Acrylamide: Mechanism of Acrylamide: Mechanism of Formation in Heated FoodsFormation in Heated Foods

David Zyzak, Ph.D.Procter & Gamble

Snacks and Beverage Analytical and Microbiology Cincinnati, Ohio

ACRYLAMIDE SHOCK ACRYLAMIDE SHOCK Press Release April 24, 2002Press Release April 24, 2002

Stockholm University/Swedish NFA revealed acrylamide presence in variety of cooked foods.

Food Product Acrylamide (ppb)Toasted English Muffin, 5 min 50

Tortilla Chips 97

Baby Food Potatoes 101

Banana Chips 125

Roasted Asparagus 143

Pretzels 196

Hearty Rye Crispbread 242

Baked Potato Chips 317

Corn Chips 331

Cooked Taco Shell 559

Blue Potato Chips 736

Kettle Potato Chips 3400

Sample Survey Results

What is Acrylamide?What is Acrylamide?

• high boiling point

CH2

CH

C

NH2

O

Acrylamide

• very hydrophilic – water loving

CH2

CH

C

NH2

O

Acrylamide

CH COOHNH2

CH2

C

NH2

O

Asparagine

carbonyl

CH2

CH

C

OH

O

Acrylic Acid

NH3

X X

CH2

CH

C

H

O

Acrolein

NH3

Initial Thoughts on the Mechanism of Acrylamide Formation

CH COOHNH2

CH2

CH2

C

NH2

O

Glutamine

Effectiveness of Amino Acids and Dextrose to Form Acrylamide

Acrylamide Formation– Potato starch <50 ppb– Potato starch + dextrose <50 ppb– Potato starch + asparagine 117 ppb– Potato starch + dextrose + asparagine 9270

ppb

Potato Starch + Water

Amino acid Reducing sugar Variety of ingredients

+fry

Measure Acrylamide

Model System

Other Amino Acids–Alanine <50 ppb Arginine <50 ppb–Aspartic A. <50 ppb Cysteine <50 ppb–Lysine <50 ppb Methionine <50 ppb–Threonine <50 ppb Valine <50 ppb–Glutamine 156 ppb Asparagine 9270 ppb

Amino Acid Composition in Potatoes

Approximately 50% of amino acids are in the free state (not incorporated into protein).

Asparagine is roughly half of the free amino acid content.

Free vs. Bound Asparagine

Asparagine occurring as component of protein does not have an accessible primary amine group for Schiff base formation, and would not be expected to participate in the production of acrylamide. Blocking the amine group in asparagine, N-acetyl asparagine, is an effective analogue to test.

Result: No acrylamide formation observed

CH COOHNH

CH2

C

NH2

O

C

O

CH3

+ dextrose Acrylamide?

N-acetyl asparagine

Dose/Response: DextroseDose/Response: DextroseAcrylamide Formation: Dextrose Kinetics

Dextrose [%]

0.0 0.5 1.0 1.5 2.0 2.5

Acry

lam

ide

[p

pm

]

0.0

0.5

1.0

1.5

2.0

2.5

Asparagine is 10 g in the model system

Acrylamide = k [Dextrose]0.99

r2 = 0.99

Asparagine at 1.25%

Other Carbonyl Sources Which Produce Acrylamide

Also: ribose

All of these carbonyl sources produce significant acrylamide in the model system with asparagine.

O

O

H

H CH2OH

CHOH

C OH

GLYCERALDEHYDEGLYOXAL

CHOH

CH2

C OH

CHOH

CHOH

CH2OH

2-DEOXYGLUCOSE

Use of Isotopes to Understand the Mechanism Use of Isotopes to Understand the Mechanism of Acrylamide Formation from the Reaction of of Acrylamide Formation from the Reaction of

Asparagine and Dextrose and Dextrose

CH2

CH

C

NH2

O

Acrylamide

CH COOHNH2

CH2

C

NH2

O

Asparagine

carbonyl

Label Expt #1: AmideLabel Expt #1: Amide1515N-Labeled AsparagineN-Labeled Asparagine

ISOTOPE 15N AMIDE

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00Time0

100

%

0

100

%

jul1109 SIR of 2 Channels ES+ 73

6.20e53.22

0.370.04

2.51

jul1109 SIR of 2 Channels ES+ 72

6.20e5

1.93 3.20

m/z 73Mono-labeled Acrylamide

m/z 72 Unlabeled Acrylamide

97+ % of Total Acrylamide Response

CH2

CH

C

15NH2

O

CH COOHNH2

CH2

C

15NH2

O + dextrose m/z 73

Label Expt #2: Amine Label Expt #2: Amine 1515N-Labeled AsparagineN-Labeled Asparagine

ISOTOPE AMINE

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00Time0

100

%

0

100

%

JUL0833 SIR of 2 Channels ES+ 73

1.27e5

0.18 0.70 1.59 2.75 3.18

JUL0833 SIR of 2 Channels ES+ 72

1.27e53.24

1.97

0.042.33

m/z 72 Unlabeled Acrylamide

m/z 73

CH2

CH

C

NH2

O

CH COOH15NH2

CH2

C

NH2

O+ dextrose m/z 72

Label Expt #3: Uniformly Labeled AsparagineLabel Expt #3: Uniformly Labeled Asparagine

