Meat Science 122 (2016) 715

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Meat Science
journal homepage: www.elsevier.com/locate/meatsci

Effects of dietary natural and fermented herb combination on growth

performance, carcass traits and meat quality in grower-nisher pigs
Sonia Tabasum Ahmed, Hong-Seok Mun, Md. Manirul Islam, Seok-Young Ko, Chul-Ju Yang
Department of Animal Science and Technology, Sunchon National University, Suncheon, Jeonnam 540950, Republic of Korea

a r t i c l e

i n f o

Article history:
Received 21 August 2015
Received in revised form 15 June 2016
Accepted 20 July 2016
Available online 22 July 2016
Keywords:
Fermented herb combination
Meat composition
Fatty acid prole
Oxidative stability
Grower-nisher pigs

a b s t r a c t
The effects of an herb combination (pomegranate, Ginkgo biloba, licorice) in natural (NPGL) or fermented (FPGL)
form administered as 0.4% of the basal diet on the performance and meat quality of grower-nisher pigs were
evaluated. Dietary supplementation with NPGL or FPGL reduced the feed intake and back fat thickness of pigs,
while increasing lean production. Serum IgG was higher in the FPGL supplemented group. Remarkably, ingestion
of NPGL and FPGL reduced the ether extract in the longissimus dorsi muscle (LDM) with increased moisture,
whereas the cholesterol was lower in the NPGL group. Dietary supplementation of NPGL and FPGL increased
the n3 fatty acid in LDM with a reduced ratio of n6/n3. Both NPGL and FPGL signicantly reduced the
TBARS value of pig meat when fresh and after 2 and 3 weeks of storage. Overall, dietary NPGL and FPGL improved
the quality of pig meat by increasing the n3 fatty acid levels while reducing the ether extract and TBARS value.
2016 Published by Elsevier Ltd.

1. Introduction
Many scientic studies have recently been conducted to improve the
composition and nutritional quality of meat. Special attention has been
given to fatty acid composition since it is associated with meat quality
(including shelf life and avor) and of human health concerns (especially saturated fatty acid). Feed additives such as vitamins, minerals and
antioxidants have been reported to improve pork nutritional characteristics and oxidative stability (Nuernberg et al., 2002). However, consumer concerns over safety and toxicity regarding synthetic
antioxidants and other chemical additives in animal feedstuff
(Coronado, trout, Dunsea, & Shah, 2002), motivated current nutritional
studies on examination and development of different natural feed additives that have functional properties. A number of herbs and medicinal
plant by-products have received attention from animal scientists as
feed additives for livestock, because of their functional components
and functional activities (Zhang et al., 2013; Reddy, Gupta, Jacob,
Khan, & Ferreira, 2007). Phytochemical analyses exposed the bioactive
components of pomegranate (Punica granatum L.) peels [source of the
polyphenols and avonoids], leaves of Ginkgo biloba L. [source of avonoids, polysaccharides and terpenoids], and licorice (Glycyrrhiza glabra
L.) root [source of saponins, triterpenes (glycyrrhizin) and avonoids
(liquiritin, isoavonoids)] along with their antioxidant, immunomodulatory, cholesterol lowering and anti-inammatory properties (Rajan
et al., 2011; Ross, Selvasubramanian, & Jayasundar, 2001; Cao, Zhang,
Corresponding author.
E-mail address: yangcj@scnu.kr (C.-J. Yang).

http://dx.doi.org/10.1016/j.meatsci.2016.07.016
0309-1740/ 2016 Published by Elsevier Ltd.

Yu, Zhao, & Wang, 2009; Zhou, Wang, Ye, Chen, & Tao, 2015; Fukai et
al., 1998; Katamaya et al., 2011; Asan-Ozusaglam & Karakoca, 2014).
Several scientic studies have been conducted to evaluate the dietary
effects of pomegranate, Ginkgo biloba and licorice alone on broilers,
pigs and cattle, especially on growth performance and immunity; however, few of these studies have investigated its effects on meat quality
(Cao, Zhang, Yu, Zhao, & Wang, 2012; Katamaya et al., 2011; Shabtay
et al., 2008). Moreover, the combined effects of these herbs on growth
performance and meat quality have not yet been studied. Combination
of these herbs are expected to exert their benecial effects through their
combined chemical and pharmaceutical properties.
An alternative approach to sub-therapeutic antibiotics is use of benecial microorganisms that are capable of modifying gastrointestinal
microbial ecosystems and improving the growth performance of pigs
(Dierck, 1989). Recently, fermentation of plant materials with different
benecial microorganisms such as Lactobacillus spp., Saccharomyces
cerevisiae, and Bacillus spp. has been widely adopted to develop novel
functional feed additives for livestock. It is believed that the process of
fermentation promotes functional activities such as antioxidant and antimicrobial activity (Lee, Yang, & Mau, 2008; Cao et al., 2012) and increases the vitamins, enzymes and growth factors of fermented
products (Ng, Wang, Wang, Tzeng, & Shyu, 2011). Fermented feed contains large numbers of Lactobacilli with high concentrations of lactic acid
and other volatile fatty acids and has a low pH. Additionally, fermented
medicinal plants or herbs could be better than medicinal plants or benecial bacteria alone, since animals would benet from the bioactive
components of such plants and the presence of benecial bacteria in
their digestive tract. Several scientists have also reported the benecial

