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Author's personal copy
Food Chemistry 129 (2011) 822–829
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Effect of dietary supplementation of gallic acid and linoleic acid mixture
or their synthetic salt on egg quality
Samooel Jung a, Byung Hee Han b, Kichang Nam c, Dong U. Ahn d,e, Jun Heon Lee a, Cheorun Jo a,⇑
a
Department of Animal Science and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea
Department of Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea
c
Department of Animal Science and Technology, Sunchon National University, Suncheon 540-742, Republic of Korea
d
Department of Agricultural Biotechnology, Major in Biomodulation Seoul National University, Seoul 151-921, Republic of Korea
e
Department of Animal Science, Iowa State University, Ames, IA 50011-3150, USA
b
a r t i c l e
i n f o
Article history:
Received 4 November 2010
Received in revised form 23 March 2011
Accepted 5 May 2011
Available online 11 May 2011
Keywords:
Egg
Gallic acid
Linoleic acid
Antioxidative potential
Cholesterol
a b s t r a c t
The effect of a dietary supplementation of gallic acid and linoleic acid mixture (MGL) and their synthetic
salt, sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate (NGL), on egg
quality was investigated. A total of 120 laying hens were allotted into five groups over 4 weeks of the
experimental period. Birds were fed the following diets: (1) control [commercial diet (CD)], (2) 0.05%
MGL (w/w, GA:LA = 1:1, equal molar ratio), (3) 0.1% MGL, (4) 0.05% NGL, (5) 0.1% NGL. The performance
of the hen, the anti-oxidative potential of egg albumen and yolk, and the fatty acid composition and cholesterol content of egg yolk were measured. The TBARS value of egg yolk from hens fed 0.1% MGL and
0.05% NGL was lower than that fed control diet after storage for 14 days. The ABTS+ reducing activity
of egg albumen was significantly improved by MGL and NGL, but only NGL had an effect on yolk
(p < 0.05). The dietary supplementation of 0.05% or 0.1% MGL, and 0.05% NGL raised the PUFAs composition in egg yolk. The cholesterol content of egg yolk from hens fed control diet was higher than those fed
0.1% MGL, 0.05% or 0.1% NGL (p < 0.05). In conclusion, a diet consisting of MGL and NGL can improve the
antioxidative potential of egg and the fatty acid quality of egg yolk while lowering the cholesterol level.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Although eggs are an important source of nutrients due to their
high quality protein and variety of vitamins and minerals, it is often recommended that people restrict their consumption since egg
yolk contains nearly 30% lipids and high cholesterol content
(Milinsk, Murakami, Gomes, Matsushita, & de Souza, 2003;
Weggemans, Zock, & Katan, 2001). Egg consumption is also
believed to raise the risk of cardiovascular disease by increasing
blood cholesterol levels (Weggemans et al., 2001). However, previous studies have found that dietary cholesterol does not contribute
to high blood cholesterol, and there has been little evidence that
egg ingestion causes increases in serum lipid or total cholesterol
levels (McNamara, 2002).
On the other hand, polyunsaturated fatty acids (PUFAs) contained in the lipids of egg yolk are regarded as a substantial nutrient as they prevent coronary heart disease and other chronic
diseases (Russo, 2009). A high ratio of unsaturated fatty acids to
saturated fatty acids could diminish the negative effects of high
cholesterol intake (Milinsk et al., 2003). For this reason, several
studies had been conducted to raise PUFA content in eggs by using
⇑ Corresponding author. Tel.: +82 42 821 5774; fax: +82 42 825 9754.
E-mail address: cheorun@cnu.ac.kr (C. Jo).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.05.030
dietary fat sources, such as natural and fish oil containing PUFAs
(Gonzalez-Esquerra & Leeson, 2000; Lawlor, Gaudette, Dickson, &
House, 2010). However, increased content of PUFAs in egg may result in oxidation since they are the primary targets of free radicals
prior to initiation of peroxidation (Scislowski, Bauchart, Gruffat,
Laplaud, & Durand, 2005). The oxidation products of PUFAs lead
to deterioration of food quality, such as flavour, colour, texture,
and nutritional value (Mielnik, Olsen, Vogt, Adeline, & Skrede,
2006). While lipid peroxidation of fresh shell egg is not readily generated, it is facilitated when eggs are preserved and cooked. Therefore, antioxidants of a kind of those natural polyphenols and
tocopherols have been used as dietary supplementation (Gladinea,
Morandb, Rockb, Baucharta, & Duranda, 2007; Meluzzi, Sirri,
Manfreda, Tallarico, & Franchini, 2000).
