12-14 September 2017. Ghent, Belgium. 1st MYCOKEY International Conference. Presentation by: Adekoya Ifeoluwa*, Njobeh P.B., Obadina A.O., Chilaka A.C., Okoth S., De Boevre M., and De Saeger S. *Department of Biotechnology and Food Technology, University of Johannesburg, South Africa

1. MULTI-MYCOTOXIN CONTAMINATION IN SELECTED FERMENTED
FOODS AND THE PERCEPTION OF MYCOTOXIN CONTAMINATION
IN NIGERIA MARKETS
Adekoya Ifeoluwa*, Njobeh P.B., Obadina A.O., Chilaka A.C.,Okoth
S., De Boevre M., and De Saeger S.
*Department of Biotechnology and Food Technology, University of
Johannesburg, South Africa
Food Safety:
Everybody’s Business

2. • Introduction
– Food Processing
– Fermentation
– Mycotoxins
– Objectives
• Methodology
• Results
• Conclusion and
recommendations
2

3. FOOD PROCESSING
BENEFITS
Reduced postharvest losses
Income Generation
Value Addition
Improved safety
Improved nutrition, etc.
3
Food Processing Technologies
Simple Sophisticated
Fermentation

4. FERMENTATION
4
• Fermentation: Oxidation of carbohydrates
through microbial action
• Low cost processing technology widely adopted
in Africa
• Types of fermentation and microorganisms
involved
• Fermented Foods: fufu, kenkey, ogi, iru, etc.
• Production: manufactured mostly in homes
under spontaneous conditions with different
methods.
THEIR SAFETY REMAINS A

5. MYCOTOXINS
5
• Toxic secondary fungal metabolites
• 25% of the global food output is
contaminated by mycotoxins, causing
significant economic losses (Ostry, 2017)
• Hazardous to human and animal health
• Examples: fumonisins, aflatoxins
• Occurrence has been documented in
commodities worldwide including
fermented foods

6. • Strategies for reduction:
Awareness creation and
enlightenment of
people on mycotoxins
6
Table 1: Occurrence of mycotoxins in some African foods
Mycotoxins Food Category Country Authors
Aflatoxins, fumonisin B1,
ochratoxin A,
deoxynivalenol,
zearalenone
Maize, beans, soybeans,
peanut, rice
Cameroon Njobeh et al. (2010)
Fumonisins, aflatoxins, etc Maize, groundnut,
sorghum, rice
Nigeria Adetunji et al. (2017); Chilaka et al. (2016);
Ezekiel et al. (2014); Makun et al. (2011)
Aflatoxins, fumonisins Sorghum, traditional beer,
maize
Malawi Matumba et al. (2014); (2015)
Aflatoxins, fumonisins,
ochratoxin A,
deoxynivalenol,
zearalenone, etc.
Maize Zimbabwe Hove et al. (2016)
Aflatoxins Maize Kenya Okoth et al. (2012), Lewis (2005)
Fumonisins Maize, beer South Africa Phoku et al. (2012), Shephard et al. (2004)

7. OBJECTIVES
• Selected Fermented Foods: fermented African oil bean seed
(ugba), fermented locust beans (iru), fermented melon
(ogiri), fermented maize gruel (ogi) and fermented sorghum
gruel (ogibaba)
• Objectives: To determine the occurrence of multiple
mycotoxins in these fermented foods in order to access their
safety.
• Investigate practices, understanding and perceived health
risk of fungal and mycotoxin contamination amongst their
sellers
7

8. METHODOLOGY
• Awareness studies: A descriptive cross-sectional study was carried out
amongst fermented food sellers (n=86) in Feburary 2015 using a
questionnaire.
• Location: South-West Nigeria
• Sampling: ogi (n=35), iru (n=60),
ogibaba (n=35), ugba (n=30), ogiri (n=31)
Period: Feburary 2015 – May 2016
Total: 191 samples
• Storage: -18oC
• Mycotoxin analysis: Laboratory of Food
Analysis, Ghent University, Belgium.
88

