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International Food Research Journal 24(4): 1636-1643 (August 2017)
Journal homepage: http://www.ifrj.upm.edu.my
1.2*Rohadi, 2Raharjo, S., 3Falah, I.I. and 2Santoso, U.
1Department of Processing Agricultural Product, Faculty of Agricultural Technology, Semarang
University, Jl. Arteri Soekarno-Hatta, Semarang 50196, Indonesia
2Department of Food and Agricultural Product Technology, Faculty of Agricultural Technology,
Gadjah Mada University, Jl. Flora No. 1, Bulaksumur , Yogyakarta 55281, Indonesia
3Department of Chemistry, Faculty of Mathematics and Natural Science, Gadjah Mada University,
Jl. Sekip Utara, Yogyakarta 55281 Indonesia
Methanolic extract of Java Plum (Syzygium cumini Linn.) seeds as a natural
antioxidant on lipid oxidation of oil-in-water emulsions
Abstract
Fish oil is considered as a great nutritional importance due to a high content of essential n-3
polyunsaturated fatty acids (PUFAs) such as linolenic acid, docosahexaenoic acid (DHA),
and eicosapentaenoic acid (EPA). However, PUFAs are susceptible to oxidative deterioration.
Methanolic extract of Java plum seed was a source of strong antioxidant polyphenols against
DPPH as well as strong Ferric (Fe3+) reducing agent. The aim of the research was to evaluate
the effect of methanolic extract of Java Plum seed (MEJS) on lipid oxidation of sh oil from
striped catsh (Pangasius hypothalamus) in oil-in-water emulsions as a food model at 35±2ºC
during 144 h of storage. The results showed that MEJS has stronger inhibition activity than
Grape Seed Extract (GSE) but lower than BHA (p<0.05) according to conjugated dienes
(CD), peroxide values (PoV), and thiobarbituric acid reactive substances (TBARS). Inhibition
activity of MEJS on lipid oxidation ranged between 28% and 38% at concentrations of 50-800
mg/L during 144 h of storage.
Introduction
Fish oil is considered as a great nutritional
importance. This is due to its naturally high content
of essential n-3 polyunsaturated fatty acids (PUFAs),
such as α-linolenic acid (C18:3), eicosapentaenoic
acid (C20:5) and docosahexaenoic acid (C22:6).
These fatty acids have benecial effects for heart’s
health (Simopoulos, 2008). However, PUFAs are
susceptible to oxidation which leads to the production
of short-chain chemical compounds associated with
decreasing food quality attributes such as rancidity as
well as textural and nutritional modication (Hsieh
and Kinsella, 1989; McClements and Decker, 2000;
Maqsood, 2010). The rate oxidation of PUFAs in
bulk oil increases equal to their unsaturated degree.
A high-PUFAs food product has more susceptible
to oxidative deterioation (McClements and Decker,
2000) such as sh oil from farmed freshwater Tra
catsh (Pangasius hypothalamus) (Ho and Paul,
2009).
Previous research showed that methanolic extract
of Java Plum (Syzygium cumini Linn.) seed (MEJS)
has a strong antioxidant activity according to RSA-
DPPH (radical scavenging activity 2,2’diphenyl-1-
picrilhydrazil) and FRAP (ferric reducing antioxidant
power) (Rohadi et al., 2016). In addition that a polar
hydrophilic antioxidant is less effective in an oil-in-
water emulsion compared to a non-polar lipophilic
and vice versa. The polar antioxidants such as trolox,
ascorbic acid, rosmarinic acid, and carnosic acid are
more effective in bulk oil due to its accumulation
in the air-oil interphase area while in an emulsion
system, the non-polar ones such as α-tocophorol,
ascorbyl palmitate, and carnosol are more effective
(McClement and Decker, 2000).
