Stability of the Trichothecene,

Deoxynivalenol in Processed Foods

and Wheat Flake Cereal

Ken Voss & Maurice Snook

Toxicology & Mycotoxin Research Unit,

USDA Agricultural Research Service,

Athens, GA

Contributors

Jim Bair, North American Millers Association

Wu Li, Frito-Lay, Inc.

Brian Strouts, American Institute of Baking

Thomas Trautman, General Mills

Jeff Barach, Nancy Rachman, Grocery Manufacturers

Association

Pigs [Schlatter, Toxicol. Lett, (2004)]• Feed refusal, vomiting, poor production

• NOAEL 40 – 60 µg/kg BWt

Mice [Iverson et al., Teratog. Carcinog. Mutag. (1995)]

• Reduced feed intake; males fed > 5 ppm

• Decreased body weight; > 1 ppm

• Immunoglobulin levels altered

• NOAEL = 100 μg/kg BWt/day

Mice [Pestka et al.; Tryphonas et al.; others] & rats • Decreased protein synthesis (ribotoxic response)

• Disrupted signaling for growth

• Immune (IgA) nephropathy in mice

• Disrupted signaling for immune functions,

cell survival & replication

DON Toxicity in Animals

DON & Humans

Effects uncertain

• Reports of acute illness, particularly from Asia

> Gastrointestinal symptoms

> General discomfort, dizziness etc.

People are exposed:

• DON excretion in urine & consumption of cereal

products correlated (Turner et al. EHP 116, 2008)

• Some exposures exceed EU TDI of 1 μg/kg BWt/day

• DON found in cereal products (breads, pasta, beer, etc)

Concern for infants and adolescents

Guidelines / Regulations for Food

US FDA Guidance for Industry = 1 ppm (finished products)

Japan = 1.1 ppm in unpolished wheat

EU = Maximum allowed levels are variable:

Item Max ppm

• Unprocessed durum wheat 1.75

• Unprocessed wheat 1.25

• Cereal flour, pasta 0.75

• Various products (bread) 0.50

• Processed foods for infants 0.20

Rationale

Minimizing DON in foods desirable

-- food safety

-- guidances / limits

DON is water soluble and boiling reduces amount in

noodles

DON is generally heat stable under routine baking

conditions

BUT reported results for breads vary: specific

conditions likely important

Use conditions that are relevant to US food industry

Other products not well studied

Purpose: to determine the chemical stability of DON during the production of common cooked wheat products under commercially-relevant conditions.

Flour

Sugar Cookies (AIB)

Snack Crackers (AIB)

White Pan Bread (AIB)

(sponge & dough)

Pretzels (FL)

Cake Donuts (AIB)

Whole Wheat

Cereal Flakes

Intermediate products

• Dough

• Pellets

* Provided by General Mills

Cookies: Bake 25-30 min at 163 oC

Crackers: Bake ~6 min at 227-252 oC

(top), 193-232 oC (bottom)

Pretzels: Proprietary (dough, extrusion,

bake, brown in caustic bath, bake)

Donuts: Fried 45 sec/side at 190 oC

Bread: Fermentation (4 hr at RT); Bake

18 min at 227 oC.

Flour Products:

Cooking Conditions

Wheat Series: Product Stream(proprietary)Wheat

Grind, mix ingredients

Dough

Extrude

Pellets

Roll

Cereal Flakes

n=2 n=1 (300 g) n=3

Extract 1 (25 g)

Extract 2

Extract 3

Triplicate

(duplicate)

injections

EXPERIMENTAL OVERVIEW

Item (AIB, Frito-Lay or General Mills)

Subsamples

ANALYSIS PROTOCOL

300 g Flour, Wheat & Products

1. Freeze-dry - defat with hexane – dry

2. Mix & reduce - ball mill - recombine

6. Quantification (ppm) : Gas chromatography - EC

3. Extract with CH3N:H20 (84:16) (100 ml)

4. Cleanup: Romer MycoSep® 227 Trich+

5. Dry & reconstitute with TMS derivitizing agent

Mass balance estimation

25 g Sample

Spike/Recovery & Product Yield(Flour Products)

Spike/Recovery*

All flour based items = 80%

Flour = 86% (AIB) 68% (FL)**

Cookies = 91%

Crackers = 108%

Pretzels = 68%

Donuts = 52%

Bread = 82%

* triplicate

** same result obtained after

correction for recovery

Yield ***

1.0

1.6

1.1

0.96

2.8 (fat uptake)

1.6

*** relative amount

of product produced

per g flour in recipe

Spike/Recovery & Product Yield(Wheat Series)

Spike/Recovery *

All items = 90%

Wheat = 91%

Dough = 82%

Pellets = 90%

Flakes = 95%

* triplicate

Yield **

1.0

1.47

1.47

1.06

** relative amount

of product produced

per g flour in recipe

min0 5 10 15 20 25

uV

0

10000

20000

30000

40000

50000

UNK1 A, TRACE GC (D10407\002F0201.D)