9. 9
Mycotoxin Analysis
• All reagents and chemicals were of analytical grade.
• Sample Preparation
5g of sample +
Internal standards
Calibration curve
construction
Extraction (1 hr)
(ACN/CH3COOH/H2O)
79/1/20
Defatting (2x)
N-Hexane
SPE
Purification
Centrifugation
Filtration and
Purification
(Multisep 226
Columns)
Evaporation and
redissolution and
filtration
LC-MS/MS
Analysis
Fig 1. Flowchart for sample preparation for mycotoxin analysis

10. 10
Mycotoxin Analysis
• 23 Mycotoxins: AFB1, AFB2, AFG1, AFG2, FB1, FB2, FB3, DON, 15-ADON, 3-
ADON, NEO, OTA, AOH, AME, ZEA, NIV, DON, STE, ROQ C, ENN B, FUS-X, HT-
2, T-2, and DAS.
• Identification and quantification: Waters Acquity UPLC apparatus + Quattro
Premier XE Tandem Mass Spectrometer. Identification criteria (EC, 2006)
• Method validation: limit of quantification, limit of detection and apparent
recovery (EC, 2006)
• Data analysis: Descriptive statistics (mean, range, frequencies, and
percentages). Kendall’s tau-b test for correlation of the degree of awareness
of fungal and mycotoxin contamination amongst the fermented food sellers
and their education level.

11. RESULTS
11

12. 12
Awareness Studies
Parameters Incidence (%) Parameters Incidence (%) Parameters Incidence (%)
Sociodemographic Variables
Gender
Male
Female
6 (7)
80 (93)
Education level
None
Primary
Secondary
Tertiary
9 (11)
52 (61)
23 (27)
2 (2)
Age
<30 years
31-50 years
>50 years
6 (7)
74 (86)
6 (7)
Fermented Food
Characteristics
Mode of consumption
Direct consumption
Food Ingredient
Both
28 (33)
46 (54)
12 (14)
Food type
Ogi
Iru
Ogiri
Ugba
28 (33)
21 (24)
19 (22)
18 (21)
Food source
Home processed
Market
Processors
4 (5)
32 (37)
50 (58)
Storage Variables
Storage method of raw
materials
Bags
Containers
Not applicable
13 (15)
5 (6)
68 (79)
Storage duration of raw
materials
1-3 months
>3 months
Not applicable
13 (15)
5 (6)
68 (79)
Average shelf life of
raw material
1-4 weeks
>4 weeks
Not applicable
1 (1)
17 (20)
68 (79)
Storage method of finished
product
Polyethylene bags
Containers
Paper
Leaves
Wooden Boxes
49 (57)
14 (16)
3 (4)
17 (20)
3 (4)
Storage duration of
finished product
1-7 days
>7 days
86 (100)
-
Average shelf life of
finished product
1-3 days
3 -7 days
> 7 days
14 (16)
42 (49)
30 (35)
Table 2. Descriptive statistics and knowledge of fungal and mycotoxin contamination amongst
fermented food sellers (n=86)

13. 13
Table 2 (Contd.). Descriptive statistics and knowledge of fungal and mycotoxin contamination
amongst fermented food sellers (n=86)
Parameters Incidence (%) Parameters Incidence (%) Parameters Incidence (%)
Knowledge of Fungi and
Mycotoxins
Knowledge of fungi
Yes
No
Not sure
63 (73)
16 (19)
7 (8)
Identification of fungal
contamination in food
Yes
No
59 (68)
27(32)
Frequency of
contamination
Rarely
Frequently
Not applicable
36 (42)
19 (22)
31 (36)
Perception of reasons of fungi
occurrence
Storage
Bad raw materials
Insect infestation
All of the Above
Not sure
21 (24)
19 (22)
18 (21)
21 (24)
7 (8)
Knowledge of health risk
associated with fungal
contamination
Yes
No
7 (8)
79 (92)
Knowledge of
production of toxins
by fungi
Yes
No
3 (4)
83 (96)
Knowledge of mycotoxin
contamination
Yes
No
2 (2)
84 (98)
Willingness to attend
training on mycotoxin
mitigation
Yes
No
83 (97)
3 (3)