Synthetic antioxidants such as BHA (Butylated
Hydroxy Anisole), BHT (Butylated Hydroxy
Toluene) and TBHQ (tert-butylhydroquinone) were
more effective to inhibit food deterioration due to
lipid oxidation during processing and storage (Frankel
1998; Maqsood, 2010). However, application of
those antioxidants raised doubt in terms of toxicity-
related problems of synthetic antioxidants (Madavi
and Salunke, 1995; Buxiang and Fukuhara, 1997;
Baydar et al., 2007; Vayupharp and Laksanalamal,
Keywords
Java Plum seed
Natural antioxidant
Lipid oxidation
Oil-in-water emulsions
Article history
Received: 27 April 2016
Received in revised form:
15 July 2016
Accepted: 24 July 2016
1637 Rohadi et al./IFRJ 24(4): 1636-1643
2012). In the other hand, consumer’s demand for
environmentally friendly natural antioxidants has
greatly increased in recent years (Perumalla et al.,
2011). The last decades researches have been focused
on the effectiveness of natural antioxidants as a chain-
breaking agent in lipid oxidation, such as tocopherol,
plant extracts (rosemary, blackberries, blueberries,
cherries, grapes, raspberries, strawberries, tea), and
kiam wood extract (Kanner et al., 1994; Frankel,
1996; Maqsood, 2010; Perumalla et al., 2011).
Zhang and Lin (2009) reported that Java plum
(Syzygium cumini Linn.) extract has high tannin
content and antioxidant activity toward DPPH and
FRAP test, therefore, it has a high potential as a
natural antioxidant. Among part of the plant, the seed
has the highest polyphenols content (Vasi and Austin,
2009; Rydlewsky, 2013; Saha et al., 2013). Rohadi
et al. (2016) reported that yield of Java Plum seed
extract using 50% methanol (v/v) was 16.29% (db)
and contain 45.99±0.25% total phenolic compound
expressed as g gallic acid equivalents (GAE/100 g
dry extract), 2.28±0.07% total avonoid expressed
as g quercetin equivalents (QE/100 g dry extract),
and 26.9±0.07% total tannin as expressed as g tannic
acid equivalents (TAE/100 g dry extract). This result
showed that extract of Java plum seed has a high
potential as a natural antioxidant. Several studies
have reported the natural polyphenol of plant extracts
were more effective to inhibit lipid oxidation in an
oil-in-water emulsion (Roedig-Penman and Gordon,
1997; Maqsood and Benjakul, 2010; Skowyra
et al., 2014; Azman et al., 2016). However, the
scientic information of Java plum seed as a natural
antioxidant on lipid oxidation of sh oil in an oil-
in-water emulsion is still limited. Therefore, the
aim of this research was to evaluate the effect of
methanolic extract of Java Plum seed (MEJS) on lipid
oxidation of sh oil from striped catsh (Pangasius
hypothalamus) in an oil-in-water emulsion as a food
model during storage.
Materials and Methods
Materials
Live striped catsh (Pangasius hypothalamus)
were obtained from “Mina Jaya” farmer group in
Margo Mulyo Village, Seyegan, Sleman, Yogyakarta.
Java plum (Syzygium cumini Linn. var. Genthong) were
collected from Semarang, Central Java, Indonesia
during December 2014. Grape seed extract (GSE)
contained 95% oligoprotoanthocianidin (OPC) (Bulk
powder, UK), 2,2’-diphenyl-1-picrilhydrazil (DPPH)
and 1,1,3,3-tetra methoxypropane (TMP) (Aldrich
Chemical Co.), and butylatedhydroxyanisole (BHA)
(Sigma Chemical Co.) were an analytical grade.
Preparation of methanolic extract of Java plum seed
The seed was separated from the fruit pulp and
then was sliced with stainless steel knife, dried using
cabinet dryer at 47±3º C for 24 h. The dried seed
was ground into powder using a grinder then sieved
through 80 mesh. Twenty ve grams of Java Plum
seed powder were exhaustively three times extracted
with aqueous-methanol 50% (v/v) at a ratio of 1:10
(w/v) using the maceration method for 6 h at ambient
temperature according to (Vasi and Austin, 2009)
with slight modication. The extracts were collected
and concentrated using a rotary vacuum evaporator
(IKA-RV 10 Basic Germany) then the solid crude
extract was freeze-dried for solvent removal and kept
in the refrigerator for further analysis. This extraction
procedure was repeated three times.