Octadeca-9,12-dienyl-3,4,5-hydroxybenzoate (GA–LA) is synthesized from gallic acid (GA) and linoleic acid (LA; C18:2n-6) (Jo,
Jeong, Lee, Kim, & Byun, 2006). GA is a representative natural
polyphenol found in wine, grapes, and tea (Hogan, Zhang, Li,
Zoecklein, & Zhou, 2009; Kim et al., 2006). As a metabolite of
propyl gallate, GA is known to possess several pharmacological
and biological activities, including anti-oxidative, anti-carcinogenic, anti-mutagenic, anti-allergic, and anti-inflammatory
activities (Jo et al., 2006). LA is an essential fatty acid as well as
the primary precursor of all n-6 PUFAs (Russo, 2009). It is
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octadeca-9,12-dienyl-3,4,5-hydroxybenzoate (GA–LA, compound
4), the sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate (NGL) was synthesized.
A solution of sodium methoxide (1.3 mM) and compound 4
(3 mM) was mixed in with methanol (10 ml) at room temperature.
After stirring the solution at room temperature for 4 h, the solvent
was removed under reduced pressure. The crude compound was
purified by column chromatography (SiO2; elution with hexane).
The final product was obtained as dark green powder with 97%
yield; 1H NMR (400 MHz, CDCl3) d 0.9 (t, J = 6.9 Hz, 3H, H18),
1.29 (m, 16H, H3–7, H15–17), 1.73 (m, 2H, H2), 2.05 (m, 4H, H8,
H14), 2.78 (dd, J = 6.1, 6.1 Hz 2H, H11), 4.26 (t, J = 6.3 Hz, 2H, –
OCH2), 5.35 (m, 4H), 5.8 (s, 2H, –OH), 7.26 (s, 1H, aromatic), 7.27
(s, 1H, aromatic); IR (CHCl3) 3570, 3357, 3013, 2918, 2865, 1691,
1615, 1465, 1390, 1242, 1198 cm1.
The estimated water solubilities of NGL were 82.0% and 62.4% at
1000 and 10,000 ppm, respectively.
converted to arachidonic acid (AA; C20:4n-6) in animal tissues and
has been shown to have anti-inflammatory effects by decreasing
the secretion of interleukin (IL)-6 and -1b as well as that of tumour
necrosis factor a (Zhao et al., 2005). Jo et al. (2006) reported that
synthesized GA–LA possesses greater biological function than GA,
including antioxidative activity by free radical scavenging ability,
skin whitening by tyrosinase inhibition which is more powerful
than ascorbic acid, and anti-inflammatory effects by cyclooxygenase 1 and 2 inhibition. Jang et al. (2008) also reported that GA–LA
showed a hypolipidemic activity in mice induced by high-fat diet.
Jang et al. (2009) indicated that GA–LA had a strong and synergistic
inhibitory effect on cancer cell proliferation (in vitro), and had higher enzyme inhibition ability, such as tyrosinase, hyaluronidase, and
xanthine oxidase when compared with those of tocopherol. Furthermore, synthesized GA–LA as well as a mixture of GA and LA
(MGL) decreases serum triglycerides as well as the total serum cholesterol levels (in vivo) (Jang et al., 2008, 2009). Further, a previous
study showed that dietary supplementation with MGL improved
the antioxidative potential as well as the ratio of unsaturated fatty
acids to saturated fatty acids in broiler meat (Jung et al., 2010).
To be used more conveniently as a functional material, high solubility and bioavailability are key properties in pharmaceutical
compounds (Serajuddin, 2007). Therefore, in the present study,
the salt form of GA–LA was developed (NGL), and the effect of dietary supplementation with NGL or MGL was investigated on hen
performance and egg quality attributes.
2.3. Animals and experimental design
A total of 120 laying hens (18 wk old, Led Lohman) were obtained from a commercial flock. Laying hens of similar body weight
(1.7 ± 0.1 kg) were randomly distributed into five groups (12 hens/
treatment, 2 hens/cage). They were reared in accordance with university guidelines for animal experimentation and were maintained under standard conditions of temperature, humidity, and
ventilation with 16 h of fluorescent lighting for the entire experimental period. Birds were fed a commercial diet until 90% egg production was obtained. Each treatment consisted of six replicates
containing four birds each. During the 4 week experimental period,
birds were fed the following diets: (1) control [commercial diet
(CD)], (2) 0.05% MGL (w/w, GA:LA = 1:1, equal molar ratio), (3)
0.1% MGL, (4) 0.05% NGL, (5) 0.1% NGL. The control diet was a typical commercial diet consisting of approximately 15.2% crude protein, 4.5% crude fat, 3.1% crude fibre, and 2700 ME kcal/kg
(Chunhajeil Feed Co., Daejeon, Korea). The treatment diets were
based on the commercial diet supplemented with 0.05% MGL,
0.1% MGL, 0.05% GA–LA, or 0.1% GA–LA. Clean drinking water
was supplied ad libitum. The experimental diets were mixed every
3 days, at which point the feed intake was calculated. The eggs
were gathered daily and weighed individually.
