be isolated from Tanzanian fermented gruel (Togwa)
were used to ferment maize gruel, and their ability to remove aflatoxins in maize gruel after 24 h
is indicated in Table 2.

62

Table 2: Effect of fermentation on reduction of aflatoxin B1 in maize gruel
Concentration of AFB1 in gruel

% Reduction of
2
AFB1

1

Culture
0h
Natural fermentation (NF)
Back Slopping (BS)
AS

3

4

MC

5

40.0±0.00
39.3±0.26
35.5±1.27

4h
a

a

a

37.4± 0.30

a

Pediococcus pentosaceus (PP)

39.1±0.59

Lactobacillus plantarum (LP)

38.0 ±0.03

Lactobacillus casei (LC)

35.7±1.19

Lactobacillus fermentum (LF)

37.7±0.26

1
2
3

a

a

a

a

24 h

36.7±1.20
28.5±0.28
27.9±0.54
28.8±0.14
26.6±0.31
28.7±0.17
29.7±0.33
27.3±0.33

a

b

b

b

b

b

b

b

4h

24 h

c

8.4

55.9

d

28.8

68.0

d

30.2

68.3

c

28.1

53.9

c

33.4

45.0

c

28.3

54.8

c

25.9

51.7

c

31.7

55.1

17.7±0.33
12.8±0.41
12.7±0.20

18.5±0.24
18.4±0.30
18.1±0.15
19.3±0.24
18.0±0.08

Values are means of three replicate measurements;
Change is expressed as %, relative to readings recorded at 0 hour;
Back-sloped with 10% (v/v) previously fermented gruel, (BS);

4

Co-culture of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri;
(AS);
5
Co-culture of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus lactis,
(SR3.54); (MC)
Means in the same column with the same superscript are not significantly different (p<0.05).

63

The duration of fermentation was critical in determining the ability of LAB in detoxifying AFB1,
with longer fermentation time being more effective (Table 2). It was observed that for all
examined strains, the percentage of aflatoxins AFB1 removal in fermented maize-based gruel
increased with fermentation period (24 h) (Table 2). Removal of AFB1 was slower through the
natural fermentation (NF), in which only 8% of the initial AFB1 concentration was reduced after
4 h. All the other methods reduced more than 25% of AFB1 within the same time period.

There was no significant difference (p<0.05) amongst the bacterial cultures in reducing AFB1
after 4 h, suggesting that the synergistic effect of mixed cultures compared to single bacterial
strains was not clearly visible at the beginning of fermentation (Table 2). This could be partly
due to the fact that during this initial stage of fermentation, the bacteria were still actively
metabolizing to produce lactic acid and other fermentation by-products, as it establishes a
suitable environment for survival.

However, after 24 h, the ability of LAB strains to significantly repress AFB1 in samples was
observed. All bacterial cultures showed a significant ability (p<0.05) to remove AFB1 after 24 h
of fermentation (Table 2). Even natural fermentation removed more than half of the initial AFB1
concentration in the gruel samples after 24 h (Table 2). It was also observed that all the
Lactobacillus strains tested removed between 52 – 55% of the AFB1 from the gruel samples
while the Pediococcus pentosaceus had the lowest toxin reduction (45%) after 24 h of
64

fermentation (Table 2). Since bacterial activity on substrates differ considerably, this suggested
that bacterial effect on AFB1 reduction during fermentation processes could be strain specific
and therefore there is need for further exploration with other different lactic acid producing
bacterial strains. Some researchers also reported that a number of LAB vary in their AFB1
detoxification rate, and some strains had higher percentage of AFB1 reduction than others [29].
It could also be argued that the synergistic effect of mixed bacterial strains on AFB1 reduction
was more effective in the presence of Lactobacillus buchneri than in the presence of
Lactobacillus lactis (Table 2). This is because a commercially mixed culture containing
Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri (AS) significantly
reduced AFB1 from the gruel samples by 68% while a similar co-culture of Enterococcus
faecium, Lactobacillus plantarum and Lactobacillus lactis, (SR3.54); (MC), reduced AFB1 by
54.9%, after 24 h of fermentation (Table 2).

Surprisingly, back-slopping, a typical traditional procedure used in fermented dough process
using the sponge-method, and is also used by some communities in fermenting their gruel, had
similar effect in reducing AFB1 as the commercial co-culture containing Enterococcus faecium,
Lactobacillus plantarum and Lactobacillus buchneri (AS) (Table 2). Our results indicated that
back-slopping reduced AFB1 by 68% in maize-based gruel fermented for 24 h, which was
significantly higher (p<0.05) than that of monobacterial strain cultures tested (Table 2).

65

Furthermore, back-slopping was even superior to natural fermentation in reducing AFB1 in
maize gruel samples after 24 h at 30° C (Table 2).

3.3.2.3 Effect of fermentation on fumonisin B1 levels
In many developing countries including Tanzania, often maize grains for consumption are not
screened for the presence of mycotoxins such as, fumonisin B1. This poses a challenge because
when these grains are used in food preparation, the exposure levels to these potentially harmful
mycotoxins are unknown. However, the fermentation process, particularly, by using lactic acid
bacteria, has been reported to detoxify fumonisin B1 [30] and thus may potentially be capable of
reducing levels of this toxin in fermented maize-based gruel.
In this study, removal of fumonisin B1 appeared to be time dependent. The results indicated that
removal of fumonisin B1 increased with time of fermentation (Table 3). The use of LAB starter
cultures was superior at removing fumonisin B1 within the first 4 h of fermentation compared to
natural fermentation or back-slopping (Table 3). Fermenting maize-based gruel using LAB
cultures at 30°C reduced the levels of fumonisin B1 by about 10 – 17% within 4 h, while natural
fermentation reduced levels by 7%. However, reduction of fumonisin B1 in maize based gruel
was more substantial after 24 h in the presence of LAB (14 – 27% reduction), indicating that
longer fermentation time is essential in achieving greater elimination of mycotoxins in fermented
foods. It was also observed that a co-culture of Enterococcus faecium, Lactobacillus plantarum
and Lactobacillus buchneri, (AS) was superior (removed 27%) to that of Enterococcus faecium,
Lactobacillus plantarum and Lactobacillus lactis (SR3.54) (MC) (removed 14%) of the