14. 14
Table 3. Kendall’s tau-b correlation between education and awareness level of fungi and mycotoxins
amongst respondents
Correlations Level of
Education
Do you
know what
fungi is
Can you
identify
food with
fungi
Does fungi
contamination
of foodstuffs
cause health
problems
Do you
know fungi
produce
toxins
Have you
heard of
mycotoxin
contamination
Level of Education R 1.000 .296** -.172 .014 .048 .308**
Sig. (2-tailed) . .003 .084 .888 .643 .003
N 86 86 86 86 86 86
Do you know what
fungi is
R .296** 1.000 .355** .249* .075 -.139
Sig. (2-tailed) .003 . .000 .015 .471 .186
N 86 86 86 86 86 86
Can you identify food
with fungi
R -.172 .355** 1.000 .069 .190 .100
Sig. (2-tailed) .084 .000 . .495 .064 .332
N 86 86 86 86 86 86
Does fungi
contamination of
foodstuffs cause health
problems
R .014 .249* .069 1.000 .122 .221*
Sig. (2-tailed) .888 .015 .495 . .240 .035
N 86 86 86 86 86 86
Do you know fungi
produce toxins
R .048 .075 .190 .122 1.000 .109
Sig. (2-tailed) .643 .471 .064 .240 . .307
N 86 86 86 86 86 86
Have you heard of
mycotoxin
contamination
R .308** -.139 .100 .221* .109 1.000
Sig. (2-tailed) .003 .186 .332 .035 .307 .
N 86 86 86 86 86 86

