Journal of Food Processing and Preservation ISSN 1745-4549

EFFECTIVENESS OF CLOVE ESSENTIAL OIL AND GRAPE SEED

EXTRACT COMBINATION ON MICROBIAL AND LIPID
OXIDATION CHARACTERISTICS OF RAW BUFFALO PATTY
DURING STORAGE AT ABUSE REFRIGERATION TEMPERATURE jfpp_736 31..38

HOSSEIN TAJIK1, AZRA FARHANGFAR1, MEHRAN MORADI2,3 and SEYED MEHDI RAZAVI ROHANI1
1
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
2
Department of Food Science and Technology, Ahar Faculty of Agriculture, University of Tabriz, Tabriz 51664-16471, Iran

3
Corresponding author. ABSTRACT
TEL: +98-426-2237717;
FAX: +98-426-2237718; EMAIL: The effect of clove essential oil (CEO) (0.1%) and grape seed extract (GSE) (0.1
mmoradi@tabrizu.ac.ir; and 0.2%) on spoilage populations (mesophilic and psychrotrophic viable count,
moradi.mehran@yahoo.com lactic acid bacteria, Pseudomonas spp. and yeast), Listeria monocytogenes and lipid
oxidation of raw buffalo patties during storage at 8C for 9 days was examined.
Received for Publication November 14, 2011
Accepted for Publication April 29, 2012
CEO significantly affected the growth of Pseudomonas spp., psychrotrophic and
lactic acid bacteria and did not affect growth of mesophilic bacteria. Combining
doi:10.1111/j.1745-4549.2012.00736.x CEO with GSE (0.1 and 0.2%) yielded any significant effect on mesophilic and
Pseudomonas spp. Mesophilic and psychrotrophic bacteria were the most resistant
and sensitive groups to the agents by 00.5 and 0.8 log cycles reduction, respec-
tively. GSE had no inhibitory effect on the growth of L. monocytogenes, while CEO
inhibited the pathogen growth, significantly. Samples with 0.1% CEO had the
lowest degrees of lipid oxidation, which was 73% lower than the control.

PRACTICAL APPLICATIONS
The combination of one or more ingredients provides an enhanced antimicrobial/
antioxidant activity in a concentration, which does not produce undesirable
changes in the flavor or aroma. It could be suggested that additive, synergistic or
antagonistic effects of combined use of two agents in food is certainly related to
the type and concentrations of compounds. Results of this study will contribute
significantly to efforts to reduce the problem of Listeria monocytogenes and oxida-
tion on buffalo patties.

are a wide range of natural products of plants, animals and

INTRODUCTION
Safety and shelf life of meat products is of great importance
for consumers, the food industry and the health authorities.
Among the different meat products, ground and commi-
nuted meat products are highly perishable because of their
nutrient-dense medium for the growth of many microor-
ganisms. Microbial growth (spoilage or pathogens) and oxi-
dative rancidity are known as the major problems causing
the shortening of the shelf life of such products (Shan et al.
2009).
Biopreservatives are useful in extending the shelf life of
foods reducing or eliminating pathogenic bacteria and
increasing overall quality of food products. Biopreservatives
microorganisms that can be useful in extending foods shelf
life (Ponce et al. 2011). Within different naturally occurring
antimicrobial/antioxidant ingredients, the essential oils
(EOs) and various extracts of plants are outstanding alter-
natives to chemical preservatives and their use in foods
meets consumer demands for minimally processed natural
products while providing some extra functional benefits to
both food and consumer (Burt 2004; Pajohi et al. 2011).
Given to the partially hydrophobic nature of EO, they are
able to work more effectively at the interface of the lipid-
and water-compatible portions of meat, which make them
suitable ingredients for use in meat systems (Shan et al.
2009).