66

fumonisin B1 from gruel within 24 h (Table 3), indicating the possibility of the strain-strain
interaction relationship in elimination of fumonisins.
Table 3: Effect of LAB on fumonisin B1 levels in fermented maize based gruel
Culture

% Reduction of
2
FB1

1

Concentration of FB1 (ppb)
0h
Natural fermentation (NF)
Back Sloping (BS)
AS

3

4

MC

5

1.00±0.00
1.00±0.00
0.93±0.03
1.00±0.00

Pediococcus pentosaceus (PP)

1.00±0.00

Lactobacillus plantarum (LP)

0.96±0.03

Lactobacillus casei (LC)

0.93±0.03

Lactobacillus fermentum (LF)

1.00±0.00

4h
a
a
a
a
a
a
a
a

0.93±0.03
0.93±0.03
0.83±0.03
0.90±0.00
0.80±0.00
0.86±0.03
0.90±0.03
0.86±0.03

24 h
a
a
a
a
a
a
a
a

24 h

a

7.0

20.0

a

7.0

30.0

a

17.0

27.0

a

10.0

14.0

a

20.0

24.0

a

14.0

24.0

a

10.0

17.0

a

14.0

17.0

0.80±0.03

4h

0.70±0.03
0.73±0.06
0.86±0.03
0.76±0.03
0.76±0.03
0.83±0.03
0.83±0.03

1

Values are means of three replications

2

Change is expressed as %, relative to readings recorded at 0 hour

3

Back-sloped with 10% (v/v) previously fermented gruel, (BS)

4

Co-culture of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri; (AS)

5

Co-culture of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus lactis,
(SR3.54); (MC)
Means in the same column with the same superscript are not significantly different (p<0.05).

At the end of fermentation period, it was also observed that back-slopping was superior (30%
reduction) to natural fermentation process (20% reduction) in the elimination of fumonisins from

67

fermented gruel within 24 h. This might be due to the fact that inoculation with previously
fermented gruel samples (back-slopping) might have facilitated selection of strains which are
best adapted to the food substrate. Other authors [31] also reported a 13% reduction of fumonisin
in ogi (a fermented suspension of wet milled maize in water) when fermented for 24 h. Even
though time was critical for fumonisin B1 in gruel samples and highest reduction occurred at 24
h , but the reduction was not statistically significant (p<0.05).

3.3.3 pH
Time permitting, lactic acid bacteria are capable of producing significant levels of lactic acid
when conditions are right, thus lowering pH of their environments which confers protection to
foods against spoilage agents and pathogens, for example in sauerkraut and fermented cereal
gruels [32]. Therefore, pH drop is a key indicator of bacterial activity in fermented foods. The
change in pH during lactic acid bacteria fermentation of Tanzania maize gruel is presented in
Table 4. At the beginning of the fermentation process, all the maize based gruel samples had pH
levels around 5.8. After 4 h of fermentation, natural fermentation (NF) method had significantly
(p<0.05) slower pH drop compared to the other methods when considered against time 0 h
(Table 4). This corresponded with the lower reduction of AFB1 and FB1 by natural fermentation
method as indicated in Table 2 and 3, within the same time period, and suggested that NF had
slower bacterial activity in the initial stages of fermentation. However, the slow production of
lactic acid by Lactobacillus casei compared to the other Lactobacillus strains after 4 h at 30° C
(Table 4) could be an indication of selectivity of bacteria on substrates.

68

Table 4: pH levels during lactic acid fermentation of maize based gruel

pH

Culture
0h
Natural fermentation (NF)
Back-slopped (BS)
AS

2

3

MC

4

4h

5.76±0.04
5.80±0.00
5.80±0.00
5.80±0.00

Pediococcus pentosaceus (PP)

5.80±0.00

Lactobacillus plantarum (LP)

5.80±0.00

Lactobacillus casei (LC)

5.80±0.00

Lactobacillus fermentum(LF)

5.80±0.00

1
2

1

a

a

a

a

a

a

a

a

24 h

5.36±0.17
4.67±0.05
4.28±0.03
4.33±0.00
4.26±0.01
4.27±0.04

b

b

b

b

b

5.42±0.01
4.43±0.01

a

a

b

3.74±0.10
3.52±0.03
3.44±0.06
3.47±0.05
3.46±0.03
3.41±0.07
3.68±0.07

c

c

c

c

c

c

c

3.50± 0.53

c

Values are means of replicate measurements
Back-slopped with 10% (v/v) previously fermented gruel, (BS)

3

Commercial mixed culture of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus buchneri; (AS)
4
Commercial mixed culture of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus lactic, (SR3.54); (MC)
Means in the same column with the same superscript are not significantly different (p<0.05).

Within the first 4h, using commercially mixed cultures was not significantly superior at
producing lactic acid compared to back-slopping method (Table 4), which also corresponded

69

with their respective ability to reduce AFB1 and FB1 reported in Tables 2 and 3, suggesting
fermentation rate and detoxification potential of lactic acid bacteria were not influenced by the
process but by the types of bacteria used. This information is crucial in the determination of
bacterial strains for recommendation when fermentation time is critical and specific organoleptic
properties are desired.

