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

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

Bachelor’s degree in Applied Biology and Biopharmaceutical

Science.

Submitted: 18th March 2016

©Mirella Amarachi Ejiugwo

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DECLARATION

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

I cited and referred to other authors’ works.

…………………………………………..

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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 -20˚C (in the

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

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

37˚C. 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.

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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.

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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

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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

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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 -20°C for a week, against DPPH radical. .......................................................... 24


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


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

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

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


(NFC), stored at 37°C 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. ............................................................. 27

Figure 14 Effect of dilution on the free radical scavenging activity of commercial orange juice

(NFC) at different storage temperatures for 7 days. ..................................................................... 28

Figure 15 Effect of dilution on the scavenging activity of commercial orange juice (NFC)

stored at -20˚C for 14 days. .............................................................................................................. 28


stored at 4˚C for 14 days. ................................................................................................................. 29

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stored at 25˚C for 14 days. ............................................................................................................... 29





Figure 20 Effect of different temperatures, ranging from -20 to 37 °C, on the scavenging

activity of undiluted commercial orange juice (NFC) stored for 14 days. .................................. 31

Figure 21 Effect of different temperatures, ranging from -20 to 37 °C, on the antioxidant

activity of 1:2 diluted commercial orange juice (NFC) stored for 14 days. ................................ 31

Figure 22 Effect of different temperatures, ranging from -20 to 37 °C, on the antioxidant

activity of 1:5 diluted commercial orange juice (NFC) stored for 14 days. ................................ 32

Figure 23 Effect of dilution and storage temperature on the scavenging activity of

commercial orange juice (NFC), stored for 14 days. .................................................................... 32

Figure 24 Effect of storage time on the scavenging capacity of undiluted commercial orange

juice (NFC) stored for 7 days (in blue) and 14 days(in red). ....................................................... 33

Figure 25 Effect of storage time on the scavenging capacity of 1:2 diluted commercial

orange juice (NFC) stored for 7 days (in blue) and 14 days (in red). ......................................... 33


orange juice (NFC) stored for 7 days (in blue) and 14 days (in red). ......................................... 34

Figure 27 Progressive decolouring of DPPH at increasing concentrations of ascorbic acid

standards (the range of 0 to 1000 µM, from left to right) in triplicate: this corresponds to an

increasing order of antioxidant activity of ascorbic acid towards DPPH reagent (60 µM). ..... 40

Figure 28 The scavenging activity seen by the decolouring of DPPH in set-up dilutions of

commercial orange juice NFC (from L-R: undiluted, I:2 and 1:5) stored at different

temperatures, from left to right: at -20˚C (in the freezer), 4˚C(in the fridge), 25˚C (at room

temperature), 30˚C and 37˚C. .......................................................................................................... 40

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LIST OF TABLES

Table 1 Antioxidant activity data of freshly squeezed and commercial orange juices,

expressed in VCAEC (µM). ...................................................................................... 20

Table 2 Antioxidant activity of prepared dilutions of freshly squeezed orange juice.

Each value is the mean of triplicate determinations per trial. ................................... 20

Table 3 Antioxidant activity of set up dilutions of commercial orange juice, performed

in triplicate per trial. Each value is the mean of triplicate determinations per trial. ... 20

Table 4 Antioxidant activity data, expressed in Vitamin C Equivalent Antioxidant

Capacity (VCEAC) of commercial orange juice (NFC), not from concentrate, stored

at different temperatures for 7 days.......................................................................... 35

Table 5 Antioxidant activity data, expressed in Vitamin C Equivalent antioxidant

Capacity (VCEAC), of commercial orange juice (NFC) stored at different

temperatures for 14 days. ........................................................................................ 35

Table 6 Ascorbic acid standard curve data for determining the equivalent scavenging

capacity/activity of prepared samples (FSOJ and NFC) towards DPPH radical. ...... 41

Table 7 Corresponding antioxidant activity of ascorbic acid standards. The IC50 of the

range of prepared ascorbic acid standards equals to 762.20 µM. ............................ 41

Table 8 DPPH Inhibition data of Irish commercial orange juice (NFC) stored at

different temperatures for 7 days. ............................................................................ 42

Table 9 DPPH inhibition data of Irish commercial orange juice (COJ) stored at

different temperatures for 14 days. .......................................................................... 43

Table 10 Completed risk assessment form of the present research work. ............... 44

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ABBREVIATIONS

1,1-diphenyl-2-picrylhydrazyl - DPPH

Butylated Hydroxytoluene – BHT

Antioxidant activity – AA

Free Radical Scavenging Activity – FRSA

Methanol – MetOH

Distilled water – DW

Commercial orange juice - COJ

Freshly squeezed orange juice - FSOJ

Vitamin C Equivalent Antioxidant Capacity – VCEAC

Not from concentrate - NFC

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INTRODUCTION

Antioxidants are stable low molecular weight reducing agents, capable of donating

electrons to and neutralizing reactive nitrogen species (RNS) and reactive oxygen

species (ROS). Some antioxidants are found both in the intracellular and extracellular

environment of most eukaryotes (they are defined as endogenous antioxidants) –

produced during physiological metabolic reactions. On the other hand, other

antioxidants, which cannot be biosynthesized, are supplied through the diet (which aid

in scavenging excess free radicals), such as: vitamin C (ascorbic acid), vitamin E,

lycopene, β-carotene, flavonoids, selenium, polyphenols –likewise called exogenous

antioxidants.

The major sources of dietary antioxidants are fruit and vegetables, namely: red

grapes, strawberries, raisins, prunes, blueberries, oranges, cherries, kiwi, lemon, kale,

beetroots, spinach, broccoli flowers, Brussel sprouts, red bell peppers, eggplants, etc.

Antioxidants are capable of delaying or inhibiting cellular damage by scavenging free

radical metabolic by-products. Generally, in vivo, an antioxidant acts as either a radical

scavenger, hydrogen donor, electron donor, peroxide decomposer, singlet oxygen

quencher, enzyme inhibitor, synergist, or a metal-chelating agent (Frie B, 1988).

This said, antioxidants work specifically to safeguard cellular integrity within biological

systems, against free radicals: by single electron transfer and hydrogen atom transfer

mechanisms. Free radicals refer to any molecular species capable of independent

existence that contains one/more unpaired electrons in the outer atomic orbital; they

are generated as waste products of normal cell aerobic respiration (Victor R. Preedy,

2013). The presence of an unpaired electron in free radicals results in certain common

properties shared by most radicals: instable and highly reactivity. They can either

donate an electron to or accept an electron from other molecules, therefore behaving

as either oxidants or reductants, respectively (Cheeseman KH, 1993). Other sources

of free radicals are the immune system, stress, pollution, dietary factors, inflammation,

toxins and drugs.