Journal of Food Processing and Preservation 38 (2014) 3138 2012 Wiley Periodicals, Inc. 31
EFFECT OF CLOVE AND GSE ON BUFFALO PATTY
Syzygium aromaticum flower bud, commonly known as
clove, is a perennial tropical plant. In the food industry,
cloves are often used in the form of ground, extracted EOs
or oleoresin in a small amount because of their intense
flavor (Chaieb et al. 2007). The clove EO (CEO) possesses
significant quantity of eugenol, which is well-known for its
antimicrobial and antioxidant properties (Lee and Shiba-
moto 2001; Jirovetz et al. 2006; Chaieb et al. 2007). Its
inhibitory action against various gram-positive and gram-
negative bacteria in meat and meat products were recorded
(Zhang et al. 2009; Miladi et al. 2010) Grape seed extract
(GSE) is a by-product derived from the grape seeds (Vitis
vinifera), which is commercially available as a dietary
supplement and also has a Generally Recognized As Safe
status approved by the Food and Drug Administration
(Over et al. 2010). GSE is of special interest because of its
high flavonoid content with phenolics natures, particularly
resveratrol, which are responsible for antioxidant and anti-
microbial activities of the extract. Resveratrol (trans-3, 4
5-trihydroxystilbene), a phenolic compound produced pri-
marily in the grapevine, is present in wines and various
parts of the grape (Kulkarni et al. 2011). When compared
with other antioxidants, the following order of strength has
been demonstrated: butylated hydroxyanisole (BHA) >
resveratrol > propyl gallate > tripolyphosphate > vanillin >
phenol > butylated hydroxytoluene > alpha-tocopherol
(Murcia and Martinez-Tome 2001). The antimicrobial and
antioxidant activity of GSE in ground chicken (Brannan
2009), raw frozen vacuum-packaged beef (Rojas and Brewer
2008) and ground beef (Ahn et al. 2004) has been reported.
The practical application of several EOs in foods is
limited because of the strong flavor they impart to foods
and also to their interaction with some food ingredients.
Encapsulation of the EOs seems to be an attractive new
approach, but the best solution will be the use of two or
more active compounds with each other in lower concen-
trations (Stojanovic et al. 2011). The combination of one or
more ingredients provides an enhanced antimicrobial/
antioxidant activity in a concentration that does not
produce undesirable changes in the flavor or aroma (Burt
2004). The aim of the present study was to investigate the
effect of CEO and GSE alone or in combination in concen-
trations below sensorial thresholds on the microbial and
lipid oxidation characteristics of raw buffalo patty during 9
days of storage at 8C.
MATERIAL AND METHODS
Extraction and Chemical Analysis of EO
S. aromaticum flower buds (clove) were purchased from the
local groceries and authenticated by Hosseinlar at the Insti-
tute of Medicinal Plants (Karaj, Iran). The comminuted
32 Journal of Food Processing and Preservation 38 (2014) 3138 2012 Wiley Periodicals, Inc.
H. TAJIK ET AL.
buds were subjected to hydro-distillation for 2.5 h using a
clevenger-type apparatus. The water traces of extracted oil
were removed with anhydrous sodium sulfate followed by
filtering via 0.22-mm filters and stored at 4C before use.
Chemical composition of CEO was analyzed using a gas
chromatograph (GC) (Hewlett-Packard [Santa Clara,
CA] 6890N) equipped with a column HP-5MS (30 m
length 0.25 mm i.d., film thickness 0.25 mm) coupled to a
mass spectrometer (MS) (Hewlett-Packard 5973N). The
chromatographic conditions were as follows: helium flow
