INTRODUCTION
Fish meat is an ideal source of animal protein which
has a high nutritional value. Nowadays, consumers are
interested in healthy food [1]. Nevertheless, convenience
food, including burgers, has remained common all over
the world [2]. Ready-to-cook fish products is becoming
popular among consumers due to their high nutritional
value and short time of cooking [2]. Still, to preserve its
quality, fish meat should be processed properly [3].
In recent years, changing socioeconomic factors,
namely, an increase in the number of employed women,
have led to an increased demand for convenience
products. Therefore, some efforts have been made to
extend the shelf life of ready-to-eat food [4, 5].
Surimi, stabilised myofibrillar proteins of fish
muscle, can be made of both sea-water and fresh-water
fish. To obtain surimi, fish fillet is minced, washed by
water, and stabilised by blending with cryoprotectants.
A cryoprotectant mix, containing sugar, sorbitol, and
phosphates, is added to the minced fish [6]. Surimi is
an important ingredient for food production in many
countries due to its technological properties [6].
Currently, there are a number of ways to control
the growth of pathogenic microorganisms in food
products. One of the ways is the use of essential oils
(EOs). EOs are aromatic oily extracts obtained from
different parts of plants, such as flowers, leaves, wood,
bark, roots, seeds, or peel, which exhibit bactericidal
or bacteriostatic properties [7]. EOs are considered
as natural preservatives for raw or mildly processed
food [8]. EOs have a wide spectrum of antimicrobial
properties. As an antimicrobial agent, EOs destroy both
the lipid bi-layer of cell membranes and enzyme systems
as well as inactivate the genetic material of bacteria [9].
EOs display their antimicrobial action against
pathogenic microorganisms, including gram-positive and
gram-negative, as well as mold and parasites [10–14].
In addition, EOs are reported to have antioxidant
properties [15–17]. Natural antioxidants have an
advantage over artificial ones because of their high
content in phenolic compounds as well as other active
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components which can effectively inhibit oxidative
reactions [17, 18].
Cumin (Cuminum cyminum L.) is a flowering plant in
the family Apiaceae. Its seeds have been commonly used
for centuries as a spice [19]. Thyme (Zataria Multiflora
Boiss.) is an aromatic perennial evergreen herb beloning
to the family Labiateae and used in cooking [20]. In
addition, there is data on the successful use of thyme EO
as an antimicrobial agent in chicken meat patties [21].
The aim of this work was to find a way to prolong
the characteristics and shelf life of novel burgers made
of chicken meat and surimi, as well as to investigate
chemical and microbiological changes in the burgers
stored at 2°C for 27 days.
STUDY OBJECTS AND METHODS
Preparation of minced chicken meat and surimi.
Fresh chicken and silver carp (Hypophthalmichthys
molitrix L.) were purchased from a local market in
Ahvaz, Khuzestan Province, Iran. The chicken was
minced and then kept at –18°C until used. Fresh fish was
transported on ice into a laboratory, washed, beheaded,
gutted, and filleted. The fillet obtained was thoroughly
washed, put through a meat mincer with 4 mm diameter
holes (EG-1200-EBS, Jahan Ava, Iran) for 2 min.
The minced fish was washed with a triple volume of
water (4°C) and stirred for 10 min. The washed minced
fish was filtered through two layers of cheesecloth and
then subsequently dewatered by using a manual juicer
extractor. Washing was performed three times. The
third washing was carried out with 0.5% NaCl (Merck,
Germany) solution. A ratio of the minced fish to NaCl
was 1:3 (w/w).
After dewatering, the minced fish was mixed with
cryoprotectants, i.e. sucrose 3% (Merck, Germany) and
sorbitol 3% (Merck, Germany), for 60 s and frozen using
a blast freezer. The surimi obtained was kept at –18°C
until used.
Preparation of combined burgers and treatments.
Before burgers preparation, frozen surimi and minced
chicken meat were put in a refrigerator (at 4°C) at night.
