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Evaluation of Food Processing and Storage Conditions on Antioxidant Activity Levels (Using DPPH Assay)

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  • EVALUATION OF FOOD PROCESSING AND

    STORAGE CONDITIONS ON ANTIOXIDANT

    ACTIVITY LEVELS

    (USING DPPH ASSAY)

    A thesis submitted to the Department of Life and Physical Sciences of

    Galway-Mayo Institute of Technology as a partial fulfilment of the

    Bachelors degree in Applied Biology and Biopharmaceutical

    Science.

    Submitted: 18th March 2016

    Mirella Amarachi Ejiugwo

  • 1

    DECLARATION

    I hereby declare that the submitted work was composed originally by me, unless where

    I cited and referred to other authors works.

    ..

  • 2

    ABSTRACT

    Epidemiological studies so far have discovered the beneficial properties of

    antioxidants, found predominantly in fruit and vegetables, on human health: they

    primarily safeguard against the development of chronic and degenerative diseases.

    A good number of in vitro and in vivo assays exist to quantify the antioxidant activity

    present in various fruit and vegetables, among which DPPH method was selected in

    this context, due to its simplicity, rapidity and applicability to a wide variety of food and

    beverages.

    One of the two objectives of this research study was to compare the scavenging

    activity of antioxidants present in commercial orange juice (not from concentrate) and

    freshly squeezed orange juice using the DPPH method. It was found that freshly

    squeezed orange juice, compared to any packaged orange-based beverage in the

    market, has a greater antioxidant activity. Thus, encouraging direct consumption of

    fresh fruit, rich in antioxidants, rather than their commercial derivatives (which are

    subject to antioxidant activity decline over time).

    The other aim of the present work was to determine the effect of storage conditions

    on the antioxidant activity present in commercial orange juice: at predefined

    temperatures for different lengths of time. The obtained result was that the optimum

    storage temperatures for commercial orange juice resulted to be at -20C (in the

    freezer), followed by at 4C (when refrigerated) - as the antioxidant activity values were

    relatively high, compared to those gotten at 25C (at room temperature), 30C and

    37C. It was proved that high temperatures deteriorate the antioxidant content of fruit

    juice. Furthermore, it was observed that scavenging activity of the commercial orange

    juice decreased with longer storage time, even at its optimum temperatures.

  • 3

    ACKNOWLEDGEMENTS

    My immense gratitude goes chiefly to God Almighty, who made it possible for this

    project to be realized.

    Likewise, I wish to express to appreciate every person that helped in the course of

    this research work, primarily: my supervisor Dr. Sheila Faherty and the laboratory

    technician, Mr. Michael.

    Finally, of equal importance: I wish to appreciate my husband, Pastor David

    Richman Olayinka, for his continual encouragement and helping hand, and our son,

    Answer Samuel, who had to stay off me sometimes for long while deeply engrossed

    in this project till the end that this work was completed successfully.

  • 4

    TABLE OF CONTENTS

    ABSTRACT ................................................................................................................ 2

    ACKNOWLEDGEMENTS .......................................................................................... 3

    TABLE OF CONTENTS ............................................................................................. 4

    LIST OF FIGURES ..................................................................................................... 6

    LIST OF TABLES ....................................................................................................... 8

    ABBREVIATIONS ...................................................................................................... 9

    INTRODUCTION ...................................................................................................... 10

    MATERIALS AND METHODS ................................................................................. 16

    Apparatus & Reagents .......................................................................................... 16

    Preparation of DPPH Reagent .............................................................................. 16

    Preparation of Ascorbic Acid Standards and Positive Controls ............................. 16

    Preparation of Samples ........................................................................................ 17

    DPPH Radical Scavenging Activity ....................................................................... 17

    Spectrophotometric Reading of Ascorbic Acid Standards, Samples and Positive

    Controls................................................................................................................. 18

    Effect of Storage Conditions on Antioxidant Activity of Orange Juice ................... 18

    Statistical Analysis ................................................................................................ 18

    EXPERIMENTAL RESULTS .................................................................................... 19

    Antioxidant Activity of Freshly Squeezed and Commercial Orange(NFC) Juices . 19

    Effect of Storage Conditions on the Antioxidant Activity of Commercial Orange

    Juice (Not From Concentrate) ............................................................................... 23

    DISCUSSION ........................................................................................................... 36

    CONCLUSION ......................................................................................................... 39

  • 5

    APPENDICES .......................................................................................................... 40

    Additional Figures, Tables and Illustrations of Performed DPPH Assay ............ 40

    Effect of Storage Time on scavenging capacity of Selected Irish Commercial

    Orange Juice (NFC) ........................................................................................... 42

    RISK ASSESSMENT ......................................................................................... 44

    SAFETY DATA SHEETS ................................................................................... 45

    BIBLIOGRAPHY ...................................................................................................... 67

  • 6

    LIST OF FIGURES

    Figure 1 Redox reaction between DPPH and an antioxidant (AH). ........................................... 13

    Figure 2 Generated ascorbic acid calibration curve showing the decreasing absorbance of

    DPPH in function of increasing ascorbic acid concentration. ...................................................... 19

    Figure 3 Graphical representation of the antioxidant activity of prepared dilutions of freshly

    squeezed orange juice. ..................................................................................................................... 21

    Figure 4 Graphical representation of scavenging activity exhibited by prepared dilutions of

    commercial orange juice (NFC) towards DPPH. ........................................................................... 22

    Figure 5 Graphical comparison between the scavenging activity of prepared dilutions of

    freshly squeezed (in blue) and NFC (in orange) juices. An overall higher free radical

    scavenging activity is evident in the dilutions of freshly squeezed orange juice, compared to

    those of the commercial orange product. ....................................................................................... 22

    Figure 6 Effect of dilution on the scavenging activity of analyzed commercial orange juice

    (NFC), stored at -20C for a week, against DPPH radical. .......................................................... 24

    Figure 7 Effect of dilution on the scavenging activity of analyzed commercial orange juice

    (NFC), stored at 4C for a week. ..................................................................................................... 24

    Figure 8 Effect of dilution on the scavenging activity of analyzed commercial orange juice

    (NFC), stored at 25C for a week. ................................................................................................... 25

    Figure 9 Effect of dilution on the antioxidant activity of analyzed commercial orange juice,

    stored at 30C for a week. ................................................................................................................ 25

    Figure 10 Effect of dilution on the scavenging activity of analyzed commercial orange juice

    (NFC), stored at 37C for a week. ................................................................................................... 26

    Figure 11 Effect of storage temperature on the scavenging activity of undiluted commercial

    orange juice (NFC), after 7 days. ..................................................................................................... 26

    Figure 12 Effect of storage temperature on the scavenging activity of 1:2 diluted commercial

    orange juice, upon 7 days, towards DPPH. ................................................................................... 27

    Figure 13 Effect of different storage temperatures on the free radical scavenging activity of

    1:5 diluted commercial orange juice (NFC) after 7 days

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