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Effect of Ascorbic Acid on the Oxidative Stability of Water-In-Oil Emulsion in

the Presence of Lipophilic Antioxidants

Article  in  International Journal of Food Properties · February 2015

DOI: 10.1080/10942912.2012.675609

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 International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: http://www.tandfonline.com/loi/ljfp20

Effect of Ascorbic Acid on the Oxidative Stability of

Water-In-Oil Emulsion in the Presence of Lipophilic
Antioxidants

Sameera Rege, Shamim Momin & Dipti Bhowmick

To cite this article: Sameera Rege, Shamim Momin & Dipti Bhowmick (2015) Effect of Ascorbic
Acid on the Oxidative Stability of Water-In-Oil Emulsion in the Presence of Lipophilic Antioxidants,
International Journal of Food Properties, 18:2, 259-265, DOI: 10.1080/10942912.2012.675609

To link to this article: https://doi.org/10.1080/10942912.2012.675609

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International Journal of Food Properties, 18:259–265, 2015
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print/1532-2386 online
DOI: 10.1080/10942912.2012.675609

Effect of Ascorbic Acid on the Oxidative Stability of

Water-In-Oil Emulsion in the Presence of Lipophilic
Antioxidants

Sameera Rege, Shamim Momin, and Dipti Bhowmick

Department of Oils, Oleochemicals and Surfactants Technology, Institute of Chemical Technology,
Nathalal Parekh Marg, Matunga, Mumbai, India

Emulsions undergo an oxidation process because of the presence of water. The undesired phenomenon
of autoxidation needs to be prevented as the lipidic fraction in the emulsion undergoes degradation. The
surface active compounds so formed in the process disturb the stability of the emulsion. Accordingly,
the characteristics of the emulsions are altered. This autoxidation process can be prevented by means of
antioxidant. In the present study, a well known hydrophilic antioxidant, ascorbic acid, was examined at
various concentrations to stabilize water-in-oil emulsion. The outcome showed that the concentration of
a compound played an important role in determining its antioxidant or pro-oxidant effect.

Keywords: Emulsion, Antioxidant, Autoxidation, Sunflower oil, Ascorbic acid.

INTRODUCTION

Emulsion-based delivery systems have been widely used in the food industry to protect active ingre-
dients against extreme conditions, to enhance their stability, to maintain their effectiveness, and to
mask bad odors and bitter tastes.[1] A good emulsion formulation should retain its sensorial, chemi-
cal, and physical characteristics over time.[2] The lipid oxidation in water-in-oil emulsion takes place
in the continuous phase, i.e., oil. During storage, the setting in of chemical reactions that occur in
the droplet interior, interfacial region, or continuous phase may change the properties of the system.
Newly formed surface-active molecules in the continuous phase may exchange for those adsorbed
to the droplet surface, thus changing the composition and properties of the interfacial layer. In this
context, the oxidation of the lipidic fraction of an emulsion system is one of the causes of emulsion
quality deterioration. Some lipid oxidation products are fairly surface active and, therefore, they can
modify the composition of the interface.[3]
Though water exerts a strong influence on the rate of chemical deterioration of foods, espe-
cially on lipid peroxidation, it has been suggested that lipid oxidation in water-in-oil (w/o) will
occur at a rate similar to that of oils because the surface of the lipid phase is exposed directly to

Received 4 December 2011; accepted 11 February 2012.

Address correspondence to Shamim Momin, Department of Oils, Oleochemicals and Surfactants Technology, Institute of
Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai 400 019, India. E-mail: samomin@rediffmail.com