After 24 h, all the other bacterial strains were still not significantly different (p<0.05) from the
natural fermentation process in lowering pH of the gruel (Table 4). The pH of the samples, after
24 hours at 30° C, ranged between 3.4 – 3.7, with the Lactobacillus plantarum strain having the
greatest ability to produce more lactic acid than the rest, and lowering the pH to 3.4 (Table 4).
These results agree with earlier reports that Lactobacillus plantarum strains produce lactic acid
faster than other Lactobacillus strains during fermentation of heat processed cereal substrates
[17].

3.3.4 Lactic acid production during fermentation of maize based gruel
Bacterial production of lactic acid during fermentation correlates with pH levels of the samples
in which they are inoculated and rate of substrate utilization. Table 5 shows relative amounts of
lactic acid produced during fermentation of Tanzanian maize-based gruel. Among the methods
used, the natural fermentation process had the lowest level of lactic acid production during
fermentation (Table 5), which also correlated with the slightly higher pH level indicated in Table
4, indicating it had lower metabolic activities that corresponded with the lower reduction of
AFB1 and FB1.

70

Even though Lactobacillus casei strain produced significantly (p<0.05) lower amounts of lactic
acid after 4 h of fermentation compared to other Lactobacillus species (Table 5), which
correlated with its comparatively slightly higher pH reported in Table 4, it however had greater
ability to rapidly produce lactic acid between 4 and 24 h, compared to the rest of the strains
(Table 5). Quantitatively, Lactobacillus plantarum still produced significantly (p<0.05) higher
amount of lactic acid after 24 h compared to all the other cultures (Table 5), which also
correlated with its ability to significantly lower the pH of samples in which it is present (Table
4). This implies that acid production or microbial mass might a play part in toxin reduction. This
study confirms observations [33] that maize mash fermented with Lactobacillus cultures of
Lactobacillus fermentum, Lactobacillus brevis and Lactobacillus salivarius also showed similar
levels of acid after 24 h of fermentation.

However, it is important to note that the back-slopping, Lactobacillus fermentum as well as
fermentation with commercially mixed culture of Enterococcus faecium, Lactobacillus
plantarum and Lactobacillus buchneri (AS) produced significantly (p<0.05) higher levels of
lactic acid after 4 h at 30°C (Table 5), which also correlated with significantly lower pH levels
compared to natural fermentation method reported in Table 4, indicating the rapid bacterial
activity in these samples during the initial stages of fermentation process.

71

Table 5: Lactic acid production during fermentation of maize based gruel
Lactic acid (µg/mL) × 100

Culture treatment

0h

Natural fermentation (NF)
Back-slopped (BS)
AS

2

3

MC

4

4h

0.00±0.00
0.00±0.00
0.00±0.00
0.00±0.00

Pediococcus pentosaceus (PP)

0.00±0.00

Lactobacillus plantarum (LP)

0.00±0.00

Lactobacillus casei (LC)

0.00±0.00

Lactobacillus fermentum(LF)

0.00±0.00

1
2

a

a

a

a

a

a

a

a

0.11±0.00

24 h

b

0.92±0.00
0.92±0.00
0.67±0.00
0.69±0.00
0.68±0.00

e

e

d

d

d

0.35±0.05
0.88±0.01

c

e

3.01±0.01

f

3.67±0.00
3.34±0.01
3.33±0.03
3.52±0.00

i

g

g

h

3.95±0.01
3.62±0.01
3.63±0.00

j

i

i

Values are means of replicate measurements
Back-slopped with 10% (v/v) previously fermented gruel, (BS)

3

Commercial mixed culture of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus buchneri; (AS)
4
Commercial mixed culture of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus lactis, (SR3.54); (MC)
Means in the same column with the same superscript are not significantly different (p<0.05).

3.3.5 Effect of pH and lactic acid on aflatoxin B1 reduction
Bacterial production of lactic acid is directly correlated to pH of fermented foods (Tables 4 and
5), which may result in active inhibition of spoilage and pathogenic bacteria. This inhibition is
72

not only due to lactic acid production but also to production of fermentation end products such as
diacetyl, acetaldehyde and acetic acid, which may accumulate over time to inhibitory levels.
However, lowering pH through production of lactic acid by lactic acid bacteria may also be
connected to reduction of AFB1 in fermented foods. In this study, a proportional relationship was
established between the reduction in both pH values and the corresponding reduction of
aflatoxins B1 in fermented maize based gruel samples i.e. the lower the pH value, the greater the
decrease of AFB1 content in the gruel samples tested (Figure 7A).

It seems that the rate of reduction of AFB1 in maize-based fermented gruel at the end of the
fermentation period varied with the type of bacterial strain (Figure 7A). Back-slopping (BS) and
commercially mixed culture of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus buchneri (AS) had the most extensive reduction of AFB1 between the pH ranges
of 3.4 – 3.6; both showed about 68% AFB1 removal. Within the same pH range, commercially
mixed culture of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus lactic,
SR3.54 (MC), Lactobacillus plantarum (LP) and Lactobacillus faecium (LF) removed 54, 55 and
55% of AFB1, respectively; while Pediococcus pentosaceus (PP) only removed 45% of AFB1
after 24 h of fermentation (Table 3; Figure 7A). Thus, maize-based gruel consumers in aflatoxin
prone regions such as the Eastern zone (Morogoro) and Western zone (Shinyanga) of Tanzania
would be at less health risk if their gruel was fermented with Lactobacillus bacterial species.
This would partly be due to production of significantly (p<0.05) higher levels of lactic acid by

