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