15. 15
Table 4. Method performance parameters of the fermented food matrixes
Mycotoxins Calibration range
µg/kg
Fermented Melon
(ogiri)
Fermented Locust
bean (iru)
Fermented African Oil
Bean (ugba)
Fermented Maize
Gruel (ogi)
Fermented Sorghum
Gruel (ogi baba)
LOD
(µg/kg)
LOQ
(µg/kg)
AR
(%)
LOD
(µg/kg)
LOQ
(µg/kg)
AR
(%)
LOD
(µg/kg)
LOQ
(µg/kg)
AR
(%)
LOD
(µg/kg)
LOQ
(µg/kg)
AR
(%)
LOD
(µg/kg)
LOQ
(µg/kg)
AR
(%)
Deoxynivalenol 200-800 11 22 100 4.9 9.8 99 15 30 101 7 14 97 12 24 101
Nivalenol 100-400 48 96 100 11 22 100 21 42 103 35 70 101 87 175 99
Neosolaniol 50-200 20 40 95 16 32 99 24 48 96 2.2 4.4 103 3.0 6.0 100
Fusarenon-X 100-400 39 78 97 8.1 16 101 25 50 96 21 42 100 45 90 100
3-Acetyldeoxynivalenol 25-100 2.3 4.6 96 2.0 4.0 102 1.2 2.4 101 5.0 10 105 12 24 97
15-Acetyldeoxynivalenol 12.5-50 1.7 3.5 94 3.9 7.9 101 1.8 3.7 96 10 20 95 7.0 14 99
Aflatoxin B1 10-40 2.0 4.0 96 1.2 3.3 96 1.5 3.0 100 3.8 7.5 100 5.0 10 100
Aflatoxin B2 10-40 2.3 4.6 96 1.8 3.3 94 1.4 2.8 96 1.8 3.5 99 2.5 5.0 102
Aflatoxin G1 10-40 3.9 7.8 99 1.7 3.3 95 1.9 3.9 98 1.8 3.5 98 2.5 5.0 101
Aflatoxin G2 10-40 3.7 7.4 96 1.2 2.3 91 2.2 4.4 94 3.8 7.5 100 5.0 10 99
Diacetoxyscirpenol 2.5-10 0.9 1.8 97 0.7 1.4 97 1.0 2.0 89 0.3 0.6 99 0.5 1.0 94
Alternariol 50-200 6.5 13 98 9.7 20 100 5.9 11 98 40 80 92 40 80 99
Alternariol Methyl Ether 100-400 54 107 96 5.0 10 96 4.6 9.2 98 5.0 10 109 6.3 12 96
HT-2 Toxin 50-200 6.5 13 98 7.4 14 98 15 30 94 6.5 13 85 6.5 13 95
T-2 Toxin 50-200 12 24 98 14 28 94 13 26 100 3.6 7.2 87 8.0 16 94
Fumonisin B1 200-800 24 48 97 22 44 100 38 76 97 8.2 16 87 10 20 98
Fumonisin B2 200-800 11 22 99 9.4 18 99 43 87 95 12 23 89 11 22 100
Fumonisin B3 25-100 13 26 97 21 42 97 33 66 94 14 28 89 14 28 96
Ochratoxin A 25-100 11 22 89 1.2 2.4 93 3.6 7.2 90 1.5 3.0 99 2.5 5.0 95
Sterigmatocystin 25-100 5.5 11 100 1.7 3.3 97 1.9 3.8 95 1.3 2.5 100 2.5 5.0 101
Roquefortine C 5-20 4.9 9.7 101 1.2 2.3 99 1.0 2.0 99 4.0 8.0 97 6.0 12 98
Zearalenone 50-200 9.8 20 96 2.9 5.9 92 4.4 8.8 104 3.3 6.5 102 3.8 7.6 93
Enniatin B 40-160 26 52 93 6.4 13 94 5.6 11 99 6.3 12 82 7.9 16 91

16. 1616
Table 4. Contamination of mycotoxins in fermented foods from South West, Nigeria
Mycotoxins Ogiri (N=31) Iru (N=60) Ugba (N=30) Ogi (N=35) Ogi baba ( N=35)
% +ve
samples
Range % +ve
samples
Range % +ve
samples
Range Mean % +ve
samples
Range Mean % +ve
samples
Range
Deoxynivalenol 3 (10) 20-54 4 (7) 7-118 4 (13) 36-38 37 4 (11) 8-55 32 3 (9) 32-112
Nivalenol - - - - - - - - <LOD - 3 (9) <LOD
Neosolaniol 1 (3) - - - - - - - - - -
Fusarenon-X - - 3 (5) 40-76 - - - - - - - -
3-Acetyl-DON - - 1 (2) - - - - - - - - -
15-Acetyl-DON - - - - - - - - - - - -
Aflatoxin B1 15 (48) 3-4 1 (2) - 6 (20) 3-36 20 8 (23) 4-17 6 4 (11) 9-24
Aflatoxin B2 5 (16) 3-4 1 (2) - 1 (3) - 2 7 (20) 2-7 5 - -
Aflatoxin G1 - - 2 (3) 8-8 - - - 2 (6) 1-2 1 1 (3) -
Aflatoxin G2 2 (7) 5-6 3 (5) 2-6 - - - - - - - -
ƩAflatoxins 17 (55) 3-12 7 (12) 2-8 7 (23) 3-36 18 9 (26) 2-17 5 4 (11) 6-40
Diacetoxyscirpenol 1 (3) - - - - - 0 - - - 4 (11) 1-2
Alternariol - - - - - - 0 3 (9) 40-75 25 - -
A- Methyl Ether 6 (19) 64-153 6 (10) 19-77 4 (13) 25-193 109 - - - 8 (23) 30-35
HT-2 Toxin 4 (13) 18-35 9 (15) 17-51 1 (3) - 17 3 (9) 20-21 21 - -
T-2 Toxin - - 7 (12) 19-31 1 (3) - 15 - - - - -
Fumonisin B1 - - 4 (7) 61-167 - - - 25 (71) 68-2492 384 14 (40) 16-68
Fumonisin B2 - - 4 (7) 32-42 - - - 23 (66) 94-659 250 9 (25) 15-65
Fumonisin B3 - - 4 (7) 76-89 - - - 18 (51) 42-404 112 26 (74) 20-42
Ʃ Fumonisin B1, B2 - - 8 (13) 32-167 - - - 25 (71) 68-3151 645 18 (52) 15-129
Ʃ Fumonisin B1, B2, B3 - - 12 (20) 32-167 - - -- 27 (77) 42-3555 672 29 (83) 15-168
Ochratoxin A 6 (19) 16-27 7 (12) 2-21 1 (3) - 9 - - - 2 (6) 5-6
Sterigmatocystin 6 (19) 6-9 - - 2 (7) 22 2 10 (29) 4-7 4 8 (23) 4-9
Roquefortine C - - 2 (3) 10-14 - - - - - - - -
Zearalenone 8 (25) 21-45 5 (8) 11-33 4 (13) 39-117 72 3 (9) 4-6 5 - -
Enniatin B - - - - 4 (13) 6-6 6 5 (14) 6-14 7 - -

