Food Control 40 (2014) 134e139

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Grape seed and clove bud extracts as natural antioxidants in silver

carp (Hypophthalmichthys molitrix) llets during chilled storage: Effect
on lipid and protein oxidation
Ce Shi, Jianyun Cui, Xiaofei Yin, Yongkang Luo*, Zhongyun Zhou
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing Higher Institution Engineering Research Center of Animal
Products, P.O. Box 112, Beijing 100083, PR China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 19 June 2013
Received in revised form
31 October 2013
Accepted 2 December 2013

The aim of this study was to investigate the utilization of clove bud extract (CBE) and grape seed extract
(GSE) as natural antioxidants for retarding lipid and protein oxidation in silver carp llets stored at
4
1  C. The results indicate that CBE exhibited higher total phenolic content, DPPH and Fe2-chelating
activity than GSE. GSE and a 20-times dilution of CBE were found to be effective in retarding lipid and
protein oxidation; both treatments resulted in low levels of PV and TBA, and protected against the
decrease of L*, a*, salt-soluble protein content and total sulfhydryl group. CBE20 more efciently
inhibited lipid oxidation than did GSE. The antioxidant effect of the two extracts on protein oxidation was
less pronounced than the effect on lipid oxidation. CBE20 and GSE could be used as natural antioxidants
to minimize lipid and protein oxidation and to extend shelf life of llets.
2014 Elsevier Ltd. All rights reserved.

Keyword:
Silver carp llets
Clove bud extract
Grape seed extract
Lipid and protein oxidation
Natural antioxidant

1. Introduction
With a harvest of 3,713,900 tons in 2011, silver carp (Hypophthalmichthys molitrix) is the most abundant freshwater sh in
China. It is known to be rich in protein and fat (Li, Sinclair, & Li,
2011). Due to its attractive white colour and high nutritional
value, silver carp are often used to produce ready-to-eat and high
quality salt sh products, which are well suited for human consumption. In spite of these obvious benets, one major obstacle in
using silver carp is that they are highly susceptible to oxidation
because of the relatively high content of polyunsaturated fatty acids.
Lipid oxidation leads to unpleasant odour, rancid taste and discolouration (Farvin, Grejsen, & Jacobsen, 2012). Moreover, proteins
can be modied by the compounds resulting from lipid oxidation,
which leads to nutritional changes in amino acids and a decrease of
protein functionality (solubility and hydrophobicity). Many attempts have been made to reduce lipid oxidation and pigment in
meats through synthetic or natural antioxidants. Although synthetic antioxidants such as butylated hydroxytoluene (BHT) and
butylated hydroxy anisole (BHA) can markedly delay or prevent the

* Corresponding author. Tel./fax: 86 10 62737385.

E-mail addresses: luoyongkang@263.net, luoyongkang@cau.edu.cn (Y. Luo).
0956-7135/$ e see front matter 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.12.001

oxidation of substrate, the possible carcinogenic effects of synthetic

antioxidants in foods have been found (Lindenschmidt, Tryka, Goad,
& Witschi, 1986; Shahidi, 2000). Therefore, there is a general desire
to replace synthetic antioxidants with natural ingredients.
In recent years, application of plant extracts as natural antioxidants in meat products have been attempted by different researchers. The effect of kiam wood extract on the postponement of
haemoglobin-mediated lipid oxidation of washed Asian sea bass
mince was investigated by Maqsood and Benjakul (2013). The efcacy of potato peel ethanol extracts as an antioxidant in minced
horse mackerel during chilled storage was observed by Farvin et al.
(2012). Mitsumoto, OGrady, Kerry, and Buckley (2005) showed that
tea catechins were stronger natural antioxidants compared with
vitamin C in cooked or raw beef and chicken patties during
refrigerated storage. Mansour and Khalil (2000) found that addition
of freeze-dried extracts from ginger rhizomes and fenugreek seeds
to beef patties effectively controlled lipid oxidation and colour
changes during chilled storage.
Grape seed is a waste by-product of the food industry and as
such presents a cheap source of natural antioxidant due to its
phenolic content (Spigno & De Faveri, 2007). The extracts contain
bioactive phenolic compounds which have recently been recognized for their efcacy in providing signicant antioxidant activity

