Asparaginase for acrylamide mitigation in snacks

Novozymes Switzerland AG

Benjamin Thieringer B.Sc. Technical Service Specialist

• Introduction to Novozymes

• A few words on acrylamide issue

• Asparaginase mode of action

• Application testing in snacks

• Summing up

Contents

• More than 6,000 employees, more than 50% based outside Denmark

• Global leader in industrial enzymes & microorganisms, market leader in all main industries

• Enzymes account for > 90% of revenue

• Total sales: DKK 11.23 billion (+7% in DKK, +3% in LCY, +4% organic) or EUR 1.51 billion (FY2012)

• Strong focus on R&D (~ 14% of sales invested in R&D)

Enzymes for industrial use - market size approx.* ~EUR 2.55 billion

DuPont 21%

Novozymes 47%

DSM 6%

Others 26%

Source: Novozymes 2012 estimates

Novozymes is the undisputed world leader in industrial enzymes

Acrylamide, a chemical naturally formed in many food products

• An organic chemical compound

• Used in various technical applications

• Was first raised as an issue in drinking water in the mid-1990s and has been found in food since 2002

• Acrylamide is formed naturally in foods as a by-product during cooking at temperatures in excess of 250 °F/120°C and at low moisture

• The intake of acrylamide from food depends on the quantity and frequency of the foods consumed

Median, 1st quartile, and 3rd quartile

Measured acrylamide content (ppb) in different food categories

Measured acrylamide content (ppb) in different food categories. Legends shown: Median, 1st quartile, and 3rd quartile. Source: www.irmm.jrc.be/html/activities/acrylamide

• Acrylamide has been shown to be carcinogenic in animal studies. So far, research into a possible carcinogenic link for humans has been inconclusive

• Acrylamide has been classified by the International Agency for Research on Cancer (IARC) as “probably carcinogenic for humans” (1994)

• Risks assessments indicate a human health concern for acrylamide in food

• A 3-year EU study of acrylamide (The HEATOX Project) concluded that ”Increasing toxicological evidence suggests that acrylamide in food might be a cancer risk factor” (November 2007)

• The Joint FAO/WHO Expert Committee on Food Additives (JECFA) reconfirmed that levels of dietary exposure to acrylamide indicate a “human health concern” (FAO/WHO/February 2010)

• US FDA National Toxicology Program (NTP): draft report on the toxicology and carcinogenicity of acrylamide showing “clear evidence of carcinogenic activity in animals“ (April 2011)

• In Europe, the European Commission recently asked the European Food Safety Agency (EFSA) to re-assess the risks related to the presence of acrylamide in food .

Why is acrylamide in food a concern?

Authorities’ attention increasing rapidly

• Acrylamide has been classified by the International Agency for Research on Cancer (IARC) as “probably carcinogenic for humans” (1994)

• Legislation covering acrylamide is currently limited.

• Acrylamide monitoring program is reinforced in N. America and Europe

• European Commission: industry recommendation on acrylamide published 2010/307/EC, “indicative values” to guide member states in reviewing effectiveness of acrylamide mitigation tools (February 2011). Looking into reinforcing the FoodDrinkEurope’s Toolbox and possibly reviewing “indicative values” (before summer).

• Proposition 65 (California): acrylamide added to the list of ‘chemicals known to cause cancer (90’s) and to cause reproductive toxicity’ (2011). Lawsuits filed targeting several food service companies for violating Proposition 65 law by not providing cancer warnings.

• FDA: new monitoring program on acrylamide initiated in 2011. Industry guidance expected (2013) + possibly stronger health warnings about acrylamide

• “Code of Practice for the Reduction of Acrylamide in Foods” prepared by the Codex Alimentarius Commission approved by JECFA (CAC/RCP 67-2009 )

Asparaginase – specific and effective

• Asparaginase can effectively reduce acrylamide formation by converting the precursor asparagine into aspartic acid

• Asparaginases generally have a high specificity for asparagine with very limited activity on glutamine, meaning that other amino acids are not affected

Acrylamide is formed from reducing sugar and asparagine at high temperature and low moisture.

