Comparison of manual and automatised TAC determination in fruits

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Comparison of manual and automatised TAC
determination in fruits
Institute of Applied Microbiology

• Anita Herndl
• Short Term Scientific Mission
• COST Action 863
• 9.10.-20.10.2006

University of Natural Resources and Applied Life
Sciences, Vienna
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STSM Program
Host Maurizio Battino Università Politecnica
delle Marche, Ancona

• Methods
• Extraction Methods
• TEAC
• Manual
• Flow Injection
• Total Phenolic Content (Folin-Ciocalteu)
• Total Anthocyanin Content

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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
ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulph
onic acid)
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TEAC

• - ABTS not naturally present in mammalians
• underestimation

• Widely used
• Simple and fast
• No special equipment required
• Stability of ABTS
• ABTS is soluble in aqueous and organic
  solvents
• can be automated

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FIA-TEAC

• The system consists of
• HPLC pump
• Manual injection valve with a 5 µL loop
• Reaction coil
• UV/VIS Detector
• ABTS 4C
• Reaction 35C
• 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
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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

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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
• ?max Determination by wavelength scan
• Standard predominant anthocyanin in sample
• for strawberries Pelargonidin

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Results
Hydrophilic antioxidant capacity determined by
manual TEAC and FIA-TEAC
FIA-TEAC
Variation coefficient µmol TE/g FW apple
1st Day 1,22 7,46
2nd Day 1,81 6,76
3rd Day 4,88 7,20

Inter-Day 5,04 7,10

1st Day 3,23 5,85
2nd Day 19,67 5,67

Inter-Day 12,54 5,76
Manual TEAC
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Results
Lipophilic antioxidant capacity determined by
manual TEAC and FIA-TEAC
FIA-TEAC
Variation coefficient Variation coefficient µmol TE/g FW apple
1st Day 2,69 2,69 1,93
2nd Day 7,03 7,03 1,68
3rd Day 3,09 3,09 1,84

Inter-Day 7,46 7,46 1,85

1st Day 4,89 1,09 1,09
2nd Day 22,91 0,48 0,48

Inter-Day 43,37 0,79 0,79
Manual TEAC
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Results

• Total Phenolic Content (Apple)
• Total Anthocyanin Content (Strawberry)

Variation coefficient mg GAE/100g FW
Hydrophilic components 5,90 101,88
Lipophilic components 10,50 26,42
Sample Variation coefficient mg Pg/100g FW
1 1,33 35,80
2 3,19 32,22
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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

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

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• THANK YOU FOR YOUR ATTENTION