259
260 REGE, MOMIN, AND BHOWMICK

air.[4] Saturated lipids are considerably more stable to lipid oxidation than unsaturated lipids, i.e.,
essential fatty acids (EFAs).[5] Hence, the rate of oxidation of w/o emulsion formulated using oil
containing EFAs is more than that prepared using oil containing saturated fatty acids. Consequently,
a suitable antioxidant needs to be added to protect such w/o emulsions from oxidative deteriora-
tion. Some antioxidants are effective at low concentration and behaved as a pro-oxidant at a high
concentration.[6]
Though antioxidants are useful to prevent lipid oxidation, their activity may vary widely depend-
ing upon the composition of the system. Partitioning of antioxidants, hydrogen bonding, interphase
transport, surface accessibility, and interaction of emulsifier with antioxidants are considered to
be important parameters that determine antioxidant activity in lipid-containing systems.[7] Thus,
potent antioxidants can undergo oxidation forming reactive substances and subsequently act as pro-
oxidants depending on the systems.[8] Moreover, the type of emulsifier influences the distribution of
antioxidant in emulsion[9] and thereby decreases lipid oxidation in droplets.[10]
Among natural antioxidants, ascorbic acid is used without restriction in foods because of its
potent antioxidant activity and its nutritional value as vitamin C. Besides possessing antioxidant
potential, it has owed medicinal benefits. It is one of the important water soluble vitamins that is
easily absorbed but not stored in the body. Hence, ascorbic acid has to be regularly supplemented
through diet or tablets to maintain ascorbic acid pool in the body. A deficiency of ascorbic acid leads
to scurvy. Ascorbic acid plays an important role in the maintenance of collagen, which represents
about one-third of the total body protein. Ascorbic acid is known to enhance the availability and
absorption of iron from non-heme iron sources. Ascorbic acid supplementation is found to facilitate
the dietary absorption of iron.[11]
Similarly, well known natural antioxidants, such as kalonji seeds and curcuminoids, possess ther-
apeutic applications. The seeds of kalonji are known to have a number of therapeutic uses,[12] such
as anticarcinogenic, antibacterial, anti-inflammatory, analgesic, and antipyretic. Likewise, curcumin,
a major component of curcuminoids, has medicinal properties against various diseases, such as
anorexia, coughs, diabetes, hepatic disorders, rheumatism, and Alzheimer disease.[13]
The present work aims to study the effect of ascorbic acid on the oxidative stability of w/o
emulsion formulated using refined sunflower oil (RSFO) as a source of EFAs. Polyglycerol polyri-
cinoleate (PGPR) and tween 80 were used for the emulsification process. The optimum quantity
of ascorbic acid needed to inhibit the oxidative deterioration of the emulsion was evaluated. The
work is further extended to evaluate the effect of ascorbic acid in synergism with kalonji seeds
ethanol extract (KEE) and curcuminoids. The results showed that the antioxidant is effective only at
a particular proportion.

MATERIALS AND METHODS

Materials
RSFO was received as a gift sample from M/s Cargill India Pvt. Ltd. (New Delhi, India). The oil
contained 428 ppm residual tocopherol and did not contain any synthetic antioxidants. Polyglycerol
polyricinoleate (PGPR) was procured from Fine Organics (India). Tween 80 was procured from
Croda Chemicals India Pvt. Ltd. Curcuminoids powder was obtained from Kancor India Ltd.
(Angamaly South, India). Kalonji seeds were procured from a local vendor. All other chemical
reagents and solvents were obtained from S.D. Fine-Chem Limited (Mumbai, India).

Extraction of Kalonji Seeds

Kalonji seeds were crushed and extracted using ethanol in a Soxhlet extractor with a minimum
of 14 siphons. The crude KEE was obtained after removal of the solvent using a rotary vacuum
evaporator.
 WATER-IN-OIL EMULSION IN PRESENCE OF LIPOPHILIC ANTIOXIDANTS 261

Preparation of Emulsions
Water-in-oil emulsions were prepared by dropwise addition of a 15-ml water phase containing 1%
(w/v) Tween 80 to 85 g RSFO containing 18% (w/v) PGPR. Emulsification was done using a
homogenizer (Komal Scientific, India) at 3000 rpm for 30 min. The concentration of ascorbic acid
in the water phase was varied (5, 10, 25, 50, and 100 ppm) to find its effect on the oxidative stability.
Curcuminoids (50 ppm) and KEE (2% w/v) were added to the oil phase (RSFO) and emulsions
containing various combinations of hydrophilic and lipophilic antioxidants were prepared.

Study of Antioxidant Activity

The oxidative stability of emulsions was checked at 35◦ C for 10 days at regular intervals of 2 days by
determining peroxide value as per the AOCS Official Method.[14] This temperature was selected so
as to accelerate the oxidation without changing the structure of the emulsion. Also, induction period
(IP) at 110◦ C was measured using Rancimat (Rancimat 679, Metrohm Ltd., Switzerland) with air
flow at 20 L/h. The antioxidant activities of natural antioxidants were compared with control sample
and tert-Butylhydroquinone (TBHQ (200 ppm based fat content)) under the same conditions. The
relative antioxidant activities were compared using oxidative factor (OXF) for antioxidants based on
mean peroxide value of triplicate experiments using the following formula: [15]