73

Lactobacillus species (3.62 – 3.95 mg/mL × 100) compared to Pediococcus pentosaceus (3.52
µg/mL) (Table 5; Figure 7B).
Figure 7: Effect of pH and lactic acid on aflatoxin B1 reduction during LAB fermentation
of maize gruel

Sample pH (A); and Lactic acid (B) levels. Values plotted are means of triplicate analysis.
Natural fermentation (NF); Back-slopped (BS); Co-culture of Enterococcus faecium,
Lactobacillus plantarum and Lactobacillus buchneri (AS); Co- culture of Enterococcus faecium,
Lactobacillus plantarum and Lactobacillus lactis, (SR3.54)(MC); Pediococcus pentosaceus
(PP); Lactobacillus plantarum (LP); Lactobacillus casei (LC); and Lactobacillus fermentum
(LF).

74

Lactic acid production has been reported to transform AFB1 into the nontoxic aflatoxins B2a
thus reducing the effect of the toxin as reported in fermented milk products [34], and by other
authors [35]. Lactic acid and/or lactic acid bacteria metabolites have also been suggested to
confer inhibitory effects on aflatoxins [36].

3.3.6 Effect of pH and lactic acid on reduction of fumonisin B1
The role of pH and lactic acid production in the fermented samples in reduction of FB1 was
evident in this study. For example, after 24 h, of fermentation mono-cultures of LAB strains that
had the ability to lower the pH of the fermented products to less than 3.5, i.e., Pediococcus
pentosaceus and Lactobacillus plantarum, had greater ability to reduce fumonisin B1 (24%
reduction) than the rest of the mono-cultures, i.e. Lactobacillus casei and Lactobacillus
fermentum (pH > 3.5; 14.0% FB1 reduction) (Tables 3 and 4; Figure 8A). It was inferred that
production of organic acids by these organisms during fermentation could be key to observed
mycotoxins reduction (Table 5; Figure 8B).

On the other hand, based on observations made in this study, strain specificity in reduction of
fumonisins B1 toxin could not be overlooked. For instance, the pH and the lactic acid levels in
samples fermented with mixed cultures i.e. AS and MC, were statistically similar (Tables 4 and
5); however, AS had greater ability to reduce fumonisin B1 compared to MC at the end of the
fermentation period (Table 3). It was observed that AS and MC had 27 and 14% fumonisin B1
reduction, respectively, when samples were allowed to ferment for 24 h (Table 3).

This

significant difference could be related to their microbial profiles in the final phase of
75

fermentation rather than their total bacterial population or sample pH. This could also explain
why Pediococcus pentosaceus and Lactobacillus plantarum had greater ability to reduce
fumonisin B1 than Lactobacillus casei and Lactobacillus fermentum observed in this study
(Tables 3 and 4; Figure 8A). The total microbial count in samples was found to be statistically
similar after 24 h; however, the relative cell numbers of the individual strains present in AS and
MC samples at the end of the fermentation process was not determined. Since bacterial growth
is dependent on substrate and the cultural conditions therein, the antagonistic or synergistic
effects created by these culture mixes in reducing fumonisin B1 in maize-based fermenting
process might merit further investigation.

It was also observed that the back-slopping method, due to its ability to promote faster bacterial
growth and thus greater production of lactic acid in the gruel (and lower the pH of the samples)
than natural fermentation process, also seemed to have greater fumonisin reduction potential
(Tables 4 and 5; Figures 8A and B). It was observed that when back-slopped method was used,
the level of lactic acid produced after 24 h of fermentation (Table 5; Figure 8B) was significant
enough to create conditions that favoured greater reduction of fumonisin B1 (30% reduction)
than when natural fermentation was used (20% reduction) (Table 3; Figure 8B).

76

40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0

Fumonisin B1 Reduction (%)
pH

4.0
3.9
3.8
3.7
3.6
3.5
3.4
3.3
3.2
3.1
3.0

pH

A

Fumonisin B1 Reduction (%)

Figure 8: The effect of pH and lactic acid concentration on reduction of fumonisin B1

Gruel Samples

Sample pH (A); and Lactic acid (B) levels. Values plotted are means of triplicate analysis.
Natural fermentation (NF); Back-slopped (BS); Co- culture of Enterococcus faecium,
Lactobacillus plantarum and Lactobacillus buchneri (AS); Co- culture of Enterococcus faecium,
Lactobacillus plantarum and Lactobacillus lactis, (SR3.54)(MC); Pediococcus pentosaceus
(PP); Lactobacillus plantarum (LP); Lactobacillus casei (LC); and Lactobacillus fermentum
(LF).

77

3.3.7 Microbial load during LAB fermentation
Apart from pH values and lactic acid levels, microbial cell numbers of the added cultures were
also monitored to investigate the fermentation process in Tanzanian maize- based gruel samples.
Typically, an extended storage of the gruel and malt during the fermentation period allows for
bacterial populations to increase. The bacterial activity is thus associated with pleasant
organoleptic properties of fermented gruel.

Little is known about the microbial content and biodiversity of homemade maize-based gruel in
Tanzania, but it is expected to vary depending on pre- and post- harvest handling of the crop,
initial bacterial load, bacterial strains present, fermentation time, as well as the hygiene of the
person preparing it.

A pH drop and lactic acid production are some physiological changes bacterial cultures utilize to
colonize an environment. It was observed, in this study, that variations in the rate at which
different strains of bacteria tested affected the pH and lactic acid levels in the gruel samples,
especially at the end of the fermentation period (24) (Tables 4 and 5).We therefore wanted to
determine the bacterial abundance of the LAB strains tested after 24 h of fermentation to
elucidate their intimate relationship with pH and lactic acid levels in the fermented maize based
gruel sample.