17. 1717
Figure 2. Percentage of co-occurrence of mycotoxins in fermented foods from South West,
Nigeria
DON + ZEA
*152 Positive samples

18. Potential Contamination Sources
• Carry-over from the raw materials
• Fungal contamination of the fermented foods (Adekoya et al. 2017)
• Unacceptable trade activities by processors and sellers e.g mixing
mouldy raw material/ finished products with high-quality products
to maximise profit
• Unhygienic practices/processing environments
18
• Spontaneity of fermentation process
• Post processing contamination (Fermentation: mycotoxin reduction
(Okeke et al. 2015; Karlvosky et al. 2016)
• Improper storage practices
• Poor food safety knowledge

19. CONCLUSION
• This study gave an insight into the safety of some Nigerian fermented
foods and established the awareness of the sellers towards fungal and
mycotoxin contamination.
• There exists a wide knowledge gap amongst participants on this aspect
of food safety.
• 82% of the samples had mycotoxins occurring singly or in combination
though relatively at low incidence and contamination levels.
• Ogi was the most contaminated sample (94%) and had the highest no
of co-occuring metabolites (> risk of mycotoxin exposure)
• Some of the samples exceeded the maximum limit for FB (ogi [92%]),
AF (ugba [20%]), OTA (ogiri [16%]) and ZEA (ugba [13%]) in foods as
regulated by the EC.
19

20. Going Forward………………………..SSA
AWARENESS
IMPLEMENTATION
OF MONITORING
PROGRAMS FROM
FARM TO FORK
DEVELOPMENT OF
INTERVENTION
STRATEGIES &
IMPLEMENTION OF
EXISTING ONES
ENCOURAGEMENT
OF
DIETARY DIVERSITY
20

21. Let’s Remember………………………..
Every time we eat or drink we are either feeding
disease or fighting it. So, either we are
mycotoxin experts/researchers or not……………
21
FOOD SAFETY IS EVERYONES RESPONSIBILITY

22. ACKNOWLEDGMENT
Laboratory of Food Analysis, Ghent University, Belgium
Food, Environment and Health Research Group,
University of Johannesburg, South Africa
African Women in Agricultural Research and
Development (AWARD), Kenya
Organizing Committee of the MYCOKEY Conference and the
Organization for Women in Science in the Developing World
(OWSD), Italy
22
RESEARCH
CONFERENCE ATTENDANCE

23. 23
THANKS FOR YOUR RAPT ATTENTION