Annals of Agricultural Science (2014) 59(2), 213220

H O S T E D BY
Faculty of Agriculture, Ain Shams University

Annals of Agricultural Science

www.elsevier.com/locate/aoas

Oxidative stability of ghee as aected by natural

antioxidants extracted from food processing wastes
Gehan A. El-Shourbagy *, Kahled M. El-Zahar

Food Science Department, Faculty of Agriculture, Zagazig University, 44519 Zagazig, Egypt

Received 14 August 2014; revised 25 August 2014; accepted 1 September 2014

Available online 8 December 2014

KEYWORDS Abstract In this research, bioactive compounds found in peanut skins (PS), pomegranate peels
Ghee; (PP) and olive pomace (OP) were extracted using ethanol (80%), ethyl acetate and n-hexane. Eth-
Food processing wastes; anol extract showed slightly better antioxidant characteristics compared with ethyl acetate and hex-
Natural antioxidants; ane extracts. Extracts showed varying degrees of antioxidant potential in different test systems in a
Stability indices; dose-dependent manner, whereas, antioxidant capacity of extracts was found to be in parallel with
BHA their higher phenolic contents. Total phenolic compounds (as gallic acid equivalent) ranged between
0.89 and 16.6, 1.83 and 261, and 1.56 and 124 mg gallic acid/g extract for OP, PS and PP, respec-
tively. Ethanol extracts of different by-products were added to ghee at concentrations of 200, 400
and 600 ppm, respectively. BHA was also added to ghee at a concentration of 200 ppm for compar-
ison. All samples were incubated at 63 C for 21 days. Ethanol extracts of PS, OP and PP gave good
antioxidant activity during accelerated oxidative incubation of ghee. The results revealed that
ethanolic extracts under study, at a concentration of 200 ppm, could be used to retard fat
auto-oxidation.
2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams
University. Open access under CC BY-NC-ND license.