43703

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00Time0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

SEPT0526 SIR of 5 Channels ES+ 76

1.90e53.00

0.052.33

SEPT0526 SIR of 5 Channels ES+ 75

1.90e52.330.05

0.21 1.950.43 0.80 1.471.20 2.97 3.693.80

4.66 5.305.43

SEPT0526 SIR of 5 Channels ES+ 74

1.90e54.713.212.762.511.391.020.640.05

0.43 2.063.53 4.093.85

4.205.895.38

5.976.37 6.75

SEPT0526 SIR of 5 Channels ES+ 73

1.90e50.05

0.24 1.52 2.35 2.57 3.40

SEPT0526 SIR of 5 Channels ES+ 72

1.90e5

0.051.952.11 5.70

m/z 76

m/z 75

m/z 74

m/z 73

m/z 72

Tetra-labeled Acrylamide

m/z 76

13CH2

13CH

13C

15NH2

O+ dextrose

13CH13COOH15NH2

13CH2

13C

15NH2

O

Acrylamide Formation MechanismAcrylamide Formation Mechanism

CH2

CH

NH2O

+ NH3

ACRYLAMIDEMW 71 (75)

Beta alanine amideMW 88 (93)

R1R2

O

NH2CH

CH2

NH2O

OH

O

+

NCH

CH2

NH2O

OH

O

H

R1R2

HO

NCH

CH2

NH2O

OH

OR2

R1

NCH2

CH2

NH2O

R2

R1CO2

NH

R2

R1

CH2

CH

NH2O

+

H2O

O

R2

R1

H2NCH2

CH2

NH2O

+

ACRYLAMIDEMW 71 (75)

carbonyl sourceMW (dextrose) 180 asparagine

MW 132 (138)

Schiff baseMW 294 (300)

decarboxylation

H2O

H2OH2O

MW 250 (255)

7 2 7 68 9 9 42 9 5 3 0 11 8 0 1 8 01 3 3 1 3 9

7 2 7 68 9 9 42 9 5 3 0 11 8 0 1 8 01 3 3 1 3 9

7 2 7 68 9 9 42 9 5 3 0 11 8 0 1 8 01 3 3 1 3 9

m /z

179 A t = 0

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00Time0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

SEPT0433 Scan ES+ 72

2.00e5

SEPT0433 Scan ES+ 89

2.00e5

1.940.75 2.76 4.54 4.88

SEPT0433 Scan ES+ 295

2.00e5

SEPT0433 Scan ES+ 180

2.00e52.06

SEPT0433 Scan ES+ 133

2.00e5

Monitoring Intermediates in Acrylamide Formation Monitoring Intermediates in Acrylamide Formation

13CH13COOH15NH2

13CH2

13C

15NH2

O

CH COOHNH2

CH2

C

NH2

OA B

Understanding Acrylamide Formation in Food Products

•Selective removal of asparagine with asparaginase to address these questions.

•What about other potential sources of acrylamide? methionine, glutamine, cysteine, acrolein, etc…

• Is asparagine the only precursor to acrylamide in heated foods?

Asparaginase: Mode of Action

H2O

asparagine

asparaginase

NH3+

ammonia

aspartic acid

OH

O

NH2CH

CH2

NH2O

OH

O

NH2CH

CH2

HOO

Asparaginase Experiment on Potato Product

Boil for 1 hour

Blend flesh 1:3 with distilled water

Asparaginase-treated

Microwave @ 2 min intervals for total of 10 min.Highly Cooked to Maximize Acrylamide Formation (both control and asparaginase-treated products were dry and brown)

Control45 min @ RT

Washed Russet Burbank Potatoes

Asparagine Analysis of Enzyme-Treated Potato Product

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24

Vo

lts

0.0

0.2

0.4

0.6

0.8

1.0

Vo

lts

0.0

0.2

0.4

0.6

0.8

1.0

Fluorescence Detector (Ex:260nm, Em:313nm)A202AUG01-07

Minutes0 2 4 6 8 10 12 14 16 18 20 22 24

Volts

0.0

0.2

0.4

0.6

0.8

1.0

Volts

0.0

0.2

0.4

0.6

0.8

1.0

Fluorescence Detector (Ex:260nm, Em:313nm)E102AUG01-08

Asparaginase treated

ControlA

spar

agin

e

Asp

arat

ic

acid

Asp

arag

ine

Unreacted FMOC

ISTD

 

 

 

 

   

Potato Product

Microwaved snack

Acrylamide (ppb)Control Asparaginase

20,500 164

% Reduction1

>99

1Calculated as (control – asparaginase treated)/control x 100.

Asparaginase Reduces Acrylamide in Cooked Potato Products

Acrylamide Precursors – Where to Intervene

Asparagine Reducing Sugars- Glucose- Fructose- Sucrose hydrolysis?

• Factors affecting asparagine and reducing sugars- Variety of potato- Storage conditions

ConclusionsConclusions

• Asparagine is the major source of acrylamide formation in foods.

• Carbonyl source (reducing sugars) is required in the reaction.

• Oil oxidation products and starch do not appear to be significant factors in acrylamide formation.