rate was 1.5 mL/min; temperature programmed to rise from
50 to 240C with a gradient of 3C/min (holding the initial
and final temperature for 6 min); followed by a temperature
enhancement of 15C/min up to 300C, (holding for 3 min);
injector port temperature and detector temperature were set
at 290C and 250C, respectively. The individual compounds
were identified and confirmed with those of authentic
samples and with available library data of the GC/MS
system (WILEY 2001 data software).
Preparation of GSE
Commercial GSE powder (extraction solvent: water, total
phenolics [gallic acid equivalents, dry basis] 90 g GAE/
100 g, high-performance liquid chromatography (HPLC)
phenol profile >10% Monomers, HPLC 6080% oligomers
<25% polymers, heavy metals [Pb,Cd, Sn, Hg] < 10 ppm,
arsenic <3 ppm, moisture [loss upon drying] 8.0% and
insoluble substances [1% in water] < 5%) was provided
kindly from Mega Natural Inc. (Madera, CA).
Listeria monocytogenes Cultivation and
Standardization
Lyophilized cultures of L. monocytogenes Persian Type
Culture Collection 1163 acquired from the culture collec-
tion of the Department of Food Hygiene and Quality
Control, Faculty of Veterinary Medicine Urmia University
(Urmia, Iran). The bacteria were maintained in granule
containing cryo-tubes at -70C. Inoculums of bacteria were
prepared by transferring a granule containing bacteria to
the tryptic soy agar (TSA) (Lab M, Lancs, UK) slant and
incubated at 37 1C for 24 h. TSA slants were maintained
at 4 1C and subculturing was carried out every month to
maintain bacterial viability. Prior to the experiment, the
bacterium was reactivated by two subcultures. For the
first subculture, three or four well-isolated colonies were
touched with a sterile wire loop and suspended into 10 mL
of tryptic soy broth (Lab M) and incubated at 37 1C
for 24 h. Subsequently, a second subculture was prepared
and incubated for 20 h at 37 1C. Bacterial suspensions
adjusted to approximately 9 log10 cfu/mL by visible ultra-
violet spectrophotometer at 595 nm (optical density~0.6)
H. TAJIK ET AL.
and eventually confirmed by plating on TSA and counting
viable cells after 24 h at 37 1C.
Assessment of Acceptable Level of CEO
Post-rigor buffalo muscles were purchased directly from a
local meat packaging plant and transported refrigerated
immediately to the laboratory. Under an aseptic condition,
meat was ground in a meat grinder. Ground meat was
homogenized with different concentrations of CEO (final
concentrations 0, 0.1, 0.25, 0.5, 1 and 2% v/w) in a commer-
cial mixer and then raw patties were formed using a burger
mold and evaluated by a 10-member sensory panel par-
ticipants made up of members of our department. The
participants were asked to mark the highest acceptable con-
centration on the basis of samples odor and color accord-
ing to the A 9-point hedonic scale (9 = like extremely;
8 = like very much; 7 = like moderately; 6 = like slightly;
5 = neither like nor dislike; 4 = dislike slightly; 3 = dislike
moderately; 2 = dislike very much; 1 = dislike extremely).
Meat Preparation and L. monocytogenes
Inoculation
For inoculation of L. monocytogenes, meat surface was firstly
sprayed with 95% (v/v) ethanol and then burnt. The burnt
tissue was eliminated and the inner sterile tissue was asepti-
cally ground in a meat grinder. Ground meat was inoculated
thoroughly with appropriate solution of L. monocytogenes to
obtain a starting count of 5 log10 cfu/g viable cells. The
samples were stomached at 260 rpm for 1 min to distribute
the inoculum. Prior to the inoculation of the meat, CEO
