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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Aksoy, A. 114
INTRODUCTION
Meat has a wide range of nutrients and is a food preferred
by many humans around the world as a source of animal
protein. However, meat is conducive to the reproduction of
many microorganisms, and as a result it spoils very easily. It
is also sensitive to spoiling chemical and enzymatic activ-
ity. Te decomposition of the fat, protein and carbohydrates
contained in meat result in odors, favors and appearance
that is unpleasant, making it unsuitable for human consump-
tion. Terefore, meat spoilage must be controlled in order to
maintain nutritional value, texture and taste and to extend
the shelf life (1).
As supermarkets rapidly expand and develop, it is ex-
tremely important that meat be capable of being transported
long distances without spoiling or losing characteristics like
color, texture and nutritional value (2). In addition, consumers
demand for natural, delicious and highly nutritious foods has
encouraged many researchers to examine new preservative
technologies, which has resulted in traditional approaches to
preservation being replaced today by bio-preservatives and
non-thermal techniques. Furthermore, the use of chemical
preservatives is still drawing attention (3, 4, 5).
In this study, the in vitro efect was determined on se-
lected pathogenic bacteria by essential oils (Origanum onites,
Syzygium aromaticum) of some plants used as spices, water
extracts (Rhus coiaria L., Hibiscus sabdarifa L.), the natural
Te Efect of Some Natural Antimicrobial Substances on the
Shelf Life of Beef
Aksoy, A.,
1
* Sezer, Ç.,
2
Aydin, D.B.
3
and Güven, A.
4
1
Kafkas University, Faculty of Engineering Architecture, Department of Food Engineering, TR-36100 Kars – Turkey.
2
Kafkas University, Faculty of Veterinary Medicine, Department of Nutritional Hygiene and Technology, TR-36100 Kars – Turkey.
3
Diyarbakır Provincial Directorate of Food, Agriculture and Animal Husbandry Ofce of Food and Feed Diyarbakır – Turkey.
4
Kafkas University Faculty of Arts and Sciences, Biology Department, TR-36100 Kars – Turkey.
*
Corresponding Author: Dr. Aksem Aksoy. Tel: +90 474 212 3623/102, Fax: +90 474 223 9957, Email: aksemaksoy@hotmail.com
ABSTRACT
Tis study compares the efect of natural and artifcial antimicrobial substances (nisin, lysozyme, lactic
acid, trisodium phosphate, cetylpyridinium chloride and acidifed sodium chloride) with herbal extracts
(Origanum onites, Syzygium aromaticum, Rhus coriaria L. and Hibiscus sabdarifa L.) on certain pathogenic
microorganisms and investigates the feasibility of using trisodium phosphate, nisin, oregano and hibiscus to
extend the shelf life of beef. Te essential oils of oregano and clove demonstrated the strongest antimicrobial
efect on Listeria monocytogenes, Yersinia enterocolitica O3, Salmonella enteritidis and Staphylococcus aureus,
followed by water extracts of hibiscus and sumac. No statistical diference between the results of nisin group
and physiological saline solution group with control group was found during beef shelf life trials (P>0.05).
In addition, oregano and trisodium phosphate extended the shelf life by 3 days, and hibiscus by 8 days. It was
concluded that hibiscus was a signifcant antimicrobial agent, but that it was not suitable for organoleptic
reasons because using it directly on meat caused discoloration. Te essential oil of oregano, on the other
hand, could be used as an alternative to existing chemical decontaminants in order to extend shelf life and
decontaminate beef.
Keywords: Beef, shelf life, Hibiscus sabdarifa, Oregano.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 115 Shelf Life of Beef
antimicrobial agents such as lysozyme, nisin and lactic acid
(LA) beside synthetic antimicrobial constituents: trisodium
phosphate (TSP), cetylpyridinium (CPC) and acidifed so-
dium chloride (ASC). Tereafter, the efect of oregano oil,
hibiscus infusion, nisin and trisodium phosphate (TSP)
upon extending potential of shelf-life of beefs in cold stor-
age (4±1°C) was investigated.