to human foods (Buci
c-Kojic, Planini
c, Tomas, Jakobek, & Seruga,

C. Shi et al. / Food Control 40 (2014) 134e139

2009; Gibis & Weiss, 2012; Rosales Soto, Brown, & Ross, 2012).
Clove, which is commercially cultivated in the south of China, is an
important aromatic spice. Clove bud extracts have likewise been
found to possess great antioxidant activity (Verrez-Bagnis, Ladrat,
Nolle, & Fleurence, 2002). Our numerous earlier studies have
found that extracts of grape seeds and clove buds are highly efcient in reducing lipid oxidation in minced meat and sh oil.
However, the antioxidative effect of grape seed and clove bud extracts in sh llets had not yet been studied.
The overall objective of this study was to identify whether the
addition of grape seed and clove bud extracts can retard both lipid
and protein oxidation and extend the shelf life of silver carp llets.
In addition, this study provided greater insight into the potential of
grape seed and clove bud extracts as natural and effective sources
of antioxidants for sh processing.
2. Materials and methods
2.1. Materials
2.1.1. Preparation of grape seed and clove bud extracts
Grape seeds (Merlot) were obtained from wineries located in
Beijing, China, and clove buds were obtained from Xintong Co., Ltd.,
located in Shandong province, China. Both were dried at 50
2  C
for 8 h and then ground into a ne powder.
Three extracts were obtained: grape seed extract (GSE), clove
bud extract (CBE) and 20-fold dilution of CBE (CBE20). For preparation of GSE, 10 g of grape seed powder was added to 100 ml
boiled distilled water and left for 3 h at room temperature; the
extract was obtained by ltration through Whatman Grade No. 1
lter paper. The extract was collected in a separate bottle, and the
residue (9.55
0.25 g) was re-extracted twice under the same
conditions as mentioned above. This extract was referred to as GSE.
CBE was prepared in the same manner using 10 g clove bud powder
(8.25
0.25 g of residue was used for the re-extraction). In the case
of CBE20, 1 ml CBE was diluted in 19 ml distilled water. The extract
obtained was referred to as CBE20. These extracts were kept
at 20  C prior to analysis. All extracts were analyzed for total
phenolic content, 1,1 diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging activity and Fe2-chelating ability.
2.1.2. Fish material preparation
A total of 60 silver carp (H. molitrix) weighing 274
18 g each
were purchased from a market in Beijing, China, and were transported alive to the laboratory. The fresh silver carp were slaughtered, scaled, gutted and washed in water, and each sh was
divided into two pieces. The samples were divided into three
groups. One group was brined with 1.0% salt (weight of distilled
water weight of llet) (control); the second group was brined
with a mixture of 1.0% salt (weight of distilled water weight of
llet) 2.0% GSE (weight of distilled water weight of llet) (T1);
the third group was brined with a mixture of 1.0% salt (weight of
distilled water weight of llet) 2.0% CBE20 (weight of distilled
water weight of llet) (T2). The llet to distilled water ratio was
10:1. The llets were packaged and sealed in polythene bags and
stored in refrigerated incubators at 4
1  C for temperature
equalization. Three random samples were taken from each group
for analysis at each sampling time (0, 3, 6, 9, 12, 15 and 18 d).
2.2. Methods
2.2.1. Determination of total phenolics
Determination of polyphenol content was performed by the
FolineCiocalteu method and expressed as gallic acid equivalents
(mg GAE/g) (Boligon et al., 2009). A quantity of 0.5 ml of 2 N Foline