Endogenous antioxidants are a network of enzymes that are capable of detoxifying

superoxide derivatives (generated mainly during normal metabolic processes) namely:

superoxide dismutases, catalase, ubiquinol, and glutathione.

Superoxide dismutases (SODs), a class of closely related enzymes that catalyze the

dismutation of the superoxide anion into oxygen and hydrogen peroxide, are present

in almost all aerobic cells and in extracellular fluids. There are three major families of

superoxide dismutase, based on the constituent metal cofactor: Cu/Zn (which binds

both copper and zinc), Fe and Mn types (which bind either iron or manganese), and

finally the Ni type which binds nickel (Wuerges, 2004).

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The reactive final product of the detoxifying activity of superoxide dismutases,

hydrogen peroxide, is then broken down into less reactive water and oxygen

molecules, catalyzed by the enzyme catalase.

Another important enzymatic antioxidant - the glutathione system - consists of

glutathione, glutathione reductase, glutathione peroxidases, and glutathione S-

transferases. This system is found in animals, plants, and microorganisms. Assisted

by vitamin C, it mops up potentially dangerous oxidizing radicals in aqueous parts and

surfaces of the cells (Michael B. Davies, 1991).

On the other hand, the most common non-enzymatic scavengers are: ascorbic acid,

tocopherols and tocotrienols (vitamin E), melatonin and uric acid.

Ascorbic acid (also known as “vitamin C”) is a 6-carbon lactone, synthesized

naturally from glucose by many animals and plants. However, it must be absorbed

through diet for humans. Primarily, vitamin C is an electron donor (reducing agent or

antioxidant), which accounts for possibly all its entire biochemical and molecular

functions. Hitherto, it acts as an electron donor to 11 enzymes, three of which pertain

to fungi (in the recycling process of pyrimidines and the deoxyribose moiety of

deoxynucleosides); the remaining eight enzymes are found in humans: three of which

participate in collagen hydroxylation (for the formation of collagen) and two in carnitine

biosynthesis. It is involved in aiding the absorption of inorganic iron; very significant in

vascular function and it is an important cofactor in neurotransmitter biosynthesis (K.A.,

2003).

Futhermore, vitamin C reduces tocopherol (vitamin E) radicals back to their active

form. It is capable of scavenging a whole range of known radicals, such as: hydrogen

peroxide, hydroxyl radical, superoxide radical and singlet oxygen.

Melatonin - a permeable hormone present naturally in animals, plants and some

other living organisms - is an electron-rich, potent, and broad-spectrum free radical

scavenger. Once oxidized, upon reacting with free radicals via additive reactions, it is

converted into stable end products, that are excreted in the urine - unlike other

antioxidants, which can reversibly return to their former reduced state. In scientific

terms, it does not undergo redox cycling. Thereby, melatonin is described as "suicidal

antioxidant". Furthermore, it is supposed that melatonin likely stimulate some

important antioxidative enzymes, i.e., superoxide dismutase, glutathione peroxidase

and glutathione reductase ( (Reiter RJ, 1997)

Melatonin in plants not only provides an alternative exogenous source of melatonin

for herbivores but also suggests that melatonin may be an important antioxidant in

plants which protects them from a hostile environment that includes extreme heat, cold

and pollution, all of which generate free radicals.

Finally, another non-enzymatic free radical antioxidant worth-mentioning is vitamin E:

a group of eight related tocopherols and tocotrienols, fat-soluble molecules with an

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established metabolic role in trapping free radicals in membranes and lipoproteins.

Furthermore, due to structural affinity, vitamin E is capable of stabilizing the membrane

structure.

Vitamin E, an antioxidant system with lipophilic nature due to the constituting phenol

group, functions both in vitro and in vivo: it is capable of breaking the chain reactions

of oxidative damage of polyunsaturated acids by hydroxyl radicals and superoxide (if

it remains oxidized, it can bring about disastrous consequences on the cells).

Free radicals are found within eukaryotes as by-products of metabolic reactions

within the cellular environment. Their presence is the etiological basis of chronic

diseases related to oxidation of important biomolecules (namely nucleic acids, lipids,

proteins and carbohydrates): consequently, they are believed to be the primary

mechanism underlying carcinogenesis, cardiovascular, neurologic, inflammatory,

renal disorders, gastro-intestinal diseases, pulmonary disorders, ocular disorders,

infertility and other oxidative stress-induced diseases.

Antioxidants have been found to render multiple counteractive effects against

oxidative stress-related diseases. Thus, research in this sector continues so to gain

more insight into their potential nutraceutical use. In simple terms, they are postulated

to aid in the prevention of ailments linked with genetic alteration, cellular damage and

homeostasis disruption caused by free radicals. Thus, they inhibit disequilibrium

between free radical proliferation and antioxidant defence system.

Furthermore, there are defined mechanisms of actions by which antioxidants function:

1. The first line of defense is constituted of preventive antioxidants, which

suppress the formation of radicals, most especially the metal-induced

decompositions of hydroperoxides and hydrogen peroxide. Examples:

glutathione peroxidase, glutathione-s-transferase, phospholipid hydroperoxide

glutathione peroxidase (PHGPX), and peroxidase (known to decompose lipid

hydroperoxides to corresponding alcohols).

2. The second line of defense relates to antioxidants that suppress chain initiation

and/or disrupt chain propagation reactions: vitamin C, uric acid, bilirubin,

albumin, thiols, vitamin E and ubiquinol.

3. The third line of defense are the repair and de novo antioxidants. Proteolytic

enzymes, proteinases, proteases, and peptidases - found in the cytosol and in

the mitochondria of mammalian cells - recognize, degrade, and remove

oxidatively modified proteins and inhibit the buildup of oxidized proteins. In

addition, DNA repair systems, such as glycosylases and nucleases, exist which

play an important role in the total defense system against oxidative damage of

DNA.

4. The forth line of defense, called “adaptation”, is whereby the signal for the

production and reactions of free radicals triggers biosynthesis and transport of

the suitable antioxidant to the right site.

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Biological macromolecules, such as nucleic acids, proteins, lipids and carbohydrates

are the major biological "antagonists" of free radicals.

A good number of both in vitro and in vivo methods for the determination of

antioxidant activity, ranging from fruit and vegetables to biological samples. However,

each method has a certain delimited scope, based on the measurement of certain

parameters governing the desired properties of the antioxidant activity of samples of

interest. In vitro methods are the most commonly conducted, unlike the in vivo ones,

due to their relative simplicity and quick retrieval of results.

Amongst known in vitro methods of antioxidant activity analysis, the 1,1-diphenyl-2-

picrylhydrazyl (DPPH) method is most common. The underlying principle of this

method is the reduction of the purple-coloured stable radical (DPPH) - due to the

presence of an unpaired electron at nitrogen atom of constituting hydrazil group - in

the presence of an antioxidant (which actually acts as an electron donor). Following

redox reaction with an antioxidant, the DPPH undergoes decoloring from purple to

pale yellow, coupled with a consequential absorbance decrease at its characteristic

wavelength (λ = 515 nm), and it becomes a stable diamagnetic molecule. The resulting

product is DPPH-H, in its reduced form.

