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

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

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

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

Acrolein Acrolein (2-Propenal) c H 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**

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

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 419 3 Oct. 2002, p. 449

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

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 0.5 - 1.0 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)

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 ppb Asparagine 9270 ppb From: Sanders et al. (2002)

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)

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)

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)

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)

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

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

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?

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

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)

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

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

Asparagine/glucose aqueous 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)

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)

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

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

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

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

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

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

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

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.

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