1. Formation of Acrylamide in Food
Lauren Jackson, Ph.D.
U.S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
National Center for Food Safety & Technology
Summit-Argo, IL
Food Advisory Committee
Contaminants and Natural Toxicants Subcommittee ACRYLAMIDE
December 4-5, 2002

2. OBJECTIVES
Summarize what is known about mechanisms, precursors and factors that affect acrylamide formation
Identify the research needs
Discuss the FDA Action Plan for Acrylamide- Formation
Understand the conditions that lead to the formation of acrylamide in food
Develop methods to prevent or reduce formation of acrylamide

3. What is Known About Acrylamide Formation?
How is acrylamide formed in food?
Precursors
Pathways/mechanisms
What factors affect acrylamide formation?
Food composition
Processing

4. Precursors of Acrylamide/Pathways of Formation
Acrolein
Acrylic acid
Amino acids alone
Amino acids + reducing sugars via Maillard browning/Strecker degradation

5. Acrolein Acrolein (2-Propenal) cH O
NH2 OH
(O)
(+NH3)
Acrolein
Acrylamide
Acrylic
Acid
Acrolein (2-Propenal)
Structurally similar to acrylamide
Formed in oil during frying
Also formed from thermal degradation of starches, sugars, amino acids and proteins
Disproven as the major acrylamide precursor**

6. Acrylic Acid Acrylic Acid
Structurally similar to acrylamide
Formed from thermal deamination of alpha- and beta-alanine
Formed from assorted di-acids (malic, tartaric) and amino acids (cysteine, serine)
Not believed to be the major acrylamide precursor** c
NH2 O H
(+NH3)
Acrylamide OH
Acrylic
Acid

7. Amino Acids Alone c Alanine Asparagine Glutamine Methionine
Amino acids alone not believed to be a major pathway in potatoes and grains
Relevance to acrylamide formation in other foods (coffee?) needs to be verified
H2N
Acrylamide O
NH2 OH R.
Asparagine
Glutamine c H
Stadler et al. Nature Vol Oct. 2002, p. 449

8. Amino Acids + Reducing Sugars (Maillard Reaction and Strecker Degradation)
What are the Maillard and Strecker reactions?
Reaction of amino acids with reducing sugars (glucose, fructose, ribose etc.) or other source of carbonyls
Responsible for color and flavor formation in heated foods
Reasons for suspecting mechanism
Potatoes have a relatively high levels of free amino acids
Potatoes and grain products are rich in carbohydrates (possible sources of reducing sugars and carbonyls)
Acrylamide levels in some foods tend to increase with level of browning

9. Which Amino Acids Form Acrylamide? Aqueous Model System Studies
Reaction mixture
mg acrylamide/
mole amino acid
Asparagine + glucose
221
Glycine + glucose
< 0.5
Cysteine + glucose
Methionine + glucose
Glutamine + glucose
Aspartic acid + glucose
Conditions: 0.1 mmole amino acid: 0.1 mmole glucose in 100 microliters of 0.5 M phosphate buffer (pH 5.5); 185°C, 20 min.
From: Mottram et al. (2002)

10. Which Amino Acids Form Acrylamide? Potato Chip Model System Studies
Acrylamide Formation
Potato starch <50 ppb
Potato starch + glucose <50 ppb
Potato starch + asparagine ppb
Potato starch + glucose + asparagine ppb
Other Amino Acids
Alanine <50 ppb Arginine <50 ppb
Aspartic Acid <50 ppb Cysteine <50 ppb
Lysine <50 ppb Methionine <50 ppb
Threonine <50 ppb Valine <50 ppb
Glutamine 156 ppb Asparagine ppb
From: Sanders et al. (2002)

11. Further Proof of Asparagine as Precursor of Acrylamide: Origin of Nitrogen and Carbons of Acrylamide
#1)
NH2 C
CH2
NH2 CH COOH O 15
15N-acrylamide
m/z 73
CH2
#2)
NH2 C
CH2
NH2 CH COOH O 15
Unlabeled Acrylamide
m/z 72
CH2
#3)
NH2 C
CH2
NH2 CH COOH O 15 13
15N13C13C13C-acrylamide
m/z 76
CH2
From: R.A. Sanders et al. (2002)

12. Mechanisms of Formation: A. Maillard Reaction/Strecker Degradation
Formation of acrylamide after Strecker degradation of asparagine (and methionine) in the presence of dicarbonyls (Maillard browning products)
Heating asparagine with butanedione, instead of glucose, resulted in acrylamide formation
From: Mottram et al. (2002)

13. Mechanisms of Formation: B. Formation from N-Glycosides
N-(D-glucos-1-yl)-L-asparagine
N-(D-fructos-2-yl)-L-asparagine
N-(D-glucos-1-yl)-L-glutamine
N-(D-glucos-1-yl)-L-methionine
From: Stadler et al. (2002)

14. Speculated Pathway B via Formation of N-Glycosides
(micromole per mole N-glycoside)
Acrylamide formed
Conditions: 180°C; 30 min; dry state
From: Stadler et al. (2002)

15. Formation of Acrylamide from Other Amino Acids + Sugars
Glutamine
Aspartic acid
Cysteine?
Methionine
believed to be the second most important precursor amino acid
May form acrylamide via N-glycoside formation as well as through Maillard/Strecker pathway
May be due to
impurities

