1. Isabelle Yang Antioxidant Assay Selection

2. Generally: Oxidation is loss of electrons

3. Different category Oxidation in aqueous phase; eg. Protein oxidation Oxidation in lipid phase; eg. Lipid oxidation Enzymatic reaction; eg. Browning in apple slides Non-enzymatic reaction; eg. Browning in whit wine Reactive oxygen species; (ROS) Reactive nitrogen species; (RNS) Free radical Non free radical

4. ROS – Triplet oxygen (Ground state Oxygen) - Singlet oxygen (Strong electron robber) - Superoxide (O 2.- ) - Peroxyl (ROO`) -Alkoxyl (RO`) -Hydroxyl (HO`) RNS : `NO, ONOO - Non-radicals Free radical

5. Free radical oxidation Free radicals are Any molecular species capable of independent existence, which contains one or more unpaired valence electrons not contributing to intramolecular bonding

6. Structure: - cells (compounds transfer, interface with ROS, RNS) - Phase: water/lipid; polar/non-polar - pH, temperature, metal ions Oxidation order: -how vulnerable the compounds are; -which one is going to die first; (polyphenol, linoeicacid; carotenoids.)

7. Antioxidant is - In food: protectors against oxidative stress - Dietary antioxidant: substance in foods that significantly decreases the adverse effects of reactive species, ROS/RNS, on normal physiological function in humans. (Institute of Medicine)

8. Physical barriers Metal chelators: siderphores, proteins Work on enzymes - Inhibit oxidative enzymes through pH, temperature: lipidoxidase, Polyphenol oxidase - Antioxidant enzyme cofactors (Q10, Selenium) Reducing (reductive) compounds

9. Free radical scavenger Inhibit the reactive oxidants Reducing oxidation number : ferric, cupper Different antioxidants could be mixed together (antagonistic response; synergic response)

10. Relative antioxidant efficiency (RAE) RAE= S cd S bc

11. Simulate the target food system in order to determine the Relative antioxidant efficiency HAT : Hydrogen atom transfer - Simulate free radical in food, measure antioxidants’ capacity of competing free radicals, and terminate the radical chain. SET: Single electron transfer - Measure antioxidants’ reducing capacity

13. Test radical chain breaking capacity Free radical generator / Initiator (AAPH; AMVN) Oxidizable molecular probe (UV ; fluorescence; BODIPY) antioxidant Similar food system: lipid phase (cholesterol, carotenoids, linoleic acid) TRAP : Total radical trapping antioxidant parameter ORAC : Oxygen radical absorbance capacity Crocin bleaching assay ( natural carotenoid)

15. The more vulnerable, the more competitive in sacrificial and terminate the radical chain by dying together.

17. TRAP : Total radical trapping antioxidant parameter

18. Crocin Bleaching Assay

19. Test reducing capacity. More general antioxidant capacity than HAT Probe: Oxidant with metal ion, measure reducing capacity by different color of same metal ion with different oxidation number Metal ion: Molybdenum, Ferric, Copper

20. Folin-Ciocalteu assay – total phenols Trolox Equivalent Antioxidant Capacity Assay ( ABTS) Ferric Ion Reducing Antioxidant Power Assay Total Antioxidant Potential Assay (Cu II) 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity Assay (DPPH)

21. FCR -sodium tungstate (Na 2 WO 4.2H 2 O, 100 g), sodium molybdate (Na 2 MoO 4.2H 2 O, 25 g) -Total phenolics - pH ~10

23. Ferric Ion Reducing Antioxidant Power Assay

24. Total Antioxidant Potential Assay (Cu II)

25. Trolox Equivalent Antioxidant Capacity Assay ( ABTS)

26. Trolox Equivalent Antioxidant Capacity Assay ( ABTS)

27. 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Capacity Assay (DPPH) Radical scavenging HAT or SET

30. Antioxidant for homemade guacamole - Lemon juice; ascorbic acid, low pH to inhibit Polyphenol oxidase - Package: physical barrier - Metal chelalor: EDTA, calcium disodium EDTA