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Mycotoxins in Grain and Feed IndustriesII. Best Practices in Handling and Testing

Erin Bowers, Iowa State University, Agricultural Engineering

Charles Hurburgh, Iowa State University, Agricultural Engineering

Alison Robertson, Iowa State University, Plant Pathology

Learning Objectives

• This learning module will focus on the sampling, testing, and storage of grains containing mycotoxins

• Module Objectives:

• Learn BMP for testing for mycotoxin contamination and for consignment of inbound ingredients (grains) that potentially contain toxins.

• Learn BMP for preventing the production of mycotoxins in grain or grain sample storage.

**BMP=Best Management Practices

Basics:Agriculturally Important

Mycotoxins• Fungi contaminate grains, food, and

feed worldwide• Mycotoxins are chemical compounds

produced by some fungi • More than 400 mycotoxins identified

worldwide, 30 concerning for human or animal health

• 5 principle mycotoxins affect cereal grains (corn, wheat, rye, barley, oats)

• aflatoxins, fumonisins, deoxynivalenol (vomitoxin), zearalenone, and ochratoxin A

Gibberella Ear Rot caused by Gibberella zeae

Wheat Scab

Developing Fungus is Dependent on the Environmental Conditions During Pollination and Early Grain Development

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Mycotoxin(s) FungiFavorable Conditions

Primary Grains

AflatoxinsAspergillus flavus Aspergillus parasiticus

Hot and dry, drought

Corn, Durum (in EU)

Deoxynivalenol (Vomitoxin) Zearalenone

Fusarium graminearumFusarium culmorum

Cool, wet, humid at grain fill

Corn, Wheat, Oats, Rye, Barley, Durum

FumonisinsFusarium verticillioidesFusarium proliferatum

Warm to hot, dry at and after flowering Corn

Ochratoxin APenicillium verrucosum Harvest conditions

determine Corn

• Environmental stresses increase susceptibility• Planting, harvest, and storage practices also

contribute to mycotoxin risk

• Most mycotoxins remain intact after cooking, drying, freezing or storage conditions.

• Mycotoxins at very low concentrations (ppm or ppb) are capable of causing serious health problems for humans as well as animals.

• There is usually no treatment for mycotoxin poisoning (mycotoxicosis).

Basics:Mycotoxins in Grain and Feed

ppm: parts per millionppb: parts per billion

Poisonous Ingredients in Food

Federal Food, Drug, and Cosmetic Act §402 [21 U.S.C. 342]

A food shall be deemed adulterated—(a)(1) If it bears or contains any poisonous of deleterious substance which may render it injurious to health; but in case the substance is not an added

substance such food shall not be considered adulterated under this clause if the quantity of such substance does not ordinarily

render it injurious to health.

• Most mycotoxin contamination happens in the field• Prevent contaminated grain from entering the facility

• Pre-harvest preventative controls• Scouting and pre-harvest observations• Climate and weather conditions

• At-receiving mycotoxin testing procedure

Mycotoxin Management Plan

Output

Laboratory Sample

Analysis Sample

Mycotoxin Result

Mycotoxin testing procedure

Procedural component

1) Sampling• Sample size reduction (if necessary)

2) Sample Preparation• Grinding• Mixing• Subdividing

3) Analysis

An example of a grinder that would be used to grind a laboratory sample.

• Mycotoxins are not distributed uniformly throughout fields, bins, or transportation containers• Localization of moldy grain

• High variability among individual kernels• A few mycotoxin-affected kernels can limit grain use

• Misclassification of grain

Hypothetical distribution of an incoming load of corn with an average aflatoxin level of 10 parts-per-billion (ppb)

0 ppb 0 ppb 0 ppb 0 ppb 0 ppb

0 ppb 0 ppb 0 ppb 0 ppb100 ppb

Heterogeneous distribution

Sampling (the laboratory sample)

• The process of acquiring a sample of grain (laboratory sample) that is representative of the lot • Subject to sampling bias

• Sample representativeness is increased by taking incremental samples• This is a time-consuming practice• Not practical to use for every load of grain entering a facility• Prescribed for compliance sampling (aflatoxin)

• Incremental samples are aggregated and mixed to form the laboratory sample

Sampling Equipmentx

x

xx x

x

xHand grain probe (trier)

Mechanical grain probe sampling a truckload of grain at a receiving point

An Ellis sampler can be used to manually sample grain on a conveyor

Above: An example of a probing pattern to be used for sampling a flat-bottom truck or trailer.