Introduction has been reassessed because of possible toxic or carcinogenic

Antioxidants (natural and synthetic) play a signicant role in
retarding lipid oxidation reactions in food products. The detri-
mental effects of excessive lipid oxidation such as formation of
off-avors and undesirable oxidized chemical compounds
(aldehydes, ketones and organic acids) are well known (Saad
et al., 2007). Synthetic antioxidants (e.g., TBHQ, BHA and
BHT) are widely used as food additives, but their application
* Corresponding author. Mobile: +20 01005469481.
E-mail address: gehan53@yahoo.com (G.A. El-Shourbagy).
Peer review under responsibility of Faculty of Agriculture, Ain-Shams
University.
http://dx.doi.org/10.1016/j.aoas.2014.11.008
0570-1783 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.
Open access under CC BY-NC-ND license.
components formed during their degradation (Jo et al., 2006;
Pitchaon et al., 2007). Consequently, the search for endoge-
nous protective ingredients in foods has been intensied
wherein their utilization requires only manipulation of food
formulations. A number of natural antioxidants have been
added during food processing and have elongated the shelf life
and oxidative stability of stored products (Chenn et al., 2008;
Ebrahimabadi et al., 2010; Jang et al., 2012; Xiaowei et al.,
2011).
A huge amount of plant biomass wastes are produced
yearly as by-products from the agro-food industries. These
wastes are attractive sources of natural antioxidants. The high
concentration of phenolic compounds present in peels, skins
214
and seeds supports the utilization of these residues as a source
of natural antioxidants. Phenolic compounds exhibit a wide
range of physiological properties such as anti-allergenic, anti-
atherogenic, anti-inammatory, anti-microbial, antioxidant,
anti-thermobiotic, cardio protective and vasodilatory effects
(Balasundran et al., 2006). Phenolics could be extracted by
water or solvents and the extraction conditions need to be opti-
mized with respect to solvent polarity and physical conditions
(Nepote et al., 2005). In addition, research has indicated that
natural phenolic compounds can be extracted from raw mate-
rials or waste products of food industry (Peschel et al., 2006).
Studies were conducted to investigate antioxidant proper-
ties of peanut, peanut kernels, peanut hulls and peanut-based
products (Yu et al., 2005; Wang et al., 2007). Peanut skins were
demonstrated to be rich in phenolics and other health promot-
ing compounds (Yu et al., 2005; Wang et al., 2007; Monagas
et al., 2009). The olive oil industry generates large quantities
of a deleterious by-product known as olive pomace. Olive
pomace has broad spectrum toxicity against some microorgan-
isms, plants, insects, animals and human cells (Obied et al.,
2007; Aldini et al., 2006). Nevertheless, olive pomace consid-
ered as a potential source for natural antioxidants
(Niaounakis and Halvadakis, 2004; Aldini et al., 2006). A wide
range of phenolic compounds have been identied in virgin oil
(Suarez et al., 2010) wherein only ca. 2% of the total phenols
found in olive fruits are transferred to the extracted olive oil.
The rest of phenolic compounds (98%) are retained in the olive
waste cake. Pomegranate has been used extensively in the folk
medicine of many cultures and its consumption has grown tre-
mendously especially in the last decades (Li et al., 2006; Cam
et al., 2009). The peels of some fruits have higher antioxidant
activity than pulps (Guo et al., 2003; Fuhrman et al., 2005).
Pomegranate is a good example for this type of fruits wherein
their peels constitute approximately 40% of the whole fruit
and are rich in ellagic acid derivatives (Cerda et al., 2003;
Seeram et al., 2005).
From an environmental and economical perspective, it is
very important that plant by-products produced by agro-food
industry be used in food industry. However, ghee, the most
famous traditional dairy product in Egypt and many countries
in Middle East, undergoes oxidative degradation during stor-
age, resulting in alteration of major quality parameters affect-
ing its suitability for consumption. Development of rancidity
reduces the shelf life of the product, which ultimately affects
consumer acceptability (Mehta, 2006; Mariod et al., 2010;
Pawar et al., 2012). Therefore, the objectives of this study
were; (1) to evaluate different extracts from peanut skins
(PS), pomegranate peels (PP) and olive pomace (OP) as a
source of natural antioxidants, (2) to characterize the compo-
sition and content of phenolics in different extracts and (3) to
evaluate the efciency of using agro food wastes ethanolic
extracts in improving the quality, overall acceptance and oxi-
dative stability of ghee during storage under thermal oxidative
conditions.
Materials and methods
Materials
Plant biomass wastes, as a by-product of food industries, com-
monly found in Egypt, were used in this investigation. Olive
G.A. El-Shourbagy, K.M. El-Zahar
(Olea europaea L.) pomace (OP) was obtained from Food
Technology Research Institute (Agricultural Research Center,
Giza, Egypt). Peanut (Arachis hypogaea L.) skins (PS) were
obtained from the 10th of Ramadan City. Pomegranate
(Punica granatum L.) fruits (PP), were obtained from local
market (Zagazig, Egypt), washed with distilled water and man-
ually peeled. Pomegranate peels (PP) were collected then rinsed
with distilled water and considered as a by-product. The start-
ing materials were dried in an air draft drying oven (40 C)
until the moisture content became 12% or less. By-products
were ground and sieved through 60 mesh sieve and nally
cooled or kept at 4 C until the extractions were carried out.
Butylated hydroxy anisole (BHA), 1,1-diphenyl-2-pic-
rylhydrazyl (DPPH), gallic acid and quercin were purchased
from Sigma (St. Louis, MO, USA). All other chemicals and
reagents were of the highest purity available.
Preparation of extracts
Dried materials were extracted with different solvents, namely
ethanol (80%), ethyl acetate and n-hexane at a ratio of 10:1 (v/
w, 10 mL solvent: 1 g raw material) in closed vessels by stirring
at room temperature (25 C) for 4 h followed by ltration
through Whatmann #1 lter paper. The residues were re-
extracted again under the same conditions. All vessels were
wrapped with aluminum foil to prevent light degradation dur-
ing extraction (Yu et al., 2005). N-hexane and ethyl acetate
extracts were evaporated in a rotary evaporator (Buchi-water
bath-B-480, Switzerland) below 40 C, while ethanol 80%
extracts were freeze-dried (Thermo Electron Corporation-
Heto Power Dry LL 300 Freeze Dryer, Czechoslovak). The
dried extracts after evaporation of solvents were weighed to
determine the yield and stored at 20 C until used.
Determination of total phenolic compounds (TPC)
The concentration of TPC in different extracts was measured
using UV spectrophotometer (Jenway-UVVIS Spectropho-
tometer), based on a colorimetric oxidation/reduction reac-
tion, as described by Skerget et al. (2005) using Folin
Ciocalteu reagent. Specically, 0.5 mL of diluted extract
(10 mg in 10 mL solvent) was mixed with 2.5 mL of FolinCio-
calteu reagent (diluted 10 times with distilled water) and 2 mL
of Na2CO3 (75 g/1 L). The sample was incubated for 5 min at
50 C then cooled. For a control sample, 0.5 mL of distilled
water was used. The absorbance was measured at 760 nm.
Total phenolic content expressed as gallic acid equivalent
(GAE) was calculated, and the results were expressed as a
mg GAE g1 extract.
Identication of phenolic acids using HPLC
Phenolic acids of the dried extracts were identied according to
the method described by Mattila et al. (2000). HPLC (Hewllet
Packard series 1050, USA) equipped with autosampling, injec-
tor, solvent degasser, UV detector set at 330 nm and quarter
HP pump (series 1050) was used. Column (C18 hypersil
BDS) with particle size 5 lm was used. The separation was car-
ried out with methanol and acetonitrile as a mobile phase at
ow rate of 1 mL/min. The column temperature was per-
formed at room temperature (25 C) throughout the
Oxidative stability of ghee as affected by natural antioxidants 215

experiment. Identication and quantication were carried out
based on calibrations of the standards prepared from phenolic
acids dissolved in a mobile phase. Retention time and peak
area were used for calculation of phenolic acid compounds
by the data analysis of Hewllet Packared Software.
Radical scavenging activity (RSA) of extracts
The electron donation ability of the obtained extracts was
measured by bleaching of the purple colored solution of DPPH
according to the method of Hanato et al. (1988). One hundred
lL of each extracts (10 mg extract/10 mL solvent) was added
to 3 mL of 0.1 mM DPPH dissolved in ethyl acetate, ethanol
and hexane according to the solvent used for extraction. After
incubation period of 30, 60 and 120 min at room temperature,
the absorbance was determined against a control at 517 nm
(Gulcin et al., 2004). Percentage of antioxidant activity of
DPPH was calculated as follows:
Antioxidant activity Inhibition%
Acontrol  Asample =Acontrol   100
where Acontrol is the absorbance of the control reaction and
Asample is the absorbance in the presence of extract. BHA
was used as a positive control. Samples were analyzed in
triplicate.
Stability of ghee enriched with food processing wastes ethanolic
extracts
oxidation reaction passed through deionized water, in which
conductivity values were detected. Heating block was held con-
stant at 130 C. A rate of air ow through liquid butter oil
(ghee) was 10 l/h. Prior to the testing, frozen samples of ghee
were thawed at 4050 C, and a 3 0.0020 g of ghee sample
was taken for the analysis according to AOCS (1997).
Statistical analysis
Statistical analysis for the obtained data was carried out using
SPSS version 20 computer program (Dominick and Derrick,
2001). All data were expressed by means and standard devia-
tions of three replicates and were compared using one-way
ANOVAs and least signicant difference (LSD) values with
different letters within the same column differ signicantly at
P < 0.010.05.
Results and discussion
Yield of different food processing waste extracts
The yield of antioxidant extracts with different solvents varied
from 0.1 to 42.5 g extract/100 g wastes (Fig. 1). PS, PP and OP
had the highest yield when extracted with ethanol 80% fol-
lowed by hexane and ethyl acetate, respectively. Variation in
the extraction yields of different extracts might be attributed
to differences in polarity of compounds found in plants such
differences have been reported by Prakasha et al. (2001).