and GSE were added either alone or in combination to the
samples using a micropipette and the samples were then
divided into six groups: (1) control (no addition); (2) 0.1%
CEO; (3) 0.1% GSE; (4) 0.2%; (5) 0.1% CEO + 0.1% GSE;
and (6) 0.1% CEO + 0.2% GSE. The selection of the con-
centrations of GSE was established following preliminary
microbiological analysis (determination of minimum
inhibitory concentrations [MICs] of GSE against some
meat-related bacteria) (results not shown).
After appropriate homogenization, patties were formed
using a burger mold and were separately packaged in
sterile polyethylene bags (Zipack, Tehran, Iran) and stored
in temperature-controlled laboratory refrigerator (8C) for
up to 9 days. The enumeration of L. monocytogenes was
carried out on day 0, 3, 6 and 9. At each time, 25 g
meat from each treatment were transferred into sterile
stomaching bag containing 250 mL sterile 0.1% peptone
water (Merck, Darmstadt, Germany) and stomached for
1.5 min. Decimal dilutions (1:10) in 0.1% peptone water
solution were prepared and the appropriate dilution was
spread (0.1 mL) in duplicate on Listeria Chrom agar
Journal of Food Processing and Preservation 38 (2014) 3138 2012 Wiley Periodicals, Inc.
EFFECT OF CLOVE AND GSE ON BUFFALO PATTY
(CHROMagar, Paris, France) and incubated at 37 1C for
24 h. Results were expressed as log10 cfu/g.
Enumerations of Spoilage Relate Bacteria
At each sampling day (0, 3, 6 and 9), serial dilutions of rep-
resentative un-inoculated samples in 0.1% peptone water
were prepared as described (see the section of counting
L. monocytogenes). Duplicate samples of appropriate dilu-
tions were poured on plates of the following agars: plate
count agar (PCA, Merck) for total mesophilic viable count
incubated at 35C for 24 h; PCA for total psychrotrophic
viable count incubated at 7C for 57 days; Pseudomonas
agar base (Oxoid, Hampshire, UK) supplemented with
cetrimide, fucidine and cephaloridine supplements for
Pseudomonas spp. incubated at 25C for 2 days; de Man
Rogosa Sharpe agar (Merck) incubated at 30C for 3 days
and Rose Bengal Chloramphenicol selective agar (Lio-
filchem, Roseto degli Abruzzi, Italy) for yeast incubated at
25C for 35 days. Results were expressed as log10 cfu/g.
Lipid Oxidation Measurement
Lipid oxidation was performed by an extraction thiobarbi-
turic acid (TBA) method as described by Ulu (2004) with
minor modification. A 10-g meat sample was homogenized
with 35 mL of cold (4C) extraction solution containing 4%
perchloric acid and 1 mL of BHA (1 mg/mL) at 13,500 rpm
for 1 min. The blended sample was filtered through
Whatman no. 1 filter paper into a 50-mL volumetric flask.
The filtrate was adjusted to 50 mL with 4% perchloric acid
and 5-mL aliquot of the filtrate was added to 5 mL of TBA
(0.02 M). The mixture was vortexed and then incubated
in a 100C water bath for 60 min to develop the
malonaldehyde-TBA complex. The absorbance was mea-
sured at 532 nm after the solution had been cooled with
cold tap water for 10 min. TBA values were expressed as mg
malonaldehyde/kg sample.
Statistical Analysis
All assays were performed in triplicates. One-way analysis of
variance with NewmanKeuls posttest with a significance
set at P < 0.05 was performed using GraphPad (http://
www.graphpad.com).
RESULTS AND DISCUSSION
CEO Chemical Characterization
The chromatographic analysis of CEO revealed 23 different
compounds, representing 93.2% of the total CEO. Eugenol
33
EFFECT OF CLOVE AND GSE ON BUFFALO PATTY
(59.97%), b-caryophyllene (15.36%), 2-methoxy-4-[2-