Bottom right: An illustration of the action of a diverter-type sampler. At set intervals a portion of the moving grain is captured and diverted to a sample receiving container by a moving receptacle.

Grain flow

Receptacle

Sample

Corn

shelled, whole kernel

meal

flour

snack foods

popcorn

cleaned, for masa production

dry milled bran

grits or flaking grits

cereals

baby cereals

Cottonseed

whole grain

meal

Rice

cultivated, whole grain

wild, whole grain

cereals

baby cereals

Wheat

whole kernel

whole wheat flour

white flour

bran, for human consumption

cereals

baby cereals

Oats

whole

cereals

baby cereals

Barley

whole

malt

cereals

baby cereals

Rye flour

soya-based baby food products

Aflatoxin x x x x x x x x x x

Deoxynivalenol x x x x

Fumonisin x x x x x x x

Ochratoxin A x x x x x x x x x x x x x x x x x x x

Products for Surveillance Sampling

Abbreviated list adapted from CPG 7307.001 Attachment A

Aflatoxin Compliance Sampling

Commodity Lot type Number of incremental samples

Incremental sample size (lbs)

Laboratory sample size (lbs)

CornShelled, meal, flour, grits

Bulk and consumer

10 1 10

Oilseed meale.g., cottonseed

Bulk 20 1 20

Small grainse.g., wheat, barley, sorghum

Bulk 10 1 10

Adapted from FDA Investigations Operations Manual, 2013, Ch. 4, Chart 6

• Preparing the laboratory sample for analysis

1. Grinding the entire laboratory sample• The smaller the laboratory sample, the higher the

uncertainty in the final analytical result

2. Mixing• Homogenize

3. Selecting analysis sample

Sample preparation

An example of a grinder that would be used to grind a laboratory sample.

Operating characteristics of mycotoxin sampling plans

www.fstools.org/mycotoxins/

What is the probability that a grain lot will be correctly quantified using a given sampling plan?

All parameters the same except• Green=1 lb laboratory

sample• Blue=5 lb laboratory sample• Orange=10 lb laboratory

sample

Lot Aflatoxin Concentration (ppb)

Pro

bab

ility

of A

ccep

ting

Lot

(%)

Operating characteristics of mycotoxin sampling plans

www.fstools.org/mycotoxins/

Seller’s riskCommercial-false rejection of good quality grainRegulatory-false sanctions

Buyer’s riskCommercial-false acceptance of poor quality grainRegulatory-compromises food and feed safety

Seller’s Risk

Buyer’s Risk Lot Aflatoxin Concentration (ppb)

Pro

ba

bili

ty o

f Acc

ep

ting

Lo

t (%

)

• Using an appropriate method to determine the quantity or confirm the presence or absence of mycotoxins in the analysis sample

• Qualitative or Quantitative

• Rapid test or analytical laboratory

• The choice of detection method will depend on the user’s needs and resources, the commodity being tested, and the decisions which will be made using the analytical result.

