Neogen Europe Comprehensive Mycotoxin Management

Transcription

1 Neogen Europe Comprehensive Mycotoxin Management by Rolf Steinmüller World Ethanol & Biofuels Forum - 10 November 2016, Brussels

2 Agenda General introduction to mycotoxins Definition Occurrence Impact Factors involved in their development Prevention of mould infestation & mycotoxin formation Introduction to mycotoxin analysis Sampling Critical testing elements Neogen Mycotoxins solutions

3 Mycotoxins definition Low-molecular-weight natural products (i.e., small molecules) 1 Produced as secondary metabolites by filamentous fungi (moulds) 1 Produced by moulds growing on food crops during production & subsequent storage 2 Toxigenically & chemically heterogeneous 1 Occur in a wide variety of substrates, including feeds & foods Significant impact on human &animal health, economies & international trade 3 Multiple mycotoxins can occur simultaneously, and have synergistic effects Stable molecules which are extremely difficult to remove or eradicate 4 1 Bennett JW, Klich M, Food Standards Agency, Pinotti L, Ottoboni M, Giromini C, DellÓrto V, Cheli F, Jarday G, Liboza T Mathieu F, Guyonvarch A, Lebrihi A, 2011

4 Stunning diversity Fumonisin B1 Zearalenone Aspergillus flavus Aflatoxin B1 Fusarium Conidia T2 Toxin Deoxynivalenol Penicillium verrucosum Patulin Ochratoxin A

5 Occurrence of mycotoxins Mycotoxins can occur at all stages of the processing chain processing after harvest storage consumption before harvest (on the field)

6 Impact Unavoidable occurrence in food & feed on a global level, 30% -100% of food & feed samples are co-contaminated 1 Can be formed at any time in food & feed Can cause (chronic & acute) poisoning (mycotoxicoses) (thermo) stable compounds No absolutely safe detoxification procedures known Legal thresholds, recommendations & trading contracts Negative effect on crop yield & crop quality The use of DDGS as animal feed is associated with a mycotoxin risik 2 1 Binder EM, Tan LM, Chin LJ, Handl J, Richard J, 2007; Martin S, Ramos AF, Cano-Sancho G, Sanchis V, Pinotti L, Ottoboni M, Giromini C, Dell Orto V, Cheli F, 2016

7 Factors involved in mycotoxin development biological factors - Susceptibility of crop - Compatibel toxigenic fungus enviromental factors - Temperature - Moisture - Injury/damage (insect, bird, hail, mechanical) - Fungus harvest - Crop maturity - Temperature - Moisture - Harvesting process Humans storage - Temperature - Moisture - Duration of storage - Cleanliness Distribution / Processing Animals Pestka and Casale, 1989 Aminal products

8 Prevention Farm Collection Storage Distribution Food and Feed production Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013

9 Prevention - Farm Choice of variety Use varieties recommended for a certain region Choose a variety with resistance for FHB Crop rotation Rotating Fusarium host crops with non-host crops HIGH RISK - Maize Wheat LOW RISK - Potato/ legumes/ beets Wheat Crop planning Avoid high temperatures &drought stress during seed development & maturation Avoid wet periods during early flowering Maintain recommended plant spacing to avoid overcrowding (increased humidity) HIGH RISK - Delaying harvest of infected crops may increase mycotoxin content LOW RISK - Planning to harvest the crop at low moisture & full maturity Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013

10 Prevention - Farm Soil & crop mangement HIGH RISK - Crop residues - Plant stress: drought, humidity, insects LOW RISK - Removal, destruction or burial of infected crop residues - Ploughing - Optimised plant nutrition and irrigation - Avoid insect damage and weeds (grasses) - Timely application of fungicides Harvesting HIGH RISK - Moisture content is too high (> 15%) - FHB infected parts of the field - Shrivelled, damaged or lodged grain LOW RISK - Separation for food/feed - Damp/clean/dry Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013

11 Prevention - Collection Quality monitoring - Sampling plan - Moisture content - Mycotoxin analysis Pre-selection based on quality before allocating into a certain storage facility (silo): - Avoid mixing of parties of highly different quality -Segregate based on moisture content, protein content, hectolitre weight, crop data, presence of insects... Traceability -Allocated as food or feed Drying HIGH RISK - Store grains with high moisture content (> 15%) LOW RISK - Drying moisture below ± 15 % Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013

12 Prevention - Storage Good Hygiene practice recommendations for storage operations - Quality control at intake - Avoid contamination of stored products by the environment and cross-contamination between stored products - Avoid moisture LOW RISK - Good storage conditions: cool, dry and ventilated - Pest control - Adequate cleaning and maintenance of premises Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013

13 Prevention - Distribution Good hygiene practice recommendations for dispatch/delivery and transport operations - Clean and dry containers - No residues - Avoid rain - No pests and rodents Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013