propenyl] phenyl acetate (13.21%) and a-humulene
(3.93%) were found to be the major chemical constituents
of the CEO. Further constituents were found to be in quan-
tities below 1%. The composition of EOs can vary greatly
depending upon the geographical region, the variety, the
age of the plant, the method of drying and the method
of extraction (Jerkovic et al. 2001). The phytochemical
profile found for the CEO in this study was somewhat
in agreement with the results observed by Prashar et al.
(2006), which found the content of eugenol to be 78%
with 13% b-caryophyllene, whereas Chaieb et al. (2007)
found a high concentration of eugenol (88.58%), eugenol
acetate (5.62%), b-caryophyllene (1.39%), 2-heptanone
(0.93%), ethyl hexanoate (0.66%), humulenol (0.27%) and
a-humulene (0.10%).
Effect of Clove EO and GSE on
L. monocytogenes
Mean sensory scores for odor and color attributes of raw
buffalo patties with different concentrations of CEO (0, 0.1,
0.25, 0.5, 1 and 2% v/w) are shown in Fig. 1. The 0.1% CEO
was the most preferred of the treatments showing organo-
leptically acceptable score (5), while other CEO concentra-
tions exhibited lower scores. Then, 0.1% CEO was examined
alone and in combination with GSE in further antimicro-
bial and antioxidant evaluation in patty. The selection of
the concentrations of GSE was established following pre-
8
7
6 1 % CEO
Scores of panelists
0 BUFFALO PATTIES CONTAINING DIFFERENT
Color Odour
Sensorial attributes
34 Journal of Food Processing and Preservation 38 (2014) 3138 2012 Wiley Periodicals, Inc.
H. TAJIK ET AL.
liminary microbiological analysis (determination of MICs
of GSE against some meat-related bacteria) (results not
shown). Moreover, addition of GSE in lower concentration
to meat did not significantly impact consumer preference,
tenderness, appearance or flavor (Brannan 2009; Over et al.
2010).
The rate of increase in L. monocytogenes was more rapid
in the control (without antimicrobial agents) and GSE-
formulated (either 0.1 or 0.2%) samples than others
(Fig. 2). The initial L. monocytogenes population in buffalo
patty was 5.18 log10 cfu/g; the microbial count increased
steadily during storage and reaching 6.99 log10 cfu/g after 9
days. The L. monocytogenes population count was between
6.83 and 7.02 log10 cfu/g in GSE-formulated samples alone,
5.57 log10 cfu/g in 0.1% CEO and between 5.65 and 5.88
log10 cfu/g in the combined formulation during the 9-day
storage period (Fig. 2). The results showed that CEO by
itself was more efficient on L. monocytogenes than when
used in combination with different concentrations of GSE.
According to Fu et al. (2007), combination of different
plant-originated compounds could exert additive, synergis-
tic or antagonistic effects depending on the type of micro-
organism, our results suggested that the combinations of
GSE and CEO did not have an additive or synergistic effect
on the growth and survival of L. monocytogenes.
Highly hydrophobic constituents of EOs (e.g., thyme,
mint and bay oil) may show limited effectiveness on foods
of high fat content, because EOs will partition in the lipid
fraction of food phase, but as reported in this study, the
0 % CEO
0.1 % CEO
0.25 % CEO
0.5 % CEO
2 % CEO
FIG. 1. SENSORIAL ATTRIBUTES OF RAW
LEVELS OF CLOVE ESSENTIAL OIL (CEO) AS
DETERMINED BY SENSORY PANEL
H. TAJIK ET AL.
FIG. 2. EFFECT OF CLOVE ESSENTIAL OIL
(CEO) AND GRAPE SEED EXTRACT (GSE)
AND THEIR COMBINATION ON LISTERIA
MONOCYTOGENES POPULATION IN RAW
BUFFALO PATTIES DURING STORAGE AT 8C
FOR 9 DAYS. VALUES IN THE SAME ROW