135

Ciocalteu reagent was added to 1 ml of extract. After 5 min, 2 ml of

20% Na2CO3 was added to the mixture, which was incubated for
10 min at room temperature. Absorbance was measured at 730 nm.
2.2.2. Determination of chemical compounds in grape seed and
clove bud extracts
2.2.2.1. HPLC analysis. The grape seed extract was ltered through
a 0.22 mm membrane lter and analyzed using HPLC (Shimadzu,
LC-10A Tseries, Japan) equipped with SPD-10A (V) detector, COSMOSIL 5C18-PAQ column (4.6 mm ID  250 mm). Gradient elution
programme was executed by varying the proportion of solvent A
(2% acetic acid and 9% acetonitrile) and solvent B (80% acetonitrile).
The gradient elution programme: 100% A (0 min) e 100% A (10 min)
e 68% A (25 min) e 68% A (35 min) e 100% A (40 min). System was
equilibrated 5 min before next analysis. Flow rate was 1 ml/min and
column temperature was 30  C. 50 ml of sample was injected.
The peak was detected at 280 nm. Compounds were identied by
the retention time of sample chromatographic peaks being
compared with standards samples using the same HPLC operating
conditions.
2.2.2.2. GCeMS analysis. The clove bud extract was analyzed using
a 450-GC (Varian) coupled to a 220-MS (Varian) and equipped with
a DB-5 column (30 m  0.25 mm; 0.25 mm lm). The carrier gas
was helium, at a ow rate of 1 ml/min. Column temperature was
initially 60  C for 5 min, and then increased to 180  C at the rate of
3  C/min, and then to 250  C at the rate of 10  C/min. Injector
temperature was 250  C. 1 ml samples was injected by splitting.
2.2.3. Determination of Fe2-chelating ability
Determination of Fe2-chelating ability was performed according to the method described by Ruiz-Navajas, Viuda-Martos, Sendra,
Perez-Alvarez, and Fernndez-Lpez (2013). The Fe2-chelating
ability was measured in a reaction mixture containing the extract
(1.0 ml), 99.5% ethanol (3.7 ml), 2 mM FeCl2 (0.1 ml) and 5 mM ferrozine (0.2 ml). The mixture was left at room temperature for
10 min; absorbance of the resulting solution was measured at
562 nm. (A lower absorbance indicates a stronger Fe2-chelating
ability.) The ability to chelate the ferrous ion was calculated as
follows:


Fe2 -chelating ability % 1  absorbancesample =

absorbancecontrol  100
2.2.4. Determination of DPPH radical scavenging activity
DPPH assay was performed according to the method of Banerjee
et al. (2012). The extract (1.5 ml) and 99.5% ethanol (1.5 ml) were
mixed with 0.375 ml of 0.02% (w/v) 2,2-diphenyl-1-picrylhydrazyl
in ethanol. The tubes were then incubated at room temperature
for 60 min in the dark, and the absorbance was taken at 517 nm.
The radical scavenging activity (RSA) was calculated by the
following equation:

RSA %


1  absorbancesample =absorbancecontrol  100%

2.2.5. Sensory evaluation of cooked and raw sh

Each llet was placed in a plastic bag, which was sealed and
steamed for 5 min. After 5 min the llets were served to panelists
and assessed. The samples were evaluated by nine trained panelists
and the panelists were trained according to the International
Organization for Standardization (1993). Cooked and raw sh

136

C. Shi et al. / Food Control 40 (2014) 134e139


were evaluated according to the method of Simat,
Bogdanovi
c,
Kr
zelj, Soldo, and Marsi
c-Lu
ci
c (2012). The scheme consists of
seven parameters (cooked llets: odour, avour and texture; raw
llets: colour, avour, texture and resilience). A score of 0e5 points
was given for every evaluated parameter (5 representing the
highest level of freshness, 0 representing the limit of acceptability),
for a total of 35 points. An average score of less than 21 was used as
the sensory rejection point (Zhang, Li, Lu, Shen, & Luo 2011). The
panelists were requested to evaluate and score quality on evaluation sheets by feeling, smelling and examining the samples on days
0, 3, 5, 7, 9, 12, 15 and 18 of storage.

Table 1
Chemical compounds of grape seed and clove bud extracts.