Glucose Asparagine

N H 2

O Acrylamide

O

O H

O H O H

O H

N H 2 N H 2

HOOC O

+ O H

Simplified reaction

N H 2 O H

HOOC O NH 3

Aspartic acid

Asparaginase

H 2 O

Novozymes Acrylaway® L - product information

• The Acrylaway products are produced by the submerged fermentation of a genetically modified microorganism, Aspergillus oryzae. The enzyme protein, which in itself is not genetically modified, is separated and purified from the production organism. The microorganism is self-cloned according to the EU definition Acrylaway products – Acrylaway L® is a liquid product with an activity of 3500 ASNU/g

– Acrylaway 3500 BG® is a granulated product with activity of 3500 ASNU/g

– Acrylaway BeFree 3500 L® is a liquid product with an activity of 3500 ASNU/g free of benzoate

– Acrylaway 3500 BG Wheat free ® is a granulated product with an activity of 3500 ASNU/g free of wheat

– Customized products can be made available subject to substantial volume commitments

Activity of Acrylaway ® at different pH levels

(temperature 37 ° C)

Activity of Acrylaway ® at different temperatures

(pH 7.0)

0

0.4

0.6

0.8

1

2 4 10 12 14

pH

R e l . a c t i v i t y

0.2

0 6 8

0

0.2

0.4

0.6

0.8

1

20 40 80

R e l . a c t i v i t y

0 60 Temperature ( ° C)

Novozymes Acrylaway® is today used industrially in more than 30 countries

Acrylaway used

Product category

Biscuits and cookies

Crackers

Crispbread

Honey cakes

Pretzels

Waffles

Tortilla chips

Corn-based chips

Potato-based chips

Potato-based extruded snacks

…Resulting in 40-90% reduction in acrylamide formation

Pretzels with Acrylaway® tested at Reading Bakery Systems

~90% reduction in acrylamide content No impact on the flavour was seen Acrylaway was added to the dough

Trials carried out at Reading Bakery Systems proved that Acrylaway® reduces the acrylamide levels in pretzels substantially (90% at a dosage of 145 ppm).

The same volatile aroma components for pretzels are present in both the control sample and the sample to which 290 ppm Acrylaway® has been added.

Acrylamide level in crackers produced at NZ bakeries depending on the Acrylaway® dosage

Pretzels: volatile aroma components for the control and 290 ppm Acrylaway®

Cracker recipe with Acrylaway ® tested in Novozymes’ bakeries

~66% reduction in acrylamide content No impact on the flavour was seen Acrylaway was added to the dough

Trials carried out in Novozymes’ bakeries proved that Acrylaway® reduces the acrylamide levels in a cracker recipe substantially (75% at a dosage of 60 ppm)

Acrylamide level in crackers produced at NZ bakeries depending on the Acrylaway® dosage

Crackers: volatile aroma components for the control and 290 ppm Acrylaway®

The same volatile aroma components for crackers are present in both the control sample and the sample to which 290 ppm Acrylaway has been added

Tortilla chips with Acrylaway® tested in Novozymes’ bakeries

620

380

46 51 54 51 720

100

200

300

400

500

600

700

0 100 200 300 500 1000 2000

Acr

ylam

ide

(p

pb

)

ASNU/kg flour

~92% acrylamide reduction in tortilla chips Acrylaway was added to the dough

Snack pellets

Potato tubes expanded in

frying oil

2100

1600

890

230 110

0

500

1000

1500

2000

2500

Control 1000 3500 5500 7000

Acr

ylam

ide

[p

pb

]

ASNU/kg DM

~95% reduced acrylamide content Acrylaway was added into the mixer

Pellet based potato tubes with Acrylaway® tested at Pavan Italy

Acrylaway® treatment of potato granules to reduce asparagine levels

52% to 87% asparagine reduction – control compared to BB #5 and BB #20 Acrylaway was added into the mixer

0

200

400

600

800

1000

1200

mg

/10

0g

DM

Big bag #

Asparagine Aspartic acid

Snack pellets

Potato tubes expanded in

frying oil

5800

3400

1800

1800

1000

2000

3000

4000

5000

6000

7000

Control (untreatedgranules)

Treated granules 1600 ASNU/kg 2000 ASNU/kg +treated granules

Acr

ylam

ide

[p

pb

]

~96% reduction in acrylamide content Acrylaway treated granules were used Direct addtion of Acrylaway into the mixer

Acrylamide reduction in potato tubes using Acrylaway® treated potato granules

Conclusion

• Acrylamide can be found in a wide range of cereal and potato based foods

• Acrylamide reductions of up to > 95% were achieved in a broad range of snack products

• Very specific action of asparaginase on asparagine i.e. we can get the same high consumer acceptance as we do not change the characteristic of the final product

• The asparaginase technology is used today by the industry and in more than 30 countries

• Benefits for the producers are related to food safety and brand protection

• By using Acrylaway®, health concerns are reduced – reduced acrylamide levels

• Very importantly, we reduce the concerns of producers regarding possible future legislation and potential media coverage.

Asparaginase for acrylamide mitigation in snacks

Novozymes Switzerland AG

Benjamin Thieringer B.Sc. Technical Service Specialist