Acrylamide Mitigation Strategies

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2 Acrylamide Mitigation Strategies Stephen Saunders, PhD

3 What happens when we cook food? 1. Dehydrate: drive off moisture 2. Drive formation of flavor compounds via Maillard (browning) reactions

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6 Formation of Acrylamide From Asparagine H 2 N C CH 2 CH OH OH NH 2 + O HO O COOH OH OH H 2 N N C CH 2 CH O COOH

7 Formation of Acrylamide From Asparagine H 2 N N C CH 2 CH Maillard Products O COOH E A ~25-50 kcal/mole H 2 N - CO 2 N H 2 N C CH = CH 2 O C CH 2 CH 2 Beta-elimination O E A ~70 kcal/mole

8 Effect of Temperature on AA Formation (Test Tube) Acrylamide (ppb) Temperature (C) 1% gluc, 0.2% asn in sodium phosphate at ph 7.0 for 15 minutes.

9 Effect of ph on Acrylamide Formation (Test Tube) AA (ppb) C, 40 min 150 C, 15 min ph

10 The Scope of the Problem: How do we stop this reaction and still make the product we want? ASN + Sugar Heat (time) AA Important Fact #1: This is the same Maillard reaction that produces flavor during cooking! Important Fact #2: Only FREE ASN (not ASN bound in protein) participates in this reaction!

11 Can we selectively remove ASN? ASN + Sugar Heat (time) AA ASN + Asparaginase Aspartate + NH3

12 What is Asparaginase? An enzyme is a protein the speeds up (catalyzes) a specific chemical reaction, in this case the conversion of asparagine to aspartic acid. Thus a required substrate for AA formation, ASN, is selectively removed. Enzyme activity depends on: Temperature ph Moisture Degree of mixing (how long and vigorous) Dose Currently two commercial sources: DSM and (Novozymes?)

13 Where has ASNase been reported to work? Seems to be most effective for products made from dough: Tortilla chips Fabricated PC Pellet products Crackers, cookies Bread Pretzels Breakfast cereals Other? Coffee French fries

14 PreventASe Product range PreventASe (L/M/W) Potato Snacks Infant Cereals PreventASe (L/M/W) & XR Pretzels Biscuits & Cookies PreventASe XR Tortilla Chips Crackers The DSM PreventASe range is suitable for a wide range of processing temperatures (20 C-60 C)* and ph (2 to 9). Dried Potato French Fries Cereal Snacks Breakfast Cereals Bread (Substitutes) Corn Chips The choice of optimal solution depends on endapplication, ingredients and manufacturing process. Infant Biscuits Bread (Substitutes) Dried Fruits 14 * Enzyme deactivated at 65 C

15 PreventASe Application Areas PreventASe Tortilla/Corn Chips Pretzels/Salty Sticks Infant Cereals Baby/Infant Biscuits Potato Snacks / Pellets Fabricated Snacks From Sheeting & Cutting Savory/Snack Cracker Breakfast Cereals The effectiveness of PreventASe and hence acrylamide reduction is depended on factors such as: - Mixing time & intensity - Processing times - Temperatures - Substrate accessibility Biscuits & Cookies French Fries (Crisp) Bread Specialty Products 15

16 16 DSM Food Specialties USA Inc- First point of contact: Leah Roach Phone

17 Enzyme Treatment of Potato Flakes (Dec 03) Asparaginase enzyme added to mash, post-cook, and held for 40 minutes AA Model System Asparagine Reduction Fabricated PC 960 Control 3.50% 3.00% 3.50% % % AA 93% ASN 99.8% % 1.50% % % % 0.00% 0 Enzyme Control Enzyme Control Enzyme Challenges Enzyme was very expensive ($6K to make 5 lbs. of flakes!) Flake process scale-up

