http://www.jrc.ec.europa.eu/
Joint Research Centre (JRC)
Fusarium-toxin Forum of 1 - 2 February 2010 1
Analytical Aspects of Mycotoxin Binders
Anna Kolossova & Joerg Stroka
Fusarium-toxin Forum of 1 - 2 February 2010 2
Overview
Legal background
Type of Binders (structure)
Binders available to test in the study
Design/outline of study and evaluation
Analytical methods used
Statistical evaluation
Conclusion
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Legislation first…
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Some background
My first contact with “binders”…
Humans in Ghana
Macaws in Peru
Munn, C.A., 1994. Macaws: winged rainbows. Natl. Geograph. 185:118-140.
TD Phillips is working with Don Brightsmith, Head of Rainforest Expeditions, to determine the reason for geophagy in Macaws in Peru.
http://www.inkanatura.com/macawclaylicks.asp
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What has research been focussed on?
Animal health (in vivo)
Metabolic indicators (in vivo)
Cell culture studies (in vitro)
Isotherm studies (analysis of suspensions)
Effects on analysis????
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Methods for prevention and decontamination of mycotoxins
Prevention of mycotoxin contaminationPre- and post-harvest control and harvest management
Codex Alimentarius – codes of practice for the prevention and reduction of mycotoxins in cereals, peanuts, apple products, raw materialsThe use of HACCP – to consider in the futureAlthough the prevention of mycotoxin contamination in the field is the main goal of agricultural and food industries, under certain environmental conditions the contamination of various commodities with mycotoxins is unavoidable
Detoxification of mycotoxinsRemoval from contaminated commoditiesInactivation of mycotoxins by physical, chemical or biological methodsNeither the use of chemical decontamination processes, nor the mixing of batches with the aim of decreasing the level of contamination below the maximum tolerable level are legal within the EU
Mycotoxins can not be entirely eliminated
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Mycotoxin binders
A mycotoxin binder (adsorbent/denaturant) added to the diet should be able to interact in vivo with mycotoxins and restrict their absorption in the GI tract of animals
Several requirements for a mycotoxin binder
Effective inactivation of mycotoxins of interestReduction of mycotoxin bioavailability/activityAvailability of essential nutrients to an animalNot being detrimental to animal or food productNot being a growth promoter (growth promotion may mask mycotoxins)Demonstrate verifiable positive results (improvement of zootechnicalperformance; recovery of organ status; excretion of mycotoxins via faeces; recovery of the immune status)Technical and economical feasibility
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Types of Mycotoxin Binders
AdsorbentsInorganic
Activated charcoalAluminosilicates, clays
HSCAS, bentonite, montmorillonite, zeolite, clinoptilolite…Clay minerals – layered silicates [Si2O5
2-]xyZeolite – silicate made of tetrahedrons of SiO4 and AlO4
Chemically treated silicates
OrganicChemical polymers (cholestyramine, polyvinylpyrrolidone, divinylbenzene-styrene polymers…) High costsGlucan products
Yeast and products from yeast (yeast cell wall)
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Types of Mycotoxin Binders
DenaturantMycotoxin-degrading enzymesLife microorganisms producing mycotoxin-degrading enzymes
Eubacterium sp. (BBSH 797)T. mycotoxinivorans
Organic bindersEfficient against a larger range of mycotoxins than inorganic bindersMore adapted to the most frequent cases of multi-contaminated feedsBiodegradable and do not accumulate in the environment after being excreted by animals
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Oltipraz a “Mycotoxin Binder”?
http://phm.utoronto.ca/~jeffh/jnppres.ppt
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Level of Interest for Testing Mycotoxin Binders
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Level of Interest for Testing Mycotoxin Binders
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Analytical aspects
Cascade of modern analytical Methods:
– Extraction
– Clean-up / Dilution
– Separation
– Detection
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Methods used for testing
Official Methods (or those in standardization process):
• Aflatoxin B1 EN ISO 17375:2006
• Deoxynivalenol prEN 15791:2009
• Zearalenone prEN 15792:2009
• Ochratoxin A WD N954 of TC327/WG1
• Fumonisins WD N953 of TC327/WG1
• T2 and HT2 toxins Breidbach et.al. under consideration as a prospective CEN standard.