Grape seed extract

Clove bud extract

2.2.6. Determination of colour

The colour of the silver carp llets was determined with an ADCI
colorimeter (ADCI-60-C, Beijing Chentaike Instrument Technology
Co., Ltd., Beijing, China). The results were expressed in L* (brightness), a* (redness) and b* (yellowness).
2.2.7. Determination of thiobarbituric acid value (TBA)
The TBA was determined according to Erkan and zden (2008)
with some modication. Two grams of muscle from each llet was
homogenized in a 50 ml centrifuge tube to which 16 ml of 5% (w/v)
solution of trichloroacetic acid (TCA) and 100 ml butylated
hydroxytoluene were added. The mixture was centrifuged at 3600g
for 20 min at room temperature. 1 ml of 0.01 M aqueous solution of
2-TBA was mixed with 5 ml of the supernatant. The mixture was
heated in a boiling water bath for 40 min and cooled to room
temperature. The absorbance of the resultant coloured solution was
read at 532 nm. TBA values were expressed as mg of malondialdehyde (MDA) kg1 of sample.
2.2.8. Determination of peroxide value (PV)
PV was measured according to the method of Shantha and
Decker (1994). Fillet (20 g) was homogenized in 50 ml water for
2 min. A 2 ml aliquot was reacted with 3 ml of isooctane/2propanol (3:1, v/v) and centrifuged at 2000g for 5 min to obtain
the organic solvent phase. An aliquot of this phase (400 ml) was
added to 3 ml of methanol/1-butanol (2:1, v/v), followed by 30 ml
of 30% ammonium thiocyanate and 30 ml of the ferrous chloride
solution. The absorbance of the solution was measured at 500 nm
after 20 min.
2.2.9. Determination of salt-soluble protein (SSP)
Salt-soluble protein was extracted according to the method
described by Kelleher and Hultin (1991) with some modication.
Two grams of muscle from each llet was homogenized with 30 ml
0.05 M NaCl tris-maleate (pH 7.0) for 20 s and centrifuged at
10,000g for 20 min at 4  C. This process was repeated twice. The
precipitate was collected and homogenized with 20 ml 0.6 M NaCl
tris-maleate (pH 7.0), centrifuged at 10,000g for 20 min at 4  C. The
nal supernatant liquid was SSP. Protein concentration of the SSP
was determined using the Biuret method (Torten & Whitaker,
1964).
2.2.10. Determination of total sulfhydryl (SH) group
Total SH content was determined according to the method of
Yongswawatdigul and Park (2002) with some modication. A
quantity of 0.5 ml myobrillar protein (4 mg/ml) was added to
4.5 ml of 0.2 M TriseHCl (pH 8.0) containing 8 M urea, 1% SDS and
3 mM EDTA. A 4 ml aliquot of the mixture was mixed with 0.5 ml of
0.2 M TriseHCl (pH 8.0) containing 0.1 mM DTNB. The reaction
mixture was incubated for 25 min at 40  C, and the absorbance was
measured at 412 nm. Total SH content was determined using a
molar extinction coefcient of 13,600 (M1 cm1).

Compound

(%)

Gallic acid
Procyanidin B1
Procyanidin B2
Procyanidin C1
Catechin
Epicatechin
Epicatechin gallate
Dodecane
Nonadecane
Tridecane
Tetradecane
Caryophyllene
a-Caryophyllene
Cadinene
Eugenol
Copaene
Heptacosane
Benzene, 1-ethyl-3-nitro
Cedrene
Caryophyllene oxide
Naphthalene
Hexadecenoic acid
Nonadecanoic acid
Methyl palmitate
Benzene acid, 3-(1-methylethyl)
Pentatriacontane

1.39
32.06
10.01
26.99
5.91
6.46
1.16
2.57
0.53
1.10
0.25
22.43
4.05
4.81
32.15
1.35
0.67
5.57
1.38
1.96
1.94
1.00
0.70
2.73
3.89
2.13