WITH DIFFERENT SUPERSCRIPTS MEAN THAT
THE VALUES ARE SIGNIFICANTLY DIFFERENT
(P < 0.05)
opposite may also occur with less hydrophobic EOs, such as
CEO, which was more effective against L. monocytogenes in
mediate fatty products.
The antilisteric activity of CEO was examined in minced
meat at both 30C and 7C. At concentrations of 0.5 and 1%,
CEO restricted the growth of L. monocytogenes, which was
more pronounced at a concentration of 1%. Listeria counts
in treated samples were 13 log10 cfu/g less compared with
the controls at different intervals during 15 days of storage
(Menon and Garg 2001). The effectiveness of EO applica-
tion on foods is the result of factor associations such as
applications forms, concentration applied, the way of action
and storage temperatures (Ponce et al. 2011). For example,
CEO decreased the growth of L. monocytogenes more at
higher temperature (25C) than at lower temperature (4C)
(Miladi et al. 2010). Eugenol and other major antimicrobial
components of CEO are generally less dissolved at lower
temperature and the antibacterial activity may be reduced
when used in the refrigerator. According to Burt (2004)
effective EOs in decreasing order of antimicrobial activi-
ties are: oregano > clove > coriander > cinnamon > thyme >
mint >rosemary > mustard > cilantro/sage.
Ahn et al. (2004) reported that the numbers of L. mono-
cytogenes declined by 1 log cfu/g in raw ground beef treated
with 1% GSE (ActiVin, a trademark name for the GSE) after
9 days of refrigerated storage. In our study, whereas
L. monocytogenes were susceptible to 0.1% and 0.2% GSE
during the first 3 days of storage, but the population rose
from 6.83 to 7.02 log10 cfu/g for GSE-formulated samples up
to the end of 9 days of storage following a similar trend to
the control samples. Our result indicated that antibacterial
Journal of Food Processing and Preservation 38 (2014) 3138 2012 Wiley Periodicals, Inc.
EFFECT OF CLOVE AND GSE ON BUFFALO PATTY
activity of GSE at 0.1 and 0.1% concentrations against
L. monocytogenes were not bactericidal in buffalo raw patty.
The discrepancies in findings of different reports could be
the result of the complexity of the food system and the
amount and properties of the natural agents used.
Efficiency of Clove EO and GSE on Spoilage
Microbial Populations
The particular species of bacteria that contaminate the
meat, along with the environmental conditions, will deter-
mine the spoilage profile of the stored muscle food. Table 1
shows the antimicrobial effects of CEO and GSE on the dif-
ferent spoilage microbial populations. The initial meso-
philic, psychotropic and Pseudomonas spp. counts for
control, and CEO- and GSE-treated samples were by the
range of 2.823.05, 2.953.69 and 2.522.95 log10 cfu/g,
respectively. On the other hand, the initial Lactic Acid Bac-
teria (LAB) and yeast counts were in the range of 1.992.61
and 3.483.85 and 4.34.7 log10 cfu/g, respectively. The
mean increment of all microorganisms count was 4.25 log10
cfu/g, for samples treated alone by CEO and GSE. While in
the case of combined formulations, the mean increments in
combined use of GSE and CEO was 4.26 log10 cfu/g. These
implied that combined use of CEO and GSE did not exert
any additive or synergistic effect on buffalo spoilage micro-
organisms than using the alone. The individual EO and
extract contain complex components which, when com-
bined with each other, may lead to additive, synergistic or
antagonistic effects (Fu et al. 2007). These results suggest
35
EFFECT OF CLOVE AND GSE ON BUFFALO PATTY H. TAJIK ET AL.