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

According to Kim et al, some major guidelines on the constituent parameters in the

execution of the DPPH assay, deriving from multiple scientific research papers, are as

followed:

1. DPPH solutions and samples are to be prepared in either ethanol or methanol.

2. The concentration of the DPPH working standard ideally falls within the range

of 0.05 mmol/L to 1.5 mmol/L.

3. The volume ratio of DPPPH reagent to sample should be relative to the

concentration of the DPPH solution.

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4. The incubation time for the determination of free radical scavenging activity can

vary from 1 to 120 minutes (However, the most used reaction times are 15, 20

and 30 minutes).

5. The spectrophotometric determination of free radical scavenging activity of

DPPH can be conducted at different wavelengths: between the range of 492

and 525 nm. Most common wavelengths used are 515 and 517 nm.

6. The radical scavenging activity can be determined by utilizing antioxidant

standard solutions, namely: ascorbic acid, Trolox, vitamin E, BHT and BHA.

Among these, the most commonly used are ascorbic acid, Trolox and α-

tocopherol.

7. The equation used to calculate the inhibition of DPPH varies according to

literature. Most commonly used equation is: (Acontrol – Asample)/Acontrol ×

100; whereby, Acontrol = the absorbance of the control (DPPH reagent) and

Asample = the absorbance of DPPH reagent +sample). Otherwise, antioxidant

activity is expressed as IC50, the concentration of antioxidant required to quench

50% of DPPH activity: the lower this parameter is, the higher its antioxidant

capacity.

In the present lab-scale application of the DPPH assay, Butylated Hydroxytoluene

(BHT) and propryl gallate, common synthetic antioxidants used in the food industry,

are set up as reference compounds - to attribute validity to the assay. These

compounds were purposely selected due to their significant and robust antioxidant

activity towards characteristic antagonists (be it free radicals or oxidants).

The citrus fruit orange (C.sinensis) was chosen as case study of the present

research work due to its outstanding antioxidant properties.

The main energy-generating nutrients in orange juice (OJ) are glucose, fructose and

sucrose, malic and citric acids, folate and potassium; other key nutrients, acting as

antioxidants, present are ascorbic acid, flavonoids [flavanones (hesperidin, narirutin,

poncirin and naringin] and hydroxycinnamic acids, esters of ferulic, p-couramic,

sinapic and caffeic acids), carotenoids (xanthophylls, crytoxanthins, carotenes) (Victor

R. Preedy, 2013).

Orange juice (OJ) is sold under four forms:

a. Frozen concentrate: obtained by removing water, by evaporation, from the

orange juice. It has a longer shelf life of years at -6.7˚C.

b. Unpasteurized orange juice: the orange juice is freshly squeezed from the fruit

and packaged into containers (glass, plastic or carton), without being pasteurized.

Thus, it has a shelf life of few days.

c. Reconstituted orange juice: processed OJ to obtain its frozen concentrate and,

then reconstituted by the addition of the water removed earlier from it.

d. Ready-to-drink: orange juice is subject to quick processing and pasteurization,

upon squeezing the fruit. It can be stored either chilled or frozen for a minimum of 12

months.

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The nutritional quality of OJ is associated primarily with its L-ascorbic acid and its

oxidized form, dehydroascorbic acid content (which accounts for 5% of the antioxidant

activity (AA)). Based on latest epidemiological studies, the most current daily

recommended intake/dose of vitamin C is suggested to be 100-120 mg/day, so to

attain cellular saturation and optimum risk prevention and/or reduction of heart-related

diseases, cancer, aging, macular degeneration, skin disorders and stroke in healthy

individuals (K.A., 2003).

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MATERIALS AND METHODS

Apparatus & Reagents

BioSpec mini UV-VIS Spectrophotometer (Shimadzu), L-ascorbic acid (No. A-7506,

LOT 117C-0327 by Sigma-Aldrich), DPPH (produced by Sigma-Aldrich, Germany;

D9132-5G; LOT# STBF6566V; PCode 101667308), methanol (product 20847.307;

batch # 14J280511; expiry date: 10-2019), Buthylated Hydroxytoluene (≥ 99%, cat.

W218405, LOT 21966-214, by Sigma-Aldrich), propyl gallate (produced by Fluka

analytical, China: 48710-100G-I; LOT # BCBM6373V; PCode: 101548396), sterile

plastic containers, distilled water.

The execution of the DPPH assay was according to Brand-Williams et al., with some

modifications (Brand-Williams, et al., 1995).

Preparation of DPPH Solution

0.0239 g of DPPH reagent was weighed out, dissolved and made up to 100 ml with

pure methanol: yielding a final concentration of 600 µM (DPPH stock solution). A

1:10 dilution was prepared: 10 ml of DPPH stock solution was diluted to 100 ml with

pure methanol, giving a final concentration of 60 µM (DPPH working standard

solution). A fresh stock solution of DPPH (600 µM) was prepared each week, as

DPPH degrades over time.

Preparation of Ascorbic Acid Standards and Positive Controls

A stock solution of 10 mM L-ascorbic acid was prepared: 0.0176 g of ascorbic acid

was weighed out, dissolved and made up to a final volume of 10 ml with pure methanol

(ascorbic acid stock). 500 µL of the stock was diluted in 1000 µL with distilled water (5

mM). Finally, 400 µL of 5 mM L-ascorbic acid solution was diluted to 2000 µL with

distilled water – the final concentration of 1000 µM (working standard).

17

The following concentrations of ascorbic acid standards were made in test-tubes in

triplicate, using distilled water as diluent in a final volume of 100 µL: 0, 250, 400, 500,

600, 700, 800, 900 and 1000 µM.

Butylated hydroxytoluene (BHT) and propyl gallate were set up as positive controls.

A solution of 2000 µM BHT was prepared: 0.0220 g of BHT was weighed, dissolved

and made up to 50 ml with pure methanol and aliquots of 100 µL in test tubes in

triplicate. A triplicate of 2000 µM propyl gallate was prepared also: 0.0216 g of propyl

gallate was weighed out, dissolved and made up to 50 ml with methanol. These

reference compounds serve to validate the test, by verifying the efficacy of the used

DPPH reagent.

Furthermore, a control for the determination/monitoring of DPPH inhibition was

prepared: 4 ml of DPPH 60 µM in triplicate.

Preparation of Samples

Three fresh oranges (C. sinensis) – from Spain, purchased in Dunnes Stores -

were halved and the juice content extracted manually with the aid of a juicer.

The crude juice extract was filtered through a 110 mm Whatmann filter paper –

to isolate the pulpnfrom the juice content, and obtain a clarified solution.

