Post on 12-Sep-2021
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