Carmen Boeriu Wageningen UR – Food & Biobased Research, department of Biobased Products

1. Biocatalysis in the Food Industry: Impact on Food Quality and Health Carmen Boeriu Wageningen UR – Food & Biobased Research, department of Biobased Products Bucuresti, 22-24. September 2010

2. Supervisory Board Executive Board Life long learning IAC, PHLO, WMS RIKILT CIDC a.o. 20 university departments Board of Directors LEI Board of Directors Board of Directors Board of Directors Board of Directors 16 university departments 17 university departments 10 university departments 18 university departments ID-Lelystad RIVO Plant Research International Alterra FBR PV PPO Plant Sciences Green Space Sci. Animal Sciences ATF Sciences Social sciences OrganisationWageningen UR

3. Food & Biobased Research • Bridging chains • from primary producer to consumer • from fundamental research to business development • intensive public-private cooperation • effective transfer of knowledge to industry and society

4. Agro logistics Food pretreatment Foodconversion Food production 1st Food € New Pre-treatment & conversion New production Performance materials Base&platform chemicals Performance chemicals Bio Energy • Biobased • Products • Biobased materials • Bio-based • chemicals • Bio-fuels • Bio-energy Biomass sources Agro- production Food by-products & waste Logistics&storage NL production Imports € Biomass production Existing production Existing conversion • Existing • non- food: • Feed • Compost • Waste management • . € The New Biomass value chain: a new € - game.

5. Cooperation with (inter)national industry Wageningen UR Biobased Economy: the centre of cooperation and development RUG Wetsus ECN Water & Energy CCC & B-BASIC Bio2Value DSTI RUU UT LNV; EZ; VROM; IPE CatchBio TUD B-BASIC & CatchBio International Bio-Materials DPI TU/e EU

6. Research targets Biocatalysis group New synthetic routes and/or improvement of existing routes for specialty chemicals & bioactives (includes single enzymatic steps, but also chemo-enzymatic and multi-step enzymatic cascades; biocatalyst development) High through-put screening (HTPS) for bioactivity (e.g. enzyme-substrate and enzyme-inhibitor interactions) Development of new functional compounds, e.g. bioactives, bio- polymers, building blocks, biobased surfactants …

7. Our approach Protein engineering, over-expression, CD, fermentation, DSP

8. Biocatalysis group - general • Catalysts: • Free & immobilized enzymes • whole cells • Various reaction media • watery systems • organic solvents (hydrophobic, hydrophilic) • supercritical media • solid (solid-to-solid systems)

9. Biocatalysis for fine & bulk chemicals Peptides C-terminal Peptide coupling Bioactive peptides Proteins Surface modification Controlled hydrolysis Cross-linking Carbohydrates (CH) Surfactants Reactive CH Surface modification of PS (HB/HP balance) Crosslinking Bioactive Glycolipids HAs

10. Use of enzymes (Faber, 2001)

11. PS & Enzymes for commodity chemicals

12. Enzymes in Food & Health • Quality control • Biosensors • Preservers • Antioxidants (GOX) • Promoting health • Prebiotics • Lactose intolerance • Controlling blood pressure • Diabetes (sweeteners, insulin) • Food ingredients • New routes & technologies • Food grade • Mild, Envrironmentally friendly • New ingredients • Processing aids • Bakery • Confectionery

13. New routes for food ingredients Carbohydrate based ingredients

14. Modifications Introduction • Much research is dedicated to developing new carbohydrate derivatives with improved or novel properties, starting from “commodity” carbohydrates

15. Polysaccharide modification

16. Enzymatic routes to novel polysaccharide (PS) derivatives

17. Enzymatic cross-linking Peroxidase: • Versatile enzyme, using many compounds as substrate, including phenolic derivatives • In vivo function: • Cell wall formation and reinforcement • Cross-linking of cell wall polysaccharides and glycoproteins (extensin) • Coupling of monolignans and hydroxycinnamates (lignin, suberin) • Defense mechanisms  Similar reactions might take place in vitro

18. Laccase: In vivo: lignin synthesis Enzymatic cross-linking Polyphenoloxidase: In vivo: defence

19. Enzymatic cross-linking

20. Enzymatic cross-linking • Polysaccharides that are substrates for oxidative enzymes • Arabinoxylan (feruloyl residues) • Pectins (beet)

21. Enzymatic cross-linking: PS-PS adducts • Homopolymerization of (a) pectin and (b) arabinoxylan  gels, multiple cross-linking Millan et al. 2004