Preparation of striped catsh llet and its lipid
extraction
Six live striped catsh (Pangasius hypothalamus)
is weighed of approximately 1 kg per head. They were
cleaned and lleted in 2 x 5 cm2 size using stainless
steel knife, stored in polyethylene (PE) bag, and
freezed prior to oil extraction. The lipid content of
striped catsh llet was extracted according to Bligh
and Dyer (1959). Twenty ve grams of thawed llet
cuts were ground using a meat grinder and added with
200 mL of a mixture of chloroform: methanol:distilled
water (1:2:1, v/v) then centrifuged at 3000 rpm and
4oC for 5 min. A-50 mL of chloroform and 25 mL
of distilled water were added into homogenate and
furthermore mixed for 2 min at a similar speed. The
homogenate was centrifuged at 4oC with speed 3000
rpm for 45 min using J-6B Centrifuge (Beckman,
Fullerton, CA, USA). Thereafter, the supernatant
was put into a separating funnel, which chloroform
fraction was drained off into Erlenmeyer glass
previously lled with 25 g of activated anhydrous
sodium sulphate with gentle shaken. The mixture
was ltered using Whatman lter paper No.4. The
solvent was evaporated from ltrate using a vacuum
rotary evaporator at 25ºC, and the remaining solvent
residue was removed using nitrogen gas.
Determination of fatty acid prole
Fatty acid composition of striped catsh oil was
determined as Fatty Acid Methyl Ester (FAME)
according to Latimer (2000). Into 0.5 mL of sh oil,
1.5 mL of methanolic sodium was added then heated
at 70ºC for 5-10 min while vigorously shaken. The
mixture was cooled and added with 2 mL of boron
triuoride methanoate then reheated at 70oC for
Rohadi et al./IFRJ 24(4): 1636-1643 1638
5-10 min and cooled again. Thereafter, the mixture
was extracted using 1 mL of heptane and 1 mL of
saturated NaCl. The top layer was taken and put into
Eppendorf tubes. One µL of sample was injected
into GC-2010 Shimadzu (Shimadzu, Kyoto Japan)
equipped with ame ionization detector (FID) at
260ºC. Capillary column (CP-Sil8-CB 30 m x 0.25
mm) was used. The standard retention time of FAME
was used to identify chromatogram peak of each
sample. Fatty acid content was quantied based on
peak area ratio as g fatty acid /100 g oil (%).
Preparation of sh oil emulsion
Fish oil emulsion was prepared based on Maqsood,
(2010) with slight modication. Twenty ve mL of
striped catsh oil were mixed with 225 mL of 0.1 M
buffer acetate (pH 5.4) and 2.5 mL of Tween 40 then
homogenized in 400 mL plastic jars for making 250
mL of oil-in-water emulsion. The mixture was kept
in an ice-bath (10±2ºC) during homogenization at
6500 rpm (Ultra Turrax T50, IKA Werke, Germany)
for 5 min. The methanolic extract of Java plum seed
(MEJS) powder was mixed into sh oil emulsion at
various concentration (25, 50, 100, 200, 400 and 800
mg/L) using vortex (Velp Scientica Europe) at 3000
rpm for 3 min. The mixture (10 mL) was then kept
at 35±2ºC in the dark room. The control of oil-in-
water emulsion was prepared in the same procedure
but distilled water was added instead of the extract.
BHA and grape seed extract (GSE) were used as the
reference antioxidant. Samples were taken at every
24 h for the determination of conjugated diene (CD)
value, peroxide value (PoV) and the thiobarbituric
acid reactive substances (TBARS).
Determination of lipid oxidative deterioration
The lipid oxidation was determined using
evaluation of conjugated diene (CD), peroxide value
(PoV), and thiobarbituric acid reactive substances
(TBARS) value.