Total phenolic compounds (TPC)

The butter, used for preparing ghee in the present study, was
made from pasteurized and un-ripened buffaloes cream. The
butter was converted into ghee by boiling according to the
method described by Fahmi (1961). Ghee samples were divided
into eleven portions and treated as follows:
Portion (1) was kept without additives and was considered
to be as a negative control (C).
Portion (2) was treated with 200 ppm BHA and was consid-
ered to be as positive control (C1).
TPC of different food processing waste extracts were deter-
mined on the basis that the FolinCiocalteu method measures
the reduction of the reagent by phenolic compounds via the
formation of a blue complex that can be measured at 760 nm
against GAE as a standard. The amount of TPC varied in
hexane extract ethylacetate extract ethanol 80% extract
50
Extraction yield (g/100g)

45
Portions (3, 4 and 5) were treated with 200, 400 and
40
600 ppm of PS ethanolic extract respectively, (T1, T2 35
and T3). 30
25
Portions (6, 7 and 8) were treated with 200, 400 and
20
600 ppm of PP ethanolic extract respectively, (T4, T5 15
and T6). 10
5
Portions (9, 10 and 11) were treated with 200, 400 and
0
600 ppm of OP ethanolic extract respectively, (T7, T8 Olive pomace Pomegranate peels Peanut skin
and T9).
Fig. 1 Yield of antioxidant extracts (g/100 g) for different food
All samples were incubated in an oven at 63 1 C to processing wastes.
accelerate the oxidation for 21 days. Samples were analyzed
every three days for peroxide value (PV), acid value (AV),
and 2-thiobarbituric acid (TBA) value. AV and PV were deter- Table 1 Total phenolic compounds (mg gallic acid/g extract)
mined according to AOAC (2005), while TBA was assessed in different extracts.
according to Fernandez-Lopez et al. (2005).
By-product Concentration (mg gallic acid/g extract)
Hexane Ethyl acetate Ethanol 80%
Oxidation stability test of ghee using Rancimat equipment
Olive pomace (OP) 16.63 0.89 12.23
Pomegranate peels (PP) 1.56 12.49 124.23
Determination of an oxidative stability of ghee by Rancimat Peanut skins (PS) 1.83 5.69 261.69
equipment (USA, model 617), was based on volatile acids from
216 G.A. El-Shourbagy, K.M. El-Zahar

Zero time after 30 min after 1 hour after 2 hours

Table 2 Concentration of identied phenolic compounds
120
(mg/g dry matter) in food processing waste extracts as (A)

Antioxidant activity %
100
determined by HPLC.
80
Peanut skin Olive pomace Pomegranate peels
60
Gallic 0.07 0.01 0.0
Pyrogallol 0.90 0.27 12.64 40
Chlorogenic 2.58 0.06 0.84 20
Protocatechuic 5.07 1.0 1.86
Catechol 1.86 0.0 0.0 0
Olive pomace Pomegranate Peanut skins BHA
Caeic 0.54 0.0 0.0 peels
Vanillic 0.19 0.04 0.34 Hexane extracts
Catachin 10.64 0.0 0.0
Caeine 0.69 0.04 0.21 120
(B)

Antioxidant activity %
Ellagic 1.40 0.46 5.90 100
P-Coumaric 0.0 0.0 0.05
80
Benzoic 0.0 0.32 0.0
Syringic 0.0 0.01 0.0 60

40

20

the different extracts, ranging from 0.89 to 261.69 mg GAE g1 0

Olive pomace Pomegranate Peanut skins BHA
extract (Table 1). In general, the results stated that ethanol peels
80% and ethyl acetate were better than hexane in extracting Ethylacetate extracts
phenolics from PP and PS owing to their higher polarity and
good solubility (Siddhuraju and Becker, 2003; Kequan and Antioxidant activity % 120
Liangli, 2004). On the other side, hexane extracted the highest 100 (C)
amount of phenolics from OP followed by ethanol 80% as 80
shown in Table 1.
60

Identied phenolic compounds of food processing wastes 40

20

Table 2 shows the percentage of identied phenolic com- 0

Olive pomace Pomegranate Peanut skins BHA
pounds in PS, PP and OP. There was a great variation among peels
the components identied in each waste by-product. Phenolic Ethanol 80% extracts
compounds identied in PS were pyrogallol, protocatechuic,
catachin and ellagic acid with amounts ranging from 0.07 to Fig. 2 Radical scavenging activity (RSA) of hexane (A), ethyl
10.64 mg/g. The main phenolics identied in OP were pyrogal- acetate (B) and ethanol 80% (C) waste extracts in DPPH radical
lol, gallic acid, vanillic acid, protocatechuic, ellagic acid with scavenging activity system compared with BHA.