Meat for burgers was prepared from surimi (63%) and
minced chicken meat (37%).
The meat was then blended with toasted flour, 8.2%;
wheat flour, 2%; soy flour, 3%; sunflower oil, 1%; freshly
grated onion, 7%; garlic powder, 1%; sodium chloride,
1%; white pepper, 0.5%; lemon juice, 1%; and sodium
tri-polyphosphate, 0.3% (Merck, Germany).
All the ingredients in combination with 125 mg/L
of nisin (Sigma Aldrich, England) were ground through
a blender with a 5 mm plate (Gosonic, Turkey) for
4–5 min. Nisin solution, which was added to avoid
the growth of Clostridium botulinum, was prepared
by dissolving a required amount of nisin powder in
sterilised 0.02N HCl solution. Burgers (25 g in weight,
50–60 mm in diameter, and 1 cm in thickness) were
formed by a burger-maker according to [22].
RSM (response surface methodology) was used to
optimise the formulation. The results were analysed
using Design Expert 6.0.2 software, and each of the
dependent variables in the form of a quadratic regression
model was presented as follows:
y = β0 +Σ βi Xi+ Σ βii Xi2+ ΣΣi<j βij XiXj
k
i=1
k
i=1
% Cooking yield = cooked weight
raw weight × 100 (2)
% Shrinkage = ( raw thickness− cooked thickness)+ (raw diameter− raw thickness+ raw diameter % Moisture retention = (cooked weight× % moisture in cooked burger)
raw weight× % moisture in raw burger × (1)
where β0, βi, βii and βij are regression coefficients,
and Xi and Xj are coded independent variables. The
formula was selected based on the results of the sensory
evaluation of the burgers that were stored at 2°C before
testing. The test was performed with the help of RSM
software.
As control samples were used burgers made without
essential oils. They were objected to analyses of
proximate composition and cooking characteristics. The
control samples included burgers with 100, 300, and
Table 1 Composition of thyme essential oil
Number of
component
Component Retention
time, min
Amount,
%
1 α-Thujene 15.24 0.49
2 α-Pinene 15.42 2.28
3 Camphene 15.73 0.15
4 β-Pinene 16.37 0.52
5 3-Octanone 16.65 0.82
6 β-Myrecene 16.80 0.91
7 3-Octanol 17.00 0.20
8 α-Phellandrene 17.56 0.15
9 α-Terpinene 18.15 1.20
10 p-Cymene 20.31 16.13
11 Limonene 20.56 0.65
12 1,8-Cineole 21.76 0.92
13 β-ocimene 21.86 0.08
14 γ-Terpinene 22.07 2.43
15 Trans-sabinene hydrate 23.44 0.19
16 Linalool 24.69 6.92
17 Hotrienol 25.75 0.11
18 Borneol 26.84 0.42
19 4-Trpineol 29.16 0.81
20 α-Trpineol 30.63 0.64
21 Thymol methyl ether 32.11 1.51
22 Carvacrol methyl ether 33.13 2.64
23 Thymol 35.88 20.48
24 Carvacrol 36.23 29.61
25 Thymol acetate 37.52 0.13
26 Carvacryl acetate 39.05 0.15
27 β-caryophyllene 41.52 2.37
28 Aromadendrene 42.77 1.18
29 α-humulene 43.23 0.13
30 Allo-Aromadendrene 43.89 0.33
31 Ledene 45.05 0.56
32 Spatulenol 47.24 0.58
33 Caryophyllene oxide 47.96 1.34
Total: 98.03
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500 mg/L of both cumin essential oil (Barij Essen, Iran)
and thyme essential oil (Barij essence, Iran). Based on
the sensory evaluation results, an optimal concentration
for each of the EOs was selected.
Tables 1 and 2 demonstrate results of the
composition analysis of thyme and cumin EOs. The
analysis was carried out by Barij Essence Company
(Iran) by means of gas chromatography-mass
spectrometry (GC-MS).
The burgers were subdivided into two groups.
