Formation, Occurrence and Strategies to Address Acrylamide in Food Robert Brown, Ph.D.

Possible mechanism for formation of acrylamide from asparagine OH OH C – CH2 – CH NH2 COOH O H2N HO + O OH OH C – CH2 – CH N COOH O H2N By now we all know that this is the reaction we are dealing with. Although it has been suggested that other pathways are theoretically possible, no data to support this. It seems likely that this reaction is a part of, or off-shoot of the Maillard reaction. This is a proposed reaction pathway from Mottram.

Possible mechanism for formation of acrylamide from asparagine C – CH2 – CH N COOH O H2N Maillard Products EA~25 - 50 kcal/mole - CO2 C – CH = CH2 O H2N C – CH2 – CH2 N O H2N Beta-elimination By now we all know that this is the reaction we are dealing with. Although it has been suggested that other pathways are theoretically possible, no data to support this. It seems likely that this reaction is a part of, or off-shoot of the Maillard reaction. This is a proposed reaction pathway from Mottram. EA ~70 kcal/mole

Insights The chemical pathway leading to AA is a low yield pathway with high activation energy.

Summary of Acrylamide Values in Food Realize that this slide is difficult to read, but all data are publicly available on various websites. Want to make the points that: Widespread across many different food types – clearly not a “potato issue” Measured amounts vary greatly within and across food categories This is only what we know so far- as additional foods are examined the list will surely grow longer. Any interventions we can contemplate to address this question will have widespread effects.

Calories and Nutrient Intake Foods tested and found to contain acrylamide (so far) constitute: 38% of calories 33% of carbohydrates 36% of fiber 28% of fat 20% of calcium 47% of iron 25 to 35% of other micronutrients 15% of vitamin A 34% of vitamin E 22 to 44% of B, C and folate vitamins

Insights The chemical pathway leading to AA is a low yield pathway with high activation energy. The AA question affects a large fraction of the food supply, calories and nutrients commonly consumed.

Dealing With The Acrylamide Issue Remove Acrylamide after formation Remove Reactants Disrupt Reaction Like many people my initial reaction to the story out of Sweden was that it must be a mistake. As events unfolded last summer and it became clear that the results were real, F-L formed a project team of some of our best scientists and engineers to look into this question. Fairly quickly our thinking coalesced into 4 distinct boxes, and we divided our team up to work in each of these areas. We could try to get rid of the substrates for the reaction. We could possibly do something to disrupt the reaction and prevent AA from forming. This was the successful approach used to deal with nitrosamines in meat (ascorbate). AA is supposedly reactive, so could we do something to get rid of it after it was formed. Finally, under Toxicology we could have titled this box “What does it all mean?” This may be the most important box because it is the perceived hazard that drives this issue, not the mere fact that it is present in food. Toxicology

Dealing With The Acrylamide Issue Remove Acrylamide after formation Remove Reactants Disrupt Reaction Toxicology

Summary table of results - CSL 12000 12800 Over cooked 3500 Cooked 100 200 Frozen frying chips as sold 2800 Chipped & fried Nd <10 boiled <30 King Edward potatoes raw 350 310 nd Boiled LC-MS-MS GC-MS Baking potatoes raw SNFA result (µg/kg) Acrylamide concentration (µg/kg) Sample Acrylamide formation influenced by starting raw material This slide was kindly supplied by Don Mottram, and shows some data from the UK. The finding that two different varieties of potato, prepared similarly, have vastly different AA levels is an important clue to what may be going on, and leads to some potential intervention strategies. Clearly the starting material has some impact.

Asparagine in Various Crops Cheese 40 – 300 Asparagus 5.4 – 108 Cocoa (raw) 30.9 - roasted @ 125C 14.5 - roasted @ 135C 9.4 Potato 0.5 – 10 mg/g Rye 0.2 – 2.8 Wheat 0.02 – 2 Corn 0.6 – 1 Also in peanuts, soybeans, onions, coffee, tomatoes, fruits, etc. From Ellin Doyle, Ph.D., Food Research Inst., U. Wisc. Wide range of results within and across crops. Not understood at this time how much is genetic variation vs. effects of storage conditions.