MATERIAL AND METHODS
Bacteria cultures
Te following cultures were obtained from the Refk Saydam
Culture Collection (RSKK) Center (Ankara, Turkey) for use
in the study: Listeria monocytogenes (RSKK 475), Yersinia en-
terocolitica O3 (RSKK 920), Salmonella Enteritidis (RSKK
538), Staphylococcus aureus (RSKK 25923).
Meat samples
Te cattle beef (musculus longissimus dorsi) which was cut in
the abattoir and ofered for sell to a commercial enterprice
(in Kars, Turkey) was brought to the laboratory under cold
chain conditions. Tereafter aseptic conditions were adhered
too. Te beef was used by cutting to slices of approximately
1 cm thicknesses and 100 g in weight.
Antimicrobial compounds
In previous studies, the essential oils of some spices have been
shown to have a signifcant antimicrobial efect while for
other studies spices water extracts were efective (6, 7). Tis
study used the essential oils of the herbs oregano (Origanum
onites) and clove (Syzygium aromaticum) and the water ex-
tracts of the herbs sumac (Rhus coriaria L.) and fruits and
hibiscus (Hibiscus sabdarifa L.) fowers. Clove, sumac and
hibicus plants were obtained from a spice market in Kars
(Bağdat Ticaret Gıda Limited Şirketi Gimat Toptancılar
Sitesi, Ankara, Turkey). Te oregano EO (O. onites) was
provided by Türer Tarım Ltd., Şti. (Türer Tarım ve Orman
Ürünleri İthalat İhracat Sanayii ve Ticaret Limited Şirketi,
Kavaklıdere Köyü, Bornova, İzmir, Turkey). Te results of
previous studies and the amounts which can be used legally
were taken into consideration in determining the concentra-
tions of the decontaminant solutions. Table 1 describes the
methods by which the antimicrobial solutions were prepared.
Determination of antimicrobial activity
Each of the bacteria cultures were inoculated in Brain Heart
Infusion Broth (BHI, Oxoid CM 225, Basingstoke, UK) and
incubated for 18 hours at 30°C. Active cultures with bacterial
density of at least 1x10
6
-1x10
7
cfu/ml were used. In order to
determine the antimicrobial efects of the herbal extracts on
test microorganisms, 10 µl of 18-hour culture was added to
each test tube containing 5 ml of antimicrobial agent while
the control group contained 5 ml of physiological saline (PS).
Te test tubes were left at room temperature for 5 minutes,
1 hour and 24 hours to determine antimicrobial efcacy. At
the end of this period, decimal dilutions of each test tube
were prepared, and parallel inoculations were made into
specifc media to identify the microorganisms. Inoculation
was performed using the spread method with LSA (Listeria
Selective Agar Base, Oxoid CM 856, Basingstoke, UK) for
L. monocytogenes, YSA (Yersinia Selective Agar Base, Oxoid
CM 653, Basingstoke, UK) for Y.enterocolitica O3, BGA
(Brilliant Green Agar, Oxoid CM 329B, Basingstoke, UK)
for S. enteritidis, and BP (Baird Parker Agar Base, Oxoid
CM 0275+ Egg Yolk Tellurite Emulsion Oxoid SR 0054,
Basingstoke, UK for 24 hours at 37
°
C) for S. aureus. LSA,
Table 1: Preparation of antimicrobial solutions
Solution Preparation
Hibiscus
extract
50 ml of tap water at 90-100°C was added to 5g of
hibiscus fower. Tis was kept enclosed at 20±1°C for
half an hour and then drained.
Sumac extract 50 ml of tap water at 90-100°C was added to 5 g of
sumac fruit. Tis was kept enclosed at 45±1°C for half
an hour. Te individual pieces were crushed and then it
was drained. Te drained liquid was pasteurized for one
minute at 85°C.
Essential oil
of clove
500 ml of distilled water was added to 50 g of the
ground herb and then placed in a Clevenger device.