2. Materials and methods
2.1. Chemicals
Linoleic acid ethyl ester, gallic acid, diisobutylaluminum hydride (DIBAL-H), tetrahydrofuran (THF), anisole, 2-naphthalene
sulphonic acid, sodium methoxide, 2-thiobarbituric acid (TBA),
and trichloroacetic acid (TCA) were purchased from Sigma–Aldrich
Co. (St. Louis, MO, USA).
2.2. Synthesis
The synthesis was performed by the modified method of Jo et al.
(2006), as described by the following steps (Fig. 1). From
step 1.
O
i)
OCH 2CH3
OH
1
2
O
step 2.
O
HO
HO
OH
O
ii)
+
2
HO
HO
OH
OH
3
4
O
step 3.
HO
iii)
4
+
O
Sodium Methoxide
HO
O
Na
5
sodium2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate
Fig. 1. Scheme for sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate. 1, linoleic acid ethyl ester; 2, octadeca-9,12-diene-ol; 3, gallic acid; 4,
octadeca-9,12-dienyl-3,4,5-trimethoxybenzoate; 5, sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate. Reagents and reaction conditions: (i)
diisobutylaluminum hydride (2–3 M eq. of compound 1) CHCl2 at 78 °C; (ii) 2-naphthalene sulphonic acid (0.05 M eq.), anisole at 150 °C for 17 h; (iii) sodium methoxide
(0.43 M eq.), methanol at room temperature for 4 h.
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2.4. Laying hens performance and egg quality
Daily feed intake was recorded, and the feed conversion ratio
(FCR, feed intake:mass production) was calculated during the
4 week feeding periods. Daily egg production and individual egg
weights were recorded, and the egg mass production (g/d/hen)
was also determined. Whole eggs from each group of laying hens
were collected at the 27 and 28 day feeding periods in order to
measure egg quality, such as yolk colour and Haugh units (HU),
during storage for 14 days at room temperature. The yolk colour
and HU were determined using the GCM+ System (Technical Services and Supplies, York, England).
2.5. Blood collection and analysis
At the end of the experiment, a hen from each cage was randomly selected. Blood was collected via wing veinpuncture using
a syringe (Greenject-5, Doowon MediTec, Co., Gimje, Korea), placed
into vacuum tubes containing ethylenediaminetetraacetic acid,
and stored at 4 °C. Samples were centrifuged (Union 32R, Hanil
Co., Ltd., Inchun, Korea) at 1500g for 15 min after which the serum
were separated. The separated serum was used to measure the
contents of glutamic oxaloacetic transaminase (GOT), glutamic
pyruvic transaminase (GPT), total cholesterol (T. chol), triglyceride
(TG), high-density lipoprotein cholesterol (HDL-C), and lowdensity lipoprotein cholesterol (LDL-C).
2.6. 2-Thiobarbituric acid-reactive substances (TBARS) value
After measurement of the egg quality, the egg yolks were separated and pooled for analysis of TBARS, 2,2-azinobis-(3 ethylbenzothiazoline-6-sulphonic acid) (ABTS+) reducing activity, fatty acid
composition, and cholesterol content.
The egg yolk sample (5 g) in 15 ml of distilled water was
homogenised (T25b, Ika Works (Asia), Sdn, Bhd, Malaysia) at
1130g for 1 min. The sample homogenate (1 ml) was transferred
to a test tube, and the lipid oxidation was determined as the TBARS
value by following the method described by Liu et al. (2009a).
Briefly, 50 ll of butylated hydroxyanisol (7.2%) and 2 ml of TBATCA solution (20 mM TBA in 15% TCA) were added to the test tube.
Tubes were then heated (90 °C) in a boiling water bath for 30 min,
cooled, and then centrifuged at 2090g for 15 min. Absorbance of
the supernatant was measured at 532 nm with a spectrophotometer (DU530, Beckman Instruments Inc., Fullerton, CA, USA). The
TBARS value was reported as lg of malondialdehyde per g of egg
yolk.
2.7. 2,2-Azinobis-(3 ethylbenzothiazoline-6-sulphonic acid) (ABTS+)
reducing activity
Egg albumen and yolk (3 g) were homogenised (T25b, Ika
Works) in 15 ml of distilled water at 1130g for 1 min, respectively.