Three commercial orange juices (not from concentrate (NFC),100% pressed

fruit; produced in Donegal, Ireland; expiry date: September 2016), preserved in

the fridge prior to use, were used, immediately after opening.

The following dilutions were performed for both samples, using methanol: 1:5, 1:2

and 1:10. Each dilution was prepared into test tubes (100 µL/test-tube) in triplicate.

DPPH Radical Scavenging Activity

Upon preparation of ascorbic acid standards, positive controls (BHT and propyl

gallate) and samples, 3.9 ml of fresh DPPH working standard solution (60 µM) was

added to all test tubes, and were incubated in the dark (wrapped in aluminum foil) for

15 minutes – to measure their respective free radical scavenging ability.

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Spectrophotometric Reading of Ascorbic Acid Standards, Samples and

Positive Controls

The used spectrophotometer was earlier switched on, left to warm for circa 15

minutes, set at 515 nm and auto-zeroed with 4 ml of pure methanol (blank).

Upon the reaction time, all contents of test tubes were measured

spectrophotometrically at 515 nm, using 4-ml plastic cuvettes. The corresponding

DPPH inhibition (extent of antioxidant activity of antioxidants in analyzed samples

against DPPH) was calculated using the following formula:

DPPH Inhibition (%) = (Acontrol – Asample)/Acontrol × 100.

Whereby, Acontrol = absorbance of DPPH reagent (60 µM) and Asample =

absorbance of 100 µL sample + 3900 µL DPPH reagent (60 µM) - upon storing in the

dark for 15 minutes.

Effect of Storage Conditions on Antioxidant Activity of Orange Juice

Orange juice (NFC) aliquots were stored at different temperatures (namely, at -20, 4,

25, 30 and 37˚C1) for 7 and 14 days in sterile plastic containers. They were equilibrated

at room temperature, prior to the DPPH assay. Few dilutions of incubated commercial

orange juice were carried out (namely, 1:2 and 1:5 dilutions), for the DPPH assay (final

volume of sample needed: 100 µL) – using methanol as diluent.

Statistical Analysis

All determinations were performed in triplicate per trial, and each study was repeated

thrice for validation reasons. Results were expressed as mean ± standard deviation,

with the aid of Microsoft Office Excel 2016.

1 The temperatures of the used (2) incubators, freezer, fridge and the laboratory environment are within standard conditions, as

the laboratory supervisor monitors them periodically: 37, 30, -20, 4 and 25˚C, respectively.

19

EXPERIMENTAL RESULTS

Antioxidant Activity of Freshly Squeezed and Commercial Orange (NFC)

Juices

Figure 2 Generated ascorbic acid calibration curve showing the decreasing

absorbance of DPPH in function of increasing ascorbic acid concentration.

The absorbance of DPPH reagent (60 µM) declines with increasing concentration of

ascorbic acid2. The absorbance of prepared control (solely DPPH reagent 60 µM, 4

ml) was an average absorbance of 0.694 (used for the determination of DPPH

inhibition (%) of samples).

The absorbance readings of the set-up reference compounds BHT and propyl gallate

(2000 µM) result within the range of the generated calibration graph above; the

antioxidant activity herein was apparent by the decoloring from purple to pale yellow

that occurred: 0.439 ± 0.019 and 0.033 ±0.000, respectively.

Using the equation of the ascorbic acid standard curve (y = -0.0005x + 0.7246), upon

the DPPH assay, the vitamin C equivalent antioxidant capacity (VCAEC) of the

undiluted selected commercial Irish orange juice(Not From Concentrate) resulted

2 The standard curve (Figure 2) generated serves to express antioxidant activity results as VCEAC

(Vitamin C Equivalent Antioxidant Capacity) in µM.

y = -0.0005x + 0.7246R² = 0.9967

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

0 100 200 300 400 500 600 700 800 900 1000 1100

Ab

so

rba

nc

e a

t 5

15

nm

Ascorbic Acid Concentration (µM)

L-ASORBIC ACID STANDARD CURVE

20

1173.4 µM, whereas for the undiluted freshly squeezed orange juice (FSOJ), a

corresponding value of 1363.4 µM (refer to Table 1). In other terms, a higher

scavenging capacity was exhibited by the freshly squeezed than the commercial

orange juice (NFC) - circa 13.9 %.

Table 1 Antioxidant activity data of freshly squeezed and commercial orange juices,

expressed in VCAEC (µM). Each value is the mean of triplicate determinations per

trial.

Freshly Squeezed OJ Commercial OJ

Dilution Factor VCEAC Conc. (µM) VCEAC Conc. (µM)

1:10 502.8 409.0

1:5 743.2 577.6

1:2 1361.0 996.8

Undiluted 1363.4 1173.4

Table 2 Antioxidant activity of prepared dilutions of freshly squeezed orange juice.

Each value is the mean of triplicate determinations per trial.

Dilution

Factor

1st Trial

DPPH

Inhibition

Value (%)

2nd Trial

DPPH

Inhibition

Value (%)

3rd Trial

DPPH

Inhibition

Value (%)

Average

DPPH

Inhibition

Value (%)

Standard

Deviation

1:10 31.1 26.8 28.3 28.7 2.2

1:5 56.3 40.2 44.1 46.8 8.4

1:2 92.4 92.0 95.7 93.4 2.1

Undiluted 92.3 93.1 95.2 93.5 1.5

Table 3 Antioxidant activity of set up dilutions of commercial orange juice (NFC),

performed in triplicate per trial. Each value is the mean of triplicate determinations per

trial.

Dilution

Factor

1st Trial

DPPH

Inhibition

Value (%)

Mean

2nd Trial

DPPH

Inhibition

Value (%)

Mean

3rd Trial

DPPH

Inhibition

Value (%)

Mean

DPPH

Inhibition (%)

Mean

Standard

Deviation

1:10 37.1 1.8 26.1 21.7 14.7

1:5 51.8 8.3 43.0 34.4 18.8

1:2 78.8 27.4 91.6 65.9 27.7

Undiluted 78.0 66.4 93.3 79.2 11.0

21

Another equivalent expression of antioxidant activity – DPPH inhibition (%), which

indicates the percentile of DPPH reduced by antioxidants present in samples – gives

a confirmatory note to the quantified VCAEC values (see Tables 2 and 3). As the

concentration of samples of interest, FSOJ and NFC, increases their respective

scavenging activity towards the fixed concentration of DPPH radical (60 µM) increases

likewise - represented by their corresponding increasing DPPH inhibition % values.

Furthermore, per each performed dilution, freshly squeezed orange juice had a

higher antioxidant activity/ DPPH inhibition value – towards the free radical DPPH –

than the commercial orange-based beverage (not from concentrate, 100% pressed

fruit). The freshly squeezed orange juice exhibited an overall scavenging ability of

about 20.4% greater than its commercial product.