Analysis of conjugated diene (CD)
Determination of conjugated diene was carried
out according to Frankel et al. (1996) in (Maqsood,
2010). Fish oil emulsion (0.1 mL) was dissolved in
5 mL of methanol and the absorbance was measured
at λ=234 nm using spectrophotometer UV-1601
(Shimadzu, Kyoto Japan). The absorbance of λ 234
nm indicated CD value.
Analysis of peroxide value (PoV)
Determination of peroxide value was done
according to Sakanaka et al. (2004). Fish oil
emulsion (50 μL), 2.35 mL of 75% ethanol, 50 μL
of 30% ammonium thiocyanate, 50 μL of 20 mM
ferrous chloride (FeCl2) in 3.5% HCl were mixed and
shaked using a vortex. After 3 min the absorbance
of the sample was measured using spectrophotometer
UV-1601 (Shimadzu, Kyoto Japan) at λ = 500 nm.
An increase absorbance at λ = 500 nm indicated the
formation of peroxide. The percent inhibition of
peroxidation of sh oil-in water emulsion of MEJS
was calculated as follow:
Analysis of thiobarbituric acid reactive substances
(TBARS)
Determination of TBARS was done according
to (Buege and Aust, 1978) in (Maqsood, 2010). Fish
oil emulsion (0.5 mL), 2.5 mL of TBA (0,375%
thiobarbituric acid, 15% trichloroacetic acid-TCA
and 0.25 N HCl) was mixed and shaked using a
vortex. The mixture was heated in boiling water
for 10 min until pink color appeared then cooled in
water ow and mixed using a vortex at 3000 rpm
(25ºC) for 10 min and the absorbance was measured
at λ 532 nm. A standard curve was conducted using
1.1.3.3-tetramethoxypropane (TMP) at various
concentrations ranged 0.61-20.9 μM/L. TBARs value
was expressed as μM–MDA equivalent/L.
Statistical analysis
All data was presented as a mean value±standard
deviation (SD). Analysis of the variance was
determined and the mean comparison was examined
by the Duncan’s Multiple Range Test (DMRT) at
the signicance level of 0.05 using SPSS statistic
software series 22 for Windows (SPSS Inc. Chicago,
USA). Regression analysis was applied to determine
correlation among variables.
Results and Discussion
Lipid content and fatty acid composition of striped
catsh oil
The lipid content of striped catsh llet
(Pangasius hypothalamus) was 9.45%, similar to
previous research reported by (Maqsood, 2010)
which showed lipid content of striped catsh from
Thailand was 9.2%. Meanwhile, (Ho and Paul, 2009)
reported lipid content of Tra catsh from Mekong
River delta in Vietnam was only 2.55%.
In the present study, striped catsh contains 43.57%
saturated fatty acid (SFA), 40.01% monounsaturated
fatty acid (MUFA), and 16.42% polyunsaturated fatty
acid (PUFA) (Table 1). Unsaturated fatty acid content
1639 Rohadi et al./IFRJ 24(4): 1636-1643
(56.43%) was 1.3 times higher than saturated fatty
acid content (43.57%), which indicated that lipid
content of the sh was vulnerable to oxidation. The
high percentage of SFA (43.57%) was similar to the
previous study reported by (Ho and Paul, 2009). The
MUFAs content was 36.71% cis9 oleic acid (C18:1
n-9), 2.54% palmitoleat acid, and 0.43% oleic acid
isomer (trans-9 oleic acid). These values showed that
oleic acid was abundant in striped catsh oil.
The PUFAs content of striped sh oil consisted of
8.28% linoleic acid (C18:2 n-6), 2.42% eicosatrienoic
acid (C20:3 n-6), 2.01% linolenic acid (C18:3 n-3),
1.51% eicosatetraenoic acid (C20:4 n-6), and 1.34%
eicosadienoic acid (C20:2 n-6). Eicosapentaenoic
acid (C20:5 n-3) or EPA content was 0.4%, higher
than reported by Ho and Paul, (2009) and Maqsood,
(2010), i.e. 0.31% and 0.13%, respectively. However,
the striped catsh oil did not contain docosahexaenoic
acid (C22:6 n-3), which was similar to the previous
studies (Ho and Paul, 2009; Maqsood, 2010). The
difference of lipid content and fatty acid composition
of striped catsh could be contributed to a different
method of oil extraction, kind of feed, and type of
sh farmed.