Te oils obtained after three hours of distillation were
put into dark-colored enclosed bottles and kept in a
refrigerator at 4°C until they were used in the trials. In
the trials, they were used at a concentration of 1.5% in
distilled water with 0.1% Tween.
Essential oil
of oregano
Te oregano EO (O. onites) was provided by Türer
Tarım Ltd.
Lysozyme 500 mg/l in distilled water
Nisin 1000 IU/ml in 0.02 N HCl
LA 2% in distilled water
TSP 12% in distilled water
CPC 0.5% in distilled water
ASC 12% in 0.9% citric acid
LA: lactic acid, TSP: trisodium phosphate, CPC: cetylpyridinium, ASC:
Acidifed sodium chloride.
Reserch Articles
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Aksoy, A. 116
BGA and BP plates were incubated at 37°C and YSA plates
at 30°C for 48 hours.
Shelf Life Trials
Essential oil of oregano and hibiscus infusion, as well as
nisin and TSP were used in the shelf life trials. Te es-
sential oil of oregano was sprayed onto the meat while
immersion was used for the infusion fuids. A total of six
trial groups were prepared for the study. Te control group
consisted of meat samples that had not been processed in
any way. One meat sample (1 cm thick weighing about 100
grams) was placed in each dish of polystyrene foam with
an absorbing pad. It was then covered with polyethylene
flm and put in cold storage at 4°C. Ten meat samples for
the PS group were put in sterile bags that contained PS
(200 ml) and after fve minutes of this treatment, the meat
samples were removed from the liquid and drained as much
as possible. Ten, they were packaged in the same way as
the control group and put in cold storage at 4°C. Te same
experimental arrangement set up for PS was prepared, but
instead of PS a nisin group was formed using a nisin so-
lution (1000 IU/ml), a TSP group using a TSP solution
(12%) and a hibiscus group using a hibiscus infusion (10%).
In the oregano group, application was made by spraying
instead of immersion because essential oil of oregano was
used to prevent the essential oils from having a negative
efect on the smell and taste of the meat as they are more
intense than the infusion solutions. Besides, methods of
obtaining the essential oil are difcult and costly. After 5
ml of essential oil of oregano with a concentration of 1.5%
was sprayed on absorbent pads in distilled water with 0.1%
Tween 80, one meat sample was placed on each pad, and
then they were packaged in the same way as the control
group and put in cold storage (4±1°C). Microbiological,
chemical and organoleptic analysis was conducted on all
of the groups on the following days of cold storage: 0, 3, 7,
8, 9, 10, 11, 12 and 17 days.
Microbiological Analysis
Ten grams of the samples were weighed out under aseptic
conditions and placed in sterile “Stomacher” bags for the
microbiological analysis. Ten, 90 ml of sterile physiological
saline was added and homogenized for two minutes. After
decimal dilutions of the samples had been prepared, they were
inoculated on media specifc to that group of microorganisms
using the spread and pour plate techniques. Incubation was
performed as follows: for total mesophilic aerobic bacteria,
Plate Count Agar (PCA, Oxoid CM 325, Basingstoke, UK)
for 48 hours at 30°C; for total psychrotrophic aerobic bacte-
ria, Plate Count Agar (PCA, Oxoid CM 325, Basingstoke,
UK) for 10 days at 7°C; for Pseudomonas spp., Pseudomonas
Agar Base (Oxoid CM 559,Basingstoke, UK ) and C-F-C
Supplement (Oxoid SR 103, Basingstoke, UK) for 48 hours
at 30°C; for lactic acid bacteria (LAB), de Man Rogosa
Sharpe Agar (MRS, Oxoid CM 361, Basingstoke, UK)
for 3-5 days at 30°C; for Enterobacteriaceae,Violet Red Bile
Glucose Agar (VRBG, Oxoid CM 485, Basingstoke, UK)
for 48 hours at 35°C; for coliform group bacteria, Violet Red
Bile Lactose Agar (VRBL, Oxoid CM 107, Basingstoke,
UK) for 24 hours at 37°C; for Fecal Coliform Group
Bacteria, Violet Red Bile Lactose Agar (VRBL, Oxoid CM
107, Basingstoke, UK) for 24-48 hours at 44.5°C; for S. au-
reus, Baird Parker Agar (BP Oxoid CM 0275+ Egg Yolk
Tellurite Emulsion Oxoid SR 0054, Basingstoke, UK ) for
24 hours at 37
°
C; for Enterococci, Slanetz and Bartley Agar
(SB, CM 0377, Basingstoke, UK ) for 24 hours at 37°C;
for Brochotrix thermosphacta, STAA Agar (Oxoid CM0881,
Basingstoke, UK ) for 24-48 hours at 25°C (8, 9).
