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Neogen EuropeComprehensive
MycotoxinManagement
by Rolf Steinmüller
World Ethanol & BiofuelsForum - 10 November 2016,
Brussels
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
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 storage2
Toxigenically & chemically heterogeneous1
Occur in a wide variety of substrates, including feeds & foods
Significant impact on human &animal health, economies & international trade3
Multiple mycotoxins can occur simultaneously, and have synergistic effects
Stable molecules which are extremely difficult to remove or eradicate4
1Bennett JW, Klich M, 20032Food Standards Agency, 20163Pinotti L, Ottoboni M, Giromini C, DellÓrto V, Cheli F, 20164Jarday G, Liboza T Mathieu F, Guyonvarch A, Lebrihi A, 2011
Stunning diversity
Aflatoxin B1
Deoxynivalenol
Fumonisin B1 Zearalenone
T2 Toxin
Ochratoxin APatulin
Aspergillus flavus
Fusarium Conidia
Penicillium verrucosum
before harvest (on the field)
after harvest
storage
processing
consumption
Occurrence of mycotoxins
Mycotoxins can occur at all stages of the processing chain
Impact
Unavoidable occurrence in food & feedon a global level, 30% -100% of food & feed samples are co-contaminated1
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 risik2
1Binder EM, Tan LM, Chin LJ, Handl J, Richard J, 2007; Martin S, Ramos AF, Cano-Sancho G, Sanchis V, 2013 2Pinotti L, Ottoboni M, Giromini C, Dell’Orto V, Cheli F, 2016
Factors involved in mycotoxin development
Pestka and Casale, 1989
biologicalfactors
- Susceptibility of crop
- Compatibel toxigenic
fungus
enviromentalfactors
- Temperature
- Moisture
- Injury/damage
(insect, bird, hail, mechanical)
- Fungus
harvest
- Crop maturity
- Temperature
- Moisture
- Harvesting process
storage- Temperature
- Moisture
- Duration of storage
- Cleanliness
Distribution / Processing
Humans Animals
Aminal products
Prevention
Farm Collection Storage Distribution
Food and
Feed
production
Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013
Prevention - Farm
Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013
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
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
HarvestingHIGH 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
Prevention - Collection
Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013
Quality monitoring- Sampling plan - Moisture content - Mycotoxin analysis
Pre-selection based on qualitybefore allocating into a certain storage facility (silo):
- Avoid mixing of parties of highly different quality -Segregate based on moisture content, protein content, hectolitreweight, crop data, presence of insects...
Traceability-Allocated as food or feed
DryingHIGH RISK - Store grains with high moisture content (> 15%) LOW RISK - Drying moisture below ± 15 %
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
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
Mycotoxin detection
The search for the mycotoxins literally corresponds to the search of the "needle in a haystack"
Mycotoxin detection
5 senses: hearing - sight- touch- smell – tasteDo not help us here!
Mycotoxin scaleTrace analysis has different scales
ppm
ppb
Satterfield ZPE, Black J, 2004
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 (260-38.000 ppb B1 + B2)1
1 kernels within 1.000 (0,1%) is contaminatedin a lot of raw-shelled peanuts2
Miss all hotspots and underestimate true lot concentration
Select only from hotspot and overestimate true lot concentration
1Shotwell OL, Goulde ML, Bothast RJ, Hesseltine CW, 19752Whitaker et al., 1974
Test procedureDiagram 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
Test variabilityTotal variability = sampling + sample preparation + analytical variancesassociated with each step of the mycotoxin test procedure.
Total Error
SampleLot Sample Preparation Analysis
SamplingError
SamplingPreparation
Error
AnalyticalError
ppb / ppm
Cast report, 2003
Sampling and Variation
Sampling can account for as much as 90% of sampleto sample variability
Robust sampling plan to aim for high precision andhigh 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)
Mycotoxin analyses
Eeckhout M, Landschoot S, Deschuyffeleer N, De Laethauwer S, Haesaert G, 2013
Method Mycotoxins Advantage Disadvantage
Lateral flow test
Aflatoxin
Deoxynivalenol
Fumonisin
Ochratoxin A
T-2 toxin
Zearalenone
Very fast (15-20 min)
Suitable as a single test (for
example at the goods receipt)
Simple
Portable
No clean-up needed
Cross reactivity
Extensive validation needed
Single mycotoxin detection
not suitable for the analyse of
processed cereal products
ELISA
Aflatoxins
Citrinin
Deoxynivalenol
Fumonisins
Ochratoxin A
T-2 toxin
Zearalenone
Rapid
High throughput (screening
method)
Low sample volume
Simple
Portable
Often no clean-up needed
Cross reactivity
Extensive validation needed
Single mycotoxin detection
Matrix dependent
HPLC
Aflatoxins
Fumonisins
Ochratoxin A
Trichothecenes
Zearalenone
Sensitive
Reliable
Minimum variability
Time-consuming
Expensive
Substantial clean-up necessary
LC-MS
Aflatoxins
Enniatins
Ergot alkaloids
Fumonisins
Moniliformin
Ochratoxins
Trichothecenes
Zearalenone
High sample throughput
High selectivity
High sensitivity
Multiple analysis (tandem MS)
Matrix interference (sample
clean-up necessary) Expensive
Time-consuming
Expensive
Quantitative
Lateral Flow
Qualitative
Neogen Test Kit Applications
Clean-up column
Veratox® ProcedureTest 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.
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
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+
Quantitative Test Kits
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
Reveal Q+ - Incoming Grain
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+)
Validation Reports
Method Approvals
Aflatoxin MAX
Validated Commodities
Proficiency Test Scheme Regular PT schemas for the most
common mycotoxin (Afla, DON, Fum,OTA, T2-/HT2 & ZEA)
Validate the efficiency your testingprocedure
Additional Services
Sample testing service
Equipment loans for repair, calibration & demos
On-site training
Technical service
Reference materials
Proficiency Test / ring trials