The aqueous supernatant was separated by centrifugation (Union
32R, Hanil Co., Ltd., Inchun, Korea) at 2090g for 15 min. The ABTS+
reducing activity was determined by the method described by Erel
(2004). ABTS was dissolved in distilled water to a concentration of
7 mM. The ABTS radical cation was produced by mixing the ABTS
stock solution with 2.45 mM potassium persulfate (final concentration) in the dark at room temperature for 12–16 h, in order to
initiate the radical generation. This solution was then diluted with
ethanol so that its absorbance was adjusted to 0.70 ± 0.02 at
734 nm. The diluted ABTS+ solution (3 ml) was added to 20 ll of
aqueous supernatant, after which the absorbance was measured
using a spectrophotometer (Beckman) at 734 nm, with ethanol as
a blank. The percentage inhibition was calculated by the following
equation:
ABTSþ reducing activity ð%Þ ¼ ½ðabsorbance of control
absorbance of sampleÞ=
absorbance of control 100:
2.8. Fatty acid composition
The total lipid of the samples was extracted using chloroform–
methanol (2:1, v/v) according to the procedure of Folch, Lees, and
Sloane Stanley (1957). Fatty acid methyl esters were prepared from
the extracted lipids with BF3-methanol (Sigma–Aldrich), followed
by separation on a gas chromatograph (HP-6890N, Palo Alto, CA,
USA). A split inlet (split ratio, 50:1) was used to inject the samples
into a capillary column (Omegawax 320, Supelco, Bellefonte, PA,
USA; 30 m 0.25 mm 0.25 lm), and ramped oven temperature
was used (150 °C for 3 min, increased to 180 °C at 2.5 °C/min and
maintained for 5 min, then increased to 220 °C at 2.5 °C/min and
maintained for 25 min). The inlet temperature was 210 °C. Air
served as the carrier gas at a constant flow rate of 0.7 ml/min.
2.9. Cholesterol content
Egg yolks (1 g) were saponified with 20 ml of 33% ethanolic
KOH in tightly-capped tubes placed in a 60 °C water bath for 1 h.
The mixture was then cooled in ice water, and 5 ml of distilled
water was added. Cholesterol in unsaponifiable fractions was extracted twice with 5 ml of hexane. The resulting aliquot of hexane
containing cholesterol was dried under nitrogen, redissolved in
5 ml of hexane, and injected into a gas chromatograph (HP6890 N, Palo Alto, CA, USA). 5a-cholestane (Sigma–Aldrich) was
used as an internal standard. A split inlet (split ratio, 100:1) was
used to inject samples into a capillary column (HP-5, Agilent, Steven, CA, USA; 30 m 0.53 mm 0.5 lm), and the ramped oven
temperature was 270 °C isothermal, detector temperature was
300 °C, and inlet temperature was 210 °C. N2 served as the carrier
gas at a constant flow rate of 1.0 ml/min.
2.10. Statistical analysis
The experiment was conducted under the completely randomised design. Analysis of variance was performed using the raw data,
and the mean values and standard errors of the means (SEM) were
calculated by the Statistical Analysis System (SAS, 2000). Differences amongst the means were determined by the Duncan’s multiple range test with a significance defined at p < 0.05.
3. Results
3.1. Blood profile of laying hen
The levels of serum GOT (also known as aspartate aminotransferase, AST) and GPT (known as alanine aminotransferase, ALT) in
the hens fed MGL or NGL were not significantly different from that
of the hens fed control diet. Concentrations of T. col, TG, HDL-C,
and LDL-C in serum were not affected by the diet supplemented
with MGL or NGL (Table 1).
3.2. Hen performance and egg quality
The treatments did not influence egg production, egg weight,
egg mass, or feed intake of hens, whereas FCR was significantly
lower (p < 0.05) in the hens fed a diet supplemented with 0.1%
MGL compared to that of the other treatments during the 28 day
feeding periods (Table 2).
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S. Jung et al. / Food Chemistry 129 (2011) 822–829
Table 1
Effect of dietary supplementation of gallic acid and linoleic acid mixture (MGL), and their synthetic salt (NGL)a on blood profile of laying hens over a 28 day period.
Treatment
GOTc (U/l)
Control
MGL 0.05%
MGL 0.1%
NGL 0.05%
NGL 0.1%
SEMb
x,y
GPT (U/l)
x,y
191
179y
206x,y
187y
211x
6.8
2.3
2.0y
3.3x
3.3x
2.6x,y
0.30
T. chol (mg/dl)
TG (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
48
64
64
46
51
6.2
310
400
368
234
228
62.0
31
35
33
28
33
2.8
16
15
15
10
11
2.7
a
Sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate.
Standard error of means (n = 30).
Glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), total cholesterol (T. chol), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C),
low-density lipoprotein cholesterol (LDL-C).
x,y
Different letters within the same column differ significantly (p < 0.05).
b
c
Table 2
Effect of dietary supplementation of gallic acid and linoleic acid mixture (MGL), and their synthetic salt (NGL)a on egg production, egg weight, egg mass, feed intake, and feed
efficiency of laying hens over a 28 day period.b
Treatment
Egg production (%)
Egg weight (g)
Egg massc (g/d/hen)
Feed intake (g/d/hen)
Feed conversion efficiencyd
Control
MGL 0.05%
MGL 0.1%
NGL 0.05%
NGL 0.1%
SEMe
97.64
97.82
97.97
98.59
97.97
0.668
57.63
57.11
58.54
58.10
57.86
0.551
56.29
55.86
57.35
57.28
56.68
0.691
123.11
118.96
117.00
120.15
121.46
2.164
2.19x
2.13x,y
2.04y
2.09x,y
2.14x,y
0.692
a
Sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate.