S.T. Ahmed et al. / Meat Science 122 (2016) 715

effects of fermented medicinal plants on growth performance, immune

system and meat quality of broilers and pigs (Kim et al., 2012; Jeong &
Kim, 2015; Zhou et al., 2015).
In this study, we supplemented pig diets with an herb combination
(pomegranate peel extract, Ginkgo biloba L. leaves and licorice root) in
natural (NPGL) or fermented (FPGL) form. We then investigated the effects of NPGL and FPGL on the carcass characteristics, fatty acid composition and oxidative stability of Longissimus dorsi muscle (LDM). We also
investigated the growth performance and serum immunoglobulins in
grower-nisher pigs.
2. Materials and methods
2.1. Preparation and characterization of natural and fermented herb
combination
To prepare the herbal combination, peels of pomegranate
(Goheung-gun cultivar, Korea) were collected, cleaned and rinsed in
distilled water (1:1 ratio) to prepare the liquid extract according to
the method described by Devatkal, Jaiswal, Jha, Bharadwaj, and Viswas
(2013). Green leaves of Ginkgo biloba L. and roots of licorice were
cleaned, air-dried and powdered using a kitchen grinder. The herb combination contained 30% pomegranate peel extract, 4.5% Ginkgo biloba
leaf power and 0.5% licorice root powder that was mixed with 35%
wheat bran and 30% defatted rice bran. After mixing the ingredients,
one part of the combination (used as natural) was dried in an air circulatory tray drier at 60 C for 48 h to reduce the moisture contents. Another portion of the combination was inoculated with 30% (v/w)
Lactobacillus plantarum KCTC 3099 and fermented for 2 days at 37 C
while maintaining 40% moisture in a commercial fermenter (W-1000;
Wonbalhyo Industry Co., Incheon, South Korea). The fermented medium was then again inoculated with 30% (w/v) Saccharomyces cerevisiae
KCTC 7904 and fermented for 3 days at 37 C. The fermented sample
was subsequently dried in a forced air oven (Doori TEC, Doori TEC, FA,
Co., Ltd) at 32 C for 2 days to reduce the moisture levels. Finally, the
natural and fermented herb combination were stored in an air-tight
plastic bag until mixed with basal diet. The microbial concentration of
FPGL was determine after diluting 1 g with 9 mL of double distilled
water (DDW). Approximately 1 h later, 1 mL of the dilution was serially
diluted 10-fold in 0.85% NaCl solution, cultured in agar media and the
number of colonies were counted. Proximate composition of NPGL
and FPGL [crude protein (CP), ether extract (EE), moisture, and total
ash] were analyzed according to the method described by the Association of Ofcial Analytical Chemists (AOAC, 2000). Trace mineral contents of NPGL and FPGL were determined using an Atomic Absorption
Flame Emission Spectrophotometer (Model AA-6200, Shimadzu,
Japan). The fatty acid composition was determined by a direct method
for fatty acid methyl ester (FAME) synthesis using a gas chromatograph.
The pH of natural and fermented herb combination were analyzed using
a digital pH meter (Docu-pH + meter, Sartorius, USA). The type and
concentration of fermentable sugars [quantied by HPLC using external
standards (Supleco, Belafonte, PA)] and organic acids [quantied by Gas
Chromatography (Hewlett Packard HP 6890 GC System, Santa Clara,
CA)], concentration of total polyphenols, tannic acid and avonoids contents (quantied by colorimetric analysis) in NPGL and FPGL were analyzed by a commercial analytical company; the Foundation of
Agricultural Technology Commercialization and Transfer (FACT,
Suwon-si, Gyeonggi-do, Korea). The analytical results are presented in
Tables 1 and 2.
2.2. Animal and experimental design
The experimental protocols and care and management of animals
were carried out in accordance with the guidelines of the Institutional
Animal Care and Use Committee, Sunchon National University. A total
of ninety-six crossbred (Durox Landrace Yorkshire) growing pigs

Table 1
Microbial concentration and nutrient composition of natural and fermented herb
combination.
Itema
Microbial stains in FPB, cfu/g
Lactobacillus plantarum KCTC 3099
Saccharomyces cerevisiae KCTC 7904
Chemical composition, % dry matter
Moisture
Crude protein
Crude fat
Crude ber
Crude ash
Trace minerals, g/kg
Calcium
Iron
Magnesium
Sodium
Fatty acids, g/100 g
Saturated fatty acid
Monounsaturated fatty acid
Polyunsaturated fatty acid
n6 fatty acid
Linoleic acid (C18:2n 6)
Arachidonic acid (C20:4n 6)
n3 fatty acid
Alpha-linolenic acid (C18:3n3)
Eicosapentaenoic acid (C20:5n 3)

NPGL

FPGL

2.1 108
1.0 107

7.75
10.98
2.63
11.90
2.62

19.36
12.08
2.41
9.83
19.39

11.50
0.07
0.89
0.72

13.17
0.06
2.71
1.40

61.78
17.00
21.00
29.53
27.54
0.67
1.12
0.50
ND

46.79
22.43
30.64
17.11
14.16
1.07
3.89
0.69
1.45

ND, not detected.

a
Data are the means of three replicate analysis.

(Barrows, average 39.28 1.04) were randomly allotted to three dietary treatments groups (four replicates with eight pigs per replication)
according to initial body weight for a 10 week experiment. The dietary
treatments were a control (basal diet), 0.4% natural herb combination
(NPGL) with basal diet and 0.4% fermented herb combination (FPGL)
with basal diet. Commercially available corn, wheat and soybean meal
based grower and nisher diets were used as the basal diet, which
contained all nutrients in the levels recommended by NRC (2012). The
additives were added at the expense of equal amount of basal diet in a
two days interval. The nutrient composition of the experimental diets
are shown in Table 3. All pigs were housed in an environmentally controlled, slatted pig house in 12 adjacent pens (3.0 3.0 m) and provided
with ad libitum access to feed and water.
2.3. Measurements and analyses
2.3.1. Growth performance
Individual pig body weights was recorded at the beginning and end
of the experiment to calculate the average daily gain (ADG). The feed

Table 2
Concentrations of fermentable sugars, pH, organic acids, phenolics and avonoids of natural and fermented herb combination.
Itema
Fermentable sugars, %
Glucose
Fructose
Sucrose
Lactose
Maltose
pH
Organic acids, mg/kg
Lactic acid
Acetic acid
Propionic acid
Total polyphenols, mg/kg
Tannic acid, mg/kg
Total avonoids, mg/kg
a

NPGL

FPGL

5.25
5.92
0.58
Not detected
Not detected
4.92

2.61
6.09
0.00
Not detected
Not detected
3.77

316.9
210.5
Not detected
6521.9
1876.2
6301.7

2643.8
544.9
Not detected
6010.1
1193.4
5979.1

Data are the means of three replicate analysis.