Analysis

• High Performance Liquid Chromatography (HPLC)• Require trained personnel to maintain and

operate• Produces results with high

• Precision• Accuracy• Repeatability• Sensitivity

• Standard for mycotoxin confirmation analysis in compliance samples

AnalysisThe image below shows an example of an HPLC instrument set up in a lab

• Rapid test methods• Rapid relative to analytical chemistry methods• Common on-site analysis methods

• Examples of rapid test methods• Enzyme-linked immunosorbent assay (ELISA)• Lateral flow assay• Flow through assay

Analysis

Left: an example of an ELISA test Right: examples of lateral flow assays

Analysis

Left: an example of an ELISA test Right: examples of lateral flow assays

GIPSA provides outside laboratory validation of rapid test kits submitted by manufacturers

• Not mandatory to use a GIPSA-approved kit• More information,

http://www.gipsa.usda.gov/fgis/insp_weigh/raptestkit.html

Analysis

• Blacklighting (IOM 427.04g)• Screening purposes

(aflatoxin only)• Few false negatives, but

many false positives• Direct quantitative testing to

at-risk-loads• Thin-Layer Chromatography

(TLC)

Above: an example of bright greenish-yellow fluorescence

produced when Aspergillus flavus-infected corn is viewed under

blacklight

• Benefits of rapid tests vs HPLC• Ease of use • Cost• Compact (size)• Minimal calibration• Less equipment maintenance/upkeep• Faster

• Downfalls of rapid tests• Reduced sensitivity• Narrow operating range• Higher limits of detection• Not approved for regulatory decision-making

• Confirmation analysis necessary by accepted analytical method• Compliance Policy Guide Manual, section 555.400

Analysis

• FSMA: Industry action required based upon reasonable suspicion of food/feed safety threat• Documentation to verify safe handling, processing, storage, and end

use• Performance Monitoring: Check-samples ensure

appropriateness of mycotoxin management (best practice)• Especially in facilities producing finished feed or food products

Analysis

• Time requirement for testing is significant• Can’t test/sample every load• Can’t test for every mycotoxin

• Know the risks and be strategic• Weather and climate are good indicators of mycotoxin risk

• Cool, wet conditions- risk of deoxynivalenol and zearalenone• Hot, drought conditions- risk of aflatoxins• Warm, drought conditions-risk of fumonisins

• Use this as a predictive tool to direct sampling and testing

• Composite testing is good practice to monitor overall quality of incoming grain and of that which has been accepted

Practical Considerations

• Reduce the ability of fungi to survive and grow• Shelf life of grain is impacted by

• Quality of stored grain• Water availability

• Grain moisture• Grain temperature

• Drying: Reduce grain moisture to stop fungal growth and mycotoxin production• Dry promptly: minimize time between harvest and drying to maximize

shelf life of grain• Dry carefully: prevent stress cracks and reduce breakage

• Kernel temperature ≤110°F• Grain moisture in equilibrium with 65% RH will prevent fungal activity

Storage and Handling

• Temperature is an important factor for grain storage• Cool temperatures (<60°F) minimize fungal growth • Aeration can be used to facilitate temperature control

• Temperature uniformity• Minimize moisture movement

• Monitor grain for changes in quality

Storage and Handling

Blending• Aflatoxin is a food/feed adulterant• Grain >20 ppb cannot be deliberately blended• FDA blending dispensations have been allowed in rare

instances when a large portion of the U.S. corn supply is affected• State by state basis• Resultant grain used only for specific livestock feed• >500 ppb aflatoxin grain has never been allowed to be blended

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Mycotoxins in grain handling facilities

• Mycotoxin management can be accomplished using proper storage and handling practices, and through preventing contaminated grain from entering a facility

• Testing domestic grain for mycotoxins is discretionary, export is mandatory for aflatoxin only

• Each facility will likely handle mycotoxin management uniquely, depending on their individual structure, capabilities, and contract requirements

• Look for evidence of proactive action• Strategic, informed plans and actions

Proactive approaches

• Contract terms for suppliers• Attention to growing conditions in the area• Pre-harvest scouting/survey• A mycotoxin sampling plan

• Sampling• Sample preparation• Analysis

• Monitoring the effectiveness of the sampling plan• Finished product checks and verification• Check sample analysis (third party laboratory)

• Composite sample testing

} in combination with a threshold for acceptance

This training was a joint effort of

*Funding for this Grain and Feed Mill Operations course was made possible, in part, by the Food and Drug Administration through grant (1U54FD004333-01), views expressed in written materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.*

www.grains.ksu.eduwww.iowagrain.org