All measurements were done on a fresh basis; values are means for six repetitions (four laying hens per repetition).
Egg mass = (egg production egg weight)/100.
d
Feed conversion efficiency = feed intake/egg mass (g:g).
e
Standard error of the means (n = 30).
x,y
Different letters within the same column differ significantly (p < 0.05).
b
c
The general results of egg quality are shown in Table 3. The results show significant differences (p < 0.05) in the Haugh unit. Specifically, the Haugh units of the eggs from the hens treated with
0.1% MGL, 0.05 or 0.1% NGL were higher than that from the hens
fed a control diet after storage for 7 days, and that from the hens
fed 0.05% and 0.1% NGL were higher compared to the control diet
after storage for 14 days. Although the changes in yolk colour after
treatment showed no regular trend, the yolk colour values of the
eggs were significantly reduced (p < 0.05) in the eggs from hens
fed 0.1% MGL and 0.1% NGL (8.83 and 8.58 after storage for
Table 3
Effect of dietary supplementation of gallic acid and linoleic acid mixture (MGL), and
their synthetic salt (NGL)a on egg quality during storage.
Treatment
Haugh unit
Control
MGL 0.05%
MGL 0.1%
NGL 0.05%
NGL 0.1%
SEMa
Yolk colour (%)
Control
MGL 0.05%
MGL 0.1%
NGL 0.05%
NGL 0.1%
SEMb
a
b
c
d–f
g–i
3.3. ABTS+ reducing activity
The ABTS+ reducing activity of egg albumen and yolk are shown
in Fig. 2. The albumen of eggs from hens fed 0.1% MGL (42.4%), and
0.05 (49.9%) or 0.1% NGL (48.0%) had significantly higher ABTS+
reducing activity (p < 0.05) compared to the control (38.6%) after
28 days of feeding. Further, the ABTS+ reducing activity of egg yolk
from the hens fed 0.05% (20.8%) and 0.1% NGL (19.2%) was significantly higher than that of the control (8.1%).
SEMc
Storage (day)
0
7
14
99.3d
100.7a
101.6a
103.8a
103.1a
1.64
65.7e,y
66.7e,h
75.1e,g
81.3e,g
80.2e,g
2.37
57.5f,h
61.5e,g,h
60.1f,g,h
64.3f,g
63.2f,g
1.67
9.25d,g
9.08d,g,h
8.83d,h,i
8.92d,g–i
8.58d,i
0.115
0 day), 0.1% NGL (7.33 after storage for 7 days), and 0.05% NGL
(7.25 after storage for 14 days) compared to that of the control
(9.25, 7.75, and 8.00, respectively).
7.75e,g,h
8.08e,g
7.58e,g,h
7.67e,g,h
7.33f,h
0.163
8.00e,g
7.92e,g
7.75e,g,h
7.25e,h
8.00e,g
0.180
1.62
2.12
2.01
1.69
2.12
0.147
0.131
0.149
0.187
0.156
Sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate.
Standard error of the means (n = 60).
(n = 36).
Different letters within the same row differ significantly (p < 0.05).
Different letters within the same column differ significantly (p < 0.05).
Fig. 2. ABTS+ reducing activity (%) of egg albumen and yolk from laying hens fed a
dietary gallic acid and linoleic acid mixture (MGL), and sodium 2,3-dihydroxy-5(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate (NGL). a–cDifferent letters
within albumen differ significantly (p < 0.05). x–yDifferent letters within yolk differ
significantly (p < 0.05).
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3.4. 2-Thiobarbituric acid-reactive substances (TBARS) of egg yolk
Dietary supplementation with both MGL and NGL inhibited the
development of lipid oxidation in egg yolk (Table 4). The TBARS
values of egg yolk from hens fed 0.05% and 0.1% MGL as well as
0.05% and 0.1% NGL showed significantly lower values (p < 0.05)
than those of the control after storage for 7 days at room temperature. However, only the egg yolk from the hens fed 0.1% MGL and
0.05% NGL differed from that of control after 14 days of storage.