Table 3 Effect of ethanol extracts at different concentration on the PV (meq.O2/kg fat) of ghee during storage.*
Treatments Storage period (days)
0 3 6 9 12 15 18 21
a a a a a a a
C 0.87 0.02 1.95 0.2 2.22 0.04 3.65 0.06 4.42 0.05 5.33 0.03 7.61 0.02 10.21a 0.02
C1 0.85a 0.01 0.93d 0.01 1.13de 0.03 1.26de 0.02 1.82e 0.03 2.43d 0.03 3.16de 0.01 5.32d 0.03
T1 0.85a 0.01 0.92d 0.03 1.11ef 0.02 1.24de 0.05 1.82e 0.06 2.43d 0.4 3.14de 0.04 5.32d 0.05
T2 0.85a 0.02 0.91d 0.02 1.08f 0.02 1.23de 0.03 1.80e 0.03 2.41d 0.04 3.15de 0.05 5.28d 0.04
T3 0.86a 0.02 0.91d 0.01 1.08f 0.02 1.22e 0.05 1.80e 0.03 2.40d 0.03 3.12e 0.06 5.27d 0.03
T4 0.85a 0.04 0.91d 0.02 1.12def 0.04 1.25de 0.05 1.80e 0.06 2.41d 0.05 3.15de 0.03 5.32d 0.04
T5 0.86a 0.02 0.92d 0.03 1.09ef 0.03 1.24de 00.1 1.81e 0.08 2.40d 0.03 3.15de 0.04 5.29d 0.02
T6 0.86a 0.02 0.92d 0.03 1.09ef 0.02 1.22e 0.03 1.79e 0.03 2.38d 0.02 3.14de 0.03 5.28d 0.02
T7 0.85a 0.03 1.22b 0.01 2.00b 0.03 3.03b 0.02 3.93b 0.04 4.42b 0.04 5.14b 0.02 8.17b 0.04
T8 0.86a 0.01 1.03c 0.02 1.37c 0.02 2.32c 0.03 2.41c 0.04 3.61c 0.03 4.81c 0.6 6.29c 0.02
T9 0.86a 0.03 0.93d 0.04 1.16d 0.02 1.30d 0.02 1.93d 0.05 2.43d 0.04 3.19d 0.03 5.33d 0.04
LSD 0.037 0.040 0.042 0.061 0.082 0.061 0.063 0.055
C: control without antioxidants.
C1: ghee treated with 200 ppm BHA (positive control).
T1, T2, T3: ghee treated with 200, 400 and 600 ppm PS extracts, respectively.
T4, T5, T6: ghee treated with 200, 400 and 600 ppm PP extracts, respectively.
T7, T8, T9: ghee treated with 200, 400 and 600 ppm OP extracts, respectively.
*
Means with different letters within the same column differ signicantly at P < 0.010.05.
Oxidative stability of ghee as affected by natural antioxidants 217

amount ranging from 0.01 to 1.0 mg/g. The major phenolic

compounds identied in PP were pyrogallol, gallic acid, cou-

0.101a 0.004
0.053a 0.004
0.058a 0.004
0.056a 0.003
0.052a 0.004
0.058a 0.003
0.056a 0.003
0.210a 0.007
0.085a 0.002
0.079a 0.002
0.061a 0.002
maric acid, caffeine with amount ranging from 0.05 to
12.64 mg/g. Balasundran et al. (2006) stated that the antioxi-
dant activity of phenolic compounds depends on the structure,

0.14159
in particular the number and positions of the hydroxyl groups
21

of the nature of substitution on the aromatic rings. Moure

et al. (2001) reported that the antioxidant compounds from
residual sources could be used for increasing the stability of
0.048cd 0.003
0.049cd 0.004
0.049cd 0.003

0.052cd 0.004
0.050cd 0.001
0.048d 0.001

0.048d 0.003
0.071b 0.004
0.068b 0.002
0.086a 0.002

0.053c 0.003
foods by preventing lipid peroxidation and also for protecting
oxidative damage in living systems by scavenging oxygen rad-

0.00496
icals. It is well known that total antioxidant activity of waste
extracts was lineearly proportional to the concentration of
18

total phenolics.
0.062ab 0.004

0.054ab 0.003
0.051ab 0.004

RSA against DPPH

0.041b 0.003
0.041b 0.003
0.042b 0.004
0.040b 0.004
0.043b 0.002
0.043b 0.003
0.040b 0.002

0.177a 0.002

The results of RSA of various extracts are represented in

0.11641

Fig. 2. The results clearly indicated that all extracts exhibited

Effect of ethanol extracts at different concentration on the TBA (mg malonaldehyde/kg fat) of ghee during storage.*

15

antioxidant activity. The extracts that contained high amount

of TPC (Table 1) showed high RSA. In general, ethanolic
extracts followed by hexane then ethyl acetate extracts showed
0.053ab 0.002

0.050ab 0.001
0.042ab 0.003
0.035b 0.002

0.035b 0.002
0.034b 0.004
0.038b 0.004
0.036b 0.002
0.033b 0.002

0.039b 0.002
0.135a 0.002

RSA as strong as that of BHA (Fig. 2AC). It has been proved

that the antioxidant activity of plant extracts is mainly ascrib-
0.08617

able to the concentration of phenolic compounds in the plant

(Heim et al., 2002). The extract RSA with different solvents
12

varied from 91.4% to 5.50% after 120 min incubation. The

highest RSA was observed with PP ethanol 80%, hexane and
0.030 cd 0.003
0.027cd 0..002
0.027cd 0.002
0.030cd 0.005
0.029cd 0.004

0.031cd 0.003

ethyl acetate extracts with respective values of 80%, 38%

0.026d 0.001

0.041b 0.003
0.040b 0.002
0.051a 0.006

0.032c 0.003

and 35%, respectively. Ethanol 80%, hexane and ethyl acetate

extracts of PS had values of 85%, 22% and 19%, respectively.
0.00554

In addition, hexane, ethanol 80% and ethyl acetate extracts of

Means with different letters within the same column differ signicantly at P < 0.010.05.