The microbial levels during fermentation of maize-based gruel samples fermented at 30° C with
different LAB strains for 24 hours is shown in Table 6. The high counts of the starter cultures
9

ensured high final levels of cell counts in the medium: 10 CFU/mL. These cell concentrations

78

6

7

were above the bacterial levels required for probiotic products (10 –10 CFU), which would
ensure a sufficient number of living cells reaching the colon in order to be effective.

There was no significant difference (p<0.05) between the bacterial abundance of all the LAB
cultures tested at the beginning of the fermentation period (0 h) (Table 6). This provided a
statistically good reference for comparison purposes across the strains when pH and lactic acid
production potential were considered. However, after 24 h of fermentation, the number all the
LAB strains tested increased by about 2 log units(Table 6), with a concurrent decrease in pH
(Table 4), and increase in acidity (lactic acid levels) (Table 5) of the maize-based fermented
gruel within the same time period.

Comparison of LAB strains counts at the end of the fermentation period (24 h) indicated
significant (p<0.05) predominance of Lactobacillus plantarum (LP) with an increase of about 2.1
log units (Table 6). This suggested it was highly favoured by the fermentation conditions
(temperature, substrate, etc.). This strain also produced significantly (p<0.05) higher lactic acid
(3.95 mg/mL × 100) (Table 5) with a concurrent decrease in pH (Table 4) of the gruel samples
after 24 h, indicating Lactobacillus plantarum had better fermenting properties in maize-based
substrates than the other strains tested in this study.

Compared to the mono-culture of Lactobacillus species tested, it could not be ascertained
whether there was any antagonism between the LAB strains within the co-cultures AS
(Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri) and MC
(Enterococcus faecium, Lactobacillus plantarum and Lactobacillus lactis, SR3.54.
79

This is

because the relative abundance of the individual LAB strains was not investigated. Additionally,
there was no statistical difference in the total bacterial load of AS and MC after 24 h of
fermentation in the maize-based gruel (Table 6). These results also correlate with the observed
pH (Table 4) and lactic acid (Table 5) levels between these two cultures, after 24 h of
fermentation.

Table 6: Microbial count (log CFU/ml) during fermentation of maize based gruel
Microbial count during fermentation (log CFU/ml)

1

Bacterial strain
0h

AS

2

MC

7.26±0.00

3

7.25±0.01

Pediococcus pentosaceus (PP)

7.29±0.00

Lactobacillus plantarum (LP)

7.27±0.00

Lactobacillus casei (LC)

7.27±0.00

Lactobacillus fermentum(LF)

7.27±0.00

1

24 h

a

a

a

a

a

a

9.24±0.00
9.24±0.00
9.27±0.00

b

b

b

9.36±0.03

9.29±0.00

9.30±0.00

c

b

b

Values are means of three replicate counts

2

Commercial mixed culture of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus buchneri; (AS)
3
Commercial mixed culture of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus lactis, (SR3.54); (MC)
Means in the same column with the same superscript are not significantly different (p<0.05).

80

3.4 Discussion
In humans, aflatoxin B1 and fumonisin B1 are currently unavoidable contaminants of foods, and
among the most dangerous mycotoxins due to their carcinogenic property [38]. Highly aflatoxin
and fumonisin susceptible human foods are those cultivated in the tropics and subtropics such as
maize [38], especially in developing countries. Homemade staple diets in developing countries,
and especially those made from contaminated maize crops, for instance, fermented maize based
gruel, may therefore be a significant source of aflatoxin and fumonisin contamination [39].

Additionally, animals are also at high risk of aflatoxin and fumonisin poisoning when they
ingest feeds contaminated with some common moulds such as Aspergillus flavus, A. parasiticus
and Fusarium verticillioides [40]. Due to their carcinogenic properties, AFB1 and FB1 also
represent a greater global health risk because of their possible accumulation over time and
linkage to DNA [41]. Agronomic strategies to reduce toxic contamination, for example, the use
of transgenic crops, may have a significant impact in high exposure populations, such as the
Western and Eastern regions of Tanzania, but are resource intensive and more importantly,
unlikely to eliminate aflatoxin and fumonisins exposure. Maize is typically ground into flour for
various uses and thus must be processed before consumption. Therefore, intervention strategies
must also encompass and promote economically feasible processing methods, such as
fermentation, which have great potential of reducing mycotoxins before being ingested.

The popularity of fermented human foods, such as yoghurt and fermented gruel, will continue to
grow. This is because not only do they possess the beneficial probiotic effects of lactic acid
bacteria (LAB) strains used in the fermentation process but also pleasant organoleptic properties.
81

Lactic acid bacteria are also popular in food systems due to their Generally Recognized As Safe
(GRAS) status. Additionally, LAB strains are known to bind aflatoxins and other mycotoxins to
their surface [42], thus have the ability to reduce the bioavailability of these toxins in foods.In
this study, all the Lactobacillus spp tested were found to remove between 45 – 55% of AFB1 and
between 14–27% of FB1 in maize-based gruel fermented for 24 hours at 30° C. Our results show
that single strains can be used to remove single compounds, such as AFB1 and FB1 from maizebased gruel. Different single bacterial strains have previously been reported to bind these toxic in
significantly different amounts [43]. A 23% reduction of fumonisin was reported in South
African fermented stiff porridge obtained from whole maize meal [44]. In addition, a 13%
fumonisin B1 reduction was reported in back-slopped ogi (fermented maize thick paste popular
in West African countries) [31].