Physicochemical Analysis
Te Eber’s reagent was used to qualitatively determined
spoilage of the samples (10, 11). Two to three ml of pre-
pared Eber’s reagent was added to test tubes. A meat sam-
ple about the size of a chickpea (1x1cm) was inserted into
the test tube using forceps and kept there for few seconds
without coming into contact with the reagent. Te emis-
Table 2: Organoleptic evaluation form
Evaluation Criteria
Scoring:
1: Poor
9: excellent
Te aroma arisen after addition of antimicrobial agent to
the meat
Te efect of antimicrobial agent to the natural color of
the meat
Signs of spoiling or
putrefaction in meat
Odor of the meat
Color of the meat
Stickiness of meat
Structural degeneration
Efect of the antimicrobial agent on the general
organoleptic characteristics of the meat
Reserch Articles
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 117 Shelf Life of Beef
sion of smoke was accepted as a positive reaction (12).
Te pH values of meat homogenates from each group
were measured using a pH meter (Termo-Orion 3 Star,
Termoscıentıfıc-Singapore).
Organoleptic Analysis
Organoleptic analyses were performed by four female indi-
viduals of 30 to 35 years of age who were working in depart-
ment of food hygiene and technology. Te criteria used as the
basis of the organoleptic assessment and the rating system
are provided in Table 2. A nine-point hedonic scale (1: poor;
9: excellent) was used in the evaluation (13).
Statistical Analysis
Antimicrobial efcacy trials were repeated at fve diferent
times, shelf life trials were repeated three times and their aver-
ages were calculated and transformed to log
10
base. Te data was
analyzed using one-way analysis of variance (ANOVA). Test
of homogeneity of variances was carried out. Te LSD (Least
Signifcant Diference) test was used to determine diference
between the groups. Statistical analyses were conducted with the
SPSS (SPSS 16.0, Inc., Chicago, IL, USA) package program. P
value< 0.05 was considered to be statistically signifcant.
RESULTS
Antimicrobial efcacy trials
Te in vitro antimicrobial efcacy of natural and artifcial
antimicrobial agents against four tested pathogens was de-
termined at diferent intervals. Te in vitro antibacterial ef-
fcacy of the antimicrobial agents against the test bacteria are
provided in Tables 3, 4, 5 and 6. Te TSP, CPC, LA and nisin
Table 3 :Antibacterial efcacy of the trial groups against Y. enterocolitica
Bacteria counts (log cfu/ml) (mean ± SD) in the trial groups
Period Control TSP CPC LA ASC Nisin Lysozyme Oregano Clove Sumac Hibiscus
5
th
minute 6.36±0.01
a
<1
b
<1
b
<1
b
3.60±0.09
c
<1
b
6.32±0.13
a
<1
b
<1
b
4.12±0.07
d
<1
b
1
st
h 6.58±0.01
a
<1
b
<1
b
<1
b
2.55±0.07
c
<1
b
4.58±0.23
d
<1
b
<1
b
<1
b
<1
b
24
th
h 6.87±0.01
a
<1
b
<1
b
<1
b
<1
b
<1
b
3.51±0.16
c
<1
b
<1
b
<1
b
<1
b
Table 4: Antibacterial efcacy of the trial groups against S. aureus
Bacteria counts (log cfu/ml) (mean ± SD) in the trial groups