S.T. Ahmed et al. / Meat Science 122 (2016) 715

Table 3
Nutrient composition of the experimental grower-nisher diets.
Grower diet

Finisher diet

Items

Control NPGL FPGL Control NPGL FPGL

Calculated composition
Metabolizable energy (kcal/kg)
Digestible lysine (g/kg)
Digestible methionine +

3194
7.21
4.56

3198
7.22
4.56

3201 3196
7.22 7.22
4.56 4.57

3208
7.23
4.56

3204
7.22
4.56

cysteine (g/kg)
Analyzed composition (g/kg dry
matter)
Dry matter
Crude protein
Crude fat
Crude ber
Ash
Calcium
Available phosphorous
Lysine
Methionine

879
180
43
39
79
6.0
5.5
10.1
3.2

876
180
44
35
78
5.9
5.4
10.6
3.0

879
180
44
31
78
6.0
5.5
10.5
3.1

881
161
42
32
76
5.0
4.5
9.1
2.8

876
160
41
34
76
5.1
4.6
9.0
2.8

883
160
41
38
81
5.0
4.5
9.0
2.9

intake of each replicate pen was recorded and the average daily feed intake (ADFI) and gain:feed ratio (G:F) were calculated.
2.3.2. Serum immunoglobulin analysis
At the end of the experiment, blood samples (10 mL) were collected
from three randomly selected pigs from each replicate pen for serum
immunoglobulins quantication. Samples were collected via anterior
vena cava puncture using a 22-gauge sterile needle. Blood samples
were collected into BD Vacutainer tubes (Becton Dickinson, Franklin
Lakes, NJ, USA) containing K3EDTA (tripotassium ethylenediaminetetraacetic acid). The serum was then separated from the blood by centrifugation for 15 min at 1610 g and 4 C, then stored at 20 C until
immunoglobulin analysis was performed. The concentrations of serum
IgG, IgM, and IgA were assayed using Pig IgG (Cat. No. E100-104), IgM
(Cat. No. E100-100), and IgA (Cat. No. E100-102) ELISA Quantitation
Kits (Bethyl Laboratories Inc., Montgomey, TX), respectively, according
to the manufacturer's instructions. Each experiment was run in duplicate, and the results represent the means of three experiments. The absorbance of each well was measured within 30 min using a microplate
autoreader (Thermo Lab Systems, Finland) at 450 nm (correction wavelength, 570 nm). The results were expressed as mg/mL of serum.
2.3.3. Carcass traits
At the end of the experiments, all pigs were transferred to a commercial slaughter house and slaughtered by exsanguination after
being electrically stunned. Feed was withheld for 24 h before
slaughtering and the pigs were laired for 4 h with free access to water
before slaughter. Live weight at slaughter and hot carcass weight
(HCW) were recorded to calculate the dressing percentage. The carcass
quality grade was determined according to the Korea Institute for Animal Products Quality Evaluation (KAPE, 2010). The quality of pork carcasses was graded as A grade = 3, B grade = 2, C grade = 1 and nondescript = 0 based on the marbling, lean color and conditions of belly
streaks. Back fat thickness (measured with an A-mode ultrasound,
Lean-Meater, Renco Corporation, Minneapolis, MN) and loin eye area
(LEA) at the 10th rib were measured. The lean % was calculated by
using the following formula after calculating the pounds of fat free
lean according to Ray (1982):
Lean% pounds of fat free lean  warm carcass weight  100:
The Longissimus dorsi muscles (LDM) of the pig carcasses (three pigs
per replicate) were removed at the last lumbar vertebra, vacuumpacked, and stored after dividing into two parts (one for analysis of
proximate composition and fatty acid composition at 20 C and another for oxidative stability analysis at 4 C).

2.3.4. Chemical composition, cholesterol and trace mineral analysis

The CP (990.03), EE (991.36), moisture (930.15), and total ash
(942.05) contents of LDM samples were analyzed according to the
method described by the AOAC (2000).
Cholesterol from the fat using 5 g of ground meat which was homogenized with 15 mL of DDW, 0.5 mL of 5-cholesterol (reference material) and 200 mL of Folch solution (chloroform and methanol mixture at
2:1 vol:vol). The homogenate was then ltered after which added
with 50 mL of 0.5% NaOH. The samples were then saponied at 85 C
for 60 min through 10 mL of 2 N ethanolic KOH solution. After cooling
at room temperature, 5 mL of DDW was added to each sample. The
non-saponiable fraction was extracted four times using 10 mL of hexane. After drying by 95% nitrogen, the extracts were redissolved in 1 mL
hexane and transferred to small ample for analysis. Separated cholesterol samples were then exposed to chromatographic analysis in a DS 6200
gas chromatograph (Donam Co., Seongnam, Gyeonggi-do, Korea)
equipped with a ame ionization detector and a Hewlett Packard HP5 capillary column (J&W Scientic, USA) 30 m long with a 0.32 mm internal diameter and 0.25 m polyethylene glycol-lm. The chromatographic conditions were as follows: oven temperature: 250 C (held
for 2 min), followed by an increase to 290 C at 15 C/min (held for
10 min) and then to 310 C at 10 C/min (held for 10 min). During analysis, the injector and detector temperature were 280 C, the carrier gas
was nitrogen, the split ratio was 50:1, and the sample volume injected
was 2 L. The cholesterol content was expressed as mg/100 g meat.
Trace mineral contents were determined using an Atomic Absorption Flame Emission Spectrophotometer (Model AA-6200, Shimadzu,
Japan) with stock solutions of calcium (Ca), iron (Fe), magnesium
(Mg) and sodium (Na) containing 1000 mg/L of each element (Merck,
Darmstadt, Germany). Analytical calibration standards were prepared
over 0.52.0 ppm for Ca and Fe and 0.10.4 ppm for Mg and Na by suitable dilution with deionized water. Approximately 2.5 g of sample were
placed in a crucible, dried at 105 C, and then burned in a mufe furnace
at 550 C until it became grayish white, after which the crucible was
allowed to cool in a desiccator. Next, 10 mL of primary reagent (HCl:
DDW = 1:1) was added and evaporated while stirring on a hot plate,
after which 10 mL of secondary solution (HCl: DDW = 1:3) was
added and evaporated. Following evaporation, 100 mL of sample was
ltered through Whatman No. 6 lter paper using deionized water.
The samples were further diluted to ensure that the expected concentration fell within the calibration range. The absorbance levels were
measured by comparison with a calibration curve and the results were
expressed as mg/100 g of meat.
2.3.5. Fatty acid analysis
The fatty acid compositions of LDM were determined by fatty acid
methyl ester (FAME) synthesis according to the method described by
O'Fallon, Busboom, Nelson, and Gaskins (2007). Concisely, 1 g of minced
meat was taken into a Falcon tube (15-mL), after which 0.7 mL of 10 N
KOH in water and 6.3 mL of methanol were added. The tube was then
incubated for 1.5 h in a 55 C water bath with vigorous hand-shaking
for 10 S every 30 min to properly permeate, dissolve and hydrolyze
the sample. After cooling to room temperature, 0.58 mL of 24 N H2SO4
in water was added to the sample. The tube was then mixed by inversion and again incubated in a 55 C water bath for 1.5 h with vigorous
hand-shaking for 10 S every 30 min. After FAME synthesis, the tube
was cooled in a cold water bath. Following, 3 mL of hexane was added
and vortex-mixed for 5 min on a multitube vortexer. The tube was
then centrifuged for 5 min at 3000 g (HANIL, Combi-514R, Korea),
after which the top (hexane) layer containing the FAME was
dehydrated through the anhydrous Na2SO4. The extracted and
dehydrated hexane was concentrated to 1.5 mL and placed into a GC
vial for analysis.
The fatty acid composition of the FAME was determined using a gas
chromatograph (Agilent, 7890 A series, USA) equipped with a ame
ionization detector and a Hewlett Packard HP-88 capillary column