Effect of MEJS on lipid oxidation of sh oil
emulsion
Methanolic Java plum seed extract (MJES)
was prepared from dry Java plum seed powder
which extrated using methanol 50% (v/v), rather
than ethanol 50% (v/v) and ethyl acetate 85% (v/v).
Among the extracts, methanolic extract of Java
Table 1. Fatty acid prole of lipid extracted from striped catsh
(Pangasius hyphotalamus)
Table 2. The phenolic compounds of Syzygium cumini
seed were extracted using three different solvents.
Different subscript letters within the same kind of phenolic
compound mean signicant differences ( p < 0.05) (n=3).
Rohadi et al./IFRJ 24(4): 1636-1643 1640
Plum seed (MEJS) has stronger antioxidant activity
according to DPPH and Feric (Fe3+) reducing capacity
and a moderate activity for linoleic acid oxidation
inhibition (Rohadi et al., 2016). The high antioxidant
activity of Java Plum seed extract could be associated
with the high content of phenolic compounds (Table
2). The phenolic compound acts both as primary
antioxidants donate hydrogen to scavenge alkoxy and
peroxy radicals to form unreactive antioxidant radical
(chain breaking antioxidants), and also as secondary
antioxidants, such as metal chelating agents, singlet
oxygen quenchers, and oxygen scavengers (Frankel,
1998; Brewer, 2011; Saha et al., 2013).
A model emulsion was used to assess the
deterioration of lipids at two stages of oxidation,
i.e. primary oxidation products (CD and PoV) and
secondary oxidation products (TBARS). Maqsood
and Benjakul, (2010) reported that the effectiveness
of four phenolic compounds to inhibit lipid oxidation
in Menheden sh oil emulsion system during 168 h of
storage. Meanwhile, Azman et al. (2016) investigated
that the potential of bearberry leaf extract as a natural
antioxidant to inhibit lipid oxidation of sunower oil
emulsion during 40 days of storage.
Effect of MEJS on conjugated diene (CD) formation
The effect of MEJS (50 ppm) on lipid oxidation
of striped catsh oil emulsion during storage based
on conjugated diene (CD) and peroxide value (PoV)
is presented in Figure 1(a) and 1(b). The CD values
of samples gradually increased until 144 h (p<0.05),
whereas PoV increased up to 96 h (p<0.05) then
decreased slightly until the end of the storage period
(except control). A similar pattern was also found in
TBARS value (Figure 1c). TBARS value reects the
concentration of lipid oxidation secondary products
such as aldehyde (malondialdehyde, 4-hydroxy
nonenal) equivalent to malonaldehyde (µM-MDA
eq/L).
The addition of MEJS can inhibit CD formation of
an oil-in-water emulsion as well as BHA, moreover,
signicantly higher than grape seed extract (GSE)
(Figure 1a). This result could be associated with the
stronger free radical scavenging activity of BHA and
MEJS during initiation or propagation stage, therefore,
it can inhibit free radical formation in further stages.
CD values of the sample were higher than earlier
study (Maqsood and Benjakul, 2010) (ranged of 0.8-
2.0 at λ 234 nm). The difference in CD value could
Figure 1(a). Effect of methanolic extract of Java Plum
(Syzygium cumini Linn.) seed (MEJS) on lipid oxidation
products formation, conjugated diene (CD) in striped
catsh (Pangasius hyphotalamus) oil-in-water emulsion
at 35±2oC during 144 h of storage, compared to BHA
and grape seed extract (GSE). For all treatments, a nal
concentration of 50 mg/L was used in the system (n=3).
Figure 1(b). Effect of methanolic extract of Java Plum
(Syzygium cumini Linn.) seed (MEJS) on lipid oxidation
products formation, peroxide value (PoV) in striped
catsh (Pangasius hyphotalamus) oil-in-water emulsion
at 35±2oC during 144 h of storage, compared to BHA
and grape seed extract (GSE). For all treatments, a nal
concentration of 50 mg/L was used in the system (n=3).