Period Control TSP CPC LA ASC Nisin Lysozyme Oregano Clove Sumac Hibiscus
5
th
minute 6.87±0.01
a
4.26±0.08
b
<1
b
<1
b
6.60±0.28
d
3.47±0.29
e
6.73±0.40
ad
<1
b
<1
b
3.76±0.13
f
6.69±0.04
ad
1
st
h 6.91±0.01
a
<1
b
<1
b
<1
b
4.79±0.12
c
<1
b
6.50±0.04
a
<1
b
<1
b
3.47±0.12
d
4.73±0.11
c
24
th
h 8.12±0.04
a
<1
b
<1
b
<1
b
1.82±0.27
c
<1
b
4.69±0.32
d
<1
b
<1
b
<1
b
<1
b
Table 5: Antibacterial efcacy of the trial groups against L. monocytogenes
Bacteria counts (log cfu/ml) (mean ± SD) in the trial groups
Period Control TSP CPC LA ASC Nisin Lysozyme Oregano Clove Sumac Hibiscus
5
th
minute 7.02±0.02
a
0.94±0.87
b
<1
b
<1
b
6.75±0.17
a
3.44±0.09
d
6.90±0.08
a
<1
b
<1
b
<1
b
6.70±0.36
a
1
st
h 7.11±0.01
a
<1
b
<1
b
<1
b
5.45±0.24
c
<1
b
6.53±0.06
d
<1
b
<1
b
<1
b
5.17±0.13
e
24
th
h 6.14±0.01
a
<1
b
<1
b
<1
b
2.34±0.24
c
<1
b
<1
b
<1
b
<1
b
<1
b
<1
b
Table 6: Antibacterial efcacy of the trial groups against S. typhimurium.
Bacteria counts (log cfu/ml) (mean ± SD) in the trial groups
Period Control TSP CPC LA ASC Nisin Lysozyme Oregano Clove Sumac Hibiscus
5
th
minute 6.61±0.01
a
<1
b
<1
b
<1
b
6.58±0.11
a
<1
b
6.60±0.02
a
<1
b
<1
b
4.83±0.08
c
<1
b
1
st
h 6.53±0.04
a
<1
b
<1
b
<1
b
6.56±0.23
a
<1
b
6.55±0.03
a
<1
b
<1
b
<1
b
<1
b
24
th
h 6.71±0.02
a
<1
b
<1
b
<1
b
2.87±0.06
c
<1
b
4.40±0.22
d
<1
b
<1
b
<1
b
<1
b
abcd:
Tose with a diferent superscript from the average on the same line were statistically diferent (p<0.05)
LA: Lactic acid; TSP: trisodium phosphate; CPC: cetylpyridinium; ASC: Acidifed sodium chlorite
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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Aksoy, A. 118
solutions tested at the end of the fve-minute waiting period
applied during the trials completely inhibited strains of Y.
enterocolitica and S. typhimurium. While CPC and LA solu-
tions completely inhibited strains of S. aureus and L. monocy-
togenes, TSP and nisin achieved a signifcant reduction in S.
aureus and L. monocytogenes counts, demonstrating a signif-
cant diference over the control group (p<0.05). Lysozyme,
however, was not efective against any of the tested strains
(p>0.05). ASC, on the other hand, reduced the counts of Y.
enterocolitica and S. aureus strains (p<0.05), but had no sig-
nifcant antimicrobial efect against L. monocytogenes and S.
typhimurium (p>0.05).
When the antimicrobial efcacy of herbal extracts was
evaluated, the essential oils of oregano and clove had a sig-
nifcant antimicrobial efect on all of the bacteria tested at
the end of the 5-minute waiting period (p<0.05). While the
sumac infusion completely inhibited the L. monocytogenes bac-
teria, a signifcant reduction was also observed with the other
test bacteria (p<0.05). Te hibiscus infusion had a signifcant
antimicrobial efect against Y. enterocolitica and S. typhimuri-
um bacteria (p<0.05). However it was not efective against S.
aureus and L. monocytogenes in the frst 5 minutes (p>0.05), but
was efective at the end of 1 and 24 hours periods (p<0.05).