TABLE 1. ANTIMICROBIAL EFFECTS OF CEO AND GSE AND THEIR COMBINATION ON DIFFERENT SPOILAGE MICROORGANISMS IN RAW BUFFALO
PATTIES DURING STORAGE AT 8C FOR 9 DAYS

Storage time 0.1% 0.1%

(days) Control 0.1% CEO 0.1% GSE 0.2% GSE CEO + 0.1% GSE CEO + 0.2% GSE
TMVC
0 2.82* 0.15g 3.05 0.21g 2.94 0.12g 2.95 0.15g 2.94 0.15g 2.83 0.17g
3 5.81 0.18d 4.99 0.15ef 5.08 0.19def 5.45 0.20de 4.83 0.14f 5.06 0.10ef
6 7.06 0.15abc 6.71 0.17c 7.06 0.20abc 6.91 0.18bc 7.00 0.07abc 6.78 0.10c
9 7.60 0.05a 7.11 0.15abc 7.57 0.16a 7.62 0.15a 7.36 0.20abc 7.41 0.10ab
TPVC
0 3.66 0.14k 2.95 0.12l 3.69 0.15k 3.58 0.14k 3.30 0.12kl 3.45 0.15k
3 6.65 0.15i 6.22 0.15j 6.34 0.10i 6.36 0.21i 6.30 0.15i 6.41 0.10i
6 8.27 0.20cd 7.12 0.15h 8.05 0.10de 7.79 0.20ef 7.51 0.15g 7.55 0.12fg
9 8.73 0.12a 8.07 0.10de 8.62 0.18bc 8.78 0.15b 8.20 0.13d 7.94 0.18def
Pseudomonas spp.
0 2.92 0.05i 2.79 0.08ij 2.95 0.11j 2.81 0.05ij 2.52 0.04j 2.76 0.10i
3 6.14 0.15f 5.16 0.10h 5.49 0.12gh 5.78 0.10fg 5.22 0.10h 5.69 0.13g
6 7.99 0.09de 7.81 0.15e 5.49 0.18e 5.78 0.14e 5.22 0.12e 5.69 0.13e
9 8.74 0.16ab 8.33 0.20cd 8.46 0.21bc 8.91 0.20a 8.49 0.20bc 8.43 0.17bc
Lactic acid bacteria
0 2.61 0.10i 2.10 0.05j 2.32 0.16ij 1.99 0.09j 2.03 0.04j 2.00 0.14bc
3 4.76 0.09f 3.47 0.10h 4.18 0.15g 4.48 0.10fg 4.36 0.08g 4.23 0.21g
6 5.90 0.11cd 5.50 0.10e 5.76 0.10de 6.31 0.15b 5.45 0.12e 5.72 0.12de
9 6.68 0.12a 6.23 0.15bc 6.08 0.18bcd 6.99 0.14a 6.27 0.14bc 6.02 0.10bcd
Yeast
0 3.61 0.01klm 3.48 0.03m 3.85 0.08k 3.75 0.04kl 3.50 0.12lm 3.62 0.05klm
3 5.23 0.02ef 4.54 0.08i 4.85 0.10h 5.0 0.10fgh 4.12 0.10j 4.31 0.13j
6 5.59 0.06bcd 5.12 0.14fg 5.38 0.10de 5.45 0.09cde 4.97 0.12gh 4.99 0.10fgh
9 6.17 0.06a 5.52 0.05cd 5.7 0.18bc 5.79 0.21b 5.60 0.11bcd 5.61 0.08bcd

All values are mean standard deviation of three replicates.

Values in the same row with different superscripts mean that the values are significantly different (P < 0.05).
* Log10 cfu/g.
CEO, clove essential oil; GSE, grape seed extract; TMVC, total mesophilic viable count; TPVC, total psychrotrophic viable count.

that use of CEO with GSE together provided no additive or
synergistic effect in controlling microbial growth.
However, results also showed that the incorporation of
0.1% CEO significantly affected the growth of Pseudomonas
spp., psychrotrophic and LAB (P < 0.05), but it did not
affect growth of mesophilic bacteria (P > 0.05). Moreover,
the inhibitory effects of GSE on microbial populations were
not significant in 0.2% than 0.1% concentration with the
exception of Pseudomonas spp. and LAB populations. Lower
counts for LAB and yeast to those of mesophilic, psychotro-
pic and Pseudomonas spp., could be explained by low sugar
concentration of the meat which mainly dominated gram-
negative microorganisms in its spoilage. Pseudomonas spp.
cause quality deterioration of meat products due to the pro-
duction of extracellular proteases and lipases at low tem-
peratures. However, gram-positive bacteria generally tend to
be more sensitive to spices than gram-negative bacteria
(Zhang et al. 2009).
Combining CEO (0.1%) and GSE (0.1 and 0.2%) yielded
no significant effect on mesophilic bacteria and Pseudomo-
nas spp., when compared with usage in alone formulation.
During the storage of patties, mesophilic and psy-
chrotrophic bacteria were most resistant and sensitive
spoilage groups to the both agents by 00.5 and 0.8 log
cycles reduction compared with the control treatment,
respectively.
Efficiency of Clove EO and GSE on
Meat Oxidation
Ground meat is prone to lipid oxidation because of its large
surface area, which is easily reached by oxygen (Ahn et al.
2004). Therefore, the use of spices with potential antioxi-
dant activity might effectively decline oxidative deteriora-
tion. The TBA (mg malondialdehyde/kg sample) values
during 9 days of storage of patties containing CEO and GSE
are shown in Fig. 3. During 9 days of storage, the TBA
values of treated samples were significantly lower than that
of the control one (P < 0.05). The control samples rapidly
increased in TBA values throughout the refrigerated storage
whereas samples formulated with 0.1% CEO made TBA
value of 0.59 which was 27.539% lower than the TBA