3.5. Fatty acid composition and cholesterol content of egg yolk
There were significant differences in the fatty acid composition
amongst the treatments (Table 5, p < 0.05). Dietary supplementation of MGL and NGL decreased the contents of palmitic and palmitoleic acid composition of egg yolk (p < 0.05). The content of
stearic acid in the egg yolk from the hens fed 0.1% MGL (10.58%),
and 0.05% or 0.1% NGL (10.62% and 11.17%, respectively) were
higher than that fed the control diet (10.21%) (p < 0.05). The dietary
supplementation with 0.1% MGL and 0.1% NGL increased the content of oleic acid in egg yolk compared to the control diet
(p < 0.05). The linoleic acid content of egg yolk was increased by
0.05% MGL (15.93%) and 0.05% NGL (16.02), but 0.1% MGL
(14.97%) and 0.1% NGL (14.96%) decreased the linoleic acid content
of egg yolk when compared to the control diet (15.24%) (p < 0.05).
The dietary supplementation with 0.1% MGL decreased the
linolenic acid content of egg yolk, however, 0.05% NGL increased
the linolenic acid content of egg yolk compared to other treatments
(p < 0.05). Arachidonic and docosahexaenoic acids were not influenced by the dietary treatments. Regarding changes in the fatty
acid composition, the ratio of unsaturated:saturated fatty acid of
egg yolk was higher in the samples fed 0.05% or 0.1% MGL (1.68%
and 1.67, respectively) and 0.05% NGL (1.69) than the control
(1.62) (p < 0.05). The cholesterol contents of egg yolk were affected
by dietary supplementation with MGL or NGL (Fig. 3). Specifically,
cholesterol content of egg yolk from hens fed control diet was
17.69 mg/g, whereas that of egg yolk from hens fed 0.1% MGL,
and 0.05% or 0.1% NGL were 15.4, 14.6, and 15.5 mg/g of egg yolk,
respectively, which were significantly lower than that of the control diet (p < 0.05).
4. Discussion
The water solubility of a functional material is vital for its wider
application. Sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12dienyloxy)carbonyl)phenolate (NGL), which is the salt form of
octadeca-9,12-dienyl-3,4,5-hydroxybenzoate (GA–LA), is water
soluble even though GA–LA is an insoluble material.
It is important to determine the effects of feed additives on the
biochemistry of the animals when new materials are added to
feedstuffs. Serum GOT and GPT activities can be used as biochemical indicators for liver function; an increase in these enzyme activities indicates hepatocellular damage (Han, Huang, Li, Jiang, & Xu,
2008). In this work, serum GOT and GPT activities were not affected by dietary supplementation with either MGL or NGL. This
result implies that dietary MGL and NGL may not have a negative
Table 4
2-Thiobarbituric acid-reactive substances (TBARS) of egg yolk from laying hens fed a
dietary gallic acid and linoleic acid mixture (MGL), and their synthetic salt (NGL)a
during storage.
Treatment
Control
MGL 0.05%
MGL 0.1%
NGL 0.05%
NGL 0.1%
SEMb
a
b
c
d–f
g–i
SEMc
Storage (day)
0
7
14
0.23e
0.23f
0.26e
0.23e
0.21f
0.016
0.49d,g
0.41e,h
0.42d,h
0.39d,h,i
0.33e,i
0.019
0.51d,g
0.48d,g,h
0.45d,h
0.46d,h
0.47d,g,h
0.014
0.010
0.021
0.018
0.021
0.009
Sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate.
Standard error of the means (n = 15).
(n = 9).
Different letters within the same row differ significantly (p < 0.05).
Different letters within the same column differ significantly (p < 0.05).
Fig. 3. Cholesterol content of egg yolk from laying hens fed a dietary gallic acid and
linoleic acid mixture (MGL), and sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12dienyloxy)carbonyl)phenolate (NGL). a,bDifferent letters within albumen differ
significantly (p < 0.05).
Table 5
Fatty acid composition of egg yolk from laying hens fed a dietary gallic acid and linoleic acid mixture (MGL), and their synthetic salt (NGL).a
Fatty acid
a
b
c–e
SEMb
Treatment
Control
MGL 0.05%
MGL 0.1%
NGL 0.05%
NGL 0.1%
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
C20:4
C22:6
27.90c
3.52c
10.21e
37.98e
15.24d
0.25d
3.56
1.34
27.28d
3.01d
10.04e
38.72d,e
15.93c
0.27d
3.36
1.38
26.89d
2.94d
10.58d
39.61c
14.97e
0.19e
3.48
1.32
26.58f
2.94d
10.62d
38.58d,e
16.02c
0.33c
3.51
1.41
26.67e,f
2.90d
11.17c
39.11c,d
14.96e
0.27d
3.57
1.33
0.073
0.043
0.116
0.245
0.065
0.013
0.069
0.034
Saturated
Monounsaturated
Polyunsaturated
Unsaturated: saturated
38.11c
41.50d
20.39d
1.62e
37.33d,e
41.74c,d
20.94c
1.68c,d
37.48d,e
42.56c
19.97e
1.67c,d
37.20e
41.52d
21.28c
1.69c
37.84c,d
42.02c,d
20.14d,e
1.64d,e
0.177
0.258
0.121
0.013
Sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate.