10

S.T. Ahmed et al. / Meat Science 122 (2016) 715

(60 m 0.52 mm 0.20 m). Samples were injected using an auto-sampler (Agilent Technologies 7693, USA). The chromatographic conditions
were as follows: oven temperature, initial 125 C (held for 1 min), increased to 145 C at 10 C/min (held for 26 min), then further increased
to 220 C at 2 C/min (held for 2 min). The carrier gases were puried air
and H2 at 400 mL/min and 40 mL/min ow, respectively. The makeup
gas was helium at 40 mL/min, the injector and detector temperature
were 260 C and the split ratio was 30:1. Fatty acids were identied
by comparison of their retention times to those of a standard FAME mixture (Supelco 37 Component FAME Mix, 10 mg/mL in CH2Cl2, Catalogue Number 47885-U. Supelco, Bellefonte, PA, USA). Sums and ratios
useful for evaluating the nutritional value and healthiness of the fatty
acid prole were also determined; specically, the sum of saturated
fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids ( PUFA), n 3 fatty acids (n 3) and n 6
fatty acids (n 6), as well as the ratios of PUFA to SFA (PUFA/SFA)
and n6 to n3 (n6/n3) fatty acid.

2.3.6. Oxidative rancidity, glutathione peroxidase enzyme activity, pH and

microbial analysis of meat
To determine the oxidative stability of LDM, samples were preserved
in a refrigerator at 4 C and the thiobarbituric acid reactive substances
(TBARS) values of fresh, 1 week, 2 week and 3 week old meat were determined. About 4 g of meat were homogenized at full speed for 1.5 min
(Ultra-Turrax T-25 Basic, IKA Werke, GMBH & CO. KG, Staufen, Germany) with 10 mL of solution containing 20% trichloroacetic acid (TCA)
in 2 M phosphoric acid and 10 mL DDW. The homogenized mixture
was then ltered through Hyundai Micro No. 60 (Hyundai Micro Co.,
Ltd.) lter paper. Equal amounts (2 mL) of the ltrate and 2-thiobarbituric acid (98% 4, 6 Dihydroxy-2-mercaptopyrimidine, 0.005 M in
DW) were mixed and incubated in a shaking water bath at 80 C for
30 min. After cooling to room temperature, the absorbance of the samples was measured at 530 nm using a VIS-Spectrophotometer (Libra
S22, Biochrom Ltd. Cambridge, England). The amount of TBARS was calculated and expressed as micromoles of malondialdehyde (MDA) per
100 g of meat. The glutathione peroxidase enzyme activity (GSHPx U/
mg of protein) was analyzed from a commercial analytical company
(FACT, Suwon-si, Gyeonggi-do, Korea) using commercial Glutathione
Peroxidase Reductase Assay Kit (Trevigen, Inc., Gaithersburg, MD, USA).
The pH of the meat samples was measured using a digital pH meter
(Docu-pH+ meter, Sartorius, USA) by blending 2 g of meat with 18 mL
of DDW in a homogenizer. To evaluate the microbial quality, three replicate samples from each storage day with or without treatment were
used. A 25 g meat sample was homogenized with 225 mL of 0.85%
(W/V) NaCl solution and 10 fold serial dilutions (using 0.85% NaCl solution) were made before inoculating into plate count agar for aerobic
plate counts (APC) and lactic acid bacteria (LAB). For each dilution, duplicate plates were incubated at 37 C for 48 h and the colonies were
counted immediately after incubation.