One group was packaged in high density polyethylene
Microbiological analyses. Twenty five grams
of burger sample was added into 225 mL of sterile
peptone water and blended using a Stomacher lab
blender (Interscience Bag Mixer, China) for 1 min.
Homogenates of various concentrations were prepared
for the microbial test. Cultured Plate Count Agar (PCA)
(Merck, Darmstadt, Germany) was incubated at 7°C for
10 days for psychrotrophic bacteria count and at 30°C
for 48 h for total viable count (TVC) [26]. Lactic acid
bacteria (LAB) were determined on de Man Rogosa
Sharpe Agar (MRS) (Q Lab, Canada) incubated at 30°C
for 72 h [27]. Sulfite-reducing clostridia were grown
on Sulphite Polymyxin Sulfadiazine Agar (Merck,
Darmstadt, Germany) [28] incubated at 30°C for 48 h
in a plastic anaerobic AnaeroGen sachet (Anaerobic
gas pack A, Merck, Darmstadt, Germany). All
microbiological analyses were performed in triplicate,
Table 2 Composition of cumin essential oil
Number of
components
Component Retention
time, min
Amount,
%
1 β-Pinene 9.362 10.52
2 β-Myrcene 9.796 0.75
3 δ-3-Carene 10.30 0.36
4 α-Terpinene 10.523 1.20
5 o-Cymene 10.855 16.03
6 Phellandral 10.912 0.33
7 1,8-Cineole 10.975 4.90
8 γ-Terpinene 11.856 20.89
9 α-Thujene 12.640 0.21
10 Terpinene-4-ol 15.593 0.75
11 Cuminlaldehyde 17.338 38.48
12 Carvacrol 18.872 0.20
13 Trans-β-Farnesene 22.717 0.26
14 Caryophyllene oxide 25.881 0.10
15 Carotol 26.201 0.58
16 Trans-Caryophyllene 27.870 0.19
Total: 95.75
Table 3 Experimental design of burgers with thyme and cumin
EOs in air and vacuum packaging
Sample Packaging
Air packaging
(AP)
Vacuum
packaging (VP)
Control (without EOs)
With thyme EO, mg/L
With cumin EO, mg/L
–
500
500
–
500
500
y = β0 +Σ βi Xi+ Σ βii Xi2+ ΣΣi<j βij XiXj
k
i=1
k
i=1
% Cooking yield = cooked weight
raw weight × 100 (2)
% Shrinkage = ( raw thickness− cooked thickness)+ (raw diameter− cooked diameter)
raw thickness+ raw diameter × 100 (3)
% Moisture retention = (cooked weight× % moisture in cooked burger)
raw weight× % moisture in raw burger × 100 (4)
(3)
y = β0 +Σ βi Xi+ Σ βii Xi2+ ΣΣi<j βij XiXj
k
i=1
k
i=1
% Cooking yield = cooked weight
raw weight × 100 (2)
% Shrinkage = ( raw thickness− cooked thickness)+ (raw diameter− cooked diameter)
raw thickness+ raw diameter × 100 (3)
% Moisture retention = (cooked weight× % moisture in cooked burger)
raw weight× % moisture in raw burger × 100 (4)
(4)
(HDPE) bags under vacuum, and another group – in
bags (aerobically), six burgers in each bag. Each group
included control, thyme EO and cumin EO samples.
The packaged burger samples were stored at 2°C for
27 days. Microbiological and chemical evaluation of
three different batches was carried out on day 0, 3, 6, 9,
12, 15, 18, 21, 24, and 27 of storage.
Sensory analysis. Sensory evaluation was performed
by a panel of seven experienced (laboratory-trained)
judges. To optimise the fish burger formulation, the
panellists were asked to evaluate taste, colour, aroma,
and overall quality of burgers on a nine-point scale. The
scale points were: excellent, 9; very good, 8; good, 7;
acceptable, 5–6; unacceptable, 1–4 [23].