Standard error of the means (n = 15).
Different letters within the same row differ significantly (p < 0.05).
Author's personal copy
S. Jung et al. / Food Chemistry 129 (2011) 822–829
effect on the biochemical function of laying hens. Dietary MGL and
NGL did not significantly impact the concentrations of serum T.
chol, TG, HDL-C, or LDL-C in the hens. These results are in disagreement with a previous study that showed that dietary GA–LA and
the mixed form of GA and LA (MGL) reduced the levels of serum
TG (22% and 26%) and LDL-C (17% and 26%) in mice fed a high
fat diet supplemented with 1.0% GA–LA and 1.0% MGL (Jang
et al., 2008). Additionally, the serum TG level decreased by 56%
when dietary 1.0% MGL was fed to the broilers (Jung et al., 2010).
The FCR of hens was improved by dietary supplementation with
0.1% MGL compared to that of control, whereas egg production, egg
weight, egg mass, and feed intake were not influenced by either
MGL or NGL diets. FCR is generally related to the energy concentration of the diet. In addition, the amount of antioxidants in the
diet also affects the FCR by preventing the decrease in utilisation
of nutrients caused by environmental stress (Frikha, Safaa, Serrano,
Arbe, & Mateos, 2009; Sahin et al., 2006). A diet containing MGL
has linoleic acid, which may raise the energy content. However, Safaa et al. (2008) reported that increasing the linoleic acid concentration of a diet from 1.12% to 1.60% had no effect on the FCR.
However, the level of dietary linoleic acid in the present study
was lower than that of the previous study. Therefore, this improved FCR was not due to the addition of linoleic acid but instead
from the antioxidative activity of gallic acid in the MGL diet.
The best index of internal egg quality is the Haugh unit (HU),
which is calculated by measuring the height of the inner thick
albumen and the egg weight. A reduced HU indicates deterioration
of egg freshness. In the present study, the experimental diets did
not affect the HU of eggs on the day of lay, but differences in HU
were observed in the eggs from hens fed 0.1% MGL or both 0.05
and 0.1% NGL compared to the eggs from hens fed control diet. This
result is consistent with previous studies. The HU on the day of lay
was not affected by diet supplemented with ‘tallow, sunflower oil,
or flaxseed oil as the fatty acid source, or ‘lycopene or vitamin E as
the antioxidant’ (Celebi & Macit, 2008; Sahin et al., 2006). Hayat,
Cherian, Pasha, Khattak, and Jabbar (2009) reported that the albumen height and HU of the egg were affected by a diet supplemented with flax and antioxidants. However, a reduction in HU
in egg during storage was delayed by feeding with organic
selenium as an antioxidant (Pappas, Acamovic, Sparks, Surai, &
McDevitt, 2005). A reduction in HU in egg caused by storage has already been described by many researchers (Rovinson & Monsey,
1972; Silversides & Scott, 2001). The increased pH of albumen
due to the loss of carbon dioxide causes the albumen structure
(ovomusin-lysozyme complex) to break (Silversides & Scott,
2001). Further, Liu et al. (2009b) informed that oxidation of proteins in egg upon irradiation stimulates the denaturation and scission of egg white proteins, resulting in a severe decrease in HU. In
this work, the albumen of the eggs from hens fed 0.1% MGL or both
0.05% and 0.1% NGL possessed higher antioxidative activity (ABTS+
reducing activity) than that of eggs from hens fed control diet. Gallic acid contained in the MGL and NGL diets is a natural polyphenol
(Kim et al., 2006), which have been shown to bind free radicals that
play a key role in oxidation and leads to their inactivation (Hogan
et al., 2009). A previous study reported that dietary phenolic
compounds, such as oregano, rosemary, and grape pomace could
improve the antioxidative activities of lamb and broiler meat
(Goñi et al., 2007; Simitzis et al., 2008). The results of the present
study show that the delayed reduction of HU during storage may
hinder protein oxidation by improving the antioxidative activity
of egg albumen from hens fed MGL or NGL diets.
The ABTS+ reducing activity of egg yolk was different amongst
the treatments, and it reflected the level of lipid oxidation of egg
yolk in this study. Lipid oxidation of egg yolk was increased by
dietary PUFAs source and was inhibited around 55% based on the
TBARS value by dietary supplementation of 0.08% vitamin E as an
827
antioxidant (Qi & Sim, 1998). Shahryar, Salamatdoust,
Chekani-Azar, Ahadi, and Vahdatpoor (2010) reported that the
TBARS values of stored shell eggs with enriched PUFAs were alleviated around 24% and 33% after refrigerated storage for 30 and
60 days, respectively, by addition of 0.12% vitamin E in the diet.