3. Results
3.1. Characterization of natural and fermented herb combination
The microbial composition, nutrient contents, pH, fermentable
sugars (glucose, fructose, sucrose, lactose and maltose), organic acids
(lactic acid, acetic acid and propionic acid), and phenolic contents of
the NPGL and FPGL are shown in Tables 1 and 2. The fermentation process increased the CP, Ca, Mg and Na content of FPGL by 10.0%, 14.5%,
204.5% and 94.4%, respectively, while it reduced the EE and crude ber
by 8.4% and 17.4%, respectively (Table 1). The SFA content in FPGL was
reduced by 24.3%, while the MUFA and PUFA contents were increased
by 31.9% and 45.9%, respectively. Interestingly, the proportion of n6
fatty acid in FPGL was reduced by 42.1%, while the n3 fatty acid proportion was increased by 247.3% as a result of fermentation. As shown in
Table 2, the FPGL had a low pH than NPGL. Fermentation process also reduced the concentration of glucose (5.25% vs 2.61%) and sucrose (0.58%
vs 0.00%), while increased the concentration of lactic acid (316.9 mg/kg
vs 2643.8 mg/kg) and acetic acid (210.5 mg/kg vs 544.9 mg/kg) in FPGL.
The concentrations of total polyphenol, tannic acid, and avonoids in
NPGL vs FPGL were 6521.9 vs 6010.1 mg/kg, 1876.2 vs 1193.4 mg/kg
and 6301.7 vs 5979.1 mg/kg, respectively.
3.2. Growth performance
The growth performance of pigs fed diets containing NPGL and FPGL
are shown in Table 4. Dietary supplementation of NPGL and FPGL significantly reduced the feed intake of pigs (P b 0.05) without affecting the
weight gain. In addition, the G:F ratio of pigs was higher in the NPGL
or FPGL supplemented groups (P b 0.05) than the control.
3.3. Serum immunoglobulins
As shown in Table 4, dietary supplementation with FPGL signicantly increased the levels of IgG in the pig serum relative to the control and
NPGL group (P = 0.007). A tendency for higher IgM was also found in
the FPGL supplemented group (P b 0.10); however, the IgA levels
were unaffected.
3.4. Carcass traits
The dressing yield, carcass grade and loin eye area of pigs were unaffected by the dietary treatments (Table 5); however, supplementation
with both NPGL and FPGL signicantly reduced the back fat thickness
(P b 0.035) while increasing the lean production (P b 0.05).
Table 4
Growth performance and serum immunoglobulin concentrations of pigs fed natural or
fermented herb combination (pomegranate + Gingko biloba + licorice) supplemented diet from growing to nishing period.
Parameter

2.4. Statistical analyses

Statistical analysis of the gathered data was performed using the
General Linear Model with the Statistical Analysis System (SAS,
2003, Version 9.1, SAS Institute, Cary, NC, USA). Each pen was used
as the experimental unit for growth performance and carcass
characteristics, whereas a group of three pigs served as the
experimental unit for serum immunoglobulins, meat composition,
fatty acid prole, oxidative stability and pH analysis. The means
were calculated using the least square method and presented with
the standard error of the mean (SEM). Differences among means
were determined by the Student's t-test. A P 0.05 was considered
to indicate signicance for all analyses, while a P b 0.10 was
considered as a tendency.

Growth performance
Initial body weight, kg
Final body weight, kg
Weight gain, g/day
Feed intake, g/day
Gain:feed ratio (G:F)
Serum immunoglobulins (mg/mL)
IgG
IgM
IgA
ab

Treatments1
1

SEM2

P value

Control

NPGL

39.30
103.0
909.4
3247a
0.28b

39.24
100.9
880.5
2846b
0.31a

39.29
102.5
903.6
2932b
0.31a

1.04
1.89
20.6
41.8
0.01

0.999
0.717
0.587
b0.0001
0.027

225.95b
17.34
9.42

227.13b
17.85
9.15

234.94a
21.33
9.62

1.29
1.10
1.43

0.007
0.085
0.978

FPGL

Means with different superscripts in the same row differ signicantly (P b 0.05).
1
Treatments: control (no supplementation); natural herb combination (NPGL);
fermented herb combination.
2
SEM: standard error of the mean.

S.T. Ahmed et al. / Meat Science 122 (2016) 715

Table 5
Carcass characteristics of nishing pigs fed natural or fermented herb combination (pomegranate + Gingko biloba + licorice) supplemented diet.
Treatments1

Parameter

Body weight, kg
Carcass weight, kg
Dressing yield, %
Carcass grade3
Back fat thickness tenth rib, cm
Loin eye area, cm2
Lean production, %

Control

NFPGL

FPGL

103.0
83.50
81.27
1.75
2.30a
25.79
44.07b

100.9
80.83
80.43
2.17
1.93b
24.41
45.24a

102.5
83.00
81.15
2.00
1.97b
26.05
45.56a

Table 7
Effect of natural or fermented herb combination (pomegranate + Ginkgo biloba +
licorice) on the fatty acid composition of Longissimus dorsi muscle.

SEM2

P value

Fatty acid (g/100 g)

Treatments1
Control

NPGL

FPGL

1.89
1.89
2.01
0.14
0.10
1.23
0.30

0.717
0.543
0.956
0.127
0.026
0.596
0.004

Capric acid (C10:0)

Myristic acid (C14:0)
Pentadecylic acid (C15:0)
Palmitic acid (C16:0)
Stearic acid (C18:0)
Arachidic acid (C20:0)
SFA3
Palmitoleic acid (C16:1)
Ginkgolic acid (C17:1)
Oleic acid (C18:1)
Eicosenoic acid (C20:1)
MUFA3
Linoleic acid (C18:2n6)
Arachidonic acid (C20:4n6)
Alpha-linolenic acid (C18:3n3)
Eicosapentaenoic acid (C20:5n3)
PUFA3
n6 PUFA3
n3 PUFA3
PUFA/SFA
n6/n3

0.12
1.21
1.74a
23.11
10.57ab
0.23a
36.98
2.80
0.59
42.62b
0.17
46.17b
10.36
3.44a
0.64b
0.69b
15.12
13.79
1.33b
0.41
10.50a

0.10
1.23
1.43ab
22.73
11.18a
0.16b
36.83
2.89
0.42
44.30ab
0.14
47.74ab
9.70
2.68ab
0.66b
0.77a
13.81
12.37
1.43a
0.38
8.82ab

0.10
1.38
0.86b
23.26
10.27b
0.24a
36.11
3.11
0.29
46.71a
0.27
50.38a
9.66
1.77b
0.83a
0.79a
13.05
11.43
1.62a
0.36
7.06b

ab

Means with different superscripts in the same row differ signicantly (P b 0.05).
1
Treatments: control (no supplementation); natural herb combination (NPGL);
fermented herb combination.
2
SEM: standard error of the mean.
3
Carcass grade: grade A = 3, grade B = 2, grade C = 1, non-descript = 0.

