Comparison of manual and automatised TAC determination in fruits Anita Herndl Short Term Scientific Mission COST Action University of Natural Resources and Applied Life Sciences, Vienna Institute of Applied Microbiology

STSM Program Methods: Extraction Methods TEAC: Manual Flow Injection Total Phenolic Content (Folin- Ciocalteu) Total Anthocyanin Content Host: Maurizio Battino Università Politecnica delle Marche, Ancona

TEAC Trolox Equivalent Antioxidant Capacity Scavenging of the colored ABTS radical cation (ABTS + ) ABTS + is generated chemically or enzymatically Antioxidants in the sample directly react with ABTS + forming ABTS (colorless) Decolorisation is measured at 734 nm at a fixed time point Results are expressed relative to TROLOX ABTS: 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) ABTS

TEAC + Widely used Simple and fast No special equipment required Stability of ABTS + ABTS + is soluble in aqueous and organic solvents can be automated - ABTS + not naturally present in mammalians underestimation

FIA-TEAC The system consists of: HPLC pump Manual injection valve with a 5 µL loop Reaction coil UV/VIS Detector ABTS +: 4°C Reaction: 35°C Measurment: after 1,3 min Bompadre S., Leone L., Politi A., Battino M. Improved FIA-ABTS method for antioxidant capacity determination in different biological samples. Free Radic Res. 2004; 38(8):831-8

Total Phenol Content Folin-Ciocalteu-Reagent Molybdotungstonphosphate heteropolyanion Blue reaction products Measurment at 760 nm Gallic acid as reference standard Results expressed as mg Gallic acid equivalent/ 100 g FW

Total Anthocyanin Content Anthocyanin pigments change color with a change in pH Colored at pH 1 Colorless at pH 4,5 Sample equilibrated at different pH λ max : Determination by wavelength scan Standard: predominant anthocyanin in sample for strawberries: Pelargonidin

Results Variation coefficient [%] µmol TE/g FW apple 1 st Day1,227,46 2 nd Day1,816,76 3 rd Day4,887,20 Inter-Day5,04 7,10 1 st Day3,235,85 2 nd Day19,675,67 Inter-Day12,54 5,76 FIA-TEAC Manual TEAC Hydrophilic antioxidant capacity determined by manual TEAC and FIA-TEAC

Results Variation coefficient [%] µmol TE/g FW apple 1 st Day2,691,93 2 nd Day7,031,68 3 rd Day3,091,84 Inter-Day7,461,85 1 st Day4,891,09 2 nd Day22,910,48 Inter-Day43,370,79 FIA-TEAC Manual TEAC Lipophilic antioxidant capacity determined by manual TEAC and FIA-TEAC

Results Total Phenolic Content (Apple) Total Anthocyanin Content (Strawberry) Variation coefficient [%]mg GAE/100g FW Hydrophilic components5,90101,88 Lipophilic components10,5026,42 SampleVariation coefficient [%]mg Pg/100g FW 11,3335,80 23,1932,22

Concluding Remarks Training on analytical methods measuring the antioxidant capacity of fruits Hydrophilic and lipophilic components Comparison of a manual and an automated assay FIA-TEAC: relative parameters efficently controlled higher reproducibility

Thanks to COST Action 863 Chairman Bruno Mezzetti Maurizio Battino Sara Tulipani Margit Laimer

THANK YOU FOR YOUR ATTENTION