MBAA – RMD and ASBC Wild West Chapter Meeting November 8, 2007

1. MBAA – RMD and ASBC Wild West Chapter MeetingNovember 8, 2007 Electron Paramagnetic Resonance (EPR) Practical Applications in Brewing, Beer Flavor Stability, and Hop Antioxidants by Bob Foster

2. Outline EPR Chemistry and Method Metrics Practical EPR Malt, Hops and Brewing Applications Malt Extract and Wort Boiling Studies Blend/Finishing EPR Process Surveys EPR Finished Product and Sensory Packaging EPR Plastic Bottles and O2 Scavenging Crowns Hop Bitter Acids, Oregon State EPR Study, Antioxidants, and Fruit Juices Conclusions, Acknowledgements, and Q&A

3. (ElectronSpin Resonance) or (ElectronParamagnetic Resonance) atom electron spin electron • The total magnetic moment of an electron ( J ) is comprised of spin quantum numbers ( L ) & ( S ). • L is magnetism due to angular orbital momentum as the electron orbits the nucleus. • Sis due to the spin of the electron itself, pointing either “up” or “down”s-type orbitals are spherical so ( L ) is zero, thus the spin ( S ) is the only contributor to the magnetic moment. • 2. Historically, many chemists only studied samples with s-type orbitals, hence the development of the acronym ESR. The term EPR includes the contribution from both ( L )and ( S ).

4. What is a Free Radical? • A free radical is a molecule or atom with an unpaired electron. • They are also the “free electrons” that have just been lost. • They are catalyzed by transition metals ions: Fe, Cu, Mn, Co, Ni, • Example (losing electron): EPR Measures

5. The EPR spectrometer provides: a linear field sweep exposes the beer sample to a fixed frequency of microwave irradiation drives the electrons from the parallel state to the anti-parallel state measures the EPR absorption or signal. Energy Magnetic field strength where resonance occurs (“field for resonance”). Magnetic field strength (B0)

6. EPR Signal Intensity, Free Radicals, and Spin Traps As of free radicals increase in beer, the EPR signal intensity increases. Spin traps are compounds used to “trap” short-lived free radicals. A covalent bond forms between the free radical and spin trap The stabilized “radical adduct” is detected by EPR. EPR intensity R = beer derived free radical trapped by PBN. ( N-tert-Butyl-a-phenylnitrone) Magnetic field strength (Bo)

7. EPR Intensity Time (min.) EPR and Finished Dark and Light Beers Dark Beers T150 Y-axis >Ox Zero Lagtime Pale Beers Lagtime X-axis

9. Transition Metal Oxidation Catalyst in Beer • O2 and metals are integral players in catalyzing free radical chain reactions. • Scheme for producing reactive oxygen species (ROS) Transition metal catalyzed reduction O2-. Dismutation O2 HOO . FeIII FeII H+ H2O2, O2 FeII OH . HO- + FeIII Major initiating oxidant (Andersen and Skibsted, 1998)

10. Hydroxyl Radical Reaction with Ethanol HO . + CH3CH2OH Hydroxyl radical Ethanol + H2O +H2O 85% 13% .CH2CH2OH2-hydroxyethyl CH3C.HOH1-hydroxyethyl radical radical O2 O2 OO. .OOCH2CH2OH CH3CHOH Bimolecular rxs CH3CHO + HOO. HOCH2CH2OH + HOCH2CHO + CH2O + O2 + HOO. + H2O2 Acetaldehyde + hydroperoxyl radical

12. e- e- e- e- dx = 5 dx = 20 e- e- Time dx = 40 dx = 80

13. Three Classes of Antioxidants: • True Antioxidants • Retardants • Deactivators

14. Characteristics of a Good Chain-breaking Antioxidant • react with peroxyl radicals (ROO.) • when present in small amounts, inhibit oxidation of the bulk • do not add O2 to make additional peroxyl radicals • and in biological systems are renewed • trap 2 chain-carrying peroxyl radicals by H-atom abstraction (Eqn. 1a) • and by rapid recombination of the radicals formed (Eqn. 1b) kinh Ar-CH + R-O-O. R-O-O-H + ArO. (Eqn. 1a) fast R-O-O. + ArO. Nonradical products (Eqn.1b)