(PVfinal − PVinitial ) antioxidant

OXF = .
(PVfinal − PVinitial ) control

Protection coefficient (Pc ) was calculated as the ratio of IP with antioxidant to control sample,[16]

(IP) antioxidant
PC = .
(IP) control

RESULTS AND DISCUSSION

Effect of Ascorbic Acid on Oxidative Stability of Emulsion

The oil oxidation, i.e., autoxidation is a radical chain process involving initiation (radical formation),
propagation (oxygen absorption to from peroxides), and termination (formation stable products)
reaction. The radical formation is catalyzed by metal ions. Certain antioxidants like TBHQ work by
deactivating these radicals, whereas certain antioxidants like lecithin work by chelating metal ions.
In either of the cases, there is an induction period in which antioxidants continue to inhibit radical
formation without much increase in peroxide formation. After the induction period, radicals continue
to absorb oxygen to form peroxides. The rate of peroxide formation mainly depends on oxygen
supply. Hence, higher induction period implies better radical inhibition power of an antioxidant.
The effect of various concentrations of ascorbic acid on the oxidative stability of the emulsions
is shown in Table 1. The data indicated that as the concentration increased, the rate of peroxide
formation was increased. But at the same time, induction period was also increased. By evaluating
the antioxidant activity of ascorbic acid for various concentrations, a 5 ppm concentration was found
to be better in terms of peroxide formation, and antioxidant activity was decreased with an increase
in concentration. In the case of emulsions, the presence of the aqueous phase generally decreases
the activity of antioxidants because hydrogen-bonded complexes are ineffective hydrogen donors
to lipid radicals.[17] Due to the hydroperoxides being more polar than the starting lipid, the peroxy
radicals tend to direct to the aqueous phase of the emulsion where they get scavenged by ascorbate
anion AscH−[18] according to the reaction,
262 REGE, MOMIN, AND BHOWMICK

TABLE 1
Effect of ascorbic acid on oxidative stability of emulsions at 35◦ C

OXF

Antioxidant used IP† (h) Pc Day 2 Day 4 Day 6 Day 8 Day 10

Control 2.40 ± 0.06 — — — — — —

TBHQ 3.79 ± 0.09 1.58 0.65 0.43 0.45 0.49 0.54
5 ppm ascorbic acid 2.07 ± 0.10 0.86 0.85 0.85 0.84 0.87 0.86
10 ppm ascorbic acid 2.38 ± 0.10 0.99 1.01 1.03 1.02 1.02 0.98
15 ppm ascorbic acid 2.41 ± 0.07 1.00 0.90 0.94 0.92 0.91 1.00
25 ppm ascorbic acid 2.45 ± 0.14 1.02 0.84 0.95 0.96 0.91 0.99
50 ppm ascorbic acid 2.84 ± 0.18 1.18 0.81 0.90 0.91 0.90 0.96
100 ppm ascorbic acid 3.40 ± 0.09 1.42 0.53 1.21 1.11 1.12 1.25
† The values given are means of three consecutive experiments ± standard deviations.

ROO• + AscH− → ROOH + Asc•−,

or their ability to reduce hydroperoxides to more stable compounds that do not continue the radical
chain reaction:[19,20]

Asc•− + ROO• → non radical combination products.

Two ascorbyl radical anions (Asc•– ) can react to form a dimer and further undergo a reversible
disproportionation reaction (Fig. 1) to form stable dehydroascorbic acid (DHAs) and ascorbate anion
(AscH− ).[21] This would regenerate ascorbic acid in its active form.[22,23] At a higher concentration
of ascorbic acid, an excess amount of ascorbate anion (AscH− ) was present to deactivate radi-
cals forming ascorbyl radical anion (Asc•– ). Thus, at higher concentrations, the induction period
was more as compared to lower concentrations. The ascorbyl radical anion was further degraded
reversibly to form dehydroascorbic acid (DHAs) and ascorbate anion (AscH− ) in acidic medium.
As the concentration of dehydroascorbic acid (DHAs) increased, the rate of degradation of ascorbyl
radical anion decreased resulting in higher concentration of ascorbyl radical anion, which led to
higher peroxide formation. At a higher concentration of ascorbic acid, more dehydroascorbic acid
(DHAs) was formed and more ascorbyl radical anions were present in the system. Hence, the rate
of peroxide formation was also more in this case (Fig. 2a). At a lower concentration, the amount of
ascorbic acid was insufficient to scavenge radicals resulting in a lower induction period. But at the
same time, the disproportionation reaction (Fig. 1) of ascorbyl radical anion is mainly in forward

FIGURE 1 Disproportionation reaction of ascorbyl radical anion.

