Masked Mycotoxins

Masked Mycotoxins
Susan MacDonald
UK NRL Mycotoxins
Food Quality and Safety Programme
Overview
• What are Masked Mycotoxins
• Formation
• Toxicity
• Analysis
• Occurrence
• Future needs / conclusions
Berthiller, F., Crews, C., Dall'Asta, C.,
Saeger, S. D., Haesaert, G., Karlovsky,
P., Oswald, I. P., Seefelder, W., Speijers,
G. and Stroka, J. (2013),
Masked mycotoxins: A review. Mol. Nutr.
Food Res., 57: 165–186. doi:
0.1002/mnfr.201100764
Mycotoxins
• Low molecular weight, toxic, secondary metabolites
of moulds, >400 known
• Fusarium, Aspergillus, Penicillium, Alternaria,
Claviceps sp. Most important for food/feed crops
• Annual losses estimated several hundred million tons
of food worldwide (FAO - possibly 25% food supply affected)
• Regulations for their control in food and feed exist in
>100 countries
• BUT still a lot is unknown – synergistic effects, long
term exposure
What are Masked Mycotoxins?
• Bound, conjugated, hidden versions of parent toxin
• Bound – covalently bound to complex carbohydrates, cellulose,
proteins, can be produced during food processing
• Conjugated – addition of glucoside, sulphate, amino acid,
generally as result of plant detoxification mechanism
• Hidden – complexation or physical entrapment by
macromolecules
• Why is this a problem?
Masked mycotoxins may elude analysis leading to underestimates
of exposure
• First described in 1980s, Gareis named ZENGlucoside ‘masked mycotoxin’ to emphasise it was not
found by normal method but probably contributed to overall toxicity
Toxicity
• Conjugation = detoxification, so masked forms
less acutely toxic? YES and NO!
• Bioavailability of conjugated forms may be
different to parent.
• ZEN14G converted to ZEN in pigs (and humans?)
• No specific studies for fumonisins but potentially
poor bioavailability could be increased.
• OTA metabolites – if present in food could have
increased availability and add to toxic effect.
Toxicity
• D3G resistant to acid hydrolysis and human
glucosidase but released DON in presence of
gut bacteria such as Enterococcus and
Lactobacillus (gut microbiota)
F. Berthiller, R. Krska, K.J. Domig, W. Kneifel, N. Juge, R. Schuhmacher, G. Adam (2011)
Hydrolytic fate of deoxynivalenol-3-glucoside during digestion. Tox. Lett.206, 264– 267.
• D3G released DON in presence of human gut
microbiota from 5 volunteers, microbiota from
one volunteer also converted DON to DOM-1
S.W. Gratz, G. Duncan, A.J. Richardson (2013).The Human Fecal Microbiota Metabolizes
Deoxynivalenol and Deoxynivalenol-3-Glucoside and May Be Responsible for Urinary DeepoxyDeoxynivalenol, Appl. Environ. Microbiol. March 2013 vol. 79 no. 6 1821-1825
Possible increased bioavailability and partial
reactivation during digestion mean masked mycotoxins
are potential health threat to mammals
Analysis of masked mycotoxins
• Mycotoxins quantified usually by either
chromatographic or immuno-analytical methods
• Traditionally these are targeted and specific
• indirect methods for masked mycotoxins:
- enzymatic hydrolysis to parent toxin
- acidic/alkaline hydrolysis
- efficiency of hydrolysis difficult to assess
- can be done without standards – by difference or
equivalence to parent.
• direct methods:
- standards
required
Special issue: Masked mycotoxins, Volume 5, Number 3 / August 2012
- no immuno-analytical methods available
- differentiation between forms possible
Analytical issues
• Traditional methods can underestimate total
potential toxin in samples. Dall’Asta et al
demonstrated CRM contained ca 8000µg/kg fumonisins
(certified value is ca 3000µg/kg)
• Methods can overestimate. Cross reaction
can cause overestimation in ELISAs.
• Indirect methods may miss some forms.
• Standards not available.
• Reference materials, interlaboratory validated
methods and PTs not available.