Proximate composition. Protein, moisture, ash and
fat contents were measured by AOAC method [24].
Cooking characteristics. The thickness and
diameter of raw burgers were estimated at room
temperature. The burgers were fried in sunflower oil at
170°C for 5 min until an inner temperature of 72°C was
reached [25]. Cooking yield, shrinkage and moisture
reyt e=n tβio0n + wΣereβ di eXteir+m Σinedβ biiy X thi2e +fo ΣlloΣwi<inj βgi je qXuiXatjions:
k
i=1
k
i=1
% Cooking yield = cooked weight
raw weight × 100 (2)
% Shrinkage = ( raw thickness− cooked thickness)+ (raw diameter− cooked raw thickness+ raw diameter % Moisture retention = (cooked weight× % moisture in cooked burger)
raw weight× % moisture in raw burger × 100 (2)
and results were expressed as logarithm colony forming
unit (log CFU)/g sample.
Mold and yeast were counted on Yeast Extract Agar
(Merck, Darmstadt, Germany) incubated at 25°C for
5 days [29]. The experiment was performed in duplicate.
Chemical analysis. pH value was determined
using a digital pH meter on the first homogenised
concentration of samples (Sartorius, USA) [30]. Total
volatile base nitrogen (TVB-N) content was quantified
by the method of Malle and Poumeyrol [31], while
thiobarbituric acid (TBA) amount was calculated by the
method of Tsironi et al. [32].
Peroxide value (PV) was determined according to the
method described by AOAC [33]. All chemical analyses
were performed in triplicate.
Statistical Analysis. Statistical analysis was carried
out with the help of SPSS 19 (SPSS, 2010) software
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and one-way variance. Results were expressed as
mean values and standard deviation (S.D.). Analysis of
variance (ANOVA) data were subjected to determining
significant differences (P < 0.05).
RESULTS AND DISCUSSION
Sensory analysis. Average scores of sensory
characteristics were evaluated using RSM method. The
results of the analysis are shown in Table 4. The optimal
burger formulation was selected, which contained 63% of
surimi and 37% of minced chicken meat. Also, based on
average scores of sensory evaluation, a concentration of
500 mg/L for each EO was selected as optimal (Table 5).
Proximate analysis. Proximate composition was
performed in burgers made without EOs before storage.
Samples had moisture of 70.40% and contained 19.98%
of protein, 4.27% carbohydrate, 3.35% fat, and 2.0% ash.
Our results are in good agreement with those obtained
by Vanitha et al. [34].
Cooking characteristics. The cooking
characteristics of samples with no EOs were determined
before storage. Cooking yield, shrinkage, and moisture
retention were found to be 94.73, 10.19, and 80.98%,
respectively. These data are in accordance with those
of Heydari et al., who measured cooking properties in
camel burgers during freezer storage [25].
Microbiological analysis. Analysis of variance
showed that both packaging and EOs used had a
significant effect on the microbial characteristics of
burgers (P < 0.001).
Figure 1a demonstrates changes in TVC of the
burgers under study during storage. Results indicate
a significant effect (P < 0.001) of storage time, EOs
addition and packaging conditions on TVC. The
maximum TVC value obtained (107 CFU/g) was
acceptable for fresh and frozen fish [35].
The initial (day 0) TVC of burgers in air packaging,
with and without the EOs, was 4.05–4.38 log CFU/g.
For burgers in vacuum packaging, with or without
cumin/thyme EO, these values were 4.46–4.82 log
CFU/g. These results are consistent with those obtained
by Cózar et al. for fish burgers (4 log CFU/g) and
indicate a good burger quality [36]. Eventually, by day
27, TVC was 8.39–8.78 and 6.13–6.74 log CFU/g in air
packaged and in vacuum packaged burgers, respectively.
As one can see in Fig. 1a, burgers with thyme EO
in vacuum packaging demonstrated the least microbial
growth, which indicates inhibitory properties of
thyme EO. Similar results were found in an edible film
containing 0.10% of oregano and 0.15% of thyme EO in
fresh chicken sausages [17, 21].