3.5. Meat composition, trace mineral, and fatty acid prole

Dietary supplementation with NPGL or FPGL did not affect the CP
and total ash content of LDM, but did signicantly reduce the EE content
(P = 0.004) as the amount of moisture increased (P b 0.01) in the LDM
of pigs (Table 6). Dietary supplementation with NPGL also reduced the
cholesterol content of pig meat compared to the control and FPGL
(P b 0.05). The Ca content of LDM tended to be higher in the FPGL
group (P b 0.06), while the Fe content was higher in the LDM of the
NPGL supplemented group (P b 0.02).
The effects of dietary NPGL and FPGL on the fatty acid composition of
the longissimus dorsi muscle of pigs are shown in Table 7. Dietary supplementation with FPGL tended to reduce the concentration of strearic
acid (P = 0.062) while signicantly reducing the amount of arachidic
acid (P = 0.01) from LDM without affecting the sum of SFA. The concentration of oleic acid (P = 0.016) and MUFA (P = 0.023) was higher in
the FPGL supplemented group than the control. The concentration of arachidonic acid (C20:4n 6) was lower (P = 0.007), while the linolenic acid (C18:3n 3) concentration was higher (P = 0.002) in
the FPGL group. The eicosapentaenoic acid (C20:5n3) concentration
was higher in both the NPGL and FPGL group (P = 0.0004). Although
there was no signicant difference, the n6 fatty acid proportion was
numerically lower in the supplemented group than the control. In contrast, the n 3 fatty acid proportion was signicantly increased (P =
0.0003) in response to both NPGL and FPGL supplementation
(P b 0.05). The PUFA/SFA ratio was not affected by dietary treatments;
however, the n 6/n 3 ratio was lower in the FPGL group (P =
0.021) than the control.

Table 6
Effect of natural or fermented herb combination (pomegranate + Ginkgo biloba +
licorice) on the composition, cholesterol and trace mineral contents of Longissimus dorsi
muscle.
Parameter

Crude protein, %
Ether extract, %
Moisture, %
Ash, %
Cholesterol, mg/100 g
Calcium, mg/100 g
Iron, mg/100 g
Magnesium, mg/100 g
Sodium, mg/100 g
ab

Treatments1
Control

NPGL

FPGL

24.88
5.35a
68.12b
1.20
88.03a
6.03b
0.89b
21.74
32.38ab

24.51
3.25b
71.05a
1.19
62.70b
6.87ab
1.23ab
23.26
37.82a

24.39
4.03b
70.18a
1.21
100.7a
7.81a
1.43a
23.69
26.81b

11

SEM2

P value

0.32
0.38
0.60
0.03
4.37
0.40
0.10
1.76
2.21

0.545
0.004
0.011
0.872
b0.0001
0.056
0.019
0.785
0.028

Means with different superscripts in the same row differ signicantly (P b 0.05).
1
Treatments: control (no supplementation); natural herb combination (NPGL);
fermented herb combination.
2
SEM: standard error of the mean.

SEM2

P value

0.001
0.08
0.24
0.53
0.24
0.01
0.54
0.22
0.09
0.78
0.04
0.89
0.56
0.29
0.03
0.01
0.73
0.75
0.04
0.02
0.70

0.217
0.323
0.097
0.775
0.062
0.011
0.500
0.649
0.161
0.016
0.114
0.023
0.701
0.007
0.002
0.0004
0.207
0.158
0.0003
0.417
0.021

ab

Means with different superscripts in the same row differ signicantly (P b 0.05).
1
Treatments: control (no supplementation); natural herb combination (NPGL);
fermented herb combination.
2
SEM: standard error of the mean.
3
SFA = total saturated fatty acid; MUFA = total monounsaturated fatty acid;
PUFA = total polyunsaturated fatty acid; n6 PUFA = total omega 6 polyunsaturated fatty acid; n3 PUFA = total mega 3 polyunsaturated fatty acid.

3.6. Oxidative stability, GSHPx, pH and microbiology of pig meat

The effects of dietary treatments on the TBARS value of pig meat are
shown in Fig. 1 (A). Both NPGL and FPGL signicantly reduced the MDA
production in fresh meat and meat that had been stored for 2 and
3 weeks (P b 0.05). Surprisingly, at 1 week, the TBARS value was higher
in the NPGL group (P b 0.05). Higher GSHPx activities were noted (P =
0.006) in meat of both NPGL (51.13 U/mg of protein) and FPGL (57.63 U/
mg of protein) supplemented group compared to control (43.87 U/mg
of protein).
The Dietary treatments had no signicant effect on meat pH in any of
the storage periods [Fig. 1 (B)], except week 3, when a lower pH was
found in the LDM of the FPGL supplemented group (P b 0.05). The aerobic plate count and lactic acid bacteria concentration in LDM of both
NPGL and FPGL supplemented group was lower during week 3 (Fig. 2).

4. Discussion
Microbial fermentation has been identied as a fruitful process for
improving the health-promoting properties of medicinal plants. The fermentation process can alter the original bioactivities and nutritional
composition of herbs (Ng et al., 2011; Yamamoto, Saleh, Tahir,
Ohtsuka, & Hayashi, 2007), reduce the anti-nutrient effects (Deacon,
2005) and enhance the original treatment efcacy of active ingredients
(Dei, Rose, Mackenzie, & Amarowicz, 2008) by the action of enzymes
produced by bacteria, yeast and molds. In this study, fermentation of
the experimental herb combination with L. plantarum and S. cerevisiae
increased the concentration of CP, minerals (Ca, Mg, Na), MUFA and
PUFA content of FPGL, while reducing the SFA. Lactobacillus plantarum
and S. cerevisiae have been reported to possess protease and tannase activity (Mugula, Srhaug, & Stepaniak, 2003; Sturley & Young, 1988) and
produce many extracellular enzymes (Sumengen, Dincer, & Kaya,
2013). The increase in CP content after fermentation can be attributed
to bacterial growth and proliferation, as well as secretion of enzymes

12

S.T. Ahmed et al. / Meat Science 122 (2016) 715

Fig. 1. Effect of diet supplemented with natural (NPGL) or fermented (FPGL) herb combination (pomegranate + Ginkgo biloba + licorice) on TBARS (A) and pH (B) values of pork during
refrigerated storage. Bars within a particular time point not sharing a common letter differ signicantly (P b 0.05).