22. Enzymatic cross-linking: PS-protein conjugates

23. Cross - linking incubation of model systems incubation of model systems HRP and H HRP and H O O FA, 2 2 2 2 Tyr - peptides Monitoring of reaction by UV and fluorescence spectroscopy Kinetic description of substrate conversion Optimizing reaction conditions LC - MS and SE - HPLC analysis of reaction products Characterization of reaction products Application of Application of optimized optimized conditions conditions Peroxidase cross-linking of PS and proteins Arabinoxylan Proteins Cross Cross - - linking linking incubation of model systems incubation of model systems HRP and H HRP and H O O 2 2 2 2 Tyr - peptides Monitoring of reaction by UV and fluorescence spectroscopy Monitoring of reaction by UV and fluorescence spectroscopy Kinetic description of substrate conversion Kinetic description of substrate conversion Optimizing Optimizing reaction conditions reaction conditions LC LC - - MS and SE MS and SE - - HPLC analysis of reaction products HPLC analysis of reaction products Characterization of reaction products Characterization of reaction products Protein studies: Boeriu et al., JAFC, 2004 Peptide studies: Oudgenoeg et al., JAFC, 2001 Oudgenoeg et al., J. Biol. Chem, 2002 Application of Application of optimized optimized conditions conditions

24. Characterization of -casein-AX adducts AE 1: eluted with void volume AE 2: eluting by running buffer AE 3: eluting with the salt gradient

25. -casein-AX adducts

26. Enzymatic cross-linking: Applications

27. Enzymatic synthesis of amphiphilic polysaccharides

28. Surfactants World market surfactants Surfactants: past and present

29. Natural-like surfactants

30. Enzymatic synthesis of PS amphiphiles

31. Enzymatic synthesis of PS amphiphiles • Enzymes that can catalyze ester synthesis: • Lipases (EC 3.1.1.3) • Serine proteases (EC 3.4.21.62) • Esterases (EC 3.1.1.1) • In non-conventional media, they catalyze the reverse reaction, i.e. esterification

32. Enzymatic synthesis of oligo- and polysaccharides • Specific issues: • Enzymatic (trans)esterification requires water free media • Lipases and other hydrolases are active in hydrophobic solvents, but are easily inactivated in hydrophilic solvents • Carbohydrates are soluble only in hydrophilic solvents • Solutions: • Solvent mixtures • Solubilisation of substrates by prior modification with easy leaving groups • Solubilisation of enzyme in organic phase • New solvents, i.e. ionic liquids Sagis et al., 2007; Ter Haar et al., submitted

33. Enzymatic esterification of PS in two-solvent system Inulin Inulin oligosaccharides DP 2-9 C11H23COOC2H3, Novozym 435 Two-solvent system: DMSO/ButOH 20:80 – 40:60 (v/v)

34. Lipase mediated synthesis of lauryl esters of inulin • DS ~ 0.20 calculated via integration; confirmed by GC-FAME, after saponification • 2D-NMR : not-conclusive, fractionation of product needed

35. 20% DMSO Lauryl esters of inulin:characterization A=20% DMSO, B=40% DMSO, *=K adducts, Na adducts eg 5/1 = DP5 with one LA  High regioselectivity Ter Haar et al., J. Mol. Cat. Enzymatic 2010

36. ○ inulin;■inulin laurate; ▲sucrose monolaurate Lauryl esters of inulin: properties • Surface properties comparable with commercial SDS and sucrose monolaurate • Higher dilatational modulus than SDS and sucrose monolaurate

37. Foaming Lauryl esters of inulin: properties Product characterization: functional properties • uniform bubbles • stable foam: after 2h no change observed in foam height • drainage of liquid is quite fast A) After 55 min. B)After 1st rehydration, at 60 min. C) After 2nd rehydration at 75 min. • Foaming properties of the lauryl esters of inulin laurate with DS  0.2 are comparable with those of derivatives with lower DS (DS  0.1, previously produced) • Better and more stable foam than SDS

38. Inulin esters: Concluding remarks • Enzymatic esterification is a promising technology for synthesis of novel biobased surfactants • Amphiphilic oligo- and polysaccharides • Biodegradable • Biocompatible • Potential application of amphiphilic PS • Biodegradable, food grade emulsifiers • Compatibilizers • Injection-moulding operations • Detergents • Time-release drug delivery systems

39. Acknowledgments WUR-Biobased Products Cees van Dijk Guus Frissen Ben van den Broedk Marinella van Leeuwen WU Ruud ter Haar Henk Schols Leonard Sagis Willem van Berkel Harry Gruppen Gideon Oudgenoeg Fons Voragen Sponsors Ministry Economic Affairs Ministry of Agriculture, Nature and Food Quality EU-FP6; IP Bioproduction

40. Invitation 2nd International EPNOE Polysaccharide Conference 29 August – 2 September 2011 Wageningen, The Netherlnads

41. Thank you for your attention!