Surface Plot of AA/Substrate Relationship 0.0 10000 20000 AA (ppb) 0.1 0.2 0.3 0.4 ASN 20000 30000 0.2 0.5 0.0 0.2 0.8 0.6 GLU 0.4 0.2 1.0 AA = -245.2 - 427.9*(ASN) + 460.1*(GLU) + 60582.7*(ASN)*(GLU) R-Squared = .97

Insights The chemical pathway leading to AA is a low yield pathway with high activation energy. The AA question affects a large fraction of the food supply, calories and nutrients commonly consumed. The chemical reaction of asparagine and glucose is second order when the substrates are approximately equal. When one is substantially lower it becomes rate-limiting.

Dealing With The Acrylamide Issue Remove Acrylamide after formation Remove Reactants Disrupt Reaction Toxicology

Summary table of results - CSL 12000 12800 Over cooked 3500 Cooked 100 200 Frozen frying chips as sold 2800 Chipped & fried Nd <10 boiled <30 King Edward potatoes raw 350 310 nd Boiled LC-MS-MS GC-MS Baking potatoes raw SNFA result (µg/kg) Acrylamide concentration (µg/kg) Sample Yield of acrylamide increases substantially with browning

Effect of Temperature on AA Formation 1% gluc, 0.2% asn in sodium phosphate at pH 7.0 for 15 minutes.

AA Formation at 15 Minutes as a function of Temperature 380 400 420 440 Temperature (Kelvin) 5000 10000 15000 20000 Acrylamide (ppb) AA Formation at 15 Minutes as a function of Temperature AA = 442.3 * e (.07930*(Temp-383))

Insights The chemical pathway leading to AA is a low yield pathway with high activation energy. The AA question affects a large fraction of the food supply, calories and nutrients commonly consumed. The chemical reaction of asparagine and glucose is second order when the substrates are approximately equal. When one is substantially lower it becomes rate-limiting. AA formation is temperature critical and occurs well below temps at which food is commonly cooked. It will probably not be possible to cook food without forming at least some AA.

Effect of pH on Acrylamide Formation

Prevent Asparagine and Glucose Reaction The Idea + + Raw Reaction Cooking Reduced Food Inhibitor Acrylamide Watchout: The inhibitor(s) must be food safe.

Kinetic model (Wedzicha & Mottram) g l u c o s e I 1 2 m a n i d ( r ) f v t k 3 4 , 5 . y Rate constants Allow the rate of each step to be quantified in terms of reaction variables: pH, T, concentration of glucose and amino acid

Dealing With The Acrylamide Issue Remove Acrylamide after formation Remove Reactants Disrupt Reaction We have investigated a couple of possibilities here but have no success. Supercritical CO2 extraction removes aa but destroys the product. Have looked at light energy of various wavelengths, the theory being that we could force aa to react in situ. Although an interesting idea, nothing has worked in either the test-tube or in products. Toxicology

Remove After Formation Supercritical CO2 removes everything but destroys the product UV light no effect, several wavelengths including visible

Dealing With The Acrylamide Issue Remove Acrylamide after formation Remove Reactants Disrupt Reaction Toxicology

Relative Exposure to Acrylamide in U.S. Food French Fries & Potatoes Coffee Cakes Dried Foods Pop Corn Salty Snacks Chocolate Products Nuts/Seeds/Butters Breads Potato Chips Cereal Biscuit / Cookies All Other Foods

Relative Exposure – All Potato Products Zero Coffee Cakes Dried Foods Pop Corn Salty Snacks Chocolate Products Nuts/Seeds/Butters Breads Cereal Biscuit / Cookies All Other Foods

Food For Thought The notion of “carcinogens” in food is not new (cooked meat, NAS report, “Ames/Gold” list). Humans have eaten these foods for millennia. There are no obvious “quick fixes” or magic bullets. Much of what we have learned looks interesting, but the solution has not been found.

Feasibility Analysis Removal of substrates must take into account kinetics of formation along with importance of other constituents. Low temperature intervention will require development of new cooking methods. Some foods will be impossible to cook at low temperature. No universal “magic bullets” have been found. Addition of substances may work for some products but with variable efficacy. There is no precedent for an intervention into the food supply on this scale

Final Thoughts The issue affects a large portion of the food supply. Lowering acrylamide in one or a few foods has no effect- everything must be changed. Food cooked at home and in restaurants is a big challenge, and a significant source of acrylamide exposure. What does victory look like? Given the magnitude of change to the food supply we need to fully understand two things: the nature of the low dose hazard to humans, and the impact of any proposed interventions. Are there any unintended consequences to public health?