Initial counts of psychrotrophic bacteria in samples
in air and vacuum packaging were 4.34–4.76 log CFU/g,
which reached 7.04–8.79 log CFU/g by day 27 of storage
(Fig. 1b).
Kilinc et al. observed an increase in TVC and
psychrotrophic bacteria count in sardine patties from
2.50 and 2.60 log CFU/g to 6.72 and 6.98 log CFU/g on
day 7 of storage [37]. According to Pavelková et al., the
initial TVC value in control chicken breast was 4.72 log
CFU/g, while after 18 days of storage at 4 ± 0.5°C, it was
3.68 and 4.05 log CFU/g for samples with oregano and
thyme EOs in vacuum packaging [38].
In our research, thyme EO acted as a synergist to
vacuum packaging, combinations of air packaging +
cumin EO and air packaging + thyme EO were less
effective in inhibiting microbial growth. Soni et al. also
reported lower psychrophilic bacteria counts in chicken
patties containing 0.10% of oregano and 0.15% of thyme
Eos [21]. Similar results were obtained by Sharma et al.
in fresh chicken sausages during storage [17].
This inhibitory effect was also apparently due to
large amounts of phenolic substances and flavonoids in
thyme and cumin EOs.
Initially, a lacto acid bacteria (LAB) amount was
3.16 log CFU/g. By the end of the storage, it was
recorded to be 7.47–7.98 for burgers in air packaging
and 4.15–4.40 log CFU/g for those in vacuum packaging
(Fig. 1c). In [39], the initial LAB concentration in control
minced goat meat was 2.75 log CFU/g, which increased
to 6 log CFU/g by the end of vacuum storage at 4°C.
Also, Fratianni et al. reported that thyme essential oil
decreased total viable bacteria count and lactic acid
bacterial growth in chicken breast; total microbial
content reduced down to 50% compared to the control
samples [40].
In the work of Pavelková et al, the LAB count in a
control chicken breast fillet was within the range from
4.31 (day 3) to 2.62 log CFU/g (day 15), while the best
result was observed in the vacuum packing + thyme EO
Table 4 Average scores of sensory characteristics of burger
samples (surimi percentages predicted by RSM)
Surimi, % Colour Taste Aroma Texture Overall quality
50 7.17 6.00 7.57 5.43 6.14
100 7.57 5.14 8.29 7.71 5.43
75 7.83 5.57 7.00 6.14 5.71
0 7.85 5.71 7.14 6.86 5.71
100 7.42 5.29 8.14 7.57 5.57
0 7.71 5.57 7.28 6.71 5.85
25 7.30 5.57 8.28 5.43 5.71
Table 5 Average scores of sensory characteristics of burger
samples for selecting proper concentrations of cumin and
thyme EOs
Essential oil Concentration, mg/L Taste Aroma
Cumin EO 100 5.71 7.71
300 6.43 7.29
500 6.43 8.14
Thyme EO 100 6.86 7.00
300 7.14 7.71
500 7.00 8.14
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group (the highest count was 4.29 log CFU/g, on day 3,
and the lowest count was 1.43 log CFU/g, on day 6) [38].
The authors found that addition of 0.20% (v/w) of thyme
EO and storage of samples in vacuum allowed shelf life
of the chicken breast fillet to be extended.
Clearly, it can be concluded that vacuum packaging
inhibits LAB growth. Of the samples examined in this
study, the vacuum packaging + thyme EO sample had
the maximum impact on the LAB growth. LAB are one
of the main components of meat product microflora that
decreases pH of meat product through carbohydrate
fermentation [41].
We found that, due to the antibacterial properties
of cumin and thyme EOs, the shelf life of burgers with
the EOs in vacuum packaging increased. The cause of
that can be the presence of phenolic compounds such as
thymol and carvacrol in thyme and cuminaldehyde in
cumin.