 WATER-IN-OIL EMULSION IN PRESENCE OF LIPOPHILIC ANTIOXIDANTS 263

FIGURE 2 Effect of (a) ascorbic acid and (b) ascorbic acid with KEE and curcuminoids on oxidative stability
(peroxide formation) of emulsions at 35◦ C.

direction thereby facilitating the removal radicals for the system to form stable compounds. This
reduced the peroxide formation.

Effect of Ascorbic Acid in Synergism with Lipophilic Antioxidants

As discussed in a previous paper,[24] KEE and curcuminoids as such and in synergism with each
other showed antioxidant activity in w/o emulsions. Based on the OXF (Table 2), the emulsion con-
taining 50 ppm curcuminoids with 100 ppm ascorbic acid also showed marginal pro-oxidant activity,
264 REGE, MOMIN, AND BHOWMICK

TABLE 2
Effect of ascorbic acid on antioxidant activity of KEE and curcuminoids in emulsions at 35◦ C

OXF

Day Day Day Day Day

Antioxidant used IP† (h) Pc 2 4 6 8 10

Control 2.40 ± 0.06 — — — — — —

TBHQ 3.79 ± 0.09 1.58 0.65 0.43 0.45 0.49 0.54
50 ppm curcuminoids 3.64 ± 0.11 1.52 0.29 0.73 0.67 0.73 0.84
50 ppm curcuminoids + 5 ppm ascorbic acid 4.74 ± 0.18 1.98 0.73 0.72 0.79 0.84 0.83
50 ppm curcuminoids + 100 ppm ascorbic acid 4.10 ± 0.17 1.71 0.41 0.97 0.89 0.94 1.11
2% KEE 3.96 ± 0.17 1.65 0.65 0.67 0.59 0.60 0.64
2% KEE + 5 ppm ascorbic acid 4.74 ± 0.05 1.98 0.31 0.46 0.61 0.63 0.66
2% KEE + 50 ppm curcuminoids 4.07 ± 0.16 1.69 0.65 0.72 0.65 0.66 0.69
2% KEE + 50 ppm curcuminoids + 5 ppm ascorbic 4.88 ± 0.12 2.03 0.37 0.53 0.63 0.67 0.72
acid
2% KEE + 50 ppm curcuminoids + 100 ppm 5.26 ± 0.14 2.19 0.53 0.67 0.72 0.79 0.87
ascorbic acid
† The values given are means of three consecutive experiments ± standard deviations.

whereas the emulsion containing 2% KEE, 50 ppm curcuminoids, and 100 ppm ascorbic acid gave
a slight antioxidant effect that was lower than the emulsion containing TBHQ. The Pc indicated that
the stability of emulsion was increased by addition of 50 ppm curcuminoids to 100 ppm ascorbic
acid that was enhanced by further addition of 2% KEE. The results were consistent with that of
OXF. It is to be noted that even though OXF are lower for TBHQ, induction periods were higher
for emulsions containing 100 ppm ascorbic acid along with lipophilic antioxidants, i.e., KEE and
curcuminoids.
As is consistent with the previous discussion, a lower concentration (5 ppm) of ascorbic acid
showed better antioxidant activity than a higher concentration (100 ppm) even in the presence of
lipophilic antioxidants (Table 2, Fig. 2b). Also, the addition of 5 ppm ascorbic did not influence the
activity of lipophilic antioxidants as seen by OXF but there was a marginal increase in Pc with the
addition of 5 ppm ascorbic acid.

CONCLUSIONS

The antioxidant activity is concentration dependent. Ascorbic acid imparts antioxidant activity at a
much lower concentration (5 ppm) in w/o emulsion. The lipophilic antioxidants were more active
in w/o emulsions to increase oxidative stability of emulsions than lipophobic antioxidants. The
study has given an approach for the development functional food using essential fatty acids. The
optimisation of the concentration of the antioxidant is crucial in developing a functional food.

ACKNOWLEDGMENTS

This study was financially supported by Technical Education Quality Improvement Programme
(TEQIP), Government of India and World Bank.
 WATER-IN-OIL EMULSION IN PRESENCE OF LIPOPHILIC ANTIOXIDANTS 265

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