36 Journal of Food Processing and Preservation 38 (2014) 3138 2012 Wiley Periodicals, Inc.
H. TAJIK ET AL. EFFECT OF CLOVE AND GSE ON BUFFALO PATTY

FIG. 3. THIOBARBITURIC ACID (TBA) VALUES

OF RAW BUFFALO PATTIES FORMULATED
WITH CLOVE ESSENTIAL OIL (CEO) AND
GRAPE SEED EXTRACT (GSE) DURING
STORAGE AT 8C FOR 9 DAYS. VALUES IN THE
SAME ROW WITH DIFFERENT SUPERSCRIPTS
MEAN THAT THE VALUES ARE SIGNIFICANTLY
DIFFERENT (P < 0.05)

values of 0.1% GSE (0.95) and 0.2% GSE (0.82) containing values of all treated samples remained less than 1.0 for
samples. This result was in agreement with Ahn et al. (2004) the entire 9 days of storage time. TBA values lower than 1.0
who revealed that GSE (ActiVin) effectively retard lipid oxi- was usually associated with rancid flavor/odor by sensory
dation in raw ground meat. However, at the end of storage panelists.
samples packaged by 0.1% CEO had lowest degrees of
lipid oxidation (0.59 mg malondialdehyde/kg sample)
which was 73% lower than control samples (2.16 mg CONCLUSIONS
malondialdehyde/kg sample). Rojas and Brewer (2008)
The results revealed some degrees of variation in the anti-
reported that, in raw, frozen vacuum-packaged beef, 0.02%
oxidant and antimicrobial activity of CEO and GSE on
GSE reduced TBA by about 12% compared with the
refrigerated patty. We demonstrated that CEO alone was
control. Reduction of lipid oxidation and peroxidation of
more effective on both spoilage microbial population and
meat products by GSE is in a concentration-dependent
L. monocytogenes than using in combination with different
manner (Murcia and Martinez-Tome 2001).
concentrations of GSE. The mixed use of plant-originated
Our results revealed that CEO possessed higher antioxi-
antimicrobial agents seems a promising approach for incor-
dant activity than GSE in raw buffalo patties. TBA value for
poration them into various food products for which a
the 0.1% CEO treatment was 0.59, compared with 0.95,
natural antimicrobial additive is desired. But the results of
0.82, 0.67 and 0.89 for 0.1% GSE, 0.2 GSE, 0.1%
this study indicated that GSE and CEO did not have an
GSE + 0.1% CEO and 0.2% GSE + 0.1% CEO, respectively
additive or synergistic antimicrobial effect on buffalo meat
(Fig. 3). These data indicated no synergistic antioxidant
patty. It could be suggested that additive, synergistic or
activity between CEO and GSE when used together in the
antagonistic effects of combined use of two agents in food is
buffalo patty. Cloves exhibited a higher antioxidant activity
certainly related to the type and concentrations of com-
than BHA, propyl gallate, ascorbic acid or cinnamon in
pounds as the combined use of GSE and CEO did not have
refrigerated stored meats of sheep, beef and pork (Jayathila-
any significant advantages on oxidative deterioration of
kan et al. 2007). Eugenol, the major component identified in
buffalo meat than using of them alone.
CEO (59.97%) has shown antioxidant activities in various
studies (Lee and Shibamoto 2001). The CEO was found to
inhibit the generation of secondary products from lipid per-
ACKNOWLEDGMENTS
oxidation. The generation of secondary products from lipid
autoxidation was also inhibited. As reported, CEO could be The authors express their sincere appreciation to Hadi
applied as an antioxidant even at the later stages of lipid Ghasemmahdi for his technical assistance and the Mega
oxidation (Jirovetz et al. 2006). As shown at Fig. 3, TBA Natural Inc. (Madera, CA) for providing GSE.

Journal of Food Processing and Preservation 38 (2014) 3138 2012 Wiley Periodicals, Inc. 37
EFFECT OF CLOVE AND GSE ON BUFFALO PATTY
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