In this study, initial mold and yeast counts were
approximately 2 log CFU/g and reached 6.49–6.95
and 2.03–3.08 log CFU/g in samples stored in air and
vacuum packaging, respectively (Fig. 1d). Lower mold
and yeast counts in test samples compared to control
indicates the presence of EOs antifungal constituents in
meat products [42].
As for sulfite-reducing clostridia, they were not
detected in any of the samples throughout the storage
peroid.
Chemical analysis. Figure 2a demonstrates a
significant decrease in pH values of control and treated
samples during storage (P < 0.001). The initial pH
value in burger samples was 6.41. By day 27, their pH
values were 4.34–4.53 for all samples in air packaging
and 4.71–4.98 for all samples in vacuum packaging.
This decrease can be due to a reduced oxygen
content as a result aerobic microflora growth and CO2
production. Another cause of the pH decrease can be
sugar contained in the burgers, which is utilised as a
cryoprotectant.
According to Bingol and Ergun, pH diminishes by
the end of storage [43]. They also reported that the pH
of meat is influenced by various factors however the
(a) (b)
D21 D24 D27
AP + thyme EO
VP + thyme EO
0
2
4
6
8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Mold and yeast count, log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
D21 D24 D27
AP + thyme EO
VP + thyme EO
0
2
4
6
8
10
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Total viable count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 TVN-B, mg/100 g burger
AP VP 0.2
0.4
0.6
0.8
TBA, mg MDA/kg of burger
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D 0 PV, meq /kg lipid
AP VP 0
2
4
6
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
pH
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Mold and yeast count, log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Lactic acid bacteria, log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Total viable count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
pH
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Mold and yeast count, log 10 CFU/g
Storage time, days
AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Lactic acid bacteria, log 10 CFU/g
Storage time, days
0
2
4
6
8
10
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Total viable count,
log 10 CFU/g
Storage time, days
(c) (d)
Figure 1 Effects of vacuum packaging (VP) and thyme and cumin essential oils on: (a) TVC, (b) psychrophilic count, (c) LAB,
and (d) mold and yeast count in burgers stored at 2°C
0
2
4
6
8
10
12
D0 D3 D6 D9 Psychrotrophic bacteria count,
log 10 CFU/g
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TVN-B, mg/100 g burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27
PV, meq /kg lipid
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
TVN-B, mg/100 g burger
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
PV, meq /kg lipid
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
10
15
20
25
TVN-B, mg/100 g burger
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
10
PV, meq /kg lipid
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TVN-B, mg/100 g burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27
PV, meq /kg lipid
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
(1) (2)
(3) (4) (5)
(1) (2) (3)
(4) (5)
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TVN-B, mg/100 g burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27
PV, meq /kg lipid
Storage time, days
AP AP + cumin EO AP + thyme EO
(6) VP VP + cumin EO VP + thyme EO
(6)
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
10
15
20
25
TVN-B, mg/100 g burger
AP VP 0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
10
PV, meq /kg lipid
(1) (2)
(3)
(4) (5) (6)
(1) (2) (3)
(4) (5) (6)
(1)
(2)
(5) (3)
(4)
(6)
(1) (2) (3)
(4) (5) (6)
(1) (2)
(3)
(5)
(4)
(6)
(1) (2) (3)
(4) (5) (6)
306
Rashidimehr A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 301–310
major one is lactic acid bacteria growth resulted from
lactic acid production. Similar results were also obtained
by Soni et al. in regard to chicken patties stored at
refrigerator temperature [21].
Total volatile base nitrogen (TVB-N) content is
often used as an index to determine a degree of meat
decomposition. As one can see in Figure 2b, TVB-N
values of burgers increased significantly during storage
(P < 0.001). TVN concentration was determined to be
between 5 and 25 mg N/100g [44].