2.3. Statistical analysis

All experiments were performed in triplicate. Data were subjected
to analysis of variance (ANOVA). The least signicant difference (LSD)
procedure was used to test the differences among means, with signicance dened at P < 0.05 (SAS 9.2 Institute, Cary, NC, USA).
3. Results and discussion
3.1. Antioxidant potential of grape seed and clove bud extracts
The major chemical compounds in grape seed and clove bud
extract are shown in Table 1. Grade seed extract contained high
levels of procyanidin (69.06%), followed by epicatechin and catechin. Clove bud extract have mostly eugenol (32.15%), caryophyllene (22.43%) and benzene (5.57%).
Results of the evaluation of antioxidant properties are shown in
Table 2. The data show that between CBE and GSE, CBE had higher
antioxidant activity.
Phenolic compounds, which are secondary metabolites in
plants, are known to have antioxidant effects. As shown in Table 2,
CBE contained higher levels of phenolics. Total phenolic content in
GSE was signicantly (P < 0.05) lower than that of CBE but similar
to that of CBE20.
As Fe2 is believed to cause the production of oxyradicals and lipid
peroxidation, minimizing Fe2 concentration affords protection
Table 2
Total phenolic content, Fe2-chelating activity and DPPH radical scavenging activity
of grape seed and clove bud extracts.
Parameter
Total phenolic
content (mg GAE/g)
Fe2-chelating
activity (%)
DPPH radical
scavenging activity (%)

Grape seed
extract

Clove bud
extract

20-Fold dilution
of clove bud extract

6.1
0.1b

106
1.4a

5.9
0.4b

76.2
5.2b

99.0
1.4a

70.0
1.1b

76.2
1.5b

94.3
10.0a

92.7
3.9a

Mean
SE with different small letter (aec) superscripts on the same row are
signicantly different (P < 0.05).

C. Shi et al. / Food Control 40 (2014) 134e139

137

(P < 0.05) decline with increasing storage time. There was no signicant (P > 0.05) difference in the organoleptic characteristics of
all samples during the rst 6 days of storage. After 6 days of storage,
sensory score of T1 and T2 were higher than that of control. In
contrast, no signicant (P > 0.05) difference in sensory scores was
found between T1 and T2 during the whole storage period. After 9
days of storage, a general fading of colours (for example, a yellowish
slime on the surface of the llets), loss of esh rmness and
reduction of avour were observed in control samples. In contrast,
T1 and T2 failed to produce rancid odours prior to day 12, though a
dark colour was observed. T1 and T2 maintained better sensory
quality than those of untreated samples throughout storage. These
results are in accordance with Barbosa-Pereira et al. (2013), who
found that antioxidant lms extended shelf life of salmon.
Fig. 1. Effect of grape seed and clove bud extracts on sensory score of silver carp llets
during storage (4
1  C) (control: brined with 1.0% salt; T1: brined with 1.0% salt and
2.0% GSE (grape seed extract); T2: brined with 1.0% salt and 2.0% CBE20 (20-fold
dilution of clove bud extract)). Different small letters (aec) on the same day indicate
signicant difference at P < 0.05.

against oxidative damage. Table 2 shows the chelating effect of GSE

and CBE on ferrous ions. The chelating efciency of GSE was signicantly (P < 0.05) less than that of CBE, but it was no different from
that of CBE20. The higher Fe2-chelating activity in CBE may have
been due to the presence of specic phenolic compounds, which
have been reported to be good chelating agents (Robards, Prenzler,
Tucker, Swatsitang, & Glover, 1999).
The DPPH radical scavenging activities were 76.2%, 94.3% and
92.7% in GSE, CBE and CBE20, respectively. DPPH radical scavenging
activity of CBE20 was similar to that of CBE, which suggests no linear
correlation between radical scavenging ability and polyphenolic
content. Similar ndings were reported by Robards et al. (1999),
who concluded that antioxidant activity did not correlate with
phenolic content in berry and fruit wines.
Results of this study indicate that GSE and CBE20 exhibited
similar phenolic compounds and Fe2-chelating activity. Consequently, these two extracts were used to brine silver carp llets for
protein and lipid oxidative stability studies.
3.2. Effect of GSE and CBE20 on sensory evaluation of silver carp
llets
Fig. 1 shows the total sensory scores of silver carp stored at 4  C
for 18 days. Sensory quality for all llets showed a signicant

