Formation of Acrylamide in Food

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. H O H Acrolein c c c H H (O) H O H c c c Acrylic Acid OH H (+NH3) H O H Acrylamide c c c NH2 H Acrolein • 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. H O H c c c Acrylic Acid OH H (+NH3) H O H Acrylamide c c c NH2 H 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**

7. NH2 O OH H2N H2N OH O NH2 O O Asparagine Glutamine R. H O H c c c NH2 H Acrylamide Amino Acids Alone • 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 Stadler et al. Nature Vol 419 3 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 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 117 ppb • Potato starch + glucose + asparagine 9270 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 ppbAsparagine 9270 ppb From: Sanders et al. (2002)

11. 15 15 NH2 NH2 #1) C C O O CH2 CH2 NH2 CH COOH NH2 NH2 #2) C O C O CH2 CH2 15 NH2 CH COOH 15 NH2 15 NH2 #3) 13 C O 13 C O 13 CH2 13 CH2 13 13 15 NH2 CH COOH 13 Further Proof of Asparagine as Precursor of Acrylamide:Origin of Nitrogen and Carbons of Acrylamide 15N-acrylamide m/z 73 CH2 Unlabeled Acrylamide m/z 72 CH2 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 Bvia Formation of N-Glycosides Acrylamide formed (micromole per mole N-glycoside) 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. Foods high in asparagine/sugars tend to have greater acrylamide formation upon cooking Which Mechanism(s) Occur in Food?

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, 2002- 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., 2002- 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., 2002- 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. Effect of Temperature Asparagine/glucose aqueous model system (closed) In simple model systems: • At temperatures 120-170°C, acrylamide levels increase with processing temperature • Acrylamide forms at 120-140°C • May degrade at temperatures > 170°C? From Mottram et al. (2002)

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

25. 3.5min 12 ppb 180°C; 3.5 min 12 ppb 180°C; 4.0 min 46 ppb 180°C; 4.5 min 227 ppb 180°C; 5.0 min 973 ppb Effect of Time • 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 • FDA Research • CFSAN Survey of Acrylamide Levels in Food • Worldwide Research: WHO/JIFSAN clearing house

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