Moulds and Mycotoxins

Dr. Tom Gräfenhan Grain Research Laboratory, Canadian Grain Commission

Storage Moulds on Grains

Spoilage of grain during storage Degradation of

nutrients Odour Change of grain

properties Contamination

with mycotoxins

Where is the water coming from?

Water availability key! Condensation and moisture content: 17%-18% critical Risk of ‘wet pockets’ or ‘hot

spots’ Fall scenario Spring scenario

Impurities: carry over, dockage, weeds, damaged kernels

Autumn

Spring

PAMI – Facts about Grain Aeration

http://pami.ca/wp-content/uploads/2013/09/Grain-Aeration-Factsheet.pdf

Storage Moulds on Grains

Agar plating of kernels Identification of

toxigenic species Key groups or

genera often identified: Aspergillus Penicillium

Important Species of Storage Moulds

Penetration of wheat kernels

Mycelia and conidiophores underneath the bran

Mycelia on cell walls of endosperm

Penicillium verrucosum

Main producer of ochratoxins in cooler climates Occasionally recovered

from cereal grains Ubiquitous, widely

distributed Carry over of inoculum Slow growing

Identification of OTA producing moulds

Selective culture media e.g. dichloran yeast sucrose agar (DYSG) or coconut agar fluorescence ≅ OTA production by Penicillium

verrucosum

Ochratoxin A (OTA)

NH

O

O

OH

Cl

O

CH3

OHO

relatively stable bioaccumulative detected in human milk and serum

potentially carcinogenic to humans, neurotoxicity, immunotoxicity

Human Exposure to OTA cereals and cereal-based foods major contributor to

OTA intake 60- 75% of estimated dietary exposure for Canadians

(Kuiper-Goodman et al. 2010)

International Limits for OTA

Worldwide regulations for mycotoxins in food and feed in 2003 (FAO, 2004)

CFIA: swine diets (kidney damage) 0.2 mg/kg swine diets (reduced weight gain) 2 mg/kg poultry diets 2 mg/kg

Health Canada's proposed Maximum Limits and Guidance Values for OTA

Proposed Value (µg/kg) (under consideration as a guideline) •Raw cereal grains (i.e., wheat, barley, oats, rice) 5 µg/kg

Proposed Maximum Limits (ML) (µg/kg) •Wheat bran 7 µg/kg •Directly consumed cereal grains (e.g. bulgur wheat, rice, oats, pearled barley) and cereal-derived products (e.g. flour; finished foods such as bread and breakfast cereals) 3 µg/kg •Grape juice 2 µg/kg

•Dried vine fruit (e.g. raisins) 10 µg/kg •Infant formulas and cereal-based foods for infants and young children 0.5 µg/kg http://www.hc-sc.gc.ca/fn-an/securit/chem-chim/toxin-natur/summary-resume-eng.php

Occurrence of OTA in Canadian Grains

cereals

< LOQ

1 – 5 µg/kg

> 5 µg/kg

oilseeds and pulses

< LOQ

1 – 5 µg/kg

> 5 µg/kg

OTA in wheat shipments vary throughout the year

Loading date

Jan Jul Jan Jul Jan Jul Jan Jul Jan Jul Jan

mea

n O

TA (n

g/g)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Production of OTA during Grain Storage

Not a disease of field crops Different from DON issue P. verrucosum growth and

OTA production is driven by moisture content / water

availability temperature

(Abramson et al. 1990)

<LOQ set to 0

Ergosterol (mg/kg)

0 1000 2000 3000 4000 5000 6000 7000

OTA

+ O

TB (m

g/kg

)

0

50

100

150

200

oats wheat Plot 1 Regr

b22.3778746483m0.0161245625r ²0.3344330222

b-3.4532320036m0.084628713r ²0.4533044545

Ochratoxin Production vs. Fungal Biomass Wheat vs. Oats

Mould growth not a good indicator for contamination of grain with OTA and other mycotoxins

Production of OTA by different strains of Penicillium verrucosum

Penicillium verrucosum strain

OTA

(ng/

g)

0

20

40

60

80

100

Intensity of Fluorescence on Agar Media

C27-1

CIT – OTA –

C1136-1

CIT +++ OTA ++

C84-2

CIT – OTA +++

C618-2

CIT + OTA –

Heterogeneity Issues for OTA

analysis of 100 single wheat kernels from incubation experiment for OTA

OTA (ng/g) n < 20 22 20 - 100 14 100 - 1000 42 1000 - 2000 18 2000 – 10,000 3 > 10,000 1

Relationship between OTA contamination and colonization by P. verrucosum

Relationship between OTA and Penicillium verrucosum concentrations in wheat

R2 = 0.6488

0

200

400

600

800

1000

1200

1400

1600

1800

0 1 2 3 4 5 6 7

OTA (ppb)

Pv (p

pm)

sampleLinear (sample)

Bulk Sampling — Heterogeneity of Distribution

70 – 75,000 tonnes

whole grain incremental samples

composite sample 10 kg

ground test portion 10 – 100 g

Storage in Grain Silos

Optimized aeration: Understanding the Equilibrium Moisture Content (EMC) Critical control

points include: Aerated floors,

elevator & augers, man hole, down spouts

Grain Storage in Bags Ground drainage crucial, drainage tiles under plastic Top surface smooth to aid in drainage Condensation under plastic needs to be controlled with

aeration Run piles north – south to allow sun to dry off sides Plastic sheeting needs replacement after one season

Management of (Temporary) Grain Storage is Extremely Important (NDSU) Check frequently for heating, sweating, moisture

accumulation and general condition of stored grain Fence in the pile to keep animals away and plastic

sheeting intact Cool temperatures and aeration minimize mould

growth, limit moisture migration, and control insects Temporary grain storage usually is for less than 6-7

months! After that grain should be sold or moved to permanent storage before warm spring weather arrives

Additional Sources of Information

http://www.grainscanada.gc.ca/storage-entrepose/ota/ota-eng.htm http://www.grainscanada.gc.ca/storage-

entrepose/ssg-de-eng.htm http://www.usask.ca/agriculture/plantsci/winter_cere

als/winter-wheat-production-manual/chapter-23.php http://www.ag.ndsu.edu/publications/crops/grain-

drying-storage/ http://lancaster.unl.edu/ag/crops/storage.shtml

Photograph acknowledgement

http://www.slu.se/sv/institutioner/mikrobiologi/forskning/eukaryot-mikrobiologi/xerofila-svampar/ http://www.omafra.gov.on.ca/english/engineer/facts/8

5-003.htm http://www.kryan.com/Baggers.html http://www.johnbob.ca/images/ http://www.versacorporation.com/