OP had values of 24%, 20% and 18%, respectively. The results

9

of the DPPH radical scavenging assay suggest that compo-

nents involving the extracts are capable of scavenging free rad-
0.015a 0.002
0.015a 0.002
0.015a 0.002
0.013a 0.003
0.013a 0.002
0.015a 0.002
0.015a 0.001
0.015a 0.001
0.015a 0.001
0.015a 0.001
0.015a 0.002

icals via electron- or hydrogen-donating mechanisms and thus

might be able to prevent the initiation of deleterious free rad-
T7, T8, T9: ghee treated with 200, 400 and 600 ppm OP extracts, respectively.
T4, T5, T6: ghee treated with 200, 400 and 600 ppm PP extracts, respectively.
T1, T2, T3: ghee treated with 200, 400 and 600 ppm PS extracts, respectively.
0.0028

ical mediated chain reactions in susceptible matrices. This fur-

ther shows the capability of the extracts to scavenge different
6

free radicals in different systems, indicating that they may be

useful therapeutic agents for treating radical-related patholog-
0.013a 0.001
0.041a 0.001
0.013a 0.001
0.013a 0.003
0.013a 0.001
0.014a 0.002
0.012a 0.001
0.011a 0.002
0.052a 0.006
0.013a 0.001
0.013a 0.001

ical damage. The effect of antioxidants on DPPH radical-

scavenging is thought to be due to their hydrogen-donating
ability, DPPH is a stable free radical and accepts an electron
0.0431

C1: ghee treated with 200 ppm BHA (positive control).

Storage period (days)
3
0.013a 0.001
0.014a 0.001
0.013a 0.001
0.014a 0.001
0.011a 0.001
0.012a 0.002
0.012a 0.002
0.011a 0.002
0.013a 0.002
0.013a 0.001
0.013a 0.001
0.02602
C: control without antioxidants.
0
Treatments
Table 4

LSD

Fig. 3 Rancimat of ghee treated with different food processing

C1
T1
T2
T3
T4
T5
T6
T7
T8
T9

*
C

waste extracts at 130 C.

218 G.A. El-Shourbagy, K.M. El-Zahar

or hydrogen radical to become a stable molecule (Gulcin et al.,

2004). Free radicals involved in the process of lipid

0.101a 0.004
0.053a 0.004
0.058a 0.004
0.056a 0.003
0.052a 0.004
0.058a 0.003
0.056a 0.003
0.210a 0.007
0.085a 0.002
0.079a 0.002
0.061a 0.002
peroxidation are considered to play a major role in numerous
chronic pathologies such as cancer and cardiovascular diseases
(Dorman et al., 2003).

0.14159
21
Oxidative stability of ghee enriched with food processing wastes
ethanolic extracts peroxide values

0.048cd 0.003
0.049cd 0.004
0.049cd 0.003

0.052cd 0.004
0.050cd 0.001
0.048d 0.001

0.048d 0.003
0.071b 0.004
0.068b 0.002
0.086a 0.002

0.053c 0.003
Data presented in Table 3 show that the PV of control ghee
samples increased during the accelerated incubation up to

0.00496
21 days. The others samples enriched with BHA and natural
antioxidants had the lower PV values than the control sample

18
during storage under accelerated incubation at 63 C for
21 days. The results obtained in this work reected the impact

0.062ab 0.004

0.054ab 0.003
0.051ab 0.004
0.041b 0.003
0.041b 0.003
0.042b 0.004
0.040b 0.004
0.043b 0.002
0.043b 0.003
0.040b 0.002

0.177a 0.002
of these extracts, as natural antioxidants, in retarding of ghee
oxidation. The order of efciency in inhibiting oxidation was

0.11641
in the order PS, PP and nally OP extract. These results are
in agreement with Puravankara et al. (2000) and Pankaj

15
et al. (2013) who concluded that addition of ethanolic extract
of T. arjuna bark at 7% by the weight was highly effective in

0.053ab 0.002

0.050ab 0.001
0.042ab 0.003
retarding the auto-oxidation of both cow and buffalo ghee

0.035b 0.002

0.035b 0.002
0.034b 0.004
0.038b 0.004
0.036b 0.002
0.033b 0.002

0.039b 0.002
0.135a 0.002
during storage and ethanolic extract of Arjuna has signicant
Effect of ethanolic extracts at different concentrations on AV (mg KOH/g fat) of ghee during storage.*
ability to enhance the antioxidant potential of ghee. The onset

0.08617
of rancidity in ghee is mainly due to the oxidation of unsatu-
rated glycerides leading to development of peroxides and/or
due to hydrolysis of glycerides resulting in increased levels of 12
free fatty acids (FFA).
0.027cd 0.002
0.030cd 0.005
0.029cd 0.004

0.030cd 0.003
0.027cd 0.002

0.031cd 0.003
0.026d 0.001

0.041b 0.003
0.040b 0.002
0.051a 0.006

0.032c 0.003
Thiobarbituric acid (TBA) value

0.00554
It is well known that TBA values are taken as an index to eval-

Means with different letters within the same column differ signicantly at P < 0.010.05.
9

uate the advance of oxidation changes occurred in oil and fats.