3.3. Effect of GSE and CBE20 on colour values of silver carp llets
Colour expressed as L*, a* and b* of silver carp llets during
refrigerated storage is shown in Table 3. The L* value of control and
T1 decreased during storage, whereas L* value of T2 decreased from
46.0 (day 0) to 45.3 (day 9), and then increased to 46.9 (day 18).
There was no signicant (P > 0.05) change in L* value over time in
the two treated groups. However, L* values of T1 and T2 were
signicantly (P < 0.05) higher than that of control during the whole
storage. Similarly, Valencia, OGrady, Ansorena, Astiasarn, and
Kerry (2008) had also reported an increase in L* value in fresh
pork sausage containing green tea catechins and green coffee antioxidants compared to the control.
There was a signicant (P < 0.05) reduction in a* value of all
samples with increased storage time, though the control had the
lowest a* value at the end of storage time. This indicates that the
decrease of redness in the control could be due to haemoglobin lipid
oxidation and the accumulation of brownish met-haemoglobin
(Wetterskog & Undeland, 2004). Tesoriere, Butera, Gentile, and
Livrea (2007) found that phenolic extracts from capers effectively
inhibited the initiation of myoglobin to its hypervalent state of
ferrylmyoglobin, indicating a potential interaction between some
phenolic compounds and heme protein redox reactions.
A signicant (P < 0.05) increase in b* value of control was
observed during the entire storage period, whereas b* values of T1
and T2 decreased during the rst 6 days of storage and then
increased from 0.26 to 1.8 for T1 and from 0.53 to 4.4 for T2 from
day 6 to day 18, respectively. The b* values of T1 and T2 were higher
than that of control during the whole storage, which may have been

Table 3
Effect of grape seed and clove bud extracts on colour values of silver carp llets during storage (4
1  C)
Parameter

Storage time (d)

0

L* value
Control
T1
T2

44.1
1.2aB
46.9
2.2aA
46.0
1.2abA

42.4
0.90cB
44.0
0.28cA
45.7
1.5bA

a* value
Control
T1
T2

8.8
0.77aB
8.3
1.4aC
9.4
0.27aA

b* value
Control
T1
T2

3.3
0.28dB
1.7
0.57abA
2.1
0.06dA

7.8
0.28bC
8.1
0.91aB
8.9
0.61abA

1.1
0.59cB
1.3
0.72bA
2.1
0.89dA

12

15

18
42.4
0.01cC
45.9
0.91aB
46.9
0.09aA

42.3
2.1cB
44.2
0.03bA
45.3
0.17bA

42.3
0.84cB
44.3
1.3bA
45.3
1.6bA

42.1
4.1cB
44.8
2.0bA
45.8
0.71bA

43.4
0.06bB
44.3
5.4bB
47.1
0.06aA

8.3
0.90aB
8.1
0.61aB
9.5
0.71aA

6.5
0.63cC
7.1
0.41bB
8.0
0.13bA

6.5
0.49cC
7.3
0.58bB
7.7
0.34cA

5.9
0.71dC
6.5
0.06cA
6.3
0.43eB

5.8
0.08dC
6.8
0.07bcB
7.2
0.69dA

0.76
0.30bcC
0.42
0.07bB
3.4
0.43cA

1.9
0.39aB
1.9
0.17aB
4.9
0.53aA

1.4
0.11abC
1.8
0.66abB
4.4
0.25bA

0.89
0.06cC
0.26
0.05cB
0.53
0.04eA

0.42
0.02bcC
0.45
0.09cB
2.00
0.46dA

Control: brined with 1.0% salt; T1: brined with 1.0% salt and 2.0% GSE (grape seed extract); T2: brined with 1.0% salt and 2.0% CBE20 (20-fold dilution of clove bud extract).
Mean
SD with different small letter (aec) superscripts on the same row are signicantly different (P < 0.05). Mean
SD with different capital letter (AeC) superscripts on the
same column are signicantly different (P < 0.05).