Combining LAB strains together is not only practiced when desirable organoleptic
characteristics are targeted, such as in yoghurt, but may also be used to remove several toxic
compounds together [45]. In this study, however, co-cultures, AS and MC, were tested for their
ability to remove AFB1 and FB1, in maize-based gruel fermented for 24 h. AS and MC culture
mixes differed in that AS contained a Lactobacillus buchneri strain, while MC contained a
Lactobacillus lactis strain; but both had Enterococcus faecium and Lactobacillus plantarum
strains. It was observed that AS showed greater ability to remove both AFB1 and FB1 (as was
observed with back-slopping method) than MC at the end of the fermentation period (24 h) in
maize-based gruel (Tables 2 and 3), suggesting possible strain specificity in removing these

82

toxins, as was previously observed amongst the pure strains tested (Tables 2 and 3). Other
studies have also reported a wide range of genus, species and strain specific binding capacities of
several LAB strains [46]. These results show that fermentation method influences AFB1 and FB1
removal, with the use of Pediococcus pentosaceus as a starter culture showing the least ability to
remove aflatoxin B1 and co- culture comprised of Enterococcus faecium, Lactobacillus
plantarum and Lactobacillus lactis, (SR 3.54); MC showed the

least ability to remove

fumomisin B1; while back-slopping and the use of mixed cultures containing Enterococcus
faecium, Lactobacillus plantarum strains and Lactobacillus buchneri (AS) strains had greater
ability to reduce aflatoxins B1 and fumonisins B1 in maize-based gruel fermented for 24 h.
Since removal of mycotoxins is intimately linked to bacterial cell wall and / or conversion of
mycotoxins by LAB into less toxic compounds, it seems therefore, that higher amounts of AFB1
and FB1 would be removed by higher bacterial cell counts. From our results, an increase in
bacterial count by 2.0 log (Table 6) seemed sufficient to remove significant amount of AFB1 and
FB1 in maize-based fermented gruel (Table 3), thus longer fermentation time (24 h) that allowed
for bacterial multiplication is beneficial (Table 6). Other authors [42] also reported that viable
9

cell populations greater than 1×10 CFU/mL were necessary for significant removal of AFB1.
These authors reported that approximately 10

10

CFU/mL of F. aurantiacum was capable of

removing about 80% AFB1. Similarly, other authors also observed that AFB1 was rapidly
removed by both Lactobacillus rhamnosus strain GG and L. rhamnosus strain LC-705 strains to

83

about 80% of their initial levels at 24 h [37]. Thus, cell count is directly related to toxic removal
efficiency.

As bacteria metabolize substrates during growth, they produce several by-products including
lactic acid, which reduce the pH of the product and modify its sensory properties. In this study, a
proportional relationship was established between LAB ability to reduce pH (by producing lactic
acid) and the corresponding reduction of aflatoxins B1 and FB1 in maize-based fermented gruel
(Tables 2, 3, 4 and 5; and Figures 7 and 8). These results agree with other authors who reported
that higher reduction of toxic content in yoghurt occurred at reduced pH values [24].

It has been suggested that pH may contribute to reducing mycotoxins content by transforming
mycotoxins to its less toxic compounds [24]. In this study, however, we could not determine with
100% certainty that the observed AFB1 and FB1 removal was only influenced by pH reduction,
since other factors such as higher bacteria populations after 24 hours of fermentation could also
have been involved. Additionally, inhibition of aflatoxin B1 and fumonisins B1 accumulation
could also be related to production of low-molecular-weight metabolites produced by the lactic
acid bacteria at the exponential growth phase [48]. However, pH reduction (lactic acid
production) may not be solely responsible for removal or inhibition of AFB1 and FB1 in foods.
Some authors had previously reported inhibitory effects conferred by other different metabolites
other than organic acid [36]. It would therefore be interesting to establish the association
between the levels of other metabolites produced by different LAB strains and removal of AFB1
and FB1 during the fermentation of maize based gruel.
84

3.5 Conclusion
The ability of LAB strains used in this study to reduce AFB1 and FB1 from maize-based
fermented gruel has not been reported. Factors such as LAB ability to produce lactic acid, lower
pH and increase in bacteria number were observed to influence the toxin removal process.
Significant strain differences in the production of lactic acid in fermented maize-based gruel
were noted, but this did not cause any significant differences (p<0.05) in pH values after 24 h
when the pH values of all LAB strains tested were compared (Table 4). Direct correlation
between pH and mycotoxins removal was therefore not possible. However, since pH values
ranged from 3.4 to 3.7 at 24 h, it suggested that the ability to lower pH was strain specific. This
indicated that production of lactic acid was also strain specific. A proportional relationship
between ability of LAB to produce lactic acid and lower pH of the gruel samples (Tables 4 and
5); as well as between amounts of lactic acid produced and the corresponding reduction of AFB1
and FB1 in maize based gruel was established (Figures 7B and 8B).

The removal of AFB1 and FB1 by all the LAB strains tested was also time-dependent, with
greater reduction occurring after 24 h of fermentation than after 4 h (Tables 2 and 3). This also
corresponded with the greater mycotoxin reduction observed under higher lactic acid levels
(Table 5) and higher bacterial cell counts when fermentation was allowed to proceed for 24 h
(Table 6). This implied that adequate fermentation time was beneficial in not only conferring
preservation and organoleptic properties but also had greater potential in removing AFB1 and

85

FB1 from lactic acid fermented gruel. The ability of LAB co-cultures to remove AFB1 and FB1
from maize-based gruel was also observed to be dependent on constituent strains (Table 3).