TVB-N content was the highest (P < 0.001) in
samples in air packaging, which indicates that air
packaging alone, even without EOs, can significantly
increase TVB-N formation. Erkan investigated TVB-N
in vacuum-packaged filleted hot smoked rainbow trout
[45]. By day 27 of storage at 2°C, the TVB-N content
increased to 33.82 and 24.16 mg/100 g flesh in untreated
and treated with thyme EO samples, respectively. Also,
Eskandari et al. reported that a TVB-N value in fish
samples treated with black cumin remained below its
acceptable limit by day 27 [46].
According to hygienic standards, the TVB-N
acceptable limit in fish muscle is 20 mg/100 g. Thus, the
results of this study demonstrated that TVB-N values in
vacuum packaged samples with thyme and cumin EOs
were below the limit during storage.
Fat oxidation is the main cause of fish putrefaction;
an increasing amount of thiobarbituric acid (TBA) and
peroxide leads to rancidity. A steady increase in TBA
in burgers was observed during 27 days of storage
(Fig. 2c). Vacuum packaging effectively protected the
burgers from zero days, keeping TBA scores lower
than 1 mg MDA/kg during the storage period. EOs
in the combination with vacuum packaging displayed
a positive effect on the inhibition of oxidation. Köse
et al. found that a TBA level in surimi was acceptable
up to day 15, while a TVB-N concentration reached
38.2 mg/100 g by day 13, which exceed the limit of
acceptability [5].
Karabagias et al. reported that thyme did not protect
lamb meat in air packaging from oxidation, at least
not within its normal shelf life [47]. This finding is in
contrast to the results of Botsoglou et al. who observed
a three-fold reduction in a degree of lipid oxidation in
turkey in air packaging [48].
0
2
4
6
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
pH
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Mold and yeast count, log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Lactic acid bacteria, log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Total viable count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TVN-B, mg/100 g burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27
PV, meq /kg lipid
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TVN-B, mg/100 g burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27
PV, meq /kg lipid
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TVN-B, mg/100 g burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27
PV, meq /kg lipid
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
(a) (b)
(c) (d)
Figure 2 Effects of vacuum packaging (VP) and thyme and cumin essential oils on: (a) pH, (b) TVN-B, (c) TBA,
and (d) PV of burgers at 2°C
0
2
4
6
8
10
12
D0 D3 D6 D9 D27
Psychrotrophic bacteria count,
log 10 CFU/g
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TVN-B, mg/100 g burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
0 D 3 D 6 D 9 D 12 D 15 18 21 D 24 D 27
PV, meq /kg lipid
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
TVN-B, mg/100 g burger
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
PV, meq /kg lipid
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
TVN-B, mg/100 g burger
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger Storage time, days
AP AP + cumin EO AP + thyme EO
VP + thyme EO
PV, meq /kg lipid
0
2
4
6
8
10
12
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
Psychrotrophic bacteria count,
log 10 CFU/g
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
5
10
15
20
25
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TVN-B, mg/100 g burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
0.2
0.4
0.6
0.8
D0 D3 D6 D9 D12 D15 D18 D21 D24 D27
TBA, mg MDA/kg of burger
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
0
2
4
6
8
10
D 0 D 3 D 6 D 9 D 12 D 15 D 18 D 21 D 24 D 27
PV, meq /kg lipid
Storage time, days
AP AP + cumin EO AP + thyme EO
VP VP + cumin EO VP + thyme EO
(1) (2)
(3)
(5)
(4)
(6)
(1) (2) (3)
(4) (5) (6)
(1)
(2)
(3) (5)
(4)
(6)
(1) (2) (3)
(4) (5) (6)
(1)
(2) (3)
(5)
(4)
(6)
(1) (2) (3)
(4) (5) (6)
(1)
(2)
(3)
(4) (5)
(6)
(1) (2) (3)
(4) (5) (6)
307
Rashidimehr A. et al. Foods and Raw Materials, 2019, vol. 7, no. 2, pp. 301–310
According to Liu et al., TBA increased from
0.16 mg/kg (day 0) to 0.42 mg/kg (day 35) in samples
stored at –1°C. In [45], the initial TBA index value for
hot smoked rainbow trout fillets was 0.77 mg MDA/kg
and reached 1.5 mg MDA/kg by day 27. The lower
production of TBA in vacuum packing + thyme samples
can contribute to the antioxidant properties of thyme oil.