Beef shelf life trials
Te results of microbiological analysis conducted on diferent
days for the six groups, which included the control group and
the PS group, are provided in Figures 1-8.
On the frst day (day 0) total mesophilic bacteria count in
the control group nisin and TSP samples was determined to
be 3.63 log cfu/g, whereas the hibiscus and oregano groups
were found to be under the detectable limit (<1 log cfu/g)
(P<0.01). On day seventeen of cold storage, the total me-
sophilic bacteria count was lowest in the hibiscus group
4.82 log cfu/g), while the control group, nisin, TSP and
oregano groups were 11.42, 11.19, 8.31, and 8.40 log cfu/g,
respectively.
On the initial day (day 0) total psychrotrophic bacte-
ria count in the control group, nisin and TPS was 2.75 log
cfu/g, TSP, whereas in the hibiscus and oregano groups it
was found to be below the detectable limit (<1 log cfu/g)
(P<0.01). On day 17 of cold storage, the total psychrotrophic
bacteria count of the meats treated with hibiscus was found
to be 4.50 log cfu/g. On the frst day (day 0) Pseudomonas
spp. Total counts in the control group was 2.50 log cfu/g,
and the other groups were found to be under the detectable
limit (<1 log cfu/g) (P<0.01). In samples from the hibiscus
group on day 17, the Pseudomonas spp. total count was 4.43
log cfu/g while samples from the control, nisin, TSP and
oregano groups were 11.15, 10.95, 8.10 and 8.20 log cfu/g
respectively.
On the frst day (day 0) lactic acid bacteria (LAB)
count was found to be below the detectable limit (<1 log
cfu/g) in all groups including the control group. In samples
from the control and nisin group on day 17, the LAB total
count was 9.53 and 9.41 log cfu/g while samples from the
Figure 1: Total mesophilic bacteria count (log 10 cfu/g) during cold
storage (4±1°C).
Figure 2: Psychrotrophic bacteria count (log 10 cfu/g) during cold
storage (4±1°C).
K: control group; H: hibiscus group; N: nisin group; T: trisodium phosphate group; Ke: oregano group; F: physiological saline group
Reserch Articles
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 119 Shelf Life of Beef
Figure 3: Pseudomonads bacteria count (log 10 cfu/g) during cold
storage (4±1°C).
Figure 4: Lactic acid bacteria count (log 10 cfu/g) during cold storage
(4±1°C).
Figure 5: Brochotrix thermosphacta bacteria count (log 10 cfu/g) during
cold storage (4±1°C).
Figure 6: Enterobacteriaceae bacteria count (log 10 cfu/g) during cold
storage (4±1°C).
Figure 7: Coliform bacteria count (log 10 cfu/g) during cold storage
(4±1°C).
Figure 8: Staphylococcus aureus bacteria count (log 10 cfu/g) during
cold storage (4±1°C).
K: control group; H: hibiscus group; N: nisin group; T: trisodium phosphate group; Ke: oregano group; F: physiological saline group
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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Aksoy, A. 120
TSP, the hibiscus and oregano groups were found to have
total counts of 7.51, 4.20 and 7.55 log cfu/g respectively
(P>0.05). On the frst day (day 0) B. thermosphacta the to-
tal count in the control group was 3.32 log cfu/g, and in
the other groups it was below the detectable limit (<1 log
cfu/g). On the initial day (day 0) Enterobacteriaceae total
count was found to be below the detectable limit (<1 log
cfu/g) in all groups. In samples from the control and nisin
group, the Enterobacteriaceae total count was 5.92 and 5.64
log cfu/g respectively on day 17. In samples from the TSP
group, the total count was 3.41 log cfu/g, but it was below
the detectable limit (<1 log cfu/g) in the hibiscus and oreg-
ano groups, resulting in a signifcant diference compared
to the other groups (P<0.01). On the frst day (day 0) coli-
form counts were under the detectable limit (<1 log cfu/g)
in all groups just as was the case with Enterobacteriaceae.