138

C. Shi et al. / Food Control 40 (2014) 134e139

Fig. 2. Effect of grape seed and clove bud extracts on peroxide value (PV) and thiobarbituric acid (TBA) values of silver carp llets during storage (4
1  C) (A control:
brined with 1.0% salt (PV); - T1: brined with 1.0% salt and 2.0% GSE (grape seed
extract) (PV); : T2: brined with 1.0% salt and 2.0% CBE20 (20-fold dilution of clove bud
extract) (PV). > Control: brined with 1.0% salt (TBA); , T1: brined with 1.0% salt and
2.0% GSE (TBA); 6 T2: brined with 1.0% salt and 2.0% CBE20 (TBA)). Different small
letters (aec) on the same day indicate signicant difference at P < 0.05.

due to the presence of slight colour compounds in T1 and T2.

Maqsood, Benjakul, and Balange (2012) similarly reported that the
addition of kiam wood extract resulted in slightly darker sh
emulsion sausages. Gibis and Weiss (2012) have also reported
observing discolouration of fried beef patties with addition of
natural antioxidants such as grape seed extracts.
Although the colour changes associated with lipid oxidation
are widely reported, the changes observed in this study cannot
be denitively attributed to lipid or protein oxidation because
of the colours of the extracts themselves (black yellow for
extracts).
3.4. Effect of GSE and CBE20 on PV and TBA of silver carp llets
The impact of GSE and CBE20 on lipid oxidation of silver carp
llets during refrigerated storage over 18 days is shown in Fig. 2. An
increase in PV was observed in all samples during the rst 6 days of
refrigerated storage. On day 6, the PV of control was signicantly
(P < 0.05) higher than that of both T1 and T2. The continuous increase in PV was observed up to day 18 for T2, while the PV of
control and T1 decreased with increasing storage time. Control and
T1 displayed higher PV compared with T2 during the whole storage. The increase in PV of T2 indicates that the samples were in
propagation stage of lipid oxidation with a lower rate of decomposition of hydroperoxide. The decrease in PV of control and T1 was
probably due to the decomposition of hydroperoxide into other

oxygenated compounds, which are considered secondary oxidation

products (Boselli et al., 2005).
During the 18 days of storage, higher PV was found in control
than in T1 and T2. The results indicate that CBE20 and GSE were
effective in retarding the formation of hydroperoxide. Other authors have similarly found that phenolic compounds of clove
demonstrate strong antioxidant properties (Verrez-Bagnis et al.,
2002).
TBA value of all samples increased signicantly with the
advancement of storage period. Compared with treated samples,
control samples showed higher formation of TBA throughout storage.
T2 showed comparatively lower TBA value but did not differ significantly from T1 during the rst 9 days of storage. Similarly, Tajik,
Farhangfar, Moradi, and Razavi Rohani (2012), who observed strong
antioxidant effects from clove and grape seed extract, reported that
the antioxidant potential of this extract signicantly restrained TBA
value in silver carp llets.
3.5. Effect of GSE and CBE20 on protein oxidation of silver carp
llets
In foods, oxidative reactions can easily transfer from lipids to
proteins due to the strong interactions between these molecules.
Radicals, hydroperoxides and secondary compounds resulting from
lipid oxidation also react with proteins, leading to the loss of protein functionality (Farvin et al., 2012). The SSP content and SH
content of different treatments of silver carp llets during refrigeration storage are shown in Table 4. At day 0, there was no signicant (P > 0.05) difference in SSP content among the three
groups. As storage progressed, protein oxidation was evident by a
decrease in SSP content of the three groups. At the end of the
storage period, the control showed lower SSP content than did T1
and T2, which suggests that the extracts provided some protection
against protein oxidation. However, the similar reduction in SSP
content in control, T1 and T2 suggests that the extracts were not
effective in reducing the rate of protein solubility in silver carp
llets. Furthermore, these data suggest that there is no direct correlation between lipid oxidation and protein aggregation in
refrigerated storage of silver carp llets.
Change of total SH content showed a similar trend with SSP
content. A signicant (P < 0.05) decrease in total SH content was
observed during the rst 12 days of storage for control. A slight
decrease in total SH content was observed during the entire storage
period for T1 and T2. Eymard, Baron, and Jacobsen (2009), who
studied horse mackerel mince, similarly found that SH content
decreased during storage for all products. The decrease in total SH
content is due to the formation of disulphide bonds through oxidation of SH groups or disulphide interchanges (Thawornchinsombut &
Park, 2005). Among different silver carp llets, T1 and T2 had