A comparative graphical representation of the two samples (see Figure 5) gives a

concise and simplified understanding of the obtained results.

In particular, significant antioxidant activity was most evident in the undiluted aliquots

of both samples (FSOJ and NFC) – confirmed by their relatively lowest absorbance

readings at 515 nm and corresponding DPPH inhibition (%) values, compared to their

respective prepared dilutions, namely: 1:2, 1:5 and 1:10 (see Figures 3-5).

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

freshly squeezed orange juice.

28.746.8

93.4 93.5

0.0

20.0

40.0

60.0

80.0

100.0

1:10 1:5 1:2 Undiluted

DP

PH

In

hib

itio

n (

%)

Dilution Factor

Antioxidant Activity of Different Dilutions of Freshly Squeezed Orange Juice

22

Figure 4 Graphical representation of scavenging activity exhibited by prepared

dilutions of commercial orange juice (NFC) towards DPPH.

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.

21.7

34.4

65.9

79.2

0.010.020.030.040.050.060.070.080.090.0

100.0


DP

PH

In

hib

itio

n (

%)

Dilution Factor

Scavenging Activity of Different Dilutions of Commercial Orange Juice (NFC)

28.7

46.8

93.4 93.5

21.7

34.4

65.9

79.2

0.010.020.030.040.050.060.070.080.090.0

100.0


DP

PH

In

hib

itio

n (

%)

Dilution Factor

Comparison Between Scavenging Capacity of Freshly Squeezed and NFC Juices

Freshly Squeezed OJ DPPH InhibItion (%)

Packaged OJ DPPH inhibition (%)

23

Effect of Storage Conditions on the Antioxidant Activity of Commercial

Orange Juice (Not From Concentrate)

Generally, increasing scavenging activity was observed as the analyzed commercial

orange juice (NFC) became more concentrated; despite the period and temperature

of storage (see Figures 6-10, 15-19).

The obtained optimum storage temperature for the analyzed NFC, indicated by a

corresponding relative maximum scavenging activity, resulted to be at -20˚C: in the

freezer (see Figures 14 and 23) – independent of storage period and dilution –

compared to 4, 25, 30 and 37ºC.

The obtained scavenging activity of DPPH values (%) of undiluted NFC assessed

immediately (see Figure 4), compared to those gotten for NFC stored for 7 days (see

Figure 6) and for 14 days (see Figure 15) at the optimum temperature (-20ºC) was

greater and resulted: 79.2 % against 75.3% and 66.7%, respectively.

Therefore, as the length of storage time increased, a slight decline in its scavenging

capacity of COJ (stored at -20ºC) took place (see Figures 24-26): an average decrease

of about 3.3% per week. The second best storage temperature was observed at 4˚C.

From 25 to 37˚C, fluctuation of antioxidant potential occurred and, thus, inconsistent

and incomparable results were obtained (see Figures 24-26).

Upon 7 days of storage of NFC, a particular trend was noticed in the stored samples:

their experimental DPPH inhibition results showed that at 30˚, NFC had a higher

antioxidant activity than at 25ºC (at room temperature) and 37˚C (see Figures 8-10).

As the storage time continued to 14 days, the mentioned trend remained solely

towards 25˚C (see Figures 17-19): DPPH inhibition of 28.4 % against 24.2%,

respectively.

24

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

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


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

27.2

55.0

75.3

0.010.020.030.040.050.060.070.080.090.0

100.0

1:5 1:2 Undiluted

DP

PH

In

hib

itio

n (

%)

Dilution Factor

Effect of Dilution on Scavenging Acitivity of NFC Stored at -20°C for 7 Days

20.0

33.6

51.3

0.010.020.030.040.050.060.070.080.090.0

100.0

1:5 1:2 Undiluted

DP

PH

In

hib

itio

n (

%)

Dilution Factor

Effect of Dilution on Scavenging Activity of NFC Stored at 4˚C for 7 Days

25


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

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

juice, stored at 30°C for a week.

5.6 9.2

25.5

0.010.020.030.040.050.060.070.080.090.0

100.0

1:5 1:2 Undiluted

DP

PH

In

hib

itio

n (

%)

Dilution Factor


6.514.0

31.9

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

1:5 1:2 Undiluted


26

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

orange juice (NFC), stored at 37°C for a week.

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

commercial orange juice (NFC), after 7 days.

9.114.6

25.2

0.010.020.030.040.050.060.070.080.090.0

100.0

1:5 1:2 Undiluted

Effect of Dilution on Scavenging Activity of Commercial Orange Juice Stored at 37˚C for 7 Days

75.3

51.3

25.531.9

25.2

0.010.020.030.040.050.060.070.080.090.0

100.0

-20 4 25 30 37

DP

PH

In

hib

itio

n (

%)

Storage Temperature (˚C)

Effect of Storage Temperature on Scavenging Activity of Undiluted NFC Stored for 7 Days

27

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

commercial orange juice, upon 7 days, towards DPPH.

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

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

55.0

33.6

9.214.0 14.6

-20.0-10.0

0.010.020.030.040.050.060.070.080.090.0

100.0

-20 4 25 30 37

DP

PH

In

hib

itio

n (

%)


Effect of Storage Temperature on Scavenging Activity of 1:2 Diluted NFC Stored for 7 Days

27.220.0

5.6 6.5 9.1

-30.0

-20.0

-10.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

-20 4 25 30 37

DP

PH

In

hib

itio

n (

%)


Effect of Storage Temperature on Scavenging Activity of 1:5 Diluted NFC Stored for 7 Days

28

Figure 14 Effect of dilution on the free radical scavenging activity of commercial

orange juice (NFC) at different storage temperatures for 7 days.

Figure 15 Effect of dilution on the scavenging activity of commercial orange juice

(NFC) stored at -20˚C for 14 days.

75.3

51.3

25.531.9

25.2

55.0

33.6

9.2 14.0 14.6

27.220.0

5.6 6.5 9.1

-30.0-20.0-10.0

0.010.020.030.040.050.060.070.080.090.0

100.0

-20 4 25 30 37DP

PH

In

hib

itio

n (

%)


Effect of Dilution on Scavenging Capacity of NFC Stored At Varying Storage Temperatures for 7 Days

Undiluted Sample DPPH Inhibition (%)

1:2 Diluted Sample DPPH Inhibition (%)

1:5 Diluted Sample DPPH Inhibition (%)

22.7

38.8

66.7

0.010.020.030.040.050.060.070.080.090.0

100.0

1:5 1:2 Undiluted

DP

PH

In

hib

itio

n (

%)

Dilution Factor

Effect of Dilution on Scavenging Activity of NFC Stored at -20˚C for 14 Days

29


(NFC) stored at 4˚C for 14 days.



