Tips on how best to use enzymes in your formulations 4/15/2017 2009-08016-01

Test methodology and examples 4/15/2017 NOVOZYMES PRESENTATION content Formulation advice Test methodology and examples Industrial applications Summary

Composition examples of household powder detergents 4/15/2017 NOVOZYMES PRESENTATION Composition examples of household powder detergents Ingredient Laundry Detergent Dishwashing Detergent Anionic Surfactants 5 - 18% -- Nonionic Surfactants 2 - 8% 1-5% Soap 1 - 5% Builders (zeolite, carbonates, silicates) 12 - 85% 32-90% Bleaches (perborate/activator) 0 - 30% 3-15% Polymers 0 - 5% 5-10% Enzymes: protease amylase lipase cellulase Mannaway 0.7 - 1.2% 1.0 - 1.5% 0.4 - 0.8% 0.2 - 0.5% 0.2 – 0.5% 1-1-5% 1-2% Fillers (sodium sulfate) 0 - 20% Water + others to 100%

Composition examples of household liquid detergents 4/15/2017 NOVOZYMES PRESENTATION Composition examples of household liquid detergents Ingredient Laundry Detergent Prespotter Anionic Surfactants 5 - 20% 0 - 5% Nonionic Surfactants 3 - 15% 4 - 12% Soap 0 - 12% -- Coupling Agents (xylene sulfonate) Builders (citrate, carbonate) 2 - 8% 0 - 2% Buffers (triethanolamine) Enzymes: protease amylase lipase 0.3 - 0.8% 0.2 - 0.6% 0.4 - 0.6% Enzyme Stabilizers 0 - 8% Water & other to 100%

Formulation guidelines 4/15/2017 NOVOZYMES PRESENTATION Formulation guidelines . Anionic surfactant system (charged surfactants) Negative influence on enzyme stability AES = None > MES > AOS ³ LAS > Soap Non-ionic surfactant system AEO6 > NP12 > NP4 > None Amides and Alkyl polyglucosides are ok Recommendation Preferably use non-ionic surfactants Or use a combination of nonionic and anionic surfactants

4/15/2017 NOVOZYMES PRESENTATION Compatibility of enzymes with materials found in typical detergent formulations Materials Proteases Amylases Lipases Cellulases Enzymes May self-hydrolyse; other have no effect Need stabilizer when proteases present; otherwise ok in general Anionic surfactants Best: AES, MES. Other: AOS and LAS tend to interact unfavorably with enzymes. Nonionic surfactants Ok in general. High level may reduce effect. Best if anionic/nonionic ratio of ¼- ¾ Best if anionic/nonionic ratio of ¼ - ¾ Cationic surfactants May decrease stability Coupling agents / hydrotrope MPG best. Others ok in General, SXS level low general MPG best; others ok in Buffers (prefer pH < 9 in liquids) Ok to use Borate, TEA or MEA. Use <5%w TEA Keep pH at 7 - 9 for optimal stability. Recommended Stabilizers Borate, MPG, sodium formate (low pH). Calcium may help Calcium (e.g. CaCl2) best. Borate, MPG, or sodium formate when protease present. present Builders Ok in general except for strong chelating agents (e.g. EDTA, TKPP, NTA). Keep pH at 7 - 9 for optimal stability Ok in general. Keep pH at 7 - 9 for optimal stability Preservatives Most ok (e.g. Proxel, Kathon, Dowicide, etc.) except formaldehyde Bleach Percarbonate or perborate ok to use in powders. Not recommended overall in liquids. Chlorine containing type not recommended at all Other Comments Keep water content between 20 - 60% for optimal stability in liquids; keep powders from adsorbing moisture for optimal stability (may use moisture barriers) Recommendations are based on shelf-stability of enzymes in liquid formulations unless otherwise stated

Factors influencing enzyme stability in a liquid detergent 4/15/2017 NOVOZYMES PRESENTATION Factors influencing enzyme stability in a liquid detergent Water content pH Type and amount of enzyme stabilizers Time Temperature Enzyme dose Major components such as surfactants, builders Other components in the reaction mix (e.g., preservative, bleach) These factors interact and each enzyme has a specific range of operating parameters for its optimum performance.