Dall’Asta et al., 2009, Anal. Bioanal Chem, 395:1335-1345
LC-MS/MS Multi
Mycotoxin Method
Chromatography Conditions
Chromatograph &
Autosampler
Column
Acquity UPLC
Injection volume
1 µL (neutral) / 2 µl (acidic)
Acquity HSS T3 1.8 µm
(100 x 2.1 mm)
Mass Spectrometry Conditions
Mass spectrometer
Xevo TQ-S triple quadrupole
Ionisation mode
Electrospray (ES) polarity switching
positive / negative
3 kV (ES+) / 1 kV (ES-)
Capillary voltage
DON-3-glucoside in wheat
All results µg/kg
Sample
ID
DON
DON-3-G
(%DON)
NIV
EB
EB1
EA1
BEA
917
3020
300 (10)
180
59
130
30
180
918
2530
160 (6)
800
1630
380
400
919
5050
300 (6)
35
335
211
21
41
920
4390
159 (4)
9230
137
208
38
921
1360
341
26700
19600
2000
922
11600
900 (8)
150
280
170
923
1320
150 (11)
212
628
140
924
12
20
*Also contained alpha and beta-ZOL
MON
ZON
55*
300*
2450
14
480*
70
115*
Mycotoxins in maize samples
DON
Ac-DON
DON-3-G
Ethanol/DDGS production flow chart
• Analyzed samples:
•
•
5 batches of commercial ethanol/DDGS production
Naturally contaminated samples of input corn
ICT Prague RESULTS
•
•
Date of samples collection: June 2012 – February 2013
Positively detected mycotoxins: DON, D3G, FB1,
Enniatins, BEA, ZON, α-ZOL, β-ZOL
Concentration of mycotoxins [µg/kg]
Analyzed material
Batch No. 1
Starting material
After distillation
After centrifugation
and pre-concentration
of thin stillage
Final product
Batch No. 2
DON
D3G
FB1
DON
corn
milled corn
686
787
114
138
43
45
410
412
whole stillage
3200
710
312
1902
syrup
7232
1368
373
4573
WDGS
561
98
71
642
DDGS
724
117
80
435
ICT Prague RESULTS: Batch No.1
Re-calculated results, 3fold matrix pre-concentration
32/31/56%
100%
Corn
Saccharification & Fermentation
Products
ICT Prague CONCLUSIONS
•
Only Fusarium mycotoxins DON, D3G and FB1 were detected
in all intermediates and final DDGS (57 toxins tested).
•
Significant increase of all three analytes during the first phase
of fermentation occurred (the release of toxins from starch matrix
caused by enzymatic activity).
•
Decrease of concentrations within the second part of
fermentation process occurred (likely caused by yeast activity).
•
Further and significant decrease of mycotoxins during the
final DDGS drying occurred (possible thermal degradation of
compounds due to high drying temperatures 400–500°C).
Occurrence in food
• Few data collected
• Several studies report D3G in beer at equal (or
higher!) levels than DON
• D3G, ZEN-14-glucoside and ZEN-14-sulphate natural
occurrence reported in cereals and cereal products
• Hidden fumonisins reported in maize and maize
products
• Masked (glucosides) NIV, T-2 and HT-2 toxin, FUS-X
reported in literature. OTA in cell culture. Others?
ILSI review concluded more data required
to allow risk assessments
Request to EFSA - Metabolites
and masked toxins
• A scientific opinion on the risks to animal and human
health related to the presence of metabolites and the
masked or bound forms of fumonisins, zearalenone, T-2
and HT-2 toxin and nivalenol in food and feed.
(DON/D3G included in separate opinion – DON comprehensive)
•
•
•
•
•
•
Toxicity compared to parent
Co-occurrence in food and feed
Estimation of dietary exposure EU population, inc vulnerable
groups and compared to parent toxins.
Assessment of exposure in different animal species
Assessment of human health risk for the EU population as
consequence of estimated dietary exposure to the
metabolites/masked/bound forms and parent mycotoxins
Assessment of animal health risk
UK Project on Masked Mycotoxins
• Funded by UK Food Standards Agency
Collaborators:
Fera and University of Aberdeen, Rowett Research Centre
• Aims
- to conduct survey of UK retail cereal products for masked mycotoxins.
(Results will be submitted to EFSA)
- in-vitro hydrolysis of masked mycotoxins using human gut microbiota
- identify metabolites / products of hydrolysis experiments
Conclusions
• ‘Hot topic’ in mycotoxin research
• Some progress in methods, but lack of validated
methods
• Lack of analytical standards and reference materials
impedes routine analysis
• Toxicological significance not fully understood
• Not currently taken into account by Regulators
• EFSA opinion on masked mycotoxins imminent conclusions will inform some next steps
Thank You
• Colleagues at Fera:
Julie Clough, Simon Hird, Philip Jennings, Jennifer Leak, Irene
Leon, Monika Sehnalova, Joanna Stratton
• Colleagues at VSCHT ICT Prague:
Jana Hajslova, Monika Tomaniova, Z. Dzuman, M. Vaclavikova
• Franz Berthiller and Rudi Krska
(IFA Tulln, Austria)
Thank you for listening.

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