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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
Bachelor’s 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 -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.
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 -20°C for a week, against DPPH radical. .......................................................... 24
Figure 7 Effect of dilution on the scavenging activity of analyzed commercial orange juice
(NFC), stored at 4°C for a week. ..................................................................................................... 24
Figure 8 Effect of dilution on the scavenging activity of analyzed commercial orange juice
(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
Figure 10 Effect of dilution on the scavenging activity of analyzed commercial orange juice
(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
Figure 16 Effect of dilution on the scavenging activity of commercial orange juice (NFC)
stored at 4˚C for 14 days. ................................................................................................................. 29
7
Figure 17 Effect of dilution on the scavenging activity of commercial orange juice (NFC)
stored at 25˚C for 14 days. ............................................................................................................... 29
Figure 18 Effect of dilution on the scavenging activity of commercial orange juice (NFC)
stored at 30˚C for 14 days. ............................................................................................................... 30
Figure 19 Effect of dilution on the scavenging activity of commercial orange juice (NFC)
stored at 37˚C for 14 days. ............................................................................................................... 30
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
Figure 26 Effect of storage time on the scavenging capacity of 1:5 diluted commercial
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
8
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
9
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
10
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
12
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.
13
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.
14
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.
15
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.
18
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
1:10 1:5 1:2 Undiluted
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
1:10 1:5 1:2 Undiluted
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.
Figure 7 Effect of dilution on the scavenging activity of analyzed commercial orange
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
Figure 8 Effect of dilution on the scavenging activity of analyzed commercial orange
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
Effect of Dilution on Scavenging Activity of NFC Stored at 25˚C for 7 Days
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
Effect of Dilution on Scavenging Activity of NFC Stored at 30˚C for 7 Days
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 (
%)
Storage Temperature (˚C)
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 (
%)
Storage Temperature (˚C)
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 (
%)
Storage Temperature (˚C)
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
Figure 16 Effect of dilution on the scavenging activity of commercial orange juice
(NFC) stored at 4˚C for 14 days.
Figure 17 Effect of dilution on the scavenging activity of commercial orange juice
(NFC) stored at 25˚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
Effect of Dilution on Scavenging Activity of NFC Stored at 4˚C for 14 Days
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
Figure 18 Effect of dilution on the scavenging activity of commercial orange juice
(NFC) stored at 30˚C for 14 days.
Figure 19 Effect of dilution on the scavenging activity of commercial orange juice
(NFC) stored at 37˚C for 14 days.
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
Effect of Dilution on Scavenging Activity of NFC Stored at 30˚C for 14 Days
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
Effect of Dilution on Scavenging Activity of NFC Stored at 37˚C for 14 Days
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.
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.
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 (
%)
Storage Temperature (˚C)
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 (
%)
Storage Temperature (˚C)
Scavenging Activity of 1:2 Diluted NFC Stored for 14 Days at Varying Storage Temperatures
32
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.
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 (
%)
Storage Temperature (˚C)
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).
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).
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 (
%)
Storage Temperature (˚C)
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
Figure 26 Effect of storage time on the scavenging capacity of 1:5 diluted commercial
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
Inhibition (%) Standard Deviation
-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
Inhibition (%) Standard Deviation
-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
Storage Temperature (˚C)
DPPH Inhibition (%)
First Trial
DPPH Inhibition (%) Second Trial
DPPH Inhibition (%)
Third Trial
Average DPPH
Inhibition (%) Standard Deviation
-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
Storage Temperature (˚C)
DPPH Inhibition (%)
First Trial
DPPH Inhibition (%) Second Trial
DPPH Inhibition (%)
Third Trial
Average DPPH
Inhibition (%) Standard Deviation
-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
Storage Temperature (˚C)
DPPH Inhibition (%)
First Trial
DPPH Inhibition (%) Second 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.
67
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