DON

DON Standard @ 0.55 ng/uL

min0 5 10 15 20 25

uV

0

10000

20000

30000

40000

50000

UNK1 A, TRACE GC (D10407\009F3101.D)

DON CRACKER

Re

sp

on

se

GC-EC CHROMATOGRAMS

“Spiking” & mass spectral analysis of peak:

results consistent with DON

1 1 . 0 0 1 2 . 0 0 1 3 . 0 0 1 4 . 0 0 1 5 . 0 0 1 6 . 0 0 1 7 . 0 0 1 8 . 0 0 1 9 . 0 0 2 0 . 0 00

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

3 0 0 0 0

3 5 0 0 0

4 0 0 0 0

T i m e - - >

A b u n d a n c e

T I C : D 5 1 7 0 6 E . D

C r a c k e r E x t r a c t

1 1 . 0 0 1 2 . 0 0 1 3 . 0 0 1 4 . 0 0 1 5 . 0 0 1 6 . 0 0 1 7 . 0 0 1 8 . 0 0 1 9 . 0 0 2 0 . 0 00

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

2 2 0 0 0

T i m e - - >

A b u n d a n c e

T I C : D 5 1 7 0 6 D . D

D O N S t a n d a r d @ 4 . 5 4 n g / u l

Cracker

DON Standard

m/z

a; =512

b; =422 GC-MS

c; =295 (SIM)

d; =235

1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

3 5 0 0

4 0 0 0

4 5 0 0

5 0 0 0

5 5 0 0

6 0 0 0

6 5 0 0

7 0 0 0

7 5 0 0

m / z - - >

A b u n d a n c e

S c a n 3 1 2 ( 1 3 . 0 5 1 m i n ) : D 5 1 7 0 6 D .D1 0 3

2 3 5

2 9 5

4 2 2

5 1 2

D O N @ 4 . 5 4 n g /u l

ab

cd

1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 00

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

6 0 0 0

7 0 0 0

8 0 0 0

9 0 0 0

1 0 0 0 0

1 1 0 0 0

1 2 0 0 0

1 3 0 0 0

1 4 0 0 0

1 5 0 0 0

1 6 0 0 0

m / z - - >

A b u n d a n c e

S c a n 3 0 3 ( 1 2 . 9 6 4 m i n ) : D 5 1 7 0 6 E . D1 0 3

2 3 5

2 9 5

4 2 2

5 1 2

C r a c k e r

GC-MASS SPECTROMETRY

DON CONCENTRATIONS(Finished Flour Products)

Flour Cookie Cracker Pretzel Donut Bread

DO

N (

g g

-1)

0.0

0.2

0.4

0.6

0.8

1.0

##

#

#

ppm (= µg g-1)

----------------------------

Flour = 0.46 + 0.06

Cookie = 0.28 + 0.03

Cracker = 0.32 + 0.04

Pretzel = 0.51 + 0.05

Donut = 0.20 + 0.07

Bread = 0.14 + 0.01

-----------------------------

n = 9Each value = mean + SD, n = 3

#

DO

N M

ass B

ala

nce (

g g

-1 f

lou

r eq

uiv

ale

nt

basis

)

0.0

0.2

0.4

0.6

0.8

1.0

#

Flour Cookie Cracker Pretzel Donut Bread

#

ppm (= µg g-1)

----------------------------

Flour = 0.46 + 0.06

Cookie = 0.44 + 0.05

Cracker = 0.35 + 0.05

Pretzel = 0.49 + 0.07

Donut = 0.55 + 0.15

Bread = 0.23 + 0.02

-----------------------------

n = 9

MASS BALANCE ESTIMATE(Finished Flour Products)

Each value = mean + SD, n = 3

DO

N (

g g

-1)

0.0

0.2

0.4

0.6

0.8

1.0

Wheat Dough Pellets Flakes

#

#

DON CONCENTRATIONS(Wheat Series)

ppm (= µg g-1)

----------------------------

Wheat = 0.40 + 0.11

Dough = 0.34 + 0.10

Pellets = 0.29 + 0.08

Cereal

Flakes = 0.55 + 0.17

-----------------------------

n = 9

Each value = mean + SD, n = 3

Wheat Dough Pellets FlakesDO

N M

as

s B

ala

nc

e (

g g

-1 f

lou

r e

qu

iva

len

t b

as

is)

0.0

0.2

0.4

0.6

0.8

1.0#

ppm (= µg g-1)

----------------------------

Wheat = 0.40 + 0.11

Dough = 0.50 + 0.14

Pellets = 0.42 + 0.12

Cereal

Flakes = 0.58 + 0.18

----------------------------

n = 9

MASS BALANCE ESTIMATE(Wheat Series)