These results are similar to the present study, which found that
dietary supplementation of 0.1% MGL and 0.1% NGL inhibited the
increase of the TBARS value (approximately 22% and 33%, respectively) in egg yolk after storage for 7 days, and that the increase
of the TBARS values at 14 days was suppressed by the dietary supplementations of 0.1% MGL (12%) and 0.05% NGL (10%). Galobart,
Barroeta, Baucells, and Guardiola (2001) found that although the
TBARS values were not significantly different, the amount of lipid
hydroperoxide (primary oxidation products) in fresh eggs
increased upon treatment with sunflower oil compared to linseed
oil since the level of natural antioxidants in dietary oil was higher
than in linseed oil.
The modification of the fatty acid composition of egg yolk has
been attempted ever since PUFAs have been known as a beneficial
substance for human health. Celebi and Macit (2008) reported that
the content of n-3 and n-6 PUFAs in egg yolk increased by dietary
fat sources of n-3 and n-6 PUFAs, respectively. The conjugated linoleic acid in egg yolk is increased by a dietary source of conjugated
linoleic acid (Raes et al., 2002). In addition, Güçlü, Uyanik, and
_ßcan (2008) showed that the diet including 4% of n-6 sources, such
Is
as sunflower oil, maize oil, sesame oil, and cottonseed oil, and 4% of
n-3 source, such as fish oil, raised the contents of linoleic and arachidonic acid and docosahexaenoic acid in egg yolk, respectively.
These studies agree that the fatty acid composition of egg yolk is
reflected in the fatty acid composition of the diet. However, the results of the present study on the fatty acid composition of egg yolk
did not agree with previous studies. Although the linoleic acid concentrations were higher in egg yolk from hens fed 0.05% MGL and
0.05% NGL, those of egg yolk from hens fed 0.1% MGL and NGL were
lower compared to the control (p < 0.05). This may be due to the
effect of gallic acid in the MGL and NGL diets. Lipids from egg yolk
are derived and synthesized in the hen liver through the D9- and
D6-desaturase pathway, and the activity of D9- and D6-desaturase
is affected by dietary antioxidants (Ozkan, Yllmaz, Ozturk, & Ersan,
2005). Hayat et al. (2009) reported that dietary antioxidants, such
as vitamin E and butylated hydroxyl toluene alter the contents of
saturated, monounsaturated, and n-6 and n-3 polyunsaturated
fatty acids in egg.
The dietary supplementation of 0.1% MGL and NGL increased
the stearic acid content of egg yolk. This result concurred with
Celebi and Macit (2008) and Jiang, Ahn, and Sim (1991), who
confirmed that high levels of linoleic acid in sunflower increased
the stearic acid composition of egg yolk. Nonetheless, the effect
of dietary MGL and NGL on the fatty acid composition of egg yolk
remains unclear due to the observation that dietary 0.1% MGL and
0.1% NGL increased the oleic acid content of egg yolk when
compared with that of the control.
Although cholesterol is essential for the development of the embryo, many researchers have tried to decrease the amount of cholesterol in egg yolk due to its risk for cardiovascular disease in
humans (Viveros, Centeno, Arija, & Brenes, 2007). Naber and
Biggert (1989) reported the cholesterol-lowering effect of PUFAs,
which affect the activities of enzymes related to lipogenesis,
whereas González-Muñoz et al. (2009) observed that the cholesterol levels of egg were higher from the hens fed saturated fat
sources than from the hens fed unsaturated fat sources. The cholesterol levels of egg yolk were decreased by dietary 0.1% MGL as well
as 0.05% and 0.1% NGL compared to that of control, which is in
agreement with previous results. Kang, Kim, Park, and Jang
(2006) reported that 5% corn oil, composed of high linoleic acid
(57%), decreased the cholesterol content of egg yolk. However,
Author's personal copy
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S. Jung et al. / Food Chemistry 129 (2011) 822–829
the dietary supplementation of sunflower oil with high linoleic
acid (58%) did not influence the cholesterol content of egg yolk
(Kücükersan, Yesßilbağ, & Kücükersan, 2010). Azeke and Ekpo
(2009) suggested that natural polyphenols, catechins, and flavonols
have a cholesterol lowering effect on egg by scavenging reactive
oxygen species and chelating metal ions. The dietary supplementation of MGL and NGL may result in a synergistic effect of gallic acid
and linoleic acid in decreasing the cholesterol content of egg yolk.
5. Conclusions
NGL, a synthetic salt of gallic acid and linoleic acid, showed lower sensory changes and greater improvement in antioxidative potential and cholesterol-lowering activity compared to MGL.
Improving the antioxidative potential of egg and changing the lipid
composition of egg yolk by dietary supplementation of laying hens
with MGL and NGL could be profitable for the industry and advantageous for human nutrition.
Acknowledgement
This work was supported by a grant from the Next-Generation
BioGreen 21 Program (Project Number PJ0081330), Rural Development Administration, Republic of Korea.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.foodchem.2011.05.030.
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