On the initial day (day 0), day 12 and day 17 S. aureus
counts were under the detectable limit (<1 log cfu/g) in
all groups. No microorganisms from the fecal coliform or
enterococci groups could be isolated in any of the control
or trial groups.
Physicochemical Changes
According to the results of the Eber’s test conducted on the
groups, the meat samples in the control, PS and nisin groups
were spoiled on day 7, the meat samples from the TSP and
oregano groups on day 11. Te test results conducted on day
17 for the hibiscus group were negative. Te pH values of the
samples treated with TSP and hibiscus difered signifcantly
from that of the control group (p<0.01). Te pH values of
the samples were recorded for the duration of cold storage
and are presented in Figure 9.
Organoleptic Analysis
When the efect of the antimicrobial substances used in the
groups was examined, TSP, hibiscus and nisin did not afect
the odor of the meat at all, while in the oregano group, the
specifc aroma of oregano was intense, especially during the
frst days of storage. In subsequent days, oregano oil was al-
most non-detectable because of its volatile nature and was
judged to have no efect that would bother the consumer. An
examination of the efect that the antimicrobial substances
used in the groups had on the color of the meat showed that
TSP, nisin and oregano did not afect the color of the meat,
but in the hibiscus groups, the pale reddish color of hibiscus
was pronounced at the beginning but towards the end of
the storage period, the meat samples developed a dark red
or purple color. On the eighth day of storage, spoilage and
putrefaction had commenced in the control, PS and nisin
groups. Tere was discoloration and the meat samples which
developed a sticky surface. Samples from the nisin group
were spoiling on day 8 and on day 12 in the TSP and oregano
groups while the hibiscus group showed no evidence of spoil-
age even on day 17.
DISCUSSION
Tis study examined the efectiveness of some natural and ar-
tifcial antimicrobial substances (nisin, lysozyme, lactic acid,
trisodium phosphate, cetylpyridinium chloride and acidifed
sodium chloride) as well as some herbal extracts (Origanum
onites, Syzygium aromaticum, Rhus coriaria L. and Hibiscus
sabdarifa L.), consumed as spices or teas, as antibacterial
agents against food-based pathogenic microorganisms. Te
feasibility of using hibiscus infusion, nisin, trisodium phos-
phate and essential oil of oregano, which all had a signif-
cant antimicrobial efect, to extend the shelf life of beef were
deemed promising and the study was continued in this di-
rection. Te antimicrobial efects of essential oils from herbs
have been examined in several in vitro studies (3, 7, 14). From
this research, it was clear that very diferent results could be
obtained. Tese diferences may be attributed to the type of
essential oil, its composition and concentration, the type and
number of microorganisms it afects, the composition of the
substrate and storage conditions (15).
Figure 9: pH values during cold storage (4±1°C).
K: control group; H: hibiscus group; N: nisin group; T: trisodium
phosphate group; Ke: oregano group; F: physiological saline group
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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 121 Shelf Life of Beef
In this study, 1.5% concentration of oregano and 1.5%
concentration of clove oil had a signifcant antibacterial ef-
fect against all of the tested bacteria. Hammer et al. (14),
reported that only clove oil in concentrations > 2% was ef-
fective against S. typhimurium and against S. aureus bacteria
at concentrations < 2%. Baydar et al. (7), reported that even
0.5-1% concentrations of oregano oil were quite efective
against pathogens. Tis study found that the hibiscus water
extract inhibited all of the test bacteria at the end of the
24-hour period. Wong et al. (16), reported that hibiscus ex-
tract had a broad spectrum efect against Gram positive and
Gram negative bacteria, and that it was important for future
research as a natural antimicrobial agent. It has been claimed
that TSP is more efective against Gram negative pathogens
like Salmonella, Campylobacter and E. coli than it is against
Gram positive bacteria such as L. monocytogenes (17). In this
in vitro study, it exhibited a signifcant efect against Gram
negative bacteria (Y. enterocolitica and S. typhimurium), inhib-
iting them completely at the end of a fve-minute period. A
study that examined the antimicrobial efect of nisin in beef
showed that it provided an initial reduction but that its efect
weakened over time (18, 19). On its own, nisin failed to pro-
vide efective preservation, but it is said to be more efective
when combined with other compounds. Govaris et al. (20),
reported that nisin at a rate of 500 and 1000 IU/g had no
efect against S. enteritidis in minced sheep meat, but that it
was efective when combined with 0.9% oregano oil. In this
study, nisin reduced the initial microorganism count but was
not efective on days 3, 5, 7, 9, 11, 12 and 17 of storage.