Figure 1(c). Effect of methanolic extract of Java
Plum (Syzygium cumini Linn.) seed (MEJS) on
lipid oxidation products formation, thiobarbituric
acid reactive substances (TBARS) in striped catsh
(Pangasius hyphotalamus) oil-in-water emulsion at
35±2oC during 144 h of storage, compared to BHA and
grape seed extract (GSE). For all treatments, a nal
concentration of 50 mg/L was used in the system (n=3).
1641 Rohadi et al./IFRJ 24(4): 1636-1643
be contributed to a different fatty acid composition
of sh oil and phenolic compounds. Maqsood and
Benjakul, (2010) investigated Menheden sh oil and
single phenolic compund (not plant extract).
A strong correlation between RSA-DPPH activity
of MEJS or GSE and CD formation during initial 72
h was 0.98 and 0.84, respectively. Moreover, a strong
correlation was also found between MEJS or GSE
and CD formation using FRAP method, i.e. 0.96 and
0.95, respectively.
Effect of MEJS on peroxide value (PoV) formation
Peroxide value (PoV) reects the development of
primary oxidation products in emulsion food model.
The PoV of emulsion with or without 50 ppm MEJS
during storage was presented in Figure 1(b). PoV
of the samples gradually increased during 72 h then
decreased until the end of storage period. The PoV of
the samples with MEJS, BHA, and GSE were lower
than control (p<0.05). The effectiveness of MEJS
and BHA to inhibit PoV formation during initial
72 h of storage were similar, even higher than GSE
(p<0.05). Previous studies of the effectiveness of
emulsion containing plant extract antioxidant have a
similar pattern. Roedig-Penman and Gordon, (1997)
reported that emulsions containing green tea extract
required 8 days to reach the end of the induction time.
Meanwhile, Maqsood and Benjakul, (2010) found
that an emulsion containing 100 mg/L of the tannic
acid took 96 h to reach more than 4.5 (λ=500 nm). An
emulsion containing 48 µg/mL of Tara extract took
13 days to reach more than 10 meq hydroperoxide/
kg (Skowyra et al., 2014). Furthermore, Azman et al.
(2016) noted that the sample containing bearberry leaf
extract (BL) 1 g/kg reached the end of the induction
time after 20 days, while BHT samples after 36 days
of storage.
Inhibition of lipid oxidation among three
antioxidant agents (i.e. MEJS, GSE, BHA) at all
concentration (50-800 ppm) differed signicantly
(p<0.05). BHA has the highest of oxidation inhibition
(45-55%), followed by MEJS (38-40%), and the lowest
GSE (18-22%) (Figure 2). Phenolic composition of
MEJS which consisted of 45.99±0.25% total phenolic
(g GAE/100g), 2.28±0.07% avonoid (g QE/100g),
and 26.9±0.07% total tannin (g TAE/100g) may be
strongly contributed on inhibition of lipid oxidation
in sh oil emulsion system. Meanwhile, GSE
contained 95% of oligoproto-anthocianidin (OPC)
family (Bulkpowders, 2015).
Maqsood, (2010) reported that tannic acid was
very effective to inhibit CD and PoV in menheden
sh oil emulsion system. Tannic acid can be strongly
inhibited the formation of hydroperoxide and a
conjugated diene, which have a strong correlation
with DPPH and ABTS analysis. MEJS has also rich
in tannin acid and exhibited a strong inhibition of
lipid oxidation according to RSA-DPPH and FRAP
methods (Rohadi et al., 2016). A strong correlation
between RSA-DPPH activity of MEJS or GSE (25-
800 mg/L) and PoV formation during initial 72 h was
found, i.e. y = 5.67x +1.002, R2 = 0.87 for MEJS
and y = 0.296x + 1.89, R2 = 0.966 for GSE. A similar
pattern was also obtained using FRAP methods, i.e.
y = -10.95x + 20.26, R2 = 0.82 for MEJS and y =
-18.52x + 36.75, R2 = 0.875 for GSE. Mielnik et al.