18 PreventASe Asparagine Reduction - Tortilla Significant asparagine reduction (up to 84%); Dosage recommendation 45 ppm Asparagine Level (g/kg) Masa Flour (ph 8.0) Asparagine Reduction (%) Masa Flour (ph 8.0) % 83% 83% % % CONTROL PREVENTASE XR 10 PPM PREVENTASE XR 20 PPM PREVENTASE XR 30 PPM PREVENTASE XR 40 PPM PREVENTASE XR 50 PPM CONTROL PREVENTASE XR 10 PPM PREVENTASE XR 20 PPM PREVENTASE XR 30 PPM PREVENTASE XR 40 PPM PREVENTASE XR 50 PPM 18

19 PreventASe Significant acrylamide reduction (up to 88%); Dosage recommendation 45 ppm Acrylamide Amount (µg/kg) Tortilla Chips (ph 7.2) Acrylamide Reduction (%) Tortilla Chips (ph 7.2) % 88% 87% 87% CONTROL PREVENTASE XR 45 PPM PREVENTASE XR 60 PPM PREVENTASE XR 90 PPM PREVENTASE XR 100 PPM CONTROL PREVENTASE XR 45 PPM PREVENTASE XR 60 PPM PREVENTASE XR 90 PPM PREVENTASE XR 100 PPM Acrylamide Amount (µg/kg) Tortilla Chips (ph 8.0) Acrylamide Reduction (%) Tortilla Chips (ph 8.0) % 87% CONTROL PREVENTASE XR 45 PPM PREVENTASE XR 90 PPM CONTROL PREVENTASE XR 45 PPM PREVENTASE XR 90 PPM 19

20 Other Strategies to Reduce ASN? Variety selection But remember, only free ASN participates in reaction. Most published values for ASN content of grains is hydrolyzed, total ASN. Growing conditions For example, wheat grown under low sulfur conditions shown to increase free ASN Storage and handling Potatoes Breeding/GMO Blanching, washing, etc. So far, no selective way to wash or leach out ASN; this will remove sugar and other things you might need to produce flavor

21 The Scope of the Problem: How do we stop this reaction and still make the product we want? ASN + Sugar Heat (time) AA Is removing sugar an option?

22 Is Removing Glucose An Option? Recall that we need some sugar to produce Maillard flavor compounds during cooking Depending on the product/process could theoretically reduce sugar and still make a good product Low sugar chipping potatoes vs. baking potatoes Corn, other grain varieties? Classically, fermentation with yeast converts glucose into EtOH Glucose oxidase Converts glucose to gluconic acid Washing, blanching

23 The Scope of the Problem: How do we stop this reaction and still make the product we want? ASN + Sugar Heat (time) AA Can we disrupt the reaction?

24 Disrupt the Reaction? ph-dependent: below about 4 (in test tube) no AA formation (but also inhibits Maillard) Divalent cations Ca ++, Mg ++ disrupt reaction by tying up glucose Will also disrupt Maillard Heat Uncontrolled heat is the enemy: process control is your friend! How and where is heat applied in your process? Most AA is formed at the end: moisture decreases as finished product temperature increases. Remove burnt and out-of-spec bits from finished product stream

25 Controlling Heat: Vacuum Frying Takes advantage of relationship between BP of water and pressure Thus can fry product at lower temperature and boil off water (dehydrate) But, Maillard is not affected by pressure!

26 AA Formation at 20 Minutes as a function of Temperature Acrylamide (ppb) AA = * e (.04878*(Temp-373)) 384 K = 111 C = 232 F Temperature (Kelvin)

27 AA Formation during Frying at various final Moistures AA - ppb % Moisture Temp 353 Temp 300F Power (Temp 353) Power (Temp 300F) Rate of AA formation very steep between 1% - 2% AA profile in fryer indicates most AA is formed between 1% - 3% moisture.

28 % Moisture AA - ppb AA =? Product? Most snacks have finished product moisture in the 1 2% range What if we pull product out here, and then finish dry at low temp?

