Analytical Aspects of Mycotoxin Binders

Fusarium-toxin Forum of 1-2 February 2010 1 Joint Research Centre (JRC) Analytical Aspects of Mycotoxin Binders Anna Kolossova & Joerg Stroka http://www.jrc.ec.europa.eu/

Overview Fusarium-toxin Forum of 1-2 February 2010 2 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

Legislation first Fusarium-toxin Forum of 1-2 February 2010 3

Some background Fusarium-toxin Forum of 1-2 February 2010 4 My first contact with binders http://www.inkanatura.com/macawclaylicks.asp 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. Humans in Ghana

What has research been focussed on? Fusarium-toxin Forum of 1-2 February 2010 5 Animal health (in vivo) Metabolic indicators (in vivo) Cell culture studies (in vitro) Isotherm studies (analysis of suspensions) Effects on analysis????

Methods for prevention and decontamination of mycotoxins Fusarium-toxin Forum of 1-2 February 2010 6 Prevention of mycotoxin contamination Pre- 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 materials The use of HACCP to consider in the future Although 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 mycotoxins Removal from contaminated commodities Inactivation of mycotoxins by physical, chemical or biological methods Neither 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

Mycotoxin binders Fusarium-toxin Forum of 1-2 February 2010 7 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 interest Reduction of mycotoxin bioavailability/activity Availability of essential nutrients to an animal Not being detrimental to animal or food product Not being a growth promoter (growth promotion may mask mycotoxins) Demonstrate verifiable positive results (improvement of zootechnical performance; recovery of organ status; excretion of mycotoxins via faeces; recovery of the immune status) Technical and economical feasibility

Types of Mycotoxin Binders Fusarium-toxin Forum of 1-2 February 2010 8 Adsorbents Inorganic Activated charcoal Aluminosilicates, clays HSCAS, bentonite, montmorillonite, zeolite, clinoptilolite Clay minerals layered silicates [Si 2 O 5 2- ] xy Zeolite silicate made of tetrahedrons of SiO 4 and AlO 4 Chemically treated silicates Organic Chemical polymers (cholestyramine, polyvinylpyrrolidone, divinylbenzene-styrene polymers ) High costs Glucan products Yeast and products from yeast (yeast cell wall)

Types of Mycotoxin Binders Fusarium-toxin Forum of 1-2 February 2010 9 Denaturant Mycotoxin-degrading enzymes Life microorganisms producing mycotoxin-degrading enzymes Eubacterium sp. (BBSH 797) T. mycotoxinivorans Organic binders Efficient against a larger range of mycotoxins than inorganic binders More adapted to the most frequent cases of multi-contaminated feeds Biodegradable and do not accumulate in the environment after being excreted by animals

Oltipraz a Mycotoxin Binder? Fusarium-toxin Forum of 1-2 February 2010 10 http://phm.utoronto.ca/~jeffh/jnppres.ppt

Level of Interest for Testing Mycotoxin Binders Fusarium-toxin Forum of 1-2 February 2010 11

Level of Interest for Testing Mycotoxin Binders Fusarium-toxin Forum of 1-2 February 2010 12

Analytical aspects Fusarium-toxin Forum of 1-2 February 2010 13 Cascade of modern analytical Methods: Extraction Clean-up / Dilution Separation Detection

Methods used for testing Fusarium-toxin Forum of 1-2 February 2010 14 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.

Repeatability Data of the Test Methods Fusarium-toxin Forum of 1-2 February 2010 15 Mycotoxin DON AFB1 ZEA T2 & HT2 toxins FUM B1 + B2 OTA Average RSD r (%) 10.6 6.9 7.7 9.8 3.2 4.1

Tested Binders in this Study Fusarium-toxin Forum of 1-2 February 2010 16 Code Type of main product(s) ph value Amount added [%] 1 Yeast cell wall 2.9 0.2 2 Clay + organic acid 7.9 0.1 3 Clay 8.4 0.1 4 Clay 4.6 0.25 5 Yeast cell wall 6.2 0.2 6 Yeast cell wall 6.7 0.2 7 Yeast cell wall 4.7 0.25 8 Mixture organic + mineral 6.2 0.25 9 Yeast cell wall 5.0 0.2 10 Mixture organic + mineral component 8.6 0.25 11 Mixture organic + mineral component 6.9 0.25 12 Clay 9.1 0.5 13 Clay 8.9 1 14 Fibres 6.7 1 15 Montmorillonite 10.4 0.4 16 Montmorillonite 10.6 0.4 17 HSCAS 8.8 0.5 18 HSCAS 9.8 0.5 19 HSCAS 9.8 0.5 20 Fibres (lignocellulose) 5.1 2.5

Some Statistics Fusarium-toxin Forum of 1-2 February 2010 17 Data analysis Student 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: t = S X1 X 2 1 1 + n1 n2 Statistical Advisor: A. Breidbach

Aflatoxin B1 Fusarium-toxin Forum of 1-2 February 2010 18 120 100 Retrieval of aflatoxin B 1, % 80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Binder

DON Fusarium-toxin Forum of 1-2 February 2010 19 120 Retrieval of deoxynivalenol, % 100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Binder

ZON (combined binders) Fusarium-toxin Forum of 1-2 February 2010 20 100 Retrieval of zearalenone, % 80 60 40 20 0 A B C D E Binder group

OTA (combined binders) Fusarium-toxin Forum of 1-2 February 2010 21 100 Retrieval of ochratoxin A, % 80 60 40 20 0 A B C D E Binder group

Fumonisins (combined binders) Fusarium-toxin Forum of 1-2 February 2010 22 100 Retrieval of fumonisins B 1 & B 2, % 80 60 40 20 0 A B C D E Binder group

T-2 & HT-2 toxins Fusarium-toxin Forum of 1-2 February 2010 23 120 Retrieval of T-2 & HT-2 toxins, % 100 80 60 40 20 0 A B C D E Binder group

Recent Findings (FAC-A) on AfB1 Fusarium-toxin Forum of 1-2 February 2010 24

Recent Findings (FAC-A) on DON Fusarium-toxin Forum of 1-2 February 2010 25

AfB1 to AfM1 transformation in the presence of binders Fusarium-toxin Forum of 1-2 February 2010 26

Conclusion Fusarium-toxin Forum of 1-2 February 2010 27 The addition of the tested binders (at the level indicated by the manufacturer) did not result in lower analytical values for any mycotoxin currently 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.

Collaborators Fusarium-toxin Forum of 1-2 February 2010 Ouissam Abbas and Vincent Baeten In collaboration with Alexandro Rodriguez Walloon Agricultural Research Centre Quality of Agricultural Products Department Chaussée de Namur, 24 B 5030 GEMBLOUX Tél :++ 32 (0) 81/62.03.50 Fax : ++ 32 (0) 81/62.03.88 baeten@cra.wallonie.be http://cra.wallonie.be 28

IRMM staff behind the scene Fusarium-toxin Forum of 1-2 February 2010 Massimo Ambrosio Katy Kroeger Katrien Bouten 29

Open Call for Collaborators Fusarium-toxin Forum of 1-2 February 2010 30 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.htm www.irmm.jrc.be/html/job_opportunities/index.htm