13.018.8

28.2

0.010.020.030.040.050.060.070.080.090.0

100.0

1:5 1:2 Undiluted

DP

PH

In

hib

tio

n (

%)

Dilution Factor


13.4 16.324.2

0.010.020.030.040.050.060.070.080.090.0

100.0

1:5 1:2 Undiluted

DP

PH

In

hib

ion

(%

)

Dilution Factor

Effect of Dilution on Scavenging Activity of (NFC) Stored at 25˚C for 14 Days

30





15.0

23.128.4

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

1:5 1:2 Undiluted

DP

PH

In

hib

itio

n (

%)

Dilution Factor


13.817.9

31.9

0.010.020.030.040.050.060.070.080.090.0

100.0

1:5 1:2 Undiluted

DP

PH

In

hib

itio

n (

%)

Dilution Factor


31

Figure 20 Effect of different temperatures, ranging from -20 to 37 °C, on the

scavenging activity of undiluted commercial orange juice (NFC) stored for 14 days.


antioxidant activity of 1:2 diluted commercial orange juice (NFC) stored for 14 days.

66.7

28.2 24.2 28.4 31.9

0.010.020.030.040.050.060.070.080.090.0

100.0

-20 4 25 30 37

DP

PH

In

hib

itio

n (

%)


Scavenging Capacity of Undiluted NFC Stored for 14 Days at Varying Storage Temperatures

38.8

18.8 16.323.1

17.9

0.010.020.030.040.050.060.070.080.090.0

100.0

-20 4 25 30 37

DP

PH

In

hib

itio

n (

%)


Scavenging Activity of 1:2 Diluted NFC Stored for 14 Days at Varying Storage Temperatures

32


antioxidant activity of 1:5 diluted commercial orange juice (NFC) stored for 14 days.

Figure 23 Effect of dilution and storage temperature on the scavenging activity of

commercial orange juice (NFC), stored for 14 days.

22.7

13.0 13.4 15.0 13.8

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

-20 4 25 30 37

DP

PH

In

hib

itio

n (

%)


Scavenging Activity of 1:5 Diluted NFC Stored for 14 Days at Varying Temperatures

22.7

13.0 13.4 15.0 13.8

38.8

18.8 16.323.1

17.9

66.7

28.224.2

28.431.9

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

13.8 4 25 30 37

Effect of Dilution and Storage Temperature on Scavenging Activity of NFC Stored for 14 Days

1:5 Diluted Sample 1:2 Diluted Sample Undiluted Sample

33

Figure 24 Effect of storage time on the scavenging capacity of undiluted commercial

orange juice (NFC) stored for 7 days (in blue) and 14 days(in red).


orange juice (NFC) stored for 7 days (in blue) and 14 days (in red).

75.3

51.3

25.5

31.9

25.2

66.7

28.224.2

28.4 31.9

0.010.020.030.040.050.060.070.080.090.0

100.0

-20 4 25 30 37

DP

PH

In

hib

itio

n (

%)

Storage Temperature

Effect of Storage Time on Antioxidant Activity of Undiluted NFC at Different Temperatures

Undiluted OJ Sample (Stored for 7 days)

Undiluted OJ Sample (Stored for 14 days)

55.0

33.6

9.214.0 14.6

38.8

18.8 16.323.1

17.9

0.010.020.030.040.050.060.070.080.090.0

100.0

-20 4 25 30 37

DP

PH

In

hib

itio

n (

%)


Effect of Storage Time on Antioxidant Activity of 1:2 Diluted NFC at Different Temperatures

1:2 Diluted OJ Sample (Stored for 7 days) 1:2 Diluted Sample (Stored for 14 days)

34


orange juice (NFC) stored for 7 days (in blue) and 14 days (in red).

27.220.0

5.6 6.5 9.1

22.7

13.0 13.4 15.0 13.8

-30.0

-20.0

-10.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

-20 4 25 30 37

Effect of Storage Time on Antioxidant Activity of 1:5 Diluted NFC at Different Temperatures

1:5 Diluted OJ Sample (Stored for 7 days)

1:5 Diluted Sample (Stored for 14 days)

35

Table 4 Antioxidant activity data, expressed in Vitamin C Equivalent Antioxidant

Capacity (VCEAC) of commercial orange juice (COJ), not from concentrate (NFC),

stored at different temperatures for 7 days. Each value is the mean of three trials and

each trial was performed in triplicate.

Undiluted NFC 1:2 Diluted NFC 1:5 Diluted NFC

Storage Temperature (˚C) VCEAC (µM) VCEAC (µM) VCEAC (µM)

-20 1120.8 851.9 482.8

4 802.5 567.4 387.4

25 460.1 243.4 195.4

30 545.4 291.4 207.9

37 456.5 315.4 241.6

Table 5 Antioxidant activity data, expressed in Vitamin C Equivalent antioxidant

Capacity (VCEAC), of commercial orange juice (COJ), not from concentrate (NFC),

stored at different temperatures for 14 days.

Undiluted NFC 1:2 Diluted NFC 1:5 Diluted NFC

Storage Temperature (˚C) VCEAC (µM) VCEAC (µM) VCEAC (µM)

-20 1007.2 636.5 421.9

4 495.9 452.5 293.9

25 442.5 337.9 298.5

30 498.5 427.9 319.9

37 545.2 359.2 304.5

36

DISCUSSION

A quantitative comparison between the obtained scavenging capacity values of

freshly squeezed orange juice (FSOJ) and not-from-concentrate (NFC) orange

beverage showed that the former has higher antioxidant activity levels than the latter

towards the free radical DPPH (an exemplary of potential free radicals). This confirms

the well-known fact that the antioxidant activity content of fruit juices reduces during

processing and storage (Preedy, 2014); however, such is minimized by the use of

specifically designed cartons, having multiple layers of oxygen and light barriers, for

packaging – to avoid loss of ascorbic acid and flavour, and to prolong its shelf life. Few

examples of high barrier materials employed are glass or foil laminates in brick packs,

nitrogen flushing and improving gas barrier of polyethylene terephthalate by blending

with aromatic polyamides. On the other hand, freshly squeezed orange juice has been

proved to have a brief shelf life (the period required to attain 50% loss of vitamin C) at

25˚C and 4˚C: 1 and 6 days, respectively.

This said, based on obtained results, it can be assumed that freshly squeezed

orange juice is healthier and more capable of scavenging free radicals, compared to

its deriving beverage. From a health perspective, this can be interpreted that the

consumption of fresh raw orange fruit is capable of attributing higher anti-oxidizing

effects towards unstable and reactive metabolic byproducts (that cause chronic

ailments) than its corresponding beverages.

However, it has been established that actually the antioxidant activity in freshly

squeezed juice is reduced because of the presence of certain oxidative enzymes

(cytochrome oxidase, ascorbic acid oxidase and peroxidase) that act on vitamin C, the

principal antioxidant in orange fruit.