29 The Scope of the Problem: How do we stop this reaction and still make the product we want? ASN + Sugar Heat (time) AA Most of what we have talked about today can be found in the FoodDrinkEurope publication ( Other useful information can be found in Guidance to Industry from FDA (

30 Step 1: Clarify your situation What are you trying to accomplish? A reduction of xx%? A specific target of xx ppb? Do you have a clear idea of how much AA is your product(s)? Ideally you will have data over time- across all shifts and lines Do you know where in your process AA forms? Do ingredients contribute to AA in the final product? Molasses BBQ seasoning Olive oil others (FDA, HC, EFSA databases)

31 Step 2: Develop Your Strategy: Raw Materials and Ingredients Can we source raw materials with lower ASN and/or sugar? Product quality impact Cost/benefit Do we have a problem with GMO in our product? What if we have to label (Europe)? What about ingredients? Molasses reported as high as 900 ppb Canned olives up to 1000 ppb (olive oil?) BBQ Too many to list here, check FDA for where AA has been found

32 Step 2: Develop Your Strategy: Process Interventions For each unit operation where AA forms, identify potential interventions Is the enzyme an option (Plan A)? More likely to be successful if tested first on benchtop (small scale) Identify optimal conditions for enzyme activity: dose, time, temperature, moisture, ph Requires ability to measure ASN before and after treatment Even if the enzyme is effective, is it necessary? Can we achieve the desired reduction through other means? If the enzyme is not effective, Plan B!

33 Step 2: Develop Your Strategy (Plan B) Can we lower the ph during the process step where AA forms? What agents will we use to acidify? If we need to raise ph back to neutral, what will we use? How does this affect product quality? What about divalent cations? Ca++, Mg++ What about adding other amino acids to compete with ASN? Cysteine reported to work in some products What about a combination of several agents? Unfortunately creates a large matrix of possibilities At this point don t get hung up on cost or technical feasibility- just trying find out if something works!

34 Step 3: Refine the approach An iterative approach, as more data come in we can reject things that are not going to work and zero in on those that show promise. In the beginning don t worry about technical feasibility or effects on product quality- just see if you can find things that work. Once you have some idea of what works, now begin to zero in on an approach with eye towards product quality, technological feasibility and cost Prepare yourself: there is always a trade-off of some sort.

35 Step 4: Scale-Up We have an approach that works on small scale. Now: Do we need new unit op(s)? Introduce enzyme, build in time and conditions for optimal activity Introduction of acids, salts and/or other compounds Temperature controls Whatever we do, we need to meter, measure, and monitor effectiveness Regulatory considerations IMHO, enzyme is a processing aid and therefore does not need to be declared on label. FDA has confirmed GRAS status in several products Enzyme is derived from microbe genetically-modified to over-express genes for ASNase. European considerations? Impact of other additives on label? (Might be able to make a calcium claim!)

36 Case Study: Stevie Thunder s Fruit Energy Bar The situation: The ST Company has received notice under CA Prop 65 from a plaintiff s group. They inform your fruit bar contains 450 ppb. Negotiations with the plaintiff s has resulted in a proposed settlement agreement that requires A reduction of your average AA from 450 to 200 ppb Periodic compliance checks A commitment to continually work towards lowering AA even further Plaintiff s legal fees and other expenses

37 Step 1: Clarify the situation We pull 3 samples per shift (beginning, middle and end), 2 shifts/day for 5 days = 30 samples. Results come back with an average of 450 ppb with a range of ppb. Sample variability between the samples of each shift is good (+/- 10%), but you notice a jump in values in Thursday s samples from an average around 325 ppm up to an average of about 575 ppm, but again with a tight range of +/- 10%. Your VP of Manufacturing reports back that all equipment was operating within spec, but they switched over to a new lot of molasses from an alternate supplier beginning with shift 1 on Thursday.