14 Mycotoxin detection The search for the mycotoxins literally corresponds to the search of the "needle in a haystack"

15 Mycotoxin detection 5 senses: hearing - sight- touch- smell taste Do not help us here!

16 Mycotoxin scale Trace analysis has different scales ppm ppb Satterfield ZPE, Black J, 2004

17 Uneven distribution Distribution of contaminated grain is heterogeneous throughout lot, even within single ear or head Affected grains/kernels in hotspots Of 140 corn kernels analyzed for Aflatoxin, 16 had Aflatoxin ( ppb B 1 + B 2 ) 1 1 kernels within (0,1%) is contaminated in a lot of raw-shelled peanuts 2 Miss all hotspots and underestimate true lot concentration Select only from hotspot and overestimate true lot concentration 1 Shotwell OL, Goulde ML, Bothast RJ, Hesseltine CW, Whitaker et al., 1974

18 Test procedure Diagram of general steps involved in sampling, sample preparation, and analysis of mycotoxins in agricultural commodities. Lot Test sample Sample preparation (size, reduction, mixing, etc.) Subsample Analysis Mycotoxin test result Cast report, 2003

19 Test variability Total variability = sampling + sample preparation + analytical variances associated with each step of the mycotoxin test procedure. Total Error Sampling Error Sampling Preparation Error Analytical Error Lot Sample Sample Preparation Analysis ppb / ppm Cast report, 2003

20 Sampling and Variation Sampling can account for as much as 90% of sample to sample variability Robust sampling plan to aim for high precision and high accuracy Sample plans Sampling, size and number of increments Sample preparation, homogenization of aggregate Quantification Studies show variance is decreased: Increase sample size Increase in grind degree, smaller particle size Increase number of replicates (more extractions & testing in duplicates)

21 Mycotoxin analyses Lateral flow test ELISA HPLC LC-MS Method Mycotoxins Advantage Disadvantage Aflatoxin Very fast (15-20 min) Cross reactivity Deoxynivalenol Suitable as a single test (for Extensive validation needed Fumonisin example at the goods receipt) Single mycotoxin detection Ochratoxin A Simple not suitable for the analyse of T-2 toxin Portable processed cereal products Zearalenone No clean-up needed Aflatoxins Citrinin Deoxynivalenol Fumonisins Ochratoxin A T-2 toxin Zearalenone Aflatoxins Fumonisins Ochratoxin A Trichothecenes Zearalenone Aflatoxins Enniatins Ergot alkaloids Fumonisins Moniliformin Ochratoxins Trichothecenes Zearalenone Rapid High throughput (screening method) Low sample volume Simple Portable Often no clean-up needed Sensitive Reliable Minimum variability High sample throughput High selectivity High sensitivity Multiple analysis (tandem MS) Cross reactivity Extensive validation needed Single mycotoxin detection Matrix dependent Time-consuming Expensive Substantial clean-up necessary Matrix interference (sample clean-up necessary) Expensive Time-consuming Expensive Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013

22 Neogen Test Kit Applications Quantitative Qualitative Lateral Flow Clean-up column

23 Veratox Procedure Test procedure (1) Add 100 μl conjugate to each red marked mixing well. (2) Add 100 μl controls and samples to their respective wells. (3) Mix. Transfer 100 μl to antibody wells. Incubate at room temperature for 5 minutes, sliding microwell holder back and forth gently for first 20 seconds. (4) Dump liquid from antibody wells. (5) Wash wells thoroughly with deionised water. Repeat wash step five times. (6) Tap out water on absorbent paper towel. (7) Transfer 100 μl substrate from the reagent boat to the antibody wells. Incubate at room temperature for 5 minutes, sliding microwell holder back and forth gently for first 20 seconds. (8) Transfer 100 μl Red Stop from reagent boat to antibody wells. (9) Read results using a microwell reader with a 650 nm filter.

24 Quantitative Test Kits Veratox - Finished Product Benefits Accurate, Precise, Rapid Wide Acceptance Little training required Ideal for batching Rapid results Full quantification Veratox software for results

25 Reveal Q+ Procedure Test procedure (1) Prepare by entering the QR code into the AccuScan Gold reader. (2) Obtain a representative sample. Grind and weigh out a 10 g sample. (3) Add extraction solution. (4) Shake vigorously for 3 minutes, or blend for 1 minute. (5) Settle, then filter. (6) Add sample diluent to red dilution cup. (7) Add 100 μl sample extract to red dilution cup and mix up and down 5 times. (8) Transfer 100 μl to sample cup. (9) Place a new Reveal Q+ strip into the sample cup. Set a timer. (10) Remove promptly and interpret results using the AccuScan Gold reader. (11) Example Results: DON Q+

26 Quantitative Test Kits Reveal Q+ - Incoming Grain Benefits Fully quantifiable lateral flow test Accurate, Precise, Rapid Easy to use, minimal training Room temperature storage and incubation Single, Aqueous extraction with MAX Data Manager software for results Screen incoming grains - accept/reject load

27 Critical Testing Elements Sampling technique ( representative sample ) Sample grind size Grinder clean-out Water quality Insufficient extraction Sample ph Wrong pipetting Insufficient washing (ELISA) Not updating QR code (Reveal Q+)

28 Validation Reports

29 Method Approvals Aflatoxin MAX

30 Validated Commodities

31 Proficiency Test Scheme Regular PT schemas for the most common mycotoxin (Afla, DON, Fum, OTA, T2-/HT2 & ZEA) Validate the efficiency your testing procedure

32 Additional Services Sample testing service Equipment loans for repair, calibration & demos On-site training Technical service Reference materials Proficiency Test / ring trials

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