(all enzymes are protein) or release of bound proteins by the breakdown of all protein complexes (Lohlum, Forcados, Chuku, Agida, &
Ozele, 2014). Improvement in crude protein content of medicinal plants
in response to fermentation was also reported by Heong, Bhupinder,
Karim, and Fazilah (2011). The microbial fermentation also reduced
the concentration of total polyphenols, avonoids and tannic acid in
FPGL (Table 2) which may subsequently increase the availability of minerals (Mohite, Chaudhari, Ingale, & Mahajan, 2013). A study conducted
by Achinewhu (1986) revealed reduced SFA and increased total unsaturated fatty acid in fermented African oil bean seed relative to nonfermented seeds, which supports our results. The fermentation process
reduced the concentration of glucose and sucrose in FPGL, which can be
explained by their conversion to organic acids, mainly lactic acid (Table
2).
Supplementation of the diets of pigs with NPGL or FPGL signicantly
reduced the intake of the experimental pigs with higher G:F ratios. A
previous study conducted by Jeong & Kim (2015) reported no signicant effect of fermented herb combination on feed intake of growing
pigs with improvement of the G:F ratio. Zhou et al. (2015) recently reported that fermented Gingko biloba L. residues did not affect feed intake, while the G:F ratio increased when it was used to supplement
the diet of weaned piglets. In the present study, the reduction in feed intake in response to NPGL and FPGL can be explained by the presence of
complex plant polyphenols including considerable amounts of tannic
acid and avonoids in the natural and fermented herb combination
(Table 2), which are astringent, and their presence in non-ruminant
diets described to reduce the palatability and feed intake (Jansman,
1993). The higher G:F ratio instead of lower feed intake suggests that
plant polyphenols and probiotic bacteria may improve utilization of
diet energy by manipulating the gut microora (Jami et al., 2012). The
present study also conrms the study of Fiesel, Gessner, Most, and
Eder (2014) who reported that plant polyphenols are effective in increasing the gain:feed ratio in growing pigs by manipulating the intestinal microora.

Serum IgG, IgM and IgA are the key components of mammalian humoral immunity that act as safeguards for the extravascular compartment against pathogenic viruses and microorganisms. Many studies
have demonstrated the potential for herbs and probiotic feed additives
to enhance humoral immune response against specic model antigens.
However, in this study we measured the humoral immune status at a
systematic level, but not against a specic antigen. The results showed
that dietary supplementation of FPGL signicantly increased the levels
of IgG in the pig serum with a tendency for higher IgM. The immune enhancing activity of pomegranate peel powder, Ginkgo biloba L. leaf, and
licorice root have been reported in different scientic studies (Ross,
Selvasubramaniana, & Jayasundar, 2001; Zhao, Zhang, Cao, Sun, &
Wang, 2013; Katamaya et al., 2011). However, the natural herb
combination in this study did not show any signicant effects on the
immune system of pigs. Therefore, fermentation with L. plantarum and
S. cerevisiae may improve the immune enhancing activity of fermented
products (Rizzello et al., 2013), since probiotic bacteria can modulate
the systematic immune response via modulation of intestinal
microbiota (Snchez, Gueimonde, Pea, & Bernardo, 2015),
improvement of lymphocyte proliferation (Gill, 1998) and subsequent
elevation of serum immunoglobulin production. Mizumachi, Aoki,
Ohmori, Saeki, and Kawashima (2009) also reported higher IgG and
IgM levels in the serum of weaned piglets fed L. plantarum LQ80
fermented liquid feed. Fermentation of broiler feed with S. cerevisiae
also reportedly increased IgG and IgM production in the serum of
supplemented broilers (Gao et al., 2008).
Supplementation with both NPGL and FPGL signicantly reduced
back fat thickness while increasing lean production without affecting
the dressing yield, carcass grade and loin eye area of pigs. Wang et al.
(2014) found that dietary polyphenols prevent obesity though the following possible mechanisms: lower feed intake, decrease lipogenesis,
stimulation of lipolysis, reduced viability of adipocytes and proliferation
of preadipocytes, suppressed adipocyte differentiation and triglyceride
accumulation, stimulation of fatty acid -oxidation, and reduced

Fig. 2. Effect of diet supplemented with natural (NPGL) or fermented (FPGL) herb combination (pomegranate + Ginkgo biloba + licorice) on aerobic plate count (A) and lactic acid bacteria
(B) concentrations of pork during refrigerated storage. Bars within a particular time point not sharing a common letter differ signicantly (P b 0.05).