(2006) reported that commercial GSE (Grapines-High
Potency) at 0.4-1.6 g/kg concentration was relatively
effective to prevent lipid oxidation in cooked meat.
The difference in PoV inhibition among samples
could be inuenced by the difference of polarity.
McClement and Decker, (2000) reported that
hydrophilic antioxidant was less effective in oil-in-
water (o/w) emulsion system than the lipophilic and
vice versa.
Effect MEJS on TBARS formation
TBARS (thiobarbituric acid reactive substances)
method is widely used to measure malondialdehyde
(MDA), a secondary product of lipid oxidation, which
is reacted with thiobarbituric acid (TBA), to give a
red color at λ 532 nm. Malondialdehyde (MDA) are
responsible for the formation off avor, rancid odor
and the undesirable taste in the foods. The effect of
MEJS on TBARS formation in the emulsion system
is presented in Figure 1(c). TBARS of the control
sample was greater than 3.0 µM-MDA/L (p<0.05) in
72 h of storage, whereas, the samples treated with
antioxidant were less than 2.5 µM-MDA/L emulsions.
Over 144 h of storage, TBARS of all of the samples
were less than 4.0 µM-MDA/L (approximately 0.25
Figure 2. Effect of methanolic extract of Java Plum
(Syzygium cumini Linn.) seed on inhibition of lipid oxidation
in striped catsh (Pangasius hyphotalamus) oil-in-water
emulsion stored at 35±2ºC for 144 h, compared to BHA and
grape seed extract (GSE). For all of the treatment, a nal
concentration of 200 mg/L was used in the system, (n=3).
Rohadi et al./IFRJ 24(4): 1636-1643 1642
mg MDA/L), and may not change the physical and
visual properties of the emulsion. The addition of
BHA in the emulsion could inhibit lipid oxidation and
exhibited the lowest TBARS value throughout the
storage period. Meanwhile, the addition of MEJS in
the emulsion exhibited the lower of TBARS than the
sample with GSE (p<0.05). In addition, the product
was quantied using standard curve equation of y =
0.1509x + 0.029, R2 = 0.9971 (x-axis = concentration
μM-MDA/L; y-axis = absorbance). Nollet and
Toldra, (2011) reported that the acceptable limits of
TBARS value in fat products was 1.0 mg MDA/kg (1
mg-MDA/kg≈ 13.7055 µM-MDA/kg).
During the initial stage of oxidation, TBARS
concentration was 0.6 µM-MDA/L, then increased up
to the peak at 72 h ranged between 3.1 µM-MDA/L
(GSE and control) and 1.97 µM-MDA/L (BHA and
MEJS). After the peak, TBARS decreased in all of
the samples, except the control. The decreasing of
TBARS could be associated with the inhibition of
secondary product of lipid oxidation, particularly
the volatile short-chain carbon compounds which
easily evaporate. The interaction of thiobarbituric
acid (TBA) with many other carbonyl-containing
compounds such as carbohydrates, pigment, and
amino acid could be also contributing to TBARS
values. Malondialdehyde and the short-chain
compound as a lipid oxidation product were also
labile and easily decomposed into alcohol and
organic acids thus, undetectable using TBA analysis
(Borneo et al., 2009; Maqsood, 2010). Azman et
al. (2016) reported that the addition of lyophilized
bearberry leaf extract (1 g/kg) as an antioxidant in
the emulsion food model signicantly inhibited lipid
oxidation during 20 days of storage.
Conclusion
Methanolic extract of Java Plum seeds (MEJS)
had a great potential as the natural antioxidant to
inhibit the oxidative deterioration of oil-in-water
(o/w) emulsion system. Inhibition activity of lipid
peroxidation of MEJS was 28-38%, lower than BHA
(40-45%) but stronger than grape seed extract (GSE)
18-23%. MEJS could be signicantly able to suppress
the TBARS generation due to lipid oxidation.
Acknowledgement
The authors would like to thank the Directorate
General of Higher Education Ministry of Research
Technology and Higher Education of Republic of
Indonesia for nancial support.
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