15. True Antioxidant - Vitamin E & C Recycling Reaction with lipid peroxides: LOO + TOH  LOOH + TO TO a-tocopherol radical Recycling reaction with ascorbate Resonance stabilized free radical

16. Antioxidant Class EPR Effect - no recycling Oxidation without antioxidant Oxidation with small amounts of antioxidant [ Antioxidant ] EPR Signal Intensity (Oxidation) True Antioxidants = Lagtime Time

17. Typical Retardants Interfere with the Oxidation Rate • The uninhibited autoxidation (Eqn. 2a) and the kr, • rate of H-atom abstraction to propagating peroxyl radicals (Eqn. 2b) • define this after true antioxidant depletion period. R. (substrate) + O2 R-O-O. (Eqn. 2a) R’-H+ R-O-O.R’. + R-O-O-H (Eqn. 2b) kr

18. Typical Retardants (continued) • Retardant interference can also be initiated by partial chain termination by rapid cross over termination reactions with chain-carrying peroxyl radicals. (Eqn. 3a) • Retardant suppressed oxidation by slow reactions with chain carrying peroxyl radicals so that chain termination occurs by the bimolecular self reaction of peroxy radicals. (Eqn.3b) fast R.+ R-O-O.R-O-O-R (Eqn. 3a) R-O-O.+ R-O-O.R-O-O-R + O2 (Eqn 3b)

19. Retardant Class EPR Effect Oxidation without retardant Oxidation with retardant [ Retardant ] Oxidationwith huge amounts of retardants EPR Signal Intensity (Oxidation) Retardants = Shallow Slope Time

20. Deactivator Class EPR Effect [ Deactivator ] Oxidation without Deactivator effect Less EPR au EPR Signal Intensity (Oxidation) Oxidationwith Deactivators Deactivators = Less Final EPR Signal Time

21. EPR Beer-Antioxidant Method Summary • Pipette 5.0 mL of beer (or fruit juice) into a 15 mL amber vial. • Beer: Add 100 uL of 50 mM PBN, dissolve and degas by sonication. • Clarified Wort: Add 200 uL of PBN, warm, dissolve, and mix well. • Load the EPR heating block (60 °C) and run for 2-3 hours. • Analyze the data and EPR curves. • (M. Uchida & M. Ono, 1996)

22. Practical EPR – Malt, Wort, and Beer Surveys • Malt Extracts have an EPR lagtime due to active antioxidants. • Boiled Worts do not have EPR lagtime values and rely on T120, T150, or other metric values to access flavor stability potential. • Reducing boiling time reduces energy costs and prevents “scorching” and oxidation. • Brewers have preached, “When you move beer, you bruise it.” Blend/filtration/finishing processes pick-up oxygen

23. BV5 BV4 BV2 BV3 BV1 EPR and Malt Extract Studies-Barley Breeding Deactivators Antioxidants Retardants

24. EPR Malt Extract Metrics

27. EPR Wort Boiling Study – Sierra Nevada Brewing

28. Post Fermentation/Blend/Finishing EPR Process Surveys

29. EPR - Finished Product and Sensory

30. EPR and Sensory Fresh Flavor Lagtime

31. EPR, Total In-Package Oxygen (TIPO), and Sensory

32. 3. Packaging Applications with Plastic Bottles and Different Crowns

33. EPR Lagtime and Plastic Bottles

34. Crown Liner Trials – Sierra Nevada Brewing Co.

35. EPR and O2 Scavenging Crowns

36. Hop Bitter Acids, Oregon State Hop Polyphenols Study, Antioxidants and Chelating Agents

37. Chelating Agents and EPR Antioxidant Comparisons of Hop Components in Beer and Fruit Juices : • Isoxanthohumol • Bulk Xanthohumol Enriched Hop Powder • Xanthohumol • Iso-Alpha Acids Hop Extract (28.9% v/v) • Hop Polyphenol Fractions (Oregon State University-Coors) • Fruit Juices

