# Presentation: 948246

## 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)
- 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****

- c

- c

- H

- c

- O

- H

- H

- H

- c

- c

- NH2

- c

- O

- H

- H

- H

- c

- c

- OH

- c

- O

- H

- H

- H

- (O)

- (+NH3)

- Acrylamide

- 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

- c

- NH2

- c

- O

- H

- H

- H

- (+NH3)

- Acrylamide

- c

- c

- OH

- c

- O

- H

- H

- H

- Acrylic
- Acid

## 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*

- Acrylamide

- H2N

- O

- NH2

- O

- OH

- H2N

- O

- NH2

- O

- OH

- R.

- Asparagine

- Glutamine

- Stadler et al. Nature  Vol 419  3 Oct. 2002, p. 449

- c

- c

- NH2

- c

- O

- H

- H

- H

## 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 | < 0.5 |
| Methionine + glucose | < 0.5 |
| Glutamine + glucose | 0.5 - 1.0 |
| Aspartic acid + glucose  | 0.5 - 1.0 |

- 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)

- #2)

- #3)

- 15N-acrylamide
- m/z 73

- Unlabeled Acrylamide
- m/z 72

- 15N13C13C13C-acrylamide
- m/z 76

- Further Proof of Asparagine as Precursor of Acrylamide:Origin of Nitrogen and Carbons of Acrylamide

**From:  R.A. Sanders et al. (2002)**

- NH2

- C

- CH2

- NH2  CH  COOH

- O

**15**

- NH2

- C

- CH2

-        CH2

- O

**15**

- NH2

- C

- CH2

- NH2  CH  COOH

- O

**15**

- NH2

- C

- CH2

-        CH2

- O

- NH2

- C

- CH2

- NH2  CH  COOH

- O

**15**

**15**

**13**

**13**

**13**

**13**

- NH2

- C

- CH2

-        CH2

- O

**15**

**13**

**13**

**13**

## 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

- From:  Stadler et al. (2002)

*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

## Speculated Pathway Bvia Formation of N-Glycosides

- From:  Stadler et al. (2002)

- Conditions:  180°C; 30 min; dry state

- Acrylamide formed
- (micromole per mole N-glycoside)

## 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?

- Foods high in asparagine/sugars tend to have greater acrylamide formation upon cooking

| 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 + |

## 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

## Processing Conditions
Temperature- Yes
Time- Yes

- Temperature- Yes
- Time- Yes

**What Factors Affect Acrylamide Formation?**

## Effect of Temperature

- 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)

- Asparagine/glucose aqueous
- model system (closed)

- In simple model systems:

## Effect of Temperature

- From: Tareke et al. (2002)

- 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-cooked French fries

- Oven Temperature (°C)

## Effect of Temperature

- Acrylamide levels increased with frying oil temperature

- 160°C; 4 min
- 27 ppb

- 170°C; 4 min
- 70 ppb

- 180°C; 4 min
- 326 ppb

## Effect of Time

- Acrylamide levels increased with frying time

- 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

## 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
- FDA Research
- CFSAN Survey of Acrylamide Levels in Food
- Worldwide Research:  WHO/JIFSAN clearing house

## 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