What does shelf stability mean? 4/15/2017 NOVOZYMES PRESENTATION What does shelf stability mean? Stability of individual enzymes in a detergent, especially liquid Stability of enzymes in the presence of protease

Enzyme stability depends upon conditions 4/15/2017 NOVOZYMES PRESENTATION Enzyme stability depends upon conditions . 1 year 1 hour 16 weeks

Guidelines to improve enzyme stability in formulations 4/15/2017 NOVOZYMES PRESENTATION Guidelines to improve enzyme stability in formulations Use stabilizers CaCl2.2H2O to ~0.08 % w – protease and amylase stability borate + mono propylene glycol – protease inhibitors sodium formate – protease inhibitor especially at pH < 8 4-FPBA (4-formyl phenyl boronic acid) – protease inhibitor Moderate the water content to 50-60% w Lower the anionic surfactant content, especially LAS (AS) Examine antagonistic detergent components (e.g. bleach, chelating agents) Adjust pH to 9 or less , best if 7.5 to 8.5 Use the Novozymes’ Ultra proteases containing 4-FPBA Alcohols/hydroxy groups stabilize proteases & facilitate solubilization of surfactants in water phase Propylene-glycol > Ethanol >> Sorbitol > None Acetamide, glycine, maltodextrin may be added to improve enzymatic stability. Dosage: 0 - 5 % (w/w) Not compatible: Glutamic acid (Phase sep.)

4-FPBA - highly effective protease inhibitor reference 4/15/2017 NOVOZYMES PRESENTATION 4-FPBA - highly effective protease inhibitor reference reference: Inform 13 p.731 (2002) Molecular structure of 4-Formylphenyl Boronic Acid (4FPBA) C=O H-O B H Highly efficient protease inhibitor compared to borax/propylene glycol combination Since 4FPBA is already added to the liquid protease, added stabilizers are not needed or are greatly reduced, with minor formulation adjustments including lowering of pH Cost-savings on added stabilizers Good stability of protease and other enzymes in the presence of a protease Savinase® Ultra and Alcalase® Ultra are now available

4-FPBA effect in typical US model detergent (pH 7) 4/15/2017 NOVOZYMES PRESENTATION 4-FPBA effect in typical US model detergent (pH 7) 25 50 75 100 7 14 21 Days Residual Activity (%) No protease Savinase 16L Savinase Ultra 16L 25 50 75 100 7 14 21 Days Residual Activity (%) Savinase 16L Savinase Ultra 16L . incubation at 30°C

Stability of Savinase® at different pH values 4/15/2017 NOVOZYMES PRESENTATION Stability of Savinase® at different pH values The pH of the liquid detergent can also affect the stabilization of enzyme activities as illustrated in Slide 15. At increasing pH values from 9 to 10, there is a drastic decrease in residual activity for Savinase including 4-FPBA from 65% to 8%. With no inhibitor present, the decrease is from 15% to 8% revealing effect of 4-FPBA at pH 9 -9.5 but not at pH 10. Other stabilizers in addition to 4-FPBA will be needed to achieve acceptable stability at the higher pH values. Experience has shown that the level of additional stabilizer such as boric acid is not as high as used if no 4-FPBA is present. US liquid base; residual activity after 8 weeks storage at 30°C Effect of 4-FPBA decreases with increasing pH

Enzyme applications not approved due to safety considerations 4/15/2017 NOVOZYMES PRESENTATION Enzyme applications not approved due to safety considerations . Carpet cleaners and prespotters - household and institutional Hard surface cleaners using high pressure delivery system - may generate breathable aerosols Laundry prespotters using trigger sprays that deliver aerosol particles below 50 micron hence can be breathed in

Enzymes have multifunctional properties 4/15/2017 NOVOZYMES PRESENTATION Enzymes have multifunctional properties . Boost the general cleaning performance Stain removal of organic origin (detergency) Soil release (particulate soil removal) Whitening (prevent soil redeposition, help with film removal) Color Brightening (color care) Enzymes are high performance ingredients that can replace chemicals in industrial production Enzymes are readily biodegradable

In summary Enzyme offer High performance qualities 4/15/2017 NOVOZYMES PRESENTATION In summary Enzyme offer High performance qualities Enzymes are Environmentally-friendly ingredients Enzymes ensure Lower wash temperature performance