stability of the trichothecene, deoxynivalenol in processed foods
TRANSCRIPT
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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
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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
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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
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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
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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
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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
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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
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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
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Wheat Series: Product Stream(proprietary)Wheat
Grind, mix ingredients
Dough
Extrude
Pellets
Roll
Cereal Flakes
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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
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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
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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
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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
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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
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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
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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
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#
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
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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
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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
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% 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
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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
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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
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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
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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
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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
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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
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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
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Working Hypothesis
DON 3-glucoside Other conjugates,
bound forms
Free DON
norDONs
Other
decomposition
productsMatrix
binding?
Bioavailability & Toxicity
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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)
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THANKS
FOR YOUR ATTENTION
Voss KA, Snook ME (2010)
Food Additives and Contaminants 27:1694-1700