38 Step 1: Clarify the situation: Raw materials and ingredients Ingredients: Fig Prune Strawberry Smoked almonds Molasses Wheat, etc. (cake outer shell) Process: Ingredients are loaded into a mixer where they are batch mixed, loaded into a hopper which feeds into a conditioner, basically a 20-ft tube with an auger in the middle that propels the mixture. This unit op is heated to an internal temperature of 220F, measured at the exit. Product exits through an extruder that forms it into a ribbon which is cut to length. Next enrobed in wheat dough and then baked at 350F to a final product moisture of 5%. Product is cooled before entering packaging unit op.

39 Step 2: Develop Your Strategy: Raw Materials and Ingredients The first question to answer: How much AA is coming in ingredients vs. how much is forming in the process? AA Contribution Fig ND 20% n/a Prune % 75 ppb Strawberry ND 20% n/a Smoked almonds 500 5% 25 Molasses (supplier 1) % 50 Molasses (supplier 2) 100 5% 5 Wheat, etc. ND 20% n/a Potential contribution from ingredients = ppb

40 Step 2: Develop Your Strategy: Process Interventions Do we have an opportunity to attempt mitigation by changing ingredients/suppliers? YOU BET! You notice on the FDA website: Prune juice can be variable. Why is there a difference? They are all made from prunes, so what gives? Smoked almonds are 500 ppb, but roasted almonds are around 240 ppb. Even better sliced cashews and peanuts are both ND! Can we reformulate and still make an acceptable product? acceptable not identical! Molasses- your investigation reveals that supplier 1 is using ASNase to lower AA formation; supplier 2 is not.

41 Step 2: Develop Your Strategy: Process Interventions Ingredients are accounting for only about 1/3 of the finished product AA, so we must be forming it somewhere. More testing of the in-process stream: Product at the mixer/hopper = ppb; differences correlate with molasses supplier Product exiting at the extruder = 300 to 600 ppb, again differences correlate with molasses We are obviously forming AA in the conditioner. How? The exit temp is 220F, well below the reported threshold of 250F How is the heat applied? Becalski et al Food Addit Contam Low temp AA formation in prunes Finally, we are baking to a finished product moisture of 5%. But this is measured based on total product weight. The outer wheat shell has a much lower moisture content and is baked to a crispy, golden brown.

42 Step 2: Develop Your Strategy (Plan B) Do we need a Plan B, or do we have enough in terms of mitigation? This is why it so important to identify exactly what is your target. In this case, I think we have enough to proceed. An iterative process- if we can t meet our goal we can always circle back and investigate using ASNase in the conditioner, altering ph, cations, etc.

43 Step 3: Refine the approach Ingredients All of our ingredients have alternative suppliers with lower AA values Can we reformulate away from smoked almonds to just roasted? If needed can we use artificial flavor to replace smoked notes contributed by almonds? Even better can we substitute cashews or peanuts for some or all of the almonds? Acceptable vs. identical Conditioner- exit temp is 220F, but heat is supplied by multiple steam injection ports which deliver steam at F. Everywhere this steam hits product AA is forming. The wheat crust (crispy golden brown) is contributing to AA. We can look at adding ASNase to the wheat dough before baking or consider making a softer rather that a hard, crispy crust.

44 Step 4: Scale-Up We are ready to scale-up. Our mitigation plan consists of: Changing suppliers or leaning on existing to improve AA numbers in raw materials and ingredients Re-design of conditioner unit op, or could be cheaper to look at a clean sheet redesign of the process to replace this unit with something else that doesn t generate AA Adding ASNase to wheat dough if we decide to stick with the crispy shell, or redesign the product for an outer shell with a softer bite. Other considerations Some minor label changes If we use ASNase need to consider GMO aspect If we decide to change the product of course have to prepare the consumer

45 Putting it all together - final thoughts Vital to know what success looks like: Would you tell me, please, which way I ought to go from here? That depends a good deal on where you want to get to. I don't much care where Then it doesn't matter which way you go. Lewis Carroll, Alice in Wonderland You probably have more tools at your disposal than you realize. Look at your process; uncontrolled heat is your enemy! Tighten up raw material and ingredient specs Leverage knowledge of suppliers and vendors If you need more, there are more tools in the toolboxes.