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Repeatability Data of the Test Methods
4.1OTA
3.2FUM B1 + B2
9.8T2 & HT2 toxins
7.7ZEA
6.9AFB1
10.6DON
Average RSDr (%)Mycotoxin
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Tested Binders in this Study
2.55.1Fibres (lignocellulose)20
0.59.8HSCAS19
0.59.8HSCAS18
0.58.8HSCAS17
0.410.6Montmorillonite16
0.410.4Montmorillonite15
16.7Fibres14
18.9Clay13
0.59.1Clay12
0.256.9Mixture organic + mineral component11
0.258.6Mixture organic + mineral component10
0.25.0Yeast cell wall9
0.256.2Mixture organic + mineral8
0.254.7Yeast cell wall7
0.26.7Yeast cell wall6
0.26.2Yeast cell wall5
0.254.6Clay4
0.18.4Clay3
0.17.9Clay + organic acid2
0.22.9Yeast cell wall1
Amount added [%]pH valueType of main product(s)Code
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Some Statistics
Data analysisStudent’s t-test (independent two-sample t-test with unequal sample sizes and equal variance). Differences were assumed to be significant at p < 0.05.
Critical t-values (p = 0.05) were compared with those obtained by the Student’s t-test according to the following formula:
21
11
21
nnS
XXt+×
−=
Statistical Advisor: A. Breidbach
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Binder
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Ret
rieva
l of a
flato
xin
B1,
%
0
20
40
60
80
100
120
Aflatoxin B1
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DON
Binder
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Ret
rieva
l of d
eoxy
niva
leno
l, %
0
20
40
60
80
100
120
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ZON (combined binders)
Binder group
A B C D E
Ret
rieva
l of z
eara
leno
ne, %
0
20
40
60
80
100
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OTA (combined binders)
Binder group
A B C D E
Ret
rieva
l of o
chra
toxi
n A
, %
0
20
40
60
80
100
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Fumonisins (combined binders)
Binder group
A B C D E
Ret
rieva
l of f
umon
isin
s B
1 & B
2, %
0
20
40
60
80
100
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T-2 & HT-2 toxins
Binder group
A B C D E
Ret
rieva
l of T
-2 &
HT-
2 to
xins
, %
0
20
40
60
80
100
120
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Recent Findings (FAC-A) on AfB1
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Recent Findings (FAC-A) on DON
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AfB1 to AfM1 transformation in the presence of binders
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Conclusion
The addition of the tested binders (at the level indicated by the manufacturer) did not result in lower analytical values for any mycotoxincurrently regulated in feed.
The combined effect of processing such as wet heating, pH-change, etc. in the presence of mycotoxin binders was not investigated, but might be a subject of interest for other studies.
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Collaborators
Ouissam Abbas and Vincent BaetenIn collaboration with
Alexandro Rodriguez
Walloon Agricultural Research CentreQuality of Agricultural Products Department
Chaussée de Namur, 24B – 5030 GEMBLOUX
Tél :++ 32 (0) 81/62.03.50Fax : ++ 32 (0) 81/62.03.88
[email protected]://cra.wallonie.be
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IRMM staff behind the scene
Massimo AmbrosioKaty KroegerKatrien Bouten
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Open Call for Collaborators
IRMM is looking for collaborators for projects on:characterisation studies homogeneity and stability studies, and feasibility studies
As well as for scientific staff (e.g. post docs)
www.irmm.jrc.be/html/calls/call_for_collaborators/index.htmwww.irmm.jrc.be/html/job_opportunities/index.htm