In this study it was established that fermentation of maize-based gruel by back-slopping
enhanced production of lactic acid (Table 5) and reduction of AFB1 and FB1 more than the
natural fermentation (Tables 2 and 3). Back-slopping induced faster bacterial growth, and this is
an added advantage considering that AFB1 and FB1 bind to bacteria cell walls and/ or convent
into less toxic metabolites, hence the more the bacteria present in gruel the higher the amount of
AFB1 and FB1 would be removed by 24 h (Tables 2, 3 and 6).

The results of this study indicated that back-slopping is a cost-effective and user friendly way of
removing AFB1 and FB1 in lactic acid fermented gruel. Thus, households and small-scale food
processors in Tanzania who might find it difficult to access afford and manage bacteria starter
cultures could use simple and widely acceptable fermentation methods such as back-slopping.
More investigation is still needed on the interactions between strains within the mixed LAB
cultures and pH to provide insight on how fermentation conditions can be optimized to achieve
greater mycotoxins reduction capacity of probiotic microorganisms.

86

APPENDICES

87

Appendix A: ANOVA output for LAB and fermentation time on aflatoxin B1
Table 7: ANOVA for effect of LAB fermentation on aflatoxin B1 reduction in gruel
Source

Degree
of
freedom

Sum of
Square

Mean Square

F Value

Pr (>F)

Time

2

5301.4

2650.71

3313.032

<.0001

Treatments

7

183.4

26.20

32.747

<.0001

Time × Treatments

14

223.3

15.95

19.934

<.0001

Residuals

48

38.4

0.80

Response: aflatoxin B1

88

Appendix B: ANOVA output for LAB and fermentation time on fumonisin B1
Table 8: ANOVA for effect of LAB fermentation on fumonisin B1 reduction in gruel
Degree
of
freedom

Sum of
Square

Mean Square

F Value

Pr (>F)

Time

2

0.44111

0.220556

79.4000

<.0001

Treatments

7

0.05542

0.007917

2.8500

0.01436

Time × Treatments

14

0.07667

0.005476

1.9714

0.04144

Residuals

48

0.13333

0.00277

Response: FB1

89

Appendix C: ANOVA output for LAB and fermentation time on pH levels
Table 9: ANOVA for effect of LAB fermentation on pH levels in gruel
Source

Degree of
freedom

Sum of
Square

Mean Square

F Value

Pr (>F)

Time

2

59.200

29.6000

679.4619

<.0001

Treatments

7

3.431

0.4901

11.2509

<.0001

Time × Treatments

14

3.686

0.2633

6.0443

<.0001

Residuals

48

2.091

0.0436

Response: pH

90

Appendix D: ANOVA output for LAB and fermentation time on lactic acid levels
Table 10: ANOVA for effect of LAB fermentation on lactic acid levels in gruel
Source

Degree
of
freedom

Sum of
Square

Mean Square

F Value

Pr (>F)

Time

2

111.495

55.748

163163.76

<.0001

Treatments

7

1.127

0.161

471.05

<.0001

Time × Treatments

14

1.156

0.083

241.69

<.0001

Residuals

48

0.008

0.000

Response: Lactic acid

91

Appendix E: ANOVA output for LAB and fermentation time on microbial count
Table 11: ANOVA for effect of LAB fermentation on microbial counts in gruel
Source

Degree
of
freedom

Sum of
Square

Mean
Square

F Value

Pr (>F)

Time

1

24.4505

24.4505

116460

<.0001

Treatments

5

0.0082

0.0016

7.809

0.0017

Time × Treatments

5

0.0168

0.0034

15.981

<.0001

Residuals

12

0.0025

0.0002

Response: CFU

92

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98

CHAPTER FOUR
GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
4.1 General discussion
In Tanzania, maize is a staple food for the majority of the population and is grown largely under
subsistence farming with limited inputs and infrastructure for proper handling and storage.
Heavy reliance on maize for people’s daily calorie needs – 41% of the calorie intake of
Tanzanian diets – is the biggest risk factor in Tanzania. The crop is traditionally consumed in
various forms, including as a main dish (stiff porridge), gruel (as a weaning food for children and
refreshment for adults), and a snack (both roasted and steamed).

In Tanzania, maize is grown almost in every part of the country under different environmental
conditions. There are aflatoxins and fumonisin in Tanzanian maize; little contamination is found
in the South and Southern Highlands, while a higher prevalence for aflatoxin and fumonisin
contamination occurs in the Western and Eastern zones. Yet consumers’ awareness of aflatoxin
knowledge is still very poor, simply because aflatoxin and fumonisin contamination is not
readily visible on the crop.

To protect consumers from illness associated with mycotoxins (aflatoxin and fumonisin), the
Tanzanian Bureau of Standard (TBS) has set regulatory standards for total aflatoxins in maize at
10 ppb, and for aflatoxin B1 at 5 ppb. The Tanzanian Food and Drugs Authority (TFDA) is
tasked with enforcing these standards, but real enforcement is unfortunately limited to formally
packaged foods destined for trade. The TFDA conducted aflatoxin testing for 254 maize samples
in Tanzania and found that more than 40% of all maize sampled from the Eastern (Morogoro)
99

and the Western (Shinyanga) regions had more than 5 ppb aflatoxin [24]. Since most of the
maize produced is consumed domestically, the mycotoxin-contaminated maize can easily enter
the human food chain, increasing the risk of adverse health impacts. Infants and school going
children living in Morogoro and Shinyanga regions, for example, are at higher risk of higher
aflatoxin exposure due to their relatively higher energy needs, and this exposure can cause
stunting [1].