Table 4
Effect of grape seed and clove bud extracts on salt-soluble protein (SSP) content and total sulfhydryl (SH) content of silver carp llets during storage (4
1  C).
Parameter

SSP (mg/g)
Control
T1
T2

Storage time (d)

0

12

15

18

122
0aA
122
1aA
122
5aA

116
1abA
119
12abA
122
5aA

112
2bcA
114
2bcA
115
8bA

106
2cdA
112
4bcA
112
2bcA

108
0cdA
112
4bcA
112
1bcA

106
4cdA
108
2cA
108
0cA

103
10dA
108
3cA
110
6bcA

Total sulfhydryl (SH) group(mol/105 g)

Control
7.2
1.0aA
T1
7.2
0.8aA
T2
7.7
0.3aA

6.4
0.5abA
6.7
0.4abA
6.6
0.9bA

6.1
0.3bcA
6.6
0.4abcdA
6.5
0.5bcA

5.9
0.8bcA
6.5
0.2bcdB
6.5
0.1bcB

5.6
0.7bcA
6.7
0.2abcB
6.3
0.8bcdB

5.3
0.1cA
5.9
0.8dB
6.1
0.7cdB

5.4
0.1cA
6.1
0.8cdB
5.9
1.0dB

Control: brined with 1.0% salt; T1: brined with 1.0% salt and 2.0% GSE (grape seed extract); T2: brined with 1.0% salt and 2.0% CBE20 (20-fold dilution of clove bud extract).
Mean
SD with different small letter (aec) superscripts on the same row are signicantly different (P < 0.05). Mean
SD with different capital letter (AeC) superscripts on the
same column are signicantly different (P < 0.05).

C. Shi et al. / Food Control 40 (2014) 134e139

signicantly higher (P < 0.05) SH content from day 9 to day 18. Lower
loss of SH in T1 and T2 may have been due to lower oxidation caused
by the abundance of phenolic compounds in GSE and CBE20.
Phenolic compounds have previously been suggested to inhibit
protein oxidation, retard lipid oxidative reactions, bind to proteins
and form complexes with them (Siebert, Troukhanova, & Lynn,1996).
4. Conclusion
The addition of GSE and CBE20 retarded the increase of PV and
TBA; protected against decreases of L* value, a* value, salt-soluble
protein content and total sulfhydryl group; and extended by 3
days the sensory shelf life of silver carp llets compared to the
control. The high efcacy of the grape seed and clove bud extracts
correlated with high total phenolics and signicant Fe2-chelating
ability and DPPH radical scavenging activity. It clearly demonstrated the antioxidant effects of grape seed and clove bud extracts
on silver carp llets. Between the two treatment groups, T2 was
more effective in inhibiting the rate of decomposition of hydroperoxide in silver carp llets. This might be due to lower phenolic
contents in grape seed extracts compared to clove bud extracts. The
antioxidant effect of both extracts on lipid oxidation was more
pronounced than was the effect on protein oxidation. Therefore,
GSE and CBE20 can be employed as natural antioxidants to prevent
lipid and protein oxidation and extend shelf life of silver carp llets
in chilled storage.
Acknowledgements
This study was supported by the earmarked fund for China
Agriculture Research System (CARS-46) and National Natural Science Foundation of China (award nr 30871946).
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