Each value = mean + SD, n = 3

% F

lou

r V

alu

e

0

20

40

60

80

100

120

140

Concentration

Mass Balance

Flour

Cooki

es

Cra

cker

s

Pre

tzel

s

Donuts

Bre

ad

Summary: Finished Flour Products

What Happened in Bread? Don’t know: chemical fate was not pursued

Thermal decomposition likely

-- norDON A

-- norDON B (Bretz et al., 2006)

-- norDON C

Found in cookies, crackers, breads (Germany)

-- norDON A most prevalent; found in 66% of the

items at concentrations ~ 10–25 % of DON

-- norDON compounds not toxic in an in vitro

assay using IHKE cells at 100 x EC50 of DON

Suggests that – at least in this case – degradation

might be beneficial

Does not rule out production of unknown toxins

What Happened in Bread?

Fermentation step also unique to bread in the

series of flour products examined

-- 2.5 % baker’s yeast, 4 hr at RT

Neira et al. (1997), Samar et al. (2001) reported

that fermenting contributed to reducing DON

in bread (41 – 56 %).

% I

nit

ial

(Fo

ur

Eq

uiv

ale

nts

)n

ot

co

rre

cte

d f

or

rec

ove

ry

0

20

40

60

80

100

120Shorter (20-45 min)

Longer (46-90 min) Fermentation time:

30 40 50 30 40 50

Fermentation Temp. 0

C

French Vienna

X

X = our result

What Happened in Bread?Bergamini et al. (2010) studied baking and

fermentation effects on DON during bread making

1. Fermentation time 2. Fermentation temperature

3. Baking time 4. Baking temperature

-- Fermentation increased DON in the dough

-- Baking time/temperature determined reduction

-- Initial DON concentration mattered; more reduction

was found at higher initial concentrations **

Industrial experiment: flour DON = 0.33 μg g-1;

fermentation conditions held constant (32 oC/56 min);

varied baking time/temperature from 170 – 225 oC for

3 – 13 min.

-- Final DON in breads ranged from 0.27 -0.32 μg g-1;

corresponded to 81 to 95 % of initial DON amount

** conflicts conclusion of Neira et al. 1997

Bread Studies Comparison

% Remaining

El-Banna et al. (1983): 115

Scott et al. (1983,84): 102, 107

Abbas et al. (1985): 36, 84

Seitz et al. (1986): 53-125

Neira et al. (1997): 56

Konishi et al. (2006): 108

Sugiyama et al. (2009): 74 (10 - >300)

Valle-Agarra et al. (2009): 52 (spiked wheat)

Bergamini et al. (2010): 81-96

This study (2010): 50

Summary: Wheat Series%

Wh

eat

Valu

e

0

20

40

60

80

100

120

140

160

Concentration

Mass Balance

Whea

t

Dough

Pel

lets

Flake

s

What Happened in Cereal?

Don’t know: chemical fate was not pursued

Release of DON from conjugates is possible

-- DON-glucoside (Berthiller et al., 2009)

Occur naturally in wheat (23/23 samples positive)

-- variable amounts reported

-- 12%-46% relative to DON (Berthiller et al. 2005; 2009)

Hypothesis: DON can be released from conjugates

under some conditions – should be tested.

CAUTION: The variation (large SD) found in our

analysis does not rule out that the increase was

incidental (heterogeneity of DON in wheat or

from sampling protocol) – needs more work

SUMMARY & CONCLUSIONS

Significant amounts of DON survive during

production of most products

Reduced DON in bread

• Concentration (ppm) = ~ 70% reduction

• Mass balance = 50% reduction

• Both “loss” and dilution contribute

Reduced DON (ppm) in finished donuts due

entirely to dilution and fat uptake

DON stable or increased somewhat in finished

wheat flake cereal

We have a lot of work to do to understand

• DON-food matrix interactions

• identity, occurrence, toxicity of decomposition

products

Working Hypothesis

DON 3-glucoside Other conjugates,

bound forms

Free DON

norDONs

Other

decomposition

productsMatrix

binding?

Bioavailability & Toxicity

ACKNOWLEDGEMENTS

ARS Toxicology & Mycotoxin Research Unit

•Emma (Crickett) Wray

•Norma Stewart

•Trevor Mitchell

Technical Personnel

• American Institute of Baking, Manhattan, KS

• Frito-Lay Inc., Plano, TX

• General Mills Inc., Minneapolis, MN

Funding: USDA Extramural Cooperative Agreement with

Grocery Manufacturers Association, Washington, DC

(National Food Processors Association Research Foundation)

THANKS

FOR YOUR ATTENTION

Voss KA, Snook ME (2010)

Food Additives and Contaminants 27:1694-1700