Maize is an excellent substrate for the growth of mycotoxigenic fungi that produce highly potent
toxins, with aflatoxin B1 (AFB1) and fumonisin B1 (FB1) being the most toxic to humans. Thus,
several strategies to reduce aflatoxin and fumonisin levels in maize-based foods have been
proposed but are mostly based on the environmental conditions, proper crop storage and
agronomic practices [2, 3]. Biological control using competitive exclusion has been successfully
used to reduce aflatoxin contamination in various crops including maize [4]. The use of
competitive atoxigenic strains to out-compete toxigenic strains at the farm level [5] as well as
lactic acid bacteria (LAB) to detoxify mycotoxins have also been proposed [6, 7] .

LAB strains have GRAS status (generally recognized as safe) and when used in food
fermentation they contribute to the development of the desired sensory properties in the final
product. Fermentation – a low energy preservation process – is an old but important food
processing technology. It is attractive and affordable to poor farmers in Tanzania. LAB
fermentation may be an effective strategy to reduce mycotoxin contamination in maize-based
complementary foods in Tanzania.

100

In this study, it was observed that processing maize into traditional products, such as fermented
gruel, can significantly reduce the levels of mycotoxins in food. Both aflatoxin B1 and fumonisin
B1 decreased when LAB strains were used to ferment maize-based gruel at 30° C (Tables 2 and
3). Co-cultures of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri,
(AS), had greater ability to remove AFB1 (68.3% reduction) and FB1 (27% reduction) than cocultures of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus lactis, (SR 3.54),
(MC), (54% reduction), which only reduced AFB1 and FB1 by 54 and 14%, respectively, after
24 h at 30°C (Tables 2 and 3). This suggested that even though mycotoxin reduction might be
strain specific, elimination of mycotoxins in naturally contaminated maize is possible using LAB
fermentation.

Overall, the results suggested that back-slopping (which removed 68% of AFB1 and 30% of FB1
within 24 h) was the most feasible and effective method to detoxify mycotoxins in homemade
foods such as maize-based fermented gruel.
Production of lactic acid (and reduction of pH of samples) by LAB, higher bacterial cell counts
and longer fermentation time (24 h) were considered significant factors in AFB1 and FB1
removal in the fermented gruel. Reduction of mycotoxins was more substantial after 24 h of
fermentation, when acidity was lower. Careful selection of LAB strains is therefore critical. In
this study, the co-culture comprising of Enterococcus faecium, Lactobacillus plantarum and
Lactobacillus buchneri showed the best ability to remove mycotoxins in fermented gruel.

101

The ability of lactic acid bacteria to detoxify mycotoxins has previously been reported [8, 9].
The use of this strategy in reducing mycotoxins from naturally contaminated maize is thus quite
promising. The mechanisms of mycotoxin biosynthesis inhibition by lactic acid bacteria have
been reported as strain dependent and could involve the binding of mycotoxin to the cell wall of
bacteria and/or conversion of mycotoxin into less/non-toxic derivatives [10, 11]. More
investigation is still needed on how the LAB strains used in this study and pH work together to
reduce mycotoxin levels in maize-based substrates. This would provide insight on how
fermentation conditions could be optimized to achieve greater mycotoxin reduction capacity of
probiotic microorganisms.

4.2 Conclusion
Mycotoxin (especially AFB1 and FB1) contamination has become a global health issue
especially in sub-Saharan African countries and the consumption of these toxins has been
reported to cause acute and chronic effects in animals and human beings. The results of this
study indicated that lactic acid fermentation could be a part of a comprehensive mycotoxicosis
prevention strategy that can help detoxify the commonly consumed maize-based gruels in
Tanzania. Consumers could benefit from enhanced food safety, through consumption of gruel
less contaminated with mycotoxins and, might in addition, benefit from the probiotic effects of
LAB. In the scenario where LAB starter culture access and handling could prove challenging
especially at household level and to small-scale food processors, the use of back-slopping in
gruel fermentation might be advocated in order to reduce mycotoxins. However, further research
is still required to elucidate whether the mycotoxins apparently eliminated are completely
destroyed, bio-degraded or actually complexed with bacteria to non-toxic forms.
102

4.3 Recommendations
In Tanzania, chronic exposure to mycotoxins can be reduced but it will require a multidisciplinary approach, including the collaboration between the agriculture and public health
sectors, political will as well as commitment of sufficient resources by the national government.

The results in this study indicated that acute mycotoxicosis is preventable. The use of LAB
starter cultures for controlled fermentation and, promotion of traditional fermentation techniques
such as back-slopping should be encouraged. It is proposed that complementary food processors
should use fermented rather than the unfermented gruel as one of the practical ways of reducing
the risks of aflatoxicosis.

Further investigations are required on the binding mechanisms and possible stability of the
bacteria-toxin complex in vivo. This will help assess any possible adherence properties of the
bacteria-toxin complex on to the intestinal mucosa. In addition, the metabolic derivatives of
AFB1 and FB1 should be characterized and the toxicity of these metabolic derivatives needs to
be assessed. Since mycotoxin reduction by LAB could be strain specific, development of LAB
strains with greater mycotoxin reduction activity need to be explored.

This study demonstrated the ability of some strains of lactic acid bacteria to reduce the initial
concentration of AFB1 and FB1 in maize-based complementary food. However, this intervention
strategy should be rigorously tested and validated using clinical trials designed with biomarkers
serving as object end points of efficacy. The examination can be conducted by determining the
changes in blood and urinary levels of aflatoxin B1 N7-Guanine (AFB1-N7-guanine) adducts
103

and urinary fumonisin B1 (UFB1) as biomarkers of aflatoxin and fumonisin exposure,
respectively. The effects of detoxification on nutritive and sensory properties also need to be
investigated.

104

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Shetty, P. H., & Jespersen, L. (2006). Saccharomyces cerevisiae and lactic acid bacteria
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