Soni et al. noticed lower TBA values in chicken patties
containing 0.10% of oregano and 0.015% of thyme EOs.
Jayawardana et al. suggested that a cause of the reduction
of TBA values could be polyphenols present in EOs [49].
In our research, TBA values did not exceed the
acceptable limit in all samples. Similar results were
obtained by Eskandari et al. in fish treated with black
cumin [46]. Therefore, TBA cannot be used as a reliable
quality index for burgers. TBA of 2–4 mg MDA/kg
indicates a good quality of fish. TBA values in this
study were lower than 1 mg MDA/kg in all treatments
throughout the storage period. It was apparently due to a
relatively low fat content in fish (surimi).
We revealed that the antioxidant properties of cumin
and thyme EOs prolonged significantly the burger
shelf life. Sarıçoban and Yilmaz also confirmed the
antioxidant effect of cumin and thyme on TBA, which
is due to the antioxidant activity of phenolic compounds
contained in different parts of plants [44]. The main
compounds of cumin are gammaterpinene, 2-methyl-3-
phenyl-propanal, myrtenal, and glucopyranosides [44].
Figure 2d demonstrates an effect of packaging and
thyme and cumin EOs on a PV value in the burgers
under study. The initial PV value was 0.16–0.18 meq/kg
of lipid in all the burgers, while, by day 27, it reached
5.82–8.75 and 1.11–2.35 meq/kg of lipid in samples in air
and vacuum packaging, respectively (P < 0.001).
In this study, PV was increasing up to day 21 of
storage in all samples and then, by day 27, decreased.
At the end of the storage time, PV in all vacuumpackaged
samples did not reach the acceptable
limit (5 meq/kg). Similar findings were obtained by
Çoban and Keleştemur in catfish burger treated with
thyme [50]. Such findings are an evidence of EOs
inhibitory effect on microorganisms which cause
burger spoilage. The reduction of PV after day 21 can
be due to hydroperoxide degradation. The decay of
hydroperoxides results in the formation of degradation
products [51]. The reduction in PV in samples with
cumin EO can be due to cumin aldehyde, which prevents
lipid peroxidation [52].
CONCLUSION
We found that the shelf life of the novel burgers from
surimi and minced chicken meat could be extended by
using essential oils and vacuum packaging. According to
the results of the microbiological analysis, the shelf life
of the burgers was as follows: 9 days for burgers in air
packaging, 12 days for burgers with cumin and thyme
EOs in air packaging, 18 days for burgers in vacuum
packaging, and 21 days for burgers with cumin and
thyme EOs in vacuum packaging.
The shelf life for vacuum-packed burgers treated
with thyme and cumin EOs was established as 18 days
at 2°C, in compared to that for untreated burgers, which
was 6 days. In addition, vacuum packaging alone was
found to maintain burger freshness during 15 days.
Thus, burger shelf life was extended by 9 days for
the combination of thyme/cumin EO + air packaging,
15 days for vacuum-packaged samples, and 18 days
for the combination of thyme/cumin EO + vacuum
packaging. Overall, the combined use of vacuum
packaging and thyme/cumin EO demonstrated their
synergistic effect on the shelf life of the novel burgers.
These results allowed us to suggest that surimi could be
successfully used as an alternative ingredient to minced
meat in burgers production.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDEMENTS
This article was written based on the Ph.D. thesis of
Dr. Azadeh Rashidimehr. The authors wish to express
their gratitude to the research council of the Shahid
Chamran University of Ahvaz and to Mrs. P. Esfahani,
for her kind technical assistance in the food hygiene
laboratory.
FUNDING
This study was financially supported by the
Shahid Chamran University of Ahvaz (Grant
No.: 97/3/02/26247).

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