The addition of extracts as natural antioxidants to ghee
0.015a 0.002
0.015a 0.002
0.015a 0.002
0.013a 0.003
0.013a 0.002
0.015a 0.002
0.015a 0.001
0.015a 0.001
0.015a 0.001
0.015a 0.001
0.015a 0.002
retarded the oxidative changes during accelerated storage
(Table 4). This means that the formation of malonaldehyde,
which affects the formation of pink color intensity from the

T7, T8, T9: ghee treated with 200, 400 and 600 ppm OP extracts, respectively.
T4, T5, T6: ghee treated with 200, 400 and 600 ppm PP extracts, respectively.
T1, T2, T3: ghee treated with 200, 400 and 600 ppm PS extracts, respectively.
reaction of TBA material with malonaldehyde, took place at 0.0028
a relatively lower rate in treated ghee samples. However, the
6

control ghee samples showed higher TBA values throughout

0.013a 0.001
0.041a 0.001
0.013a 0.001
0.013a 0.003
0.013a 0.001
0.014a 0.002
0.012a 0.001
0.011a 0.002
0.052a 0.006
0.013a 0.001
0.013a 0.001

the accelerated incubation period. These structural require-

ments were supported by the powerful antioxidant activity of
the well known BHA. Phenolic compounds act as hydrogen
0.0431

or electron donors to the reaction mixture and therefore the

C1: ghee treated with 200 ppm BHA (positive control).
Storage period (days)

formation of hydro peroxides is decreased. The slow formation

3

of conjugated dienes and consequently the secondary products

0.013a 0.001
0.014a 0.001
0.013a 0.001
0.014a 0.001
0.011a 0.001
0.012a 0.002
0.012a 0.002
0.011a 0.002
0.013a 0.002
0.013a 0.001
0.013a 0.001

by extracts and their major compounds indicated that these

materials may be acted as hydrogen donors to proxy radicals.
Thus, retarding the autoxidation of linoleic acid by chain rad-
0.02602

ical termination has been reported by Farag et al. (1989),

Ozkanli and Kaya (2007) and Mohdali (2010).
C: control without antioxidants.
0

Acid value

Data in Table 5 show that the AV remained without noticeable

changes within the rst six days of storage at 63 C for all
treatments including the control. Slight increases in AV were
Treatments

observed until 15 days of storage period then considerable

Table 5

increase in AV was recorded up to the end of storage period

LSD

(21 days) for all samples including the control. Data presented
C1
T1
T2
T3
T4
T5
T6
T7
T8
T9