S.T. Ahmed et al. / Meat Science 122 (2016) 715

inammation. The experimental herb mix retained a complex phenols

including total polyphenols, tannic acids, and avonoids (Table 2),
which reduced the feed intake of experimental animals and may have
altered the intestinal lipid metabolism (Lei et al., 2007), thereby reducing the back fat thickness and increasing lean production. In addition,
Lactobacillus spp. and S. cerevisiae have been reported to possess lipolytic activity (Fryer, Lawrence, & Reiter, 1967; Hammes, Bantleon, & Min,
1990; Ciafardini, Zullo, Cioccia, & Iride, 2006) which may lower the carcass fat contents (Kalavathy, Abdullah, Jalaludin, Wong, & Ho, 2006) of
experimental pigs.
Dietary supplementation with NPGL or FPGL reduced the ether extract content with increasing amounts of moisture in the LDM of pigs.
The reduction in ether extract can be explained by the lipolytic activity
of plant polyphenols and avonoids present in the experimental herb
combination (Lei et al., 2007; Zarrouki et al., 2010; Nakagawa, Kishida,
Arai, Nishiyama, & Mae, 2004). Increasing moisture may be due to the
inverse relationship between meat moisture and fat content (Callow,
1948), which are related to meat juiciness. Dietary supplementation
with NPGL also reduced the cholesterol content of pig meat relative to
the control and FPGL, which may have been due to the high contents
of avonoids (Table 2). It is well known that plant avonoids can form
insoluble complexes with cholesterol in the digesta and inhibit the absorption of endogenous and exogenous cholesterol in the intestine
(Rao & Gurnkel, 2000). However, fermentation with probiotic bacteria
did not show any extra potential to reduce fat or cholesterol in LDM.
Higher Ca and Fe in the LDM can be attributed to the fermentation process, which increases their availability in fermented food (Mohite et al.,
2013) by the by reducing the anti-nutritional tannins and avonoids
that inhibit the absorption of minerals (Brune, Rossander, & Hallberg,
1989).
Substituting dietary SFA with MUFAs and PUFAs has been reported
to be associated with reduced risk of coronary heart disease by lowering
the low-density lipoprotein cholesterol (LDL-C). Dietary supplementation of plant polyphenols can effectively change the fatty acid composition by preventing the oxidation of unsaturated fatty acids (Cao et al.,
2012). In this study, dietary supplementation with FPGL reduced the
concentration of strearic acid and arachidic acid from LDM. Conversely,
the concentration of oleic acid and MUFA was higher in the FPGL supplemented group, which could be explained by the desaturation of SFAs
(stearic acid) to MUFAs (oleic acid) (Connor, 1999). Previously, Cao et
al. (2012) supplemented broiler diets with fermented Ginkgo biloba
with higher antioxidant capacity and reported a lower SFA in breast
meat. Dietary FPGL also reduced the proportion of arachidonic acid
(AA; C20:4n 6) and n 6 PUFA (arithmetically), while it increased
the proportion of -linolenic acid (C18:3n3), eicosapentaenoic acid
(EPA; C20:5n 3) and n 3 fatty acids. An increase in n 3 fatty
acid, especially -linolenic acid in muscle may cause a corresponding
decrease in AA and n6 fatty acid because -linolenic acid (precursor of EPA) compete linoleic acid (precursor of AA) for the same enzymes in their elongation and desaturation metabolism (Nuernberg et
al., 2002). Therefore, EPA is incorporated into cell membrane phospholipids at the expense of AA. The experimental FPGL had a good concentration of -linolenic acid which may compete with linoleic acid for
enzymes and thereby reduced their concentration and synthesis of AA
in meat. Raes, De Smet, and Demeyer (2004) also reviewed that, an increased n3 content in the intramuscular fat is accompanied with a decreased n 6 deposition. This ensued in a more favorable n 6/n 3
ratio in the meat while the PUFA/SFA ratio was less affected. Our ndings are in consistent with the review study. Lower SFA and higher
MUFA and n 3 PUFA indicate that consumption of meat from the
FPGL supplemented group may pose a lower risk of coronary heart
disease.
Oxidation of muscle lipids results in the production of free radicals,
which are involved in the production of off avors and off color in
meat. The reaction of 2-thiobarbituric acid with malondialdehyde
(MDA, formed as a secondary product of lipid peroxidation) has been

13

used for many years as a popular and reliable marker for lipid oxidation
(Descalzo & Sacho, 2008; Cao et al., 2012). It is well known that ascorbate plant polyphenolic compounds act as potential antioxidants by
scavenging free radicals (Singh, Marimuthu, De-Heluani, & Catalan,
2005) and play roles in cellular antioxidants systems in cooperation
with other cellular antioxidants, thereby protecting unsaturated fatty
acids from oxidative reaction (Havsteen, 2002). In this study, both
NPGL and FPGL signicantly reduced the MDA value in fresh meat and
2 and 3 week stored meat. The antioxidant capacities of pomegranate
peel (Rajan et al., 2011), Ginkgo biloba leaves (Zahradnkov, Schmidt,
Sekretr, & Jan, 2007) and licorice (Morteza-Semnani, Saeedi, &
Shahnavaz, 2003) have been reported to lower the oxidative deterioration of fat in pig meat, thereby reducing MDA production. Additionally,
fermentation with Lactobacillus spp. and S. cerevisiae has been reported
to possess strong reducing power and radical scavenging ability (Wu,
Sun, Zhang, & Xin, 2014), which may increase the antioxidant capacity
of fermented product (Ng et al., 2011; Hur, Lee, Kim, Choi, & Kim,
2014) by increasing the amount of phenolic compounds and avonoids
by microbial hydrolysis reaction (Table 2). In addition, an increase in the
activity of GSHPx in LDM of pigs fed NPGL and FPGL supplemented diets
is of interest because it allows for a greater oxidative stability of meat, as
conrmed by the lower values of MDA value in the meat of NPGL and
FPGL animals.
Meat pH is an important indicator of quality as it is related to shelflife, color and water holding capacity of meat. Microbiologically, a low
pH (b 5.6) in fresh meat is desired as it inhibits growth of undesired bacteria, lessens the rate of color change in meat during storage and thereby lengthen the self-life (Banwart, 1989). The ultimate pH of most pork
with normal glycolysis ranges from 5.3 to 5.8 (Warriss, 1982). The observed pH of pork in this study was also found within this range (5.3
to 5.6). Although statistically there was no difference (except week 3),
the pH was lower in both the treatment groups than control. This can
be explained by the antimicrobial effects of plant polyphenols and bacterial metabolites, which can reduce microbial spoilage of meat by reducing the growth of bacteria. The microbiological study of meat was
also support this explanation where we found lower APC and LAB at
week 3 in both NPGL and FPGL supplemented group. In support to our
result the study of Li, Shao, Zhu, Zhou, and Xu (2013) reported lower
APC in pork sausages supplemented with plant polyphenols. Dietary
polyphenols, especially avonoids, phenolic acids and tannins are also
able to regulate carbohydrate metabolism including glycolysis
(Mocanu, Nagy, & Szllsi, 2015) which may be responsible for almost
constant meat pH of the experimental groups during the storage period.

5. Conclusion
In conclusion, microbial fermentation of experimental herb improve
its' nutrient contents, while reduced the anti-nutrient factors. The experimental levels of NPGL and FPGL induce a decrease in back fat thickness, meat crude fat content, and TBARS of meat, while leading to
increased lean percentage in nishing pigs. In addition, supplementation with NPGL reduced the meat cholesterol, while increasing the
n3 PUFA proportion in meat. Conversely, FPGL increased the proportion of serum immunoglobulin G and M and MUFA and n3 fatty acid
in LDM with a lower ratio of n6/n3. Taken together, these ndings
indicate that dietary supplementation with FPGL was better than NPGL
as it can improve both immunity and meat quality. The negative effects
on feed intake warrant further study using different doses.

Acknowledgements
This study (Grant No. C0193546) was supported by Business for Academic-Industrial Cooperative establishments funded by the Korea
Small and Medium Business Administration in 2014.

14

S.T. Ahmed et al. / Meat Science 122 (2016) 715

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