16. Which Mechanism(s) Occur in Food?
% ASN (% of total free amino acids)
mg ASN/kg food
Levels of acrylamide in food after frying, baking or roasting
Potato 40
940
++++
Wheat flour 14
167
+++
High protein rye flour 18
173
Asparagus 30 ? ++
Almonds
34.4
Coffee (green) 12
Meat
< 5
N.D. or +
Foods high in asparagine/sugars tend to have greater acrylamide formation upon cooking

17. Which Mechanism(s) Occur in Food?
Potatoes: Asparagine/sugar; Maillard browning Strecker degradation
Use of asparaginase to treat potato (mashed) before frying decreased asparagine levels by 95% and acrylamide levels by >99% (Zyzak, 2002; personal communication)
Grain-based foods: Asparagine/sugars are believed to be precursors and Maillard browning/Strecker degradation are believed to be mechanism- Needs to be verified
Other foods:
Coffee, chocolate/cocoa, almonds- amino acid/sugar?
Meat- methionine/sugar?

18. What Factors Affect Acrylamide Formation?
Food composition
Precursors pH
Moisture
Other compounds
Processing conditions
Time
Temperature
Other

19. What Factors Affect Acrylamide Formation?
Food composition
Amino acids
ASN, MET, GLN, ASP, CYS
Other amino acids- LYS
Sugars
Fructose > glucose > sucrose (Becalski et al, personal communication)- aqueous model system
No difference in yield of acrylamide from D-fructose, D-galactose, lactose or sucrose (Stadler et al., 2002) under pyrolysis conditions pH
pH 8.0 > 5.5 > 3.0 (Becalski et al., personal communication)

20. What Factors Affect Acrylamide Formation?
Food composition
Moisture content
Effects unclear
Others
Sulfites- no effect on acrylamide formation in model systems (Zyzak et al., 2002; Becalski et al., personal communications)
Antioxidants- Rosemary extract had no effect on acrylamide production during frying (Becalski et al., 2002)
Glutathione/cysteine
Fermentation

21. What Factors Affect Acrylamide Formation?
Processing Conditions
Temperature- Yes
Time- Yes

22. Asparagine/glucose aqueous
Effect of Temperature
Asparagine/glucose aqueous
model system (closed)
In simple model systems:
At temperatures °C, acrylamide levels increase with processing temperature
Acrylamide forms at °C
May degrade at temperatures > 170°C?
From Mottram et al. (2002)

23. Effect of Temperature In food:
Oven-cooked French fries
In food:
Boiling and retorting produce little to no acrylamide
Frying and baking result in modest to high levels
Acrylamide levels increase with cooking/processing temperature
Oven Temperature (°C)
From: Tareke et al. (2002)

24. Effect of Temperature
160°C; 4 min
27 ppb
170°C; 4 min
70 ppb
180°C; 4 min
326 ppb
Acrylamide levels increased with frying oil temperature

25. Effect of Time Acrylamide levels increased with frying time 3.5min
12 ppb
180°C; 3.5 min
180°C; 4.0 min
46 ppb
180°C; 4.5 min
227 ppb
180°C; 5.0 min
973 ppb
Acrylamide levels increased with frying time

26. Summary of Research Findings
ASN/reducing sugar are important precursors for forming acrylamide in many foods; other amino acids may be important precursors in some foods
The Maillard reaction/Strecker degradation pathway is important in many foods
The acrolein pathway is unlikely
Processing conditions (time/temperature) are critical to levels of acrylamide in food

27. What Are the Research Gaps?
Measure the levels of free asparagine, other amino acids and reducing sugars in foods on a dry weight basis and correlate levels to acrylamide production during processing/cooking
Determine the mechanism(s) of formation of acrylamide in each food category
Determine the effects of time, temperature, pH, and moisture on acrylamide formation in various matrices
Measure the kinetics of acrylamide inhibition/destruction/scavenging under various reaction/process conditions

28. FDA Action Plan on Acrylamide: Formation
***Understand the food processing and cooking conditions that affect acrylamide formation, destruction, and inhibition in model systems and in food
FDA Research
CFSAN Exploratory Survey of Acrylamide Levels in U.S. Foods
FDA/NCFST work on effects of processing on acrylamide formation in food (potato products and baked grain products) and in model systems
Worldwide Research: WHO/JIFSAN clearing house

29. CFSAN Exploratory Survey of Acrylamide Levels in U.S. Foods

30. FDA Action Plan on Acrylamide: Formation
Determine the precursors/mechanisms resulting in acrylamide formation in foods
FDA Research
FDA/NCFST work on verifying precursors/mechanisms in grain products
Worldwide Research: WHO/JIFSAN clearing house
Understand the role of product composition on acrylamide levels in food
CFSAN Survey of Acrylamide Levels in Food

31. Research on Acrylamide Worldwide
U.S. - FDA; Food Industry; Trade Organizations; Academia
Canada - Health Canada
U.K. – Food Standards Agency; Univ. of Reading/Leeds; Leatherhead; Food Industry, Trade Organizations, Academia
Netherlands - Dutch Food Authority
Australia
France - AFSSA
Germany - BLL
Spain – CNCV/Univ. of Baeares/Rocasolano Institute & FIAB
Norway - MATFORSK
Switzerland - Government agencies; Nestle Research Centre
Sweden – Swedish Food Administration; Stockholm Univ.

32. Next Step
Study effects of processing time and temperature on formation of acrylamide in a model system and in food