*
C

in Table 4 show clearly that the AV of stored ghee was

Oxidative stability of ghee as affected by natural antioxidants
noticeably affected by enrichment with by-products ethanolic
extracts. PS ethanol 80% extracts (200 ppm) showed the low-
est increase in AV compared with other extracts and BHA at
same ratio (200 ppm). In general it is well known that natural
antioxidants have many favorable effects on human health,
such as inhibiting low-density protein oxidation, decreasing
the heart disease risks, and possessing anti-carcinogenic prop-
erties (Lafka et al., 2007). The mechanism of antioxidant activ-
ity of these compounds consists in their capability of radical
scavenging, metal chelating, and synergism with other antiox-
idants (Yemis et al., 2008). These results are in agreement with
Siddhuraju and Becker (2003).
Rancimat
The results of induction period are illustrated in Fig. 3. Induc-
tion time was the highest (24.3 h) for ghee enriched with BHA,
followed by ghee enriched with PS (18.4 h) and it was the low-
est for control (15.3 h). As can be seen from Fig. 3, the loga-
rithm of TRanc shows linear dependence on exothermic
temperature and can be described by the following equation:
(Log TRanc = a  t + b), where: a and b are adjustable coef-
cients and T the temperature in C. The oil stability index
(OSI) of ghee samples varied from 14.85 0.17 to
24.80 0.34 h; the higher the temperature and longer period
of time, the higher values of OSI in hours (Fig. 3). Although
some papers reported that high temperature with prolong per-
iod of time affected high yield, color and oxidative stability
tended to changed continuously. Degradation of oil was nor-
mally induced by moisture, high temperature, crust formation
and various structural, textural and chemical changes in the
product, and degradation of frying medium (Paul et al., 1997).
The oxidative stability of oils can be ranked based on the
rule the longer the induction time the more stable is the
oil. The relative resistances of the oils on their thermal oxida-
tive degradation depend on temperature. Also, these results
agree with Kucera (2011) and Suwarat and Tungjaroenchai
(2013).
Conclusion
Food rich in bioactive compounds has become an important
approach for more consumers, to achieve their desires to
reduce the risk of a specic disease or a health problem and
to treat minor illnesses. These are also important for improved
utilization of food and agricultural products. Peanut skin,
pomegranate peels and olive pomace extracts were prepared
using different solvents, and the in vitro antioxidant activity
of each extract was investigated. In general, it was observed
that extracts with higher antioxidant capacity were in parallel
with their higher phenolic contents. It could be concluded that
the obtained extracts using higher-polarity solvents were more
effective radical scavengers than those obtained using lower-
polarity solvents. Ethanol 80% showed slightly better charac-
teristics than hexane and ethyl acetate as a solvent for phenolic
compound extraction. Thus, for use in the food industry, eth-
anol 80% would be a more appropriate solvent. Furthermore,
it is notable that PS extracts exhibited strong antioxidant
capacity in all assays used, followed by PP and OP extracts.
Overall, ethanol 80%, ethyl acetate and hexane extract showed
relatively comparable activity to BHA. Therefore, these
219
extracts could be used as preservative ingredients in the food
and/or pharmaceutical industries.
References
Aldini, G., Piccoli, A., Beretta, G., Morazzoni, P., Riva, A., Marinello,
C., Facino, R., 2006. Antioxidant activity of polyphenols from
solid olive residues of Cv. Coratina. Fitoerapia 77, 121128.
A.O.A.C., 2005. Ofcial Methods of Analysis, 20th ed. Association of
Ofcial Analytical Chemists Gaithersberg, MD, USA.
A.O.C.S., 1997. Ofcial Methods and Recommended Practices of
American Oil Chemists Society, fourth ed. AOCS, Champaign, IL.
Balasundran, N., Sundram, K., Samman, S., 2006. Phenolic
compounds in plants and agri-industrial by-products: antioxi-
dant activity, occurrence and potential uses. Food Chem. 99,
191203.
Cam, M., Hisil, Y., Durmaz, G., 2009. Classication of eight
pomegranate juices based on antioxidant capacity measured by
four methods. Food Chem. 112, 721726.
Cerda, B., Liorach, R., Ceron, J.J., Espin, J.C., Tomas-Barberan,
F.A., 2003. Evaluation of the bioavailability and metabolism in the
rat of punicalagin, an antioxidant polyphenol from pomegranate
juice. Eur. J. Nutr. 42, 1828.
Chenn, G., Xiong, Y.L., Wang, L., Gomez-Basauri, J., Nicastro, F.,
2008. Effect of preventox on the storage stability of raw and
precooked pork patties. J. Muscle Foods 19, 116.
Dominick, S., Derrick, R., 2001. Theory and problems of statistics and
enconometrics, second ed. New York, USA, pp. 202220.
Dorman, H.J.D., Kosar, M., Kahlos, K., Holm, Y., Hiltunen, R.,
2003. Antioxidant properties and composition of aqueous extracts
from Mentha species, hybrids, varieties, and cultivars. J. Agric.
Food Chem. 51, 45634569.
Ebrahimabadi, A.H., Diafari-Bidogoli, Z., Mazoochi, A., Kashi, F.J.,
Batooli, H., 2010. Essential oil composition, antioxidant and
antimicrobial activity of the leaves and owers of Chaerophyllum
macropodum Boiss. Food Control 21, 11731178.
Fahmi, A.H., 1961. Samnah, Book in Arabic Language Published by
commercial modern publisher. Cairo University, Egypt.
Farag, R.S., Badel, A.Z.M., El-Baraty, G.S.A., 1989. Inuence of
thyme and clove essential oils on cotton seed oil oxidation. J. Am.
Oil Chem. Soc. 6, 800804.
Fernandez-Lopez, J., Zhi, N., Aleson-Carbonell, L., Perez-Alvarez,
J.A., Kuri, V., 2005. Antioxidant and antibacterial activities of
natural extracts: application in beef meat balls. Meat Sci. 69, 371
380.
Fuhrman, B., Volkova, N., Aviram, M., 2005. Pomegranate juice
inhibits oxidized LDL uptake and cholesterol biosynthesis in
macrophages. J. Nutr. Biochem. 16, 570576.
Gulcin, I., Kufrevioglu, O.I., Oktay, M., Buyukokuroglu, M.E., 2004.
Antioxidant, antimicrobial, antiulcer and analgesic activities of
nettle (Urtica dioica L.). J. Ethnopharmacol. 90, 205215.
Guo, C., Yang, J., Wei, J., Li, Y., Xu, J., Yiang, J., 2003. Antioxidant
activities of peel, pulp, and seed fractions of common fruits as
determined by FRAP assay. Nutr. Res. 23, 17191726.
Hanato, T., Kagawa, H., Yasuhara, T., Okuda, T., 1988. Two new
avonoids and other constituents in licorice root: their relative
astringency and radical scavenging effects. Chem. Pharm. Bull. 36,