Pohlman et al. (21) reported that a 10% concentration
of TSP reduced the total bacteria count in beef by 0.61 log.
Özdemir (22) reported that TSP achieved a reduction of
approximately 1 log in the total bacterial count in the skin of
chicken breasts on day 0 compared to the control group and
that the Pseudomonas spp. and enterobacteriaceae count was
generally below the detectable limit (< 2.0 log
10
cfu/g) for
the duration of the storage period compared to the control
group. In this study, however, the initial total mesophilic and
psychrotrophic count in the control and PS groups was 3.63
and 1.85 log cfu/g respectively, but it was below the detect-
able limit (< 1 cfu/g) in the group treated with TSP. Tere are
diferences between the results obtained from other research,
and the results of this study, and it was considered that the
reason for this diference may be due to the initial microbial
load and application methods. After all, it has been reported
that the efect of TSP varies depending on the concentration
of the solution, temperature, length and manner of treatment
(23, 24).
In this study, the initial total mesophilic bacteria count
in the samples treated with 1.5% oregano oil was beneath
the detectable limit (< 1 cfu/g), but in the samples from the
control and PS groups, this number was 3.63 and 1.85 log
cfu/g respectively. A number of studies have been conducted
on the ability of essential oil of oregano to extend the shelf
life of food, and most of them have obtained successful re-
sults in terms of its microbial efect (9, 25, 26, 27).
Tis study found that water extract of hibiscus had the
most signifcant efect on the shelf life of beef due to its
signifcant antimicrobial characteristics. Even on day 17 of
the storage period, total mesophilic bacteria, total psychro-
trophic, Pseudomonas spp., LAB and B. thermosphacta counts
were 4.82, 4.50, 4.43, 4.20 and 3.57 log cfu/g respectively
and the Enterobacteriacea, coliform and S. aureus counts were
below the detectable level (< 1 cfu/g).
As a result of the organoleptic analysis, it was determined
that the smell of oregano was intense in the frst few days
but in subsequent days decreased so that it would not be
objectionable. Even though hibiscus had a signifcant anti-
microbial efect, the organoleptic characteristics of the meat
samples in the hibiscus group were found unacceptable due
to their color. Te water extract of hibiscus (H. sabdarifa L.)
is consumed around the world either as a hot tea or a cold
drink. It is also used as a food coloring in the production of
Roselle jelly (28). Hibiscus extract is said to be very efective
against Gram positive and Gram negative bacteria (16, 29).
In this study, the hibiscus water extract exhibited a signifcant
inhibitory efect both in vitro and in shelf life trials. Shelf
life was determined to be 8 days in the control group and 17
days for samples from the hibiscus group.
In the light of the results obtained, it was concluded that
essential oil of oregano extended shelf life signifcantly. Te
water extract hibiscus was found to be promising as a natural
antimicrobial agent that could be an alternative to synthetic
food additives that extend shelf life and consequently im-
prove the microbial quality of meat if the negative discolor-
ation it causes in meat can be eliminated. Te possibility that
the color problem that hibiscus causes in fresh meat might
not be noticeable in meat products such as salami, sausages
and wieners and there is a need for detailed research of this
issue.
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Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Aksoy, A. 122
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