The stability of vitamin C is subject to certain parameters: high temperature, salt and

sugar concentration, pH, oxygen, enzymes, light, metal catalysts, and bioburden and

protection provided by its packaging. In particular, oxygen is the most destructive

agent in orange juice as it causes vitamin C content to degrade. So, during the

execution of the present research work, the analyzed commercial orange juice (NFC)

was exposed minimally to air: aliquots of NFC juice (per trial) were introduced into

sterile plastic containers, which were then closed hermetically immediately and sealed

with parafilm. However, prior to keeping in the dark during the reaction time (15

minutes), the NFC orange juice was exposed to air in course of setting up the reaction

mixture (DPPH + NFC) in test-tubes.

Apart from degrading vitamin C, dissolved oxygen in orange juice causes increased

browning and growth of aerobic bacteria and moulds (Victor R. Preedy, 2013).

Other uncontrollable determining factors of the calculated antioxidant activity of COJ

(not from concentrate), related to vitamin C loss of the originating raw orange fruit

(Citrus sinensis), are:

37

1. High doses of nitrogen fertilizer can reduce vitamin C content in oranges.

2. Locations with cool nights yield citrus fruit with higher levels of ascorbic acid,

compared to hot tropical areas.

3. As ripening proceeds, vitamin C decreases: early maturing citrus varieties have

the highest level of vitamin C than late maturing ones (Nagy, 1980).

To proceed, it has been established that a robust correlation exists between ascorbic

acid content and total antioxidant activity, since it is assumed that ascorbic acid is the

main component responsible for the scavenging activity of orange juice. In fact, 50%

of the free radical scavenging activity of orange juice is because of ascorbic acid, as

quantified by the DPPH assay (Victor R. Preedy, 2013). Thus, the obtained results of

the scavenging potential of commercial orange juice (NFC) upon storage at different

temperatures and lengths of time can be interpreted as expounded in the following

paragraphs.

The determined DPPH inhibition (%) values in the exemplary commercial orange

juice (NFC) are a fraction of that of freshly squeezed orange juice due to progressive

loss of its antioxidant content as a result of intrinsic and external factors. Primarily,

fructose, found in orange juice, is capable of breaking down vitamin C: the higher the

fructose content, the greater the degradation of vitamin C. On the other hand, higher

levels of citric and malic acids attribute stability onto vitamin C (Townsend, 2006).

Furthermore, the pasteurization process, which the orange juice (NFC) undergoes,

results, to some extent, in the destruction of ascorbic acid content and reduction of

some carotenoids levels (in particular: antheraxanthin, β-cryptoxanthin and

violaxanthin) (Lee & Coates, 2003) and, consequently, a drop-down of its antioxidant

activity .

Interestingly, it has been found that in orange juice containers, ascorbic acid loss is

caused by oxidation of an overlaying residual air layer within the packaging in course

of processing (Nagy & Smoot, 1977). In the present research work, contributing to

further vitamin C loss was also the transfer of NFC orange juice from their original

packaging to sterile transparent plastic recipients, prior to storage at different

temperatures– resulting in exposure to light and oxygen.

According to Nagy and Scott, storage temperature and time affect the amount of

vitamin C content of orange juice. This proposition was verified in the present work

with relation to the scavenging activity of NFC orange juice: when this latter was stored

at relatively high temperatures (25, 30 and 37ºC), the percentage of DPPH scavenged

decreased greatly, unlike at lower temperatures (-20ºC and 4ºC). Furthermore, at the

obtained optimum temperature (-20ºC: in the freezer), the antioxidant activity of NFC

declined moderately as the storage time increased (from 7 to 14 days), followed by

NFC stored at 4ºC (in the fridge). More so, the NFC aliquots stored at temperatures ≥

38

25˚C, had an unpleasant odour upon 7 and 14 days’ storage: an indicator of spoilage

as a result of microbial growth (which thrives best between 20-37˚C).

Irrespective of the obtained outputs, the standard deviation determined of calculated

results are averagely considerable (refer to Tables 8 and 9 in “Appendices” section),

resulting in less precise and accurate DPPH inhibition values (%). This primarily

derives from poor pipetting skills (while introducing DPPH reagent and samples

solutions into test-tubes, resulting in variation of pipetted volumes).

Applying these experimental results unto real life, it suggests that proper storage of

commercial orange juice(NFC) is necessary in order to avoid the oxidation of vitamin

C and consequential loss of antioxidant activity. In simple terms, storing orange juice

either refrigerated or frozen, and out of light, ensures it remains rich in vitamin C and

other antioxidants present and lasts longer – because less oxygen circulates in a

closed and cold system. Thereby, much vitamin C and other antioxidants and their

related benefits are obtained when it is consumed.

Finally, better alternative ways of producing commercial orange juice (NFC), so to

retain its nutritional and organoleptic features, are still in course of study, such as: use

of high pressure and pulse electric fields applications, and membrane technologies

(Preedy, 2014).

39

CONCLUSION

Conclusively, the present research work revealed that freshly-squeezed orange juice

has a greater antioxidant activity than commercial orange juice (NFC). In practice, this

confirms that intake of fresh fruit, rich in antioxidants, is certainly more beneficial

health-wise than their corresponding commercial-deriving beverages. This is because

fruit-based beverages undergo progressive loss of their antioxidant activity in course

of processing, packaging and shelf life.

The other aim of this research was to monitor the effect of temperature and dilution

on the efficacy of antioxidants present in an exemplary commercial orange juice (NFC).

It was found that increasing temperatures and simultaneously increasing dilution of

antioxidant-containing source result in the decline of its antioxidant activity. Indirectly,

this signifies that the storage temperature determines the “activity” of antioxidant

content in fruit against free radicals.

Finally, for future repetition of the employed assay in this research study, some

important modifications can be performed to optimize and to validate the DPPH assay

and, thereby, enhancing the quality of results, even as followed:

1. Initial and ongoing quantification/monitoring of ascorbic acid content: vitamin C

is a good indicator of the antioxidant status of orange and its derivatives.

2. Automatic pipetting technology can be used to minimize human-derived errors.

3. The assay can be performed with the solvent ethanol, rather than methanol (as

used in the current research work), because of discovered positive effects on

the determination of free radical scavenging activity (Marinova & Batchvarov,

2011).

4. Antioxidant activity as a function of reaction time studies can result helpful to

establish the optimum reaction time between DPPH reagent and sample of

interest.

5. The use of equal ratio of DPPH reagent to sample may most possibly confers

accuracy to results.

6. The reaction mixture (DPPH reagent + sample) could be agitated at low speed,

during reaction time (in the dark), to favour even distribution of DPPH reagent

into the sample – to ensure the redox reaction between both constituents goes

into completion, prior to spectrophotometric reading.

40

APPENDICES

Additional Figures, Tables and Illustrations of Performed DPPH Assay

Figure 27 Progressive decolouring of DPPH at increasing concentrations of ascorbic acid

standards (the range of 0 to 1000 µM, from left to right) in triplicate: this corresponds to an

increasing order of antioxidant activity of ascorbic acid towards DPPH reagent (60 µM).