20902097.
Heim, K.E., Taigliaferro, A.R., Bobilya, D.J., 2002. Flavonoid
antioxidants: chemistry, metabolism and structureactivity rela-
tionships. J. Nutr. Biochem. 13, 572584.
Jang, H.A., Kim, Y.P., Kim, H.S., 2012. Effect of natural antioxidants
on the oxidation of microencapsulated seed oil. Food Control 23,
528534.
Jo, S.C., Nam, K.C., Min, B.R., Ahn, D.U., Cho, S.H., Park, W.P.,
Lee, S.C., 2006. Antioxidant activity of Prunus mume extract in
cooked chicken breast meat. Int. J. Food Sci. Technol. 41, 1519.
220
Kequan, Z., Liangli, Y., 2004. Effects of extraction solvent on wheat
bran antioxidant activity estimation. LWT Food Sci. Technol. 37,
717721.
Kucera, M.R.R., 2011. Determination of the oxidative stability of
vegetable oil based lubricatis Ph.D. Thesis. Acta Facultatis
Technicae Zavlen Slovakia, pp. 111132.
Lafka, T.I., Sinanoglou, V., Lozos, E.S., 2007. On the extraction and
antioxidant activity of phenolic compounds from winery wastes.
Food Chem. 104, 12061214.
Li, Y., Guo, C., Yang, J., Wei, J., Xu, J., Cheng, S., 2006. Evaluation
of antioxidant properties of pomegranate peel extract in compar-
ison with pomegranate pulp extract. Food Chem. 96, 254260.
Mariod, A.A., Ali, R.T., Ahmed, Y.A., Abdel-Wahab, S.I., Abdul,
A.B., 2010. Effect of the method of processing on quality and
oxidative stability of anhydrous butter fat (samn). Afr. J. Biotech-
nol. 9, 10461051.
Mattila, P., Astola, J., Kumpulanien, J., 2000. Determination of
avonoids in plant material by HPLC with diode-array and electro-
array detections. J. Agric. Food Chem. 48, 58345841.
Mehta, B., 2006. Ragi (Coracana eleusine L.) a natural antioxidant
for ghee (butter oil). Int. J. Food Sci. Technol. 41, 8689.
Mohdali, A.A., 2010. Evaluation of some food processing by-products
as sources for natural antioxidants. Ph.D. Thesis. Faculty III
Process Science, Institute of Food Technology and Food Chem-
istry, Technical University of Berlin, Germany, pp. 6977.
Monagas, M., Garrido, I., Lebron-Aguilar, R., Gomez-Cordoves,
M.C., Rybarczyk, A., Amarowicz, R., Bartolome, B., 2009.
Comparative avan-3-ol prole and antioxidant capacity of roasted
peanut, hazelnut, and almond skins. J. Agric. Food Chem. 57,
1059010599.
Moure, A., Cruz, J.M., Francob, D., Dominguez, J.M., Sineirob, J.,
Dominguez, H., Nuinez, M.J., Parajo, J.C., 2001. Natural antiox-
idants from residual sources review. Food Chem. 72, 145171.
Nepote, V., Grosso, N., Guzman, C., 2005. Optimization of extraction
of phenolic antioxidants from peanut skins. J. Food Sci. Agric. 85,
3338.
Niaounakis, M., Halvadakis, C.P., 2004. Olive-Mill Waste Manage-
ment, rst ed. Tropthito-George Dardanos, Athens.
Obied, H.K., Bedgood, D.R., Prenzler, P.D., Robards, K., 2007.
Bioscreening of Australian olive mill waste extracts: biophenol
content, antioxidant, antimicrobial and molluscicidal activities.
Food Chem. Toxicol. 45, 12381248.
Ozkanli, O., Kaya, A., 2007. Storage stability of butter oils produced
from sheeps non-pasteurized and pasteurized milk. Food Chem.
100, 10261031.
Pankaj, P., Khamrui, K., Devaraja, H.C., Singh, R.R.B., 2013. The
effects of alcoholic extract of Arjuna (Terminalia arjuna Wight &
Arn.) bark on stability of claried butterfat. J. Med. Plants Res. 7
(35), 25452550.
Paul, S., Mittal, G.S., Chinnan, M.S., 1997. Regulating the use of
degraded oil/fat in deep-fat/oil food frying. Crit. Rev. Food Sci.
Nutr. 37, 635662.
Pawar, N., Arora, S., Bijoy, R., Wadhwa, B., 2012. The effects of
Asparagus racemosus (shatavari) extract on oxidative stability of
G.A. El-Shourbagy, K.M. El-Zahar
ghee, in relation to added natural and synthetic antioxidants. Int. J.
Dairy Technol. 65, 293299.
Peschel, W., Sanchez-Rabaneda, F., Diekmann, W., Plescher, A.,
Gartzia, I., Dimenez, J., Lamuela-Raventos, R., Buxaderas, S.,
Codina, G., 2006. An industrial approach in the search of natural
antioxidants from vegetable and fruit wastes. Food Chem. 97, 137
150.
Pitchaon, M., Maitree, S., Rungnaphar, P., 2007. Assessment of
phenolic content and free radical-scavenging capacity of some Thai
indigenous plants. Food Chem. 100, 14091418.
Prakasha, J.G.K., Singh, R.P., Sakariah, K.K., 2001. Antioxidant
activity of grape seed (Vitis vinifera L.) extracts on peroxidation
models in vitro. Food Chem. 73, 285290.
Puravankara, D., Boghra, V., Sharma, R.S., 2000. Effect of antiox-
idant principles isolated from mango (Mangifera indica L.) seed
kernels on oxidative stability of buffalo ghee (butter-fat). J. Food
Sci. Agric. 80, 522526.
Saad, B., Yong, Y.S., Mohd, A.N., Noorhasani, H., Abdussalam,
S.M.A., Muhammad, I.S., Saida, F.S., Khariuddin, M.T., Kama-
rudzaman, A., 2007. Determination of synthetic phenolic antiox-
idants in food items using reversed-phase HPLC. Food Chem. 105,
389394.
Seeram, N., Lee, R., Hardy, M., Heber, D., 2005. Rapid large scale
purication of ellagitannins from pomegranate husk, a by-product
of the commercial juice industry. Sep. Purif. Technol. 41, 4955.
Siddhuraju, P., Becker, K., 2003. Antioxidant properties of various
solvent extracts of total phenolic constituents from three different
agro-climatic origins of drumstick tree (Moringaoleifera Lam.)
leaves. J. Agric. Food Chem. 51, 21442155.
Skerget, M., Kotnik, P., Hadolin, M., Rizner-Hras, A., Simonic, M.,
Knez, Z., 2005. Phenols, proanthocyanidins, avones and avonols
in some plant materials and their antioxidant activities. Food
Chem. 89, 191198.
Suarez, M., Romero, M., Motilva, M., 2010. Development of phenol
enriched olive with phenolic compounds from olive cake. J. Agric.
Food Chem. 58, 10361043.
Suwarat, N., Tungjaroenchai, W., 2013. Characteristic of ghee
obtained from different post-clarication temperatures. Int. J.
Biosci. Biochem. Bioinform. 3 (4), 332334.
Wang, J., Xiaoping, Y., Zhengyu, J., Yuan, T., Huanlu, S., 2007. Free
radical and reactive oxygen species scavenging activities of peanut
skins extract. Food Chem. 104, 242250.
Xiaowei, M., Hongxia, W., Liain, L., Quansheng, Y., Songbiao, W.,
Rulinzhan, S., Shanshan, X., Yigang, Z., 2011. Polyphenolic
compounds and antioxidant properties in mango fruits. Sci. Hort.
129, 102107.
Yemis, O., Bakkalbasi, E., Artik, N., 2008. Antioxidantive activities of
grape (Vitis vinifers) seed extracts obtained from different varieties
grown in Turkey. Int. J. Food Sci. Technol. 43, 154159.
Yu, J., Ahmedna, M., Goktepe, I., 2005. Effects of processing methods
and extraction solvents on concentration and antioxidant activity
of peanut skin phenolics. Food Chem. 90, 199206.