38. DT Wort Boiling • Devoid of estrogenicity (De Keukeleire et al., 1997). • Strong inhibitor of cytochrome P450 procarcinogen enzymatic • activity (Miranda et al., 2000). • Induction of chinon reductase carcinogen detoxification • enzyme (Henderson et al., 2000). • Strong scavenger of ROS and interferes with HO. & • R-O-O. radicals better than Trolox(Gerhauser et al., 2002). • Inhibits LDL oxidation > a tocopherol (Miranda et al., 2000). • Exhibits anti-inflammatory properties < prostaglandins • (Gerhauser et al., 2002). • 7. Several inhibitory mechanisms in carcinogenesis • (Gerhauser et al., 2002). • 8. In the presence of xanthohumol, many cancer cells undergo • apoptosis (Gerhauser et al., 2002). • 9. More…. • Weakly estrogenic (De Keukeleire et al., 1997). • Approximately 30% of the xanthohumol level in hops • end up in the beer mainly as, isoxanthohumol • (Stevens, et al., 1999; Forster, et al., 2002). • Similar to 8-prenylnaringenin (R = H) “hopein” • which is very effective in the aggregation of • MCF-7/6 breast cancer cells = inhibits metastasis. • (Rong et al., 2001).

39. Iso-Alpha Acids = Antioxidants + cis-iso-a Acids trans-iso-a Acids a-Acids Courtesy of Prof. Leif-Alexander Garbe University of Berlin, 2007 (Strakova, L., et al., 2007)

40. Metal Deactivation: Fe(III)-EDTA Can Still Create Free Radicals Fe(III)EDTA Size -- too small, leaving a site for H2O H2O

41. Iso-Alpha Acids as Chelating Agents - - Courtesy of Prof. Leif-Alexander Garbe University of Berlin, 2007 Foster, 1997

42. Xanthohumol Enriched Hop Powder – Retardant & Deactivator

43. Deactivators = < EPR Antioxidant = Lagtime or x0 Retardants = Shallow Slope, dx or T150

45. EPR Comparison of Fruit Juices (non-alcohol) Raspberry Juice

47. Table No.1 EPR Summary of Hop Compounds Antioxidant Ranking

48. Conclusions • Hop polyphenols and acids tend to be proxidative when chelated to transition metals; whereas, “free” unbound hop phenolics tend to be good antioxidants and retardants. • Many known and unknown hop proanthocyanidin oligomers, flavan-3-ol monomers, different hop acids, and pigments may some day prove to be beneficial antioxidants with chelating treatments. • The positive and negative effect of these hop compounds in beer and fruit juice can now be measured using new antioxidant, retardant, and deactivator EPR metrics. • New and old hop compounds and products continue to show promise as beneficial’s to beer, fruit juice, and perhaps mankind.

49. Acknowledgements • I would like to thank the following for their support of this study: • Tom Shellhammer and Patricia Aron of Oregon State University and the Coors Brewing Company for permission to publish our EPR findings on hop antioxidants. • Eric Samp, Tony Gojanovic, Dana Sedin, David Barr, Alan Foster, and Hugo Patino for their helpful discussions concerning the Boltzmann function and free radical chemistry. • Mike Miller, Bob Smith, and HopSteiner Hop Company for their help with the purity analysis and the samples of the different hop compounds. • Prof. Leif-Alexander Garbe – University of Berlin for helpful review and conversations concerning various hop phenolic chemistries. • Dr. Gary R. Buettner’s (University of Iowa) permission to use some of his information on retardant antioxidants. • Sierra Nevada Brewing Company for permission to use some of their EPR slides.

50. Questions and Answers