Figure 28 The scavenging activity seen by the decolouring of DPPH in set-up dilutions of

commercial orange juice NFC (from L-R: undiluted, I:2 and 1:5) stored at different

temperatures, from left to right: at -20˚C (in the freezer), 4˚C(in the fridge), 25˚C (at room

temperature), 30˚C and 37˚C.

41

Table 6 Ascorbic acid standard curve data for determining the equivalent scavenging

capacity/activity of prepared samples (FSOJ and NFC) towards DPPH radical.

Ascorbic Acid

Concentration (µM)

Absorbance

1 at 515 nm

Absorbance 2 at

515 nm

Absorbance

3 at 515 nm

Average

Absorbance

Standard

Deviation

0 0.713 0.734 0.687 0.711 0.019

250 0.599 0.596 0.599 0.598 0.001

400 0.532 0.526 0.528 0.529 0.002

500 0.473 0.471 0.477 0.474 0.002

600 0.429 0.438 0.429 0.432 0.004

700 0.321 0.314 0.332 0.322 0.007

800 0.332 0.319 0.313 0.321 0.008

900 0.287 0.3 0.206 0.264 0.042

1000 0.262 0.172 0.165 0.200 0.044

Table 7 Corresponding antioxidant activity of ascorbic acid standards. The IC50 of the

range of prepared ascorbic acid standards equals to 762.20 µM.

Ascorbic Acid Concentration (µM) DPPH Inhibition Activity (%)

0 -7.1

250 9.9

400 20.4

500 28.7

600 34.9

700 51.5

800 51.6

900 60.2

1000 69.9

42

Effect of Storage Time on scavenging capacity of Selected Irish Commercial

Orange Juice (NFC)

DPPH Inhibition (%) = (Acontrol – Asample)/Acontrol × 100

Whereby, Acontrol = absorbance of DPPH working solution (60 µM) and Asample =

Absorbance of 100 µL sample + 3900 µL DPPH reagent.

Table 8 DPPH Inhibition data of NFC stored at different temperatures for 7 days.

The value for each trial is the mean of three determinations.

UNDILUTED

NFC

Storage

Temperature

(˚C)

DPPH

Inhibition (%)

First Trial

DPPH

Inhibition (%)

Second Trial

DPPH Inhibition (%)

Third Trial

Average DPPH

Inhibition (%) Standard Deviation

-20 70.5 79.8 75.5 75.3 4.7

4 81.1 32.6 40.2 51.3 26.1

25 0.5 46.9 29.2 25.5 23.4

30 9.9 45.7 40.2 31.9 19.3

37 9.4 34.5 31.8 25.2 13.8

1:2 DILUTED

NFC

Storage

Temperature

(˚C)

DPPH

Inhibition (%)

First Trial

DPPH

Inhibition (%)

Second Trial

DPPH

Inhibition (%)

Third Trial

Average DPPH


-20 59.8 55.1 50.2 55.0 4.8

4 44.2 28.0 28.6 33.6 9.2

25 -13.7 20.7 20.6 9.2 19.8

30 -7.3 25.2 24.1 14.0 18.5

37 -3.2 23.6 23.4 14.6 15.4

1:5 DILUTED

NFC

Storage

Temperature

(˚C)

DPPH

Inhibition (%)

First Trial

DPPH

Inhibition (%)

Second Trial

DPPH

Inhibition (%)

Third Trial

Average DPPH


-20 22.7 27.3 31.7 27.2 4.5

4 11.0 26.7 22.5 20.0 8.1

25 -19.0 15.3 20.4 5.6 21.4

30 -17.4 17.4 19.6 6.5 20.8

37 -9.5 16.5 20.2 9.1 16.2

43

Table 9 DPPH inhibition data of NFC stored at different temperatures for 14 days.

The value of each trial is the mean of three determinations.

UNDILUTED NFC


DPPH Inhibition (%)

First Trial

DPPH Inhibition (%) Second Trial

DPPH Inhibition (%)

Third Trial

Average DPPH


-20 62.8 68.6 68.7 66.7 3.4

4 30.8 25.9 28.0 28.2 2.4

25 26.4 23.6 22.6 24.2 2.0

30 34.1 23.9 27.2 28.4 5.2

37 35.4 30.6 29.8 31.9 3.0

1:2 DILUTED NFC


DPPH Inhibition (%)

First Trial


DPPH Inhibition (%)

Third Trial

Average DPPH


-20 37.1 37.2 42.0 38.8 2.8

4 18.9 18.6 18.9 18.8 0.2

25 15.5 17.5 16.0 16.3 1.1

30 29.2 21.2 18.9 23.1 5.4

37 18.5 16.4 18.8 17.9 1.3

1:5 DILUTED NFC


DPPH Inhibition (%)

First Trial


DPPH Inhibition (%)

Third Trial

Average DPPH

Inhibition (%)

Standard Deviation

-20 20.6 21.5 25.8 22.7 2.8

4 11.3 13.3 14.4 13.0 1.6

25 13.6 12.3 14.2 13.4 0.9

30 14.3 15.8 14.8 15.0 0.7

37 13.0 13.8 14.7 13.8 0.8

44

RISK ASSESSMENT

Table 10 Completed risk assessment form of the present research work.

Hazard High Medium Low Current Controls

Measures

Options

for

Improved

Controls

2,2-Diphenyl-1-picrylhydrazyl

(DPPH)

May cause an allergic skin

reaction. May cause allergy or

asthma symptoms or breathing

difficulties if inhaled.

-

Wear suitable

protective clothing,

gloves and eye/face

protection.

Methanol

Toxic if swallowed, in contact

with skin or if inhaled. Causes

damages to organs. Highly

flammable.

-

Keep away from heat,

sparks, open flames,

hot surfaces. No

smoking. Wear

protective clothing, eye

protection and face

protection.

Butylated Hydroxytoluene

(BHT)

Harmful, if swallowed. Irritating

to eyes, respiratory system

and skin.

-

Wear protective

clothing, gloves and

eye/face protection.

Propyl gallate

Harmful if swallowed. May

cause an allergic skin reaction.

-

Wear protective gloves.

UV-VIS Spectrophotometer

Normal hazards associated

with electrical equipment.

-

Follow basic rules for

laboratory safety.

Glassware (volumetric flasks,

beakers, etc.)

Cuts from broken glass.

-

Ensure any glassware

used has no cracks or

chipped bits.

45

SAFETY DATA SHEETS

1,1-Diphenyl-2-Picrylhydrazyl (DPPH)

46

47

48

49

50

51

52

Propyl Gallate

53

54

55

56

57

58

59

Butylated Hydroxytoluene

60

61

62

63

64

65

66

67

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